Metabolic Reprogramming by Lactate Unlocks a Pro-Survival Code in MSCs: The miR-195-3p/Oct4/VEGF Axis in Heart Repair

Metabolic Reprogramming by Lactate Unlocks a Pro-Survival Code in MSCs: The miR-195-3p/Oct4/VEGF Axis in Heart Repair | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Metabolic Reprogramming by Lactate Unlocks a Pro-Survival Code in MSCs: The miR-195-3p/Oct4/VEGF Axis in Heart Repair Mengying Yu, Zhao Peng, Zhichuan Huang, Cuixia Liu, Zhanyu Deng, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8443063/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Mesenchymal stem cell (MSC) therapy shows limited efficacy for myocardial infarction, primarily due to poor cell survival under ischemic stress. While hypoxia-regulated miRNAs are implicated in MSC function, the specific role of miR-195-3p and its potential modulation through metabolic preconditioning remain unexplored. Here, we performed miRNA sequencing of hypoxic MSCs to identify key regulators. The miR-195-3p/Oct4 interaction was validated via luciferase reporter assays, qPCR, and western blotting. MSC survival, apoptosis, and angiogenic capacity were assessed under hypoxia. Rat myocardial infarction models received MSCs with modified miR-195-3p/Oct4 expression or lactate preconditioning, followed by comprehensive evaluation of cardiac function, histopathology, and metabolic remodeling. Hypoxia markedly upregulated miR-195-3p while suppressing Oct4 in MSCs. Mechanistically, miR-195-3p directly targeted Oct4, impairing MSC survival under hypoxic stress. Lactate preconditioning restored Oct4 expression and enhanced MSC resilience. In infarcted hearts, lactate-preconditioned or Oct4-overexpressing MSCs significantly improved cardiac function, reduced fibrosis, and promoted angiogenesis compared to controls—benefits abolished by Oct4 knockdown. Oct4 restoration augmented glycolytic metabolism through GLUT1/HK2 upregulation and amplified VEGF/VEGFR2/Akt signaling. Conversely, miR-195-3p overexpression suppressed glycolysis and angiogenesis, effects rescued by Oct4 co-expression. Lactate preconditioning enhances MSC therapeutic efficacy by disrupting miR-195-3p-mediated Oct4 suppression, thereby promoting metabolic adaptation and VEGF-driven angiogenesis in ischemic myocardium. Targeting the miR-195-3p/Oct4/VEGF axis represents a promising strategy to optimize MSC-based cardiac regeneration. Mesenchymal stem cells Myocardial infarction Metabolic reprogramming Lactate preconditioning miR-195-3p Oct4 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Myocardial infarction (MI) persists as a leading cause of global cardiovascular mortality and heart failure, with the irreversible loss of cardiomyocytes presenting a fundamental therapeutic challenge [1] . Mesenchymal stem cell (MSC) transplantation has emerged as a promising regenerative strategy, leveraging their multipotent differentiation capacity and paracrine secretion of angiogenic factors to restore function in infarcted myocardium [2] . However, the clinical translation of this approach has been severely limited by the hostile ischemic microenvironment, where >90% of transplanted MSCs undergo rapid apoptosis within 72 hours due to hypoxia-induced metabolic stress and inflammatory signaling [3,4] . This catastrophic cell loss fundamentally cripples their reparative potential, as sustained MSC retention is prerequisite for effective cardiac repair [5] . Enhancing MSC resilience in the post-MI microenvironment therefore represents a critical barrier to advancing stem cell-based therapies. The infarct core and border zone exhibit severe metabolic perturbations, including oxygen and glucose deprivation that forces a shift to anaerobic glycolysis with consequent lactate accumulation [6] . While traditionally viewed as a metabolic waste product, lactate is now recognized as a key signaling molecule that modulates various cellular functions [7] . However, its potential role in preconditioning MSCs to enhance their survival and regenerative capacity remains poorly understood, representing an intriguing therapeutic avenue worthy of exploration [8] . Concurrent with metabolic stress, the ischemic microenvironment triggers profound alterations in gene expression orchestrated by microRNAs (miRNAs) [9] . These small non-coding RNAs (19-25 nt) function as primary epigenetic regulators of cell fate decisions through post-transcriptional gene silencing [10, 11] . Particularly relevant to stem cell biology, miRNAs establish context-dependent regulatory circuits with core pluripotency factors such as Oct4 [12, 13] . In embryonic stem cells, for instance, miR-145 suppression sustains Oct4-mediated self-renewal, while its artificial upregulation extinguishes Oct4 expression to force lineage commitment [13, 14] . This sophisticated miRNA-transcription factor interplay creates signaling networks that dictate cellular responses to environmental stresses, though their specific configuration in MSCs under ischemia remains largely unexplored. Among hypoxia-responsive miRNAs, the miR-15 family has been implicated in various pathological processes, demonstrating context-dependent functionality through targeting multiple transcription factors [15-18] . Our preliminary systematic screening specifically identified miR-195-3p, a miR-15 family member, as the most significantly upregulated miRNA in hypoxic MSCs, while Oct4 expression was concurrently suppressed. This inverse relationship, observed against the backdrop of Oct4's documented but complex role in MSC survival under stress [19] , prompted our investigation into their regulatory interplay and functional consequences. Significant gaps persist in understanding how metabolic preconditioning interfaces with miRNA-mediated regulation of MSC resilience. While various preconditioning strategies have shown promise, the potential of lactate-a naturally abundant metabolite in the ischemic niche [20] -to modulate the miR-195-3p/Oct4 axis remains completely unexplored. Furthermore, although numerous hypoxia-responsive miRNAs have been identified, a definitive pathway linking a specific miRNA through a master regulator like Oct4 to downstream metabolic and angiogenic effectors [21] in MSCs has not been established. This study was designed to bridge these critical gaps by testing our central hypothesis that miR-195-3p impairs MSC survival under hypoxic stress by inhibiting an Oct4-driven transcriptional program that enhances glycolytic metabolism and activates VEGF signaling, while lactate preconditioning exerts protective effects by disrupting this inhibitory pathway. Through this work, we aim to elucidate a complete signaling axis connecting metabolic preconditioning with epigenetic regulation to enhance MSC-based cardiac repair. Results Hypoxia-induced miR-195-3p directly targets and represses Oct4 To identify key regulators of MSC hypoxic response, we found that 72-hour hypoxia culture significantly altered miRNA expression. Bioinformatic analysis and miRNA-seq identified miR-195-3p as the most markedly elevated miRNA ( Figure 1A ), which was independently validated by RT-qPCR ( Figure 1B ). Concurrently, we observed a significant repression of the predicted target gene Oct4 ( Figure 1C ). Specifically, hypoxia led to a 34.9% increase in miR-195-3p (p=0.001; Figure 1D ) and a 42.0% decrease in Oct4 mRNA (p<0.001; Figure 1E ), with confirmed reduction at the protein level ( Figure 1F ), demonstrating an inverse correlation. We subsequently confirmed a direct targeting relationship. A luciferase reporter assay containing the wild-type Oct4 3'‑UTR showed that miR-195-3p overexpression significantly suppressed luciferase activity, an effect rescued by a miR-195-3p inhibitor ( Figure 1G ). Functionally, miR-195-3p overexpression in MSCs markedly decreased endogenous Oct4 mRNA and protein, while its inhibition attenuated this suppression ( Figure 1H , 1I ). Collectively, these results demonstrate that hypoxia elevates miR-195-3p expression, which in turn directly targets and transcriptionally represses Oct4, identifying a pivotal regulatory axis for MSC survival under hypoxia. miR-195-3p/Oct4 axis regulates MSC survival in hypoxia To validate the functional role of the miR-195-3p/Oct4 axis in MSC survival under hypoxia, we modulated the expression of miR-195-3p or Oct4 through genetic approaches and evaluated their effects on cell viability and apoptosis. After 72 hours of hypoxia, miR-195-3p mimic reduced cell viability by 34.4% ( p =0.01) and 30.4% ( p =0.003) compared to the CON and mimic NC groups, respectively. In contrast, miR-195-3p inhibitor enhanced viability by 37.3% ( p =0.01) and 46.2% (p < 0.001), respectively ( Figure S1A , S1C ). Accordingly, miR-195-3p mimic increased apoptosis by 24.1% ( p =0.004) and 24.4% ( p =0.004), whereas the inhibitor decreased it by 18.4% ( p =0.04) and 20.8% ( p =0.010), relative to CON or mimic NC groups ( Figure S1B , S1D ). Next, we investigated that Oct4 acted as the target signal of miR-195-3p and rescued the inhibition of miR-195-3p on the survival of MSCs under hypoxic condition. We performed genetic inhibition or overexpression of miR-195-3p/Oct4 by miR-195-3p inhibitor or mimic or Oct4 overexpression ( oe Oct4) or shRNA ( sh Oct4) on MSCs, respectively. After 72h hypoxia, compared with CON group, Oct4 overexpression maintained significantly higher viability, but Oct4 shRNA resulted in sustained viability loss. Moreover, Oct4 overexpression rescued the cell viability decrease in miR-195-3p-overexpressing MSCs. While miR-195-3p inhibitor caused significant increased in MSC viability, Oct4 shRNA abrogated this increase ( Figure 2A , 2D ). Ki67 staining revealed a concordant proliferative trend: relative to the CON group, oe Oct4 markedly increased the proliferation rate under hypoxia, whereas sh Oct4 significantly reduced it. Notably, oe Oct4 rescued the proliferation deficit induced by miR-195-3p mimic. Although miR-195-3p inhibitor enhanced proliferation, this effect was abolished by concurrent Oct4 silencing ( Figure 2B , 2E ). Conversely, an opposing pattern was observed in apoptosis: compared with CON, Oct4 overexpression substantially decreased apoptosis, while sh Oct4 led to a significant increase. Moreover, oe Oct4 counteracted the pro-apoptotic effect of miR-195-3p mimic. Similarly, the anti-apoptotic outcome resulting from miR-195-3p inhibition was reversed by Oct4 knockdown ( Figure 2C , 2F ). These findings collectively suggest that the miR-195-3p/Oct4 axis regulates MSC survival under hypoxic conditions. To further investigate the apoptotic pathway associated with the miR-195-3p/Oct4 axis, we employed a human cytokine array (AAH-CYT-G2000, RayBiotech) to profile the expression of survival-related cytokines in MSCs cultured under hypoxia, with or without miR-195-3p mimic or Oct4 overexpression. The results indicated that the proliferation-related factors Hsp27 and Oct4 were expressed at higher levels in the oe Oct4 group, and oe Oct4 rescued their suppression induced by the miR-195-3p mimic. In contrast, the apoptotic factors Bax and Caspase 9 were more abundant in MSCs treated with miR-195-3p mimic compared to those with Oct4 overexpression ( Figure 2G ). Based on the antibody array results, we selected Hsp27, Oct4, Bax, Caspase 9, along with the anti-apoptotic factor Bcl-2, for validation via qRT-PCR and western blotting in MSCs receiving miR-195-3p mimic or Oct4 overexpression. This aimed to confirm the array data and evaluate whether the miR-195-3p/Oct4 axis promotes anti-apoptosis. As shown in Figure 2H , Oct4 overexpression significantly suppressed the mRNA expression of the pro-apoptotic factors Bax and Caspase 9, while enhancing the mRNA levels of the anti-apoptotic factors Bcl-2 and Hsp27, as well as the proliferation marker Oct4. Conversely, miR-195-3p mimic produced the opposite effect, which was subsequently reversed by oe Oct4 ( Figure 2H ). These trends were further corroborated by western blot analysis ( Figure 2I ). Together, these results reinforce the conclusion that the miR-195-3p/Oct4 axis modulates MSC survival under hypoxia through regulating pro- and anti-apoptotic signaling. VEGF alleviates miR-195-3p inhibition to enhance Oct4-driven angiogenesis in hypoxic MSCs Of note, the results in the above-mentioned cytokine array showed that two isoforms of VEGFs, VEGFA and VEGFD, activators of angiogenesis [22] , and their effector genes (VEGFR2, Endoglin, Tie2) were significantly upregulated or downregulated by Oct4 overexpression or miR-195-3p-mimic ( Figures 2G ), indicating concomitant regulation of anti-apoptotic signaling and angiogenesis compared to CON group MSCs. These findings suggest that Oct4 activates VEGF signaling, thereby suppressing a cascade of miR-195-3p-induced events that convert hypoxic CMs into an apoptotic state, ultimately promoting MSCs survival and angiogenesis. Thereby, we next asked if VEGF mediates these effects. For this purpose, MSCs were treated with miR-195-3p mimic/Oct4 overexpression ( oe Oct4) either alone or in combination with VEGF overexpression ( oe VEGF) or shRNA ( sh VEGF). The MSCs were then cultured for seven days under hypoxic conditions to assess the effects. As demonstrated by CCK-8 assay ( Figure 3Ab ) and Ki67 staining ( Figure 3Ba ), VEGF overexpression promoted the proliferation of MSCs and counteracted the suppressive effect of miR-195-3p mimic on MSC proliferation ( Figure 3A, 3B ). FACS analysis via PI staining further revealed that VEGF overexpression reduced apoptosis in hypoxic MSCs and attenuated the pro-apoptotic effect induced by miR-195-3p mimic ( Figure 3C ). Immunofluorescence analysis using the vascular marker VWF showed that VEGF overexpression significantly enhanced the expression of Factor VIII in hypoxic MSCs, whereas miR-195-3p mimic reduced it. Notably, this reduction was reversed upon cotreatment with VEGF overexpression ( Figure 3D ). Furthermore, as illustrated in Figures 3E-J , VEGF overexpression markedly up-regulated the mRNA levels of key vascular growth factors and receptors—including VEGF, bFGF, VEGFR2, Tie2, Endoglin, and CD31—in MSCs under hypoxic conditions. Although miR-195-3p mimic abolished these promotive effects, concurrent VEGF overexpression restored their expression. These findings were corroborated by Western blot analyses of VEGFA, VEGFR2, CD31, and Tie2 ( Figure 3K ). Collectively, these results indicate that VEGF signaling attenuates apoptosis and stimulates angiogenesis in MSCs under hypoxic conditions. Moreover, the pro-apoptotic effect of miR-195-3p is reversed by VEGF overexpression, underscoring the importance of VEGF upregulation in supporting MSC survival and vascularization under hypoxia. Subsequent experiments showed that sh VEGF suppressed MSC proliferation ( Figure 4A ) and abolished the enhancing effect of Oct4 overexpression on proliferation, as assessed by CCK-8 assay ( Figure 4E ) and Ki67 staining ( Figure 4B , 4F ). VEGF downregulation also promoted apoptosis in hypoxic MSCs and diminished the anti-apoptotic influence of oe Oct4 ( Figure 4C , 4G ). In addition, oe Oct4 markedly increased VWF expression in hypoxic MSCs, and this effect was reduced by sh VEGF ( Figure 4D , 4H ). To determine whether Oct4 activation mediates VEGF-induced proliferation and angiogenesis, we measured VEGF-related cytokine expression in MSCs subjected to Oct4 overexpression, VEGF knockdown, or both, via qRT-PCR and immunofluroscence. The data revealed that oe Oct4 elevated VEGF mRNA levels ( Figure 4I ) and potentiated VEGF pathway activity, as indicated by upregulation of bFGF (Figure 4J ), VEGFR2 ( Figure 4K ), VECAD ( Figure 4L ), Endoglin ( Figure 4M ), and CD31 ( Figure 4N ) compared to controls. Cotreatment with sh VEGF blocked Oct4-induced VEGF pathway activation and proliferation, confirming the dependency of Oct4-mediated benefits on VEGF upregulation. Western blot analysis further validated that Oct4 overexpression enhanced VEGF signaling and increased expression of the anti-apoptotic protein Bcl2, both of which were negated by VEGF shRNA ( Figure 4O ). Together, these results demonstrate that Oct4 upregulation stimulates VEGF expression, thereby reactivating the VEGF signaling axis to bolster MSC proliferation and angiogenesis under hypoxia. miR-195-3p regulates MSC fibrosis/inflammation in hypoxia via Oct4/VEGF axis We further demonstrated that Oct4 counteracts miR-195-3p-induced fibrosis and inflammation primarily through upregulation of VEGF. In hypoxic MSCs, overexpression of miR-195-3p (via mimics) aggravated hypoxia-triggered fibrotic and inflammatory responses ( Figure 5A-C ) and markedly elevated the expression of key fibrotic markers—collagen I ( Figure 5D , 5I ) and α-smooth muscle actin (α-SMA, Figure 5E , 5I )—as well as key inflammatory mediators, including IL-6 ( Figure 5F , 5I ), iNOS ( Figure 5G , 5I ), and TGF-β1 ( Figure 5H , 5I ), at both mRNA and protein levels. Conversely, overexpression of Oct4 or VEGF significantly suppressed hypoxia-induced fibrosis and inflammation in MSCs and effectively reversed the pro-fibrotic and pro-inflammatory effects driven by miR-195-3p upregulation ( Figure 5B – I ), indicating that the Oct4/VEGF axis acts as a protective pathway against these pathological processes. Moreover, knockdown of VEGF recapitulated the phenotype induced by miR-195-3p overexpression, confirming VEGF as a major functional target mediating miR-195-3p-driven effects. The Oct4/VEGF axis rescues aerobic glycolysis from miR-195-3p-mediated suppression To elucidate the molecular mechanisms through which the Oct4/VEGF axis counteracts miR-195-3p-mediated phenotypic alterations in hypoxic MSCs, we conducted transcriptome sequencing (RNA-seq) comparing miR-195-3p- overexpressing cells with control cells ( Figure 6A ). Gene Set Enrichment Analysis (GSEA) identified the top ten pathways suppressed by miR-195-3p, encompassing E2F targets, DNA repair, mitotic spindle, cytokine–cytokine receptor interaction, G2M checkpoint, VEGF signaling, glycolysis, Oct4 targets, hypoxic response upregulation, and angiogenesis ( Figure 6B ). In light of the therapeutic significance of metabolic reprogramming in tumor biology, we sought to clarify the role of miR-195-3p in modulating glycolytic flux. Metabolic assessment revealed that miR-195-3p overexpression led to a marked reduction in extracellular acidification rate (ECAR, Figure 6D , 6E ) and glycolytic proton efflux rate (glycoPER, Figure 6F , 6G ). In contrast, Oct4/VEGF overexpression not only elevated both ECAR and glycoPER but also substantially reversed the miR-195-3p-driven suppression of glycolytic activity. Concordantly, miR-195-3p mimic impeded glucose uptake and lactate generation, whereas Oct4/VEGF overexpression augmented these processes in hypoxic MSCs ( Figure 6H , 6I ). RNA from MSCs was analyzed using the Rat Genome 230 2.0 Array. After normalization, DEGs were identified (fold-change ≥1.5, p<0.05). GSEA revealed significantly altered pathways, with top results visualized in a heatmap (NES, FDR). Specifically, key glycolytic enzymes ( SLC2A1/GLUT1 , HK2 , LDHA ) were significantly downregulated (fold change ≤0.5) in miR-195-3p overexpressing cells ( Figure 6J ). Notably, VEGF exhibited a weaker restorative effect on lactate metabolism relative to Oct4 in the context of miR-195-3p inhibition. Western blot analyses corroborated these trends, showing diminished protein expression of these glycolytic enzymes under miR-195-3p mimic treatment and enhanced expression upon Oct4 overexpression ( Figure 6K ). Moreover, single-sample GSEA (ssGSEA) indicated an inverse correlation between glycolysis scores and miR-195-3p levels in MSCs ( Figure 6C ). Collectively, these data establish that miR-195-3p attenuates glycolytic metabolism in hypoxic MSCs, an effect potently mitigated by the Oct4/VEGF axis, with Oct4 playing a predominant role. Oct4-dependent rescue by lactate restores MSC therapy efficacy impaired by miR-195-3p To investigate the interplay among miR-195-3p, lactate, and Oct4 in MSC-based therapy for ischemic hearts, we induced MI in Lewis rats (250–300 g) by permanent ligation of the left anterior descending coronary artery, as previously described [5] . A cell therapy model was subsequently established, wherein MSCs subjected to various treatments—including blank control, lactate, overexpression or knockdown of miR-195-3p/Oct4 ( oe miR-195-3p/ oe Oct4/ sh Oct4), or their combinations-were transplanted into the infarcted hearts of syngeneic rats. Of the 220 rats that underwent MI surgery, 20 were randomly selected immediately post-MI to receive intramyocardial injections of either phosphate-buffered saline (PBS), vehicle-treated MSCs, or MSCs pre-conditioned with lactate, oe miR-195-3p, oe Oct4, sh Oct4, lactate+ sh Oct4, or oe miR-195-3p+ oe Oct4. All animals were monitored for 30 days. While no mortality occurred during the cell transplantation procedure, 26 rats died during the follow-up period, resulting in 133 survivors that underwent terminal echocardiography ( Figure 7G ). No significant differences in mortality were observed among the groups ( Figure 7O ). Echocardiographic assessment revealed severe impairment of left ventricular (LV) function in PBS-injected MI rats compared to those receiving MSC transplantation ( Figure 7A ). Functional parameters, including LV ejection fraction (LVEF) and LV fractional shortening (LVFS), were significantly improved in animals receiving lactate- or oe Oct4-treated MSCs compared to the vehicle-treated MSC group. Conversely, administration of oe miR-195-3p- or sh Oct4-transfected MSCs led to worse functional outcomes relative to the vehicle group. Crucially, the beneficial effect of lactate was abrogated by concurrent Oct4 knockdown (lactate+ sh Oct4 group), whereas Oct4 overexpression ( oe miR-195-3p+ oe Oct4 group) rescued the functional impairment induced by miR-195-3p overexpression ( Figure 7H , 7I ). Consistent with the functional data, TTC ( Figure 7B ) and Masson's trichrome ( Figure 7C ) staining demonstrated that infarct size ( Figure 7J ) and fibrosis area ( Figure 7K ) were significantly reduced in the vehicle-MSC group compared to the PBS control. Lactate or oe Oct4 preconditioning further attenuated these pathological changes, whereas oe miR-195-3p or si Oct4 treatment exacerbated them. Again, si Oct4 inhibited the lactate-mediated reduction in infarct size and fibrosis, and oe Oct4 counteracted the detrimental effects of oe miR-195-3p. Immunofluorescence analysis further showed that MSCs from lactate- or oe Oct4-treated hearts exhibited a higher survival rate ( Figure 7D , 7L ) and a lower apoptosis rate ( Figure 7E , 7M ) than those from vehicle-, oe miR-195-3p-, or sh Oct4-treated animals. Oct4 manipulation effectively modulated the cellular effects induced by miR-195-3p or lactate. Finally, angiogenesis, assessed by CD31-positive staining, was enhanced in the vehicle-MSC group compared to the PBS group. The most robust angiogenesis was observed in the oe Oct4 group, followed by the lactate group, while it was significantly suppressed in the oe miR-195-3p and sh Oct4 groups ( Figures 7F , 7N ). Oct4 overexpression or knockdown respectively rescued or inhibited the angiogenic effects altered by oe miR-195-3p or lactate. In summary, our findings indicate that lactate enhances MSC survival and myocardial repair in an Oct4-dependent manner. In contrast, miR-195-3p exerts inhibitory effects on MSC therapy, which can be effectively reversed by Oct4 overexpression, indicating that miR-195 downregulation is necessary for Oct4's pro-angiogenic effect. Oct4 and miR-195-3p orchestrate metabolic reprogramming through VEGF. To investigate how Oct4, miR-195-3p, and metabolic reprogramming coordinately promote angiogenesis, we assessed the expression of key pathway components in myocardial tissue. As depicted in Figures 8A and 8D , rats receiving vehicle-MSCs showed higher Oct4 mRNA and protein levels than PBS-treated controls. Lactate preconditioning or oe Oct4 further increased Oct4 expression relative to the vehicle‑MSC group, whereas oe miR-195-3p or sh Oct4 significantly suppressed it. Notably, sh Oct4 abolished the lactate-mediated induction of Oct4, while oe Oct4 reversed the inhibitory effect of oe miR-195-3p. A parallel trend was observed for the metabolic markers GLUT1 (Figure 8B , 8D ) and HK2 ( Figure 8C , 8D ), whose expression rose in the vehicle-MSC group, was further elevated by lactate or oe Oct4, and was reduced by oe miR-195-3p or sh Oct4. Moreover, sh Oct4 blocked lactate-induced upregulation of GLUT1 and HK2, and oe Oct4 rescued their suppression by oe miR-195-3p. To explore the link between metabolic flux and Oct4, we isolated and cultured cardiomyocytes (CMs) from rats subjected to intramyocardial injection of PBS or MSCs preconditioned with vehicle, lactate, oe miR-195-3p, oe Oct4, sh Oct4, lactate+ sh Oct4, or oe miR-195-3p+ oe Oct4. Immunofluorescence analysis revealed: (1) Oct4 expression was low in PBS controls. Relative to vehicle, Oct4 was markedly reduced in sh Oct4 and oe miR-195-3p groups (collectively termed low Oct4), but restored in the oe miR-195-3p+ oe Oct4 group. By contrast, Oct4 was upregulated in oe Oct4 and lactate groups ( high Oct4), an effect blunted in the lactate+ sh Oct4 group; (2) GLUT1 expression was strongly induced in high Oct4 groups but repressed in low Oct4 groups; (3) Concordant with its target gene expression, Oct4 displayed enhanced nuclear accumulation in high Oct4 groups, whereas in low Oct4 groups it was sparsely distributed in both nucleus and cytoplasm ( Figure 8E ). Together, these data support a model in which Oct4 directly binds regulatory regions of GLUT1 and HK2 to control their expression. We next performed ¹³C-glucose metabolic tracing ( Figures 8F - 8H ). In line with Oct4 expression changes, high Oct4 groups showed increased extracellular lactate ( Figure 8F ) and significantly reduced citrate labeling ( Figure 8G ) , both of which were reversed in low Oct4 groups. Notably, labeling of the tricarboxylic acid (TCA) cycle intermediate succinate remained unchanged ( Figure 8H ). These results suggest that Oct4 drives metabolic reprogramming by regulating GLUT1 and HK2 expression, likely through direct DNA binding. Further supporting these findings, real-time RT-PCR and immunoblot analyses of myocardial tissue showed that hearts from rats pretreated with lactate or oe Oct4 exhibited elevated mRNA and protein levels of VEGF ( Figure 8I , 8D ) and VEGFR2 ( Figure 8J , 8 D ), whereas sh Oct4 and oe miR-195-3p suppressed their expression, indicating an angiogenic state in MI hearts following lactate or oe Oct4 treatment. Given the reported role of PI3K/Akt signaling in cell survival, proliferation, and ischemia-triggered angiogenesis—and our prior work showing that Akt and VEGF act in parallel to induce angiogenesis [24] —we also evaluated Akt expression. Consistent with VEGF changes, Akt levels increased in an Oct4-dependent manner in MI hearts, with the highest expression in rats receiving oe Oct4 or oe miR-195-3p+ oe Oct4-preconditioned MSCs, intermediate levels in the lactate-preconditioned MSC group, and the lowest in the sh Oct4 group. oe miR-195-3p substantially reduced Akt expression, which was restored by oe Oct4 ( Figure 8K , 8D ). Immunohistochemical staining further confirmed these patterns, revealing strongest VEGF and Akt staining in the oe Oct4 and oe miR-195-3p+ oe Oct4 groups, moderate staining in the lactate group, and weakest in the sh Oct4 group ( Figure 8L ). Collectively, these findings demonstrate that oe miR-195-3p suppresses glycolysis and angiogenesis, and that its inhibitory effects can be partially reversed through Oct-driven metabolic reprogramming. Discussion The therapeutic potential of MSCs in treating ischemic injuries is often limited by their poor survival and functional decline within the harsh hypoxic microenvironment of damaged tissues [25] . This study defines a core molecular circuitry that dictates this critical cellular fate. We have characterized an integrated miR-195-3p/Oct4/VEGF signaling cascade that links the cellular response to hypoxia with fundamental processes governing survival, metabolic adaptation, and paracrine activity in MSCs. Our findings establish that hypoxia-induced miR-195-3p acts as a key instigator of MSC dysfunction by directly targeting and inhibiting Oct4, a central pluripotency regulator. This inhibition sets off a series of deleterious downstream events, whereas the Oct4/VEGF pathway and its associated metabolic mediator, lactate, function as crucial compensatory mechanisms that ultimately dictate the success of MSC-based therapeutic interventions. Hypoxia triggers a deterministic suppression: The role of miR-195-3p as a molecular switch The initial observation that hypoxia robustly induces miR-195-3p while concurrently repressing Oct4 pinpoints a critical upstream event in the MSC hypoxic response. Hypoxia, a known regulator of microRNA biogenesis, drives the expression of miR-195-3p, establishing it as a principal mediator within the hypoxic signaling network [26] . This induction promotes a transition in cellular state—shifting from a phenotype focused on maintenance and repair to one geared toward stress adaptation, often compromising core stem cell attributes [27] . We further provide evidence that miR-195-3p directly targets Oct4, a finding with significant biological implications. Oct4 operates not simply as a passive marker of stemness but as a master transcriptional regulator that coordinates an extensive network of genes governing self-renewal, proliferation, and cellular robustness [28] . Its suppression, therefore, represents a fundamental disruption of MSC identity and functional capacity. The miR-195-3p-mediated inhibition of Oct4 offers a mechanistic basis for the frequently documented loss of stem cell characteristics and increased apoptosis in MSCs post-transplantation into ischemic environments. We demonstrate that the consequent impairment of MSC viability under hypoxia is directly dependent on the miR-195-3p/Oct4 axis. This acquired vulnerability can be attributed to the disintegration of Oct4-dependent transcriptional programs that promote cell survival, thereby heightening susceptibility to apoptotic triggers. Oct4 loss initiates cell-autonomous dysfunction: compromised viability and metabolic arrest The downregulation of Oct4 precipitates a signaling failure that disrupts three principal downstream pathways: survival mechanisms, paracrine communication, and metabolic homeostasis [29] . This autonomous dysfunction first emerges as a viability crisis, where Oct4-deficient cells display accelerated apoptosis and impaired viability. Beyond simply increasing susceptibility to cell death, the loss of Oct4 critically undermines cellular defenses against oxidative and ischemic stress—defects that are severely amplified under hypoxic conditions [30] . Our genetic interrogation confirms Oct4 as the indispensable mediator through which miR-195-3p controls MSC survival. Overexpression of Oct4 potently enhanced cell viability and proliferation while inhibiting apoptosis, effectively counteracting the damage induced by miR-195-3p. Molecular profiling verified that Oct4 coordinates an anti-apoptotic program through the synergistic upregulation of Bcl-2 and Hsp27, coupled with suppression of Bax and Caspase 9. Simultaneously, we identified a profound metabolic arrest in which miR-195-3p directly disrupts glycolytic flux in hypoxic MSCs. Cells lacking Oct4 exhibited severe impairments in extracellular acidification, glucose uptake, and lactate production—functional deficiencies mirrored by the coordinated downregulation of crucial glycolytic enzymes (SLC2A1, HK2, LDHA). This metabolic failure creates a pathological contradiction: although hypoxia typically induces adaptive glycolysis, the concurrent surge in miR-195-3p, via Oct4 suppression, obstructs this essential metabolic shift [32] . The resulting energy deficit leaves MSCs incapable of meeting basic metabolic requirements, directly accounting for their functional decline under ischemic stress [33, 34] . Collectively, these results position Oct4 as a master integrator of cellular autonomy, synchronizing survival control with metabolic competence. VEGF executes Oct4-directed reparative functions: Orchestrating angiogenesis and microenvironmental homeostasis Our investigation delineates a vital rescue pathway whereby Oct4, countering the inhibitory influence of miR-195-3p, directs a pro-survival and angiogenic program in hypoxic MSCs largely through VEGF upregulation. Functionally, Oct4 overexpression strongly enhanced the expression of multiple angiogenic factors and receptors (VEGF, bFGF, VEGFR2, etc.), identifying VEGF as the pivotal downstream effector of Oct4-mediated vascular programming. Critically, VEGF overexpression alone was sufficient to rectify the angiogenesis and survival defects induced by miR-195-3p, whereas VEGF knockdown nullified the beneficial effects of Oct4. This definitive genetic evidence positions VEGF as the essential executor of Oct4-mediated reparative functions. The Oct4/VEGF axis further modulates microenvironmental homeostasis by regulating fibrotic and inflammatory responses. Both Oct4 and VEGF overexpression suppressed the pathological expression of collagen I, α-SMA, IL-6, iNOS, and TGF-β1, while VEGF knockdown replicated the pro-fibrotic/inflammatory phenotype seen with miR-195-3p overexpression. This expanded functional role reveals VEGF not merely as an angiogenic factor but as a central coordinator of tissue repair programming that prevents aberrant healing [35] . Contemporary research corroborates VEGF's pleiotropic roles, including direct immune modulation and fibroblast regulation [36, 37] , consistent with the anti-fibrotic and anti-inflammatory effects we observed. Significantly, metabolic rescue experiments revealed a hierarchical organization within this axis: although Oct4/VEGF restoration recovered glycolytic function, Oct4 specifically exhibited a superior capacity in reinstating lactate metabolism. This functional hierarchy highlights the sophisticated architecture of the Oct4-directed rescue network. The emerging concept of pluripotency factor repurposing is strongly supported by our demonstration that Oct4 coordinates both cell-autonomous fitness and tissue-level repair via VEGF-mediated programs [38, 39] . Furthermore, the identification of lactate as a metabolic co-effector uncovers an elegant feed-forward mechanism whereby lactate stabilizes HIF-1α to potentiate VEGF signaling [40] . Thus, VEGF acts as the central hub of a refined repair circuit, translating Oct4 directives into coordinated tissue regeneration. Metabolic reprogramming via an Oct4-lactate circuit is essential for MSC therapeutic efficacy The growing discipline of stem cell metabolism has underscored the pivotal role of metabolic rewiring in determining the therapeutic effectiveness of transplanted cells [42] . Our results place the transcriptional regulator Oct4 at the center of a lactate-driven metabolic reprogramming that is indispensable for MSC survival and function in the ischemic myocardium. This research reveals a sophisticated regulatory pathway wherein lactate, beyond its role as a metabolic by-product, functions as a signaling molecule that reinforces an Oct4-dependent pro-survival and pro-angiogenic state, a process potently opposed by miR-195-3p. The central importance of Oct4 in this framework is conclusively demonstrated by our rescue studies. The finding that lactate preconditioning markedly improved left ventricular function, diminished infarct size and fibrosis, and boosted MSC survival and angiogenesis—with all these advantages entirely negated by simultaneous Oct4 knockdown—confirms that Oct4 is not merely associated with, but is essential for, lactate-mediated therapeutic benefits [43] . Conversely, the deleterious consequences of miR-195-3p overexpression, which replicated the functional and structural impairments observed with Oct4 knockdown, were fully remedied by Oct4 co-overexpression. This reciprocal relationship highlights a fundamental antagonism: miR-195-3p serves as a negative regulator of this reparative pathway, and its downregulation seems to be a prerequisite for activating the full pro-angiogenic potential of Oct4. The convergence of both lactate's action and miR-195-3p's inhibition on Oct4 consolidates its status as the master regulatory node in this network. The most insightful mechanistic revelation from this work is the elucidation of how Oct4 directs a metabolic shift toward glycolysis to power cardiac repair. We present multifaceted evidence supporting this model. Firstly, Oct4 directly governs the expression of key glycolytic regulators, GLUT1 and HK2. The congruent expression patterns of Oct4, GLUT1, and HK2 across experimental conditions—upregulated by lactate and oe Oct4, suppressed by miR-195-3p and sh Oct4—strongly indicate a coordinated regulatory scheme [44] . This is further reinforced by immunofluorescence data from isolated cardiomyocytes, showing that groups with high Oct4 expression were linked to pronounced GLUT1 induction and, importantly, a marked nuclear localization of Oct4 itself. This alteration in subcellular distribution suggests enhanced transcriptional activity, leading us to hypothesize a model where Oct4, upon activation by lactate or release from miR-195-3p suppression, translocates to the nucleus and directly binds to regulatory elements of the GLUT1 and HK2 genes. The functional outcomes of this transcriptional control were validated by our ¹³C-glucose metabolic tracing studies. The " high Oct4" groups ( oe Oct4 and lactate-preconditioned) displayed a classic Warburg-like metabolic profile: elevated extracellular lactate production accompanied by a substantial decrease in citrate labeling from glucose [45] . This pattern signifies a fundamental redirection of carbon flux, where glucose is preferentially shunted away from the mitochondrial TCA cycle and toward lactate fermentation [46] . The unaltered labeling of succinate implies a specific bottleneck or diversion at the TCA cycle's initial stages, consistent with increased glycolytic flow [47] . This Oct4-driven glycolytic transition is not an incidental occurrence but is functionally paramount. Glycolysis provides a more efficient ATP-yielding pathway under the hypoxic conditions of an infarcted heart, thus fostering MSC survival [48] . Moreover, the lactate generated can act as a paracrine signaling molecule and an alternative energy substrate for neighboring cardiomyocytes [49] , establishing a metabolically symbiotic microenvironment. This metabolic reprogramming does not occur in isolation; it is intrinsically connected to the angiogenic requirements of tissue repair. We identified the VEGF signaling pathway as a key downstream effector connecting Oct4-mediated metabolism to angiogenesis. The expression of both VEGF and its receptor VEGFR2 was closely correlated with Oct4 levels, being highest in the oe Oct4 and lactate groups and lowest following Oct4 knockdown. This forges a direct link from metabolic rewiring to a pro-angiogenic secretome. The concomitant upregulation of Akt, a central kinase in cell survival and proliferation [50] , further strengthens this connection. The substantial angiogenesis, evidenced by CD31 staining, in the " high Oct4/ high glycolysis" groups can therefore be ascribed to a dual mechanism: first, the direct production of VEGF stimulated by Oct4, and second, the provision of glycolytically derived energy and biosynthetic precursors necessary for endothelial cell proliferation and new vessel formation. The observation that Akt and VEGF expression patterns were nearly identical suggests they may function in a coordinated, parallel fashion, as previously reported [24] , to optimize the angiogenic response. Conclusion In ischemic myocardium, suppressed miR-195-3p activates Oct4, driving metabolic reprogramming toward glycolysis. This shift enhances MSC survival via lactate production and initiates a lactate-VEGF feed-forward loop that promotes angiogenesis and functional recovery. The Oct4-lactate axis thus represents a key therapeutic target; its potentiation through lactate preconditioning or miR-195-3p inhibition offers a promising metabolic engineering strategy to improve MSC-based cardiac repair. Study limitations Despite the significant insights provided by this study, several limitations should be acknowledged. First, the precise molecular mechanisms by which lactate influences Oct4 stabilization and nuclear translocation, while implied, require further direct experimental validation. Second, the in vivo dynamics of the proposed metabolic symbiosis, where lactate produced by MSCs is utilized by cardiomyocytes, were inferred from indirect evidence and would benefit from more direct tracing studies in the intact animal model. Third, the therapeutic strategies explored (e.g., lactate preconditioning, genetic manipulation) were applied prior to transplantation; their clinical translation would require the development of safe, efficient, and transient delivery methods for use in a potential clinical setting. Finally, while the rat model of MI is well-established, the potential differences in human MSC biology and the human ischemic microenvironment necessitate future studies using human cells and potentially more complex disease models to fully validate the translational relevance of these findings. The potential off-target effects of systemically inhibiting miR-195-3p also warrant careful future investigation. Materials and Methods An expanded description of materials and methods is provided in Supplemental Appendix. Antibodies and reagents Detailed information on primers and antibodies is provided in Supplementary Tables S1 and S2, respectively. 4’, 6-Diamidino-2-phenylindole (DAPI; Sigma-Aldrich, #28718-90-3) was used for nuclear staining. Ethical compliance All animal experiments were approved by the Animal Care and Use Committee of Ji-Nan University (Ethical Approval No. 20220923-02.), conducted in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals, and adhere to ARRIVE 2.0 guidelines. Animals Inbred Lewis rats were housed under standard conditions with ad libitum access to food and water. Cell culture and hypoxia treatment Peripheral blood-derived mesenchymal stem cells (PBMSCs) were isolated from rat abdominal aortic blood as previously described [51] and cultured in DMEM/F-12 medium supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin at 37°C under 5% CO 2 . For hypoxic exposure, passage 2 cells were placed in a modular incubator chamber (Billups-Rothenberg) infused with a gas mixture of 1% O 2 , 5% CO 2 , and balance N 2 for 24-72 hours. Normoxic controls were maintained at 21% O 2 and 5% CO 2 . miRNA sequencing and bioinformatic analysis Total RNA was extracted using TRIzol reagent (Invitrogen). miRNA sequencing libraries were prepared with the QIAseq miRNA Library Kit (Qiagen) and sequenced on an Illumina NextSeq 550 platform. Reads were processed via the miRDeep2 pipeline. Differential expression analysis was performed using DESeq2 with thresholds of |log 2 fold change| > 1 and adjusted p-value < 0.05. miRNA targets were predicted using TargetScan v8.0 and miRDB. Quantitative RT-PCR RNA was reverse transcribed using the PrimeScript RT Reagent Kit (Takara). qPCR was performed on a QuantStudio 6 Pro system (Applied Biosystems) using SYBR Green Master Mix (Roche). The 2 –ΔΔ CT method was applied with GAPDH as the endogenous control. Primer sequences are listed in Supplementary Table S1 . miRNA transfection PBMSCs were transfected with miR-195-3p mimic or inhibitor sponge (Syngentech, Shanghai, China) using Lipofectamine 3000 (Invitrogen). Scrambled miRNA or non-targeting sponge served as negative controls. Plasmid construction and transfection The precursor sequence of miR-195-3p was cloned into the pMXs vector (Addgene). A 300-bp fragment of the Oct4 3’-UTR containing the predicted binding site was inserted into the pmirGLO Dual-Luciferase vector (Promega); a mutant version was generated via site-directed mutagenesis (Q5 Kit, NEB). Coding sequences of Oct4 and VEGF were cloned into the pcDNA3.1 vector. Short hairpin RNAs (shRNAs) targeting Oct4 and VEGF were designed and cloned into the pLKO.1 vector. All constructs were verified by Sanger sequencing. Luciferase reporter assay HEK-293T cells were co-transfected with 100 ng of wild-type or mutant Oct4 3’-UTR reporter plasmid and 50 nM miR-195-3p mimic or negative control (GenePharma). After 48 hours, luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega) on a GloMax Navigator instrument (Promega). Firefly luciferase values were normalized to Renilla luciferase. Western blotting Cells were lysed in RIPA buffer with protease and phosphatase inhibitors (Roche). Protein concentration was determined via BCA assay (Pierce). Proteins (20–30 μg) were separated by 10% SDS-PAGE, transferred to PVDF membranes (Millipore), and incubated with primary antibodies (Supplementary Table S2 ) followed by HRP-conjugated secondary antibodies. Signals were detected using ECL substrate (Bio-Rad) and a ChemiDoc MP Imaging System (Bio-Rad). Cell viability and proliferation assays Viability was assessed using the Cell Counting Kit-8 (CCK-8; Dojindo). For proliferation, cells were stained with Ki67 antibody and DAPI; positive cells were counted across five random fields using a Nikon Eclipse Ti2 fluorescence microscope. Apoptosis analysis Apoptosis was evaluated using the Annexin V-FITC/PI Apoptosis Detection Kit (BD Biosciences). Samples were analyzed on a BD FACS Celesta flow cytometer, and data were processed using FlowJo v10.8. Cytokine array Cytokine expression was profiled using the AAH-CYT-G2000 Array (RayBiotech). Signals were detected by chemiluminescence and quantified with ImageJ. Immunofluorescence staining Cells or tissue sections were fixed, permeabilized, blocked, and incubated with primary antibodies (Supplementary Table S2 ), followed by Alexa Fluor-conjugated secondary antibodies (Invitrogen). Nuclei were stained with DAPI. Images were acquired using a Zeiss LSM 880 confocal microscope. Metabolic analysis Extracellular acidification rate (ECAR) and glycolytic proton efflux rate (glycoPER) were measured using a Seahorse XFe96 Analyzer and the XF Glycolysis Stress Test Kit (Agilent). Glucose uptake and lactate production were assessed using commercial kits (Cayman Chemical and Sigma-Aldrich, respectively). RNA sequencing and gene set enrichment analysis Total RNA was extracted using the RNeasy Plus Mini Kit (Qiagen). Libraries were prepared with the TruSeq Stranded mRNA Kit (Illumina) and sequenced on an Illumina NovaSeq 6000. Reads were aligned to GRCh38 using STAR, and gene counts were obtained with featureCounts. Differential expression analysis was performed using DESeq2. Gene Set Enrichment Analysis (GSEA) was conducted using Hallmark and KEGG gene sets. Myocardial infarction model Male Lewis rats (250–300 g) were anesthetized with 2% isoflurane, and MI was induced by permanent ligation of the left anterior descending coronary artery. Infarction was confirmed by myocardial blanching and ST-segment elevation. PBMSC preconditioning and EGFP labeling To determine the optimal lactate concentration for preconditioning, PBMSCs were treated with a gradient of sodium lactate (0, 5, 10, 15, and 20 mM; Sigma-Aldrich) for 48 hours under hypoxic condition. Subsequent analyses of cell viability and proliferation indicated that 10 mM sodium lactate yielded the greatest enhancement in both parameters (Supplementary Figure S1 ). Based on these results, PBMSCs were preconditioned with 10 mM sodium lactate for 24 hours under hypoxia before transplantation. Lentiviral vectors encoding miR-195-3p, Oct4, or Oct4-shRNA were packaged in HEK-293T cells. PBMSCs were transduced at an MOI of 50 with 8 μg/mL polybrene. EGFP labeling was performed via co-transfection, with >70% efficiency confirmed by fluorescence microscopy and immunoblotting. Experimental groups and cell transplantation A total of 220 rats that survived MI surgery were randomized into eight experimental groups (n = 20 per group) using a computer-generated scheme. Researchers were blinded to group assignments. Groups received intramyocardial injections of PBS, vehicle-treated MSCs, lactate-preconditioned MSCs, miR-195-3p-overexpressing MSCs, Oct4-overexpressing MSCs, Oct4-knockdown MSCs, lactate-preconditioned + Oct4-knockdown MSCs, or miR-195-3p-overexpressing + Oct4-overexpressing MSCs (5×10 6 cells per heart). Postoperative analgesia and antibiotics were administered. The work has been reported in line with the ARRIVE guidelines 2.0. Echocardiography Transthoracic echocardiography was performed 30 days post-transplantation using a Vevo 3100 system (VisualSonics). Left ventricular ejection fraction (LVEF) and fractional shortening (LVFS) were derived from M-mode recordings. Histological analysis Hearts were sectioned and stained with TTC or Masson’s trichrome. Fibrosis area was quantified using ImageJ and expressed as a percentage of total left ventricular area. Engraftment and angiogenesis assessment Engraftment was evaluated by quantifying EGFP-positive cells. Apoptosis was assessed via TUNEL staining (Roche). Angiogenesis was evaluated by CD31 staining and vascular density calculation. Metabolic tracing Isolated cardiomyocytes were incubated with [U-¹³C] glucose (Cambridge Isotope Laboratories). Metabolites were extracted and analyzed by LC-MS (Q Exactive HF, Thermo Scientific). Data were processed using Xcalibur and MetaboAnalyst 5.0. Statement. The work has been reported in line with the ARRIVE guidelines 2.0. Statistical analysis Data are expressed as mean ± SD. Analyses were performed using GraphPad Prism 9.0. Multiple groups were compared by one-way ANOVA with Tukey’s post-hoc test; two-group comparisons used two-tailed Student’s t-test. Survival was analyzed by Kaplan–Meier with log-rank test. Sample sizes were determined by power analysis (α = 0.05, power = 0.8). p < 0.05 was considered statistically significant. Declarations Acknowledgement s None Author contributions Y. M.: conceptualization, methodology, writing-original draft preparation, preparing figures 1-3. P. Z.: conceptualization, methodology, preparing figures 4-6. H. Z.：preparing figures 7, 8. L. C.: Animal model. Z. D.: data collection, preparing Table S1 and Table S2. Y. L.: methodology. W. P.: technical support. S. Z.: conceptualization, data curation, funding acquisition, investigation, supervision,validation, visualization, preparing Graphic abstract, writing – review & editing. All authors reviewed the manuscript. Funding This work was supported by Guangzhou Science and Technology Program Project funded by the Guangzhou Science and Technology Bureau (Grant No. 2023A03J0982, to SZ). Availability of data and materials The RNA-seq data supporting the results of this study have been deposited in the Sequence Read Archive under accession number [PRJNA1416857]. Declarations The authors declare that they have not used Artificial Intelligence in this study. Competing interests There is no conflict of interest between the authors. Consent for publication All authors read and approved the final manuscript. Additional information Supplementary information The online version contains supplementary material available. Correspondence and requests for materials should be addressed to Shaoheng Zhang. Data availability The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. References Barrère-Lemaire S, Vincent A, Jorgensen C, Piot C, Nargeot J, Djouad F. Mesenchymal stromal cells for improvement of cardiac function following acute myocardial infarction: a matter of timing. Physiol Rev. 2024; 104(2): 659-725. Burchfield JS, Xie M, Hill JA. Pathological ventricular remodeling: mechanisms: part 1 of 2. Circulation. 2013; 128(4): 388-400. Fomovsky GM, Holmes JW. Evolution of scar structure, mechanics, and ventricular function after myocardial infarction in the rat. Am J Physiol Heart Circ Physiol. 2010; 298(1): H221-H228. Lin M, Liu X, Zheng H, Huang X, Wu Y, Huang A, Zhu H, Hu Y, Mai W, Huang Y. IGF-1 enhances BMSC viability, migration, and anti-apoptosis in myocardial infarction via secreted frizzled-related protein 2 pathway. Stem Cell Res Ther. 2020; 11(1): 22. Zhang S, Zhao L, Wang J, Chen N, Yan J, Pan X. HIF-2α and Oct4 have synergistic effects on survival and myocardial repair of very small embryonic-like mesenchymal stem cells in infarcted hearts. Cell Death Dis. 2017; 8(1): e2548. Wang Y, Wang Q, Sui X, Guo M, Li L, Jia W, Tian Y, Lu Q, Wang B. Catalpalactone protects rats nerve function from hypoxic lesion by polarizing microglial cells toward M2 phenotype. Eur J Med Res. 2025; 30(1): 96. Babenko VA, Varlamova EG, Saidova AA, Turovsky EA, Plotnikov EY. Lactate protects neurons and astrocytes against ischemic injury by modulating Ca2+ homeostasis and inflammatory response. FEBS J. 2024; 291(8): 1684-1698. Bispo DSC, Graça ICR, Jesus CSH, Rodrigues JE, Correia MC, Atella S, Duarte IF, Goodfellow BJ, Oliveira MB, Mano JF, Gil AM. Metabolic markers detect early ostedifferentiation of mesenchymal stem cells from multiple donors. Stem Cell Res Ther. 2025; 16(1): 294. Lu R, Cai H, Liu Y, Ma G, Wang J, Yan M, Zhang Z, Yu B, Li Z, Fang S. Long non-coding RNA AK023617 orchestrates atherosclerosis by regulating the circadian rhythm of immunity-related GTPase family M protein in macrophages. Noncoding RNA Res. 2024; 11: 262-272. Xiong J, Ma F, Ding N, Xu L, Ma S, Yang A, Hao Y, Zhang H, Jiang Y. miR-195-3p alleviates homocysteine-mediated atherosclerosis by targeting IL-31 through its epigenetics modifications. Aging Cell. 2021; 20(10): e13485. Carvalho A, Ji Z, Zhang R, Zuo W, Qu Y, Chen X, Tao Z, Ji J, Yao Y, Ma G. Inhibition of miR-195-3p protects against cardiac dysfunction and fibrosis after myocardial infarction. Int J Cardiol. 2023; 387: 131128. She S, Wei Q, Kang B, Wang YJ. Cell cycle and pluripotency: Convergence on octamer‑binding transcription factor 4 (Review). Mol Med Rep. 2017; 16(5): 6459-6466. Xu N, Papagiannakopoulos T, Pan G, Thomson JA, Kosik KS. MicroRNA-145 regulates OCT4, SOX2, and KLF4 and represses pluripotency in human embryonic stem cells. Cell. 2009; 137(4): 647-658. Jauhari A, Singh T, Yadav S. Expression of miR-145 and Its Target Proteins Are Regulated by miR-29b in Differentiated Neurons. Mol Neurobiol. 2018; 55(12): 8978-8990. Guo J, Wang M, Liu X. MicroRNA-195 suppresses tumor cell proliferation and metastasis by directly targeting BCOX1 in prostate carcinoma. J Exp Clin Cancer Res. 2015; 34(1): 91. Wang M, Zhang J, Tong L, Ma X, Qiu X. MiR-195 is a key negative regulator of hepatocellular carcinoma metastasis by targeting FGF2 and VEGFA. Int J Clin Exp Pathol. 2015; 8(11): 14110-14120. Shen M, Dong C, Ruan X, Yan W, Cao M, Pizzo D, Wu X, Yang L, Liu L, Ren X, Wang SE. Chemotherapy-Induced Extracellular Vesicle miRNAs Promote Breast Cancer Stemness by Targeting ONECUT2. Cancer Res. 2019; 79(14): 3608-3621. Liu K, Ma L, Zhou F, Yang Y, Hu HB, Wang L, Zhong L. Identification of microRNAs related to myocardial ischemic reperfusion injury. J Cell Physiol. 2019; 234(7): 11380-11390. Buccini S, Haider KH, Ahmed RP, Jiang S, Ashraf M. Cardiac progenitors derived from reprogrammed mesenchymal stem cells contribute to angiomyogenic repair of the infarcted heart. Basic Res Cardiol. 2012; 107(6): 301. Qiu Z, Zhao J, Huang F, Bao L, Chen Y, Yang K, Cui W, Jin W. Myocardial fibrosis reversion via rhACE2-electrospun fibrous patch for ventricular remodeling prevention. NPJ Regen Med. 2021; 6(1): 44. Pan Z, Huang Y, Qian H, Du X, Qin W, Liu T. Superparamagnetic iron oxide nanoparticles drive miR-485-5p inhibition in glioma stem cells by silencing Tie1 expression. Int J Biol Sci. 2020 Feb 10;16(7):1274-1287. Wang H, Bai Z, Qiu Y, Kou J, Zhu Y, Tan Q, Chen C, Mo R. Empagliflozin-Pretreated MSC-Derived Exosomes Enhance Angiogenesis and Wound Healing via PTEN/AKT/VEGF Pathway. Int J Nanomedicine. 2025; 20: 5119-5136. Boueya IL, Sandhow L, Albuquerque JRP, Znaidi R, Passaro D. Endothelial heterogeneity in bone marrow: insights across development, adult life and leukemia. Leukemia. 2025; 39(1): 8-24. Zhang S, Zhao L, Shen L, Xu D, Huang B, Wang Q, Lin J, Zou Y, Ge J. Comparison of various niches for endothelial progenitor cell therapy on ischemic myocardial repair: coexistence of host collateralization and Akt-mediated angiogenesis produces a superior microenvironment. Arterioscler Thromb Vasc Biol. 2012; 32(4): 910-923. Chen X, Zhou C, Xu D, Liu X, Li S, Hou J, Zhang K, Zeng C, Zheng G, Wu H, Wu H, Wang W, Fu J, Wang T. Peptide hormone ELABELA promotes rat bone marrow-derived mesenchymal stem cell proliferation and migration by manipulating the cell cycle through the PI3K/AKT pathway under the hypoxia and ischemia microenvironment. Stem Cell Res Ther. 2022; 13(1): 32. Park HK, Hu S, Kim SY, Yoon S, Yoon NG, Lee JH, Choi W, Kong SY, Kim JH, Nam D, Kang BH. Pseudohypoxic stabilization of HIF1α via cyclophilin D suppression promotes melanoma metastasis. Signal Transduct Target Ther. 2025; 10(1): 231. Yao L, Gong X, Liu Y, Hao K, Jiang J, Luo Z. The miR-195-3p/BDNF axis is a promising therapeutic for heart failure. Int J Cardiol. 2024; 406: 131991. Lee EJ, Sun R, Kim J. The self-renewal function of Oct-4 can be replaced by the EWS-Oct-4 fusion protein in embryonic stem cells. Cell Mol Life Sci. 2025; 82(1): 166. Chen W, Wang YJ. Multifaceted roles of OCT4 in tumor microenvironment: biology and therapeutic implications. Oncogene. 2025; 44(18): 1213-1229. Schmitz DA, Okamura D, Sakurai M, Ding Y, Oura S, Ballard E, Wei Y, Yu L, Hu Y, Wu J. Dissecting Oct4 enhancer function in pluripotent stem cells and mouse embryogenesis. Stem Cell Reports. 2025: 102706. Han Y, Yang J, Fang J, Zhou Y, Candi E, Wang J, Hua D, Shao C, Shi Y. The secretion profile of mesenchymal stem cells and potential applications in treating human diseases. Signal Transduct Target Ther. 2022; 7(1): 92. Egea V. HypoxamiRs: the hidden architects of tissue adaptation in hypoxia. Cell Death Dis. 2025; 16(1): 732. Fu J, Chen X, Liu X, Xu D, Yang H, Zeng C, Long H, Zhou C, Wu H, Zheng G, Wu H, Wang W, Wang T. ELABELA ameliorates hypoxic/ischemic-induced bone mesenchymal stem cell apoptosis via alleviation of mitochondrial dysfunction and activation of PI3K/AKT and ERK1/2 pathways. Stem Cell Res Ther. 2020; 11(1): 541. Watanuki S, Kobayashi H, Sugiura Y, Yamamoto M, Karigane D, Shiroshita K, Sorimachi Y, Morikawa T, Fujita S, Shide K, Haraguchi M, Tamaki S, Mikawa T, Kondoh H, Nakano H, Sumiyama K, Nagamatsu G, Goda N, Okamoto S, Nakamura-Ishizu A, Shimoda K, Suematsu M, Suda T, Takubo K. SDHAF1 confers metabolic resilience to aging hematopoietic stem cells by promoting mitochondrial ATP production. Cell Stem Cell. 2024; 31(8): 1145-1161.e15. Kračun D, Görlach A, Snedeker JG, Buschmann J. Reactive oxygen species in tendon injury and repair. Redox Biol. 2025; 81: 103568. Cao DC, Liang Y, Guo Y, Wu DY, Wang NN, Li YM, Sun HF, Wang Q, Zhang X, Chi YL, Sun PP, Li YJ, Wang RR, Wang PY, Xie SY, Xie N. Oxygen-Generating Biomimetic Nano-Herb System for Synergistic Therapy & Pain Relief in Triple-Negative Breast Cancer via HIF-1α/VEGF Pathway. Int J Nanomedicine. 2025; 20: 7113-7132. Harada A, Yasumizu Y, Harada T, Fumoto K, Sato A, Maehara N, Sada R, Matsumoto S, Nishina T, Takeda K, Morii E, Kayama H, Kikuchi A. Hypoxia-induced Wnt5a-secreting fibroblasts promote colon cancer progression. Nat Commun. 2025; 16(1): 3653. Sun ED, Zhou OY, Hauptschein M, Rappoport N, Xu L, Navarro Negredo P, Liu L, Rando TA, Zou J, Brunet A. Spatial transcriptomic clocks reveal cell proximity effects in brain ageing. Nature. 2025; 638(8049): 160-171. Grunewald M, Kumar S, Sharife H, Volinsky E, Gileles-Hillel A, Licht T, Permyakova A, Hinden L, Azar S, Friedmann Y, Kupetz P, Tzuberi R, Anisimov A, Alitalo K, Horwitz M, Leebhoff S, Khoma OZ, Hlushchuk R, Djonov V, Abramovitch R, Tam J, Keshet E. Counteracting age-related VEGF signaling insufficiency promotes healthy aging and extends life span. Science. 2021; 373(6554): eabc8479. Wang X, Huang J, Fan W, Li N, Yang H, Yang W, Li W, Li R, Liu J, Li X, Zhou Q, Hou S. Lactate-binding protein DNMT3A in HRMECs promotes angiogenesis by upregulating VEGFA through HIF-1α lactylation. Genome Biol. 2025; 26(1): 377. Wang Y, Ge M, Wang J, Xu Y, Wang N, Xu S. Metabolic reprogramming in ischemic stroke: when glycolytic overdrive meets lipid storm. Cell Death Dis. 2025; 16(1): 788. Wu H, Xing C, Yu B, Guo L, Dou X, Gao L, Yang S, Zhang Y, Gao X, Li S, Xia B, Ma T, Hao Y, Yang Y, Gao X, Wei Y, Xue B, Zhang Q, Feng CL, Huang J. Metabolic Reprogramming of Neural Stem Cells by Chiral Nanofiber for Spinal Cord Injury. ACS Nano. 2025; 19(4): 4785-4801. Chen X, Yuan Y, Zhou F, Li L, Pu J, Zeng Y, Jiang X. Lactylation: From Homeostasis to Pathological Implications and Therapeutic Strategies. MedComm (2020). 2025; 6(6): e70226. Renikunta HV, Lazarow K, Gong Y, Shukla PC, Nageswaran V, Giral H, Kratzer A, Opitz L, Engel FB, Haghikia A, Costantino S, Paneni F, von Kries JP, Streckfuss-Bömeke K, Landmesser U, Jakob P. Large-scale microRNA functional high-throughput screening identifies miR-515-3p and miR-519e-3p as inducers of human cardiomyocyte proliferation. iScience. 2023; 26(5): 106593. Vander Heiden MG, Cantley LC, Thompson CB. Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science. 2009; 324(5930): 1029-1033. Alhasan KA, King MA, Pattar BSB, Lewis IA, Lopaschuk GD, Greenway SC. Anaplerotic filling in heart failure: a review of mechanism and potential therapeutics. Cardiovasc Res. 2024; 120(17): 2166-2178. Cordani M, Rumio C, Bontempi G, Strippoli R, Marcucci F. Oxidative and Glycolytic Metabolism: Their Reciprocal Regulation and Dysregulation in Cancer. Cells. 2025; 14(15): 1177. Popazova O, Belenichev I, Bukhtiyarova N, Ryzhenko V, Gorchakova N, Oksenych V, Kamyshnyi O. Molecular and Biochemical Mechanisms of Cardiomyopathy Development Following Prenatal Hypoxia-Focus on the NO System. Antioxidants (Basel). 2025; 14(6): 743. Li G, Cui H, Fan R, Liu G, Hou Z, Zhang Y, Che X, Wang T, Wei H, Li P. Delivery of Molecular Hydrogen for Precision Immunomodulation: Mechanisms, Detection Methods, and Applications. Adv Sci (Weinh). 2025; 12(32): e00283. Wang J, An Z, Wu Z, Zhou W, Sun P, Wu P, Dang S, Xue R, Bai X, Du Y, Chen R, Wang W, Huang P, Lam SM, Ai Y, Liu S, Shui G, Zhang Z, Liu Z, Huang J, Fang X, He K. Spatial organization of PI3K-PI(3,4,5)P3-AKT signaling by focal adhesions. Mol Cell. 2024; 84(22): 4401-4418.e9. Wang P, Deng Z, Li A, Li R, Huang W, Cui J, Chen S, Li B, Zhang S. β-Catenin promotes long-term survival and angiogenesis of peripheral blood mesenchymal stem cells via the Oct4 signaling pathway. Exp Mol Med. 2022; 54(9): 1434-1449. Additional Declarations No competing interests reported. Supplementary Files SupplementalDataResearchdesignandmethods20262.docx YusPaper1FigureS120262.tif YusPaper1FigureS2.tif YusPaper1TableS1202510primer.doc YusPaper1TableS220259.doc YusPaper1Graphicabstract.tif OriginalWesternblotimages20262.pdf ARRIVEGuidelines2.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers invited by journal 22 Mar, 2026 Editor assigned by journal 10 Feb, 2026 Submission checks completed at journal 06 Feb, 2026 First submitted to journal 05 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8443063","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":610331678,"identity":"0dd04eba-4eef-4166-bcfa-ddcc54781e6c","order_by":0,"name":"Mengying Yu","email":"","orcid":"","institution":"Guangzhou Red Cross Hospital of Ji-Nan University","correspondingAuthor":false,"prefix":"","firstName":"Mengying","middleName":"","lastName":"Yu","suffix":""},{"id":610331679,"identity":"c3eab0ef-fe7e-4da6-8304-9e5dc73eaeb2","order_by":1,"name":"Zhao Peng","email":"","orcid":"","institution":"Guizhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhao","middleName":"","lastName":"Peng","suffix":""},{"id":610331680,"identity":"9d71ebdf-a820-416b-a956-cb58549a4d75","order_by":2,"name":"Zhichuan Huang","email":"","orcid":"","institution":"Guangzhou Red Cross Hospital of Ji-Nan University","correspondingAuthor":false,"prefix":"","firstName":"Zhichuan","middleName":"","lastName":"Huang","suffix":""},{"id":610331682,"identity":"9d3490d3-43d6-4b9c-bda1-a05c29118310","order_by":3,"name":"Cuixia Liu","email":"","orcid":"","institution":"Guangzhou Red Cross Hospital of Ji-Nan University","correspondingAuthor":false,"prefix":"","firstName":"Cuixia","middleName":"","lastName":"Liu","suffix":""},{"id":610331683,"identity":"8975049b-9c88-464c-abf0-00ee0b0e7d1e","order_by":4,"name":"Zhanyu Deng","email":"","orcid":"","institution":"Guangzhou Red Cross Hospital of Ji-Nan University","correspondingAuthor":false,"prefix":"","firstName":"Zhanyu","middleName":"","lastName":"Deng","suffix":""},{"id":610331684,"identity":"4d01bfc5-6fd3-4a60-a941-13ae1d525579","order_by":5,"name":"Yunlong Li","email":"","orcid":"","institution":"Guangzhou Red Cross Hospital of Ji-Nan University","correspondingAuthor":false,"prefix":"","firstName":"Yunlong","middleName":"","lastName":"Li","suffix":""},{"id":610331685,"identity":"a40f8151-174a-4107-b7d8-cb5172641350","order_by":6,"name":"Pengzhen Wang","email":"","orcid":"","institution":"Guangzhou Red Cross Hospital of Ji-Nan University","correspondingAuthor":false,"prefix":"","firstName":"Pengzhen","middleName":"","lastName":"Wang","suffix":""},{"id":610331686,"identity":"963819ce-e6c9-477e-b8fc-6eb4af9d86e5","order_by":7,"name":"Shaoheng Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6klEQVRIiWNgGAWjYDACCQYGZgiD+fiPDwY2dqRoYUuQnFGQlkyKFh4FaZ4PhxgbCOmQn9388HFBxR27+bN7GIxtDA4wM7AfProBnxaDO8eMjWeceZbcOOfsgeQcgzt8DDxpaTfwapFIMJPmbTuczCyRl3A4x+AZM9CFZni1yM9I/ybN++9wMptEjmGzhcFhxgZCWhhu5ABtaThsxyORY8zMQIwWgxs5xcY8xw4nSEikpTH2GKQlsxHyC9BhGx/z1By2l5+RfIzhxx8bO372w8fwOwwKEhtgLDZilIOAPbEKR8EoGAWjYAQCAJs3SOxkqffqAAAAAElFTkSuQmCC","orcid":"","institution":"Guangzhou Red Cross Hospital of Ji-Nan University","correspondingAuthor":true,"prefix":"","firstName":"Shaoheng","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2025-12-24 13:23:37","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8443063/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8443063/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105387721,"identity":"3196c361-bf55-4f9b-9330-151ebcb1e7c2","added_by":"auto","created_at":"2026-03-25 12:47:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3989815,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHypoxia induces miR-195-3p expression and directly targets Oct4 in MSCs\u003c/strong\u003e. (\u003cstrong\u003eA\u003c/strong\u003e) Heatmap of the top 10 differentially expressed miRNAs in MSCs under normoxia versus hypoxia for 72 hours (red: up-regulated; blue: down-regulated). (\u003cstrong\u003eB\u003c/strong\u003e) Validation of miR-195-3p upregulation under hypoxia by qRT-PCR. (\u003cstrong\u003eC\u003c/strong\u003e) Bioinformatic prediction of Oct4 as a potential target of miR-195-3p. (\u003cstrong\u003eD\u003c/strong\u003e, \u003cstrong\u003eE\u003c/strong\u003e) qRT-PCR analysis of miR-195-3p (\u003cstrong\u003eD\u003c/strong\u003e) and Oct4 mRNA (\u003cstrong\u003eE\u003c/strong\u003e) expression in normoxic and hypoxic MSCs. (\u003cstrong\u003eF\u003c/strong\u003e) Western blot analysis of Oct4 protein levels under both conditions. (\u003cstrong\u003eG\u003c/strong\u003e) Dual-luciferase reporter assay of 293T cells co-transfected with wild-type (wt-Oct4) or mutant (mut-Oct4) 3′-UTR of Oct4, together with miR-195-3p mimic or inhibitor. (\u003cstrong\u003eH\u003c/strong\u003e) qRT-PCR analysis of Oct4 mRNA in hypoxic MSCs transfected with NC mimic, miR-195-3p mimic, NC inhibitor, or miR-195-3p inhibitor for 72 hours. (\u003cstrong\u003eI\u003c/strong\u003e) Western blot analysis of Oct4 protein in MSCs under the same transfection conditions as in (\u003cstrong\u003eH\u003c/strong\u003e). Culture conditions: Normoxia (21% O\u003csub\u003e2\u003c/sub\u003e, 5% CO\u003csub\u003e2\u003c/sub\u003e, balance N\u003csub\u003e2\u003c/sub\u003e); Hypoxia (1% O\u003csub\u003e2\u003c/sub\u003e, 5% CO\u003csub\u003e2\u003c/sub\u003e, balance N\u003csub\u003e2\u003c/sub\u003e). Data are presented as mean ± SD (n = 3 for panels \u003cstrong\u003eB\u003c/strong\u003e, \u003cstrong\u003eD\u003c/strong\u003e, \u003cstrong\u003eE\u003c/strong\u003e; n = 5 for panels \u003cstrong\u003eG\u003c/strong\u003e, \u003cstrong\u003eH\u003c/strong\u003e). Statistical significance was determined by paired t-test (\u003cstrong\u003eB\u003c/strong\u003e, \u003cstrong\u003eD\u003c/strong\u003e, \u003cstrong\u003eE\u003c/strong\u003e) or one-way ANOVA (\u003cstrong\u003eG\u003c/strong\u003e, \u003cstrong\u003eH\u003c/strong\u003e). \u003csup\u003e***\u003c/sup\u003eP \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"YusPaper1Figure120262.png","url":"https://assets-eu.researchsquare.com/files/rs-8443063/v1/70f9c137b2baa46f8389811d.png"},{"id":105387722,"identity":"381ecd14-da7f-4756-85b1-ff7cece3eed8","added_by":"auto","created_at":"2026-03-25 12:47:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":12833122,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRegulation of the miR-195-3p/Oct4 axis on MSC survival under hypoxia\u003c/strong\u003e. (\u003cstrong\u003eA\u003c/strong\u003e) Representative optical micrographs of MSCs after 72 hours of hypoxic culture following genetic inhibition or overexpression of miR-195-3p or Oct4 via miR-195-3p mimic/inhibitor, Oct4 overexpression (\u003csub\u003e\u003cem\u003eoe\u003c/em\u003e\u003c/sub\u003eOct4), or Oct4 shRNA (\u003csub\u003e\u003cem\u003esh\u003c/em\u003e\u003c/sub\u003eOct4). Scale bar: 50 µm. (\u003cstrong\u003eB\u003c/strong\u003e) Representative immunofluorescence images of Ki67 staining in MSCs under the same genetic modifications. Nuclei were co-stained with Ki67 (green) and DAPI (4′,6-Diamidino-2- phenylindole, blue). Scale bar: 50 µm. (\u003cstrong\u003eC\u003c/strong\u003e)\u0026nbsp;Apoptosis of MSCs under hypoxia for 72 hours, as measured by flow cytometry, following genetic modulation of miR-195-3p or Oct4. (\u003cstrong\u003eD\u003c/strong\u003e) Viability of MSCs under hypoxia for 72 hours assessed using CCK-8 assay under the same treatment conditions. (\u003cstrong\u003eE-F\u003c/strong\u003e) Statistical analysis of Ki67-positive cells (from \u003cstrong\u003eB\u003c/strong\u003e) and apoptosis rates (from \u003cstrong\u003eC\u003c/strong\u003e), respectively.\u0026nbsp;(\u003cstrong\u003eG\u003c/strong\u003e) Apoptosis-related cytokine levels (pg/ml) under hypoxia, as detected by antibody array in MSCs transfected with miR-195-3p mimic or \u003csub\u003e\u003cem\u003eoe\u003c/em\u003e\u003c/sub\u003eOct4. (\u003cstrong\u003eH\u003c/strong\u003e) mRNA expression levels of Bax, Caspase 9, Bcl-2, Hsp27, and Oct4 in MSCs treated with miR-195-3p mimic or \u003csub\u003e\u003cem\u003eoe\u003c/em\u003e\u003c/sub\u003eOct4 under hypoxia. (\u003cstrong\u003eI\u003c/strong\u003e) Representative Western blots showing protein levels of Bax, Caspase 9, Bcl-2, Hsp27, and Oct4 in MSCs after 72 hours of hypoxia with or without miR-195-3p mimic or \u003csub\u003e\u003cem\u003eoe\u003c/em\u003e\u003c/sub\u003eOct4. Data are presented as mean ± SD. Significance was determined by one-way ANOVA (n=5, each group). In (\u003cstrong\u003eD\u003c/strong\u003e) and (\u003cstrong\u003eH\u003c/strong\u003e): \u003csup\u003e*\u003c/sup\u003eP \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003eP \u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003eP \u0026lt; 0.001. In (\u003cstrong\u003eG\u003c/strong\u003e): \u003csup\u003e*\u003c/sup\u003eP \u0026lt; 0.05 vs. CON; \u003csup\u003e#\u003c/sup\u003eP \u0026lt; 0.05 vs. oeOct4; \u003csup\u003e†\u003c/sup\u003eP \u0026lt; 0.05 vs. miR-195-3p mimic.\u003c/p\u003e","description":"","filename":"YusPaper1Figure220262.png","url":"https://assets-eu.researchsquare.com/files/rs-8443063/v1/f1847453b8c5b041ce424066.png"},{"id":105387725,"identity":"782739cc-ac94-4a20-90a7-9266c6134830","added_by":"auto","created_at":"2026-03-25 12:47:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":13811884,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVEGF attenuates miR-195-3p-mediated apoptosis and promotes vascularization in hypoxic MSCs\u003c/strong\u003e. (\u003cstrong\u003eA\u003c/strong\u003e) (\u003cstrong\u003ea\u003c/strong\u003e) Representative micrographs of MSCs after 7 days of hypoxic culture with VEGF overexpression (\u003csub\u003e\u003cem\u003eoe\u003c/em\u003e\u003c/sub\u003eVEGF) and/or miR-195-3p mimic. Scale bar: 50 µm. (\u003cstrong\u003eb\u003c/strong\u003e) Viability of MSCs under hypoxia measured by CCK-8 assay. (B) (\u003cstrong\u003ea\u003c/strong\u003e) Immunofluorescence images and (\u003cstrong\u003eb\u003c/strong\u003e) quantitative analysis of Ki67\u003csup\u003e+\u003c/sup\u003e MSCs (green; nuclei stained with DAPI, blue). Scale bar: 50 µm. (\u003cstrong\u003eC\u003c/strong\u003e) (\u003cstrong\u003ea\u003c/strong\u003e) Apoptosis of hypoxic MSCs assessed by flow cytometry and (\u003cstrong\u003eb\u003c/strong\u003e) corresponding quantitative analysis. (\u003cstrong\u003eD\u003c/strong\u003e) (\u003cstrong\u003ea\u003c/strong\u003e) Immunofluorescence images of VEGF and VWF and (\u003cstrong\u003eb\u003c/strong\u003e) quantitative expression of Factor VIII under different treatments. Scale bar: 50 µm. (\u003cstrong\u003eE\u003c/strong\u003e-\u003cstrong\u003eJ\u003c/strong\u003e) mRNA expression levels of VEGF (\u003cstrong\u003eE\u003c/strong\u003e), bFGF (\u003cstrong\u003eF\u003c/strong\u003e), VEGFR2 (\u003cstrong\u003eG\u003c/strong\u003e), Tie2 (\u003cstrong\u003eH\u003c/strong\u003e), Endoglin (\u003cstrong\u003eI\u003c/strong\u003e), and CD31 (J) in MSCs. (\u003cstrong\u003eK\u003c/strong\u003e) Western blot analysis of VEGFA, VEGFR2, Tie2, and CD31 protein expression in MSCs under hypoxia. All data are presented as mean ± SD; \u003csup\u003e*\u003c/sup\u003ep \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003ep \u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003ep \u0026lt; 0.001; one-way ANOVA, n = 5. DAPI=4′,6-Diamidino-2- phenylindole.\u003c/p\u003e","description":"","filename":"YusPaper1Figure320262.png","url":"https://assets-eu.researchsquare.com/files/rs-8443063/v1/d099b03091e8b71cf16905d7.png"},{"id":105387728,"identity":"2326942d-970f-437b-b1c4-e81be7ebaa31","added_by":"auto","created_at":"2026-03-25 12:47:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":19376349,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOct4 upregulation promotes MSC proliferation and angiogenesis under hypoxia via activation of VEGF signaling. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Morphology of MSCs after 7 days of hypoxia following overexpression of Oct4 (\u003csub\u003e\u003cem\u003eoe\u003c/em\u003e\u003c/sub\u003eOct4) and/or VEGF knockdown (\u003csub\u003e\u003cem\u003esh\u003c/em\u003e\u003c/sub\u003eVEGF). Scale bar: 50 μm. (\u003cstrong\u003eB\u003c/strong\u003e) Representative immunofluorescence images of Ki67 (green) and DAPI (blue) in treated MSCs under hypoxia. Scale bar: 50 μm. (\u003cstrong\u003eC\u003c/strong\u003e) Apoptosis analysis by flow cytometry in MSCs after 7 days of hypoxia with indicated genetic modifications. (\u003cstrong\u003eD\u003c/strong\u003e) Immunofluorescence images of VEGF and VWF expression in MSCs treated with oeVEGF and/or miR-195-3p mimic. Scale bar: 50 μm. (\u003cstrong\u003eE\u003c/strong\u003e) Viability of MSCs under hypoxia measured by CCK-8 assay. (\u003cstrong\u003eF\u003c/strong\u003e-\u003cstrong\u003eH\u003c/strong\u003e) Quantification of Ki67‑positive cells (\u003cstrong\u003eF\u003c/strong\u003e), apoptosis rate (\u003cstrong\u003eG\u003c/strong\u003e), and VWF expression (\u003cstrong\u003eH\u003c/strong\u003e) under each condition. (\u003cstrong\u003eI\u003c/strong\u003e-\u003cstrong\u003eN\u003c/strong\u003e) mRNA expression levels of VEGF (\u003cstrong\u003eI\u003c/strong\u003e), bFGF (\u003cstrong\u003eJ\u003c/strong\u003e), VEGFR2 (\u003cstrong\u003eK\u003c/strong\u003e), VECAD (\u003cstrong\u003eL\u003c/strong\u003e), Endoglin (\u003cstrong\u003eM\u003c/strong\u003e), and CD31 (\u003cstrong\u003eN\u003c/strong\u003e) assessed by qRT‑PCR. (\u003cstrong\u003eO\u003c/strong\u003e) Protein levels of VEGFA, VEGFR2, CD31, VECAD, and Bcl2 detected by Western blot after 7 days of hypoxia with or without \u003csub\u003e\u003cem\u003eoe\u003c/em\u003e\u003c/sub\u003eOct4 or \u003csub\u003e\u003cem\u003esh\u003c/em\u003e\u003c/sub\u003eVEGF. Data are shown as mean ± SD; n = 5 per group. One-way ANOVA was used for statistical analysis. \u003csup\u003e*\u003c/sup\u003eP \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003eP \u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003eP \u0026lt; 0.001. DAPI=4′,6-Diamidino-2- phenylindole.\u003c/p\u003e","description":"","filename":"YusPaper1Figure420262.png","url":"https://assets-eu.researchsquare.com/files/rs-8443063/v1/6db7743b994138e9c839cbe4.png"},{"id":105387724,"identity":"1045c3d6-1f49-403b-91f7-0b4fe80e6bec","added_by":"auto","created_at":"2026-03-25 12:47:48","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":11055176,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emiR-195-3p regulates MSC fibrosis and inflammation under hypoxia via the Oct4/VEGF axis. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Representative immunofluorescence images showing Collagen I and IL-6 expression in MSCs following transfection with miR-195-3p mimic, \u003csub\u003e\u003cem\u003eoe\u003c/em\u003e\u003c/sub\u003eOct4, \u003csub\u003e\u003cem\u003eoe\u003c/em\u003e\u003c/sub\u003eVEGF, and/or \u003csub\u003e\u003cem\u003esh\u003c/em\u003e\u003c/sub\u003eVEGF under hypoxic conditions. Nuclei were stained with DAPI. Scale bar: 50 μm. (\u003cstrong\u003eB\u003c/strong\u003e, \u003cstrong\u003eC\u003c/strong\u003e) Quantitative analysis of Collagen I-positive (\u003cstrong\u003eB\u003c/strong\u003e) and IL6-positive (\u003cstrong\u003eC\u003c/strong\u003e) cells across experimental groups. (\u003cstrong\u003eD\u003c/strong\u003e-\u003cstrong\u003eH\u003c/strong\u003e) mRNA expression levels of collagen I (\u003cstrong\u003eD\u003c/strong\u003e), α‑SMA (\u003cstrong\u003eE\u003c/strong\u003e), IL6 (\u003cstrong\u003eF\u003c/strong\u003e), iNOS (G), and TGF‑β1 (\u003cstrong\u003eH\u003c/strong\u003e) determined by qRT-PCR. (\u003cstrong\u003eI\u003c/strong\u003e) Protein levels of collagen I, α‑SMA, IL6, iNOS, and TGF-β1 assessed by Western blot after 7 days of hypoxia with or without miR-195-3p mimic, \u003csub\u003e\u003cem\u003eoe\u003c/em\u003e\u003c/sub\u003eOct4, \u003csub\u003e\u003cem\u003eoe\u003c/em\u003e\u003c/sub\u003eVEGF, and/or \u003csub\u003e\u003cem\u003esh\u003c/em\u003e\u003c/sub\u003eVEGF treatment. All data are presented as mean ± SD; n = 5 per group. One-way ANOVA with post-hoc test was used for multiple comparisons. \u003csup\u003e*\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e***\u003c/sup\u003eP\u0026lt;0.001. DAPI=4′,6-Diamidino-2- phenylindole.\u003c/p\u003e","description":"","filename":"YusPaper1Figure520262.png","url":"https://assets-eu.researchsquare.com/files/rs-8443063/v1/f234293170ec73c8b8d40fce.png"},{"id":105387727,"identity":"ab452b33-4fd1-4194-88f6-b2f9a1faca9c","added_by":"auto","created_at":"2026-03-25 12:47:48","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":6285090,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOct4 antagonizes miR-195-3p to ameliorate glucose metabolic reprogramming in MSCs\u003c/strong\u003e. (\u003cstrong\u003eA\u003c/strong\u003e) Volcano plot displaying differentially expressed genes in hypoxic MSCs treated with miR-195-3p mimic versus control after 72 hours. (\u003cstrong\u003eB\u003c/strong\u003e) Top 10 enriched gene sets identified from the 756 downregulated genes. (\u003cstrong\u003eC\u003c/strong\u003e) Pearson correlation analysis between miR-195-3p expression and single-sample GSEA (ssGSEA) scores for glycolysis in MSCs. (\u003cstrong\u003eD\u003c/strong\u003e) Extracellular acidification rate (ECAR) measured using a Seahorse XF96 Analyzer in MSCs under the following conditions: control (CON), miR-195-3p mimic (miR-195-mimic) alone, Oct4 overexpression (\u003csub\u003e\u003cem\u003eoe\u003c/em\u003e\u003c/sub\u003eOct4) alone,\u003cem\u003e \u003c/em\u003eVEGF overexpression (\u003csub\u003e\u003cem\u003eoe\u003c/em\u003e\u003c/sub\u003eVEGF) alone, or miR-195-mimic combined with \u003csub\u003e\u003cem\u003eoe\u003c/em\u003e\u003c/sub\u003eOct4 or \u003csub\u003e\u003cem\u003eoe\u003c/em\u003e\u003c/sub\u003eVEGF. Glu, glucose; Oligo, oligomycin. (\u003cstrong\u003eE\u003c/strong\u003e) Quantification of ECAR under the treatments described in (\u003cstrong\u003eD\u003c/strong\u003e). (\u003cstrong\u003eF\u003c/strong\u003e) Glycolytic proton efflux rate (glycoPER) analyzed under the same experimental conditions as in (\u003cstrong\u003eD\u003c/strong\u003e). (\u003cstrong\u003eG\u003c/strong\u003e) Quantification of glycoPER as outlined in (\u003cstrong\u003eF\u003c/strong\u003e). (\u003cstrong\u003eH\u003c/strong\u003e, \u003cstrong\u003eI\u003c/strong\u003e) Glucose consumption (\u003cstrong\u003eH\u003c/strong\u003e) and lactate production (\u003cstrong\u003eI\u003c/strong\u003e) in MSCs across the indicated treatment groups. (\u003cstrong\u003eJ\u003c/strong\u003e) Heat map comparing mRNA expression levels of key glycolytic enzymes between miR-195-3p mimic-treated and control MSCs. (\u003cstrong\u003eK\u003c/strong\u003e) Western blot analysis of key glycolytic enzymes in MSCs treated with CON, miR-195-mimic alone, \u003csub\u003e\u003cem\u003eoe\u003c/em\u003e\u003c/sub\u003eOct4 alone,\u003cem\u003e \u003c/em\u003e\u003csub\u003e\u003cem\u003eoe\u003c/em\u003e\u003c/sub\u003eVEGF alone, or miR-195-mimic combined with \u003csub\u003e\u003cem\u003eoe\u003c/em\u003e\u003c/sub\u003eOct4 or \u003csub\u003e\u003cem\u003eoe\u003c/em\u003e\u003c/sub\u003eVEGF. All experiments were performed with three technical replicates. Statistical significance was assessed by one-way ANOVA across five biological replicates. Data are presented as mean ± SD; \u003csup\u003e*\u003c/sup\u003eP \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003eP \u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003eP \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"YusPaper1Figure620262.png","url":"https://assets-eu.researchsquare.com/files/rs-8443063/v1/9ce1b1c7c4c48e18f2223346.png"},{"id":105387734,"identity":"5b4a2b54-88fa-433b-87ce-a83b5fa6bfe1","added_by":"auto","created_at":"2026-03-25 12:47:49","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":24667111,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLactate counteracts miR-195-3p-mediated impairment of MSC therapy through an Oct4-dependent mechanism. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Representative echocardiographic images obtained 1 month after myocardial infarction (MI) in rats receiving intramyocardial injections of phosphate-buffered saline (PBS), vehicle-treated MSCs, or MSCs preconditioned with lactate, \u003csub\u003e\u003cem\u003eoe\u003c/em\u003e\u003c/sub\u003emiR-195-3p, \u003csub\u003e\u003cem\u003eoe\u003c/em\u003e\u003c/sub\u003eOct4, \u003csub\u003e\u003cem\u003esh\u003c/em\u003e\u003c/sub\u003eOct4, lactate + \u003csub\u003e\u003cem\u003esh\u003c/em\u003e\u003c/sub\u003eOct4, or \u003csub\u003e\u003cem\u003eoe\u003c/em\u003e\u003c/sub\u003emiR-195-3p + \u003csub\u003e\u003cem\u003eoe\u003c/em\u003e\u003c/sub\u003eOct4. (\u003cstrong\u003eB\u003c/strong\u003e) Transverse heart sections stained with TTC for infarct size quantification (expressed as a percentage of the entire left ventricular area). The infarcted myocardium appears pale due to the absence of red TTC staining. (\u003cstrong\u003eC\u003c/strong\u003e) High-magnification Masson’s trichrome staining of heart sections at 30 days post-MI. Viable myocardium stains bright red, fibrotic areas appear blue, and the infarcted region remains unstained (pale). Scale bars = 20 μm. (\u003cstrong\u003eD\u003c/strong\u003e,\u003cstrong\u003e E\u003c/strong\u003e) Immunofluorescence images showing engraftment of GFP-labeled MSCs (\u003cstrong\u003eD\u003c/strong\u003e, green) and TUNEL staining (\u003cstrong\u003eE\u003c/strong\u003e, red) for apoptosis in the infarct border zone at 1 month post-transplantation. Scale bars = 50 μm. (\u003cstrong\u003eF\u003c/strong\u003e) Representative immunohistochemical staining for CD31 (brown) in peri-infarct regions to assess vascular density. Scale bars = 20 μm. (\u003cstrong\u003eG\u003c/strong\u003e) Schematic diagram illustrating the animal experimental group and survival. (\u003cstrong\u003eH\u003c/strong\u003e, \u003cstrong\u003eI\u003c/strong\u003e) Quantitative echocardiographic analysis of left ventricular ejection fraction (LVEF, \u003cstrong\u003eH\u003c/strong\u003e) and fractional shortening (LVFS, \u003cstrong\u003eI\u003c/strong\u003e) at 1 month after PBS or MSC transplantation. (\u003cstrong\u003eJ\u003c/strong\u003e–\u003cstrong\u003eN\u003c/strong\u003e) Bar graphs summarizing quantitative data for infarct size (\u003cstrong\u003eJ\u003c/strong\u003e), fibrosis area (\u003cstrong\u003eK\u003c/strong\u003e), MSC engraftment rate (\u003cstrong\u003eL\u003c/strong\u003e), apoptosis rate (\u003cstrong\u003eM\u003c/strong\u003e), and microvascular density (\u003cstrong\u003eN\u003c/strong\u003e), corresponding to panels (\u003cstrong\u003eB\u003c/strong\u003e)–(\u003cstrong\u003eF\u003c/strong\u003e), respectively. Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA followed by appropriate post-hoc tests. \u003csup\u003e*\u003c/sup\u003eP \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003eP \u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003eP \u0026lt; 0.001. Sample sizes: For echocardiographic analyses (\u003cstrong\u003eH\u003c/strong\u003e, \u003cstrong\u003eI\u003c/strong\u003e): n = 15, 16, 15, 18, 20, 15, 17, and 19 for PBS-, vehicle-, lactate-, \u003csub\u003e\u003cem\u003eoe\u003c/em\u003e\u003c/sub\u003emiR-195-3p-, \u003csub\u003e\u003cem\u003eoe\u003c/em\u003e\u003c/sub\u003eOct4-, \u003csub\u003e\u003cem\u003esh\u003c/em\u003e\u003c/sub\u003eOct4-, lactate+\u003csub\u003e\u003cem\u003esh\u003c/em\u003e\u003c/sub\u003eOct4-, and \u003csub\u003e\u003cem\u003eoe\u003c/em\u003e\u003c/sub\u003emiR-195-3p+ \u003csub\u003e\u003cem\u003eoe\u003c/em\u003e\u003c/sub\u003eOct4-treated groups, respectively. For histomorphometric analyses (\u003cstrong\u003eJ\u003c/strong\u003e–\u003cstrong\u003eN\u003c/strong\u003e): n = 5 per group. (\u003cstrong\u003eO\u003c/strong\u003e) Kaplan‒Meier survival curves to compare mortality between the eight rat groups. Significance was determined by log-rank (Mantel‒Cox) test (n = 20 per group).\u003c/p\u003e","description":"","filename":"YusPaper1Figure7202511.png","url":"https://assets-eu.researchsquare.com/files/rs-8443063/v1/5d10135e38b43919300a6a3c.png"},{"id":105565384,"identity":"638f458c-7d5d-4680-8886-7b7408aedd6b","added_by":"auto","created_at":"2026-03-27 12:53:05","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":17319183,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOct4 promotes angiogenesis through miR-195-3p–dependent metabolic reprogramming of MSCs\u003c/strong\u003e. (\u003cstrong\u003eA\u003c/strong\u003e–\u003cstrong\u003eC\u003c/strong\u003e) mRNA expression of (\u003cstrong\u003eA\u003c/strong\u003e) Oct4, (\u003cstrong\u003eB\u003c/strong\u003e) GLUT1, and (\u003cstrong\u003eC\u003c/strong\u003e) HK2 in myocardial tissues from rats treated with PBS or MSCs preconditioned with vehicle, lactate, \u003csub\u003e\u003cem\u003eoe\u003c/em\u003e\u003c/sub\u003emiR-195-3p, \u003csub\u003e\u003cem\u003eoe\u003c/em\u003e\u003c/sub\u003eOct4, \u003csub\u003e\u003cem\u003esh\u003c/em\u003e\u003c/sub\u003eOct4, lactate + \u003csub\u003e\u003cem\u003esh\u003c/em\u003e\u003c/sub\u003eOct4, or \u003csub\u003e\u003cem\u003eoe\u003c/em\u003e\u003c/sub\u003emiR-195-3p + \u003csub\u003e\u003cem\u003eoe\u003c/em\u003e\u003c/sub\u003eOct4. (\u003cstrong\u003eD\u003c/strong\u003e) Representative immunoblots and quantitative analysis of Oct4, GLUT1, HK2, VEGF, VEGFR2, and Akt protein levels. (\u003cstrong\u003eE\u003c/strong\u003e) Immunofluorescence staining of Oct4 (\u003cstrong\u003egreen\u003c/strong\u003e) and GLUT1 (\u003cstrong\u003ered\u003c/strong\u003e) in rat cardiomyocytes isolated after the indicated MSC-based treatments. Nuclei were stained with DAPI (blue). Scale bar: 50 μm. (\u003cstrong\u003eF\u003c/strong\u003e–\u003cstrong\u003eH\u003c/strong\u003e) Metabolic profiling by ¹³C-glucose tracing: (\u003cstrong\u003eF\u003c/strong\u003e) Extracellular lactate production; (\u003cstrong\u003eG\u003c/strong\u003e) ¹³C-labeled citrate enrichment; (\u003cstrong\u003eH\u003c/strong\u003e) ¹³C-labeled succinate enrichment. (\u003cstrong\u003eI\u003c/strong\u003e–\u003cstrong\u003eK\u003c/strong\u003e) mRNA expression of angiogenesis-related factors: (\u003cstrong\u003eI\u003c/strong\u003e) VEGF, (\u003cstrong\u003eJ\u003c/strong\u003e) VEGFR2, and (\u003cstrong\u003eK\u003c/strong\u003e) Akt in myocardial tissues. (\u003cstrong\u003eL\u003c/strong\u003e) Immunohistochemical staining of VEGF and Akt in myocardial sections from rats receiving the indicated treatments. Scale bar: 20 μm. Data are presented as mean ± SEM; n = 5 per group. Statistical significance was determined by one-way ANOVA with appropriate post-hoc tests. \u003csup\u003e*\u003c/sup\u003eP \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003eP \u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003eP \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"YusPaper1Figure82025112.png","url":"https://assets-eu.researchsquare.com/files/rs-8443063/v1/01f878aec3bd3d64c61baa75.png"},{"id":105570088,"identity":"92e42a29-12f6-4d9a-b45a-2834b8b79d14","added_by":"auto","created_at":"2026-03-27 13:14:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":103749373,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8443063/v1/684411c1-1901-4d55-a6b7-54f57c5e4e97.pdf"},{"id":105387720,"identity":"1f0dcd52-dfbd-464a-9797-e2d28c5caac8","added_by":"auto","created_at":"2026-03-25 12:47:47","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":54489,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalDataResearchdesignandmethods20262.docx","url":"https://assets-eu.researchsquare.com/files/rs-8443063/v1/872e02f50896cdf1bcedd213.docx"},{"id":105387735,"identity":"aeb5e446-67b1-4f39-886c-f81e84394c67","added_by":"auto","created_at":"2026-03-25 12:47:49","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":11918672,"visible":true,"origin":"","legend":"","description":"","filename":"YusPaper1FigureS120262.tif","url":"https://assets-eu.researchsquare.com/files/rs-8443063/v1/44bcdbf32dc21cdc9ada0f32.tif"},{"id":105387732,"identity":"4f8d24fb-3e29-434a-9484-0bbe2ddbe63d","added_by":"auto","created_at":"2026-03-25 12:47:48","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":13611104,"visible":true,"origin":"","legend":"","description":"","filename":"YusPaper1FigureS2.tif","url":"https://assets-eu.researchsquare.com/files/rs-8443063/v1/4544b81a99521c96c15869ce.tif"},{"id":105387729,"identity":"4ba1ace8-b0fd-495a-b105-50020ac87613","added_by":"auto","created_at":"2026-03-25 12:47:48","extension":"doc","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":33792,"visible":true,"origin":"","legend":"","description":"","filename":"YusPaper1TableS1202510primer.doc","url":"https://assets-eu.researchsquare.com/files/rs-8443063/v1/4b9dd6a1697b15b070feed63.doc"},{"id":105387726,"identity":"c7277739-b552-45a7-8f0d-dfffefd0c61d","added_by":"auto","created_at":"2026-03-25 12:47:48","extension":"doc","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":76288,"visible":true,"origin":"","legend":"","description":"","filename":"YusPaper1TableS220259.doc","url":"https://assets-eu.researchsquare.com/files/rs-8443063/v1/018b349fe48f2a63b88ca612.doc"},{"id":105387730,"identity":"8e47e747-8e46-4cc1-9982-0b0e2ecd5d2c","added_by":"auto","created_at":"2026-03-25 12:47:48","extension":"tif","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":14397984,"visible":true,"origin":"","legend":"","description":"","filename":"YusPaper1Graphicabstract.tif","url":"https://assets-eu.researchsquare.com/files/rs-8443063/v1/9e0cfeeb6fc1e192dd7d293c.tif"},{"id":105387723,"identity":"ef7ae634-3cc0-433a-be24-2ae653b128fb","added_by":"auto","created_at":"2026-03-25 12:47:48","extension":"pdf","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":3316312,"visible":true,"origin":"","legend":"","description":"","filename":"OriginalWesternblotimages20262.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8443063/v1/ad61989084bbb87485ac4a2d.pdf"},{"id":105387731,"identity":"1e04d6b9-fb91-4146-aa0f-6199188f7ff0","added_by":"auto","created_at":"2026-03-25 12:47:48","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":25144,"visible":true,"origin":"","legend":"","description":"","filename":"ARRIVEGuidelines2.docx","url":"https://assets-eu.researchsquare.com/files/rs-8443063/v1/37a893f7ec30963d7f388323.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Metabolic Reprogramming by Lactate Unlocks a Pro-Survival Code in MSCs: The miR-195-3p/Oct4/VEGF Axis in Heart Repair","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMyocardial infarction (MI) persists as a leading cause of global cardiovascular mortality and heart failure, with the irreversible loss of cardiomyocytes presenting a fundamental therapeutic challenge \u003csup\u003e[1]\u003c/sup\u003e. Mesenchymal stem cell (MSC) transplantation has emerged as a promising regenerative strategy, leveraging their multipotent differentiation capacity and paracrine secretion of angiogenic factors to restore function in infarcted myocardium \u003csup\u003e[2]\u003c/sup\u003e. However, the clinical translation of this approach has been severely limited by the hostile ischemic microenvironment, where \u0026gt;90% of transplanted MSCs undergo rapid apoptosis within 72 hours due to hypoxia-induced metabolic stress and inflammatory signaling\u003csup\u003e\u0026nbsp;[3,4]\u003c/sup\u003e. This catastrophic cell loss fundamentally cripples their reparative potential, as sustained MSC retention is prerequisite for effective cardiac repair \u003csup\u003e[5]\u003c/sup\u003e. Enhancing MSC resilience in the post-MI microenvironment therefore represents a critical barrier to advancing stem cell-based therapies.\u003c/p\u003e\n\u003cp\u003eThe infarct core and border zone exhibit severe metabolic perturbations, including oxygen and glucose deprivation that forces a shift to anaerobic glycolysis with consequent lactate accumulation \u003csup\u003e[6]\u003c/sup\u003e. While traditionally viewed as a metabolic waste product, lactate is now recognized as a key signaling molecule that modulates various cellular functions\u003csup\u003e\u0026nbsp;[7]\u003c/sup\u003e. However, its potential role in preconditioning MSCs to enhance their survival and regenerative capacity remains poorly understood, representing an intriguing therapeutic avenue worthy of exploration\u003csup\u003e\u0026nbsp;[8]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eConcurrent with metabolic stress, the ischemic microenvironment triggers profound alterations in gene expression orchestrated by microRNAs (miRNAs) \u003csup\u003e[9]\u003c/sup\u003e. These small non-coding RNAs (19-25 nt) function as primary epigenetic regulators of cell fate decisions through post-transcriptional gene silencing \u003csup\u003e[10, 11]\u003c/sup\u003e. Particularly relevant to stem cell biology, miRNAs establish context-dependent regulatory circuits with core pluripotency factors such as Oct4 \u003csup\u003e[12, 13]\u003c/sup\u003e. In embryonic stem cells, for instance, miR-145 suppression sustains Oct4-mediated self-renewal, while its artificial upregulation extinguishes Oct4 expression to force lineage commitment\u003csup\u003e\u0026nbsp;[13, 14]\u003c/sup\u003e. This sophisticated miRNA-transcription factor interplay creates signaling networks that dictate cellular responses to environmental stresses, though their specific configuration in MSCs under ischemia remains largely unexplored.\u003c/p\u003e\n\u003cp\u003eAmong hypoxia-responsive miRNAs, the miR-15 family has been implicated in various pathological processes, demonstrating context-dependent functionality through targeting multiple transcription factors\u003csup\u003e\u0026nbsp;[15-18]\u003c/sup\u003e. Our preliminary systematic screening specifically identified miR-195-3p, a miR-15 family member, as the most significantly upregulated miRNA in hypoxic MSCs, while Oct4 expression was concurrently suppressed. This inverse relationship, observed against the backdrop of Oct4\u0026apos;s documented but complex role in MSC survival under stress\u003csup\u003e\u0026nbsp;[19]\u003c/sup\u003e, prompted our investigation into their regulatory interplay and functional consequences.\u003c/p\u003e\n\u003cp\u003eSignificant gaps persist in understanding how metabolic preconditioning interfaces with miRNA-mediated regulation of MSC resilience. While various preconditioning strategies have shown promise, the potential of lactate-a naturally abundant metabolite in the ischemic niche \u003csup\u003e[20]\u003c/sup\u003e-to modulate the miR-195-3p/Oct4 axis remains completely unexplored. Furthermore, although numerous hypoxia-responsive miRNAs have been identified, a definitive pathway linking a specific miRNA through a master regulator like Oct4 to downstream metabolic and angiogenic effectors \u003csup\u003e[21]\u0026nbsp;\u003c/sup\u003ein MSCs has not been established.\u003c/p\u003e\n\u003cp\u003eThis study was designed to bridge these critical gaps by testing our central hypothesis that miR-195-3p impairs MSC survival under hypoxic stress by inhibiting an Oct4-driven transcriptional program that enhances glycolytic metabolism and activates VEGF signaling, while lactate preconditioning exerts protective effects by disrupting this inhibitory pathway. Through this work, we aim to elucidate a complete signaling axis connecting metabolic preconditioning with epigenetic regulation to enhance MSC-based cardiac repair.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHypoxia-induced miR-195-3p directly targets and represses Oct4\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo identify key regulators of MSC hypoxic response, we found that 72-hour hypoxia culture significantly altered miRNA expression. Bioinformatic analysis and miRNA-seq identified miR-195-3p as the most markedly elevated miRNA (\u003cstrong\u003eFigure 1A\u003c/strong\u003e), which was independently validated by RT-qPCR (\u003cstrong\u003eFigure 1B\u003c/strong\u003e). Concurrently, we observed a significant repression of the predicted target gene Oct4 (\u003cstrong\u003eFigure 1C\u003c/strong\u003e). Specifically, hypoxia led to a 34.9% increase in miR-195-3p (p=0.001; Figure \u003cstrong\u003e1D\u003c/strong\u003e) and a 42.0% decrease in Oct4 mRNA (p\u0026lt;0.001; \u003cstrong\u003eFigure 1E\u003c/strong\u003e), with confirmed reduction at the protein level (\u003cstrong\u003eFigure 1F\u003c/strong\u003e), demonstrating an inverse correlation.\u003c/p\u003e\n\u003cp\u003eWe subsequently confirmed a direct targeting relationship. A luciferase reporter assay containing the wild-type Oct4 3\u0026apos;‑UTR showed that miR-195-3p overexpression significantly suppressed luciferase activity, an effect rescued by a miR-195-3p inhibitor (\u003cstrong\u003eFigure 1G\u003c/strong\u003e). Functionally, miR-195-3p overexpression in MSCs markedly decreased endogenous Oct4 mRNA and protein, while its inhibition attenuated this suppression (\u003cstrong\u003eFigure 1H\u003c/strong\u003e, \u003cstrong\u003e1I\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eCollectively, these results demonstrate that hypoxia elevates miR-195-3p expression, which in turn directly targets and transcriptionally represses Oct4, identifying a pivotal regulatory axis for MSC survival under hypoxia.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003emiR-195-3p/Oct4 axis regulates MSC survival in hypoxia\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo validate the functional role of the miR-195-3p/Oct4 axis in MSC survival under hypoxia, we modulated the expression of miR-195-3p or Oct4 through genetic approaches and evaluated their effects on cell viability and apoptosis. After 72 hours of hypoxia, miR-195-3p mimic reduced cell viability by 34.4% (\u003cem\u003ep\u003c/em\u003e=0.01) and 30.4% (\u003cem\u003ep\u003c/em\u003e=0.003) compared to the CON and mimic NC groups, respectively. In contrast, miR-195-3p inhibitor enhanced viability by 37.3% (\u003cem\u003ep\u003c/em\u003e=0.01) and 46.2% (p \u0026lt; 0.001), respectively (\u003cstrong\u003eFigure S1A\u003c/strong\u003e, \u003cstrong\u003eS1C\u003c/strong\u003e). Accordingly, miR-195-3p mimic increased apoptosis by 24.1% (\u003cem\u003ep\u003c/em\u003e=0.004) and 24.4% (\u003cem\u003ep\u003c/em\u003e=0.004), whereas the inhibitor decreased it by 18.4% (\u003cem\u003ep\u003c/em\u003e=0.04) and 20.8% (\u003cem\u003ep\u003c/em\u003e=0.010), relative to CON or mimic NC groups (\u003cstrong\u003eFigure\u003c/strong\u003e \u003cstrong\u003eS1B\u003c/strong\u003e, \u003cstrong\u003eS1D\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eNext, we investigated that Oct4 acted as the target signal of miR-195-3p and rescued the inhibition of miR-195-3p on the survival of MSCs under hypoxic condition. We performed genetic inhibition or overexpression of miR-195-3p/Oct4 by miR-195-3p inhibitor or mimic or Oct4 overexpression (\u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003eOct4) or shRNA (\u003cem\u003e\u003csub\u003esh\u003c/sub\u003e\u003c/em\u003eOct4) on MSCs, respectively. After 72h hypoxia, compared with CON group, Oct4 overexpression maintained significantly higher viability, but Oct4 shRNA resulted in sustained viability loss. Moreover, Oct4 overexpression rescued the cell viability decrease in miR-195-3p-overexpressing MSCs. While miR-195-3p inhibitor caused significant increased in MSC viability, Oct4 shRNA abrogated this increase (\u003cstrong\u003eFigure 2A\u003c/strong\u003e, \u003cstrong\u003e2D\u003c/strong\u003e). Ki67 staining revealed a concordant proliferative trend: relative to the CON group, \u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003eOct4 markedly increased the proliferation rate under hypoxia, whereas \u003cem\u003e\u003csub\u003esh\u003c/sub\u003e\u003c/em\u003eOct4 significantly reduced it. Notably, \u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003eOct4 rescued the proliferation deficit induced by miR-195-3p mimic. Although miR-195-3p inhibitor enhanced proliferation, this effect was abolished by concurrent Oct4 silencing (\u003cstrong\u003eFigure 2B\u003c/strong\u003e, \u003cstrong\u003e2E\u003c/strong\u003e). Conversely, an opposing pattern was observed in apoptosis: compared with CON, Oct4 overexpression substantially decreased apoptosis, while \u003cem\u003e\u003csub\u003esh\u003c/sub\u003e\u003c/em\u003eOct4 led to a significant increase. Moreover, \u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003eOct4 counteracted the pro-apoptotic effect of miR-195-3p mimic. Similarly, the anti-apoptotic outcome resulting from miR-195-3p inhibition was reversed by Oct4 knockdown (\u003cstrong\u003eFigure 2C\u003c/strong\u003e, \u003cstrong\u003e2F\u003c/strong\u003e). These findings collectively suggest that the miR-195-3p/Oct4 axis regulates MSC survival under hypoxic conditions.\u003c/p\u003e\n\u003cp\u003eTo further investigate the apoptotic pathway associated with the miR-195-3p/Oct4 axis, we employed a human cytokine array (AAH-CYT-G2000, RayBiotech) to profile the expression of survival-related cytokines in MSCs cultured under hypoxia, with or without miR-195-3p mimic or Oct4 overexpression. The results indicated that the proliferation-related factors Hsp27 and Oct4 were expressed at higher levels in the \u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003eOct4 group, and \u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003eOct4 rescued their suppression induced by the miR-195-3p mimic. In contrast, the apoptotic factors Bax and Caspase 9 were more abundant in MSCs treated with miR-195-3p mimic compared to those with Oct4 overexpression (\u003cstrong\u003eFigure 2G\u003c/strong\u003e). Based on the antibody array results, we selected Hsp27, Oct4, Bax, Caspase 9, along with the anti-apoptotic factor Bcl-2, for validation via qRT-PCR and western blotting in MSCs receiving miR-195-3p mimic or Oct4 overexpression. This aimed to confirm the array data and evaluate whether the miR-195-3p/Oct4 axis promotes anti-apoptosis. As shown in \u003cstrong\u003eFigure 2H\u003c/strong\u003e, Oct4 overexpression significantly suppressed the mRNA expression of the pro-apoptotic factors Bax and Caspase 9, while enhancing the mRNA levels of the anti-apoptotic factors Bcl-2 and Hsp27, as well as the proliferation marker Oct4. Conversely, miR-195-3p mimic produced the opposite effect, which was subsequently reversed by \u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003eOct4 (\u003cstrong\u003eFigure 2H\u003c/strong\u003e). These trends were further corroborated by western blot analysis (\u003cstrong\u003eFigure 2I\u003c/strong\u003e). Together, these results reinforce the conclusion that the miR-195-3p/Oct4 axis modulates MSC survival under hypoxia through regulating pro- and anti-apoptotic signaling.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eVEGF alleviates miR-195-3p inhibition to enhance Oct4-driven angiogenesis in hypoxic MSCs\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOf note, the results in the above-mentioned cytokine array showed that two isoforms of VEGFs, VEGFA and VEGFD, activators of angiogenesis\u003csup\u003e\u0026nbsp;[22]\u003c/sup\u003e,\u0026nbsp;and their effector genes (VEGFR2, Endoglin, Tie2) were significantly upregulated or downregulated by Oct4 overexpression or miR-195-3p-mimic (\u003cstrong\u003eFigures 2G\u003c/strong\u003e), indicating concomitant regulation of anti-apoptotic signaling and angiogenesis compared to CON group MSCs. These findings suggest that Oct4 activates VEGF signaling, thereby suppressing a cascade of miR-195-3p-induced events that convert hypoxic CMs into an apoptotic state, ultimately promoting MSCs survival and angiogenesis. Thereby, we next asked if VEGF mediates these effects. For this purpose, MSCs were treated with\u0026nbsp;miR-195-3p mimic/Oct4 overexpression (\u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003eOct4)\u003csup\u003e\u0026nbsp;\u003c/sup\u003eeither alone or in combination with\u0026nbsp;VEGF overexpression (\u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003eVEGF) or shRNA (\u003cem\u003e\u003csub\u003esh\u003c/sub\u003e\u003c/em\u003eVEGF).\u0026nbsp;The MSCs\u0026nbsp;were then cultured for seven days under hypoxic conditions\u0026nbsp;to assess the effects.\u0026nbsp;As demonstrated by CCK-8 assay (\u003cstrong\u003eFigure 3Ab\u003c/strong\u003e) and Ki67 staining (\u003cstrong\u003eFigure 3Ba\u003c/strong\u003e), VEGF overexpression promoted the proliferation of MSCs and counteracted the suppressive effect of miR-195-3p mimic on MSC proliferation (\u003cstrong\u003eFigure 3A, 3B\u003c/strong\u003e). FACS analysis via PI staining further revealed that VEGF overexpression reduced apoptosis in hypoxic MSCs and attenuated the pro-apoptotic effect induced by miR-195-3p mimic (\u003cstrong\u003eFigure 3C\u003c/strong\u003e). Immunofluorescence analysis using the vascular marker VWF showed that VEGF overexpression significantly enhanced the expression of Factor VIII in hypoxic MSCs, whereas miR-195-3p mimic reduced it. Notably, this reduction was reversed upon cotreatment with VEGF overexpression (\u003cstrong\u003eFigure 3D\u003c/strong\u003e). Furthermore, as illustrated in \u003cstrong\u003eFigures 3E-J\u003c/strong\u003e, VEGF overexpression markedly up-regulated the mRNA levels of key vascular growth factors and receptors\u0026mdash;including VEGF, bFGF, VEGFR2, Tie2, Endoglin, and CD31\u0026mdash;in MSCs under hypoxic conditions. Although miR-195-3p mimic abolished these promotive effects, concurrent VEGF overexpression restored their expression. These findings were corroborated by Western blot analyses of VEGFA, VEGFR2, CD31, and Tie2 (\u003cstrong\u003eFigure 3K\u003c/strong\u003e). Collectively, these results indicate that VEGF signaling attenuates apoptosis and stimulates angiogenesis in MSCs under hypoxic conditions. Moreover, the pro-apoptotic effect of miR-195-3p is reversed by VEGF overexpression, underscoring the importance of VEGF upregulation in supporting MSC survival and vascularization under hypoxia.\u003c/p\u003e\n\u003cp\u003eSubsequent experiments showed that \u003cem\u003e\u003csub\u003esh\u003c/sub\u003e\u003c/em\u003eVEGF suppressed MSC proliferation (\u003cstrong\u003eFigure 4A\u003c/strong\u003e) and abolished the enhancing effect of Oct4 overexpression on proliferation, as assessed by CCK-8 assay (\u003cstrong\u003eFigure 4E\u003c/strong\u003e) and Ki67 staining (\u003cstrong\u003eFigure 4B\u003c/strong\u003e, \u003cstrong\u003e4F\u003c/strong\u003e). VEGF downregulation also promoted apoptosis in hypoxic MSCs and diminished the anti-apoptotic influence of \u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003eOct4 (\u003cstrong\u003eFigure 4C\u003c/strong\u003e, \u003cstrong\u003e4G\u003c/strong\u003e). In addition, \u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003eOct4 markedly increased VWF expression in hypoxic MSCs, and this effect was reduced by \u003cem\u003e\u003csub\u003esh\u003c/sub\u003e\u003c/em\u003eVEGF (\u003cstrong\u003eFigure 4D\u003c/strong\u003e, \u003cstrong\u003e4H\u003c/strong\u003e). To determine whether Oct4 activation mediates VEGF-induced proliferation and angiogenesis, we measured VEGF-related cytokine expression in MSCs subjected to Oct4 overexpression, VEGF knockdown, or both, via qRT-PCR and immunofluroscence. The data revealed that \u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003eOct4 elevated VEGF mRNA levels (\u003cstrong\u003eFigure 4I\u003c/strong\u003e) and potentiated VEGF pathway activity, as indicated by upregulation of bFGF \u003cstrong\u003e(Figure 4J\u003c/strong\u003e), VEGFR2 (\u003cstrong\u003eFigure 4K\u003c/strong\u003e), VECAD (\u003cstrong\u003eFigure 4L\u003c/strong\u003e), Endoglin (\u003cstrong\u003eFigure 4M\u003c/strong\u003e), and CD31 (\u003cstrong\u003eFigure 4N\u003c/strong\u003e) compared to controls. Cotreatment with \u003cem\u003e\u003csub\u003esh\u003c/sub\u003e\u003c/em\u003eVEGF blocked Oct4-induced VEGF pathway activation and proliferation, confirming the dependency of Oct4-mediated benefits on VEGF upregulation. Western blot analysis further validated that Oct4 overexpression enhanced VEGF signaling and increased expression of the anti-apoptotic protein Bcl2, both of which were negated by VEGF shRNA (\u003cstrong\u003eFigure 4O\u003c/strong\u003e). Together, these results demonstrate that Oct4 upregulation stimulates VEGF expression, thereby reactivating the VEGF signaling axis to bolster MSC proliferation and angiogenesis under hypoxia.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003emiR-195-3p regulates MSC fibrosis/inflammation in hypoxia via Oct4/VEGF axis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe further demonstrated that Oct4 counteracts miR-195-3p-induced fibrosis and inflammation primarily through upregulation of VEGF. In hypoxic MSCs, overexpression of miR-195-3p (via mimics) aggravated hypoxia-triggered fibrotic and inflammatory responses (\u003cstrong\u003eFigure 5A-C\u003c/strong\u003e) and markedly elevated the expression of key fibrotic markers\u0026mdash;collagen I (\u003cstrong\u003eFigure 5D\u003c/strong\u003e, \u003cstrong\u003e5I\u003c/strong\u003e) and \u0026alpha;-smooth muscle actin (\u0026alpha;-SMA, \u003cstrong\u003eFigure 5E\u003c/strong\u003e, \u003cstrong\u003e5I\u003c/strong\u003e)\u0026mdash;as well as key inflammatory mediators, including IL-6 (\u003cstrong\u003eFigure 5F\u003c/strong\u003e, \u003cstrong\u003e5I\u003c/strong\u003e), iNOS (\u003cstrong\u003eFigure 5G\u003c/strong\u003e, \u003cstrong\u003e5I\u003c/strong\u003e), and\u0026nbsp;TGF-\u0026beta;1\u0026nbsp;(\u003cstrong\u003eFigure 5H\u003c/strong\u003e, \u003cstrong\u003e5I\u003c/strong\u003e), at both mRNA and protein levels. Conversely, overexpression of Oct4 or VEGF significantly suppressed hypoxia-induced fibrosis and inflammation in MSCs and effectively reversed the pro-fibrotic and pro-inflammatory effects driven by miR-195-3p upregulation (\u003cstrong\u003eFigure 5B\u003c/strong\u003e\u003cstrong\u003e\u0026ndash;\u003c/strong\u003e\u003cstrong\u003eI\u003c/strong\u003e), indicating that the Oct4/VEGF axis acts as a protective pathway against these pathological processes. Moreover, knockdown of VEGF recapitulated the phenotype induced by miR-195-3p overexpression, confirming VEGF as a major functional target mediating miR-195-3p-driven effects.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eThe Oct4/VEGF axis rescues aerobic glycolysis from miR-195-3p-mediated suppression\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo elucidate the molecular mechanisms through which the Oct4/VEGF axis counteracts miR-195-3p-mediated phenotypic alterations in hypoxic MSCs, we conducted transcriptome sequencing (RNA-seq) comparing miR-195-3p- overexpressing cells with control cells (\u003cstrong\u003eFigure 6A\u003c/strong\u003e). Gene Set Enrichment Analysis (GSEA) identified the top ten pathways suppressed by miR-195-3p, encompassing E2F targets, DNA repair, mitotic spindle, cytokine\u0026ndash;cytokine receptor interaction, G2M checkpoint, VEGF signaling, glycolysis, Oct4 targets, hypoxic response upregulation, and angiogenesis (\u003cstrong\u003eFigure 6B\u003c/strong\u003e). In light of the therapeutic significance of metabolic reprogramming in tumor biology, we sought to clarify the role of miR-195-3p in modulating glycolytic flux. Metabolic assessment revealed that miR-195-3p overexpression led to a marked reduction in extracellular acidification rate (ECAR, \u003cstrong\u003eFigure 6D\u003c/strong\u003e, \u003cstrong\u003e6E\u003c/strong\u003e) and glycolytic proton efflux rate (glycoPER, \u003cstrong\u003eFigure 6F\u003c/strong\u003e, \u003cstrong\u003e6G\u003c/strong\u003e). In contrast, Oct4/VEGF overexpression not only elevated both ECAR and glycoPER but also substantially reversed the miR-195-3p-driven suppression of glycolytic activity. Concordantly, miR-195-3p mimic impeded glucose uptake and lactate generation, whereas Oct4/VEGF overexpression augmented these processes in hypoxic MSCs (\u003cstrong\u003eFigure 6H\u003c/strong\u003e, \u003cstrong\u003e6I\u003c/strong\u003e). RNA from MSCs was analyzed using the Rat Genome 230 2.0 Array. After normalization, DEGs were identified (fold-change\u0026nbsp;\u0026ge;1.5, p\u0026lt;0.05). GSEA revealed significantly altered pathways, with top results visualized in a heatmap (NES, FDR). Specifically, key glycolytic enzymes (\u003cem\u003eSLC2A1/GLUT1\u003c/em\u003e, \u003cem\u003eHK2\u003c/em\u003e, \u003cem\u003eLDHA\u003c/em\u003e) were significantly downregulated (fold change\u0026nbsp;\u0026le;0.5) in miR-195-3p overexpressing cells (\u003cstrong\u003eFigure 6J\u003c/strong\u003e). Notably, VEGF exhibited a weaker restorative effect on lactate metabolism relative to Oct4 in the context of miR-195-3p inhibition. Western blot analyses corroborated these trends, showing diminished protein expression of these glycolytic enzymes under miR-195-3p mimic treatment and enhanced expression upon Oct4 overexpression (\u003cstrong\u003eFigure 6K\u003c/strong\u003e). Moreover, single-sample GSEA (ssGSEA) indicated an inverse correlation between glycolysis scores and miR-195-3p levels in MSCs (\u003cstrong\u003eFigure 6C\u003c/strong\u003e). Collectively, these data establish that miR-195-3p attenuates glycolytic metabolism in hypoxic MSCs, an effect potently mitigated by the Oct4/VEGF axis, with Oct4 playing a predominant role.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eOct4-dependent rescue by lactate restores MSC therapy efficacy impaired by miR-195-3p\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the interplay among miR-195-3p, lactate, and Oct4 in MSC-based therapy for ischemic hearts, we induced MI in Lewis rats (250\u0026ndash;300 g) by permanent ligation of the left anterior descending coronary artery, as previously described \u003csup\u003e[5]\u003c/sup\u003e. A cell therapy model was subsequently established, wherein MSCs subjected to various treatments\u0026mdash;including blank control, lactate, overexpression or knockdown of miR-195-3p/Oct4 (\u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003emiR-195-3p/\u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003eOct4/\u003cem\u003e\u003csub\u003esh\u003c/sub\u003e\u003c/em\u003eOct4), or their combinations-were transplanted into the infarcted hearts of syngeneic rats. Of the 220 rats that underwent MI surgery, 20 were randomly selected immediately post-MI to receive intramyocardial injections of either phosphate-buffered saline (PBS), vehicle-treated MSCs, or MSCs pre-conditioned with lactate, \u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003emiR-195-3p, \u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003eOct4, \u003cem\u003e\u003csub\u003esh\u003c/sub\u003e\u003c/em\u003eOct4, lactate+\u003cem\u003e\u003csub\u003esh\u003c/sub\u003e\u003c/em\u003eOct4, or \u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003emiR-195-3p+\u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003eOct4. All animals were monitored for 30 days. While no mortality occurred during the cell transplantation procedure, 26 rats died during the follow-up period, resulting in 133 survivors that underwent terminal echocardiography (\u003cstrong\u003eFigure 7G\u003c/strong\u003e). No significant differences in mortality were observed among the groups (\u003cstrong\u003eFigure 7O\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEchocardiographic assessment revealed severe impairment of left ventricular (LV) function in PBS-injected MI rats compared to those receiving MSC transplantation (\u003cstrong\u003eFigure 7A\u003c/strong\u003e). Functional parameters, including LV ejection fraction (LVEF) and LV fractional shortening (LVFS), were significantly improved in animals receiving lactate- or \u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003eOct4-treated MSCs compared to the vehicle-treated MSC group. Conversely, administration of \u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003emiR-195-3p- or \u003cem\u003e\u003csub\u003esh\u003c/sub\u003e\u003c/em\u003eOct4-transfected MSCs led to worse functional outcomes relative to the vehicle group. Crucially, the beneficial effect of lactate was abrogated by concurrent Oct4 knockdown (lactate+\u003cem\u003e\u003csub\u003esh\u003c/sub\u003e\u003c/em\u003eOct4 group), whereas Oct4 overexpression (\u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003emiR-195-3p+\u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003eOct4 group) rescued the functional impairment induced by miR-195-3p overexpression (\u003cstrong\u003eFigure 7H\u003c/strong\u003e, \u003cstrong\u003e7I\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConsistent with the functional data, TTC (\u003cstrong\u003eFigure 7B\u003c/strong\u003e) and Masson\u0026apos;s trichrome (\u003cstrong\u003eFigure 7C\u003c/strong\u003e) staining demonstrated that infarct size (\u003cstrong\u003eFigure 7J\u003c/strong\u003e) and fibrosis area (\u003cstrong\u003eFigure 7K\u003c/strong\u003e) were significantly reduced in the vehicle-MSC group compared to the PBS control. Lactate or \u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003eOct4 preconditioning further attenuated these pathological changes, whereas \u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003emiR-195-3p or \u003cem\u003e\u003csub\u003esi\u003c/sub\u003e\u003c/em\u003eOct4 treatment exacerbated them. Again, \u003cem\u003e\u003csub\u003esi\u003c/sub\u003e\u003c/em\u003eOct4 inhibited the lactate-mediated reduction in infarct size and fibrosis, and \u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003eOct4 counteracted the detrimental effects of \u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003emiR-195-3p. Immunofluorescence analysis further showed that MSCs from lactate- or \u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003eOct4-treated hearts exhibited a higher survival rate (\u003cstrong\u003eFigure 7D\u003c/strong\u003e, \u003cstrong\u003e7L\u003c/strong\u003e) and a lower apoptosis rate (\u003cstrong\u003eFigure 7E\u003c/strong\u003e, \u003cstrong\u003e7M\u003c/strong\u003e) than those from vehicle-, \u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003emiR-195-3p-, or \u003cem\u003e\u003csub\u003esh\u003c/sub\u003e\u003c/em\u003eOct4-treated animals. Oct4 manipulation effectively modulated the cellular effects induced by miR-195-3p or lactate. Finally, angiogenesis, assessed by CD31-positive staining, was enhanced in the vehicle-MSC group compared to the PBS group. The most robust angiogenesis was observed in the \u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003eOct4 group, followed by the lactate group, while it was significantly suppressed in the \u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003emiR-195-3p and \u003cem\u003e\u003csub\u003esh\u003c/sub\u003e\u003c/em\u003eOct4 groups (\u003cstrong\u003eFigures 7F\u003c/strong\u003e, \u003cstrong\u003e7N\u003c/strong\u003e). Oct4 overexpression or knockdown respectively rescued or inhibited the angiogenic effects altered by \u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003emiR-195-3p or lactate. In summary, our findings indicate that lactate enhances MSC survival and myocardial repair in an Oct4-dependent manner. In contrast, miR-195-3p exerts inhibitory effects on MSC therapy, which can be effectively reversed by Oct4 overexpression, indicating that miR-195 downregulation is necessary for Oct4\u0026apos;s pro-angiogenic effect.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eOct4 and miR-195-3p orchestrate metabolic reprogramming through VEGF.\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate how Oct4, miR-195-3p, and metabolic reprogramming coordinately promote angiogenesis, we assessed the expression of key pathway components in myocardial tissue. As depicted in \u003cstrong\u003eFigures 8A\u003c/strong\u003e and \u003cstrong\u003e8D\u003c/strong\u003e, rats receiving vehicle-MSCs showed higher Oct4 mRNA and protein levels than PBS-treated controls. Lactate preconditioning or \u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003eOct4 further increased Oct4 expression relative to the vehicle‑MSC group, whereas \u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003emiR-195-3p or \u003cem\u003e\u003csub\u003esh\u003c/sub\u003e\u003c/em\u003eOct4 significantly suppressed it. Notably, \u003cem\u003e\u003csub\u003esh\u003c/sub\u003e\u003c/em\u003eOct4 abolished the lactate-mediated induction of Oct4, while \u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003eOct4 reversed the inhibitory effect of \u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003emiR-195-3p. A parallel trend was observed for the metabolic markers GLUT1 \u003cstrong\u003e(Figure 8B\u003c/strong\u003e, \u003cstrong\u003e8D\u003c/strong\u003e) and HK2 (\u003cstrong\u003eFigure 8C\u003c/strong\u003e, \u003cstrong\u003e8D\u003c/strong\u003e), whose expression rose in the vehicle-MSC group, was further elevated by lactate or \u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003eOct4, and was reduced by \u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003emiR-195-3p or \u003cem\u003e\u003csub\u003esh\u003c/sub\u003e\u003c/em\u003eOct4. Moreover, \u003cem\u003e\u003csub\u003esh\u003c/sub\u003e\u003c/em\u003eOct4 blocked lactate-induced upregulation of GLUT1 and HK2, and \u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003eOct4 rescued their suppression by \u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003emiR-195-3p.\u003c/p\u003e\n\u003cp\u003eTo explore the link between metabolic flux and Oct4, we isolated and cultured cardiomyocytes (CMs) from rats subjected to intramyocardial injection of PBS or MSCs preconditioned with vehicle, lactate, \u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003emiR-195-3p, \u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003eOct4, \u003cem\u003e\u003csub\u003esh\u003c/sub\u003e\u003c/em\u003eOct4, lactate+\u003cem\u003e\u003csub\u003esh\u003c/sub\u003e\u003c/em\u003eOct4, or \u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003emiR-195-3p+\u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003eOct4. Immunofluorescence analysis revealed: (1) Oct4 expression was low in PBS controls. Relative to vehicle, Oct4 was markedly reduced in \u003cem\u003e\u003csub\u003esh\u003c/sub\u003e\u003c/em\u003eOct4 and \u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003emiR-195-3p groups (collectively termed \u003csup\u003elow\u003c/sup\u003eOct4), but restored in the \u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003emiR-195-3p+\u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003eOct4 group. By contrast, Oct4 was upregulated in \u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003eOct4 and lactate groups (\u003csup\u003ehigh\u003c/sup\u003eOct4), an effect blunted in the lactate+\u003cem\u003e\u003csub\u003esh\u003c/sub\u003e\u003c/em\u003eOct4 group; (2) GLUT1 expression was strongly induced in \u003csup\u003ehigh\u003c/sup\u003eOct4 groups but repressed in \u003csup\u003elow\u003c/sup\u003eOct4 groups; (3) Concordant with its target gene expression, Oct4 displayed enhanced nuclear accumulation in \u003csup\u003ehigh\u003c/sup\u003eOct4 groups, whereas in \u003csup\u003elow\u003c/sup\u003eOct4 groups it was sparsely distributed in both nucleus and cytoplasm (\u003cstrong\u003eFigure 8E\u003c/strong\u003e). Together, these data support a model in which Oct4 directly binds regulatory regions of GLUT1 and HK2 to control their expression. We next performed \u0026sup1;\u0026sup3;C-glucose metabolic tracing (\u003cstrong\u003eFigures 8F\u003c/strong\u003e-\u003cstrong\u003e8H\u003c/strong\u003e). In line with Oct4 expression changes, \u003csup\u003ehigh\u003c/sup\u003eOct4 groups showed increased extracellular lactate (\u003cstrong\u003eFigure 8F\u003c/strong\u003e) and significantly reduced citrate labeling (\u003cstrong\u003eFigure 8G\u003c/strong\u003e) , both of which were reversed in \u003csup\u003elow\u003c/sup\u003eOct4 groups. Notably, labeling of the tricarboxylic acid (TCA) cycle intermediate succinate remained unchanged (\u003cstrong\u003eFigure 8H\u003c/strong\u003e). These results suggest that Oct4 drives metabolic reprogramming by regulating GLUT1 and HK2 expression, likely through direct DNA binding.\u003c/p\u003e\n\u003cp\u003eFurther supporting these findings, real-time RT-PCR and immunoblot analyses of myocardial tissue showed that hearts from rats pretreated with lactate or \u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003eOct4 exhibited elevated mRNA and protein levels of VEGF (\u003cstrong\u003eFigure 8I\u003c/strong\u003e, \u003cstrong\u003e8D\u003c/strong\u003e) and VEGFR2 (\u003cstrong\u003eFigure 8J\u003c/strong\u003e, 8\u003cstrong\u003eD\u003c/strong\u003e), whereas \u003cem\u003e\u003csub\u003esh\u003c/sub\u003e\u003c/em\u003eOct4 and \u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003emiR-195-3p suppressed their expression, indicating an angiogenic state in MI hearts following lactate or \u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003eOct4 treatment. Given the reported role of PI3K/Akt signaling in cell survival, proliferation, and ischemia-triggered angiogenesis\u0026mdash;and our prior work showing that Akt and VEGF act in parallel to induce angiogenesis \u003csup\u003e[24]\u003c/sup\u003e\u0026mdash;we also evaluated Akt expression. Consistent with VEGF changes, Akt levels increased in an Oct4-dependent manner in MI hearts, with the highest expression in rats receiving \u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003eOct4 or \u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003emiR-195-3p+ \u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003eOct4-preconditioned MSCs, intermediate levels in the lactate-preconditioned MSC group, and the lowest in the \u003cem\u003e\u003csub\u003esh\u003c/sub\u003e\u003c/em\u003eOct4 group. \u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003emiR-195-3p substantially reduced Akt expression, which was restored by \u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003eOct4 (\u003cstrong\u003eFigure 8K\u003c/strong\u003e, \u003cstrong\u003e8D\u003c/strong\u003e). Immunohistochemical staining further confirmed these patterns, revealing strongest VEGF and Akt staining in the \u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003eOct4 and \u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003emiR-195-3p+\u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003eOct4 groups, moderate staining in the lactate group, and weakest in the \u003cem\u003e\u003csub\u003esh\u003c/sub\u003e\u003c/em\u003eOct4 group (\u003cstrong\u003eFigure 8L\u003c/strong\u003e). Collectively, these findings demonstrate that \u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003emiR-195-3p suppresses glycolysis and angiogenesis, and that its inhibitory effects can be partially reversed through Oct-driven metabolic reprogramming.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe therapeutic potential of MSCs in treating ischemic injuries is often limited by their poor survival and functional decline within the harsh hypoxic microenvironment of damaged tissues \u003csup\u003e[25]\u003c/sup\u003e. This study defines a core molecular circuitry that dictates this critical cellular fate. We have characterized an integrated miR-195-3p/Oct4/VEGF signaling cascade that links the cellular response to hypoxia with fundamental processes governing survival, metabolic adaptation, and paracrine activity in MSCs. Our findings establish that hypoxia-induced miR-195-3p acts as a key instigator of MSC dysfunction by directly targeting and inhibiting Oct4, a central pluripotency regulator. This inhibition sets off a series of deleterious downstream events, whereas the Oct4/VEGF pathway and its associated metabolic mediator, lactate, function as crucial compensatory mechanisms that ultimately dictate the success of MSC-based therapeutic interventions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHypoxia triggers a deterministic suppression: The role of miR-195-3p as a molecular switch\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe initial observation that hypoxia robustly induces miR-195-3p while concurrently repressing Oct4 pinpoints a critical upstream event in the MSC hypoxic response. Hypoxia, a known regulator of microRNA biogenesis, drives the expression of miR-195-3p, establishing it as a principal mediator within the hypoxic signaling network \u003csup\u003e[26]\u003c/sup\u003e. This induction promotes a transition in cellular state\u0026mdash;shifting from a phenotype focused on maintenance and repair to one geared toward stress adaptation, often compromising core stem cell attributes \u003csup\u003e[27]\u003c/sup\u003e. We further provide evidence that miR-195-3p directly targets Oct4, a finding with significant biological implications. Oct4 operates not simply as a passive marker of stemness but as a master transcriptional regulator that coordinates an extensive network of genes governing self-renewal, proliferation, and cellular robustness \u003csup\u003e[28]\u003c/sup\u003e. Its suppression, therefore, represents a fundamental disruption of MSC identity and functional capacity. The miR-195-3p-mediated inhibition of Oct4 offers a mechanistic basis for the frequently documented loss of stem cell characteristics and increased apoptosis in MSCs post-transplantation into ischemic environments. We demonstrate that the consequent impairment of MSC viability under hypoxia is directly dependent on the miR-195-3p/Oct4 axis. This acquired vulnerability can be attributed to the disintegration of Oct4-dependent transcriptional programs that promote cell survival, thereby heightening susceptibility to apoptotic triggers.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eOct4 loss initiates cell-autonomous dysfunction: compromised viability and metabolic arrest\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe downregulation of Oct4 precipitates a signaling failure that disrupts three principal downstream pathways: survival mechanisms, paracrine communication, and metabolic homeostasis \u003csup\u003e[29]\u003c/sup\u003e. This autonomous dysfunction first emerges as a viability crisis, where Oct4-deficient cells display accelerated apoptosis and impaired viability. Beyond simply increasing susceptibility to cell death, the loss of Oct4 critically undermines cellular defenses against oxidative and ischemic stress\u0026mdash;defects that are severely amplified under hypoxic conditions\u003csup\u003e\u0026nbsp;[30]\u003c/sup\u003e. Our genetic interrogation confirms Oct4 as the indispensable mediator through which miR-195-3p controls MSC survival. Overexpression of Oct4 potently enhanced cell viability and proliferation while inhibiting apoptosis, effectively counteracting the damage induced by miR-195-3p. Molecular profiling verified that Oct4 coordinates an anti-apoptotic program through the synergistic upregulation of Bcl-2 and Hsp27, coupled with suppression of Bax and Caspase 9.\u003c/p\u003e\n\u003cp\u003eSimultaneously, we identified a profound metabolic arrest in which miR-195-3p directly disrupts glycolytic flux in hypoxic MSCs. Cells lacking Oct4 exhibited severe impairments in extracellular acidification, glucose uptake, and lactate production\u0026mdash;functional deficiencies mirrored by the coordinated downregulation of crucial glycolytic enzymes (SLC2A1, HK2, LDHA). This metabolic failure creates a pathological contradiction: although hypoxia typically induces adaptive glycolysis, the concurrent surge in miR-195-3p, via Oct4 suppression, obstructs this essential metabolic shift \u003csup\u003e[32]\u003c/sup\u003e. The resulting energy deficit leaves MSCs incapable of meeting basic metabolic requirements, directly accounting for their functional decline under ischemic stress \u003csup\u003e[33, 34]\u003c/sup\u003e. Collectively, these results position Oct4 as a master integrator of cellular autonomy, synchronizing survival control with metabolic competence.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eVEGF executes Oct4-directed reparative functions: Orchestrating angiogenesis and microenvironmental homeostasis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur investigation delineates a vital rescue pathway whereby Oct4, countering the inhibitory influence of miR-195-3p, directs a pro-survival and angiogenic program in hypoxic MSCs largely through VEGF upregulation. Functionally, Oct4 overexpression strongly enhanced the expression of multiple angiogenic factors and receptors (VEGF, bFGF, VEGFR2, etc.), identifying VEGF as the pivotal downstream effector of Oct4-mediated vascular programming. Critically, VEGF overexpression alone was sufficient to rectify the angiogenesis and survival defects induced by miR-195-3p, whereas VEGF knockdown nullified the beneficial effects of Oct4. This definitive genetic evidence positions VEGF as the essential executor of Oct4-mediated reparative functions. The Oct4/VEGF axis further modulates microenvironmental homeostasis by regulating fibrotic and inflammatory responses. Both Oct4 and VEGF overexpression suppressed the pathological expression of collagen I, \u0026alpha;-SMA, IL-6, iNOS, and TGF-\u0026beta;1, while VEGF knockdown replicated the pro-fibrotic/inflammatory phenotype seen with miR-195-3p overexpression. This expanded functional role reveals VEGF not merely as an angiogenic factor but as a central coordinator of tissue repair programming that prevents aberrant healing \u003csup\u003e[35]\u003c/sup\u003e. Contemporary research corroborates VEGF\u0026apos;s pleiotropic roles, including direct immune modulation and fibroblast regulation \u003csup\u003e[36, 37]\u003c/sup\u003e, consistent with the anti-fibrotic and anti-inflammatory effects we observed.\u003c/p\u003e\n\u003cp\u003eSignificantly, metabolic rescue experiments revealed a hierarchical organization within this axis: although Oct4/VEGF restoration recovered glycolytic function, Oct4 specifically exhibited a superior capacity in reinstating lactate metabolism. This functional hierarchy highlights the sophisticated architecture of the Oct4-directed rescue network. The emerging concept of pluripotency factor repurposing is strongly supported by our demonstration that Oct4 coordinates both cell-autonomous fitness and tissue-level repair via VEGF-mediated programs \u003csup\u003e[38, 39]\u003c/sup\u003e. Furthermore, the identification of lactate as a metabolic co-effector uncovers an elegant feed-forward mechanism whereby lactate stabilizes HIF-1\u0026alpha; to potentiate VEGF signaling \u003csup\u003e[40]\u003c/sup\u003e. Thus, VEGF acts as the central hub of a refined repair circuit, translating Oct4 directives into coordinated tissue regeneration.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMetabolic reprogramming via an Oct4-lactate circuit is essential for MSC therapeutic efficacy\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe growing discipline of stem cell metabolism has underscored the pivotal role of metabolic rewiring in determining the therapeutic effectiveness of transplanted cells \u003csup\u003e[42]\u003c/sup\u003e. Our results place the transcriptional regulator Oct4 at the center of a lactate-driven metabolic reprogramming that is indispensable for MSC survival and function in the ischemic myocardium. This research reveals a sophisticated regulatory pathway wherein lactate, beyond its role as a metabolic by-product, functions as a signaling molecule that reinforces an Oct4-dependent pro-survival and pro-angiogenic state, a process potently opposed by miR-195-3p. The central importance of Oct4 in this framework is conclusively demonstrated by our rescue studies. The finding that lactate preconditioning markedly improved left ventricular function, diminished infarct size and fibrosis, and boosted MSC survival and angiogenesis\u0026mdash;with all these advantages entirely negated by simultaneous Oct4 knockdown\u0026mdash;confirms that Oct4 is not merely associated with, but is essential for, lactate-mediated therapeutic benefits \u003csup\u003e[43]\u003c/sup\u003e. Conversely, the deleterious consequences of miR-195-3p overexpression, which replicated the functional and structural impairments observed with Oct4 knockdown, were fully remedied by Oct4 co-overexpression. This reciprocal relationship highlights a fundamental antagonism: miR-195-3p serves as a negative regulator of this reparative pathway, and its downregulation seems to be a prerequisite for activating the full pro-angiogenic potential of Oct4. The convergence of both lactate\u0026apos;s action and miR-195-3p\u0026apos;s inhibition on Oct4 consolidates its status as the master regulatory node in this network.\u003c/p\u003e\n\u003cp\u003eThe most insightful mechanistic revelation from this work is the elucidation of how Oct4 directs a metabolic shift toward glycolysis to power cardiac repair. We present multifaceted evidence supporting this model. Firstly, Oct4 directly governs the expression of key glycolytic regulators, GLUT1 and HK2. The congruent expression patterns of Oct4, GLUT1, and HK2 across experimental conditions\u0026mdash;upregulated by lactate and \u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003eOct4, suppressed by miR-195-3p and \u003cem\u003e\u003csub\u003esh\u003c/sub\u003e\u003c/em\u003eOct4\u0026mdash;strongly indicate a coordinated regulatory scheme \u003csup\u003e[44]\u003c/sup\u003e. This is further reinforced by immunofluorescence data from isolated cardiomyocytes, showing that groups with high Oct4 expression were linked to pronounced GLUT1 induction and, importantly, a marked nuclear localization of Oct4 itself. This alteration in subcellular distribution suggests enhanced transcriptional activity, leading us to hypothesize a model where Oct4, upon activation by lactate or release from miR-195-3p suppression, translocates to the nucleus and directly binds to regulatory elements of the GLUT1 and HK2 genes. The functional outcomes of this transcriptional control were validated by our \u0026sup1;\u0026sup3;C-glucose metabolic tracing studies. The \u0026quot;\u003csup\u003ehigh\u003c/sup\u003eOct4\u0026quot; groups (\u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003eOct4 and lactate-preconditioned) displayed a classic Warburg-like metabolic profile: elevated extracellular lactate production accompanied by a substantial decrease in citrate labeling from glucose \u003csup\u003e[45]\u003c/sup\u003e. This pattern signifies a fundamental redirection of carbon flux, where glucose is preferentially shunted away from the mitochondrial TCA cycle and toward lactate fermentation\u003csup\u003e\u0026nbsp;[46]\u003c/sup\u003e. The unaltered labeling of succinate implies a specific bottleneck or diversion at the TCA cycle\u0026apos;s initial stages, consistent with increased glycolytic flow \u003csup\u003e[47]\u003c/sup\u003e. This Oct4-driven glycolytic transition is not an incidental occurrence but is functionally paramount. Glycolysis provides a more efficient ATP-yielding pathway under the hypoxic conditions of an infarcted heart, thus fostering MSC survival \u003csup\u003e[48]\u003c/sup\u003e. Moreover, the lactate generated can act as a paracrine signaling molecule and an alternative energy substrate for neighboring cardiomyocytes \u003csup\u003e[49]\u003c/sup\u003e, establishing a metabolically symbiotic microenvironment.\u003c/p\u003e\n\u003cp\u003eThis metabolic reprogramming does not occur in isolation; it is intrinsically connected to the angiogenic requirements of tissue repair. We identified the VEGF signaling pathway as a key downstream effector connecting Oct4-mediated metabolism to angiogenesis. The expression of both VEGF and its receptor VEGFR2 was closely correlated with Oct4 levels, being highest in the \u003cem\u003e\u003csub\u003eoe\u003c/sub\u003e\u003c/em\u003eOct4 and lactate groups and lowest following Oct4 knockdown. This forges a direct link from metabolic rewiring to a pro-angiogenic secretome. The concomitant upregulation of Akt, a central kinase in cell survival and proliferation \u003csup\u003e[50]\u003c/sup\u003e, further strengthens this connection. The substantial angiogenesis, evidenced by CD31 staining, in the \u0026quot;\u003csup\u003ehigh\u003c/sup\u003eOct4/\u003csup\u003ehigh\u003c/sup\u003eglycolysis\u0026quot; groups can therefore be ascribed to a dual mechanism: first, the direct production of VEGF stimulated by Oct4, and second, the provision of glycolytically derived energy and biosynthetic precursors necessary for endothelial cell proliferation and new vessel formation. The observation that Akt and VEGF expression patterns were nearly identical suggests they may function in a coordinated, parallel fashion, as previously reported \u003csup\u003e[24]\u003c/sup\u003e, to optimize the angiogenic response.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn ischemic myocardium, suppressed miR-195-3p activates Oct4, driving metabolic reprogramming toward glycolysis. This shift enhances MSC survival via lactate production and initiates a lactate-VEGF feed-forward loop that promotes angiogenesis and functional recovery. The Oct4-lactate axis thus represents a key therapeutic target; its potentiation through lactate preconditioning or miR-195-3p inhibition offers a promising metabolic engineering strategy to improve MSC-based cardiac repair.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStudy limitations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDespite the significant insights provided by this study, several limitations should be acknowledged. First, the precise molecular mechanisms by which lactate influences Oct4 stabilization and nuclear translocation, while implied, require further direct experimental validation. Second, the \u003cem\u003ein vivo\u003c/em\u003e dynamics of the proposed metabolic symbiosis, where lactate produced by MSCs is utilized by cardiomyocytes, were inferred from indirect evidence and would benefit from more direct tracing studies in the intact animal model. Third, the therapeutic strategies explored (e.g., lactate preconditioning, genetic manipulation) were applied prior to transplantation; their clinical translation would require the development of safe, efficient, and transient delivery methods for use in a potential clinical setting. Finally, while the rat model of MI is well-established, the potential differences in human MSC biology and the human ischemic microenvironment necessitate future studies using human cells and potentially more complex disease models to fully validate the translational relevance of these findings. The potential off-target effects of systemically inhibiting miR-195-3p also warrant careful future investigation.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eAn expanded description of materials and methods is provided in Supplemental Appendix.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAntibodies and reagents\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDetailed information on primers and antibodies is provided in Supplementary Tables S1 and S2, respectively. 4\u0026rsquo;, 6-Diamidino-2-phenylindole (DAPI; Sigma-Aldrich, #28718-90-3) was used for nuclear staining.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthical compliance\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were approved by the Animal Care and Use Committee of Ji-Nan University (Ethical Approval No. 20220923-02.), conducted in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals, and adhere to ARRIVE 2.0 guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAnimals\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInbred Lewis rats were housed under standard conditions with ad libitum access to food and water.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCell culture and hypoxia treatment\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePeripheral blood-derived mesenchymal stem cells (PBMSCs) were isolated from rat abdominal aortic blood as previously described\u0026nbsp;\u003csup\u003e[51]\u0026nbsp;\u003c/sup\u003eand cultured in DMEM/F-12 medium supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin at 37\u0026deg;C under 5% CO\u003csub\u003e2\u003c/sub\u003e. For hypoxic exposure, passage 2 cells were placed in a modular incubator chamber (Billups-Rothenberg) infused with a gas mixture of 1% O\u003csub\u003e2\u003c/sub\u003e, 5% CO\u003csub\u003e2\u003c/sub\u003e, and balance N\u003csub\u003e2\u003c/sub\u003e for 24-72 hours. Normoxic controls were maintained at 21% O\u003csub\u003e2\u003c/sub\u003e and 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003emiRNA sequencing and bioinformatic analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted using TRIzol reagent (Invitrogen). miRNA sequencing libraries were prepared with the QIAseq miRNA Library Kit (Qiagen) and sequenced on an Illumina NextSeq 550 platform. Reads were processed via the miRDeep2 pipeline. Differential expression analysis was performed using DESeq2 with thresholds of |log\u003csub\u003e2\u003c/sub\u003e fold change| \u0026gt; 1 and adjusted p-value \u0026lt; 0.05. miRNA targets were predicted using TargetScan v8.0 and miRDB.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eQuantitative RT-PCR\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA was reverse transcribed using the PrimeScript RT Reagent Kit (Takara). qPCR was performed on a QuantStudio 6 Pro system (Applied Biosystems) using SYBR Green Master Mix (Roche). The 2\u003csup\u003e\u0026ndash;\u0026Delta;\u0026Delta;\u0026nbsp;\u003c/sup\u003eCT method was applied with GAPDH as the endogenous control. Primer sequences are listed in Supplementary \u003cstrong\u003eTable S1\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003emiRNA transfection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePBMSCs were transfected with miR-195-3p mimic or inhibitor sponge (Syngentech, Shanghai, China) using Lipofectamine 3000 (Invitrogen). Scrambled miRNA or non-targeting sponge served as negative controls.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePlasmid construction and transfection\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe precursor sequence of miR-195-3p was cloned into the pMXs vector (Addgene). A 300-bp fragment of the Oct4 3\u0026rsquo;-UTR containing the predicted binding site was inserted into the pmirGLO Dual-Luciferase vector (Promega); a mutant version was generated via site-directed mutagenesis (Q5 Kit, NEB). Coding sequences of Oct4 and VEGF were cloned into the pcDNA3.1 vector. Short hairpin RNAs (shRNAs) targeting Oct4 and VEGF were designed and cloned into the pLKO.1 vector. All constructs were verified by Sanger sequencing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eLuciferase reporter assay\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHEK-293T cells were co-transfected with 100 ng of wild-type or mutant Oct4 3\u0026rsquo;-UTR reporter plasmid and 50 nM miR-195-3p mimic or negative control (GenePharma). After 48 hours, luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega) on a GloMax Navigator instrument (Promega). Firefly luciferase values were normalized to Renilla luciferase.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eWestern blotting\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were lysed in RIPA buffer with protease and phosphatase inhibitors (Roche). Protein concentration was determined via BCA assay (Pierce). Proteins (20\u0026ndash;30\u0026nbsp;\u0026mu;g) were separated by 10% SDS-PAGE, transferred to PVDF membranes (Millipore), and incubated with primary antibodies (Supplementary \u003cstrong\u003eTable S2\u003c/strong\u003e) followed by HRP-conjugated secondary antibodies. Signals were detected using ECL substrate (Bio-Rad) and a ChemiDoc MP Imaging System (Bio-Rad).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCell viability and proliferation assays\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eViability was assessed using the Cell Counting Kit-8 (CCK-8; Dojindo). For proliferation, cells were stained with Ki67 antibody and DAPI; positive cells were counted across five random fields using a Nikon Eclipse Ti2 fluorescence microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eApoptosis analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eApoptosis was evaluated using the Annexin V-FITC/PI Apoptosis Detection Kit (BD Biosciences). Samples were analyzed on a BD FACS Celesta flow cytometer, and data were processed using FlowJo v10.8.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCytokine array\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCytokine expression was profiled using the AAH-CYT-G2000 Array (RayBiotech). Signals were detected by chemiluminescence and quantified with ImageJ.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eImmunofluorescence staining\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells or tissue sections were fixed, permeabilized, blocked, and incubated with primary antibodies (Supplementary \u003cstrong\u003eTable S2\u003c/strong\u003e), followed by Alexa Fluor-conjugated secondary antibodies (Invitrogen). Nuclei were stained with DAPI. Images were acquired using a Zeiss LSM 880 confocal microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMetabolic analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExtracellular acidification rate (ECAR) and glycolytic proton efflux rate (glycoPER) were measured using a Seahorse XFe96 Analyzer and the XF Glycolysis Stress Test Kit (Agilent). Glucose uptake and lactate production were assessed using commercial kits (Cayman Chemical and Sigma-Aldrich, respectively).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eRNA sequencing and gene set enrichment analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted using the RNeasy Plus Mini Kit (Qiagen). Libraries were prepared with the TruSeq Stranded mRNA Kit (Illumina) and sequenced on an Illumina NovaSeq 6000. Reads were aligned to GRCh38 using STAR, and gene counts were obtained with featureCounts. Differential expression analysis was performed using DESeq2. Gene Set Enrichment Analysis (GSEA) was conducted using Hallmark and KEGG gene sets.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMyocardial infarction model\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMale Lewis rats (250\u0026ndash;300 g) were anesthetized with 2% isoflurane, and MI was induced by permanent ligation of the left anterior descending coronary artery. Infarction was confirmed by myocardial blanching and ST-segment elevation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePBMSC preconditioning and EGFP labeling\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine the optimal lactate concentration for preconditioning, PBMSCs were treated with a gradient of sodium lactate (0, 5, 10, 15, and 20 mM; Sigma-Aldrich) for 48 hours under hypoxic condition. Subsequent analyses of cell viability and proliferation indicated that 10 mM sodium lactate yielded the greatest enhancement in both parameters (Supplementary \u003cstrong\u003eFigure S1\u003c/strong\u003e). Based on these results, PBMSCs were preconditioned with 10 mM sodium lactate for 24 hours under hypoxia before transplantation. Lentiviral vectors encoding miR-195-3p, Oct4, or Oct4-shRNA were packaged in HEK-293T cells. PBMSCs were transduced at an MOI of 50 with 8 \u0026mu;g/mL polybrene. EGFP labeling was performed via co-transfection, with \u0026gt;70% efficiency confirmed by fluorescence microscopy and immunoblotting.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eExperimental groups and cell transplantation\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 220 rats that survived MI surgery were randomized into eight experimental groups (n = 20 per group) using a computer-generated scheme. Researchers were blinded to group assignments. Groups received intramyocardial injections of PBS, vehicle-treated MSCs, lactate-preconditioned MSCs, miR-195-3p-overexpressing MSCs, Oct4-overexpressing MSCs, Oct4-knockdown MSCs, lactate-preconditioned + Oct4-knockdown MSCs, or miR-195-3p-overexpressing + Oct4-overexpressing MSCs (5\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells per heart). Postoperative analgesia and antibiotics were administered. The work has been reported in line with the ARRIVE guidelines 2.0.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEchocardiography\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTransthoracic echocardiography was performed 30 days post-transplantation using a Vevo 3100 system (VisualSonics). Left ventricular ejection fraction (LVEF) and fractional shortening (LVFS) were derived from M-mode recordings.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHistological analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHearts were sectioned and stained with TTC or Masson\u0026rsquo;s trichrome. Fibrosis area was quantified using ImageJ and expressed as a percentage of total left ventricular area.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEngraftment and angiogenesis assessment\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEngraftment was evaluated by quantifying EGFP-positive cells. Apoptosis was assessed via TUNEL staining (Roche). Angiogenesis was evaluated by CD31 staining and vascular density calculation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMetabolic tracing\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIsolated cardiomyocytes were incubated with [U-\u0026sup1;\u0026sup3;C] glucose (Cambridge Isotope Laboratories). Metabolites were extracted and analyzed by LC-MS (Q Exactive HF, Thermo Scientific). Data were processed using Xcalibur and MetaboAnalyst 5.0.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eStatement.\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe work has been reported in line with the ARRIVE guidelines 2.0.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eStatistical analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are expressed as mean \u0026plusmn; SD. Analyses were performed using GraphPad Prism 9.0. Multiple groups were compared by one-way ANOVA with Tukey\u0026rsquo;s post-hoc test; two-group comparisons used two-tailed Student\u0026rsquo;s t-test. Survival was analyzed by Kaplan\u0026ndash;Meier with log-rank test. Sample sizes were determined by power analysis (\u0026alpha; = 0.05, power = 0.8). p \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAcknowledgement s\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthor contributions\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY. M.: conceptualization, methodology, writing-original draft preparation, preparing figures 1-3. P. Z.: conceptualization, methodology, preparing figures 4-6. H. Z.：preparing figures 7, 8. L. C.: Animal model. Z. D.: data collection, preparing Table S1 and Table S2. Y. L.: methodology. W. P.: technical support. S. Z.: conceptualization, data curation, funding acquisition, investigation, supervision,validation, visualization, preparing Graphic abstract, writing\u0026nbsp;–\u0026nbsp;review \u0026amp; editing. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Guangzhou Science and Technology Program Project funded by the Guangzhou Science and Technology Bureau (Grant No. 2023A03J0982, to SZ).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAvailability of data and materials\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe RNA-seq data supporting the results of this study have been deposited in the Sequence Read Archive under accession number [PRJNA1416857].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDeclarations\u003c/em\u003e\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have not used Artificial Intelligence in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCompeting interests\u003c/em\u003e\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThere is no conflict of interest between the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConsent for publication\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAdditional information\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupplementary information The online version contains supplementary material available.\u003c/p\u003e\n\u003cp\u003eCorrespondence and requests for materials should be addressed to Shaoheng Zhang.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eData availability\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the\u003c/p\u003e\n\u003cp\u003ecorresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBarr\u0026egrave;re-Lemaire S, Vincent A, Jorgensen C, Piot C, Nargeot J, Djouad F. Mesenchymal stromal cells for improvement of cardiac function following acute myocardial infarction: a matter of timing. Physiol Rev. 2024; 104(2): 659-725. \u003c/li\u003e\n\u003cli\u003eBurchfield JS, Xie M, Hill JA. Pathological ventricular remodeling: mechanisms: part 1 of 2. Circulation. 2013; 128(4): 388-400.\u003c/li\u003e\n\u003cli\u003eFomovsky GM, Holmes JW. Evolution of scar structure, mechanics, and ventricular function after myocardial infarction in the rat. Am J Physiol Heart Circ Physiol. 2010; 298(1): H221-H228.\u003c/li\u003e\n\u003cli\u003eLin M, Liu X, Zheng H, Huang X, Wu Y, Huang A, Zhu H, Hu Y, Mai W, Huang Y. IGF-1 enhances BMSC viability, migration, and anti-apoptosis in myocardial infarction via secreted frizzled-related protein 2 pathway. Stem Cell Res Ther. 2020; 11(1): 22.\u003c/li\u003e\n\u003cli\u003eZhang S, Zhao L, Wang J, Chen N, Yan J, Pan X. HIF-2\u0026alpha; and Oct4 have synergistic effects on survival and myocardial repair of very small embryonic-like mesenchymal stem cells in infarcted hearts. Cell Death Dis. 2017; 8(1): e2548. \u003c/li\u003e\n\u003cli\u003eWang Y, Wang Q, Sui X, Guo M, Li L, Jia W, Tian Y, Lu Q, Wang B. Catalpalactone protects rats nerve function from hypoxic lesion by polarizing microglial cells toward M2 phenotype. Eur J Med Res. 2025; 30(1): 96. \u003c/li\u003e\n\u003cli\u003eBabenko VA, Varlamova EG, Saidova AA, Turovsky EA, Plotnikov EY. Lactate protects neurons and astrocytes against ischemic injury by modulating Ca2+ homeostasis and inflammatory response. FEBS J. 2024; 291(8): 1684-1698. \u003c/li\u003e\n\u003cli\u003eBispo DSC, Gra\u0026ccedil;a ICR, Jesus CSH, Rodrigues JE, Correia MC, Atella S, Duarte IF, Goodfellow BJ, Oliveira MB, Mano JF, Gil AM. Metabolic markers detect early ostedifferentiation of mesenchymal stem cells from multiple donors. Stem Cell Res Ther. 2025; 16(1): 294.\u003c/li\u003e\n\u003cli\u003eLu R, Cai H, Liu Y, Ma G, Wang J, Yan M, Zhang Z, Yu B, Li Z, Fang S. Long non-coding RNA AK023617 orchestrates atherosclerosis by regulating the circadian rhythm of immunity-related GTPase family M protein in macrophages. Noncoding RNA Res. 2024; 11: 262-272. \u003c/li\u003e\n\u003cli\u003eXiong J, Ma F, Ding N, Xu L, Ma S, Yang A, Hao Y, Zhang H, Jiang Y. miR-195-3p alleviates homocysteine-mediated atherosclerosis by targeting IL-31 through its epigenetics modifications. Aging Cell. 2021; 20(10): e13485. \u003c/li\u003e\n\u003cli\u003eCarvalho A, Ji Z, Zhang R, Zuo W, Qu Y, Chen X, Tao Z, Ji J, Yao Y, Ma G. Inhibition of miR-195-3p protects against cardiac dysfunction and fibrosis after myocardial infarction. Int J Cardiol. 2023; 387: 131128. \u003c/li\u003e\n\u003cli\u003eShe S, Wei Q, Kang B, Wang YJ. Cell cycle and pluripotency: Convergence on octamer‑binding transcription factor 4 (Review). Mol Med Rep. 2017; 16(5): 6459-6466.\u003c/li\u003e\n\u003cli\u003eXu N, Papagiannakopoulos T, Pan G, Thomson JA, Kosik KS. MicroRNA-145 regulates OCT4, SOX2, and KLF4 and represses pluripotency in human embryonic stem cells. Cell. 2009; 137(4): 647-658. \u003c/li\u003e\n\u003cli\u003eJauhari A, Singh T, Yadav S. Expression of miR-145 and Its Target Proteins Are Regulated by miR-29b in Differentiated Neurons. Mol Neurobiol. 2018; 55(12): 8978-8990. \u003c/li\u003e\n\u003cli\u003eGuo J, Wang M, Liu X. MicroRNA-195 suppresses tumor cell proliferation and metastasis by directly targeting BCOX1 in prostate carcinoma. J Exp Clin Cancer Res. 2015; 34(1): 91.\u003c/li\u003e\n\u003cli\u003eWang M, Zhang J, Tong L, Ma X, Qiu X. MiR-195 is a key negative regulator of hepatocellular carcinoma metastasis by targeting FGF2 and VEGFA. Int J Clin Exp Pathol. 2015; 8(11): 14110-14120.\u003c/li\u003e\n\u003cli\u003eShen M, Dong C, Ruan X, Yan W, Cao M, Pizzo D, Wu X, Yang L, Liu L, Ren X, Wang SE. Chemotherapy-Induced Extracellular Vesicle miRNAs Promote Breast Cancer Stemness by Targeting ONECUT2. Cancer Res. 2019; 79(14): 3608-3621.\u003c/li\u003e\n\u003cli\u003eLiu K, Ma L, Zhou F, Yang Y, Hu HB, Wang L, Zhong L. Identification of microRNAs related to myocardial ischemic reperfusion injury. J Cell Physiol. 2019; 234(7): 11380-11390. \u003c/li\u003e\n\u003cli\u003eBuccini S, Haider KH, Ahmed RP, Jiang S, Ashraf M. Cardiac progenitors derived from reprogrammed mesenchymal stem cells contribute to angiomyogenic repair of the infarcted heart. Basic Res Cardiol. 2012; 107(6): 301.\u003c/li\u003e\n\u003cli\u003eQiu Z, Zhao J, Huang F, Bao L, Chen Y, Yang K, Cui W, Jin W. Myocardial fibrosis reversion via rhACE2-electrospun fibrous patch for ventricular remodeling prevention. NPJ Regen Med. 2021; 6(1): 44.\u003c/li\u003e\n\u003cli\u003ePan Z, Huang Y, Qian H, Du X, Qin W, Liu T. Superparamagnetic iron oxide nanoparticles drive miR-485-5p inhibition in glioma stem cells by silencing Tie1 expression. Int J Biol Sci. 2020 Feb 10;16(7):1274-1287. \u003c/li\u003e\n\u003cli\u003eWang H, Bai Z, Qiu Y, Kou J, Zhu Y, Tan Q, Chen C, Mo R. Empagliflozin-Pretreated MSC-Derived Exosomes Enhance Angiogenesis and Wound Healing via PTEN/AKT/VEGF Pathway. Int J Nanomedicine. 2025; 20: 5119-5136.\u003c/li\u003e\n\u003cli\u003eBoueya IL, Sandhow L, Albuquerque JRP, Znaidi R, Passaro D. Endothelial heterogeneity in bone marrow: insights across development, adult life and leukemia. Leukemia. 2025; 39(1): 8-24.\u003c/li\u003e\n\u003cli\u003eZhang S, Zhao L, Shen L, Xu D, Huang B, Wang Q, Lin J, Zou Y, Ge J. Comparison of various niches for endothelial progenitor cell therapy on ischemic myocardial repair: coexistence of host collateralization and Akt-mediated angiogenesis produces a superior microenvironment. Arterioscler Thromb Vasc Biol. 2012; 32(4): 910-923.\u003c/li\u003e\n\u003cli\u003eChen X, Zhou C, Xu D, Liu X, Li S, Hou J, Zhang K, Zeng C, Zheng G, Wu H, Wu H, Wang W, Fu J, Wang T. Peptide hormone ELABELA promotes rat bone marrow-derived mesenchymal stem cell proliferation and migration by manipulating the cell cycle through the PI3K/AKT pathway under the hypoxia and ischemia microenvironment. Stem Cell Res Ther. 2022; 13(1): 32.\u003c/li\u003e\n\u003cli\u003ePark HK, Hu S, Kim SY, Yoon S, Yoon NG, Lee JH, Choi W, Kong SY, Kim JH, Nam D, Kang BH. Pseudohypoxic stabilization of HIF1\u0026alpha; via cyclophilin D suppression promotes melanoma metastasis. Signal Transduct Target Ther. 2025; 10(1): 231.\u003c/li\u003e\n\u003cli\u003eYao L, Gong X, Liu Y, Hao K, Jiang J, Luo Z. The miR-195-3p/BDNF axis is a promising therapeutic for heart failure. Int J Cardiol. 2024; 406: 131991. \u003c/li\u003e\n\u003cli\u003eLee EJ, Sun R, Kim J. The self-renewal function of Oct-4 can be replaced by the EWS-Oct-4 fusion protein in embryonic stem cells. Cell Mol Life Sci. 2025; 82(1): 166.\u003c/li\u003e\n\u003cli\u003eChen W, Wang YJ. Multifaceted roles of OCT4 in tumor microenvironment: biology and therapeutic implications. Oncogene. 2025; 44(18): 1213-1229.\u003c/li\u003e\n\u003cli\u003eSchmitz DA, Okamura D, Sakurai M, Ding Y, Oura S, Ballard E, Wei Y, Yu L, Hu Y, Wu J. Dissecting Oct4 enhancer function in pluripotent stem cells and mouse embryogenesis. Stem Cell Reports. 2025: 102706.\u003c/li\u003e\n\u003cli\u003eHan Y, Yang J, Fang J, Zhou Y, Candi E, Wang J, Hua D, Shao C, Shi Y. The secretion profile of mesenchymal stem cells and potential applications in treating human diseases. Signal Transduct Target Ther. 2022; 7(1): 92.\u003c/li\u003e\n\u003cli\u003eEgea V. HypoxamiRs: the hidden architects of tissue adaptation in hypoxia. Cell Death Dis. 2025; 16(1): 732.\u003c/li\u003e\n\u003cli\u003eFu J, Chen X, Liu X, Xu D, Yang H, Zeng C, Long H, Zhou C, Wu H, Zheng G, Wu H, Wang W, Wang T. ELABELA ameliorates hypoxic/ischemic-induced bone mesenchymal stem cell apoptosis via alleviation of mitochondrial dysfunction and activation of PI3K/AKT and ERK1/2 pathways. Stem Cell Res Ther. 2020; 11(1): 541.\u003c/li\u003e\n\u003cli\u003eWatanuki S, Kobayashi H, Sugiura Y, Yamamoto M, Karigane D, Shiroshita K, Sorimachi Y, Morikawa T, Fujita S, Shide K, Haraguchi M, Tamaki S, Mikawa T, Kondoh H, Nakano H, Sumiyama K, Nagamatsu G, Goda N, Okamoto S, Nakamura-Ishizu A, Shimoda K, Suematsu M, Suda T, Takubo K. SDHAF1 confers metabolic resilience to aging hematopoietic stem cells by promoting mitochondrial ATP production. Cell Stem Cell. 2024; 31(8): 1145-1161.e15.\u003c/li\u003e\n\u003cli\u003eKračun D, G\u0026ouml;rlach A, Snedeker JG, Buschmann J. Reactive oxygen species in tendon injury and repair. Redox Biol. 2025; 81: 103568.\u003c/li\u003e\n\u003cli\u003eCao DC, Liang Y, Guo Y, Wu DY, Wang NN, Li YM, Sun HF, Wang Q, Zhang X, Chi YL, Sun PP, Li YJ, Wang RR, Wang PY, Xie SY, Xie N. Oxygen-Generating Biomimetic Nano-Herb System for Synergistic Therapy \u0026amp; Pain Relief in Triple-Negative Breast Cancer via HIF-1\u0026alpha;/VEGF Pathway. Int J Nanomedicine. 2025; 20: 7113-7132.\u003c/li\u003e\n\u003cli\u003eHarada A, Yasumizu Y, Harada T, Fumoto K, Sato A, Maehara N, Sada R, Matsumoto S, Nishina T, Takeda K, Morii E, Kayama H, Kikuchi A. Hypoxia-induced Wnt5a-secreting fibroblasts promote colon cancer progression. Nat Commun. 2025; 16(1): 3653.\u003c/li\u003e\n\u003cli\u003eSun ED, Zhou OY, Hauptschein M, Rappoport N, Xu L, Navarro Negredo P, Liu L, Rando TA, Zou J, Brunet A. Spatial transcriptomic clocks reveal cell proximity effects in brain ageing. Nature. 2025; 638(8049): 160-171.\u003c/li\u003e\n\u003cli\u003eGrunewald M, Kumar S, Sharife H, Volinsky E, Gileles-Hillel A, Licht T, Permyakova A, Hinden L, Azar S, Friedmann Y, Kupetz P, Tzuberi R, Anisimov A, Alitalo K, Horwitz M, Leebhoff S, Khoma OZ, Hlushchuk R, Djonov V, Abramovitch R, Tam J, Keshet E. Counteracting age-related VEGF signaling insufficiency promotes healthy aging and extends life span. Science. 2021; 373(6554): eabc8479.\u003c/li\u003e\n\u003cli\u003eWang X, Huang J, Fan W, Li N, Yang H, Yang W, Li W, Li R, Liu J, Li X, Zhou Q, Hou S. Lactate-binding protein DNMT3A in HRMECs promotes angiogenesis by upregulating VEGFA through HIF-1\u0026alpha; lactylation. Genome Biol. 2025; 26(1): 377.\u003c/li\u003e\n\u003cli\u003eWang Y, Ge M, Wang J, Xu Y, Wang N, Xu S. Metabolic reprogramming in ischemic stroke: when glycolytic overdrive meets lipid storm. Cell Death Dis. 2025; 16(1): 788.\u003c/li\u003e\n\u003cli\u003eWu H, Xing C, Yu B, Guo L, Dou X, Gao L, Yang S, Zhang Y, Gao X, Li S, Xia B, Ma T, Hao Y, Yang Y, Gao X, Wei Y, Xue B, Zhang Q, Feng CL, Huang J. Metabolic Reprogramming of Neural Stem Cells by Chiral Nanofiber for Spinal Cord Injury. ACS Nano. 2025; 19(4): 4785-4801. \u003c/li\u003e\n\u003cli\u003eChen X, Yuan Y, Zhou F, Li L, Pu J, Zeng Y, Jiang X. Lactylation: From Homeostasis to Pathological Implications and Therapeutic Strategies. MedComm (2020). 2025; 6(6): e70226.\u003c/li\u003e\n\u003cli\u003eRenikunta HV, Lazarow K, Gong Y, Shukla PC, Nageswaran V, Giral H, Kratzer A, Opitz L, Engel FB, Haghikia A, Costantino S, Paneni F, von Kries JP, Streckfuss-B\u0026ouml;meke K, Landmesser U, Jakob P. Large-scale microRNA functional high-throughput screening identifies miR-515-3p and miR-519e-3p as inducers of human cardiomyocyte proliferation. iScience. 2023; 26(5): 106593.\u003c/li\u003e\n\u003cli\u003eVander Heiden MG, Cantley LC, Thompson CB. Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science. 2009; 324(5930): 1029-1033. \u003c/li\u003e\n\u003cli\u003eAlhasan KA, King MA, Pattar BSB, Lewis IA, Lopaschuk GD, Greenway SC. Anaplerotic filling in heart failure: a review of mechanism and potential therapeutics. Cardiovasc Res. 2024; 120(17): 2166-2178.\u003c/li\u003e\n\u003cli\u003eCordani M, Rumio C, Bontempi G, Strippoli R, Marcucci F. Oxidative and Glycolytic Metabolism: Their Reciprocal Regulation and Dysregulation in Cancer. Cells. 2025; 14(15): 1177.\u003c/li\u003e\n\u003cli\u003ePopazova O, Belenichev I, Bukhtiyarova N, Ryzhenko V, Gorchakova N, Oksenych V, Kamyshnyi O. Molecular and Biochemical Mechanisms of Cardiomyopathy Development Following Prenatal Hypoxia-Focus on the NO System. Antioxidants (Basel). 2025; 14(6): 743.\u003c/li\u003e\n\u003cli\u003eLi G, Cui H, Fan R, Liu G, Hou Z, Zhang Y, Che X, Wang T, Wei H, Li P. Delivery of Molecular Hydrogen for Precision Immunomodulation: Mechanisms, Detection Methods, and Applications. Adv Sci (Weinh). 2025; 12(32): e00283.\u003c/li\u003e\n\u003cli\u003eWang J, An Z, Wu Z, Zhou W, Sun P, Wu P, Dang S, Xue R, Bai X, Du Y, Chen R, Wang W, Huang P, Lam SM, Ai Y, Liu S, Shui G, Zhang Z, Liu Z, Huang J, Fang X, He K. Spatial organization of PI3K-PI(3,4,5)P3-AKT signaling by focal adhesions. Mol Cell. 2024; 84(22): 4401-4418.e9. \u003c/li\u003e\n\u003cli\u003eWang P, Deng Z, Li A, Li R, Huang W, Cui J, Chen S, Li B, Zhang S. \u0026beta;-Catenin promotes long-term survival and angiogenesis of peripheral blood mesenchymal stem cells via the Oct4 signaling pathway. Exp Mol Med. 2022; 54(9): 1434-1449. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"stem-cell-research-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scrt","sideBox":"Learn more about [Stem Cell Research \u0026 Therapy](http://stemcellres.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/scrt/default.aspx","title":"Stem Cell Research \u0026 Therapy","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Mesenchymal stem cells, Myocardial infarction, Metabolic reprogramming, Lactate preconditioning, miR-195-3p, Oct4","lastPublishedDoi":"10.21203/rs.3.rs-8443063/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8443063/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Mesenchymal stem cell (MSC) therapy shows limited efficacy for myocardial infarction, primarily due to poor cell survival under ischemic stress. While hypoxia-regulated miRNAs are implicated in MSC function, the specific role of miR-195-3p and its potential modulation through metabolic preconditioning remain unexplored. Here, we performed miRNA sequencing of hypoxic MSCs to identify key regulators. The miR-195-3p/Oct4 interaction was validated via luciferase reporter assays, qPCR, and western blotting. MSC survival, apoptosis, and angiogenic capacity were assessed under hypoxia. Rat myocardial infarction models received MSCs with modified miR-195-3p/Oct4 expression or lactate preconditioning, followed by comprehensive evaluation of cardiac function, histopathology, and metabolic remodeling. Hypoxia markedly upregulated miR-195-3p while suppressing Oct4 in MSCs. Mechanistically, miR-195-3p directly targeted Oct4, impairing MSC survival under hypoxic stress. Lactate preconditioning restored Oct4 expression and enhanced MSC resilience. In infarcted hearts, lactate-preconditioned or Oct4-overexpressing MSCs significantly improved cardiac function, reduced fibrosis, and promoted angiogenesis compared to controls—benefits abolished by Oct4 knockdown. Oct4 restoration augmented glycolytic metabolism through GLUT1/HK2 upregulation and amplified VEGF/VEGFR2/Akt signaling. Conversely, miR-195-3p overexpression suppressed glycolysis and angiogenesis, effects rescued by Oct4 co-expression. Lactate preconditioning enhances MSC therapeutic efficacy by disrupting miR-195-3p-mediated Oct4 suppression, thereby promoting metabolic adaptation and VEGF-driven angiogenesis in ischemic myocardium. Targeting the miR-195-3p/Oct4/VEGF axis represents a promising strategy to optimize MSC-based cardiac regeneration.","manuscriptTitle":"Metabolic Reprogramming by Lactate Unlocks a Pro-Survival Code in MSCs: The miR-195-3p/Oct4/VEGF Axis in Heart Repair","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-25 12:47:38","doi":"10.21203/rs.3.rs-8443063/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2026-03-23T03:01:05+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-10T16:01:45+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-06T05:22:07+00:00","index":"","fulltext":""},{"type":"submitted","content":"Stem Cell Research \u0026 Therapy","date":"2026-02-05T13:15:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"stem-cell-research-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scrt","sideBox":"Learn more about [Stem Cell Research \u0026 Therapy](http://stemcellres.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/scrt/default.aspx","title":"Stem Cell Research \u0026 Therapy","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"be1fb5ce-824a-4096-b738-9b0732eebb3e","owner":[],"postedDate":"March 25th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-25T12:47:38+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-25 12:47:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8443063","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8443063","identity":"rs-8443063","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}