CDK9 regulates neointima formation following vascular injury via targeting transcription factor Twist1

CDK9 regulates neointima formation following vascular injury via targeting transcription factor Twist1 | 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 CDK9 regulates neointima formation following vascular injury via targeting transcription factor Twist1 Liqing Yu, Xiaoxia Zhou, Jiafeng Chen, Mengxiang Li, Ruiying Wang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8452448/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Restenosis is a major limiting factor of percutaneous coronary intervention (PCI), accompanied by abnormal proliferation, migration and phenotypic switching of vascular smooth muscle cells (VSMCs). As a key component of positive transcription factor-b (P-TEFb), CDK9 phosphorylates RNA polymerase II and regulates various genes that are involved in the regulation of diverse cellular processes including cell growth and proliferation. In this study we investigated whether and how CDK9 regulated vascular remodeling after injury in mice. Results Our study observes a marked upregulation of CDK9 and its substrate pSer2 in VSMCs during neointimal hyperplasia following carotid artery injury, and the neointimal formation can be remarkably ameliorated by the CDK9 inhibitor (iCDK9). Overexpression of CDK9 promotes phenotypic switching, proliferation and migration of VSMCs in vitro, whereas inhibition of CDK9 obtains the opposites results. Moreover, CDK9 upregulates the expression of transcription factor Twist1, which is a key inducer of epithelial-mesenchymal transition (EMT) and has been widely implicated in the pathological progression of cardiovascular diseases. Furthermore, application of the Twist1 inhibitor Harmine largely abolished the function of CDK9 in promoting VSMCs phenotypic switching, proliferation and migration in vitro. Conclusions Our findings demonstrate a crucial regulatory role of CDK9 in neointima formation after vascular injury, and strategies targeting CDK9 inhibition potentially overcome limitations of sustained efficacy following PCI. neointimal formation vascular smooth muscle cell CDK9 phenotypic switching Twist1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Cardiovascular diseases are the leading cause of human morbidity and mortality worldwide[1-3]. In recent decades, the incidence of vascular stenosis related diseases especially coronary heart disease, has been increasing greatly and seriously threatening human health[4-7]. Clinically, percutaneous coronary intervention (PCI) is one well established and effective method for the treatment of coronary artery disease[8-11]. But unfortunately, months after PCI surgery, vascular restenosis is currently a common complication, which may narrow the newly opened coronary arteries again and re-block the blood supply to the myocardium[12-14]. Repeated interventional treatments are required, which increase the physical and economic burden on patients, so restenosis is one of the big challenges of PCI surgery in the clinic. Previous studies have reported that the neointima formation primarily driven by excessive proliferation and migration of vascular smooth muscle cells (VSMCs), plays a vital role in restenosis[15-17]. Vascular smooth muscle cells (VSMCs) are a major component of blood vessels and play an important role in maintaining the physiological functions of blood vessels[18]. VSMCs are not terminally differentiated and exhibit phenotypic and functional plasticity in response to microenvironmental cues[16, 18-20]. Under normal physiological conditions, VSMCs located in the media of the vascular wall exhibit a contractile phenotype and possess low proliferative and migratory capacities[21, 22]. The quiescent contractile phenotype of VSMCs is characterized by abundant expression of contractile phenotype-associated genes, such as myocardin (MyoC), CNN1, α-smooth muscle actin(α-SMA), transgelin (SM22α), and MYH11[23, 24]. In response to vascular injury or some other pathological stimuli, VSMCs switch from a contractile phenotype to a dedifferentiated synthetic phenotype, which is characterized by a significant decrease of contractile gene expression and high proliferative and migratory abilities[25-27]. The synthetic VSMCs proliferate and migrate to the vascular intima, accompanied by massive secretion and deposit of extracellular matrix (ECM), resulting in continuous production of neointima[28-30]. Therefore, the phenotypic switching of VSMCs plays vital roles in neointima formation and vascular stenosis, and deciphering the molecular mechanisms governing VSMC phenotypic switch is important to the development of novel avenues for interventions in vascular remodeling after injury. As a key component of the positive transcription elongation factor b (P-TEFb) complex, Cyclin-dependent kinase 9 (CDK9) differs from other CDK family members in that it has little effects on regulating the cell cycle[31, 32]. Instead, it mainly regulates gene transcription through phosphorylating the C-terminal domain (CTD) of RNA polymerase II (RNA pol II) at Ser2, which is critical for RNA synthesis[33, 34]. The function of CDK9 in cancer and AIDS has been well-studied, recently an increasing number of studies have found that CDK9 is also involved in cardiovascular diseases[35-40]. For example, under pro-hypertrophic stimuli, CDK9 can be recruited to promotor regions of hypertrophic genes by other transcription factors such as BRD4 and p300/GATA4 complex, then increase the phosphorylation of RNA Pol II Ser2 (pSer2) and facilitate the transcription level of target genes, leading to cardiomyocyte proliferation and cardiac hypertrophy[35]. Additionally, the elevation of CDK9 in patients’ serum with atherosclerosis (AS) or pulmonary arterial hypertension (PAH) has been observed[40-42]. Though several studies have explored that CDK9 can promote the development of AS and PAH by regulating the proliferation and phenotypic switching of VSMCs, its molecular mechanisms are largely unknown and whether CDK9 is involved in neointima formation after vascular injury has not been studied. In this study, we explore the effect and underlying mechanism of CDK9 on neointima hyperplasia induced by vascular injury in vitro and in vivo. Our results suggest that CDK9 expression and its substrate pSer2 are positively correlated with human carotid arterial stenosis. CDK9 inhibitor iCDK9 significantly inhibits neointima formation in mice after vascular injury. Genetic knockdown and pharmacological inhibition of CDK9 resulted in a significant increase of VSMC proliferation/migration and phenotypic switching. Moreover, we identified transcription factor Twist1 as a CDK9 target gene: inhibition of Twist1 by Harmine represses the effect of CDK9 on the proliferation/migration and phenotypic switching of VSMCs. The findings of this study expand our understanding of neointimal hyperplasia and provide novel insight into the development of therapeutic strategies for restenosis. Results CDK9 expression and activity was induced in VSMCs during neointimal hyperplasia In order to investigate the role of CDK9 in neointimal formation, a mouse carotid artery injury model was established through ligation initially. As shown in Figure1A, neointimal formation gradually increased over two weeks after injury (Figure 1A). Interestingly, after carotid artery ligation in mice, the CDK9 expression was induced in a time-dependent manner in response to the injure, which was accompanied by a significant decrease of the contractile VSMC marker α-SMA (Figure 1B-C). Additionally, as a substrate of CDK9 kinase, the p-Ser2 level of RNA poll was obviously increased after injury (Figure 1D-E). Furthermore, western blot analysis confirmed the significant increase of CDK9 expression and p-Ser2 level at the sites of injury at 28 days after ligation compared with the non-ligated vessels (Figure 1F-G). As shown in Figure 1B, CDK9 was largely co-localized with α-SMA-positive VSMC. To determine whether the increase of CDK9 expression and p-Ser2 level was due to the VSMC phenotypic modulation in the process of injury-induced neointimal formation, human aortic SMCs (HA-VSMCs) were treated with PDGF-BB and Rapamycin, respectively. As expected, the α-SMA expression decreased after PDGF-BB treatment, but increased in the Rapamycin group, whereas both the mRNA and protein levels of CDK9 elevated obviously after PDGF-BB treatment, and Rapamycin inhibited the CDK9 expression level (Figure 1H-J). Meanwhile, the change of p-Ser2 level was consistent with that of CDK9(Figure 1I-J). And the same results were observed in rat-SMCs (Figure S1). Taken together, these data indicate that CDK9 is involved in the neointimal formation after vascular injury and VSMC phenotypic switching. Inhibition of CDK9 alleviates neointimal formation following vascular injury In order to confirm the role of CDK9 in neointimal formation, the specific CDK9 inhibitor CDK9-IN-2 (iCDK9) was employed in the vascular injury model. As shown in Figure 2A, mice about 10 weeks old were subjected to ligation of the left carotid artery, then iCDK9 (5 or 10mg/kg) was administered via intraperitoneal injection daily three days after the surgery. The HE staining results showed that three weeks after the ligation, the intimal hyperplasia was obvious, which can be alleviated by iCDK9 treatment in a dose-dependent manner with a significant decrease in the intimal area and a lower intima/media ratio (Figure 2B-C). Furthermore, immunohistochemical assays for contractile VSMC marker α-SMA showed that the decrease of α-SMA expression after vascular injury could be repressed by iCDK9 treatment (Figure 2D-E). As expected, the p-Ser2 level in ligated vascular was decreased greatly after iCDK9 treatment (Figure 2F-G). Taken together, these data suggest that iCDK9 can relieve neointimal hyperplasia after vascular injury in a phosphatase activity-dependent manner. CDK9 regulates the proliferation and migration of VSMCs As VSMC proliferation and migration are key processes in neointimal formation following arterial injury. So next, we aim to determine whether the effects of CDK9 on neointimal formation were due to the regulation of VSMC proliferation and migration. Firstly, we examined the potential role of CDK9 in PDGF-BB-induced cell migration and proliferation in vitro models. In order to evaluate the effect of CDK9 on VSMC migration, we performed scratch‑wound‑ healing assays. It is well known that cell motility of VSMCs can be promoted by PDGF-BB stimulation. The results revealed that iCDK9, or the selective CDK9 degrader Thal significantly retarded wound closure, under both quiescent and PDGF-BB-stimulated conditions (Figure 3A-B). Proliferationof VSMCs is another key mechanism in the process of neointimal formation. Next, we evaluated the regulation of CDK9 on VSMC proliferation. To this end, PDGF-BB-stimulated VSMCs were subjected to Edu incorporation assays. After PDGF-BB stimulation, the Edu-positive cells increased greatly; however, it can be inhibited by CDK9 inhibitors iCDK9 or degrader Thal (Figure 3C-D). To further verify the regulation of CDK9 on proliferation of VSMCs, CDK9 was knocked down in VSMCs by siRNA; the result showed that after CDK9 knockdown, the expression levels of the cell proliferation marker PCNA protein were significantly decreased (Figure 3E-F and Figure S2). However, when CDK9 was overexpressed by adenovirus infection, the level of PCNA protein increased obviously in VSMCs (Figure 3G-H). We further evaluated VSMC proliferation in vivo, and another cell proliferation marker Ki67 was tested by immuno-histochemistry (IHC). The expression level of Ki67 was higher in left carotid arteries after 3 weeks of ligation than in the unligated right carotid arteries (Sham); however, inhibition of the CDK9 activity by iCDK9 significantly repressed Ki67 expression levels in the left carotid artery compared with that in the negative control group (Figure 3I). Taken together, these results indicate that inhibition or deficiency of CDK9 in VSMCs repressed their migration and proliferation, thereby alleviating the injury-induced neointimal formation. Inhibition of CDK9 can suppress phenotypic switching of VSMCs in vitro The phenotype switching of VSMCs, from the differentiated contractile state to the dedifferentiated synthetic state, plays an important role in neointimal formation after vascular injury. Consistent with the inhibition effect of iCDK9 on neointimal formation in vivo, iCDK9 up-regulated basal levels of VSMC contractile marker α-SMA and down-regulated VSMC synthetic marker OPN and proliferation marker PCNA in VSMCs (Figure 4A). Similar result was observed when the CDK9 selective degrader Thal was employed in VSMCs. Accompanied by the decrease of CDK9 protein level after Thal treatment, contractile markers increased, but synthetic markers decreased obviously (Figure 4B). As an important dedifferentiation factor, PDGF-BB can stimulate VSMCs to undergo phenotypic switching, which is manifested as a decrease of differentiation marker genes and an increase of synthetic markers. As expected, PDGF-BB significantly increased cell proliferation marker PCNA and synthetic markers but decreased the contractile markers; however, these changes were attenuated by iCDK9 (Figure 4C-D). Besides, CDK9 degrader Thal also significantly inhibits the changes of protein expression related to VSMCs phenotypic switching induced by PDGF-BB (Figure 4E-F). In addition, immunofluorescence assays further demonstrated that iCDK9 or Thal obviously increased the basal expression level of α-SMA, and inhibited PDGF-BB-induced VSMC phenotypic switching (Figure 4G). We confirmed these results by RNAi-mediated CDK9 knockdown experiments in HA-VSMCs. The contractile phenotype-related proteins such as SM22a and CNN1 increased, whereas the synthetic markers such as Vimentin and OPN decreased in CDK9 siRNA-treated HA-VSMCs (Figure 4H). Taken together, our data suggest that CDK9 plays an important role in mediating phenotypic switching of VSMCs, and intervention of CDK9 can effectively inhibit phenotypic switching; thus, CDK9 may serve as a potential target for neointimal formation in vivo. CDK9 regulates the expression level of Twist1 We next explored the underlying mechanisms of CDK9 in phenotypic modulation of VSMCs. Since it has been reported that the process of VSMC phenotypic switching from the contractile state to the synthetic state can be induced by PDGF-BB and repressed by Rapamycin, VSMCs were treated with PDGF-BB, Rapamycin or iCDK9, respectively. Subsequently, we performed whole transcriptome analysis using RNA sequencing. 367 genes synchronously altered significantly under various treatment conditions were chosen for further analysis (Figure 5A). From previous results in our study, iCDK9 can inhibit the phenotypic switching of VSMCs as Rapamycin does, so the genes that exhibit consistent change trends in both the iCDK9 group and the Rapamycin group, yet show opposite trends to the PDGF-BB group, are considered as potential candidates for our further research. Cluster analysis of these 367 genes showed that only 96 genes significantly increased in the PDGF-BB group, but decreased in both the iCDK9 and Rapamycin groups (Figure 5B). As a key component of the positive transcription factor P-TEFb complex, CDK9 mainly regulates the transcription process of intracellular genes; thus, we focus on the changes of transcription-related proteins. GO analysis of the 96 selected genes revealed that 4 of them are transcription-related genes, including Twist1, Helz2, Jun, and Mef2c (Figure 5C). As an important transcription factor, Twist1 regulates multiple biological processes, including epithelial-mesenchymal transition (EMT), cell proliferation, migration, and extracellular matrix remodeling[43-45]. Recent studies reported that Twist1 can induce VSMC proliferation and phenotypic switching[46-48]. Therefore, we speculate whether CDK9 promotes the phenotypic switching of smooth muscle by regulating the expression of Twist1. To this end, we first validated the RNA-seq results using qRT-PCR and found that, consistent with the sequencing results, the mRNA level of Twist1 was increased in the PDGF-BB group but decreased in the Rapamycin and iCDK9 groups significantly (Figure 5D). Subsequently, the protein level of Twist1 was detected, and the results showed that its change trend was consistent with that of the mRNA level (Figure 5E-F). To further verify that CDK9 can directly regulate the gene level of Twist1, CDK9 was overexpressed in VSMCs via adenovirus infection, and the protein level of Twist1 increased significantly after CDK9 overexpression (Figure 5G-H). This funding was further supported by siCDK9; western blotting showed that in contrast to overexpression, down regulation of CDK9 by siCDK9 decreased the protein level of Twist1 in VSMCs (Figure 5I-J). Above all, the results indicated that CDK9 can regulate the expression level of Twist1 in VSMCs, which may contribute to its modulation in vascular remodeling. Harmine alleviates the promoting effect of CDK9 on proliferation, migration, and phenotypic switching of VSMCs To determine whether Twist1 is required in the CDK9-regulated proliferation and migration of VSMCs, growth and motility were examined. After being infected with adenovirus for 24 h, VSMCs were treated with the Twist1 inhibitor Harmine for another 24 h. We found that the wound healing (scratch assay) of VSMCs was significantly promoted by CDK9 overexpression, but this effect was almost abolished in the presence of Harmine (Figure 6A-B). The inhibitory effect of Harmine on VSMC proliferation was further confirmed by EdU incorporation analysis. Consistent with the wound healing assay results, the number of EdU-positive cells increased significantly following CDK9 overexpression, but this increase could be suppressed by Harmine, indicating Twist1 mediates CDK9-regulated VSMC proliferation (Figure 6C-D). Consistently, overexpression of CDK9 leads to a significant increase in the expression of the proliferation marker PCNA; however, treatment with Harmine can reverse this effect, resulting in the inhibition of PCNA expression (Figure 6E). To further investigate the role of Twist1 in CDK9-regulated phenotypic switching, the inhibition of Harmine under PDGF-BB stimulation was explored. PDGF-BB plays a key role in the phenotypic regulation of VSMCs; as expected treatment with either the CDK9 inhibitor (iCDK9) or Harmine alone could inhibit PDGF-BB-induced phenotypic switching (Figure 6F). Interestingly, Harmine could further enhance the inhibitory effect of iCDK9 on PDGF-BB-induced phenotypic switching, leading to a further significant decrease in the expression of contractile markers, including α-SMA, SM22a, and CNN1, and an increase in the expression of the proliferative marker PCNA and synthetic marker OPN in VSMCs (Figure 6G). The enhancing effect of Harmine on iCDK9-mediated inhibition of VSMCs phenotypic switching was further confirmed by qRT-PCR. Following treatment with iCDK9, the PDGF-BB-induced reduction in contractile phenotype markers such as α-SMA, SM22a, and CNN1 was reversed; additionally, the introduction of Harmine led to a further increase in the expression of these contractile phenotype markers (Figure 6H-J). Herein, results from Harmine treatment in the iCDK9-treated group and the CDK9 overexpression group showed that Harmine is capable of inhibiting CDK9-regulated proliferation, migration, and phenotypic switching in VSMCs, and Twist1 serves as the critical target mediating CDK9’s regulatory effects on VSMCs. Discussion Restenosis remains a major clinical challenge limiting the long-term efficacy of percutaneous coronary intervention (PCI), with neointimal formation driven by abnormal vascular smooth muscle cell (VSMC) proliferation, migration, and phenotypic switching being its core pathological feature[5] [16, 49]. In this study, we systematically investigated the role and molecular mechanism of cyclin-dependent kinase 9 (CDK9) in vascular injury-induced neointimal hyperplasia, and our findings identify CDK9 as a pivotal regulator of this process, with transcription factor Twist1 serving as its key downstream effector. CDK9 is well-characterized for its role in regulating RNA polymerase II (RNA pol II)-dependent gene transcription by phosphorylating the Ser2 residue of RNA pol II’s C-terminal domain (CTD, pSer2)[31, 34]. While previous studies have linked CDK9 to cardiovascular diseases such as atherosclerosis (AS) and pulmonary arterial hypertension (PAH)[40, 41], its involvement in post-injury neointimal formation remained unaddressed. Our study fills this gap by demonstrating that CDK9 expression and activity (reflected by pSer2 levels) are significantly upregulated in VSMCs during mouse carotid artery ligation-induced neointimal hyperplasia (Figure 1). This upregulation is not merely correlative: pharmacological inhibition of CDK9 with CDK9-IN-2 (iCDK9) dose-dependently reduces neointimal area and the intima/media ratio in vivo (Figure 2). These results align with recent reports that CDK9 regulates VSMC function in AS[41] , but extend this knowledge by establishing CDK9 as a direct contributor to the neointimal response to mechanical vascular injury-the primary trigger of PCI restenosis. A key insight from our in vitro studies is that CDK9 modulates multiple VSMC behaviors critical for neointimal formation. This is particularly significant because phenotypic switching of VSMCs from a quiescent, contractile state to a proliferative, synthetic state is an early and rate-limiting step in neointimal formation[28, 31]. Our data thus suggest that CDK9 promotes neointimal hyperplasia by coordinatey enhancing VSMC proliferation, migration, and dedifferentiation, three interrelated processes that drive vascular remodeling after injury. To unravel the molecular mechanism by which CDK9 regulates VSMC function, we identified Twist1 as the most promising target, as it is a well-known inducer of epithelial-mesenchymal transition (EMT) and has been linked to VSMC proliferation and phenotypic switching in cardiovascular disease[26, 28]. Furthermore, our validation experiments confirmed that Twist1 is a direct downstream target of CDK9, and the results establish a CDK9-Twist1 axis that mediates VSMC dysfunction during neointimal formation. This mechanism expands our understanding of CDK9’s transcriptional targets in vascular biology. Prior studies focused on its role in cardiomyocyte hypertrophy via genes like GATA4[35], but our work identifies Twist1 as a key mediator of CDK9’s effects in VSMCs. The clinical relevance of our findings lies in their potential to address PCI restenosis. Current strategies for restenosis prevention (e.g., drug-eluting stents) have limitations, including late stent thrombosis and incomplete inhibition of neointimal growth[12, 13]. Our study shows that targeting CDK9 (e.g., with iCDK9) effectively reduces neointimal formation in mice, suggesting that CDK9 inhibitors could be developed as novel therapeutics for restenosis. However, this study has several limitations. First, our in vivo experiments were conducted exclusively in male mice, and future studies should include female mice to assess potential sex differences, as sex is a known modifier of cardiovascular disease[4]. Second, due to the crucial role of CDK9 in the intestinal tract, it has been challenging to obtain (SMC)-specific CDK9 deficiency (CDK9-SMC-KO) mice. Therefore, it is impossible to study the regulatory role of CDK9 in intimal hyperplasia using genetically engineered mouse models. Conclusion In summary, our study demonstrates that CDK9 is upregulated in VSMCs after vascular injury and promotes neointimal formation by enhancing VSMC proliferation, migration, and phenotypic switching via the transcription factor Twist1. These findings identify the CDK9-Twist1 axis as a novel therapeutic target for preventing PCI-related restenosis and provide a new framework for understanding the molecular mechanisms of vascular remodeling. Materials and Methods Materials The CDK9 inhibitor CDK9-IN-2 (HY-16462), CDK9 degrader THAL-SNS-032(HY-123937) and Rapamycin (HY-10219) were from Med Chem Express. Human PDGF-BB protein (220-bb-010) was obtained from R&D Systems. Tamoxifen (T5648) was purchased from Sigma-Aldrich. Methods Animals All animals were maintained and used in accordance with the guidelines of the Institutional Animal Care and Use Committee of Xiamen University (approval no. XMULAC20250055). All mice were housed in the specific pathogen-free laboratory with a 12-h light/12-h dark cycle at 22 to 24° and humidity (55 ± 5%) with free access to food and water. C57BL/6J mice were purchased from Gem Pharmatec. For iCDK9 treatment in vivo, 10-week-old male mice were subjected to carotid artery ligation and three days later followed by 18-day iCDK9 oral gavage regimen as shown in Figure 2A. After the last administration, mice were euthanized and carotid arteries were collected and subjected to the following experiments. Mouse carotid artery ligation Only male mice were used in the study. For all surgical procedures, 10-week-old male mice were anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg, Sigma-Aldrich, Saint Louis, MO, USA). Then the left carotid artery (LCA) was carefully dissected and completely ligated at the proximal end of the carotid bifurcation with a 5-0 silk suture. A similar procedure without ligation on the right carotid artery (RCA) was used as a sham operation group. After 14, 21, or 28 d after surgery, carotid arteries were collected and subjected to the following experiments. Immunofluorescence staining and Immunohistochemistry Mice were euthanized by decapitation under isoflurane anesthesia, then carotid arteries were perfused and embedded in the OCT agent (4583, SAKURA). Frozen sections of 5 µm thickness and cells grown on glass coverslips were fixed with 4% paraformaldehyde (PFA) for 10 min after being washed with PBS for three times and permeabilized with 0.1% Triton X-100 in PBS for 5 min. The cells were blocked in 1% goat serum and incubated with the following primary antibodies at 4℃ overnight: anti-α-SMA (BM0002, BOSTER Biological Technology), anti-CDK9 (ab239364), and anti-pSer2 (ab5095). After incubation of second antibodies (A-11012 or A-21235, Invitrogen) for 1h at room temperature, the nuclei were stained with DAPI (P0131). The confocal immunofluorescence images were acquired by Zeiss confocal laser scanning microscopy. Mouse carotid arteries were collected and fixed with 4% PFA for 24 h before paraffin embedding. Hematoxylin-eosin (HE) staining and IHC for Ki67 were performed by Service-bio (Wuhan, China). Images were acquired with a Tissue FAX microscope (Leica, Germany). Morphometric analysis was performed using ImageJ software. Cell culture and treatment The human aortic smooth muscle cell line HA-VSMCs (HUM-iCell-c010) was purchased from iCell Bioscience (Shanghai, China). HA-VSMCs were cultured with a primary smooth muscle cell culture system (PriMed-iCell-004, iCell Bioscience) in a humidified incubator at 37 °C with 5% CO2. Primary rat vascular smooth muscle cells (rVSMCs) were isolated from the thoracic aorta of rats and routinely cultured in Dulbecco’s modified Eagle’s medium (DMEM, PM150210, procell) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Briefly, male Sprague-Dawley rats (80-100 g) were deeply anesthetized with intraperitoneal injection of sodium pentobarbital (50 mg/kg), and the adventitia and endothelium of fresh thoracic aorta vascular tissue were removed. The medial layer was cut into small pieces and cultured in DMEM containing 20% FBS. After VSMCs sprouted from the tissue block, the medium was replaced with DMEM containing 10% FBS and 1% penicillin-streptomycin. The purity of VSMCs was as identified by a specific marker, α-SMA (1:500; BM0002, BOSTER Biological Technology). Cells were used for subsequent experiments between passages 4-7. Before treatment with indicated agents, cells were starved with serum-free DMEM for 24h. Quantitative real-time PCR (qRT-PCR) Total RNA was isolated from cultured cells using Trizol reagent (Cat#: YZ-15596018, Invitrogen) following the manufacturer’s protocol. Extracted RNA was reverse transcribed using the Hifair® II 1st Strand cDNA Synthesis kit (Cat#: 11123ES60, Yeasen) to obtain cDNA. Real-time PCR was performed using a quantitative PCR kit (Cat#: 11204ES08, Yeasen) with the primer sequences listed in Supplementary Table S1 and all experiments were conducted in triplicate. Cell Proliferation Assay 5-ethynyl-2’-deoxyuridine (EdU) incorporation assay (C0078S; Beyotime Biotechnology, Shanghai, China) was preformed to assess the VSMC proliferation according to the manufacturer’s instructions. Briefly, after treatment, cells were incubated with EdU at a final concentration of 10 µM for 4 h before measurement, and total cellular nuclei were stained with Hoechst 33342. The EdU-positive cells were captured with an EVOS FL AUTO2 microscope and measured using ImageJ software. In vitro migration assay A wound-healing assay was used to measure the migration ability of VSMCs. Briefly, after starvation for 12 h, VSMCs cultured in 12-well plates were scratched with sterile plastic 10 µL micro-pipette tips to draw a straight line in the middle of wells and washed with 1xPBS twice to remove the cellular debris. Then VSMCs were treated with indicated agents for another 24 h and photographs were acquired with an EVOS FL AUTO2 microscope. The migration area was analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, United States). Recombinant adenovirus construction and transfection CDK9-overexpressing recombinant adenovirus (Adv-CDK9) and blank control recombinant adenovirus (Adv-Ctrl) were purchased from WZ Biosciences Inc. (Shandong, China). Human CDK9 cDNA was packaged into adenoviral vector pADM-CMV-mCMV-copGFP, and the empty vector was used as a control. HA-VSMCs at a 50–60% confluence were infected with adenovirus for 24 h and the mediums were changed with a fresh primary smooth muscle cell culture system for another 24 h, then harvested for subsequent experiments. Western Blot Analysis After treatment, cultured cells were harvested and lysed in RIPA buffer with complete protease inhibitors (Cat#:05892791001, Roche) and phosphatase inhibitors (Cat#:4906837001,Roche), and the supernatant was obtained through centrifugation(12000rpm, 4℃, 10 min). After determining the protein concentration by BCA Protein Assay Kit (A55864, Thermo Fisher Scientific), equal amounts of proteins were separated in SDS-PAGE gels. For tissue extraction, the mouse aorta tissues were excised and homogenized in the RIPA buffer; the supernatant was collected and processed in subsequent experiments in the same manner as cultured cells. The primary antibodies against indicated proteins are listed in Supplementary Table S2. Quantification of WB protein bands was performed using ImageJ software (National Institutes of Health, Bethesda, MD, United States). Small interfering RNA transfection The small interference RNAs (siRNAs) that target human CDK9 and siRNA control were synthesized by Gene Pharma (Shanghai, China) and sequences of siRNA were described in Supplementary Table S3. VSMCs at a 30–50% confluence were pre-incubated in the transfection medium (Opti-MEM, Cat NO.31985070, Gibco) and then transfected with siRNAs by using Lipofectamine RNAi MAX (Cat NO. 13778150, Invitrogen) according to the instructions provided by the manufacturer. Six hours after transfection, the medium was replaced with fresh serum-free DMEM medium and cultured for another 18 h, then subjected to the following experiment. Transcriptomic profiling To determine the potential target of CDK9 in regulating the VSMC phenotypic switching, rat-VSMCs were treated with DMSO (0.1%, as a control), the phenotypic switching activator PDGF-BB, the inhibitor Rapamycin or iCDK9, respectively for 24 h. The total RNA was extracted from the VSMCs using Trizol reagent (15596026, Thermo Fisher Scientific) following the manufacturer’s instructions and all RNA samples were analyzed for concentration and purity using a Nanodrop 2000. The RNA-seq transcriptome library was prepared following Illumina® Stranded mRNA Prep Ligation from Illumina (San Diego, CA) using 1μg of total RNA. RNA sequencing was carried out on NovaSeq X Plus(illumina) and the obtained sequencing reads were aligned to the reference Rattus norvegicus genome mRatBN7.2 (https://asia.ensembl.org/Rattus_norvegicus/Info/Index). To identify the significantly differentially expressed genes (DEGs), the expression level of each transcript was calculated according to the transcripts per million reads (TPM) method. RSEM was used to quantify gene abundances. DEGs with a fold change (FC) of ≥2 and a p-value <0.05 were considered as significantly differently expressed genes. Statistical Analysis Statistical analyses were performed to assess the significance of the differences and correlations observed in the experiments. All data are expressed as means±standard deviation (mean±SD) and analyzed using GraphPad Prism 10. Statistical analyses were performed using paired or unpaired Student’s t-tests (2 groups) or analysis of variance (ANOVA, > 2groups) as appropriate. A p-value < 0.05 was considered to indicate statistically significant differences. Declarations Author Contributions G.L. and J.W. designed the research and wrote the paper; LQ.Y., XX.Z., JF.C., and MX.L. performed the research; RY.W. analyzed the data. Funding The author(s) declare that financial support was received for the research, authorship, and/or publication of this article. This study was supported by the National Natural Science Foundation of China (Project #82304578) and the Fujian Provincial Natural Science Foundation (Project #2024J011419) to J. Wu. Availability of data and materials The transcriptome data generated in this study have been deposited in the National Center for Biotechnology Information in the Sequence Read Archive (SRA) database under the accession number PRJNA1357582. Additional information required to reanalyze the data reported in this paper is available from the lead contact upon request. Competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. References DALYs GBD, Collaborators H. Global, regional, and national disability-adjusted life-years (DALYs) for 359 diseases and injuries and healthy life expectancy (HALE) for 195 countries and territories, 1990-2017: a systematic analysis for the Global Burden of Disease Study 2017. Lancet. 2018; 392:1859–922. Lavie CJ. Progress in Cardiovascular Diseases Statistics 2022. Prog Cardiovasc Dis. 2022; 73:94–95. Fairweather D, Aung N, Carter RE. Editorial: Novel translational advances in artificial intelligence for diagnosis and treatment of cardiovascular diseases. Front Cardiovasc Med. 2025; 12:1604528. Goodarzynejad H, Sheikh Fathollahi M, Shafiee A. Editorial: The role of sex in coronary artery disease. Front Cardiovasc Med. 2023; 10:1220439. Anderson LL, Dai D, Miller AL, Roe MT, Messenger JC, Wang TY. Percutaneous coronary intervention for older adults who present with syncope and coronary artery disease? Insights from the National Cardiovascular Data Registry. Am Heart J. 2016; 176:1–9. Lawrie K, O'Neill S, Malik J, Balaz P, Corr M, Waldauf P. Classifications of haemodialysis vascular access-induced limb ischaemia. J Vasc Access. 2025:11297298251328428. Tachi M, Tanaka A, Riku S, Furuta T, Tobe A, Hayashi K, Murohara T. Percutaneous Treatment for Venous Stenosis Caused by a Vascular Closure Device. Cardiovasc Revasc Med. 2022; 40S:157–58. Serruys PW, Ono M, Garg S, Hara H, Kawashima H, Pompilio G, Andreini D, Holmes DR, Jr., Onuma Y, King Iii SB. Percutaneous Coronary Revascularization: JACC Historical Breakthroughs in Perspective. J Am Coll Cardiol. 2021; 78:384–407. Chacko L, J PH, Rajkumar C, Nowbar AN, Kane C, Mahdi D, Foley M, Shun-Shin M, Cole G, Sen S et al . Effects of Percutaneous Coronary Intervention on Death and Myocardial Infarction Stratified by Stable and Unstable Coronary Artery Disease: A Meta-Analysis of Randomized Controlled Trials. Circ Cardiovasc Qual Outcomes. 2020; 13:e006363. Akbari T, Al-Lamee R. Percutaneous Coronary Intervention in Multi-Vessel Disease. Cardiovasc Revasc Med. 2022; 44:80–91. Watanabe H, Yamamoto K, Shiomi H, Morimoto T, Kato E, Matsumura Y, Nakatsuma K, Takeji Y, Yaku H, Yamamoto E et al . Percutaneous coronary intervention using new-generation drug-eluting stents versus coronary arterial bypass grafting in stable patients with multi-vessel coronary artery disease: From the CREDO-Kyoto PCI/CABG registry Cohort-3. PLoS One. 2022; 17:e0267906. Fattori R, Piva T. Drug-eluting stents in vascular intervention. Lancet. 2003; 361:247–9. McNair ED, Wells CR, Mabood Qureshi A, Basran R, Pearce C, Orvold J, Devilliers J, Prasad K. Soluble receptors for advanced glycation end products (sRAGE) as a predictor of restenosis following percutaneous coronary intervention. Clin Cardiol. 2010; 33:678–85. Zhao L, Feng L, Shan R, Huang Y, Shen L, Fan M, Wang Y. Nanoparticle-based approaches for treating restenosis after vascular injury. Front Pharmacol. 2024; 15:1427651. Xu K, Al-Ani MK, Pan X, Chi Q, Dong N, Qiu X. Plant-Derived Products for Treatment of Vascular Intima Hyperplasia Selectively Inhibit Vascular Smooth Muscle Cell Functions. Evid Based Complement Alternat Med. 2018; 2018:3549312. Owens GK, Kumar MS, Wamhoff BR. Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol Rev. 2004; 84:767–801. Zhang Q, Li H, Zhuang T, Xu L, Wu W, Pi J, Zhu P, Geng L, Duan Y, Xu J et al . CCN5 suppresses injury-induced vascular restenosis by inhibiting smooth muscle cell proliferation and facilitating endothelial repair via thymosin beta4 and Cd9 pathway. Eur Heart J. 2025; 46:1645–58. Lacolley P, Regnault V, Nicoletti A, Li Z, Michel JB. The vascular smooth muscle cell in arterial pathology: a cell that can take on multiple roles. Cardiovasc Res. 2012; 95:194–204. Frismantiene A, Philippova M, Erne P, Resink TJ. Smooth muscle cell-driven vascular diseases and molecular mechanisms of VSMC plasticity. Cell Signal. 2018; 52:48–64. Mao C, Ma Z, Jia Y, Li W, Xie N, Zhao G, Ma B, Yu F, Sun J, Zhou Y et al . Nidogen-2 Maintains the Contractile Phenotype of Vascular Smooth Muscle Cells and Prevents Neointima Formation via Bridging Jagged1-Notch3 Signaling. Circulation. 2021; 144:1244–61. Chen R, McVey DG, Shen D, Huang X, Ye S. Phenotypic Switching of Vascular Smooth Muscle Cells in Atherosclerosis. J Am Heart Assoc. 2023; 12:e031121. Yap C, Mieremet A, de Vries CJM, Micha D, de Waard V. Six Shades of Vascular Smooth Muscle Cells Illuminated by KLF4 (Kruppel-Like Factor 4). Arterioscler Thromb Vasc Biol. 2021; 41:2693–707. Basatemur GL, Jorgensen HF, Clarke MCH, Bennett MR, Mallat Z. Vascular smooth muscle cells in atherosclerosis. Nat Rev Cardiol. 2019; 16:727–44. Grootaert MOJ, Bennett MR. Vascular smooth muscle cells in atherosclerosis: time for a re-assessment. Cardiovasc Res. 2021; 117:2326–39. Farina FM, Hall IF, Serio S, Zani S, Climent M, Salvarani N, Carullo P, Civilini E, Condorelli G, Elia L et al . miR-128-3p Is a Novel Regulator of Vascular Smooth Muscle Cell Phenotypic Switch and Vascular Diseases. Circ Res. 2020; 126:e120–e35. Zhao C, Shen J, Lu Y, Ni H, Xiang M, Xie Y. Dedifferentiation of vascular smooth muscle cells upon vessel injury. Int Immunopharmacol. 2025; 144:113691. Jensen LF, Bentzon JF, Albarran-Juarez J. The Phenotypic Responses of Vascular Smooth Muscle Cells Exposed to Mechanical Cues. Cells. 2021; 10. Cao G, Xuan X, Hu J, Zhang R, Jin H, Dong H. How vascular smooth muscle cell phenotype switching contributes to vascular disease. Cell Commun Signal. 2022; 20:180. Chappell J, Harman JL, Narasimhan VM, Yu H, Foote K, Simons BD, Bennett MR, Jorgensen HF. Extensive Proliferation of a Subset of Differentiated, yet Plastic, Medial Vascular Smooth Muscle Cells Contributes to Neointimal Formation in Mouse Injury and Atherosclerosis Models. Circ Res. 2016; 119:1313–23. Suna G, Wojakowski W, Lynch M, Barallobre-Barreiro J, Yin X, Mayr U, Baig F, Lu R, Fava M, Hayward R et al . Extracellular Matrix Proteomics Reveals Interplay of Aggrecan and Aggrecanases in Vascular Remodeling of Stented Coronary Arteries. Circulation. 2018; 137:166–83. Paparidis NF, Durvale MC, Canduri F. The emerging picture of CDK9/P-TEFb: more than 20 years of advances since PITALRE. Mol Biosyst. 2017; 13:246–76. Zheng B, Gold S, Iwanaszko M, Howard BC, Wang L, Shilatifard A. Distinct layers of BRD4-PTEFb reveal bromodomain-independent function in transcriptional regulation. Mol Cell. 2023; 83:2896–910 e4. Larochelle S, Amat R, Glover-Cutter K, Sanso M, Zhang C, Allen JJ, Shokat KM, Bentley DL, Fisher RP. Cyclin-dependent kinase control of the initiation-to-elongation switch of RNA polymerase II. Nat Struct Mol Biol. 2012; 19:1108–15. Zhou Q, Li T, Price DH. RNA polymerase II elongation control. Annu Rev Biochem. 2012; 81:119–43. Sunagawa Y, Morimoto T, Takaya T, Kaichi S, Wada H, Kawamura T, Fujita M, Shimatsu A, Kita T, Hasegawa K. Cyclin-dependent kinase-9 is a component of the p300/GATA4 complex required for phenylephrine-induced hypertrophy in cardiomyocytes. J Biol Chem. 2010; 285:9556–68. Sano M, Abdellatif M, Oh H, Xie M, Bagella L, Giordano A, Michael LH, DeMayo FJ, Schneider MD. Activation and function of cyclin T-Cdk9 (positive transcription elongation factor-b) in cardiac muscle-cell hypertrophy. Nat Med. 2002; 8:1310–7. Li Z, Guo Z, Lan R, Cai S, Lin Z, Li J, Wang J, Li Z, Liu P. The poly(ADP-ribosyl)ation of BRD4 mediated by PARP1 promoted pathological cardiac hypertrophy. Acta Pharm Sin B. 2021; 11:1286–99. Matrone G, Wilson KS, Maqsood S, Mullins JJ, Tucker CS, Denvir MA. CDK9 and its repressor LARP7 modulate cardiomyocyte proliferation and response to injury in the zebrafish heart. J Cell Sci. 2015; 128:4560–71. Xue Q, Zhang Q, Guo Z, Wu L, Chen Y, Chen Z, Yang K, Cao J. Therapeutic Hypothermia Inhibits Hypoxia-Induced Cardiomyocyte Apoptosis Via the MiR-483-3p/Cdk9 Axis. J Am Heart Assoc. 2023; 12:e026160. Jia Q, Hu Z, Song N, Mao W. Flavopiridol Mitigates the Progression of Monocrotaline-Induced Pulmonary Hypertension in Rats by Targeting Cyclin-Dependent Kinase 9. Cardiovasc Drugs Ther. 2023; 37:449–60. Huang S, Luo W, Wu G, Shen Q, Zhuang Z, Yang D, Qian J, Hu X, Cai Y, Chattipakorn N et al . Inhibition of CDK9 attenuates atherosclerosis by inhibiting inflammation and phenotypic switching of vascular smooth muscle cells. Aging (Albany NY). 2021; 13:14892–909. Han Y, Zhao S, Gong Y, Hou G, Li X, Li L. Serum cyclin-dependent kinase 9 is a potential biomarker of atherosclerotic inflammation. Oncotarget. 2016; 7:1854–62. Xu Y, Ma X, Ni W, Zheng L, Lin Z, Lai Y, Yang N, Dai Z, Yao T, Chen Z et al . PKM2-Driven Lactate Overproduction Triggers Endothelial-To-Mesenchymal Transition in Ischemic Flap via Mediating TWIST1 Lactylation. Adv Sci (Weinh). 2024; 11:e2406184. Yu X, He T, Tong Z, Liao L, Huang S, Fakhouri WD, Edwards DP, Xu J. Molecular mechanisms of TWIST1-regulated transcription in EMT and cancer metastasis. EMBO Rep. 2023; 24:e56902. Tu M, Ge B, Li J, Pan Y, Zhao B, Han J, Wu J, Zhang K, Liu G, Hou M et al . Emerging biological functions of Twist1 in cell differentiation. Dev Dyn. 2025; 254:8–25. Wang Y, Yang K, Jiang G, Zheng X, Cheng F, Li W. Twist1 promoting neointima hyperplasia after vascular injury in diabetes via regulating hyperglycemia-induced phenotype switching of vascular smooth muscle cells. Atherosclerosis. 2025; 409:120496. Ren J, Xiao X, Li R, Lv C, Zhang Y, Wang L, Hong T, Zhang H, Wang Y. Single-cell sequencing reveals that endothelial cells, EndMT cells and mural cells contribute to the pathogenesis of cavernous malformations. Exp Mol Med. 2023; 55:628–42. Fan Y, Gu X, Zhang J, Sinn K, Klepetko W, Wu N, Foris V, Solymosi P, Kwapiszewska G, Kuebler WM. TWIST1 Drives Smooth Muscle Cell Proliferation in Pulmonary Hypertension via Loss of GATA-6 and BMPR2. Am J Respir Crit Care Med. 2020; 202:1283–96. Yang G, Zhang Z, Ma X, Chen J, Shi H, Yang J, Han Q. Role of Carvedilol in Inhibiting the Proliferation and Migration of Vascular Smooth Muscle Cells by Upregulating microRNA-145 Expression. Physiol Res. 2025; 74:577–88. Additional Declarations No competing interests reported. Supplementary Files supplementarymaterialsCDK920251113.docx Cite Share Download PDF Status: Posted Version 1 posted 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. 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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-8452448","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":570147899,"identity":"2cb32c82-f58e-4044-bcb1-b2dd427fcac7","order_by":0,"name":"Liqing Yu","email":"","orcid":"","institution":"Xiamen Cardiovascular Hospital of Xiamen University, School of Medicine, Fujian Branch of National Clinical Research Center for Cardiovascular Diseases","correspondingAuthor":false,"prefix":"","firstName":"Liqing","middleName":"","lastName":"Yu","suffix":""},{"id":570147900,"identity":"707fa6e9-7ff9-4acd-914d-80f1a08ef01d","order_by":1,"name":"Xiaoxia Zhou","email":"","orcid":"","institution":"Xiamen Cardiovascular Hospital of Xiamen University, School of Medicine, Fujian Branch of National Clinical Research Center for Cardiovascular Diseases","correspondingAuthor":false,"prefix":"","firstName":"Xiaoxia","middleName":"","lastName":"Zhou","suffix":""},{"id":570147901,"identity":"0f919673-a878-4e54-a7ed-4f5b1bb75d66","order_by":2,"name":"Jiafeng Chen","email":"","orcid":"","institution":"Xiamen Cardiovascular Hospital of Xiamen University, School of Medicine, Fujian Branch of National Clinical Research Center for Cardiovascular Diseases","correspondingAuthor":false,"prefix":"","firstName":"Jiafeng","middleName":"","lastName":"Chen","suffix":""},{"id":570147902,"identity":"8dfb9287-0be7-4022-800b-eb128adff9b1","order_by":3,"name":"Mengxiang Li","email":"","orcid":"","institution":"Xiamen Cardiovascular Hospital of Xiamen University, School of Medicine, Fujian Branch of National Clinical Research Center for Cardiovascular Diseases","correspondingAuthor":false,"prefix":"","firstName":"Mengxiang","middleName":"","lastName":"Li","suffix":""},{"id":570147903,"identity":"c29453e0-29de-452a-9b38-ad46d651173b","order_by":4,"name":"Ruiying Wang","email":"","orcid":"","institution":"Xiamen Cardiovascular Hospital of Xiamen University, School of Medicine, Fujian Branch of National Clinical Research Center for Cardiovascular Diseases","correspondingAuthor":false,"prefix":"","firstName":"Ruiying","middleName":"","lastName":"Wang","suffix":""},{"id":570147904,"identity":"f6698ade-2ebf-4198-a97a-ba28f56b9228","order_by":5,"name":"Gang Li","email":"","orcid":"","institution":"Xiamen Cardiovascular Hospital of Xiamen University, School of Medicine, Fujian Branch of National Clinical Research Center for Cardiovascular Diseases","correspondingAuthor":false,"prefix":"","firstName":"Gang","middleName":"","lastName":"Li","suffix":""},{"id":570147905,"identity":"e79f5d4c-4330-4791-9e48-79a10e03974f","order_by":6,"name":"Jun Wu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0klEQVRIiWNgGAWjYHCCBAaGChs5fgiHmVgtZ9KMJRtI0MLAwNh2OHHDAWK1GNxIeCZdwMacuPlGdpoEQ4V1YgP72QOEtKRJz+BhM9525uw2CYYz6YkNPHkJeLWYgbTwSPDIbjveu00C5MIGCR4DIrQYSDBubuYFavlHtJYEA8UN7CBbGojQYn/mQbI1z4EEY4kzZzdbJBxLN27jycGvRbI9J/E277//cvwzcjfe+FBjLdvPfga/FgYGngQEG8RkI6AeCNgPEFYzCkbBKBgFIxsAAJSsQ/hOM+3yAAAAAElFTkSuQmCC","orcid":"","institution":"Xiamen Cardiovascular Hospital of Xiamen University, School of Medicine, Fujian Branch of National Clinical Research Center for Cardiovascular Diseases","correspondingAuthor":true,"prefix":"","firstName":"Jun","middleName":"","lastName":"Wu","suffix":""}],"badges":[],"createdAt":"2025-12-26 06:08:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8452448/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8452448/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":99692427,"identity":"473136d0-2192-4a19-934f-665d8561941d","added_by":"auto","created_at":"2026-01-07 10:35:24","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":21183902,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe expression and activity of CDK9 are induced in VSMCs during neointimal hyperplasia. (A) \u003c/strong\u003eC57BL/6 mice were subjected to carotid artery ligation, and representative images of hematoxylin and eosin (HE)-stained carotid arteries were taken at 2, 3, and 4 weeks post-injury. Scale bar, 50 μm, n=7/group. \u003cstrong\u003e(B and C)\u003c/strong\u003eRepresentative images of immunofluorescence staining and quantitative analysis for CDK9 and α-SMA in the carotid arteries at indicated time after injury. Scale bar, 50 μm, n=7/group.\u003cstrong\u003e (D and E)\u003c/strong\u003eRepresentative images of immunofluorescence staining and quantitative analysis for p-Ser2 and α-SMA in the carotid arteries at indicated time after injury. Scale bar, 50 μm, n=6/group.\u003cstrong\u003e (F and G)\u003c/strong\u003e Western blots analysis and quantification of p-Ser2 and CDK9 level in RCAs (control) and LCAs of C57BL/6 mice at 4 weeks post-injury.\u003cstrong\u003e \u003c/strong\u003eThe protein expression level was normalized to GAPDH. n=3/group.\u003cstrong\u003e(H)\u003c/strong\u003e qPCR analysis was performed to detect the mRNA level of CDK9 and α-SMA in VSMCs after treatment with PDGF-BB or Rapamycin for 24h. \u003cstrong\u003e(I and J)\u003c/strong\u003eWestern blot analysis and quantification of p-Ser2, PoII (RNA polymerase II), KLF4, CDK9, and α-SMA in VSMCs after treatment with PDGF-BB or Rapamycin for 24h. The protein expression level was normalized to GAPDH.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8452448/v1/4bba4edf32ccf2b7168ccf2a.png"},{"id":99692424,"identity":"81e4c1e1-2a81-477c-a296-217efcd0f565","added_by":"auto","created_at":"2026-01-07 10:35:24","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":11506464,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCDK9 selective inhibitor iCDK9 represses neointima formation following vascular injury. (A) \u003c/strong\u003eSchematic workflow for iCDK9 treatment in vivo. \u003cstrong\u003e(B and C) \u003c/strong\u003eRepresentative images of HE-stained carotid arteries of mice 21 d after injury in the vehicle control group or iCDK9 treatment group(B), and quantification analysis of the ratios of intima to media(C). Scale bar, 50 μm, n=7/group.\u003cstrong\u003e (D and E)\u003c/strong\u003e Carotid arteries on day 21 post-injury with or without iCDK9 treatment were subjected to immunofluorescence to test contractile marker α-SMA(D), and the relative intensity of α-SMA was quantified (E). Scale bar, 50 μm, n=5/group.\u003cstrong\u003e (F and G) \u003c/strong\u003eRepresentative immunofluorescence staining and quantification of the relative intensity of p-Ser2 in carotid arteries on day 21 post-injury with or without iCDK9 treatment. Scale bar, 50 μm, n=7/group.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8452448/v1/648aec743a4c2435f466c861.png"},{"id":99692425,"identity":"0f1373ae-3b0b-4357-906c-9e6fa50b80de","added_by":"auto","created_at":"2026-01-07 10:35:24","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":12009110,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCDK9 regulates the proliferation and migration of smooth muscle cells. (A) \u003c/strong\u003eRepresentative images of the scratch assay in VSMCs as indicated. Scale bar, 20 μm. \u003cstrong\u003e(B) \u003c/strong\u003eThe quantification results for the wound closure assay.\u003cstrong\u003e (C-D)\u003c/strong\u003e VSMCs were subjected to Edu incorporation assay as indicated, and representative images are presented.\u003cstrong\u003e \u003c/strong\u003eScale bar, 20 μm.\u003cstrong\u003e \u003c/strong\u003eQuantification of Edu-positive cells was measured by ImageJ software. \u003cstrong\u003e(E and F)\u003c/strong\u003e Western blot analysis and quantification of PCNA and CDK9 protein expressions in VSMCs treated with control or CDK9 siRNA. The protein expression level was normalized to GAPDH. \u003cstrong\u003e(G and H) \u003c/strong\u003eWestern blot analysis and quantification of PCNA and CDK9 protein expressions in VSMCs 48 h after infection of Ad-Control or Ad-CDK9.\u003cstrong\u003e (I) \u003c/strong\u003eRepresentative cross‑sections of immunohistochemically stained Ki67 (brown) in carotid arteries 21 d after injury with or without iCDK9 treatment. Scale bar, 50 μm, n=6/group.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8452448/v1/9b5600e152c6e5ae204b17c2.png"},{"id":99796561,"identity":"620947a4-d15f-4c1a-859f-fc05bd4e85aa","added_by":"auto","created_at":"2026-01-08 13:42:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":7129054,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCDK9 inhibition in VSMCs results in an increase of contractile phenotype. (A) \u003c/strong\u003eWestern blots analysis of indicated proteins in VSMCs treated with CDK9 inhibitor iCDK9 at different concentrations for 24 h.\u003cstrong\u003e (B) \u003c/strong\u003eWestern blot analysis of indicated proteins in VSMCs treated with CDK9 degrader Thal at different concentrations for 24 h. \u003cstrong\u003e(C) \u003c/strong\u003eVSMCs were treated with PDGF-BB in the presence or absence of iCDK9 for 24 h, then subjected to western blot analysis to detect the contractile markers and synthetic markers. \u003cstrong\u003e(D and E) \u003c/strong\u003eAfter treatment with PDGF-BB with/without iCDK9 or Thal for 24 h, the indicated mRNA level in VSMCs was measured by qRT-PCR. \u003cstrong\u003e(F) \u003c/strong\u003eContractile markers and synthetic markers were detected by western blot analysis after VSMCs were treated with PDGF-BB in the presence or absence of iCDK9 for 24 h.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(G) \u003c/strong\u003eRepresentative immunofluorescence staining of α-SMA in VSMCs after treatment with PDGF-BB in the presence of iCDK9 or Thal. Scale bar, 10 μm.\u003cstrong\u003e (H) \u003c/strong\u003eWestern blot analysis of SM22a, CNN1, OPN, Vimentin and CDK9 protein expressions in VSMCs treated with control or CDK9 siRNA for 48 h.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8452448/v1/c6afd54ad79c2ee975be3d1b.png"},{"id":99692420,"identity":"a880255d-2c64-424d-9c6c-4861a8e47a01","added_by":"auto","created_at":"2026-01-07 10:35:24","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3959894,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTwist1 is a potential target of CDK9 for regulating phenotypic transformation of smooth muscle cells. (A)\u003c/strong\u003e RNA sequencing was performed after VSMCs treated with PDGF-BB, Rapamycin or iCDK9 for 24 h. Venn diagram showing changes of transcripts with a rigorous criteria of absolute log 2 FC≥2 and p-value\u0026lt;0.05. \u003cstrong\u003e(B) \u003c/strong\u003eHeatmap of the 367 genes with significant changes in all the PDGF-BB, Rapmycin and iCDK9 groups. \u003cstrong\u003e(C)\u003c/strong\u003e Four transcription-related genes, including Twist1, Helz2, Jun, and Mef2c, among the 96 genes that markedly increased in the PDGF-BB group but decreased in both the Rapamycin and iCDK9 groups. \u003cstrong\u003e(D) \u003c/strong\u003eThe relative mRNA levels of Twist1 and SM22a were confirmed by qRT-PCR. \u003cstrong\u003e(E-F) \u003c/strong\u003eWestern blot analysis and quantification of Twist1 protein expression in VSMCs after treated with indicated agents for 24 h. The protein expression level was normalized to GAPDH. \u003cstrong\u003e(G and H) \u003c/strong\u003eWestern blot analysis and quantification of Twist1 and CDK9 protein expressions in VSMCs after infected with Adv-control or Adv-CDK9. The protein expression level was normalized to GAPDH. \u003cstrong\u003e(I and J) \u003c/strong\u003eWestern blot analysis and quantification of Twist1 and CDK9 protein expressions in VSMCs after transfected with siNC (negative control) or siCDK9. The protein and mRNA expression levels were normalized to GAPDH.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8452448/v1/1ee875b35a78d00efbc6e61b.png"},{"id":99796435,"identity":"e9af6e38-4100-4a4e-a265-98a02ec8e71a","added_by":"auto","created_at":"2026-01-08 13:41:46","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":5777945,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibition of Twist1 alleviates the promoting effect of CDK9 on proliferation, migration, and phenotypic switching of VSMCs. (A-B)\u003c/strong\u003e Cell migratory capacity was examined by the scratch assay in VSMCs as indicated and representative images were shown. Scale bar, 20 μm. The quantification results for the wound closure assay were measured using ImageJ.\u003cstrong\u003e (C-D)\u003c/strong\u003e Representative images and quantification of Edu-positive cells were shown in VSMCs as indicated. Scale bar, 20 μm.\u003cstrong\u003e (E-F) \u003c/strong\u003eVSMCs were infected with Adv-Ctrl or Adv-CDK9; 24 h later, cells were treated with Harmine for another 24 h, then whole cell lysates were subjected to western blot analysis to test the expression level of PCNA and CDK9. The quantification results of PCNA after Adv-CDK9 infection with or without Harmine treatment were measured via ImageJ.\u003cstrong\u003e(G)\u003c/strong\u003e VSMCs were treated with indicated agents for 24 h, then western blot analysis was performed to detect the protein levels of OPN, PCNA, α-SMA, CNN1 and SM22a. \u003cstrong\u003e(H-J) \u003c/strong\u003eThe mRNA levels of α-SMA, SM22a and CNN1 in VSMCs after treated as indicated for 24 h were determined by qRT-PCR.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-8452448/v1/fa1ff96338ddd3b06b5ac3d0.png"},{"id":99805286,"identity":"f0e20347-2389-480a-9641-efe3fe12c34b","added_by":"auto","created_at":"2026-01-08 14:16:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":61395237,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8452448/v1/cc88a74e-6ac3-425c-8870-c0e14aa48e52.pdf"},{"id":99692422,"identity":"9adb5277-7dcb-44da-b4e4-f0d14d64a41e","added_by":"auto","created_at":"2026-01-07 10:35:24","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":179289,"visible":true,"origin":"","legend":"","description":"","filename":"supplementarymaterialsCDK920251113.docx","url":"https://assets-eu.researchsquare.com/files/rs-8452448/v1/03250b23dbf0f04583d23c6d.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"CDK9 regulates neointima formation following vascular injury via targeting transcription factor Twist1","fulltext":[{"header":"Background","content":"\u003cp\u003eCardiovascular diseases are the leading cause of human morbidity and mortality worldwide[1-3]. In recent decades, the incidence of vascular stenosis\u0026nbsp;related diseases especially coronary heart disease, has been increasing greatly and seriously threatening human health[4-7]. Clinically, percutaneous coronary intervention (PCI) is one well established and effective method for the treatment of coronary artery disease[8-11]. But unfortunately, months after PCI surgery, vascular restenosis is currently a common complication, which may narrow the newly opened coronary arteries again and re-block the blood supply to the myocardium[12-14]. Repeated interventional treatments are required, which increase the physical and economic burden on patients, so restenosis is one of the big challenges of PCI surgery in the clinic. Previous studies have reported that the neointima formation primarily driven by excessive proliferation and migration of vascular smooth muscle cells (VSMCs), plays a vital role in restenosis[15-17].\u003c/p\u003e\n\u003cp\u003eVascular smooth muscle cells (VSMCs) are a major component of blood vessels and play an important role in maintaining the physiological functions of blood vessels[18]. VSMCs are not terminally differentiated and exhibit phenotypic and functional plasticity in response to microenvironmental cues[16, 18-20]. Under normal physiological conditions, VSMCs located in the media of the vascular wall exhibit a contractile phenotype and possess low proliferative and migratory capacities[21, 22]. The quiescent contractile phenotype of VSMCs is characterized by abundant expression of contractile phenotype-associated genes, such as myocardin (MyoC), CNN1, α-smooth muscle actin(α-SMA), transgelin (SM22α), and MYH11[23, 24]. In response to vascular injury or some other pathological stimuli, VSMCs switch from a contractile phenotype to a dedifferentiated synthetic phenotype, which is characterized by a significant decrease of contractile gene expression and high proliferative and migratory abilities[25-27]. The synthetic VSMCs proliferate and migrate to the vascular intima, accompanied by massive secretion and deposit of extracellular matrix (ECM), resulting in continuous production of neointima[28-30]. Therefore, the phenotypic switching of VSMCs plays vital roles in neointima formation and vascular stenosis, and deciphering the molecular mechanisms governing VSMC phenotypic switch is important to the development of novel avenues for interventions in vascular remodeling after injury.\u003c/p\u003e\n\u003cp\u003eAs a key component of the positive transcription elongation factor b (P-TEFb) complex, Cyclin-dependent kinase 9 (CDK9) differs from other CDK family members in that it has little effects on regulating the cell cycle[31, 32]. Instead, it mainly regulates gene transcription through phosphorylating the C-terminal domain (CTD) of RNA polymerase II\u0026nbsp;(RNA pol II) at Ser2, which is critical for RNA synthesis[33, 34]. The function of CDK9 in cancer and AIDS has been well-studied, recently an increasing number of studies have found that CDK9 is also involved in cardiovascular diseases[35-40]. For example, under pro-hypertrophic stimuli, CDK9 can be recruited to promotor regions of hypertrophic genes by other transcription factors such as BRD4 and p300/GATA4 complex, then increase the phosphorylation of RNA Pol II Ser2 (pSer2) and facilitate the transcription level of target genes, leading to\u0026nbsp;cardiomyocyte proliferation and cardiac hypertrophy[35]. Additionally, the elevation of CDK9 in patients’ serum with atherosclerosis (AS) or pulmonary arterial hypertension (PAH) has been observed[40-42]. Though several studies have explored that CDK9 can promote the development of AS and PAH by regulating the proliferation and phenotypic switching of VSMCs, its molecular mechanisms are largely unknown and whether CDK9 is involved in neointima formation after vascular injury has not been studied.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;In this study, we explore the effect and underlying mechanism of CDK9 on neointima hyperplasia induced by vascular injury in vitro and in vivo. Our results suggest that CDK9 expression and its substrate pSer2 are positively correlated with human carotid arterial stenosis. CDK9 inhibitor iCDK9 significantly inhibits neointima formation in mice after vascular injury. Genetic knockdown and pharmacological inhibition of CDK9 resulted in a significant increase of VSMC proliferation/migration and phenotypic switching. Moreover, we identified transcription factor Twist1 as a CDK9 target gene: inhibition of Twist1 by Harmine represses the effect of CDK9 on the proliferation/migration and phenotypic switching of VSMCs. The findings of this study expand our understanding of neointimal hyperplasia and provide novel insight into the development of therapeutic strategies for restenosis.\u003c/p\u003e"},{"header":"Results ","content":"\u003cp\u003e\u003cstrong\u003eCDK9 expression and activity was induced in VSMCs during neointimal hyperplasia\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn order to investigate the role of CDK9 in neointimal formation, a mouse carotid artery injury model was established through ligation initially. As shown in Figure1A, neointimal formation gradually increased over two weeks after injury (Figure 1A). Interestingly, after carotid artery ligation in mice, the CDK9 expression was induced in a time-dependent manner in response to the injure, which was accompanied by a significant decrease of the contractile VSMC marker α-SMA (Figure 1B-C). Additionally, as a substrate of CDK9 kinase, the p-Ser2 level of RNA poll was obviously increased after injury (Figure 1D-E). Furthermore, western blot analysis confirmed the significant increase of CDK9 expression and p-Ser2 level at the sites of injury at 28 days after ligation compared with the non-ligated vessels\u0026nbsp;(Figure 1F-G).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs shown in Figure 1B, CDK9 was\u0026nbsp;largely co-localized with α-SMA-positive VSMC. To determine whether the increase of CDK9 expression and p-Ser2 level was due to the VSMC phenotypic modulation in the process of injury-induced neointimal formation, human aortic SMCs (HA-VSMCs) were treated with PDGF-BB and Rapamycin, respectively. As expected, the α-SMA expression decreased after PDGF-BB treatment, but increased in the Rapamycin group, whereas both the mRNA and protein levels of CDK9 elevated obviously after PDGF-BB treatment, and Rapamycin inhibited the CDK9 expression level (Figure 1H-J). Meanwhile, the change of p-Ser2 level was consistent with that of CDK9(Figure 1I-J). And the same results were observed in rat-SMCs (Figure S1). Taken together, these data indicate that CDK9 is involved in the neointimal formation after vascular injury and VSMC phenotypic switching.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInhibition of CDK9 alleviates neointimal formation following vascular injury\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn order to confirm the role of CDK9 in neointimal formation, the specific CDK9 inhibitor CDK9-IN-2 (iCDK9) was employed in the vascular injury model. As shown in Figure 2A, mice about 10 weeks old were subjected to ligation of the left carotid artery, then iCDK9 (5 or 10mg/kg) was administered via intraperitoneal injection daily three days after the surgery. The HE staining results showed that three weeks after the ligation, the intimal hyperplasia was obvious, which can be alleviated by iCDK9 treatment in a dose-dependent manner with a significant decrease in the intimal area and a lower intima/media ratio (Figure 2B-C).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFurthermore, immunohistochemical assays for contractile VSMC marker α-SMA showed that the decrease of α-SMA expression after vascular injury could be repressed by iCDK9 treatment (Figure 2D-E). As expected, the p-Ser2 level in ligated vascular was decreased greatly after iCDK9 treatment (Figure 2F-G).\u0026nbsp;Taken together, these data suggest that iCDK9 can relieve neointimal hyperplasia after vascular injury in a phosphatase activity-dependent manner.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCDK9 regulates the proliferation and migration of VSMCs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs VSMC proliferation and migration are key processes in neointimal formation following arterial injury. So next, we aim to determine whether the effects of CDK9 on neointimal formation were due to the regulation of VSMC proliferation and migration. Firstly, we examined the potential role of CDK9 in PDGF-BB-induced cell migration and proliferation in vitro models. In order to evaluate the effect of CDK9 on VSMC migration, we performed scratch‑wound‑ healing assays. It is well known that cell motility of VSMCs can be promoted by PDGF-BB stimulation. The results revealed that iCDK9, or the selective CDK9 degrader Thal significantly retarded wound closure, under both quiescent and PDGF-BB-stimulated conditions (Figure 3A-B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eProliferationof VSMCs is another key mechanism in the process of\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eneointimal formation. Next, we evaluated the regulation of CDK9 on VSMC proliferation. To this end, PDGF-BB-stimulated VSMCs were subjected to Edu incorporation assays. After PDGF-BB stimulation, the Edu-positive cells increased greatly; however, it can be inhibited by CDK9 inhibitors iCDK9 or degrader Thal (Figure 3C-D). To further verify the regulation of CDK9 on proliferation of VSMCs, CDK9 was knocked down in VSMCs by siRNA; the result showed that after CDK9 knockdown, the expression levels of the cell proliferation marker PCNA protein were significantly decreased (Figure 3E-F and Figure S2). However, when CDK9 was overexpressed by adenovirus infection, the level of PCNA protein increased obviously in VSMCs (Figure 3G-H). We further evaluated VSMC proliferation in vivo, and another cell proliferation marker Ki67 was tested by immuno-histochemistry (IHC). The expression level of Ki67 was higher in left carotid arteries after 3 weeks of ligation than in the unligated right carotid arteries (Sham); however, inhibition of the CDK9 activity by iCDK9 significantly repressed Ki67 expression levels in the left carotid artery compared with that in the negative control group (Figure 3I). Taken together, these results indicate that inhibition or deficiency of CDK9 in VSMCs repressed their migration and proliferation, thereby alleviating the injury-induced neointimal formation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInhibition of CDK9 can suppress phenotypic switching of VSMCs in vitro\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe phenotype switching of VSMCs, from the differentiated contractile state to the dedifferentiated synthetic state, plays an important role in neointimal formation after vascular injury. Consistent with the inhibition effect of iCDK9 on neointimal formation in vivo, iCDK9 up-regulated basal levels of VSMC contractile marker α-SMA and down-regulated VSMC synthetic marker OPN and\u0026nbsp;proliferation marker PCNA in VSMCs (Figure 4A). Similar result was observed when the CDK9 selective degrader Thal was employed in VSMCs. Accompanied by the decrease of CDK9 protein level after Thal treatment, contractile markers increased, but synthetic markers decreased obviously (Figure 4B). As an important dedifferentiation factor, PDGF-BB can stimulate VSMCs to undergo phenotypic switching, which is manifested as a decrease of differentiation marker genes and an increase of synthetic markers. As expected, PDGF-BB\u0026nbsp;significantly increased cell proliferation marker PCNA and synthetic markers but decreased the contractile markers;\u0026nbsp;however, these changes were attenuated by iCDK9 (Figure 4C-D).\u0026nbsp;Besides, CDK9 degrader Thal also\u0026nbsp;significantly\u0026nbsp;inhibits the changes of protein expression related to VSMCs phenotypic switching induced by PDGF-BB (Figure 4E-F). In addition, immunofluorescence assays further demonstrated that iCDK9 or Thal obviously increased the basal expression level of α-SMA, and inhibited PDGF-BB-induced VSMC phenotypic switching (Figure 4G). We confirmed these results by RNAi-mediated CDK9 knockdown experiments in HA-VSMCs. The contractile phenotype-related proteins such as SM22a and CNN1 increased, whereas the synthetic markers such as Vimentin and OPN decreased in CDK9 siRNA-treated HA-VSMCs (Figure 4H). Taken together, our data suggest that CDK9 plays an important role in mediating phenotypic switching of VSMCs, and intervention of CDK9 can effectively inhibit phenotypic switching; thus, CDK9 may serve as a potential target for neointimal formation in vivo.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCDK9 regulates the expression level of Twist1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe next explored the underlying mechanisms of CDK9 in phenotypic modulation of VSMCs. Since it has been reported that the process of VSMC phenotypic switching from the contractile state to the synthetic state can be induced by PDGF-BB and repressed by Rapamycin, VSMCs were treated with PDGF-BB, Rapamycin or iCDK9, respectively. Subsequently, we performed whole transcriptome analysis using RNA sequencing. 367 genes synchronously altered significantly under various treatment conditions were chosen for further analysis (Figure 5A). From previous results in our study, iCDK9 can inhibit the phenotypic switching of VSMCs as Rapamycin does, so the genes that exhibit consistent change trends in both the iCDK9 group and the Rapamycin group, yet show opposite trends to the PDGF-BB group, are considered as potential candidates for our further research. Cluster analysis of these 367 genes showed that only 96 genes significantly increased in the PDGF-BB group, but decreased in both the iCDK9 and Rapamycin groups (Figure 5B). As a key component of the positive transcription factor P-TEFb complex, CDK9 mainly regulates the transcription process of intracellular genes; thus, we focus on the changes of transcription-related proteins. GO analysis of the 96 selected genes revealed that 4 of them are transcription-related genes, including Twist1, Helz2, Jun, and Mef2c (Figure 5C).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs an important transcription factor, Twist1 regulates multiple biological processes, including epithelial-mesenchymal transition (EMT), cell proliferation, migration, and extracellular matrix remodeling[43-45]. Recent studies reported that Twist1 can induce VSMC proliferation and phenotypic switching[46-48]. Therefore, we speculate whether CDK9 promotes the phenotypic switching of smooth muscle by regulating the expression of Twist1. To this end, we first validated the RNA-seq results using qRT-PCR and found that, consistent with the sequencing results, the mRNA level of Twist1 was increased in the PDGF-BB group but decreased in the Rapamycin and iCDK9 groups significantly (Figure 5D). Subsequently, the protein level of Twist1 was detected, and the results showed that its change trend was consistent with that of the mRNA level (Figure 5E-F). To further verify that CDK9 can directly regulate the gene level of Twist1, CDK9 was overexpressed in VSMCs via adenovirus infection, and the protein level of Twist1 increased significantly after CDK9 overexpression (Figure 5G-H).\u0026nbsp;This funding was further supported by siCDK9; western blotting showed that in contrast to overexpression, down regulation of CDK9 by siCDK9 decreased the protein level of Twist1 in VSMCs (Figure 5I-J). Above all, the results indicated that CDK9 can regulate the expression level of Twist1 in VSMCs, which may contribute to its modulation in vascular remodeling.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHarmine alleviates the promoting effect of CDK9 on proliferation, migration, and phenotypic switching of VSMCs\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine whether Twist1 is required in the CDK9-regulated proliferation and migration of VSMCs, growth and motility were examined. After being infected with adenovirus for 24 h, VSMCs were treated with the Twist1 inhibitor Harmine for another 24 h. We found that the wound healing (scratch assay) of VSMCs was significantly promoted by CDK9 overexpression, but this effect was almost abolished in the presence of Harmine (Figure 6A-B). The inhibitory effect of Harmine on VSMC proliferation was further confirmed by EdU incorporation analysis. Consistent with the wound healing assay results, the number of EdU-positive cells increased significantly following CDK9 overexpression, but this increase could be suppressed by Harmine, indicating Twist1 mediates CDK9-regulated VSMC proliferation (Figure 6C-D). Consistently, overexpression of CDK9 leads to a significant increase in the expression of the proliferation marker PCNA; however, treatment with Harmine can reverse this effect, resulting in the inhibition of PCNA expression (Figure 6E).\u003c/p\u003e\n\u003cp\u003eTo further investigate the role of Twist1 in CDK9-regulated phenotypic switching, the inhibition of Harmine under PDGF-BB stimulation was explored. PDGF-BB plays a key role in the phenotypic regulation of VSMCs; as expected treatment with either the CDK9 inhibitor (iCDK9) or Harmine alone could inhibit PDGF-BB-induced phenotypic switching (Figure 6F). Interestingly, Harmine could further enhance the inhibitory effect of iCDK9 on PDGF-BB-induced phenotypic switching, leading to a further significant decrease in the expression of contractile markers, including α-SMA, SM22a, and CNN1, and an increase in the expression of the proliferative marker PCNA and synthetic marker OPN in VSMCs (Figure 6G). The enhancing effect of Harmine on iCDK9-mediated inhibition of VSMCs phenotypic switching was further confirmed by qRT-PCR. Following treatment with iCDK9, the PDGF-BB-induced reduction in contractile phenotype markers such as α-SMA, SM22a, and CNN1 was reversed; additionally, the introduction of Harmine led to a further increase in the expression of these contractile phenotype markers (Figure 6H-J). Herein, results from Harmine treatment in the iCDK9-treated group and the CDK9 overexpression group showed that Harmine is capable of inhibiting CDK9-regulated proliferation, migration, and phenotypic switching in VSMCs, and Twist1 serves as the critical target mediating CDK9’s regulatory effects on VSMCs.\u003c/p\u003e"},{"header":"Discussion ","content":"\u003cp\u003eRestenosis remains a major clinical challenge limiting the long-term efficacy of percutaneous coronary intervention (PCI), with neointimal formation driven by abnormal vascular smooth muscle cell (VSMC) proliferation, migration, and phenotypic switching being its core pathological feature[5] [16, 49]. In this study, we systematically investigated the role and molecular mechanism of cyclin-dependent kinase 9 (CDK9) in vascular injury-induced neointimal hyperplasia, and our findings identify CDK9 as a pivotal regulator of this process, with transcription factor Twist1 serving as its key downstream effector.\u003c/p\u003e\n\u003cp\u003eCDK9 is well-characterized for its role in regulating RNA polymerase II (RNA pol II)-dependent gene transcription by phosphorylating the Ser2 residue of RNA pol II’s C-terminal domain (CTD, pSer2)[31, 34]. While previous studies have linked CDK9 to cardiovascular diseases such as atherosclerosis (AS) and pulmonary arterial hypertension (PAH)[40, 41], its involvement in post-injury neointimal formation remained unaddressed. Our study fills this gap by demonstrating that CDK9 expression and activity (reflected by pSer2 levels) are significantly upregulated in VSMCs during mouse carotid artery ligation-induced neointimal hyperplasia (Figure 1). This upregulation is not merely correlative: pharmacological inhibition of CDK9 with CDK9-IN-2 (iCDK9) dose-dependently reduces neointimal area and the intima/media ratio in vivo (Figure 2). These results align with recent reports that CDK9 regulates VSMC function in AS[41] , but extend this knowledge by establishing CDK9 as a direct contributor to the neointimal response to mechanical vascular injury-the primary trigger of PCI restenosis.\u003c/p\u003e\n\u003cp\u003eA key insight from our in vitro studies is that CDK9 modulates multiple VSMC behaviors critical for neointimal formation. This is particularly significant because phenotypic switching of VSMCs from a quiescent, contractile state to a proliferative, synthetic state is an early and rate-limiting step in neointimal formation[28, 31]. Our data thus suggest that CDK9 promotes neointimal hyperplasia by coordinatey enhancing VSMC proliferation, migration, and dedifferentiation, three interrelated processes that drive vascular remodeling after injury.\u003c/p\u003e\n\u003cp\u003eTo unravel the molecular mechanism by which CDK9 regulates VSMC function, we identified Twist1 as the most promising target, as it is a well-known inducer of epithelial-mesenchymal transition (EMT) and has been linked to VSMC proliferation and phenotypic switching in cardiovascular disease[26, 28]. Furthermore, our validation experiments confirmed that Twist1 is a direct downstream target of CDK9, and the results establish a CDK9-Twist1 axis that mediates VSMC dysfunction during neointimal formation. This mechanism expands our understanding of CDK9’s transcriptional targets in vascular biology. Prior studies focused on its role in cardiomyocyte hypertrophy via genes like GATA4[35], but our work identifies Twist1 as a key mediator of CDK9’s effects in VSMCs.\u003c/p\u003e\n\u003cp\u003eThe clinical relevance of our findings lies in their potential to address PCI restenosis. Current strategies for restenosis prevention (e.g., drug-eluting stents) have limitations, including late stent thrombosis and incomplete inhibition of neointimal growth[12, 13]. Our study shows that targeting CDK9 (e.g., with iCDK9) effectively reduces neointimal formation in mice, suggesting that CDK9 inhibitors could be developed as novel therapeutics for restenosis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHowever, this study has several limitations. First, our in vivo experiments were conducted exclusively in male mice, and future studies should include female mice to assess potential sex differences, as sex is a known modifier of cardiovascular disease[4]. Second, due to the crucial role of CDK9 in the intestinal tract, it has been challenging to obtain (SMC)-specific CDK9 deficiency (CDK9-SMC-KO) mice. Therefore, it is impossible to study the regulatory role of CDK9 in intimal hyperplasia using genetically engineered mouse models.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, our study demonstrates that CDK9 is upregulated in VSMCs after vascular injury and promotes neointimal formation by enhancing VSMC proliferation, migration, and phenotypic switching via the transcription factor Twist1. These findings identify the CDK9-Twist1 axis as a novel therapeutic target for preventing PCI-related restenosis and provide a new framework for understanding the molecular mechanisms of vascular remodeling.\u003c/p\u003e"},{"header":"Materials and Methods ","content":"\u003cp\u003e\u003cstrong\u003eMaterials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe CDK9 inhibitor CDK9-IN-2 (HY-16462), CDK9 degrader THAL-SNS-032(HY-123937) and Rapamycin (HY-10219) were from Med Chem Express. Human PDGF-BB protein (220-bb-010) was obtained from R\u0026amp;D Systems. Tamoxifen (T5648) was purchased from Sigma-Aldrich.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animals were maintained and used in accordance with the guidelines of the Institutional Animal Care and Use Committee of Xiamen University (approval no. XMULAC20250055). All mice were housed in the specific pathogen-free laboratory with a 12-h light/12-h dark cycle at 22 to 24\u0026deg; and humidity (55 \u0026plusmn; 5%) with free access to food and water. C57BL/6J mice were purchased from Gem Pharmatec. For iCDK9 treatment in vivo, 10-week-old male mice were subjected to carotid artery ligation and three days later followed by 18-day iCDK9 oral gavage regimen as shown in Figure 2A. After the last administration, mice were euthanized and carotid arteries were collected and subjected to the following experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMouse carotid artery ligation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOnly male mice were used in the study. For all surgical procedures, 10-week-old male mice were anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg, Sigma-Aldrich, Saint Louis, MO, USA). Then the left carotid artery (LCA) was carefully dissected and completely ligated at the proximal end of the carotid bifurcation with a 5-0 silk suture. A similar procedure without ligation on the right carotid artery (RCA) was used as a sham operation group. After 14, 21, or 28 d after surgery, carotid arteries were collected and subjected to the following experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence staining and Immunohistochemistry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMice were euthanized by decapitation under isoflurane anesthesia, then carotid arteries were perfused and embedded in the OCT agent (4583, SAKURA). Frozen sections of 5 \u0026micro;m thickness and cells grown on glass coverslips were fixed with 4% paraformaldehyde (PFA) for 10 min after being washed with PBS for three times and permeabilized with 0.1% Triton X-100 in PBS for 5 min. The cells were blocked in 1% goat serum and incubated with the following primary antibodies at 4℃\u0026nbsp;overnight: anti-\u0026alpha;-SMA (BM0002, BOSTER Biological Technology), anti-CDK9 (ab239364), and anti-pSer2 (ab5095). After incubation of second antibodies (A-11012 or A-21235, Invitrogen) for 1h at room temperature, the nuclei were stained with DAPI (P0131). The confocal immunofluorescence images were acquired by Zeiss confocal laser scanning microscopy. Mouse carotid arteries were collected and fixed with 4% PFA for 24 h before paraffin embedding. Hematoxylin-eosin (HE) staining and IHC for Ki67 were performed by Service-bio (Wuhan, China). Images were acquired with a Tissue FAX microscope (Leica, Germany). Morphometric analysis was performed using ImageJ software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell culture and treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe human aortic smooth muscle cell line HA-VSMCs (HUM-iCell-c010) was purchased from iCell Bioscience (Shanghai, China). HA-VSMCs were cultured with a primary smooth muscle cell culture system (PriMed-iCell-004, iCell Bioscience) in a humidified incubator at 37 \u0026deg;C with 5% CO2. Primary rat vascular smooth muscle cells (rVSMCs) were isolated from the thoracic aorta of rats and routinely cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM, PM150210, procell) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Briefly, male Sprague-Dawley rats (80-100 g) were deeply anesthetized with intraperitoneal injection of sodium pentobarbital (50 mg/kg), and the adventitia and endothelium of fresh thoracic aorta vascular tissue were removed. The medial layer was cut into small pieces and cultured in DMEM containing 20% FBS. After VSMCs sprouted from the tissue block, the medium was replaced with DMEM containing 10% FBS and 1% penicillin-streptomycin. The purity of VSMCs was as identified by a specific marker, \u0026alpha;-SMA (1:500; BM0002, BOSTER Biological Technology). Cells were used for subsequent experiments between passages 4-7. Before treatment with indicated agents, cells were starved with serum-free DMEM for 24h.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative real-time PCR (qRT-PCR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was isolated from cultured cells using Trizol reagent (Cat#: YZ-15596018, Invitrogen) following the manufacturer\u0026rsquo;s protocol. Extracted RNA was reverse transcribed using the Hifair\u0026reg; II 1st Strand cDNA Synthesis kit (Cat#: 11123ES60, Yeasen) to obtain cDNA. Real-time PCR was performed using a quantitative PCR kit (Cat#: 11204ES08, Yeasen) with the primer sequences listed in Supplementary Table S1 and all experiments were conducted in triplicate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell Proliferation Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e5-ethynyl-2\u0026rsquo;-deoxyuridine (EdU) incorporation assay (C0078S; Beyotime Biotechnology, Shanghai, China) was preformed to assess the VSMC proliferation according to the manufacturer\u0026rsquo;s instructions. Briefly, after treatment, cells were incubated with EdU at a final concentration of 10 \u0026micro;M for 4 h before measurement, and total cellular nuclei were stained with Hoechst 33342. The EdU-positive cells were captured with an EVOS FL AUTO2 microscope and measured using ImageJ software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vitro migration assay\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA wound-healing assay was used to measure the migration ability of VSMCs. Briefly, after starvation for 12 h, VSMCs cultured in 12-well plates were scratched with sterile plastic 10 \u0026micro;L micro-pipette tips to draw a straight line in the middle of wells and washed with 1xPBS twice to remove the cellular debris. Then VSMCs were treated with indicated agents for another 24 h and photographs were acquired with an EVOS FL AUTO2 microscope. The migration area was analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, United States).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRecombinant adenovirus construction and transfection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCDK9-overexpressing recombinant adenovirus (Adv-CDK9) and blank control recombinant adenovirus (Adv-Ctrl) were purchased from WZ Biosciences Inc. (Shandong, China). Human CDK9 cDNA was packaged into adenoviral vector pADM-CMV-mCMV-copGFP, and the empty vector was used as a control. HA-VSMCs at a 50\u0026ndash;60% confluence were infected with adenovirus for 24 h and the mediums were changed with a fresh primary smooth muscle cell culture system for another 24 h, then harvested for subsequent experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern Blot Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter treatment, cultured cells were harvested and lysed in RIPA buffer with complete protease inhibitors (Cat#:05892791001, Roche) and phosphatase inhibitors (Cat#:4906837001,Roche), and the supernatant was obtained through centrifugation(12000rpm, 4℃, 10 min). After determining the protein concentration by BCA Protein Assay Kit (A55864, Thermo Fisher Scientific), equal amounts of proteins were separated in SDS-PAGE gels. For tissue extraction, the mouse aorta tissues were excised and homogenized in the RIPA buffer; the supernatant was collected and processed in subsequent experiments in the same manner as cultured cells.\u0026nbsp;The primary antibodies against indicated proteins are listed in Supplementary Table S2.\u0026nbsp;Quantification of WB protein bands was performed using ImageJ software (National Institutes of Health, Bethesda, MD, United States).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSmall interfering RNA transfection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe small interference RNAs (siRNAs) that target human CDK9 and siRNA control were synthesized by Gene Pharma (Shanghai, China) and sequences of siRNA were described in Supplementary Table S3. VSMCs at a 30\u0026ndash;50% confluence were pre-incubated in the transfection medium (Opti-MEM, Cat NO.31985070, Gibco) and then transfected with siRNAs by using Lipofectamine RNAi MAX (Cat NO. 13778150, Invitrogen) according to the instructions provided by the manufacturer. Six hours after transfection, the medium was replaced with fresh serum-free DMEM medium and cultured for another 18 h, then subjected to the following experiment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTranscriptomic profiling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine the potential target of CDK9 in regulating the VSMC phenotypic switching, rat-VSMCs were treated with DMSO (0.1%, as a control), the phenotypic switching activator PDGF-BB, the inhibitor Rapamycin or iCDK9, respectively for 24 h. The total RNA was extracted from the VSMCs using Trizol reagent (15596026, Thermo Fisher Scientific) following the manufacturer\u0026rsquo;s instructions and all RNA samples were analyzed for concentration and purity using a Nanodrop 2000. The RNA-seq transcriptome library was prepared following Illumina\u0026reg; Stranded mRNA Prep Ligation from Illumina (San Diego, CA) using 1\u0026mu;g of total RNA. RNA sequencing was carried out on NovaSeq X Plus(illumina) and the obtained sequencing reads were aligned to the reference Rattus norvegicus genome mRatBN7.2 (https://asia.ensembl.org/Rattus_norvegicus/Info/Index). To identify the significantly differentially expressed genes (DEGs), the expression level of each transcript was calculated according to the transcripts per million reads (TPM) method. RSEM was used to quantify gene abundances. DEGs with a fold change (FC) of \u0026ge;2 and a p-value \u0026lt;0.05 were considered as significantly differently expressed genes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analyses were performed to assess the significance of the differences and correlations observed in the experiments. All data are expressed as means\u0026plusmn;standard deviation (mean\u0026plusmn;SD) and analyzed using GraphPad Prism 10. Statistical analyses were performed using paired or unpaired Student\u0026rsquo;s t-tests (2 groups) or analysis of variance (ANOVA, \u0026gt; 2groups) as appropriate. A p-value \u0026lt; 0.05 was considered to indicate statistically significant differences.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eG.L. and J.W. designed the research and wrote the paper; LQ.Y., XX.Z., JF.C., and MX.L. performed the research; RY.W. analyzed the data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author(s) declare that financial support was received for the research, authorship, and/or publication of this article. This study was supported by the National Natural Science Foundation of China (Project #82304578) and the Fujian Provincial Natural Science Foundation (Project #2024J011419) to J. Wu.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe transcriptome data generated in this study have been deposited in the National Center for Biotechnology Information in the Sequence Read Archive (SRA) database under the accession number PRJNA1357582. Additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDALYs GBD, Collaborators H. Global, regional, and national disability-adjusted life-years (DALYs) for 359 diseases and injuries and healthy life expectancy (HALE) for 195 countries and territories, 1990-2017: a systematic analysis for the Global Burden of Disease Study 2017. Lancet. 2018; 392:1859\u0026ndash;922.\u003c/li\u003e\n\u003cli\u003eLavie CJ. Progress in Cardiovascular Diseases Statistics 2022. Prog Cardiovasc Dis. 2022; 73:94\u0026ndash;95.\u003c/li\u003e\n\u003cli\u003eFairweather D, Aung N, Carter RE. Editorial: Novel translational advances in artificial intelligence for diagnosis and treatment of cardiovascular diseases. Front Cardiovasc Med. 2025; 12:1604528.\u003c/li\u003e\n\u003cli\u003eGoodarzynejad H, Sheikh Fathollahi M, Shafiee A. Editorial: The role of sex in coronary artery disease. Front Cardiovasc Med. 2023; 10:1220439.\u003c/li\u003e\n\u003cli\u003eAnderson LL, Dai D, Miller AL, Roe MT, Messenger JC, Wang TY. Percutaneous coronary intervention for older adults who present with syncope and coronary artery disease? Insights from the National Cardiovascular Data Registry. Am Heart J. 2016; 176:1\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003eLawrie K, O\u0026apos;Neill S, Malik J, Balaz P, Corr M, Waldauf P. Classifications of haemodialysis vascular access-induced limb ischaemia. J Vasc Access. 2025:11297298251328428.\u003c/li\u003e\n\u003cli\u003eTachi M, Tanaka A, Riku S, Furuta T, Tobe A, Hayashi K, Murohara T. Percutaneous Treatment for Venous Stenosis Caused by a Vascular Closure Device. Cardiovasc Revasc Med. 2022; 40S:157\u0026ndash;58.\u003c/li\u003e\n\u003cli\u003eSerruys PW, Ono M, Garg S, Hara H, Kawashima H, Pompilio G, Andreini D, Holmes DR, Jr., Onuma Y, King Iii SB. Percutaneous Coronary Revascularization: JACC Historical Breakthroughs in Perspective. J Am Coll Cardiol. 2021; 78:384\u0026ndash;407.\u003c/li\u003e\n\u003cli\u003eChacko L, J PH, Rajkumar C, Nowbar AN, Kane C, Mahdi D, Foley M, Shun-Shin M, Cole G, Sen S\u003cem\u003e et al\u003c/em\u003e. Effects of Percutaneous Coronary Intervention on Death and Myocardial Infarction Stratified by Stable and Unstable Coronary Artery Disease: A Meta-Analysis of Randomized Controlled Trials. Circ Cardiovasc Qual Outcomes. 2020; 13:e006363.\u003c/li\u003e\n\u003cli\u003eAkbari T, Al-Lamee R. Percutaneous Coronary Intervention in Multi-Vessel Disease. Cardiovasc Revasc Med. 2022; 44:80\u0026ndash;91.\u003c/li\u003e\n\u003cli\u003eWatanabe H, Yamamoto K, Shiomi H, Morimoto T, Kato E, Matsumura Y, Nakatsuma K, Takeji Y, Yaku H, Yamamoto E\u003cem\u003e et al\u003c/em\u003e. Percutaneous coronary intervention using new-generation drug-eluting stents versus coronary arterial bypass grafting in stable patients with multi-vessel coronary artery disease: From the CREDO-Kyoto PCI/CABG registry Cohort-3. PLoS One. 2022; 17:e0267906.\u003c/li\u003e\n\u003cli\u003eFattori R, Piva T. Drug-eluting stents in vascular intervention. Lancet. 2003; 361:247\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003eMcNair ED, Wells CR, Mabood Qureshi A, Basran R, Pearce C, Orvold J, Devilliers J, Prasad K. Soluble receptors for advanced glycation end products (sRAGE) as a predictor of restenosis following percutaneous coronary intervention. Clin Cardiol. 2010; 33:678\u0026ndash;85.\u003c/li\u003e\n\u003cli\u003eZhao L, Feng L, Shan R, Huang Y, Shen L, Fan M, Wang Y. Nanoparticle-based approaches for treating restenosis after vascular injury. Front Pharmacol. 2024; 15:1427651.\u003c/li\u003e\n\u003cli\u003eXu K, Al-Ani MK, Pan X, Chi Q, Dong N, Qiu X. Plant-Derived Products for Treatment of Vascular Intima Hyperplasia Selectively Inhibit Vascular Smooth Muscle Cell Functions. Evid Based Complement Alternat Med. 2018; 2018:3549312.\u003c/li\u003e\n\u003cli\u003eOwens GK, Kumar MS, Wamhoff BR. Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol Rev. 2004; 84:767\u0026ndash;801.\u003c/li\u003e\n\u003cli\u003eZhang Q, Li H, Zhuang T, Xu L, Wu W, Pi J, Zhu P, Geng L, Duan Y, Xu J\u003cem\u003e et al\u003c/em\u003e. CCN5 suppresses injury-induced vascular restenosis by inhibiting smooth muscle cell proliferation and facilitating endothelial repair via thymosin beta4 and Cd9 pathway. Eur Heart J. 2025; 46:1645\u0026ndash;58.\u003c/li\u003e\n\u003cli\u003eLacolley P, Regnault V, Nicoletti A, Li Z, Michel JB. The vascular smooth muscle cell in arterial pathology: a cell that can take on multiple roles. Cardiovasc Res. 2012; 95:194\u0026ndash;204.\u003c/li\u003e\n\u003cli\u003eFrismantiene A, Philippova M, Erne P, Resink TJ. Smooth muscle cell-driven vascular diseases and molecular mechanisms of VSMC plasticity. Cell Signal. 2018; 52:48\u0026ndash;64.\u003c/li\u003e\n\u003cli\u003eMao C, Ma Z, Jia Y, Li W, Xie N, Zhao G, Ma B, Yu F, Sun J, Zhou Y\u003cem\u003e et al\u003c/em\u003e. Nidogen-2 Maintains the Contractile Phenotype of Vascular Smooth Muscle Cells and Prevents Neointima Formation via Bridging Jagged1-Notch3 Signaling. Circulation. 2021; 144:1244\u0026ndash;61.\u003c/li\u003e\n\u003cli\u003eChen R, McVey DG, Shen D, Huang X, Ye S. Phenotypic Switching of Vascular Smooth Muscle Cells in Atherosclerosis. J Am Heart Assoc. 2023; 12:e031121.\u003c/li\u003e\n\u003cli\u003eYap C, Mieremet A, de Vries CJM, Micha D, de Waard V. Six Shades of Vascular Smooth Muscle Cells Illuminated by KLF4 (Kruppel-Like Factor 4). Arterioscler Thromb Vasc Biol. 2021; 41:2693\u0026ndash;707.\u003c/li\u003e\n\u003cli\u003eBasatemur GL, Jorgensen HF, Clarke MCH, Bennett MR, Mallat Z. Vascular smooth muscle cells in atherosclerosis. Nat Rev Cardiol. 2019; 16:727\u0026ndash;44.\u003c/li\u003e\n\u003cli\u003eGrootaert MOJ, Bennett MR. Vascular smooth muscle cells in atherosclerosis: time for a re-assessment. Cardiovasc Res. 2021; 117:2326\u0026ndash;39.\u003c/li\u003e\n\u003cli\u003eFarina FM, Hall IF, Serio S, Zani S, Climent M, Salvarani N, Carullo P, Civilini E, Condorelli G, Elia L\u003cem\u003e et al\u003c/em\u003e. miR-128-3p Is a Novel Regulator of Vascular Smooth Muscle Cell Phenotypic Switch and Vascular Diseases. Circ Res. 2020; 126:e120\u0026ndash;e35.\u003c/li\u003e\n\u003cli\u003eZhao C, Shen J, Lu Y, Ni H, Xiang M, Xie Y. Dedifferentiation of vascular smooth muscle cells upon vessel injury. Int Immunopharmacol. 2025; 144:113691.\u003c/li\u003e\n\u003cli\u003eJensen LF, Bentzon JF, Albarran-Juarez J. The Phenotypic Responses of Vascular Smooth Muscle Cells Exposed to Mechanical Cues. Cells. 2021; 10.\u003c/li\u003e\n\u003cli\u003eCao G, Xuan X, Hu J, Zhang R, Jin H, Dong H. How vascular smooth muscle cell phenotype switching contributes to vascular disease. Cell Commun Signal. 2022; 20:180.\u003c/li\u003e\n\u003cli\u003eChappell J, Harman JL, Narasimhan VM, Yu H, Foote K, Simons BD, Bennett MR, Jorgensen HF. Extensive Proliferation of a Subset of Differentiated, yet Plastic, Medial Vascular Smooth Muscle Cells Contributes to Neointimal Formation in Mouse Injury and Atherosclerosis Models. Circ Res. 2016; 119:1313\u0026ndash;23.\u003c/li\u003e\n\u003cli\u003eSuna G, Wojakowski W, Lynch M, Barallobre-Barreiro J, Yin X, Mayr U, Baig F, Lu R, Fava M, Hayward R\u003cem\u003e et al\u003c/em\u003e. Extracellular Matrix Proteomics Reveals Interplay of Aggrecan and Aggrecanases in Vascular Remodeling of Stented Coronary Arteries. Circulation. 2018; 137:166\u0026ndash;83.\u003c/li\u003e\n\u003cli\u003ePaparidis NF, Durvale MC, Canduri F. The emerging picture of CDK9/P-TEFb: more than 20 years of advances since PITALRE. Mol Biosyst. 2017; 13:246\u0026ndash;76.\u003c/li\u003e\n\u003cli\u003eZheng B, Gold S, Iwanaszko M, Howard BC, Wang L, Shilatifard A. Distinct layers of BRD4-PTEFb reveal bromodomain-independent function in transcriptional regulation. Mol Cell. 2023; 83:2896\u0026ndash;910 e4.\u003c/li\u003e\n\u003cli\u003eLarochelle S, Amat R, Glover-Cutter K, Sanso M, Zhang C, Allen JJ, Shokat KM, Bentley DL, Fisher RP. Cyclin-dependent kinase control of the initiation-to-elongation switch of RNA polymerase II. Nat Struct Mol Biol. 2012; 19:1108\u0026ndash;15.\u003c/li\u003e\n\u003cli\u003eZhou Q, Li T, Price DH. RNA polymerase II elongation control. Annu Rev Biochem. 2012; 81:119\u0026ndash;43.\u003c/li\u003e\n\u003cli\u003eSunagawa Y, Morimoto T, Takaya T, Kaichi S, Wada H, Kawamura T, Fujita M, Shimatsu A, Kita T, Hasegawa K. Cyclin-dependent kinase-9 is a component of the p300/GATA4 complex required for phenylephrine-induced hypertrophy in cardiomyocytes. J Biol Chem. 2010; 285:9556\u0026ndash;68.\u003c/li\u003e\n\u003cli\u003eSano M, Abdellatif M, Oh H, Xie M, Bagella L, Giordano A, Michael LH, DeMayo FJ, Schneider MD. Activation and function of cyclin T-Cdk9 (positive transcription elongation factor-b) in cardiac muscle-cell hypertrophy. Nat Med. 2002; 8:1310\u0026ndash;7.\u003c/li\u003e\n\u003cli\u003eLi Z, Guo Z, Lan R, Cai S, Lin Z, Li J, Wang J, Li Z, Liu P. The poly(ADP-ribosyl)ation of BRD4 mediated by PARP1 promoted pathological cardiac hypertrophy. Acta Pharm Sin B. 2021; 11:1286\u0026ndash;99.\u003c/li\u003e\n\u003cli\u003eMatrone G, Wilson KS, Maqsood S, Mullins JJ, Tucker CS, Denvir MA. CDK9 and its repressor LARP7 modulate cardiomyocyte proliferation and response to injury in the zebrafish heart. J Cell Sci. 2015; 128:4560\u0026ndash;71.\u003c/li\u003e\n\u003cli\u003eXue Q, Zhang Q, Guo Z, Wu L, Chen Y, Chen Z, Yang K, Cao J. Therapeutic Hypothermia Inhibits Hypoxia-Induced Cardiomyocyte Apoptosis Via the MiR-483-3p/Cdk9 Axis. J Am Heart Assoc. 2023; 12:e026160.\u003c/li\u003e\n\u003cli\u003eJia Q, Hu Z, Song N, Mao W. Flavopiridol Mitigates the Progression of Monocrotaline-Induced Pulmonary Hypertension in Rats by Targeting Cyclin-Dependent Kinase 9. Cardiovasc Drugs Ther. 2023; 37:449\u0026ndash;60.\u003c/li\u003e\n\u003cli\u003eHuang S, Luo W, Wu G, Shen Q, Zhuang Z, Yang D, Qian J, Hu X, Cai Y, Chattipakorn N\u003cem\u003e et al\u003c/em\u003e. Inhibition of CDK9 attenuates atherosclerosis by inhibiting inflammation and phenotypic switching of vascular smooth muscle cells. Aging (Albany NY). 2021; 13:14892\u0026ndash;909.\u003c/li\u003e\n\u003cli\u003eHan Y, Zhao S, Gong Y, Hou G, Li X, Li L. Serum cyclin-dependent kinase 9 is a potential biomarker of atherosclerotic inflammation. Oncotarget. 2016; 7:1854\u0026ndash;62.\u003c/li\u003e\n\u003cli\u003eXu Y, Ma X, Ni W, Zheng L, Lin Z, Lai Y, Yang N, Dai Z, Yao T, Chen Z\u003cem\u003e et al\u003c/em\u003e. PKM2-Driven Lactate Overproduction Triggers Endothelial-To-Mesenchymal Transition in Ischemic Flap via Mediating TWIST1 Lactylation. Adv Sci (Weinh). 2024; 11:e2406184.\u003c/li\u003e\n\u003cli\u003eYu X, He T, Tong Z, Liao L, Huang S, Fakhouri WD, Edwards DP, Xu J. Molecular mechanisms of TWIST1-regulated transcription in EMT and cancer metastasis. EMBO Rep. 2023; 24:e56902.\u003c/li\u003e\n\u003cli\u003eTu M, Ge B, Li J, Pan Y, Zhao B, Han J, Wu J, Zhang K, Liu G, Hou M\u003cem\u003e et al\u003c/em\u003e. Emerging biological functions of Twist1 in cell differentiation. Dev Dyn. 2025; 254:8\u0026ndash;25.\u003c/li\u003e\n\u003cli\u003eWang Y, Yang K, Jiang G, Zheng X, Cheng F, Li W. Twist1 promoting neointima hyperplasia after vascular injury in diabetes via regulating hyperglycemia-induced phenotype switching of vascular smooth muscle cells. Atherosclerosis. 2025; 409:120496.\u003c/li\u003e\n\u003cli\u003eRen J, Xiao X, Li R, Lv C, Zhang Y, Wang L, Hong T, Zhang H, Wang Y. Single-cell sequencing reveals that endothelial cells, EndMT cells and mural cells contribute to the pathogenesis of cavernous malformations. Exp Mol Med. 2023; 55:628\u0026ndash;42.\u003c/li\u003e\n\u003cli\u003eFan Y, Gu X, Zhang J, Sinn K, Klepetko W, Wu N, Foris V, Solymosi P, Kwapiszewska G, Kuebler WM. TWIST1 Drives Smooth Muscle Cell Proliferation in Pulmonary Hypertension via Loss of GATA-6 and BMPR2. Am J Respir Crit Care Med. 2020; 202:1283\u0026ndash;96.\u003c/li\u003e\n\u003cli\u003eYang G, Zhang Z, Ma X, Chen J, Shi H, Yang J, Han Q. Role of Carvedilol in Inhibiting the Proliferation and Migration of Vascular Smooth Muscle Cells by Upregulating microRNA-145 Expression. Physiol Res. 2025; 74:577\u0026ndash;88.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"neointimal formation, vascular smooth muscle cell, CDK9, phenotypic switching, Twist1","lastPublishedDoi":"10.21203/rs.3.rs-8452448/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8452448/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRestenosis is a major limiting factor of percutaneous coronary intervention (PCI), accompanied by abnormal proliferation, migration and phenotypic switching of vascular smooth muscle cells (VSMCs). As a key component of positive transcription factor-b (P-TEFb), CDK9 phosphorylates RNA polymerase II and regulates various genes that are involved in the regulation of diverse cellular processes including cell growth and proliferation. In this study we investigated whether and how CDK9 regulated vascular remodeling after injury in mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur study observes a marked upregulation of CDK9 and its substrate pSer2 in VSMCs during neointimal hyperplasia following carotid artery injury, and the neointimal formation can be remarkably ameliorated by the CDK9 inhibitor (iCDK9). Overexpression of CDK9 promotes phenotypic switching, proliferation and migration of VSMCs in vitro, whereas inhibition of CDK9 obtains the opposites results. Moreover, CDK9 upregulates the expression of transcription factor Twist1, which is a key inducer of epithelial-mesenchymal transition (EMT) and has been widely implicated in the pathological progression of cardiovascular diseases. Furthermore, application of the Twist1 inhibitor Harmine largely abolished the function of CDK9 in promoting VSMCs phenotypic switching, proliferation and migration in vitro.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur findings demonstrate a crucial regulatory role of CDK9 in neointima formation after vascular injury, and strategies targeting CDK9 inhibition potentially overcome limitations of sustained efficacy following PCI.\u003c/p\u003e","manuscriptTitle":"CDK9 regulates neointima formation following vascular injury via targeting transcription factor Twist1","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-07 10:35:19","doi":"10.21203/rs.3.rs-8452448/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6ad1604e-fc5d-4d84-84cf-f022222fdf08","owner":[],"postedDate":"January 7th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-07T17:53:58+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-07 10:35:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8452448","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8452448","identity":"rs-8452448","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}