Newly-engineered angiopoietin-1 as a cell-priming agent for CVD

Newly-engineered angiopoietin-1 as a cell-priming agent for CVD | 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 Newly-engineered angiopoietin-1 as a cell-priming agent for CVD Jeehoon Kang, Hyun Ju Seo, HyunJu Son, Minjun Kang, Jaewon Lee, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7625543/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 Human peripheral blood stem cells (PBSC) that have been most frequently used for repair or regeneration in ischemic cardiovascular disease (CVD) showed limitations in their efficacy. We previously reported that angiopoietin-1 (Ang1) is the cell-priming agent to enhance the vasculogenic potential of PBSC. The limitation was the difficulty to produce Ang1 protein with high efficiency. In this study, we engineered Ang1 structure and made FVA3-Ang1 by adding VASP and COMP sequence for stable tetramer formation as well as FALG sequence for purification in large scale production and a signal peptide derived from influenza A virus (IAV) for better protein expression. FVA3-Ang1 showed stronger effect on endothelial cells than naïve Ang1 or COMP-Ang1 in terms of gene expression of Ang1, Ang2, VEGFA, FGF2, and KDR, as well as phosphorylation of Tie2, ERK, and Akt. Then we primed PBSC with FVA3-Ang1 and examined the transcriptome analysis. Priming for 1 hour did not change whole gene expression profiles of PBSC, whereas priming for 24 hours did change the pattern from myeloid toward endotheloid lineage. In mouse models of hind-limb ischemia and myocardial infarction, FVA3-Ang1-primed PBSCs showed superior engraftment and tissue regeneration compared to non-primed cells. A clinical trial is underway to assess efficacy and safety of FVA3-Ang1-primed PBSCs when infused via the culprit coronary artery following emergent stent implantation. engraftment angiogenesis paracrine signaling recovery Tie-2 receptor regeneration Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Ischemic diseases are characterized by restricted blood supply to specific tissues, leading to necrosis and organ dysfunction 1 . Due to the limited efficacy of current treatment options, these conditions often result in long-term complications for patients, imposing a significant clinical burden. Cell therapy has emerged as a promising therapeutic strategy; however, its efficacy requires further optimization 2 . Previously, we demonstrated that intracoronary infusion of granulocyte colony-stimulating factor (G-CSF)-mobilized human peripheral blood stem cells ( mob PBSCs) from bone marrow can promote myocardial recovery in patients with acute myocardial infarction 3 , 4 . Furthermore, enhanced mobilization of peripheral blood monocyte cells (PBMCs) using a combination of erythropoietin (EPO) and G-CSF led to improved cardiac function. However, the low efficiency of engraftment in ischemic myocardium and the limited evidence of sustained cardiac function recovery remain key challenges that must be addressed before broader clinical application 5 , 6 . Angiopoietin 1 (Ang1) is a well-characterized protein that promotes angiogenesis by mediating endothelial cell migration, adhesion and survival through the Tie-2 tyrosine kinase receptor 7 – 9 . Given its therapeutic potential, Ang1 has been investigated for treating vascular pathologies, lung infections, and neurological disorders 10 – 12 . We previously identified that mob PBSCs express the Tie2 receptor on their surface and demonstrated that ex vivo Ang1 priming could enhance their therapeutic efficacy in ischemic disease treatment 13 . In this study, we developed a unique cartilage oligomeric matrix protein (COMP)-Ang1 construct for priming purposes. COMP-Ang1 replaces the central coiled-coil domain region of natural Ang1 with COMP, whose ‘ECDACG’ sequence in the C-terminal region forms disulfide bonds with other alpha-helices, thereby stabilizing its oligomeric structure 14 , 15 . However, despite promising preclinical results, COMP-Ang1 proved unsuitable for therapeutic application due to instability in large-scale production. To overcome these limitations, our study aimed to develop a more stable form of Ang1 optimized for large-scale production and clinical application. Additionally, we sought to address concerns that the priming process might alter the differentiation state of mob PBSCs or induce reprogramming 16 , potentially leading to unintended tumorigenic complications. In this study, we engineered Ang1 construct by adding VASP and COMP sequence for the stabilized tetramer formation because multimeric Ang1 is more potent than naïve monomer by clustering Tie1 receptors leading to strong angiogenic or vasculogenic signal. Furthermore, we added FLAG sequence for purification process of this protein in large scale production and added a signal peptide derived from influenza A virus (IAV) for better protein expression. We characterized this novel form of Ang1 (FVA3-Ang1) regarding its potency to stimulate endothelial cells and suitability for ex vivo priming of PBSC. Furthermore, to translate our findings into clinical practice, we initiated a clinical trial to evaluate the safety and efficacy of Ang1-primed mob PBSCs in patients with myocardial infarction. By overcoming previous challenges associated with Ang1-based cell therapy, we aim to provide a scalable and effective treatment for ischemic diseases. 2. Results 2.1 Engineering of FVA3-Ang1 protein that is more potent than naïve Ang1 We developed a novel form of Ang1 designed as a stable ex vivo priming agent. To enhance protein expression, we incorporated a signal peptide derived from either Azurocidin or influenza A virus, along with a FLAG tag at the N-terminal region 17 . Given the challenges associated with clinical-grade purification of COMP-Ang1 due to its structural fragility 18 , we explored alternative domains, including Phe14 19 and human vasodilator-stimulated phosphoprotein (VASP) 20 . VASP, predominantly expressed as a tetramer, was hypothesized to provide superior structural stability compared to COMP. Additionally, because the "ECDACG" sequence in the C-terminal region of COMP is responsible for stabilizing the oligomeric structure, we integrated this sequence into the C-terminal region of VASP, resulting in a modified construct termed VASP2 (Fig. 1 A). To achieve high expression levels, we integrated the gene into a genomic hotspot using the Cas9D10A system (Sigma-Aldrich; Supplementary Fig. 1A) and identified hotspot site 13 as the optimal location due to its strong GFP expression ( Supplementary Fig. 1B ). We constructed multiple Ang1 overexpression vectors incorporating COMP, Phe14, VASP1, and VASP2 (Table 1 ). Transient expression assays demonstrated poor oligomerization for constructs containing Phe14 and COMP, whereas the FVA3 construct (IAV-FLAG-VASP2-Ang1) consistently formed tetramers (Figs. 1 B and 1 C). To corroborate these biochemical observations and provide structural insights, we performed in silico modeling using AlphaFold3 to predict the monomeric and putative tetrameric assemblies of FVA3-Ang1 and COMP-Ang1. The modeled tetrameric architecture for FVA3-Ang1 is shown in Fig. 1 D, whereas the COMP-Ang1 models displayed less consistent interface packing. Multimer stability was quantified using PDBePISA analysis of AlphaFold-Multimer tetramer models, which revealed that FVA3-Ang1 has substantially larger buried interface areas and more negative predicted interaction energies than COMP-Ang1. FVA3-Ang1 exhibited a 10–20% larger interface area and stronger binding energy compared with COMP-Ang1, suggesting enhanced stability of the complex. Moreover, the complex significance score, which was close to 1.0, indicates that this interaction is physiologically meaningful and is predicted to form a stable tetramer (Figs. 1 E). To further validate the enhanced stability of FVA3-Ang1, we measured the half-lives of all three Ang1 variants in HUVEC culture medium using ELISA. Interestingly, all three proteins demonstrated comparable stability with no significant differences in degradation rates over the tested time course, suggesting that the superior functional performance of FVA3-Ang1 is attributed to its enhanced oligomerization rather than intrinsic protein stability in the culture environment ( Supplementary Fig. 1D ). Table 1 Ang1 overexpression vectors Vector name FCA Hot spot 13 + mCMV + Influenza A virus + FLAG + COMP + Ang1 FPA Hot spot 13 + mCMV + Influenza A virus + FLAG + Phe14 + Ang1 FPA2 Hot spot 13 + mCMV + Influenza A virus + FLAG + Phe14 with a disulfide bridge + Ang1 FVA2 Hot spot 13 + mCMV + Azurocidin + FLAG + VASP with a disulfide bridge + Ang1 FVA3 Hot spot 13 + mCMV + Influenza A virus + FLAG + VASP with a disulfide bridge + Ang1 COMP, Cartilage oligomeric matrix protein; mCMV, murine cytomegalovirus; Phe14, Phe14 Phenylalanine; VASP, vasodilator-stimulated phosphoprotein Based on these findings, the optimal configuration—hotspot site 13 with an influenza A virus signal peptide and VASP2—was designated FVA3-Ang1. For scalable production, we established a stable Ang1-expressing CHO-K1 cell line cultured in CDM4CHO medium (Cytiva™). Clonal selection was performed using ClonePix FL ( Supplementary Fig. 1C ), and the final cell line, named CHO-K1DW, was confirmed to exhibit robust growth and stability. By integrating the FVA3-Ang1 gene into hotspot site 13 of CHO-K1DW cells, we developed a reliable system for the large-scale production of Ang1. 2.2 FVA3-Ang1 is stronger than naïve Ang1 or COMP-Ang1 in stimulating endothelia cells under in vitro HUVEC experiment Preliminary in vitro analyses comparing naïve Ang1, COMP-Ang1, and FVA3-Ang1 were conducted to evaluate signal transduction, gene expression, and angiogenic potential. These comparative studies revealed that FVA3-Ang1 exhibited the most potent biological effects, followed by COMP-Ang1, whereas naive Ang1 showed baseline activity. Based on these findings and considering the limited scope of in vivo studies, we investigated the therapeutic efficacy of FVA3-Ang1 as a lead candidate. To evaluate the transcriptional responses to Ang1 variants, we measured the mRNA levels of pro-angiogenic factors in HUVECs (Fig. 2 A). Treatment of HUVEC with each Ang1 variant increased the transcript levels of ANGPT1, ANGPT2, FGF2, VEGFA, and KDR relative to the vehicle control. The increases elicited by FVA3-Ang1 and COMP-Ang1 were more pronounced than those induced by naïve Ang1, consistent with their enhanced pro-angiogenic activities. We analyzed the phosphorylation of Tie1 receptor as well as Akt and ERK in HUVECs following treatment with naïve Ang1, COMP-Ang1, or FVA3-Ang1. As shown in Fig. 2 B, all Ang1 variants induced phosphorylation of Tie2, AKT and ERK compared to the vehicle control. Phosphorylated Tie2, ERK, and AKT were quantified and normalized to their respective total protein levels. Notably, FVA3-Ang1 and COMP-Ang1 elicited stronger and more sustained activation of Tie2, ERK, and Akt than naïve Ang1. Quantitative densitometric analysis revealed that the phosphorylation levels of Tie2, ERK, and Akt were significantly higher in the FVA3-Ang1 group than in the naïve Ang1 group. These results indicate that FVA3-Ang1 preserves the signaling capacity of COMP-Ang1 and confers enhanced activation compared with naïve Ang1, supporting its potential as a functionally improved Ang1 variant ( Fig. 2 B ) . Finally, 24 hours treatment with FVA3-Ang1 promoted endothelial lineage characteristics (Fig. 2 C). The expression of the endothelial markers CD31, VE-Cadherin (VECAD), and eNOS increased in a dose-dependent manner, with the greatest upregulation observed at 400 ng/mL. Together, the transcriptional, signaling, and phenotypic data support that FVA3-Ang1 is a functionally improved Ang1 variant with robust pro-angiogenic activity in HUVECs. To assess the angiogenic activity of the three Ang1 variants, we performed a wound-healing assay using HUVECs. While naive Ang1 showed no significant difference compared to controls, both COMP-Ang1 and FVA3-Ang1 significantly promoted wound closure. Notably, FVA3-Ang1 had the most pronounced effect, leading to greater reduction in wound area at both 12 and 24 h in a dose- and time-dependent manner (Fig. 2 D-E). These results confirm that FVA3-Ang1 enhances endothelial cell function and angiogenic potential while maintaining stability and efficacy. We further evaluated angiogenic potential using a tube formation assay. HUVECs were treated with naïve Ang1, COMP-Ang1 or FVA3-Ang1 at 200 ng/mL and 400 ng/mL for either 1 hour or 24 hours before being seeded onto Matrigel (Supplementary Fig. 2A) . Tube formation was observed at both concentrations and time points, with no significant difference between 1-hour and 24-hour treatment durations (Supplementary Fig. 2B). Next, we examined endothelial cell proliferation using EdU incorporation assay. After 1 and 24 hours of stimulation, native Ang1 did not increase the proportion of EdU-positive cells compared to that in the untreated controls. In contrast, both COMP-Ang1 and FVA3-Ang1 significantly enhanced HUVEC proliferation, with FVA3-Ang1 consistently yielding the highest percentage of proliferating cells ( Supplementary Fig. 2C-2E ). To evaluate endothelial barrier integrity, we performed immunofluorescence staining for ZO-1 and Claudin-5. At both 1 and 24 h, naive Ang1 failed to improve tight junction organization compared to the controls. In contrast, COMP-Ang1 and particularly FVA3-Ang1 markedly reinforced the continuity and intensity of ZO-1 and Claudin-5 staining, indicating tighter junctional complexes ( Supplementary Fig. 2F-2K ). Quantitative analysis confirmed that FVA3-Ang1 produced the greatest improvement in tight junction integrity compared to other treatments. Consistent with these findings, FITC-dextran permeability assay demonstrated that FVA3-Ang1 treatment resulted in the most robust reduction in paracellular leakage at both 1 and 24 hours, whereas COMP-Ang1 showed intermediate effects and naive Ang1 had minimal impact ( Supplementary Fig. 2L ). Collectively, these data indicate that naïve Ang1 exerts little influence on angiogenesis or endothelial barrier stabilization in vitro, whereas FVA3-Ang1 demonstrates superior activity compared to COMP-Ang1 and represents the most potent variant in multiple functional assays. 2.3 Whole mRNA expression profiles of mob PBSC after priming with FVA3-Ang1 We evaluated the FVA3-Ang1 priming strategy with a dual focus: ensuring safety by assessing modifications in cell characteristics and enhancing therapeutic efficacy by augmenting angiogenic potential. Gene expression patterns were analyzed via total RNA sequencing of mob PBSCs collected from patients diagnosed with acute myocardial infarction (GEO. The analysis included three groups: naive mob PBSCs, 1-hour primed mob PBSCs, and 24-hour primed- mob PBSCs. Among 26,137 reliable probes, gene expression patterns in the 1-hour primed mob PBSCs closely resembled those of naïve mob PBSCs. However, in the 24-hour primed mob PBSCs, 59 genes were upregulated and 243 genes were downregulated compared to the other two groups, as illustrated in the heatmap analysis (Figs. 3 A, 3 B). Gene ontology analysis revealed that 24-hour priming altered cell characteristics by upregulating genes related to protein stabilization and growth factor receptor signaling pathway while downregulating genes associated with cell adhesion, and innate immune response (Fig. 3 C). To further evaluate the effects of FVA3-Ang1 priming, qRT-PCR analysis was performed on mob PBSCs primed for 1 hour and 24 hours, with all cells harvested at the 24-hour time point. Compared to PBMCs (negative control) and naïve mob PBSC, 1-hour priming did not significantly increase CD31, CXCR4, or VE-CAD expression, while 24-hour priming resulted in elevated CXCR4 and VE-CAD expression. The Tie2 receptor expression increased following both 1-hour and 24-hour priming (Fig. 4 A). Flow cytometry analysis demonstrated a gradual increase in CD31, CXCR4, Tie2 and VE-CAD protein expression from PBMCs to naïve mob PBSCs and further in 1-hour primed mob PBSCs. However, this increase was only statistically significant in the 24-hour primed group. Additionally, CD45 expression showed a gradual decrease, which was significant only after 24-hour priming with FVA3-Ang1. Notably, Annexin V expression remained unchanged across all groups, indicating no increase in apoptosis (Figs. 4 B, 4 C). 2.4 Regenerative efficacy of mob PBSC is improved by priming with FVA3-Ang1 in the hind-limb ischemia or myocardial infarction mice model Considering that COMP-Ang1’s in vivo efficacy was already established in our previous study, the current study evaluated only the in vivo effects of FVA3-Ang1 13 . To evaluate the therapeutic potential of 1-hour FVA3-Ang1-primed mob PBSCs, we conducted experiments in mouse models of hind-limb ischemia and myocardial infarction (Fig. 5 A). While extended priming periods (e.g., 24 hours) may potentially enhance therapeutic efficacy, we employed a 1-hour priming protocol to ensure compliance with regulatory guidelines that restrict cellular modifications that could alter fundamental cell characteristics or lineage commitment. This approach maintains the inherent properties of PBSCs while providing sufficient angiopoietin-1 priming to enhance their therapeutic potential. In the hind-limb ischemia model, the right femoral artery was ligated, followed by an injection of 1×10 5 cells into the peripheral muscle. Laser Doppler Perfusion Imaging (LDPI) was performed at 0, 3, 7, and 14 days post-surgery (Fig. 5 B). The FVA3-Ang1 primed mob PBSC group exhibited the highest efficacy in blood flow recovery and tissue regeneration (Figs. 5 C– 5 E). Immunohistochemistry analysis confirmed active neovascularization, as evidenced by increased CD31 expression (Figs. 5 F, 5 G, 5 F and 5 G). Additionally, HLA-ABC expression demonstrated superior engraftment of primed cells (Figs. 5 H, 5 I) in the FVA3-Ang1 primed mob PBSC group compared to controls. HLA-ABC fluorescence intensity was normalized to DAPI intensity for each field to correct for variations in cell number and staining. To evaluate cell therapy efficacy in a myocardial infarction model, the left anterior descending artery was ligated in mice, followed by the injection of 1 x 10 5 cells into the infarcted anterior myocardium. Echocardiographic and histologic assessments were performed on day 14 (Fig. 6 A). The overall survival rate is shown in Fig. 6 B. Echocardiographic analysis revealed superior functional improvement in the FVA3-Ang1 primed- mob PBSC group, demonstrating enhanced left ventricular function and reduced ventricular dilation (Fig. 6 C). Histologic assessment showed a significant reduction in fibrosis in the FVA3-Ang1-primed mob PBSC group (Figs. 6 D, 6 E), with superior neovascularization (Figs. 6 F, 6 G) and engraftment (Figs. 6 H, 6 I). Notably, this study was not designed to assess the transdifferentiation of PBSCs into cardiomyocytes; instead, we focused on their paracrine effects and endothelial interactions. Although a small number of engrafted cells may theoretically adopt a myogenic phenotype, we did not observe or quantify definitive cardiomyocyte differentiation or formation of striated muscle structures in the myocardial model. 2.5 Clinical trial, “MAGIC-CELL-6-PRIMING”, to evaluate the efficacy and safety of mob PBSC that were primed with FVA3-Ang1 in patients with AMI To evaluate the efficacy and safety of mob PBSC in human cardiovascular diseases, we are conducting a clinical trial titled “Myocardial Regeneration and Angiogenesis in Myocardial Infarction with Intracoronary Infusion of Mobilized Peripheral Blood Stem Cells after Priming with FVA3-Ang1, 6th version (MAGIC Cell-6 Priming).” This investigator-initiated, single-arm trial is being performed at Seoul National University Hospital, South Korea (KCT0006753, NCT06364150). The inclusion and exclusion criteria are outlined in Table 2 , and a detailed study protocol is depicted in Fig. 7 . In brief, patients diagnosed with acute myocardial infarction will undergo percutaneous coronary intervention to the culprit coronary artery in accordance with standard clinical procedures. To facilitate peripheral blood stem cell mobilization, patients will receive a single bolus infusion of long-acting EPO followed by three days of daily subcutaneous injections of G-CSF. Apheresis will then be performed to collect mob PBSC. In an ex vivo setting, mob PBSC will be primed with FVA3-Ang1 for one hour. After priming, the cells will undergo two rounds of washing to minimize residual FVA3-Ang1 in the final product, in accordance with regulatory requirements in Korea, as separate clinical safety data for this protein are not yet available. A total of 2 x10 9 FVA3-Ang1-primed mob PBSCs will then be infused into the infarct myocardium through the culprit coronary artery. The infusion cell count was determined based on previous MAGIC CELL trials 13 . Table 2 Inclusion and Exclusion criteria of the MAGIC Cell-6 study Inclusion criteria 1. Male and female aged ≥ 19 years and ≤ 80 years 2. A clinical diagnosis of acute myocardial infarction within 4 weeks from randomization 3. Successful percutaneous coronary intervention to the target lesion (TIMI flow grade 3 and residual stenosis < 30% at the target lesion) with a drug-eluting stent and/or drug-eluting balloon 4. Agreement to give written informed consent. Exclusion criteria 1. Patients with uncontrolled heart failure (Killip class ≥ grade 2, or left ventricular ejection fraction < 20%) 2. Patients with uncontrolled chest pain due to ischemia 3. Patients with uncontrolled arrythmia 4. Active malignancy, or incompletely treated malignancy 5. Active infectious disease 6. Uncontrolled hematologic disease, including coagulopathy or bleeding diathesis 7. Presence of non-cardiac comorbidity with life expectancy ≤ 1 year from randomization 8. Females with childbearing potential or breast-feeding 9. Refusal to give written informed consent 10. Other conditions that may result in protocol non-compliance by the committees During the washing process, patient plasma, prepared separately, was used as the resuspension medium. This approach mitigated cell aggregation, which was observed when saline was used for resuspension ( Supplementary Fig. 3A ). Additionally, viability testing with Trypan blue staining revealed a 20% reduction in cell viability when saline was used, alongside increased cell death and apoptosis. In contrast, resuspension in patient plasma preserved cell viability without any increase in cell death, or apoptosis ( Supplementary Fig. 3B and 3C ). The concentration of FVA3-Ang1 following the washing process is shown in Supplementary Fig. 3D , demonstrating a negligible amount of remnant FVA3-Ang1 in the final cell product. Furthermore, flow cytometric analysis of the activation markers CD69 and CD25 confirmed the absence of unintended immune responses in the treated cells ( Supplementary Fig. 3E ). The primary endpoint of the MAGIC Cell-6 trial is the improvement of infarct-related regional wall motion abnormality, assessed via echocardiography at the 12-months follow-up. Secondary endpoints are listed in Table 3 . The study protocol was approved by the institutional review board of the participating center and adheres to the principles of the Declaration of Helsinki. All patients will provide written informed consent prior to participation. Table 3 Study endpoints of the MAGIC Cell-6 study Primary endpoint Improvement of the infarct-related regional wall motion abnormality - Measured by echocardiography, at 12-months follow-up Secondary endpoints 1. Regional wall motion score index (Measured by echocardiography, at 12-months follow-up 2. Clinical outcomes (number of patients, up to 12-months follow-up) a. All-cause death b. Cardiac death c. Target lesion revascularization d. Readmission due to any cause e. Readmission due to heart failure 3. B-natriuretic peptide level (unit of pf/mL, at 12-months follow-up) 4. 6-minute walk test (unit of meters, at 12-months follow-up) 3. Discussion In this study, we aimed to improve the efficacy of PBSC therapy for myocardial infarction. Our main findings are: 1) We developed a novel engineered molecule, FVA3-Ang1, a stable and clinically scalable form of Ang1 for therapeutic applications. 2) Gene expression analysis showed that 1-hour priming with FVA3-Ang1 did not modify gene expression, whereas 24-hour priming altered gene expression, inducing an endothelial-like phenotype. 3) Protein expression analysis confirmed that endothelial lineage markers were only upregulated after 24-hour FVA3-Ang1 priming. This finding is critical, as regulatory agencies in Korea impose strict approval criteria when cell characteristics change after priming. 4) In vivo studies demonstrated that 1-hour priming with FVA3-Ang1 significantly enhanced the regenerative potential of mob PBSCs in hind-limb ischemia and myocardial infarction models. Collectively, our results suggest that short-term (1-hour) priming with FVA3-Ang1 enhances the angiogenic and regenerative potential of mob PBSCs without altering their fundamental genetic profile, thus supporting its clinical applicability in stem-cell based therapy for myocardial infarction. 3.1 Current Limitations of Stem Cell Therapy: Ang1 as a Promising Agent for Priming Strategy Current ischemic disease treatments primarily focus on revascularization, aiming to restore blood flow to occluded vessels. Metallic stents are widely used for this purpose and have improved patient outcomes. However, a major limitation remains: while revascularization prevents further ischemic damage, it does not actively promote tissue repair and regeneration 21 . Stem cell therapy has been proposed as a breakthrough strategy to address this limitation 22 . Despite promising results, previous studies have highlighted several challenges, including low therapeutic efficacy 5 , 6 . Various modifications have been explored to enhance the regenerative potential of stem cells, such as using undifferentiated stem cells, genetic modifications, and exogenous protein priming. However, the use of undifferentiated stem cells and genetic modifications raises concerns about tumorigenic risks and ethical issues 16 , 23 . A more practical and feasible approach is to use the autologous stem/progenitor cells from bone marrow. However, this strategy has two major limitations: weak regenerative potency of adult stem cells and the requirement for invasive procedures (i.e. bone marrow aspiration). To overcome these challenges, we developed a cytokine-based autologous mob PBSC therapy, which mobilizes adult stem cells from the bone marrow to peripheral blood using G-CSF injection, enabling us to obtain the same volume and quality of stem/progenitors from the peripheral blood as from bone marrow 13 , 24 . To further enhance mobilization efficiency, we used a combination-cytokine approach by adding EPO alongside G-CSF, which significantly improved stem cell yield and mobilization efficacy compared to G-CSF alone. The stem cell niche is hypothesized to be regulated by interactions between angiopoietin on niche-supporting cells and Tie2 receptors on stem cells. Our previous study confirmed that all the stem/progenitor cells mobilized into peripheral blood ( mob PBSC) express Tie2 receptors 13 . This finding led us to investigate the feasibility of ex vivo protein priming of mob PBSCs with Ang1 from bone marrow by G-CSF and EPO, demonstrating that Ang1 priming improved cell engraftment and therapeutic efficacy in ischemic disease models. However, a significant limitation in our previous study was the use of COMP-Ang1, which exhibited poor stability and high aggregation, making mass production for clinical application impractical. 3.2 Engineering of FVA3-Ang1 (IAV-FLAG-VASP2-Ang1): A More Stable and Potent Tetramer Ang1 Protein Suitable for Large Production with High Efficiency In this study, we developed a modified form of Ang1 designed to enhance structural stability, expression efficiency, and its ability to recruit and activate the Tie2 receptor as a multimer. To achieve this, we engineered Ang1 to predominantly express as tetramers, with tetramerization serving as the primary conformation in the expression profile. This structural stabilization was reinforced by incorporating VASP2, a novel variant containing a specific sequence that facilitates disulfide bond formation at the C-terminal end. Additionally, to maximize Ang1 expression, we introduced a signal peptide from influenza A virus (IAV) at the N-terminal region, leading to FVA3-Ang1 (IAV-FLAG-VASP2-Ang1) 25 – 29 . Although the measured half-life of naïve Ang1 was not substantially different from that of COMP-Ang1 or FVA3-Ang1, Ang1 instability and tendency to aggregate constrain its clinical utility ( Supplementary Fig. 1D ). COMP-Ang1 improved stability and bioactivity compared to the native protein but still fell short of clinical-grade requirements. To overcome these limitations, we developed FVA3-Ang1, a clinically manufacturable tetramer-stabilized Ang1 variant. Importantly, PBSCs treated with FVA3-Ang1 were subjected to a dedicated washing procedure to remove residual protein, thereby minimizing systemic exposure and producing favorable pharmacokinetic and tissue distribution profiles compatible with therapeutic applications. Collectively, through strategic engineering and refinement of the cultivation process, we achieved significant advancements in the production of FVA3-Ang1 oligomers, improving their stability and therapeutic potential for applications in vascular biology and angiogenesis. The functional activity of FVA3-Ang1 was subsequently validated through experiments demonstrating increased pAKT activity, enhanced CD31 and VECAD expression, and improved endothelial tube formation. When comparing FVA3-Ang1 to COMP-Ang1, it is important to note that while our in vivo experiments did not include COMP-Ang1, its angiogenic effects have been well-documented in our previous study 13 . We expanded our discussion to include the Tie2 signaling axis, PI3K/Akt activation, and the role of CXCR4 in neovascularization, as supported by existing literature 13 . Our focus with FVA3-Ang1 is supported by qPCR data showing sustained upregulation of Tie2 mRNA in mob PBSCs after 1 and 24 hours of treatment, reflecting durable Tie2 activation. These features are expected to translate into superior therapeutic efficacy. Moreover, while RNA-sequencing data for COMP-Ang1-primed cells remain unavailable, our transcriptomic analysis of FVA3-Ang1-primed mob PBSCs revealed significant functional enhancements, such as increased expression of CXCR4 and CD31. 3.3 Enhanced Function of mob PBSCs Without Genetic Modification Following Short Term Exposure to FVA3-Ang1 This study demonstrated that mob PBSCs primed with FVA3-Ang1 exhibit superior tissue regeneration potential compared to naïve mob PBSCs in the context of stem cell therapy. To obtain regulatory approval for the clinical application of FVA3-Ang1-primed mob PBSCs in patients with acute myocardial infarction, it is essential to comply with the stringent requirements set forth by the Korean FDA. A key regulatory criterion is ensuring that the priming process enhances the regenerative capacity of the cells without altering their genetic characteristics. After a short 1-hour and prolonged 24-priming strategy, we could find that the cell characteristics changed after a prolonged priming strategy, while minimal alteration was shown after a 1-hour priming, proven by total-RNA sequencing. However, even after a 1-hour priming, mob PBSC primed with FVA3-Ang1 exhibited enhanced endothelial and angiogenic properties, while maintaining the genetic profile of hematopoietic cells and showing no signs of increased cell death. Mouse ischemic disease models also confirmed the efficacy of FVA3-Ang1 primed mob PBSC, by demonstrating recovery of the myocardial infarct and improvement of cardiac function. 3.4 Translation Application: The MAGIC Cell-6-Priming trial Building on our previous experience with mob PBSC-based therapy for ischemic disease, we first demonstrated in the MAGIC Cell-3 trial that adding cell therapy to standard stent treatment significantly improved cardiac function compared with stent alone 3 . We then advanced our strategy in MAGIC Cell-5(clinicaltrials.gov ID: NCT00501917) by confirming the efficacy of combined G-CSF and darbepoetin-primed cell therapy 30 and further identified the superior effect of angiopoietin within that regimen. These findings inform the design of the MAGIC Cell-6 trial (clinicaltrials.gov ID: NCT06364150), which is currently underway to evaluate the clinical feasibility and efficacy of FVA3-Ang1-primed mob PBSCs in patients with acute myocardial infarction. This clinical protocol includes several key innovations in stem cell therapy. First, the use of autologous peripheral blood-derived cells eliminates the risk of transplant rejection and inflammatory complications. To overcome the variability in hematopoietic capability and the potentially diminished cytokine response in critically ill patients, we employed a combination of G-CSF and EPO, both of which have been proven safe for human use. mob PBSCs were isolated and concentrated via apheresis, a well-established clinical procedure. A critical challenge in the trial was that FVA3-Ang1 has not been previously approved for human use, as there are no prior clinical safety data. Consequently, we established a controlled ex vivo priming and washing process. The ex vivo priming process is performed in a GMP-certified facility at Seoul National University Hospital, guaranteeing a sterile environment free from microbial, particulate, and pyrogenic contamination. This approach enabled us to maximize the priming efficacy while minimizing the risk of hypersensitivity reactions to recombinant proteins. Another unique aspect of the trial is the washing process, which was developed in response to regulatory requirements. While we hypothesized that residual FVA3-Ang1 in the final cell preparation might provide additional therapeutic benefits to the infarcted myocardium, the regulatory agency mandated its complete removal before infusion, given the absence of prior phase-1 clinical trials in humans. Consequently, we implemented a standardized washing protocol to eliminate any residual FVA3-Ang1 from the final preparation of primed mob PBSCs. As the washing procedure involves centrifugation, which could potentially damage the cells, we assessed cell viability pre- and post-washing, confirming minimal cell damage. The washing solution was autologous plasma, derived from the supernatant of centrifuged whole blood. This approach ensures the presence of various endogenous cytokines that are beneficial for the survival of ex vivo washed mob PBSCs. We have previously demonstrated that autologous plasma contains high levels of pro-angiogenic cytokines such as IL8, IL17, PDGF, and VEGF, which may further enhance the therapeutic potential of washed mob PBSCs 31 , 32 . Importantly, in the MAGIC Cell-6 trial, six patients have been treated to date, and no acute complications related to cell injection have been observed at the immediate post-procedure state. ( Supplementary Fig. 4A-C ). Also, during the 90-day clinical follow-up, and no adverse findings were detected in these patients, reflecting the immediate safety of this technique. We are planning to perform a 1-year clinical follow-up for all patients along with evaluation of the stent patency and myocardial recovery by coronary angiogram and myocardial perfusion tests. Collectively, this trial introduces a novel, short ex vivo priming strategy that is clinically applicable and a specific washing process ensuring that only primed mob PBSCs, and not the priming agent, are delivered to the infarcted myocardium through the culprit coronary artery. This distinction is crucial for obtaining regulatory approval, as it guarantees that sterile, non-immunogenic, autologous cells, free from any foreign agents, are administered to patients. The results of the MAGIC Cell-6 trial will provide critical insights into the real-world clinical impact of FVA3-Ang1-primed mob PBSCs, offering a potentially transformative therapy for acute myocardial infarction. 4. Limitation Long-term safety assessment remains a critical consideration for this novel technique. The cells used in this study are derived from adult bone marrow, which inherently lowers the risk of tumorigenicity 33 . Moreover, the short-term exposure to FVA3-Ang1, as implemented in our 1-hour priming protocol, does not significantly alter gene expression, further minimizing the likelihood of tumorigenic transformation. Notably, previous studies involving COMP-Ang1 have not reported any evidence of tumorigenicity, and the regulatory advantages of short-term priming provide an additional safeguard against potential risks. Since the effect of COMP-Ang1, which can be considered as a control, was confirmed in previous in vivo experiments, in this study, we conducted in vivo experiments to confirm only the effect of FVA3-Ang1 13 . Nonetheless, we propose a framework for future studies to investigate the long-term risk of tumor formation. Additionally, while this study did not directly assess fibrosis-related mediators, such as TGF-β and α-SMA or the macrophage phenotypes M1/M2 balance 34 , previous literature suggests that these factors could contribute to the overall therapeutic effect. Future research should also explore the secretory profile of primed mob PBSCs, as potential paracrine mechanisms may support endothelial interactions and further enhance tissue recovery. 5. Materials and Methods 5.1 The cultivation of Angiopoietin Expression Cell Line We applied a strategic development approach to generate a stable Ang1 overexpressing cell line ( Supplementary Fig. 1A ). Initially, we adapted a Chinese Hamster Ovary cell line 35 to suspension culture and subsequently engineered a promoter system to induce Ang1 overexpression. To optimize gene expression, we identified hotspot sites mediating overexpression in the cell genome and proceeded with the development of a mass production process. Validating an Ang1 vector system mediated by euchromatin, where gene expression occurs effectively within the cell nucleus, we selected hotspot site 13 as the most efficient inducer among 8 candidates ( Supplementary Fig. 1B ). Ultimately, we employed a systematic approach utilizing Design of Experiments (DoE). Our optimized cultivation process achieved the highest expression levels and purity of Ang1 under specific conditions, including seeding cells at a density of 334.34 cells/mL, maintaining a pH of 7.4, and regulating dissolved oxygen levels at 30%. 5.2 The collection of mob PBSC and PBMCs The collection of mob PBSCs and PBMCs followed a modified protocol based on our previous report 13 . PBSCs were mobilized from patients who received subcutaneous EPO 5 µg/kg (maximum 300 µg) for one day and G-CSF 5 µg/kg twice for three days. On the fourth day, mob PBSCs were collected by apheresis (Fig. 7 ). A washing solution was independently prepared by collecting the plasma from the patient and mixing this with Dulbecco's Phosphate-Buffered Saline at a 1:1 ratio. An equivalent amount of the washing solution was added to the product of apheresis and centrifuged at 3000 RPM for 25 minutes. The isolated mob PBSCs were then treated according to the specified protocol. The washing solution was stored separately, which was later used as a solvent when injected into the mouse hind legs and myocardial infarction models. As a negative control, PBMCs were collected from a healthy control group. In a 50 mL sterile tube, 20 mL of peripheral blood was mixed with 10 mL of PBS and 12 mL of Ficoll Paqur Plus (Cytiva ™ ) that had been shaken vigorously was slowly added. Centrifugation was performed at 2500 RPM for 30 minutes at room temperature, with deceleration set to 0 RPM. The buffy coat layer located below the separated plasma was aspirated with a 1 mL pipette and placed in a new 50 mL sterile tube. The remaining volume was filled with PBS, and centrifugation was performed at 1800 RPM for 10 minutes at 4 ℃, with deceleration also set to 0 RPM. The supernatant was aspirated, followed by resuspension with 1 mL of PBS. The remaining volume was filled with PBS, and centrifugation was performed under the same conditions. From this stage, deceleration was set to 5 RPM. The supernatant was aspirated, and the PBMC was obtained by counting the cells and centrifuging under the same conditions. This experiment was conducted with the approval of the Institutional Review Board (IRB) of Seoul National University Hospital (MAGIC cell-5 trial, NCT00501917). 5.3 Priming mob PBSCs with FVA3-Ang1 and wash-out process The priming process was performed with FVA3-Ang1 at a concentration of 400 ng/mL in a 37℃ incubator for 1 hour or 24 hours, according to the protocol of confirming genetic expression characteristics. After FVA3-Ang1 priming, two washing processes were performed to ensure the remaining FVA3-Ang1. This was performed for clinical application of mob PBSC treated with FVA3-Ang1, which aimed to establish a safe washout process before infusion. Preparation of the washing solution was explained above. The remaining volume of the 50 mL sterile tube containing the primed cell solution was filled with the washing solution and centrifuged at 1000 rcf for 5 minutes. After removing the supernatant, resuspension was performed with the washing solution, and the centrifuge was done under the identical condition. Finally, the plasma isolated from the isolated mob PBSCs was used as a resuspension solution to be used in animal experiments. Ultimately, the concentration of Ang1 detected in the mob PBSC after undergoing two washing processes was measured to be below the Limit of Detection (LOD) value of 0.87 ng/mL, demonstrating its safety for human use ( Supplementary Fig. 3D ). 5.4 Total RNA-Sequencing Library preparation and sequencing libraries were prepared from total RNA using the NEBNext Ultra II Directional RNA-Seq Kit (NEW ENGLAND BioLabs, Inc., UK). rRNA was removed using RIBO COP rRNA depletion kit (LEXOGEN, Inc., Austria). The rRNA depleted RNAs were used for the cDNA synthesis and shearing, following manufacture’s instruction. Indexing was performed using the Illumina indexes 1–12. The enrichment step was carried out using of PCR. Subsequently, libraries were checked using the Agilent 2100 bioanalyzer (DNA High Sensitivity Kit) to evaluate the mean fragment size. Quantification was performed using the library quantification kit using a Step One Real-Time PCR System (Life Technologies, Inc., USA). High-throughput sequencing was performed as paired-end 100 sequencing using NovaSeq 6000 (Illumina, Inc., USA). For data analysis, a quality control of raw sequencing data was performed using FastQC. Adapter and low-quality reads (< Q20) were removed using FASTX_Trimmer and BBMap. Then the trimmed reads were mapped to the reference genome using TopHat 36 . Gene expression levels of genes, isoforms and IncRNAs were estimated using FPKM values by Cufflinks 37 . The FRKM values were normalized based on TMM + CPM method using EdgeR within R. Data mining and graphic visualization were performed using ExDEGA. 5.5 Flow cytometry analysis Antigen analysis was performed on mob PBSCs and PBMCs. Cells that had been primed with FVA3-Ang1 were washed according to the washing protocol, by filling a 50 mL sterile tube with PBS and centrifuging at 1000 rcf for 3 minutes at room temperature. The supernatant was aspirated, and the cells were resuspended in 1 mL PBS and centrifuged under the same conditions. The cells were then resuspended in FACS buffer for staining. To confirm the cell characteristics of mob PBSCs and PBMCs, FITC-conjugated anti-CD31 (BD Pharmingen), PE-conjugated anti-VE-cadherin (BD), APC-conjugated anti-CXCR4 (BD), APC-conjugated anti-CD45 (BD), PE-conjugated anti-Tie2 (BD) and FITC-conjugated anti-Annexin V (BD) antibodies and Propidium Iodide(PI) were used. 5.6 Quantitative reverse Transcriptase-Polymerase chain reaction mRNA expression was quantified by real-time RT-PCR using a 7500 real-time PCR system according to the experimenter's protocol. The real-time reaction was performed in a 96-well plate with 3 replicates at a final volume of 20 µL and was normalized with GAPDH RNA. Relative quantitative real-time PCR for CD31, CXCR4, VE-cadherin, and Tie2 was performed using the TOYOBO protocol. The PCR conditions were as follows: Quantitative real-time PCR for binding was performed using the SYBGreen protocol. The RNA solution was incubated at 65℃ for 5 minutes, then cooled sufficiently on ice. 5x RT Master Mix was added to adjust the final volume to 20 µL, and the reaction was incubated at 37℃ for 15 minutes. cDNA synthesis was completed by incubating at 50℃ for 5 minutes and 98℃ for 5 minutes. The final product should be stored at 4℃ or -20℃. 5.7 Immunofluorescent staining To measure the capillary density, the paraffin-fixing sections of each group were stained with CD31 antibodies, and the muscle structure was also stained with Laminin antibodies, and anti-HLA-ABC was used as the primary antibody for the engraftment of the injected cells. Secondary antibodies were stained for 1 hour at room temperature using alexa-488 and alexa-555. Figures were obtained using confocal microscopy (Leica). 5.8 Matrigel tube formation Matrigel tube formation was carried out to confirm the angiogenesis network of COMP-Ang1 and FVA3-Ang1. We solidified 50 µL of Basement Matrigel by placing at least 30 minutes in a 37℃ incubator at 96well. COMP-Ang1 and FVA3-Ang1 were primed by concentration and by time and confirmed under various conditions. 1) Vehicles, 2) VEGF, 3) COMP, or FVA3 were treated. Under each condition, 1×10 4 HUVECs were seeded with 100 µL of EGM-2MV media with 2% FBS. We observed tubes formed after 24 hours and total length, branching length and nodes of complete tube formed by cells were quantified using ImageJ. 5.9 Western blot Groups treated under hourly and concentration-specific conditions with Angioinetin-1 and controls were lysed with RIPA buffer containing protease/phosphatase inhibitors and centrifuged at 15,000 RPM for 30 minutes at 4°C. The isolated proteins were separated on 4–15% SDS-PAGE gels and transferred onto a polyvinylidene difluoride membrane. It was then incubated overnight at 4°C by various primary antibodies after membrane blocking and subsequently subjected to appropriate secondary antibodies. Immunoblot signals were detected by Amersham 680, and the figures were quantified by ImageJ. 5.10 Mouse Hind-limb Ischemia and Myocardial Infarction Models The neovascularization of mob PBSCs primed with FVA3-Ang1 was confirmed using a mouse hindlimb model and myocardial infarction model. Anesthesia was performed by injecting alfaxalone 100 µL and Rompun 5 µL into the intraperitoneal space. Afterwards, anesthesia was maintained at 1.5 to 2% of isoflurane through a respiratory anesthesia. A hindlimb ischemia model was created by ligating the femoral artery of Balb/c nude mice (male, 8 weeks old). Primed or non-primed mob PBSCs (cell number of 1 x 10 5 ) were injected into the muscle around the ligated vessel in a volume of 30 µL. The experimental groups were (1) PBS (n = 5), (2) mob PBSCs only (n = 6), and (3) FVA3-Ang1 primed mob PBSCs (n = 6). Laser Doppler perfusion imaging (LDPI,) was performed four times, on days 0, 3, 7, and 14, starting from the day of cell injection. A myocardial infarction model was created by a 1 cm length incision on the left side of the torso and the thoracic cavity was opened through a microscope. The left descending artery of the mouse heart is tied with a surgical thread to prevent antegrade blood flow. The experimental groups were (1) PBS (n = 4), (2) mob PBSCs only (n = 10), and (3) FVA3-Ang1 400 ng/mL 1hr priming mob PBSCs (n = 10). The skin was sealed using a 6 − 0 black suture, and the gas was removed from the abdominal cavity using a 5 mL syringe connected to the catheter to form a negative pressure, and the suture was completed. At the end of the experiments, animals were anesthetized with alfaxalone and rompun, followed by diaphragmatic incision, exsanguination, and organ harvesting for euthanasia. All experiments were approved by the Institutional Animal Care and Use Committee in Seoul National University Hospital (SNUH-IACUC) and animals were maintained in the facility accredited AAALAC International (#001169) in accordance with Guide for the Care and Use of Laboratory Animals 8th edition, NRC (2010). 5.11 ELISA assay The human Angiopoietin 1 Quantikine ELISA kit (R&D Systems, Minneapolis, USA) was used to quantify residual Angiopoietin-1 levels in the serum at three different time points: (1) immediately after priming with Ang1, (2) after the first wash, and (3) after the final wash. 5.12 Trypan blue cell viability assay To evaluate cell viability following washing and resuspension, a Trypan blue exclusion assay was conducted. The viability of mob PBSCs was assessed under three conditions: cells primed with FVA3-Ang1 for 1 hour, cells resuspended in patient plasma after washing, and cells resuspended in normal saline after washing. Cells were stained with 0.4% Trypan blue for 3 minutes, and viable cells were determined using a hemocytometer following standard protocols. The percentage of viable cells was quantified. 5.13 Scratch assay HUVECs were plated in an ibidi Culture-Insert 2-well (µ-Dish 35 mm) with 4×10 4 cells per reservoir and incubated 24 h at 37°C and 5% CO₂ to reach confluence. Inserts were removed to create a defined 500 µm gap, wells were washed once with prewarmed PBS, and serum-free medium containing vehicle (control) or Ang-1, COMP-Ang1, or FVA3-Ang1 200 or 400 ng/mL was added. Images of the same fields were acquired at 0, 12, and 24 hours by phase-contrast microscopy. Wound areas were measured in ImageJ and percent closure calculated as [(area₀ − area_t)/area₀] x 100. Statistical analysis was by two-way ANOVA (factors: treatment, time); p < 0.05 was considered significant. 5.14 EdU assay HUVECs were grown in 35 mm confocal dishes(ibidi GmbH) to 80% confluency and treated with vehicle or Ang-1, COMP-Ang1, or FVA3-Ang1 200 or 400 ng/mL for 1 or 24 hours. After each treatment period, cells were incubated with 10 µM EdU (ClickTech EdU Cell Proliferation Kit 647 for IM, BCK-EDU647) for 30 min. Cells were fixed in 4% paraformaldehyde 15 min, permeabilized with 0.5% Triton X-100 10 min, RT and processed according to the manufacturer’s instructions for the Click reaction. Nuclei were counterstained with DAPI and imaged by confocal microscopy. EdU⁺ nuclei were quantified as a proportion of total DAPI nuclei (EdU⁺/DAPI) over ≥ 5 random fields per sample. Statistical analysis was by two-way ANOVA ; p < 0.05 was considered significant. 5.15 FITC-Dextran HUVECs were seeded onto Transwell polycarbonate membrane 0.4 µm inserts (Corning, 3412) at 1×10 5 cells in insert and cultured in complete endothelial medium until a confluent monolayer was achieved. Prior to treatment, inserts were washed once and equilibrated in prewarmed Hanks’ balanced salt solution (HBSS; H8264) for 30 min at 37°C. Cells were then incubated with vehicle or Ang-1, COMP-Ang1, or FVA3-Ang1 (200 or 400 ng/mL for 1 hour or 24 hours. After each treatment period, fluorescein isothiocyanate–dextran (FITC-Dextran) in HBSS 1 mg/mL was added to the apical chamber and plates were incubated at 37°C. After 30 min, 100 µL samples were taken from the basolateral chamber and transferred to a black 96-well plate; fluorescence was read on a plate reader (Ex/Em 485/520 nm). A standard curve of FITC-dextran was used to convert fluorescence units to amount transported. Permeability was expressed relative to vehicle control. Statistical analysis was by two-way ANOVA, p < 0.05 was considered significant. 5.16 Angiopoietin-1 AlphaFold modeling and multimer stability prediction Amino-acid sequences of COMP-Ang1 and FVA3-Ang were modelled using AlphaFold-Multimer via the ColabFold/AlphFold to predict multimeric assemblies. Models were generated with default ColabFold/AlphaFold settings and five models per prediction were produced and the top model was selected by the AlphaFold multimer ranking confidence. Selected top models were submitted to PDBePISA for interface analysis. From PISA we report interface buried surface area, the Complexation Significance Score (CSS) as an index of interface relevance to assembly formation, and the solvation free-energy gain on interface formation reported by PISA as Gint, together with the number of hydrogen bonds and salt bridges across the interfaces. CSS and ΔGint (Gint) were used to evaluate predicted interface energetics in conjunction with the AlphaFold ranking confidence. 5.17 Half-life measurement of Ang1 proteins Human umbilical vein endothelial cells (HUVECs) were cultured in 24-well plates until confluent. Recombinant human Ang1, COMP-Ang1, or FVA3-Ang1 was added to the culture medium at a final concentration of 400 ng/mL. At indicated time points (0, 0.5, 1, 2, 4, 8, 12, and 24 hours), aliquots of conditioned medium were collected and centrifuged at 300 × g for 5 minutes to remove cellular debris. The supernatants were stored at − 80°C until analysis. Remaining concentrations of Ang1 protein were quantified using a human angiopoietin-1 ELISA kit (R&D, DANG10) according to the manufacturer’s instructions. Protein half-lives were calculated by plotting Ang1 concentration decay curves over time and fitting the data to a one-phase exponential decay model. 5.18 Statistical analysis Continuous variables are summarized as means (standard deviations). Group comparisons for continuous variables were conducted using unpaired t-tests. The two-way ANOVA was performed for statistical analysis, and a p-value less than 0.05 was considered significant. No statistical significance correction for multiple comparisons was applied in this study. In cases where the assumptions for parametric tests (e.g., normality) were not met, nonparametric tests were used. Specifically, the Mann-Whitney U test was performed for two-group comparisons, while the Kruskal-Wallis H test was used for comparisons involving three or more groups. These tests were chosen to analyze the differences in ranked data, ensuring the robustness of our findings even with non-normally distributed variables. All statistical analyses were conducted using SPSS, V25 (SPSS Inc.) and R programming language, version 4·2·4 (R Foundation for Statistical Computing). Declarations Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Hyun-Jai Cho and Hyo-Soo Kim ( [email protected] , [email protected] ) Data Availability Statement The data supporting the findings of this study are available from the corresponding authors upon reasonable request. All RNA sequencing datasets supporting the findings of this study are publicly available in the NCBI Gene Expression Omnibus (GEO) repository under the accession code GSE304470. Acknowledgements This research was supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: RS-2023-KH143762. HX23C1754). 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Cornea 28:795–800. 10.1097/ICO.0b013e31819839c6 Roberts A, Trapnell C, Donaghey J, Rinn JL, Pachter L (2011) Improving RNA-Seq expression estimates by correcting for fragment bias. Genome Biol 12:R22. 10.1186/gb-2011-12-3-r22 Additional Declarations The authors declare no competing interests. Supplementary Files SupplementaryNewAng1forpriming20250915.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-7625543","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":515704807,"identity":"cf7b7312-c4f1-4a48-81db-53d545a6c73a","order_by":0,"name":"Jeehoon Kang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jeehoon","middleName":"","lastName":"Kang","suffix":""},{"id":515704808,"identity":"6e82dc23-e54c-45fc-8d45-7b91e8f07636","order_by":1,"name":"Hyun Ju Seo","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Hyun","middleName":"Ju","lastName":"Seo","suffix":""},{"id":515704809,"identity":"c3ab2c5b-3c64-41f7-9eb6-4f922ee90246","order_by":2,"name":"HyunJu Son","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"HyunJu","middleName":"","lastName":"Son","suffix":""},{"id":515704810,"identity":"f6035765-7db4-458b-9ff5-f2004daf5346","order_by":3,"name":"Minjun Kang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Minjun","middleName":"","lastName":"Kang","suffix":""},{"id":515704811,"identity":"cdda0607-18d3-40b1-8452-fcbe7064fa2d","order_by":4,"name":"Jaewon Lee","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jaewon","middleName":"","lastName":"Lee","suffix":""},{"id":515704812,"identity":"2af756d0-87cf-43b4-a86c-de5904ea4dac","order_by":5,"name":"Eun Ju Lee","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Eun","middleName":"Ju","lastName":"Lee","suffix":""},{"id":515704813,"identity":"a10cea11-ce6d-4340-911f-8b2eaece5cf4","order_by":6,"name":"Hyun-Jai Cho","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4ElEQVRIiWNgGAWjYBACAxAhAYQGDMwHoGJsRGthSyBBC4TBA2MT0GIukWP2wKLGQt6cvefz58I2Bnn+Bra0D/i0WM7IMTeQOCZhuLPn7DbpmW0MhjMOsB2egddhN3LMJCTYJBIMbuRuY+ZtY2DcwMDejN8vYC3/QFpyHn8GarEnTotkG1gLgzRQS+IGBrbD+LWceVYmIdknYbjhzDEzaZ5zEskzDrMl49dyPHmbtMS3OnmD482PP/OU2dj2t7cZ49XCIJDAwCyB4AKZzPg1MDDwH2BgxBsNo2AUjIJRMAoAEF9Abfcam0AAAAAASUVORK5CYII=","orcid":"","institution":"","correspondingAuthor":true,"prefix":"","firstName":"Hyun-Jai","middleName":"","lastName":"Cho","suffix":""},{"id":515704814,"identity":"11963ad4-b46a-4723-bf18-88cc95da536a","order_by":7,"name":"Hyo-Soo Kim","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Hyo-Soo","middleName":"","lastName":"Kim","suffix":""}],"badges":[],"createdAt":"2025-09-16 03:54:29","currentVersionCode":1,"declarations":{"humanSubjects":true,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":true,"humanSubjectConsent":true,"humanSubjectClinicalTrial":true,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-7625543/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7625543/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91565567,"identity":"0ce7fcda-28aa-4cbc-b4e9-758f5070981c","added_by":"auto","created_at":"2025-09-17 19:24:06","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4120936,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDevelopment and Validation of the FVA3-Ang1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Due to the fragile clinical-grade protein purification of COMP-Ang1, various domains such as COMP, Phe14, VASP and VASP2 were constructed as an alternative of COMP. (B) Using COMP, Phe14, VASP1 and VASP2, multiple Ang1 overexpression vectors were constructed. These vectors were combined at the specifically selected Hot-spot site 13. (C) A transient expression assays with various combinations showed that Phe14 and COMP exhibited the formation of multiple oligomeric complexes, while the VASP2 exclusively formed tetramers of Ang1 without forming monomers or dimers. (D) Predicted tetrameric structures of FVA3-Ang1 and COMP-Ang1 generated using Colab/AlphaFold. (E) PDBePISA analysis of AlphaFold3-predicted multimeric structures showing the interface stability metrics for FVA3-Ang1 and COMP-Ang1 tetramers.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7625543/v1/52b3fa2592f2e42bb0b263a7.png"},{"id":91564919,"identity":"d8aad68f-0893-460e-a354-83c3c7114565","added_by":"auto","created_at":"2025-09-17 19:16:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1985457,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of COMP-Ang1 and FVA3-Ang1 in vitro\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) In quantitative RT-PCR analysis, the RNA expression levels of ANGP1, ANGP2, VEGFA, FGF2, and KDR in HUVECs significantly increased only by 24-hour FVA3-Ang1 treatment (n=3) and then by naïve Ang1 treatment (n=3). Control group was treated with PBS (n=3). (****: p\u0026lt;0.0001 using Mann-Whitney test, Mean ± SEM) (B) To test the priming effect of the novel FVA3-Ang1 protein, HUVECs were treated with FVA3-Ang1 and COMP-Ang1 at 200 ng/mL and 400 ng/mL for 1 hour (n=4). (**: p\u0026lt;0.01 using Mann-Whitney test, Mean ± SEM). Phosphorylation of AKT, ERK and Tie2 showed a concentration-dependent relationship, which was superior with FVA3-Ang1 than with COMP-Ang1 and naïve Ang1. (C) Priming HUVECs with FVA3-Ang1 protein at 200 ng/mL and 400 ng/mL for 24 hours showed enhanced protein expression of endothelial cell lineage markers (CD31, VE-CAD, and eNOS n=3) ) (*: p\u0026lt;0.05, **: p\u0026lt;0.01 using Mann-Whitney test, Mean ± SEM). (D) A wound healing assay was performed to confirm the angiogenic potential induced by Ang1 treatment. HUVECs were treated with Ang1, COMP-Ang1, or FVA3-Ang1 at concentrations of 200 ng/mL or 400 ng/mL, for 1 hour or 24 hours. (E) Quantification of wound area percentage at 0, 12, and 24 hours in HUVECs wound healing assays. Statistical analysis was performed using two-way ANOVA (n=3). (*: p\u0026lt;0.05, **: p\u0026lt;0.01, ***: p\u0026lt;0.001 using two-way ANOVA).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7625543/v1/5c76c57c62328a51cf37134f.png"},{"id":91564923,"identity":"a3ecb770-4716-4e00-a3c6-037bd6ee9f73","added_by":"auto","created_at":"2025-09-17 19:16:06","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":502202,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenetic assay of FVA3-Ang1 primed \u003c/strong\u003e\u003csup\u003e\u003cstrong\u003emob\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003ePBSC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A, B) Gene expression patterns were confirmed through total-RNA sequencing. Of the 26,137 reliable probes, the gene expression pattern was similar for the 1-hour primed \u003csup\u003emob\u003c/sup\u003ePBSCs and naïve \u003csup\u003emob\u003c/sup\u003ePBSCs, while the pattern was distinct in 24-hour primed \u003csup\u003emob\u003c/sup\u003ePBSCs. In a heatmap analysis, 59 genes were up-regulated genes, and 243 genes were down-regulated, compared to the other two groups(n=3). (C) By a genetic ontology analysis, the up-regulated and down-regulated genes were characterized.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7625543/v1/01683cfef2107f52819d5812.png"},{"id":91564918,"identity":"9a795339-7627-4ea2-be5c-acfa8d165090","added_by":"auto","created_at":"2025-09-17 19:16:06","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":930852,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRNA and protein expression modification by FVA3-Ang1 priming\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) In quantitative RT-PCR analysis, the RNA expression level of CXCR4 and VE-CAD in \u003csup\u003emob\u003c/sup\u003ePBSC significantly increased only by 24-hour FVA3-Ang1 priming (n=3). The Tie2 level was increased by a 1-hour(n=3) and 24-hour priming. Control groups consisted of PBMC (n=4) and \u003csup\u003emob\u003c/sup\u003ePBSC (n=3). (**: p\u0026lt;0.01 using Mann-Whitney test, Mean ± SEM). (B, C) In a flow cytometry analysis, \u003csup\u003emob\u003c/sup\u003ePBSC primed with FVA3-Ang1 for 1 hour (n=5) and 24 hours (n=4) exhibited a trend of increase in CD31, CXCR4, VE-CAD protein expression, a decrease in CD45, and no difference in Annexin V expression. Control groups consisted of PBMC (n=3) and \u003csup\u003emob\u003c/sup\u003ePBSC (n=5) (*: p\u0026lt;0.05, **: p\u0026lt;0.01, ***: p\u0026lt;0.001 using Mann-Whitney test, Mean ± SEM).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7625543/v1/1007c54a3d2953584cd0db89.png"},{"id":91564922,"identity":"e9a6cb44-fe35-44fd-a6b2-db54b2466585","added_by":"auto","created_at":"2025-09-17 19:16:06","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1739226,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCell therapy for the mouse hindlimb ischemia model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) The scheme of \u003cem\u003ein vivo\u003c/em\u003e animal experiments. After successful revascularization for a patient diagnosed as acute myocardial infarction, the patient will receive a systemic injection of EPO, followed by subcutaneous administration of G-CSF for the subsequent three days. \u003csup\u003emob\u003c/sup\u003ePBSCs are collected by apheresis, and the collected \u003csup\u003emob\u003c/sup\u003ePBSCs will be undergo \u003cem\u003eex vivo\u003c/em\u003e FVA3-Ang1 priming for one hour. The FVA3-Ang1 primed \u003csup\u003emob\u003c/sup\u003ePBSCs were then injected into mouse models of hindlimb ischemia and myocardial infarction. (B) For the mouse hindlimb ischemia model, three groups were tested; the vehicle group (n=5), the naïve \u003csup\u003emob\u003c/sup\u003ePBSC group (n=6), and the FVA3-Ang1 primed \u003csup\u003emob\u003c/sup\u003ePBSC group (n=6). After the right femoral arterial was ligated, an injection of 1×10\u003csup\u003e5\u003c/sup\u003e cells to the peripheral muscle was performed. LDPI was performed at 0, 3, 7, and 14 days after surgery, and histologic assessment was performed at day 14. (C) Gross results showed an improvement in limb salvage in the FVA3-Ang1 primed \u003csup\u003emob\u003c/sup\u003ePBSC group. (D, E) LDPI results demonstrated the best recovery of blood flow in FVA3-Ang1 primed \u003csup\u003emob\u003c/sup\u003ePBSC group (**: p\u0026lt;0.01 using Mann-Whitney test, Mean ± SEM). (F, G) Immunohistochemistry analysis confirmed superior active neovascularization by expression of CD31 (PBS; n=6, \u003csup\u003emob\u003c/sup\u003ePBSC; n=5, FVA3-Ang1 primed \u003csup\u003emob\u003c/sup\u003ePBSC; n=5, Mean ± SEM). (H, I) Immunohistochemistry analysis confirmed superior engraftment by expression of HLA-ABC in the FVA3-Ang1 primed \u003csup\u003emob\u003c/sup\u003ePBSC group (PBS; n=6, \u003csup\u003emob\u003c/sup\u003ePBSC; n=5, FVA3-Ang1 primed \u003csup\u003emob\u003c/sup\u003ePBSC; n=5, Mean ± SEM). (*: p\u0026lt;0.05, **: p\u0026lt;0.01 using Mann-Whitney test, Mean ± SEM).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7625543/v1/c3091f6ccb8d134be0a78ac8.png"},{"id":91564921,"identity":"351f844a-c0c6-4f90-92a4-3f71e1ac1e21","added_by":"auto","created_at":"2025-09-17 19:16:06","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1808024,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCell therapy for the mouse myocardial infarction model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) For the mouse myocardial infarction model, the left anterior descending artery was ligated and 1 x 10\u003csup\u003e5\u003c/sup\u003e cells were injected into the infarcted anterior myocardium, and echocardiographic and histologic assessment was performed on day 14. Three groups were tested; the vehicle group (n=10), the naïve \u003csup\u003emob\u003c/sup\u003ePBSC group (n=10), and the FVA3-Ang1 primed \u003csup\u003emob\u003c/sup\u003ePBSC group(n=10). (B) The overall survival is showed that the FVA3-Ang1 primed \u003csup\u003emob\u003c/sup\u003ePBSC group had the best survival (*: p\u0026lt;0.05 using Mann-Whitney test, Mean ± SEM). (C) Echocardiographic measurements revealed that FVA3-Ang1 primed \u003csup\u003emob\u003c/sup\u003ePBSC group (n=10) demonstrated superior results in terms of LVESD, LVEDD, LVFS, and LVEF compared to the PBS (n=4) and \u003csup\u003emob\u003c/sup\u003ePBSC groups (n=10). (*. p\u0026lt;0.05. **: p\u0026lt;0.01, ***: p\u0026lt;0.001 using Mann-Whitney test, Mean ± SEM). (D, E) Three groups were tested; the vehicle group (n=10), the naïve \u003csup\u003emob\u003c/sup\u003ePBSC group (n=10), and the FVA3-Ang1 primed \u003csup\u003emob\u003c/sup\u003ePBSC group(n=10). Masson-Trichrome staining of the myocardial infarction heart showed that the fibrosis area was reduced in the FVA3-Ang1 primed \u003csup\u003emob\u003c/sup\u003ePBSC group (*: p\u0026lt;0.05 using Mann-Whitney test, Mean ± SEM). (F, G) Immunohistochemistry analysis of heart tissue used cTnT and confirmed superior active neovascularization by expression of CD31 (n=6) (**: p\u0026lt;0.01 using Mann-Whitney test, Mean ± SEM). (H, I) Immunohistochemistry analysis (n=6) confirmed superior engraftment by expression of HLA-ABC in the FVA3-Ang1 primed \u003csup\u003emob\u003c/sup\u003ePBSC group. (**: p\u0026lt;0.01 using Mann-Whitney test, Mean ± SEM)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAbbreviations:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLVEDD, left ventricular end diastolic diameter; LVEF, left ventricular ejection fraction; LVESD, left ventricular end systolic diameter; LVFS, left ventricular fractional shortening.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7625543/v1/04c8d24778d481a22b4ae152.png"},{"id":91565568,"identity":"eb5c6f78-11f8-4326-9780-469f1b0d5d5a","added_by":"auto","created_at":"2025-09-17 19:24:06","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":619380,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProtocol of the MAGIC Cell-6 clinical trial\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Patients diagnosed with acute myocardial infarction, who received successful percutaneous coronary intervention will be enrolled in the MAGIC Cell-6 trial (clinicaltrials.gov ID: NCT06364150). After systemic injection of erythropoietin and G-CSF, \u003csup\u003emob\u003c/sup\u003ePBSC will be collected by an apheresis process. In a GMP facility, \u003cem\u003eex vivo\u003c/em\u003e FVA3-Ang1 priming will be done at 400 ng/ml for 1 hour at 37℃. The plasma of the patient will be separately collected, for cell washing and suspension. After FVA3-Ang1 priming, \u003csup\u003emob\u003c/sup\u003ePBSC will be washed twice to remove remnant FVA3-Ang1. For the final product, the cell number will be counted, and an Endotoxin and Mycoplasma analysis will be performed to screen contamination. Then, a total of 2 X 10\u003csup\u003e9\u003c/sup\u003e cells will be suspended into 10 mL of the patients plasma, followed by intracoronary infusion.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7625543/v1/eb6b0b6db52cfa8c5b7cf515.png"},{"id":91566383,"identity":"1b519910-1287-4fdb-951b-d63959f0345f","added_by":"auto","created_at":"2025-09-17 19:40:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":13169952,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7625543/v1/ec98d371-a7ed-4bc6-93fc-30defc4b10ad.pdf"},{"id":91565570,"identity":"ecb0a9cf-438f-4aa9-9251-764566a4a252","added_by":"auto","created_at":"2025-09-17 19:24:06","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":8659050,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryNewAng1forpriming20250915.docx","url":"https://assets-eu.researchsquare.com/files/rs-7625543/v1/eb76c84fecd5593a3a0d236f.docx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eNewly-engineered angiopoietin-1 as a cell-priming agent for CVD\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eIschemic diseases are characterized by restricted blood supply to specific tissues, leading to necrosis and organ dysfunction \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Due to the limited efficacy of current treatment options, these conditions often result in long-term complications for patients, imposing a significant clinical burden. Cell therapy has emerged as a promising therapeutic strategy; however, its efficacy requires further optimization \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Previously, we demonstrated that intracoronary infusion of granulocyte colony-stimulating factor (G-CSF)-mobilized human peripheral blood stem cells (\u003csup\u003emob\u003c/sup\u003ePBSCs) from bone marrow can promote myocardial recovery in patients with acute myocardial infarction \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eFurthermore, enhanced mobilization of peripheral blood monocyte cells (PBMCs) using a combination of erythropoietin (EPO) and G-CSF led to improved cardiac function. However, the low efficiency of engraftment in ischemic myocardium and the limited evidence of sustained cardiac function recovery remain key challenges that must be addressed before broader clinical application \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAngiopoietin 1 (Ang1) is a well-characterized protein that promotes angiogenesis by mediating endothelial cell migration, adhesion and survival through the Tie-2 tyrosine kinase receptor \u003csup\u003e\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Given its therapeutic potential, Ang1 has been investigated for treating vascular pathologies, lung infections, and neurological disorders \u003csup\u003e\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. We previously identified that \u003csup\u003emob\u003c/sup\u003ePBSCs express the Tie2 receptor on their surface and demonstrated that \u003cem\u003eex vivo\u003c/em\u003e Ang1 priming could enhance their therapeutic efficacy in ischemic disease treatment \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. In this study, we developed a unique cartilage oligomeric matrix protein (COMP)-Ang1 construct for priming purposes. COMP-Ang1 replaces the central coiled-coil domain region of natural Ang1 with COMP, whose \u0026lsquo;ECDACG\u0026rsquo; sequence in the C-terminal region forms disulfide bonds with other alpha-helices, thereby stabilizing its oligomeric structure \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. However, despite promising preclinical results, COMP-Ang1 proved unsuitable for therapeutic application due to instability in large-scale production. To overcome these limitations, our study aimed to develop a more stable form of Ang1 optimized for large-scale production and clinical application. Additionally, we sought to address concerns that the priming process might alter the differentiation state of \u003csup\u003emob\u003c/sup\u003ePBSCs or induce reprogramming \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, potentially leading to unintended tumorigenic complications.\u003c/p\u003e\u003cp\u003eIn this study, we engineered Ang1 construct by adding VASP and COMP sequence for the stabilized tetramer formation because multimeric Ang1 is more potent than na\u0026iuml;ve monomer by clustering Tie1 receptors leading to strong angiogenic or vasculogenic signal. Furthermore, we added FLAG sequence for purification process of this protein in large scale production and added a signal peptide derived from influenza A virus (IAV) for better protein expression.\u003c/p\u003e\u003cp\u003eWe characterized this novel form of Ang1 (FVA3-Ang1) regarding its potency to stimulate endothelial cells and suitability for \u003cem\u003eex vivo\u003c/em\u003e priming of PBSC. Furthermore, to translate our findings into clinical practice, we initiated a clinical trial to evaluate the safety and efficacy of Ang1-primed \u003csup\u003emob\u003c/sup\u003ePBSCs in patients with myocardial infarction. By overcoming previous challenges associated with Ang1-based cell therapy, we aim to provide a scalable and effective treatment for ischemic diseases.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Engineering of FVA3-Ang1 protein that is more potent than na\u0026iuml;ve Ang1\u003c/h2\u003e\u003cp\u003eWe developed a novel form of Ang1 designed as a stable \u003cem\u003eex vivo\u003c/em\u003e priming agent. To enhance protein expression, we incorporated a signal peptide derived from either Azurocidin or influenza A virus, along with a FLAG tag at the N-terminal region \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Given the challenges associated with clinical-grade purification of COMP-Ang1 due to its structural fragility \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, we explored alternative domains, including Phe14 \u003csup\u003e19\u003c/sup\u003e and human vasodilator-stimulated phosphoprotein (VASP) \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. VASP, predominantly expressed as a tetramer, was hypothesized to provide superior structural stability compared to COMP. Additionally, because the \"ECDACG\" sequence in the C-terminal region of COMP is responsible for stabilizing the oligomeric structure, we integrated this sequence into the C-terminal region of VASP, resulting in a modified construct termed VASP2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo achieve high expression levels, we integrated the gene into a genomic hotspot using the Cas9D10A system (Sigma-Aldrich; Supplementary Fig.\u0026nbsp;1A) and identified hotspot site 13 as the optimal location due to its strong GFP expression (\u003cb\u003eSupplementary Fig.\u0026nbsp;1B\u003c/b\u003e). We constructed multiple Ang1 overexpression vectors incorporating COMP, Phe14, VASP1, and VASP2 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Transient expression assays demonstrated poor oligomerization for constructs containing Phe14 and COMP, whereas the FVA3 construct (IAV-FLAG-VASP2-Ang1) consistently formed tetramers (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). To corroborate these biochemical observations and provide structural insights, we performed in silico modeling using AlphaFold3 to predict the monomeric and putative tetrameric assemblies of FVA3-Ang1 and COMP-Ang1. The modeled tetrameric architecture for FVA3-Ang1 is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, whereas the COMP-Ang1 models displayed less consistent interface packing. Multimer stability was quantified using PDBePISA analysis of AlphaFold-Multimer tetramer models, which revealed that FVA3-Ang1 has substantially larger buried interface areas and more negative predicted interaction energies than COMP-Ang1. FVA3-Ang1 exhibited a 10\u0026ndash;20% larger interface area and stronger binding energy compared with COMP-Ang1, suggesting enhanced stability of the complex. Moreover, the complex significance score, which was close to 1.0, indicates that this interaction is physiologically meaningful and is predicted to form a stable tetramer (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). To further validate the enhanced stability of FVA3-Ang1, we measured the half-lives of all three Ang1 variants in HUVEC culture medium using ELISA. Interestingly, all three proteins demonstrated comparable stability with no significant differences in degradation rates over the tested time course, suggesting that the superior functional performance of FVA3-Ang1 is attributed to its enhanced oligomerization rather than intrinsic protein stability in the culture environment (\u003cb\u003eSupplementary Fig.\u0026nbsp;1D\u003c/b\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eAng1 overexpression vectors\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVector name\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFCA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHot spot 13\u0026thinsp;+\u0026thinsp;mCMV\u0026thinsp;+\u0026thinsp;Influenza A virus\u0026thinsp;+\u0026thinsp;FLAG\u0026thinsp;+\u0026thinsp;COMP\u0026thinsp;+\u0026thinsp;Ang1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFPA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHot spot 13\u0026thinsp;+\u0026thinsp;mCMV\u0026thinsp;+\u0026thinsp;Influenza A virus\u0026thinsp;+\u0026thinsp;FLAG\u0026thinsp;+\u0026thinsp;Phe14\u0026thinsp;+\u0026thinsp;Ang1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFPA2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHot spot 13\u0026thinsp;+\u0026thinsp;mCMV\u0026thinsp;+\u0026thinsp;Influenza A virus\u0026thinsp;+\u0026thinsp;FLAG\u0026thinsp;+\u0026thinsp;Phe14 with a disulfide bridge\u0026thinsp;+\u0026thinsp;Ang1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFVA2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHot spot 13\u0026thinsp;+\u0026thinsp;mCMV\u0026thinsp;+\u0026thinsp;Azurocidin\u0026thinsp;+\u0026thinsp;FLAG\u0026thinsp;+\u0026thinsp;VASP with a disulfide bridge\u0026thinsp;+\u0026thinsp;Ang1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFVA3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHot spot 13\u0026thinsp;+\u0026thinsp;mCMV\u0026thinsp;+\u0026thinsp;Influenza A virus\u0026thinsp;+\u0026thinsp;FLAG\u0026thinsp;+\u0026thinsp;VASP with a disulfide bridge\u0026thinsp;+\u0026thinsp;Ang1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"2\"\u003eCOMP, Cartilage oligomeric matrix protein; mCMV, murine cytomegalovirus; Phe14, Phe14 Phenylalanine; VASP, vasodilator-stimulated phosphoprotein\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eBased on these findings, the optimal configuration\u0026mdash;hotspot site 13 with an influenza A virus signal peptide and VASP2\u0026mdash;was designated FVA3-Ang1. For scalable production, we established a stable Ang1-expressing CHO-K1 cell line cultured in CDM4CHO medium (Cytiva\u0026trade;). Clonal selection was performed using ClonePix FL (\u003cb\u003eSupplementary Fig.\u0026nbsp;1C\u003c/b\u003e), and the final cell line, named CHO-K1DW, was confirmed to exhibit robust growth and stability. By integrating the FVA3-Ang1 gene into hotspot site 13 of CHO-K1DW cells, we developed a reliable system for the large-scale production of Ang1.\u003c/p\u003e\u003cp\u003e\u003cb\u003e2.2 FVA3-Ang1 is stronger than na\u0026iuml;ve Ang1 or COMP-Ang1 in stimulating endothelia cells under\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e \u003cb\u003eHUVEC experiment\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePreliminary in vitro analyses comparing na\u0026iuml;ve Ang1, COMP-Ang1, and FVA3-Ang1 were conducted to evaluate signal transduction, gene expression, and angiogenic potential. These comparative studies revealed that FVA3-Ang1 exhibited the most potent biological effects, followed by COMP-Ang1, whereas naive Ang1 showed baseline activity. Based on these findings and considering the limited scope of in vivo studies, we investigated the therapeutic efficacy of FVA3-Ang1 as a lead candidate. To evaluate the transcriptional responses to Ang1 variants, we measured the mRNA levels of pro-angiogenic factors in HUVECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Treatment of HUVEC with each Ang1 variant increased the transcript levels of ANGPT1, ANGPT2, FGF2, VEGFA, and KDR relative to the vehicle control. The increases elicited by FVA3-Ang1 and COMP-Ang1 were more pronounced than those induced by na\u0026iuml;ve Ang1, consistent with their enhanced pro-angiogenic activities. We analyzed the phosphorylation of Tie1 receptor as well as Akt and ERK in HUVECs following treatment with na\u0026iuml;ve Ang1, COMP-Ang1, or FVA3-Ang1. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, all Ang1 variants induced phosphorylation of Tie2, AKT and ERK compared to the vehicle control. Phosphorylated Tie2, ERK, and AKT were quantified and normalized to their respective total protein levels. Notably, FVA3-Ang1 and COMP-Ang1 elicited stronger and more sustained activation of Tie2, ERK, and Akt than na\u0026iuml;ve Ang1. Quantitative densitometric analysis revealed that the phosphorylation levels of Tie2, ERK, and Akt were significantly higher in the FVA3-Ang1 group than in the na\u0026iuml;ve Ang1 group. These results indicate that FVA3-Ang1 preserves the signaling capacity of COMP-Ang1 and confers enhanced activation compared with na\u0026iuml;ve Ang1, supporting its potential as a functionally improved Ang1 variant \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. Finally, 24 hours treatment with FVA3-Ang1 promoted endothelial lineage characteristics (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The expression of the endothelial markers CD31, VE-Cadherin (VECAD), and eNOS increased in a dose-dependent manner, with the greatest upregulation observed at 400 ng/mL. Together, the transcriptional, signaling, and phenotypic data support that FVA3-Ang1 is a functionally improved Ang1 variant with robust pro-angiogenic activity in HUVECs. To assess the angiogenic activity of the three Ang1 variants, we performed a wound-healing assay using HUVECs. While naive Ang1 showed no significant difference compared to controls, both COMP-Ang1 and FVA3-Ang1 significantly promoted wound closure. Notably, FVA3-Ang1 had the most pronounced effect, leading to greater reduction in wound area at both 12 and 24 h in a dose- and time-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-E). These results confirm that FVA3-Ang1 enhances endothelial cell function and angiogenic potential while maintaining stability and efficacy. We further evaluated angiogenic potential using a tube formation assay. HUVECs were treated with na\u0026iuml;ve Ang1, COMP-Ang1 or FVA3-Ang1 at 200 ng/mL and 400 ng/mL for either 1 hour or 24 hours before being seeded onto Matrigel \u003cb\u003e(Supplementary Fig.\u0026nbsp;2A)\u003c/b\u003e. Tube formation was observed at both concentrations and time points, with no significant difference between 1-hour and 24-hour treatment durations \u003cb\u003e(Supplementary Fig.\u0026nbsp;2B).\u003c/b\u003e Next, we examined endothelial cell proliferation using EdU incorporation assay. After 1 and 24 hours of stimulation, native Ang1 did not increase the proportion of EdU-positive cells compared to that in the untreated controls. In contrast, both COMP-Ang1 and FVA3-Ang1 significantly enhanced HUVEC proliferation, with FVA3-Ang1 consistently yielding the highest percentage of proliferating cells (\u003cb\u003eSupplementary Fig.\u0026nbsp;2C-2E\u003c/b\u003e). To evaluate endothelial barrier integrity, we performed immunofluorescence staining for ZO-1 and Claudin-5. At both 1 and 24 h, naive Ang1 failed to improve tight junction organization compared to the controls. In contrast, COMP-Ang1 and particularly FVA3-Ang1 markedly reinforced the continuity and intensity of ZO-1 and Claudin-5 staining, indicating tighter junctional complexes (\u003cb\u003eSupplementary Fig.\u0026nbsp;2F-2K\u003c/b\u003e). Quantitative analysis confirmed that FVA3-Ang1 produced the greatest improvement in tight junction integrity compared to other treatments. Consistent with these findings, FITC-dextran permeability assay demonstrated that FVA3-Ang1 treatment resulted in the most robust reduction in paracellular leakage at both 1 and 24 hours, whereas COMP-Ang1 showed intermediate effects and naive Ang1 had minimal impact (\u003cb\u003eSupplementary Fig.\u0026nbsp;2L\u003c/b\u003e). Collectively, these data indicate that na\u0026iuml;ve Ang1 exerts little influence on angiogenesis or endothelial barrier stabilization in vitro, whereas FVA3-Ang1 demonstrates superior activity compared to COMP-Ang1 and represents the most potent variant in multiple functional assays.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Whole mRNA expression profiles of \u003csup\u003emob\u003c/sup\u003ePBSC after priming with FVA3-Ang1\u003c/h2\u003e\u003cp\u003eWe evaluated the FVA3-Ang1 priming strategy with a dual focus: ensuring safety by assessing modifications in cell characteristics and enhancing therapeutic efficacy by augmenting angiogenic potential. Gene expression patterns were analyzed via total RNA sequencing of \u003csup\u003emob\u003c/sup\u003ePBSCs collected from patients diagnosed with acute myocardial infarction (GEO. The analysis included three groups: naive \u003csup\u003emob\u003c/sup\u003ePBSCs, 1-hour primed \u003csup\u003emob\u003c/sup\u003ePBSCs, and 24-hour primed-\u003csup\u003emob\u003c/sup\u003ePBSCs. Among 26,137 reliable probes, gene expression patterns in the 1-hour primed \u003csup\u003emob\u003c/sup\u003ePBSCs closely resembled those of na\u0026iuml;ve \u003csup\u003emob\u003c/sup\u003ePBSCs. However, in the 24-hour primed \u003csup\u003emob\u003c/sup\u003ePBSCs, 59 genes were upregulated and 243 genes were downregulated compared to the other two groups, as illustrated in the heatmap analysis (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Gene ontology analysis revealed that 24-hour priming altered cell characteristics by upregulating genes related to protein stabilization and growth factor receptor signaling pathway while downregulating genes associated with cell adhesion, and innate immune response (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further evaluate the effects of FVA3-Ang1 priming, qRT-PCR analysis was performed on \u003csup\u003emob\u003c/sup\u003ePBSCs primed for 1 hour and 24 hours, with all cells harvested at the 24-hour time point. Compared to PBMCs (negative control) and na\u0026iuml;ve \u003csup\u003emob\u003c/sup\u003ePBSC, 1-hour priming did not significantly increase CD31, CXCR4, or VE-CAD expression, while 24-hour priming resulted in elevated CXCR4 and VE-CAD expression. The Tie2 receptor expression increased following both 1-hour and 24-hour priming (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Flow cytometry analysis demonstrated a gradual increase in CD31, CXCR4, Tie2 and VE-CAD protein expression from PBMCs to na\u0026iuml;ve \u003csup\u003emob\u003c/sup\u003ePBSCs and further in 1-hour primed \u003csup\u003emob\u003c/sup\u003ePBSCs. However, this increase was only statistically significant in the 24-hour primed group. Additionally, CD45 expression showed a gradual decrease, which was significant only after 24-hour priming with FVA3-Ang1. Notably, Annexin V expression remained unchanged across all groups, indicating no increase in apoptosis (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e2.4 Regenerative efficacy of\u003c/b\u003e \u003csup\u003e\u003cb\u003emob\u003c/b\u003e\u003c/sup\u003e\u003cb\u003ePBSC is improved by priming with FVA3-Ang1 in the hind-limb ischemia or myocardial infarction mice model\u003c/b\u003e\u003c/p\u003e\u003cp\u003eConsidering that COMP-Ang1\u0026rsquo;s in vivo efficacy was already established in our previous study, the current study evaluated only the in vivo effects of FVA3-Ang1\u003csup\u003e13\u003c/sup\u003e. To evaluate the therapeutic potential of 1-hour FVA3-Ang1-primed \u003csup\u003emob\u003c/sup\u003ePBSCs, we conducted experiments in mouse models of hind-limb ischemia and myocardial infarction (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). While extended priming periods (e.g., 24 hours) may potentially enhance therapeutic efficacy, we employed a 1-hour priming protocol to ensure compliance with regulatory guidelines that restrict cellular modifications that could alter fundamental cell characteristics or lineage commitment. This approach maintains the inherent properties of PBSCs while providing sufficient angiopoietin-1 priming to enhance their therapeutic potential. In the hind-limb ischemia model, the right femoral artery was ligated, followed by an injection of 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells into the peripheral muscle. Laser Doppler Perfusion Imaging (LDPI) was performed at 0, 3, 7, and 14 days post-surgery (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The FVA3-Ang1 primed \u003csup\u003emob\u003c/sup\u003ePBSC group exhibited the highest efficacy in blood flow recovery and tissue regeneration (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC\u0026ndash;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Immunohistochemistry analysis confirmed active neovascularization, as evidenced by increased CD31 expression (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). Additionally, HLA-ABC expression demonstrated superior engraftment of primed cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI) in the FVA3-Ang1 primed \u003csup\u003emob\u003c/sup\u003ePBSC group compared to controls. HLA-ABC fluorescence intensity was normalized to DAPI intensity for each field to correct for variations in cell number and staining.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo evaluate cell therapy efficacy in a myocardial infarction model, the left anterior descending artery was ligated in mice, followed by the injection of 1 x 10\u003csup\u003e5\u003c/sup\u003e cells into the infarcted anterior myocardium. Echocardiographic and histologic assessments were performed on day 14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The overall survival rate is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB. Echocardiographic analysis revealed superior functional improvement in the FVA3-Ang1 primed-\u003csup\u003emob\u003c/sup\u003ePBSC group, demonstrating enhanced left ventricular function and reduced ventricular dilation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Histologic assessment showed a significant reduction in fibrosis in the FVA3-Ang1-primed \u003csup\u003emob\u003c/sup\u003ePBSC group (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE), with superior neovascularization (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG) and engraftment (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI). Notably, this study was not designed to assess the transdifferentiation of PBSCs into cardiomyocytes; instead, we focused on their paracrine effects and endothelial interactions. Although a small number of engrafted cells may theoretically adopt a myogenic phenotype, we did not observe or quantify definitive cardiomyocyte differentiation or formation of striated muscle structures in the myocardial model.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e2.5 Clinical trial, \u0026ldquo;MAGIC-CELL-6-PRIMING\u0026rdquo;, to evaluate the efficacy and safety of\u003c/b\u003e \u003csup\u003e\u003cb\u003emob\u003c/b\u003e\u003c/sup\u003e\u003cb\u003ePBSC that were primed with FVA3-Ang1 in patients with AMI\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo evaluate the efficacy and safety of \u003csup\u003emob\u003c/sup\u003ePBSC in human cardiovascular diseases, we are conducting a clinical trial titled \u0026ldquo;Myocardial Regeneration and Angiogenesis in Myocardial Infarction with Intracoronary Infusion of Mobilized Peripheral Blood Stem Cells after Priming with FVA3-Ang1, 6th version (MAGIC Cell-6 Priming).\u0026rdquo; This investigator-initiated, single-arm trial is being performed at Seoul National University Hospital, South Korea (KCT0006753, NCT06364150). The inclusion and exclusion criteria are outlined in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, and a detailed study protocol is depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e. In brief, patients diagnosed with acute myocardial infarction will undergo percutaneous coronary intervention to the culprit coronary artery in accordance with standard clinical procedures. To facilitate peripheral blood stem cell mobilization, patients will receive a single bolus infusion of long-acting EPO followed by three days of daily subcutaneous injections of G-CSF. Apheresis will then be performed to collect \u003csup\u003emob\u003c/sup\u003ePBSC. In an \u003cem\u003eex vivo\u003c/em\u003e setting, \u003csup\u003emob\u003c/sup\u003ePBSC will be primed with FVA3-Ang1 for one hour. After priming, the cells will undergo two rounds of washing to minimize residual FVA3-Ang1 in the final product, in accordance with regulatory requirements in Korea, as separate clinical safety data for this protein are not yet available. A total of 2 x10\u003csup\u003e9\u003c/sup\u003e FVA3-Ang1-primed \u003csup\u003emob\u003c/sup\u003ePBSCs will then be infused into the infarct myocardium through the culprit coronary artery. The infusion cell count was determined based on previous MAGIC CELL trials \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eInclusion and Exclusion criteria of the MAGIC Cell-6 study\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eInclusion criteria\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1. Male and female aged\u0026thinsp;\u0026ge;\u0026thinsp;19 years and \u0026le;\u0026thinsp;80 years\u003c/p\u003e\u003cp\u003e2. A clinical diagnosis of acute myocardial infarction within 4 weeks from randomization\u003c/p\u003e\u003cp\u003e3. Successful percutaneous coronary intervention to the target lesion (TIMI flow grade 3 and residual stenosis\u0026thinsp;\u0026lt;\u0026thinsp;30% at the target lesion) with a drug-eluting stent and/or drug-eluting balloon\u003c/p\u003e\u003cp\u003e4. Agreement to give written informed consent.\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eExclusion criteria\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1. Patients with uncontrolled heart failure (Killip class\u0026thinsp;\u0026ge;\u0026thinsp;grade 2, or left ventricular ejection fraction\u0026thinsp;\u0026lt;\u0026thinsp;20%)\u003c/p\u003e\u003cp\u003e2. Patients with uncontrolled chest pain due to ischemia\u003c/p\u003e\u003cp\u003e3. Patients with uncontrolled arrythmia\u003c/p\u003e\u003cp\u003e4. Active malignancy, or incompletely treated malignancy\u003c/p\u003e\u003cp\u003e5. Active infectious disease\u003c/p\u003e\u003cp\u003e6. Uncontrolled hematologic disease, including coagulopathy or bleeding diathesis\u003c/p\u003e\u003cp\u003e7. Presence of non-cardiac comorbidity with life expectancy\u0026thinsp;\u0026le;\u0026thinsp;1 year from randomization\u003c/p\u003e\u003cp\u003e8. Females with childbearing potential or breast-feeding\u003c/p\u003e\u003cp\u003e9. Refusal to give written informed consent\u003c/p\u003e\u003cp\u003e10. Other conditions that may result in protocol non-compliance by the committees\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eDuring the washing process, patient plasma, prepared separately, was used as the resuspension medium. This approach mitigated cell aggregation, which was observed when saline was used for resuspension (\u003cb\u003eSupplementary Fig.\u0026nbsp;3A\u003c/b\u003e). Additionally, viability testing with Trypan blue staining revealed a 20% reduction in cell viability when saline was used, alongside increased cell death and apoptosis. In contrast, resuspension in patient plasma preserved cell viability without any increase in cell death, or apoptosis (\u003cb\u003eSupplementary Fig.\u0026nbsp;3B and 3C\u003c/b\u003e). The concentration of FVA3-Ang1 following the washing process is shown in \u003cb\u003eSupplementary Fig.\u0026nbsp;3D\u003c/b\u003e, demonstrating a negligible amount of remnant FVA3-Ang1 in the final cell product. Furthermore, flow cytometric analysis of the activation markers CD69 and CD25 confirmed the absence of unintended immune responses in the treated cells (\u003cb\u003eSupplementary Fig.\u0026nbsp;3E\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eThe primary endpoint of the MAGIC Cell-6 trial is the improvement of infarct-related regional wall motion abnormality, assessed via echocardiography at the 12-months follow-up. Secondary endpoints are listed in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The study protocol was approved by the institutional review board of the participating center and adheres to the principles of the Declaration of Helsinki. All patients will provide written informed consent prior to participation.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eStudy endpoints of the MAGIC Cell-6 study\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePrimary\u003c/p\u003e\u003cp\u003eendpoint\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eImprovement of the infarct-related regional wall motion abnormality\u003c/p\u003e\u003cp\u003e- Measured by echocardiography, at 12-months follow-up\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eSecondary endpoints\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1. Regional wall motion score index (Measured by echocardiography, at 12-months follow-up\u003c/p\u003e\u003cp\u003e2. Clinical outcomes (number of patients, up to 12-months follow-up)\u003c/p\u003e\u003cp\u003ea. All-cause death\u003c/p\u003e\u003cp\u003eb. Cardiac death\u003c/p\u003e\u003cp\u003ec. Target lesion revascularization\u003c/p\u003e\u003cp\u003ed. Readmission due to any cause\u003c/p\u003e\u003cp\u003ee. Readmission due to heart failure\u003c/p\u003e\u003cp\u003e3. B-natriuretic peptide level (unit of pf/mL, at 12-months follow-up)\u003c/p\u003e\u003cp\u003e4. 6-minute walk test (unit of meters, at 12-months follow-up)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eIn this study, we aimed to improve the efficacy of PBSC therapy for myocardial infarction. Our main findings are: 1) We developed a novel engineered molecule, FVA3-Ang1, a stable and clinically scalable form of Ang1 for therapeutic applications. 2) Gene expression analysis showed that 1-hour priming with FVA3-Ang1 did not modify gene expression, whereas 24-hour priming altered gene expression, inducing an endothelial-like phenotype. 3) Protein expression analysis confirmed that endothelial lineage markers were only upregulated after 24-hour FVA3-Ang1 priming. This finding is critical, as regulatory agencies in Korea impose strict approval criteria when cell characteristics change after priming. 4) \u003cem\u003eIn vivo\u003c/em\u003e studies demonstrated that 1-hour priming with FVA3-Ang1 significantly enhanced the regenerative potential of \u003csup\u003emob\u003c/sup\u003ePBSCs in hind-limb ischemia and myocardial infarction models. Collectively, our results suggest that short-term (1-hour) priming with FVA3-Ang1 enhances the angiogenic and regenerative potential of \u003csup\u003emob\u003c/sup\u003ePBSCs without altering their fundamental genetic profile, thus supporting its clinical applicability in stem-cell based therapy for myocardial infarction.\u003c/p\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Current Limitations of Stem Cell Therapy: Ang1 as a Promising Agent for Priming Strategy\u003c/h2\u003e\u003cp\u003eCurrent ischemic disease treatments primarily focus on revascularization, aiming to restore blood flow to occluded vessels. Metallic stents are widely used for this purpose and have improved patient outcomes. However, a major limitation remains: while revascularization prevents further ischemic damage, it does not actively promote tissue repair and regeneration \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Stem cell therapy has been proposed as a breakthrough strategy to address this limitation \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Despite promising results, previous studies have highlighted several challenges, including low therapeutic efficacy \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Various modifications have been explored to enhance the regenerative potential of stem cells, such as using undifferentiated stem cells, genetic modifications, and exogenous protein priming. However, the use of undifferentiated stem cells and genetic modifications raises concerns about tumorigenic risks and ethical issues \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eA more practical and feasible approach is to use the autologous stem/progenitor cells from bone marrow. However, this strategy has two major limitations: weak regenerative potency of adult stem cells and the requirement for invasive procedures (i.e. bone marrow aspiration). To overcome these challenges, we developed a cytokine-based autologous \u003csup\u003emob\u003c/sup\u003ePBSC therapy, which mobilizes adult stem cells from the bone marrow to peripheral blood using G-CSF injection, enabling us to obtain the same volume and quality of stem/progenitors from the peripheral blood as from bone marrow \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. To further enhance mobilization efficiency, we used a combination-cytokine approach by adding EPO alongside G-CSF, which significantly improved stem cell yield and mobilization efficacy compared to G-CSF alone.\u003c/p\u003e\u003cp\u003eThe stem cell niche is hypothesized to be regulated by interactions between angiopoietin on niche-supporting cells and Tie2 receptors on stem cells. Our previous study confirmed that all the stem/progenitor cells mobilized into peripheral blood (\u003csup\u003emob\u003c/sup\u003ePBSC) express Tie2 receptors \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. This finding led us to investigate the feasibility of \u003cem\u003eex vivo\u003c/em\u003e protein priming of \u003csup\u003emob\u003c/sup\u003ePBSCs with Ang1 from bone marrow by G-CSF and EPO, demonstrating that Ang1 priming improved cell engraftment and therapeutic efficacy in ischemic disease models. However, a significant limitation in our previous study was the use of COMP-Ang1, which exhibited poor stability and high aggregation, making mass production for clinical application impractical.\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.2 Engineering of FVA3-Ang1 (IAV-FLAG-VASP2-Ang1): A More Stable and Potent Tetramer Ang1 Protein Suitable for Large Production with High Efficiency\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn this study, we developed a modified form of Ang1 designed to enhance structural stability, expression efficiency, and its ability to recruit and activate the Tie2 receptor as a multimer. To achieve this, we engineered Ang1 to predominantly express as tetramers, with tetramerization serving as the primary conformation in the expression profile. This structural stabilization was reinforced by incorporating VASP2, a novel variant containing a specific sequence that facilitates disulfide bond formation at the C-terminal end. Additionally, to maximize Ang1 expression, we introduced a signal peptide from influenza A virus (IAV) at the N-terminal region, leading to FVA3-Ang1 (IAV-FLAG-VASP2-Ang1) \u003csup\u003e\u003cspan additionalcitationids=\"CR26 CR27 CR28\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Although the measured half-life of na\u0026iuml;ve Ang1 was not substantially different from that of COMP-Ang1 or FVA3-Ang1, Ang1 instability and tendency to aggregate constrain its clinical utility (\u003cb\u003eSupplementary Fig.\u0026nbsp;1D\u003c/b\u003e). COMP-Ang1 improved stability and bioactivity compared to the native protein but still fell short of clinical-grade requirements. To overcome these limitations, we developed FVA3-Ang1, a clinically manufacturable tetramer-stabilized Ang1 variant. Importantly, PBSCs treated with FVA3-Ang1 were subjected to a dedicated washing procedure to remove residual protein, thereby minimizing systemic exposure and producing favorable pharmacokinetic and tissue distribution profiles compatible with therapeutic applications.\u003c/p\u003e\u003cp\u003eCollectively, through strategic engineering and refinement of the cultivation process, we achieved significant advancements in the production of FVA3-Ang1 oligomers, improving their stability and therapeutic potential for applications in vascular biology and angiogenesis. The functional activity of FVA3-Ang1 was subsequently validated through experiments demonstrating increased pAKT activity, enhanced CD31 and VECAD expression, and improved endothelial tube formation.\u003c/p\u003e\u003cp\u003eWhen comparing FVA3-Ang1 to COMP-Ang1, it is important to note that while our \u003cem\u003ein vivo\u003c/em\u003e experiments did not include COMP-Ang1, its angiogenic effects have been well-documented in our previous study \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. We expanded our discussion to include the Tie2 signaling axis, PI3K/Akt activation, and the role of CXCR4 in neovascularization, as supported by existing literature \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Our focus with FVA3-Ang1 is supported by qPCR data showing sustained upregulation of Tie2 mRNA in \u003csup\u003emob\u003c/sup\u003ePBSCs after 1 and 24 hours of treatment, reflecting durable Tie2 activation. These features are expected to translate into superior therapeutic efficacy. Moreover, while RNA-sequencing data for COMP-Ang1-primed cells remain unavailable, our transcriptomic analysis of FVA3-Ang1-primed \u003csup\u003emob\u003c/sup\u003ePBSCs revealed significant functional enhancements, such as increased expression of CXCR4 and CD31.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Enhanced Function of \u003csup\u003emob\u003c/sup\u003ePBSCs Without Genetic Modification Following Short Term Exposure to FVA3-Ang1\u003c/h2\u003e\u003cp\u003eThis study demonstrated that \u003csup\u003emob\u003c/sup\u003ePBSCs primed with FVA3-Ang1 exhibit superior tissue regeneration potential compared to na\u0026iuml;ve \u003csup\u003emob\u003c/sup\u003ePBSCs in the context of stem cell therapy. To obtain regulatory approval for the clinical application of FVA3-Ang1-primed \u003csup\u003emob\u003c/sup\u003ePBSCs in patients with acute myocardial infarction, it is essential to comply with the stringent requirements set forth by the Korean FDA. A key regulatory criterion is ensuring that the priming process enhances the regenerative capacity of the cells without altering their genetic characteristics. After a short 1-hour and prolonged 24-priming strategy, we could find that the cell characteristics changed after a prolonged priming strategy, while minimal alteration was shown after a 1-hour priming, proven by total-RNA sequencing. However, even after a 1-hour priming, \u003csup\u003emob\u003c/sup\u003ePBSC primed with FVA3-Ang1 exhibited enhanced endothelial and angiogenic properties, while maintaining the genetic profile of hematopoietic cells and showing no signs of increased cell death. Mouse ischemic disease models also confirmed the efficacy of FVA3-Ang1 primed \u003csup\u003emob\u003c/sup\u003ePBSC, by demonstrating recovery of the myocardial infarct and improvement of cardiac function.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Translation Application: The MAGIC Cell-6-Priming trial\u003c/h2\u003e\u003cp\u003eBuilding on our previous experience with \u003csup\u003emob\u003c/sup\u003ePBSC-based therapy for ischemic disease, we first demonstrated in the MAGIC Cell-3 trial that adding cell therapy to standard stent treatment significantly improved cardiac function compared with stent alone \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. We then advanced our strategy in MAGIC Cell-5(clinicaltrials.gov ID: NCT00501917) by confirming the efficacy of combined G-CSF and darbepoetin-primed cell therapy \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e and further identified the superior effect of angiopoietin within that regimen. These findings inform the design of the MAGIC Cell-6 trial (clinicaltrials.gov ID: NCT06364150), which is currently underway to evaluate the clinical feasibility and efficacy of FVA3-Ang1-primed \u003csup\u003emob\u003c/sup\u003ePBSCs in patients with acute myocardial infarction. This clinical protocol includes several key innovations in stem cell therapy. First, the use of autologous peripheral blood-derived cells eliminates the risk of transplant rejection and inflammatory complications. To overcome the variability in hematopoietic capability and the potentially diminished cytokine response in critically ill patients, we employed a combination of G-CSF and EPO, both of which have been proven safe for human use. \u003csup\u003emob\u003c/sup\u003ePBSCs were isolated and concentrated via apheresis, a well-established clinical procedure. A critical challenge in the trial was that FVA3-Ang1 has not been previously approved for human use, as there are no prior clinical safety data. Consequently, we established a controlled \u003cem\u003eex vivo\u003c/em\u003e priming and washing process. The \u003cem\u003eex vivo\u003c/em\u003e priming process is performed in a GMP-certified facility at Seoul National University Hospital, guaranteeing a sterile environment free from microbial, particulate, and pyrogenic contamination. This approach enabled us to maximize the priming efficacy while minimizing the risk of hypersensitivity reactions to recombinant proteins. Another unique aspect of the trial is the washing process, which was developed in response to regulatory requirements. While we hypothesized that residual FVA3-Ang1 in the final cell preparation might provide additional therapeutic benefits to the infarcted myocardium, the regulatory agency mandated its complete removal before infusion, given the absence of prior phase-1 clinical trials in humans. Consequently, we implemented a standardized washing protocol to eliminate any residual FVA3-Ang1 from the final preparation of primed \u003csup\u003emob\u003c/sup\u003ePBSCs. As the washing procedure involves centrifugation, which could potentially damage the cells, we assessed cell viability pre- and post-washing, confirming minimal cell damage. The washing solution was autologous plasma, derived from the supernatant of centrifuged whole blood. This approach ensures the presence of various endogenous cytokines that are beneficial for the survival of \u003cem\u003eex vivo\u003c/em\u003e washed \u003csup\u003emob\u003c/sup\u003ePBSCs. We have previously demonstrated that autologous plasma contains high levels of pro-angiogenic cytokines such as IL8, IL17, PDGF, and VEGF, which may further enhance the therapeutic potential of washed \u003csup\u003emob\u003c/sup\u003ePBSCs \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eImportantly, in the MAGIC Cell-6 trial, six patients have been treated to date, and no acute complications related to cell injection have been observed at the immediate post-procedure state. (\u003cb\u003eSupplementary Fig.\u0026nbsp;4A-C\u003c/b\u003e). Also, during the 90-day clinical follow-up, and no adverse findings were detected in these patients, reflecting the immediate safety of this technique. We are planning to perform a 1-year clinical follow-up for all patients along with evaluation of the stent patency and myocardial recovery by coronary angiogram and myocardial perfusion tests. Collectively, this trial introduces a novel, short \u003cem\u003eex vivo\u003c/em\u003e priming strategy that is clinically applicable and a specific washing process ensuring that only primed \u003csup\u003emob\u003c/sup\u003ePBSCs, and not the priming agent, are delivered to the infarcted myocardium through the culprit coronary artery. This distinction is crucial for obtaining regulatory approval, as it guarantees that sterile, non-immunogenic, autologous cells, free from any foreign agents, are administered to patients. The results of the MAGIC Cell-6 trial will provide critical insights into the real-world clinical impact of FVA3-Ang1-primed \u003csup\u003emob\u003c/sup\u003ePBSCs, offering a potentially transformative therapy for acute myocardial infarction.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Limitation","content":"\u003cp\u003eLong-term safety assessment remains a critical consideration for this novel technique. The cells used in this study are derived from adult bone marrow, which inherently lowers the risk of tumorigenicity \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Moreover, the short-term exposure to FVA3-Ang1, as implemented in our 1-hour priming protocol, does not significantly alter gene expression, further minimizing the likelihood of tumorigenic transformation. Notably, previous studies involving COMP-Ang1 have not reported any evidence of tumorigenicity, and the regulatory advantages of short-term priming provide an additional safeguard against potential risks. Since the effect of COMP-Ang1, which can be considered as a control, was confirmed in previous in vivo experiments, in this study, we conducted in vivo experiments to confirm only the effect of FVA3-Ang1\u003csup\u003e13\u003c/sup\u003e. Nonetheless, we propose a framework for future studies to investigate the long-term risk of tumor formation. Additionally, while this study did not directly assess fibrosis-related mediators, such as TGF-β and α-SMA or the macrophage phenotypes M1/M2 balance \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, previous literature suggests that these factors could contribute to the overall therapeutic effect. Future research should also explore the secretory profile of primed \u003csup\u003emob\u003c/sup\u003ePBSCs, as potential paracrine mechanisms may support endothelial interactions and further enhance tissue recovery.\u003c/p\u003e"},{"header":"5. Materials and Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e5.1 The cultivation of Angiopoietin Expression Cell Line\u003c/h2\u003e\u003cp\u003eWe applied a strategic development approach to generate a stable Ang1 overexpressing cell line (\u003cb\u003eSupplementary Fig.\u0026nbsp;1A\u003c/b\u003e). Initially, we adapted a Chinese Hamster Ovary cell line \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e to suspension culture and subsequently engineered a promoter system to induce Ang1 overexpression. To optimize gene expression, we identified hotspot sites mediating overexpression in the cell genome and proceeded with the development of a mass production process. Validating an Ang1 vector system mediated by euchromatin, where gene expression occurs effectively within the cell nucleus, we selected hotspot site 13 as the most efficient inducer among 8 candidates (\u003cb\u003eSupplementary Fig.\u0026nbsp;1B\u003c/b\u003e). Ultimately, we employed a systematic approach utilizing Design of Experiments (DoE). Our optimized cultivation process achieved the highest expression levels and purity of Ang1 under specific conditions, including seeding cells at a density of 334.34 cells/mL, maintaining a pH of 7.4, and regulating dissolved oxygen levels at 30%.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e5.2 The collection of \u003csup\u003emob\u003c/sup\u003ePBSC and PBMCs\u003c/h2\u003e\u003cp\u003eThe collection of \u003csup\u003emob\u003c/sup\u003ePBSCs and PBMCs followed a modified protocol based on our previous report \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. PBSCs were mobilized from patients who received subcutaneous EPO 5 \u0026micro;g/kg (maximum 300 \u0026micro;g) for one day and G-CSF 5 \u0026micro;g/kg twice for three days. On the fourth day, \u003csup\u003emob\u003c/sup\u003ePBSCs were collected by apheresis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). A washing solution was independently prepared by collecting the plasma from the patient and mixing this with Dulbecco's Phosphate-Buffered Saline at a 1:1 ratio. An equivalent amount of the washing solution was added to the product of apheresis and centrifuged at 3000 RPM for 25 minutes. The isolated \u003csup\u003emob\u003c/sup\u003ePBSCs were then treated according to the specified protocol. The washing solution was stored separately, which was later used as a solvent when injected into the mouse hind legs and myocardial infarction models.\u003c/p\u003e\u003cp\u003eAs a negative control, PBMCs were collected from a healthy control group. In a 50 mL sterile tube, 20 mL of peripheral blood was mixed with 10 mL of PBS and 12 mL of Ficoll Paqur Plus (Cytiva\u003csup\u003e\u0026trade;\u003c/sup\u003e) that had been shaken vigorously was slowly added. Centrifugation was performed at 2500 RPM for 30 minutes at room temperature, with deceleration set to 0 RPM. The buffy coat layer located below the separated plasma was aspirated with a 1 mL pipette and placed in a new 50 mL sterile tube. The remaining volume was filled with PBS, and centrifugation was performed at 1800 RPM for 10 minutes at 4 ℃, with deceleration also set to 0 RPM. The supernatant was aspirated, followed by resuspension with 1 mL of PBS. The remaining volume was filled with PBS, and centrifugation was performed under the same conditions. From this stage, deceleration was set to 5 RPM. The supernatant was aspirated, and the PBMC was obtained by counting the cells and centrifuging under the same conditions. This experiment was conducted with the approval of the Institutional Review Board (IRB) of Seoul National University Hospital (MAGIC cell-5 trial, NCT00501917).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e5.3 Priming \u003csup\u003emob\u003c/sup\u003ePBSCs with FVA3-Ang1 and wash-out process\u003c/h2\u003e\u003cp\u003eThe priming process was performed with FVA3-Ang1 at a concentration of 400 ng/mL in a 37℃ incubator for 1 hour or 24 hours, according to the protocol of confirming genetic expression characteristics. After FVA3-Ang1 priming, two washing processes were performed to ensure the remaining FVA3-Ang1. This was performed for clinical application of \u003csup\u003emob\u003c/sup\u003ePBSC treated with FVA3-Ang1, which aimed to establish a safe washout process before infusion. Preparation of the washing solution was explained above. The remaining volume of the 50 mL sterile tube containing the primed cell solution was filled with the washing solution and centrifuged at 1000 rcf for 5 minutes. After removing the supernatant, resuspension was performed with the washing solution, and the centrifuge was done under the identical condition. Finally, the plasma isolated from the isolated \u003csup\u003emob\u003c/sup\u003ePBSCs was used as a resuspension solution to be used in animal experiments. Ultimately, the concentration of Ang1 detected in the \u003csup\u003emob\u003c/sup\u003ePBSC after undergoing two washing processes was measured to be below the Limit of Detection (LOD) value of 0.87 ng/mL, demonstrating its safety for human use (\u003cb\u003eSupplementary Fig.\u0026nbsp;3D\u003c/b\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e5.4 Total RNA-Sequencing\u003c/h2\u003e\u003cp\u003eLibrary preparation and sequencing libraries were prepared from total RNA using the NEBNext Ultra II Directional RNA-Seq Kit (NEW ENGLAND BioLabs, Inc., UK). rRNA was removed using RIBO COP rRNA depletion kit (LEXOGEN, Inc., Austria). The rRNA depleted RNAs were used for the cDNA synthesis and shearing, following manufacture\u0026rsquo;s instruction. Indexing was performed using the Illumina indexes 1\u0026ndash;12. The enrichment step was carried out using of PCR. Subsequently, libraries were checked using the Agilent 2100 bioanalyzer (DNA High Sensitivity Kit) to evaluate the mean fragment size. Quantification was performed using the library quantification kit using a Step One Real-Time PCR System (Life Technologies, Inc., USA). High-throughput sequencing was performed as paired-end 100 sequencing using NovaSeq 6000 (Illumina, Inc., USA).\u003c/p\u003e\u003cp\u003eFor data analysis, a quality control of raw sequencing data was performed using FastQC. Adapter and low-quality reads (\u0026lt;\u0026thinsp;Q20) were removed using FASTX_Trimmer and BBMap. Then the trimmed reads were mapped to the reference genome using TopHat\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Gene expression levels of genes, isoforms and IncRNAs were estimated using FPKM values by Cufflinks\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. The FRKM values were normalized based on TMM\u0026thinsp;+\u0026thinsp;CPM method using EdgeR within R. Data mining and graphic visualization were performed using ExDEGA.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e5.5 Flow cytometry analysis\u003c/h2\u003e\u003cp\u003eAntigen analysis was performed on \u003csup\u003emob\u003c/sup\u003ePBSCs and PBMCs. Cells that had been primed with FVA3-Ang1 were washed according to the washing protocol, by filling a 50 mL sterile tube with PBS and centrifuging at 1000 rcf for 3 minutes at room temperature. The supernatant was aspirated, and the cells were resuspended in 1 mL PBS and centrifuged under the same conditions. The cells were then resuspended in FACS buffer for staining. To confirm the cell characteristics of \u003csup\u003emob\u003c/sup\u003ePBSCs and PBMCs, FITC-conjugated anti-CD31 (BD Pharmingen), PE-conjugated anti-VE-cadherin (BD), APC-conjugated anti-CXCR4 (BD), APC-conjugated anti-CD45 (BD), PE-conjugated anti-Tie2 (BD) and FITC-conjugated anti-Annexin V (BD) antibodies and Propidium Iodide(PI) were used.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e5.6 Quantitative reverse Transcriptase-Polymerase chain reaction\u003c/h2\u003e\u003cp\u003emRNA expression was quantified by real-time RT-PCR using a 7500 real-time PCR system according to the experimenter's protocol. The real-time reaction was performed in a 96-well plate with 3 replicates at a final volume of 20 \u0026micro;L and was normalized with GAPDH RNA. Relative quantitative real-time PCR for CD31, CXCR4, VE-cadherin, and Tie2 was performed using the TOYOBO protocol. The PCR conditions were as follows: Quantitative real-time PCR for binding was performed using the SYBGreen protocol. The RNA solution was incubated at 65℃ for 5 minutes, then cooled sufficiently on ice. 5x RT Master Mix was added to adjust the final volume to 20 \u0026micro;L, and the reaction was incubated at 37℃ for 15 minutes. cDNA synthesis was completed by incubating at 50℃ for 5 minutes and 98℃ for 5 minutes. The final product should be stored at 4℃ or -20℃.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e5.7 Immunofluorescent staining\u003c/h2\u003e\u003cp\u003eTo measure the capillary density, the paraffin-fixing sections of each group were stained with CD31 antibodies, and the muscle structure was also stained with Laminin antibodies, and anti-HLA-ABC was used as the primary antibody for the engraftment of the injected cells. Secondary antibodies were stained for 1 hour at room temperature using alexa-488 and alexa-555. Figures were obtained using confocal microscopy (Leica).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e5.8 Matrigel tube formation\u003c/h2\u003e\u003cp\u003eMatrigel tube formation was carried out to confirm the angiogenesis network of COMP-Ang1 and FVA3-Ang1. We solidified 50 \u0026micro;L of Basement Matrigel by placing at least 30 minutes in a 37℃ incubator at 96well. COMP-Ang1 and FVA3-Ang1 were primed by concentration and by time and confirmed under various conditions. 1) Vehicles, 2) VEGF, 3) COMP, or FVA3 were treated. Under each condition, 1\u0026times;10\u003csup\u003e4\u003c/sup\u003e HUVECs were seeded with 100 \u0026micro;L of EGM-2MV media with 2% FBS. We observed tubes formed after 24 hours and total length, branching length and nodes of complete tube formed by cells were quantified using ImageJ.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e5.9 Western blot\u003c/h2\u003e\u003cp\u003eGroups treated under hourly and concentration-specific conditions with Angioinetin-1 and controls were lysed with RIPA buffer containing protease/phosphatase inhibitors and centrifuged at 15,000 RPM for 30 minutes at 4\u0026deg;C. The isolated proteins were separated on 4\u0026ndash;15% SDS-PAGE gels and transferred onto a polyvinylidene difluoride membrane. It was then incubated overnight at 4\u0026deg;C by various primary antibodies after membrane blocking and subsequently subjected to appropriate secondary antibodies. Immunoblot signals were detected by Amersham 680, and the figures were quantified by ImageJ.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e5.10 Mouse Hind-limb Ischemia and Myocardial Infarction Models\u003c/h2\u003e\u003cp\u003eThe neovascularization of \u003csup\u003emob\u003c/sup\u003ePBSCs primed with FVA3-Ang1 was confirmed using a mouse hindlimb model and myocardial infarction model. Anesthesia was performed by injecting alfaxalone 100 \u0026micro;L and Rompun 5 \u0026micro;L into the intraperitoneal space. Afterwards, anesthesia was maintained at 1.5 to 2% of isoflurane through a respiratory anesthesia.\u003c/p\u003e\u003cp\u003eA hindlimb ischemia model was created by ligating the femoral artery of Balb/c nude mice (male, 8 weeks old). Primed or non-primed \u003csup\u003emob\u003c/sup\u003ePBSCs (cell number of 1 x 10\u003csup\u003e5\u003c/sup\u003e) were injected into the muscle around the ligated vessel in a volume of 30 \u0026micro;L. The experimental groups were (1) PBS (n\u0026thinsp;=\u0026thinsp;5), (2) \u003csup\u003emob\u003c/sup\u003ePBSCs only (n\u0026thinsp;=\u0026thinsp;6), and (3) FVA3-Ang1 primed \u003csup\u003emob\u003c/sup\u003ePBSCs (n\u0026thinsp;=\u0026thinsp;6). Laser Doppler perfusion imaging (LDPI,) was performed four times, on days 0, 3, 7, and 14, starting from the day of cell injection.\u003c/p\u003e\u003cp\u003eA myocardial infarction model was created by a 1 cm length incision on the left side of the torso and the thoracic cavity was opened through a microscope. The left descending artery of the mouse heart is tied with a surgical thread to prevent antegrade blood flow. The experimental groups were (1) PBS (n\u0026thinsp;=\u0026thinsp;4), (2) \u003csup\u003emob\u003c/sup\u003ePBSCs only (n\u0026thinsp;=\u0026thinsp;10), and (3) FVA3-Ang1 400 ng/mL 1hr priming \u003csup\u003emob\u003c/sup\u003ePBSCs (n\u0026thinsp;=\u0026thinsp;10). The skin was sealed using a 6\u0026thinsp;\u0026minus;\u0026thinsp;0 black suture, and the gas was removed from the abdominal cavity using a 5 mL syringe connected to the catheter to form a negative pressure, and the suture was completed. At the end of the experiments, animals were anesthetized with alfaxalone and rompun, followed by diaphragmatic incision, exsanguination, and organ harvesting for euthanasia. All experiments were approved by the Institutional Animal Care and Use Committee in Seoul National University Hospital (SNUH-IACUC) and animals were maintained in the facility accredited AAALAC International (#001169) in accordance with Guide for the Care and Use of Laboratory Animals 8th edition, NRC (2010).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e5.11 ELISA assay\u003c/h2\u003e\u003cp\u003eThe human Angiopoietin 1 Quantikine ELISA kit (R\u0026amp;D Systems, Minneapolis, USA) was used to quantify residual Angiopoietin-1 levels in the serum at three different time points: (1) immediately after priming with Ang1, (2) after the first wash, and (3) after the final wash.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e5.12 Trypan blue cell viability assay\u003c/h2\u003e\u003cp\u003eTo evaluate cell viability following washing and resuspension, a Trypan blue exclusion assay was conducted. The viability of \u003csup\u003emob\u003c/sup\u003ePBSCs was assessed under three conditions: cells primed with FVA3-Ang1 for 1 hour, cells resuspended in patient plasma after washing, and cells resuspended in normal saline after washing. Cells were stained with 0.4% Trypan blue for 3 minutes, and viable cells were determined using a hemocytometer following standard protocols. The percentage of viable cells was quantified.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003e5.13 Scratch assay\u003c/h2\u003e\u003cp\u003eHUVECs were plated in an ibidi Culture-Insert 2-well (\u0026micro;-Dish 35 mm) with 4\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells per reservoir and incubated 24 h at 37\u0026deg;C and 5% CO₂ to reach confluence. Inserts were removed to create a defined 500 \u0026micro;m gap, wells were washed once with prewarmed PBS, and serum-free medium containing vehicle (control) or Ang-1, COMP-Ang1, or FVA3-Ang1 200 or 400 ng/mL was added. Images of the same fields were acquired at 0, 12, and 24 hours by phase-contrast microscopy. Wound areas were measured in ImageJ and percent closure calculated as [(area₀ \u0026minus; area_t)/area₀] x 100. Statistical analysis was by two-way ANOVA (factors: treatment, time); p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003e5.14 EdU assay\u003c/h2\u003e\u003cp\u003eHUVECs were grown in 35 mm confocal dishes(ibidi GmbH) to 80% confluency and treated with vehicle or Ang-1, COMP-Ang1, or FVA3-Ang1 200 or 400 ng/mL for 1 or 24 hours. After each treatment period, cells were incubated with 10 \u0026micro;M EdU (ClickTech EdU Cell Proliferation Kit 647 for IM, BCK-EDU647) for 30 min. Cells were fixed in 4% paraformaldehyde 15 min, permeabilized with 0.5% Triton X-100 10 min, RT and processed according to the manufacturer\u0026rsquo;s instructions for the Click reaction. Nuclei were counterstained with DAPI and imaged by confocal microscopy. EdU⁺ nuclei were quantified as a proportion of total DAPI nuclei (EdU⁺/DAPI) over \u0026ge;\u0026thinsp;5 random fields per sample. Statistical analysis was by two-way ANOVA ; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\u003ch2\u003e5.15 FITC-Dextran\u003c/h2\u003e\u003cp\u003eHUVECs were seeded onto Transwell polycarbonate membrane 0.4 \u0026micro;m inserts (Corning, 3412) at 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells in insert and cultured in complete endothelial medium until a confluent monolayer was achieved. Prior to treatment, inserts were washed once and equilibrated in prewarmed Hanks\u0026rsquo; balanced salt solution (HBSS; H8264) for 30 min at 37\u0026deg;C. Cells were then incubated with vehicle or Ang-1, COMP-Ang1, or FVA3-Ang1 (200 or 400 ng/mL for 1 hour or 24 hours. After each treatment period, fluorescein isothiocyanate\u0026ndash;dextran (FITC-Dextran) in HBSS 1 mg/mL was added to the apical chamber and plates were incubated at 37\u0026deg;C. After 30 min, 100 \u0026micro;L samples were taken from the basolateral chamber and transferred to a black 96-well plate; fluorescence was read on a plate reader (Ex/Em 485/520 nm). A standard curve of FITC-dextran was used to convert fluorescence units to amount transported. Permeability was expressed relative to vehicle control. Statistical analysis was by two-way ANOVA, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e\u003ch2\u003e5.16 Angiopoietin-1 AlphaFold modeling and multimer stability prediction\u003c/h2\u003e\u003cp\u003eAmino-acid sequences of COMP-Ang1 and FVA3-Ang were modelled using AlphaFold-Multimer via the ColabFold/AlphFold to predict multimeric assemblies. Models were generated with default ColabFold/AlphaFold settings and five models per prediction were produced and the top model was selected by the AlphaFold multimer ranking confidence. Selected top models were submitted to PDBePISA for interface analysis. From PISA we report interface buried surface area, the Complexation Significance Score (CSS) as an index of interface relevance to assembly formation, and the solvation free-energy gain on interface formation reported by PISA as Gint, together with the number of hydrogen bonds and salt bridges across the interfaces. CSS and ΔGint (Gint) were used to evaluate predicted interface energetics in conjunction with the AlphaFold ranking confidence.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e\u003ch2\u003e5.17 Half-life measurement of Ang1 proteins\u003c/h2\u003e\u003cp\u003eHuman umbilical vein endothelial cells (HUVECs) were cultured in 24-well plates until confluent. Recombinant human Ang1, COMP-Ang1, or FVA3-Ang1 was added to the culture medium at a final concentration of 400 ng/mL. At indicated time points (0, 0.5, 1, 2, 4, 8, 12, and 24 hours), aliquots of conditioned medium were collected and centrifuged at 300 \u0026times; g for 5 minutes to remove cellular debris. The supernatants were stored at \u0026minus;\u0026thinsp;80\u0026deg;C until analysis. Remaining concentrations of Ang1 protein were quantified using a human angiopoietin-1 ELISA kit (R\u0026amp;D, DANG10) according to the manufacturer\u0026rsquo;s instructions. Protein half-lives were calculated by plotting Ang1 concentration decay curves over time and fitting the data to a one-phase exponential decay model.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003e5.18 Statistical analysis\u003c/h2\u003e\u003cp\u003eContinuous variables are summarized as means (standard deviations). Group comparisons for continuous variables were conducted using unpaired t-tests. The two-way ANOVA was performed for statistical analysis, and a p-value less than 0.05 was considered significant. No statistical significance correction for multiple comparisons was applied in this study. In cases where the assumptions for parametric tests (e.g., normality) were not met, nonparametric tests were used. Specifically, the Mann-Whitney U test was performed for two-group comparisons, while the Kruskal-Wallis H test was used for comparisons involving three or more groups. These tests were chosen to analyze the differences in ranked data, ensuring the robustness of our findings even with non-normally distributed variables. All statistical analyses were conducted using SPSS, V25 (SPSS Inc.) and R programming language, version 4\u0026middot;2\u0026middot;4 (R Foundation for Statistical Computing).\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eLead contact\u003c/p\u003e\n\u003cp\u003eFurther information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Hyun-Jai Cho and Hyo-Soo Kim ([email protected], [email protected])\u003c/p\u003e\n\u003cp\u003eData Availability Statement\u003c/p\u003e\n\u003cp\u003eThe data supporting the findings of this study are available from the corresponding authors upon reasonable request. All RNA sequencing datasets supporting the findings of this study are publicly available in the NCBI Gene Expression Omnibus (GEO) repository under the accession code GSE304470.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by a grant from the Korea Health Technology R\u0026amp;D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health \u0026amp; Welfare, Republic of Korea (grant number: RS-2023-KH143762.\u0026nbsp;HX23C1754).\u003c/p\u003e\n\u003cp\u003eThis research was supported by the Bio \u0026amp; Medical Technology Development Program of the National Research Foundation (NRF)\u0026amp; funded by the Korean government (MSIT) (No. RS-2022-NR067329).\u003c/p\u003e\n\u003cp\u003eAuthor contributions\u003c/p\u003e\n\u003cp\u003eJH Kang and HJ Seo wrote the manuscript. HJ Son, MJ Kang, and\u0026nbsp;JW\u0026nbsp;Lee performed the\u0026nbsp;research\u0026nbsp;and analyzed the\u0026nbsp;data. EJ Lee analyzed the data. HJ Cho and HS Kim provided approval of the final manuscript. All\u0026nbsp;authors have\u0026nbsp;read and approved the final\u0026nbsp;manuscript.\u003c/p\u003e\n\u003cp\u003eDeclaration of Interests\u003c/p\u003e\n\u003cp\u003eThe authors have no conflicts of interest to disclose.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWelt FGP, Batchelor W, Spears JR, Penna C, Pagliaro P, Ibanez B, Drakos SG, Dangas G, Kapur NK (2024) Reperfusion Injury in Patients With Acute Myocardial Infarction: JACC Scientific Statement. 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Genome Biol 12:R22. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/gb-2011-12-3-r22\u003c/span\u003e\u003cspan address=\"10.1186/gb-2011-12-3-r22\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Seoul National University Hospital","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":"engraftment, angiogenesis, paracrine signaling, recovery, Tie-2 receptor, regeneration","lastPublishedDoi":"10.21203/rs.3.rs-7625543/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7625543/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHuman peripheral blood stem cells (PBSC) that have been most frequently used for repair or regeneration in ischemic cardiovascular disease (CVD) showed limitations in their efficacy. We previously reported that angiopoietin-1 (Ang1) is the cell-priming agent to enhance the vasculogenic potential of PBSC. The limitation was the difficulty to produce Ang1 protein with high efficiency.\u003c/p\u003e\u003cp\u003eIn this study, we engineered Ang1 structure and made FVA3-Ang1 by adding VASP and COMP sequence for stable tetramer formation as well as FALG sequence for purification in large scale production and a signal peptide derived from influenza A virus (IAV) for better protein expression. FVA3-Ang1 showed stronger effect on endothelial cells than na\u0026iuml;ve Ang1 or COMP-Ang1 in terms of gene expression of Ang1, Ang2, VEGFA, FGF2, and KDR, as well as phosphorylation of Tie2, ERK, and Akt.\u003c/p\u003e\u003cp\u003eThen we primed PBSC with FVA3-Ang1 and examined the transcriptome analysis. Priming for 1 hour did not change whole gene expression profiles of PBSC, whereas priming for 24 hours did change the pattern from myeloid toward endotheloid lineage.\u003c/p\u003e\u003cp\u003eIn mouse models of hind-limb ischemia and myocardial infarction, FVA3-Ang1-primed PBSCs showed superior engraftment and tissue regeneration compared to non-primed cells. A clinical trial is underway to assess efficacy and safety of FVA3-Ang1-primed PBSCs when infused via the culprit coronary artery following emergent stent implantation.\u003c/p\u003e","manuscriptTitle":"Newly-engineered angiopoietin-1 as a cell-priming agent for CVD","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-17 19:16:01","doi":"10.21203/rs.3.rs-7625543/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":"8073ecc0-1dba-4b00-8324-0cb11a8d3bd2","owner":[],"postedDate":"September 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-17T19:16:01+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-17 19:16:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7625543","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7625543","identity":"rs-7625543","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}