Preconditioning With TGF-β Inhibitors Enhances Therapeutic Efficacy of Endothelial Progenitor Cells for Wound Healing in Diabetic Mice

Abstract

Diabetic wound (DW) represent a common complication of diabetes. Despite advances in regenerative repair utilizing endothelial progenitor cells (EPCs), challenges such as low survival and impaired angiogenic function of EPCs remain. Herein, we explored an effective method to induce injury-induced protection for EPCs and improves their function. This was achieved through cell preconditioning under conditions of nutrient deprivation and high glucose (NDHG), combined with sb431542, a transforming growth factor beta (TGF-β) signaling inhibitor. Specifically, after three generations of cell passage during preconditioning, umbilical cord-derived endothelial cells (ECs) exhibited characteristics resembling those of EPCs, with over 80% of the cells expressed CD34, a typical marker of EPCs. Notably, these preconditioned EPC-like cells (pEPCs) showed tolerance to pathological environment, as evidenced by robust cell viability, improved antioxidant capacity, and stable tube-forming ability under NDHG condition. The protective effect of preconditioning in pEPCs is partly achieved by activating the PI3K/AKT pathway to upregulate the expression of Nrf2 and HIF-1α. Importantly, pEPCs exhibited therapeutic potential in two diabetic mouse models-limb ischemia and skin wounds by enhancing blood vessel formation and facilitating tissue repair. Overall, this preconditioning method induced the generation of functionally enhanced pEPCs, providing an alternative source of cells for treating DWs.

Keywords

angiogenesis / cell therapy / diabetic wound / endothelial progenitor cells / preconditioning / TGF-β signaling

References

[1] G. Theocharidis, B. E. Thomas, D. Sarkar, et al., “Single Cell Transcriptomic Landscape of Diabetic Foot Ulcers,” Nature Communications 13, no. 1 (2022): 181.

[2] W. Jin, Y. LI, and M. YU, “Advances of Exosomes in Diabetic Wound Healing,” Burns Trauma 13 (2025): tkae078.

[3] Y. Yao, Y. LI, and Q. SONG, “Angiogenic Factor AGGF1-Primed Endothelial Progenitor Cells Repair Vascular Defect in Diabetic Mice,” Diabetes 68, no. 8 (2019): 1635-1648.

[4] J. Xiong, H. HU, R. GUO, et al., “Mesenchymal Stem Cell Exosomes as a New Strategy for the Treatment of Diabetes Complications,” Frontiers in Endocrinology 12 (2021): 646233.

[5] K. Huang, B. MI, Y. Xiong, et al., “Angiogenesis During Diabetic Wound Repair: From Mechanism to Therapy Opportunity,” Burns Trauma 13 (2025): tkae052.

[6] J. Han, L. LUO, O. Marcelina, et al., “Therapeutic Angiogenesis-Based Strategy for Peripheral Artery Disease,” Theranostics 12, no. 11 (2022): 5015-5033.

[7] Y. Fujita, M. Kinoshita, Y. Furukawa, et al., “Phase II Clinical Trial of CD34+ Cell Therapy to Explore Endpoint Selection and Timing in Patients With Critical Limb Ischemia,” Circulation Journal 78, no. 2 (2014): 490-501.

[8] T. Pan, Z. WEI, and Y. FANG, “Therapeutic Efficacy of CD34(+) Cell-Involved Mononuclear Cell Therapy for No-Option Critical Limb Ischemia: A Meta-Analysis of Randomized Controlled Clinical Trials,” Vascular Medicine 23, no. 3 (2018): 219-231.

[9] M. Besnier, S. Gasparino, R. Vono, et al., “miR-210 Enhances the Therapeutic Potential of Bone-Marrow-Derived Circulating Proangiogenic Cells in the Setting of Limb Ischemia,” Molecular Therapy 26, no. 7 (2018): 1694-1705.

[10] X. Dai, X. Yan, and J. Zeng, “Elevating CXCR7 Improves Angiogenic Function of EPCs via Akt/GSK-3β/Fyn-Mediated Nrf2 Activation in Diabetic Limb Ischemia,” Circulation Research 120, no. 5 (2017): e7-e23.

[11] J. Yao, Z. Shi, and X. Ma, “lncRNA GAS5/miR-223/NAMPT Axis Modulates the Cell Proliferation and Senescence of Endothelial Progenitor Cells Through PI3K/AKT Signaling,” Journal of Cellular Biochemistry 120, no. 9 (2019): 14518-14530.

[12] R. Guo, Z. Wu, and A. Liu, “Hypoxic Preconditioning-Engineered Bone Marrow Mesenchymal Stem Cell-Derived Exosomes Promote Muscle Satellite Cell Activation and Skeletal Muscle Regeneration via the miR-210-3p/KLF7 Mechanism,” International Immunopharmacology 142, no. Pt B (2024): 113143.

[13] B. Isildar and S. Ozkan, D. Neccar, et al., “Preconditioning and Post-Preconditioning States of Mesenchymal Stem Cells With Deferoxamine: A Comprehensive Analysis,” European Journal of Pharmacology 996 (2025): 177574.

[14] L. A. Ahmed, S. M. Rizk, and S. A. El-Maraghy, “Pinocembrin Ex Vivo Preconditioning Improves the Therapeutic Efficacy of Endothelial Progenitor Cells in Monocrotaline-Induced Pulmonary Hypertension in Rats,” Biochemical Pharmacology 138 (2017): 193-204.

[15] P. Zhou, Y. Z. Tan, H. J. Wang, et al., “Hypoxic Preconditioning-induced Autophagy Enhances Survival of Engrafted Endothelial Progenitor Cells in Ischaemic Limb,” Journal of Cellular and Molecular Medicine 21, no. 10 (2017): 2452-2464.

[16] Y. J. Kim, S. T. JI, D. Y. Kim, et al., “Long-Term Priming by Three Small Molecules Is a Promising Strategy for Enhancing Late Endothelial Progenitor Cell Bioactivities,” Molecules and Cells 41, no. 6 (2018): 582-590.

[17] M. R. Hamczyk, R. M. Nevado, P. Gonzalo, et al., “Endothelial-to-Mesenchymal Transition Contributes to Accelerated Atherosclerosis in Hutchinson-Gilford Progeria Syndrome,” Circulation 150, no. 20 (2024): 1612-1630.

[18] Y. Chen, Y. Zhu, X. Ren, et al., “Endothelial Cell Senescence in Marfan Syndrome: Pathogenesis and Therapeutic Potential of TGF-β Pathway Inhibition,” Journal of the American Heart Association 14, no. 9 (2025): e037826.

[19] K. Kokoroishi, A. Nakashima, S. Doi, et al., “High Glucose Promotes TGF-β1 Production by Inducing FOS Expression in Human Peritoneal Mesothelial Cells,” Clinical and Experimental Nephrology 20, no. 1 (2016): 30-38.

[20] Y. Yao, Q. SONG, C. HU, et al., “Endothelial Cell Metabolic Memory Causes Cardiovascular Dysfunction in Diabetes,” Cardiovascular Research 118, no. 1 (2022): 196-211.

[21] D. James, H. S. Nam, M. Seandel, et al., “Expansion and Maintenance of Human Embryonic Stem Cell-Derived Endothelial Cells by TGFbeta Inhibition Is Id1 Dependent,” Nature Biotechnology 28, no. 2 (2010): 161-166.

[22] Q. Nie, L. Zhu, L. Zhang, et al., “Astragaloside IV Protects Against Hyperglycemia-Induced Vascular Endothelial Dysfunction by Inhibiting Oxidative Stress and Calpain-1 Activation,” Life Sciences 232 (2019): 116662.

[23] S. Zhou, H. Z. Chen, Y. Z. Wan, et al., “Repression of P66Shc Expression by SIRT1 Contributes to the Prevention of Hyperglycemia-induced Endothelial Dysfunction,” Circulation Research 109, no. 6 (2011): 639-648.

[24] A. Robson, “Lovastatin Improves Endothelial Cell Function in LMNA-Related DCM,” Nature Reviews Cardiology 17, no. 10 (2020): 613.

[25] K. Sobierajska and M. E. Wawro, “Oxidative Stress Enhances the TGF-β2-RhoA-MRTF-A/B Axis in Cells Entering Endothelial-Mesenchymal Transition,” International Journal of Molecular Sciences 23, no. 4 (2022): 2062.

[26] M. Komaki, Y. Numata, C. Morioka, et al., “Exosomes of Human Placenta-Derived Mesenchymal Stem Cells Stimulate Angiogenesis,” Stem Cell Research & Therapy 8, no. 1 (2017): 219.

[27] M. S. Shah and M. Brownlee, “Molecular and Cellular Mechanisms of Cardiovascular Disorders in Diabetes,” Circulation Research 118, no. 11 (2016): 1808-1829.

[28] Y. Xiang, X. Yao, and X. Wang, “Houshiheisan Promotes Angiogenesis via HIF-1α/VEGF and SDF-1/CXCR4 Pathways: In Vivo and in Vitro,” Bioscience Reports 39, no. 10 (2019): BSR20191006.

[29] C. Zhang, Y. Lin, and K. Zhang, “GDF11 Enhances Therapeutic Functions of Mesenchymal Stem Cells for Angiogenesis,” Stem Cell Research & Therapy 12, no. 1 (2021): 456.

[30] Y. C. Hsu, I. S. Yu, and Y. F. Tsai, “A Preconditioning Strategy to Augment Retention and Engraftment Rate of Donor Cells During Hepatocyte Transplantation,” Transplantation 105, no. 4 (2021): 785-795.

[31] K. Ehab, O. Abouldahab, A. Hassan, et al., “Alvogyl and Absorbable Gelatin Sponge as Palatal Wound Dressings Following Epithelialized Free Gingival Graft Harvest: A Randomized Clinical Trial,” Clinical Oral Investigations 24, no. 4 (2020): 1517-1525.

[32] W. Tian, L. Zhang, and Y. Wang, “Tibial Transverse Transport Promotes Wound Healing in Diabetic Foot Ulcers by Stimulating Endothelial Progenitor Cell Mobilization and Homing Mediated Neovascularization,” Annals of Medicine 56, no. 1 (2024): 2404186.

[33] S. Liu, G. Wan, and T. Jiang, “Engineered Biomimetic Nanovesicles-Laden Multifunctional Hydrogel Enhances Targeted Therapy of Diabetic Wound,” Materials Today Bio 29 (2024): 101330.

[34] Z. Shen, Wang W, J. Chen, et al., “Small Extracellular Vesicles of Hypoxic Endothelial Cells Regulate the Therapeutic Potential of Adipose-derived Mesenchymal Stem Cells via miR-486-5p/PTEN in a Limb Ischemia Model,” Journal of Nanobiotechnology 20, no. 1 (2022): 422.

[35] Y. Yoshimatsu, I. Wakabayashi, S. Kimuro, et al., “TNF-α Enhances TGF-β-induced Endothelial-to-Mesenchymal Transition via TGF-β Signal Augmentation,” Cancer Science 111, no. 7 (2020): 2385-2399.

[36] W. M. Han, X. C. Chen, and G. R. LI, “Acacetin Protects Against High Glucose-Induced Endothelial Cells Injury by Preserving Mitochondrial Function via Activating Sirt1/Sirt3/AMPK Signals,” Frontiers in Pharmacology 11 (2020): 607796.

[37] Y. Yao, W. Liao, and R. Yu, “Potentials of Combining Nanomaterials and Stem Cell Therapy in Myocardial Repair,” Nanomedicine 13, no. 13 (2018): 1623-1638.

[38] X. Fan, L. Cyganek, and I. Akin, “Functional Characterization of Human Induced Pluripotent Stem Cell-Derived Endothelial Cells,” International Journal of Molecular Sciences 23, no. 15 (2022): 8507.

[39] E. Helle, M. Ampuja, L. Antola, et al., “Flow-Induced Transcriptomic Remodeling of Endothelial Cells Derived From Human Induced Pluripotent Stem Cells,” Frontiers in Physiology 11 (2020): 591450.

[40] J. K. Han, Y. SHIN, and M. H. SOHN, “Direct Conversion of Adult Human Fibroblasts Into Functional Endothelial Cells Using Defined Factors,” Biomaterials 272 (2021): 120781.

[41] L. Kurian, I. Sancho-martinez, and E. Nivet, “Conversion of Human Fibroblasts to Angioblast-Like Progenitor Cells,” Nature Methods 10, no. 1 (2013): 77-83.

[42] G. Farber, Y. Dong, and Q. Wang, “Direct Conversion of Cardiac Fibroblasts Into Endothelial-Like Cells Using Sox17 and Erg,” Nature Communications 15, no. 1 (2024): 4170.

[43] W. Srifa, N. Kosaric, and A. Amorin, “Cas9-AAV6-Engineered Human Mesenchymal Stromal Cells Improved Cutaneous Wound Healing in Diabetic Mice,” Nature Communications 11, no. 1 (2020): 2470.

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