Exosomes derived from fibroblasts enhance skin wound angiogenesis by regulating HIF-1α/VEGF/VEGFR pathway

Abstract

Background: Angiogenesis is vital for tissue repair but insufficient in chronic wounds due to paradoxical growth factor overexpression yet reduced neovascularization. Therapeutics physiologically promoting revascularization remain lacking. This study aims to investigate the molecular mechanisms underlying fibroblast-derived exosome-mediated angiogenesis during wound repair. Methods: To assess the effects of fibroblasts derived exosomes on wound healing and angiogenesis, a full-thickness mouse skin injury model was established, followed by pharmacological inhibition of exosome secretion. The number and state of blood vessels in wounds were assessed by immunofluorescence, immunohistochemistry, hematoxylin–eosin staining, and laser Doppler imaging system. The high-throughput miRNA sequencing was carried out to detect the miRNA profiles of fibroblast-derived exosomes. The roles of candidate miRNAs, their target genes, and relevant pathways were predicted by bioinformatic online software. The knockdown and overexpression of candidate miRNAs, co-culture system, matrigel assay, pharmacological blockade, cell migration, EdU incorporation assay, and cell apoptosis were employed to investigate their contribution to angiogenesis mediated by fibroblast-derived exosomes. The expression of vascular endothelial growth factor A (VEGFA), vascular endothelial growth factor receptor 2 (VEGFR2), hypoxia-inducible factor 1α (HIF-1α), von Hippel–Lindau (VHL), and proline hydroxylases 2 was detected by western blot, co-immunoprecipitation, immunofluorescence, real-time quantitative polymerase chain reaction, flow cytometry, and immunohistochemistry. Furthermore, a full-thickness mouse skin injury model based on type I diabetes mellitus induced by streptozotocin was established for estimating the effect of fibroblast-derived exosomes on chronic wound healing. Results: Pharmacological inhibition of exosome biogenesis markedly reduces neovascularization and delays murine cutaneous wound closure. Topical administration of fibroblast-secreted exosomes rescues these defects. Mechanistically, exosomal microRNA-24-3p suppresses VHL E3 ubiquitin ligase levels in endothelial cells to stabilize hypoxia-inducible factor-1α and heighten vascular endothelial growth factor signaling. MicroRNA-24-3p-deficient exosomes exhibit attenuated pro-angiogenic effects. Strikingly, topical application of exosomes derived from fibroblasts onto chronic wounds in diabetic mice improves neovascularization and healing dynamics. Conclusions: Overall, we demonstrate central roles for exosomal miR-24-3p in stimulating endothelial HIF-VEGF signaling by inhibiting VHL-mediated degradation. The findings establish fibroblast-derived exosomes as promising acellular therapeutic candidates to treat vascular insufficiency underlying recalcitrant wounds.

Keywords

Fibroblasts / Exosomes / Angiogenesis / Wound healing / MicroRNA-24-3p

References

[1] Eelen G, Treps L, Li X, Carmeliet P. Basic and therapeutic aspects of angiogenesis updated. Circ Res 2020; 127: 310-29. https://doi.org/10.1161/circresaha.120.316851.

[2] Shpichka A, Butnaru D, Bezrukov EA, Sukhanov RB, Atala A, Burdukovskii V, et al. Skin tissue regeneration for burn injury. Stem Cell Res Ther 2019; 10: 94. https://doi.org/10.1186/s13287-019-1203-3.

[3] Qin Q, Lee S, Patel N, Walden K, Gomez-Salazar M, Levi B, et al. Neurovascular coupling in bone regeneration. Exp Mol Med 2022; 54: 1844-9. https://doi.org/10.1038/s12276-022-00899-6.

[4] Singer AJ, Clark RA. Cutaneous wound healing. N Engl J Med 1999; 341: 738-46. https://doi.org/10.1056/nejm199909023411006.

[5] Augustin HG, Koh GY. Organotypic vasculature: from descriptive heterogeneity to functional pathophysiology. Science (New York, NY) 2017; 357: eaal2379. https://doi.org/10.1126/science.aal2379.

[6] Dudley AC, Griffioen AW. Pathological angiogenesis: mechanisms and therapeutic strategies. Angiogenesis 2023; 26: 313-47. https://doi.org/10.1007/s10456-023-09876-7.

[7] Herbert SP, Stainier DY. Molecular control of endothelial cell behaviour during blood vessel morphogenesis. Nat Rev Mol Cell Biol 2011; 12: 551-64. https://doi.org/10.1038/nrm3176.

[8] Ding BS, Nolan DJ, Butler JM, James D, Babazadeh AO, Rosenwaks Z, et al. Inductive angiocrine signals from sinusoidal endothelium are required for liver regeneration. Nature 2010; 468: 310-5. https://doi.org/10.1038/nature09493.

[9] Brem H, Tomic-Canic M. Cellular and molecular basis of wound healing in diabetes. J Clin Invest 2007; 117: 1219-22. https://doi.org/10.1172/jci32169.

[10] Geudens I, Gerhardt H. Coordinating cell behaviour during blood vessel formation. Development (Cambridge, England) 2011; 138: 4569-83. https://doi.org/10.1242/dev.062323.

[11] Orecchia P, Conte R, Balza E, Petretto A, Mauri P, Mingari MC, et al. A novel human anti-syndecan-1 antibody inhibits vascular maturation and tumour growth in melanoma. Eur J Cancer 2013; 49: 2022-33. https://doi.org/10.1016/j.ejca.2012.12.019.

[12] Kalluri R.Basement membranes: structure, assembly and role in tumour angiogenesis. Nat Rev Cancer 2003; 3: 422-33. https://doi.org/10.1038/nrc1094.

[13] Tkach M, Théry C. Communication by extracellular vesicles: where we are and where we need to go. Cell 2016; 164: 1226-32. https://doi.org/10.1016/j.cell.2016.01.043.

[14] György B, Szabó TG, Pásztói M, Pál Z, Misják P, Aradi B, et al. Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles. Cell Mol Life Sci 2011; 68: 2667-88. https://doi.org/10.1007/s00018-011-0689-3.

[15] Maas SLN, Breakefield XO, Weaver AM. Extracellular vesicles: unique intercellular delivery vehicles.Trends Cell Biol 2017; 27: 172-88. https://doi.org/10.1016/j.tcb.2016.11.003.

[16] Kalluri R, LeBleu VS. The biology, function, and biomedical applications of exosomes. Science 2020; 367: 367. https://doi.org/10.1126/science.aau6977.

[17] Sheldon H, Heikamp E, Turley H, Dragovic R, Thomas P, Oon CE, et al. New mechanism for notch signaling to endothelium at a distance by Delta-like 4 incorporation into exosomes. Blood 2010; 116: 2385-94. https://doi.org/10.1182/blood-2009-08-239228.

[18] Zhang X, Yuan X, Shi H, Wu L, Qian H, Xu W. Exosomes in cancer: small particle, big player. J Hematol Oncol 2015; 8: 83. https://doi.org/10.1186/s13045-015-0181-x.

[19] Aliotta JM, Pereira M, Wen S, Dooner MS, del Tatto M, Papa E, et al. Exosomes induce and reverse monocrotaline-induced pulmonary hypertension in mice. Cardiovasc Res 2016; 110: 319-30. https://doi.org/10.1093/cvr/cvw054.

[20] Stickney Z, Losacco J, McDevitt S, Zhang Z, Lu B. Development of exosome surface display technology in living human cells. Biochem Biophys Res Commun 2016; 472: 53-9. https://doi.org/10.1016/j.bbrc.2016.02.058.

[21] Eming SA, Martin P, Tomic-Canic M. Wound repair and regeneration: mechanisms, signaling, and translation. Sci Transl Med 2014; 6: 265sr6. https://doi.org/10.1126/scitranslmed.3009337.

[22] Li WW, Li VW, Hutnik M, Chiou AS. Tumor angiogenesis as a target for dietary cancer prevention. J Oncol 2012, 2012: 879623. https://doi.org/10.1155/2012/879623, 1, 23

[23] Smith AN, Willis E, Chan VT, Muffley LA, Isik FF, Gibran NS, et al. Mesenchymal stem cells induce dermal fibroblast responses to injury. Exp Cell Res 2010; 316: 48-54. https://doi.org/10.1016/j.yexcr.2009.08.001.

[24] Kachgal S, Putnam AJ. Mesenchymal stem cells from adipose and bone marrow promote angiogenesis via distinct cytokine and protease expression mechanisms. Angiogenesis 2011; 14: 47-59. https://doi.org/10.1007/s10456-010-9194-9.

[25] Zhang Y, Hao Z, Wang P, Xia Y, Wu J, Xia D, et al. Exosomes from human umbilical cord mesenchymal stem cells enhance fracture healing through HIF-1α-mediated promotion of angiogenesis in a rat model of stabilized fracture. Cell Prolif 2019; 52: e12570. https://doi.org/10.1111/cpr.12570.

[26] Zhou Y, Zhang XL, Lu ST, Zhang NY, Zhang HJ, Zhang J, et al. Human adipose-derived mesenchymal stem cells-derived exosomes encapsulated in pluronic F127 hydrogel promote wound healing and regeneration. Stem Cell Res Ther 2022; 13: 407. https://doi.org/10.1186/s13287-022-02980-3.

[27] Hu L, Wang J, Zhou X, Xiong Z, Zhao J, Yu R, et al. Exosomes derived from human adipose mensenchymal stem cells accelerates cutaneous wound healing via optimizing the characteristics of fibroblasts. Sci Rep 2016; 6: 32993. https://doi.org/10.1038/srep32993.

[28] Shabbir A, Cox A, Rodriguez-Menocal L, Salgado M, Van Badiavas E. Mesenchymal stem cell exosomes induce proliferation and migration of normal and chronic wound fibroblasts, and enhance angiogenesis in vitro. Stem Cells Dev 2015; 24: 1635-47. https://doi.org/10.1089/scd.2014.0316.

[29] Zhang J, Guan J, Niu X, Hu G, Guo S, Li Q, et al. Exosomes released from human induced pluripotent stem cells-derived MSCs facilitate cutaneous wound healing by promoting collagen synthesis and angiogenesis. J Transl Med 2015; 13: 49. https://doi.org/10.1186/s12967-015-0417-0.

[30] Reinert RB, Brissova M, Shostak A, Pan FC, Poffenberger G, Cai Q, et al. Vascular endothelial growth factor-a and islet vascularization are necessary in developing, but not adult, pancreatic islets. Diabetes 2013; 62: 4154-64. https://doi.org/10.2337/db13-0071.

[31] Veith AP, Henderson K, Spencer A, Sligar AD, Baker AB. Therapeutic strategies for enhancing angiogenesis in wound healing. Adv Drug Deliv Rev 2019; 146: 97-125. https://doi.org/10.1016/j.addr.2018.09.010.

[32] De Lorenzi F, Hansen N, Theek B, Daware R, Motta A, Breuel S, et al. Engineering mesoscopic 3D tumor models with a self-organizing vascularized matrix. Adv Mater 2023; 36: e2303196. https://doi.org/10.1002/adma.202303196.

[33] Johnson LR, Lee DY, Eacret JS, Ye D, June CH, Minn AJ. The immunostimulatory RNA RN7SL1 enables CAR-T cells to enhance autonomous and endogenous immune function. Cell 2021; 184: 4981-4995.e14. https://doi.org/10.1016/j.cell.2021.08.004.

[34] Simons M, Gordon E, Claesson-Welsh L. Mechanisms and regulation of endothelial VEGF receptor signalling. Nat Rev Mol Cell Biol 2016; 17: 611-25. https://doi.org/10.1038/nrm.2016.87.

[35] Olsson AK, Dimberg A, Kreuger J, Claesson-Welsh L. VEGF receptor signalling-in control of vascular function. Nat Rev Mol Cell Biol 2006; 7: 359-71. https://doi.org/10.1038/nrm1911.

[36] Rey S, Semenza GL. Hypoxia-inducible factor-1-dependent mechanisms of vascularization and vascular remodelling. Cardiovasc Res 2010; 86: 236-42. https://doi.org/10.1093/cvr/cvq045.

[37] Shi Y, Yang X, Xue X, Sun D, Cai P, Song Q, et al. HANR promotes lymphangiogenesis of hepatocellular carcinoma via secreting miR-296 exosome and regulating EAG1/VEGFA signaling in HDLEC cells. J Cell Biochem 2019; 120: 17699-708. https://doi.org/10.1002/jcb.29036.

[38] Shou X, Wang Y, Jiang Q, Chen J, Liu Q. miR-126 promotes M1 to M2 macrophage phenotype switching via VEGFA and KLF4. PeerJ 2023; 11: e15180. https://doi.org/10.7717/peerj.15180.

[39] Xu L, Fu T, Wang Y, Ji N. Diagnostic value of peripheral blood miR-296 combined with vascular endothelial growth factor B on the degree of coronary artery stenosis in patients with coronary heart disease. J Clin Ultrasound 2023; 51: 520-9. https://doi.org/10.1002/jcu.23433.

[40] Riazifar M, Mohammadi MR, Pone EJ, Yeri A, Lässer C, Segaliny AI, et al. Stem cell-derived exosomes as nanotherapeutics for autoimmune and neurodegenerative disorders. ACS Nano 2019; 13: 6670-88. https://doi.org/10.1021/acsnano.9b01004.

[41] Babapoor-Farrokhran S, Qin Y, Flores-Bellver M, Niu Y, Bhutto IA, Aparicio-Domingo S, et al. Pathologic vs. protective roles of hypoxia-inducible factor 1 in RPE and photoreceptors in wet vs. dry age-related macular degeneration. Proc Natl Acad Sci USA 2023; 120: e2302845120. https://doi.org/10.1073/pnas.2302845120.

[42] Sagini K, Costanzi E, Emiliani C, Buratta S, Urbanelli L. Extracellular vesicles as conveyors of membrane-derived bioactive lipids in immune system. Int J Mol Sci 2018; 19: 19. https://doi.org/10.3390/ijms19041227.

[43] Batrakova EV, Kim MS. Using exosomes, naturally-equipped nanocarriers, for drug delivery. J Control Release 2015; 219: 396-405. https://doi.org/10.1016/j.jconrel.2015.07.030.

[44] Isaac R, Reis FCG, Ying W, Olefsky JM. Exosomes as mediators of intercellular crosstalk in metabolism. Cell Metab 2021; 33: 1744-62. https://doi.org/10.1016/j.cmet.2021.08.006.

[45] Kalinec GM, Gao L, Cohn W, Whitelegge JP, Faull KF, Kalinec F. Extracellular vesicles from auditory cells as nanocarriers for anti-inflammatory drugs and pro-resolving mediators. Front Cell Neurosci 2019; 13: 530. https://doi.org/10.3389/fncel.2019.00530.

[46] Lamichhane TN, Sokic S, Schardt JS, Raiker RS, Lin JW, Jay SM. Emerging roles for extracellular vesicles in tissue engineering and regenerative medicine. Tissue Eng B Rev 2015; 21: 45-54. https://doi.org/10.1089/ten.TEB.2014.0300.

[47] Wilson SE. Corneal wound healing. Exp Eye Res 2020; 197: 108089. https://doi.org/10.1016/j.exer.2020.108089.

[48] Davis GE, Kemp SS. Extracellular matrix regulation of vascular morphogenesis, maturation, and stabilization. Cold Spring Harb Perspect Med 2023; 13: 13. https://doi.org/10.1101/cshperspect.a041156.

[49] Jandl K, Radic N, Zeder K, Kovacs G, Kwapiszewska G. Pulmonary vascular fibrosis in pulmonary hypertension-the role of the extracellular matrix as a therapeutic target. Pharmacol Ther 2023; 247: 108438. https://doi.org/10.1016/j.pharmthera.2023.108438.

[50] Yang B, Chen Y, Shi J. Exosome biochemistry and advanced nanotechnology for next-generation theranostic platforms. Adv Mater 2019; 31: e1802896. https://doi.org/10.1002/adma.201802896.

[51] Mendt M, Kamerkar S, Sugimoto H, McAndrews KM, Wu CC, Gagea M, et al. Generation and testing of clinical-grade exosomes for pancreatic cancer. JCI Insight 2018; 3: 3. https://doi.org/10.1172/jci.insight.99263.

[52] Thompson EM, Keir ST, Venkatraman T, Lascola C, Yeom KW, Nixon AB, et al. The role of angiogenesis in group 3 medulloblastoma pathogenesis and survival. Neuro-Oncology 2017; 19: 1217-27. https://doi.org/10.1093/neuonc/nox033.

[53] Liu C, Yan X, Zhang Y, Yang M, Ma Y, Zhang Y, et al. Oral administration of turmeric-derived exosome-like nanovesicles with anti-inflammatory and pro-resolving bioactions for murine colitis therapy. J Nanobiotechnol 2022; 20: 206. https://doi.org/10.1186/s12951-022-01421-w.

[54] Xu Y, Ouyang L, He L, Qu Y, Han Y, Duan D. Inhibition of exosomal miR-24-3p in diabetes restores angiogenesis and facilitates wound repair via targeting PIK3R3. J Cell Mol Med 2020; 24: 13789-803. https://doi.org/10.1111/jcmm.15958.