Effects of exosomes derived from normoxic or hypoxic human umbilical vein endothelial cells on hypoxia-induced cardiomyocyte injury and cardiac fibroblast activation in vitro

doi:10.12122/j.issn.1673-4254.2026.08.18

Abstract

Abstract:

Objective To investigate the effects of exosomes derived from human umbilical venous endothelial cells (HUVECs) cultured in normoxic and hypoxic conditions (N-exo and H-exo, respectively) on hypoxia-induced injury of human cardiomyocyte AC16 cells and activation of rat cardiac fibroblasts (RCFs) in vitro. Methods N-exo and H-exo were extracted from HUVECs by differential centrifugation and sucrose density gradient centrifugation and characterized. AC16 cells and RCFs in normoxic culture or hypoxic culture (1% O₂ for 24 h) were treated with N-exo or H-exo, and the changes in protein and mRNA levels were examined with Western blotting and RT-qPCR. Intracellular ROS levels and apoptosis in AC16 cells were assessed by DCFH-DA staining and TUNEL staining, and the changes in proliferation of RCFs were evaluated with EdU assay. Results N-exo and H-exo with densities of 1.5×10⁹ and 7.2×10⁸ mL^-1 were obtained, respectively, which showed abundant protein components and expressed the exosomal marker HSP70. In AC16 cells, hypoxic exposure significantly upregulated CK-MB mRNA and downregulated Cx43 and GPX4 mRNA levels, causing also enhanced expressions of ALP, IL-1β and cTnT proteins and increased intracellular ROS content and cell apoptosis. These changes were effectively alleviated by treatment with 10⁷ mL^-1 N-exo but worsened after treatment with H-exo. In RCFs, hypoxia significantly increased the expressions of collagen I, collagen III and α‑SMA and cell proliferation, which were suppressed by N-exo treatment but further enhanced by H-exo treatment. Hypoxia exposure increased HIF-1α and VEGF expression and phosphorylation level of AKT in both AC16 cells and RCFs; N-exo obviously inhibited while H-exo further enhanced these changes. Px-478, a specific HIF-1α inhibitor, significantly inhibited hypoxia-induced AC16 cell injury and RCF activation. Conclusion N-exo derived from HUVECs inhibits while H-exo exacerbates hypoxia-induced AC16 cell injury and RCF activation possibly via the HIF-1α pathway.

Key words: human umbilical vein endothelial cells, cardiac fibroblasts, cardiomyocyte, hypoxia-inducible factor

Xiaoshuan XIONG, Tao YANG, Junyu CHEN, Xiaoqing YANG, Shiyu CHEN, Huanjun RUAN, Xiaoqing WANG, Weining ZHANG, Fangzhou CHENG, Xiao KE. Effects of exosomes derived from normoxic or hypoxic human umbilical vein endothelial cells on hypoxia-induced cardiomyocyte injury and cardiac fibroblast activation in vitro[J]. Journal of Southern Medical University, 2026, 46(8): 1905-1914.

Figures/Tables 7

Fig.1 Isolation, purification and identification of HUVECs-derived exosomes. A: Particle size, concentration and transmission electron microscopy (Original magnification: ×18 500) of exosomes derived from HUVECs. B: Protein band profile of HUVECs-derived exosomes stained with Coomassie brilliant blue. C: Western blotting for detection of exosomal marker proteins in HUVECs-derived exosomes.

Fig.2 Effect of HUVECs-derived exosomes on hypoxia-induced AC16 cells. A: mRNA expression levels in AC16 cells detected by RT‑qPCR. B: Western blotting for detecting protein expression levels in AC16 cells. C: Intracellular ROS levels detected by DCFH-DA staining (×40). D: Cell apoptosis detected by TUNEL assay (×40). Data are presented as Mean±SD (n=3). *P<0.05, **P<0.01, ***P<0.001 vs Hypoxia group.

Fig.3 Effect of HUVECs-derived exosomes on hypoxia-induced RCFs. A: mRNA expression levels in RCFs cells detected by RT‑qPCR. B: Western blotting for detecting protein expression levels in RCFs cells. C: Proliferative ability of RCFs detected by EdU assay (×40). Data are presented as Mean±SD (n=3). *P<0.05, **P<0.01, ***P<0.001 vs Hypoxia group.

Fig.4 Effect of HUVEC-derived exosomes on AC16 cells and RCFs and the mediating role of the HIF-1α signaling pathway. A: Western blotting for detecting protein expression levels in AC16 cells. B: Western blotting for detecting protein expression levels in RCFs cells. Data are presented as Mean±SD (n=3). *P<0.05, **P<0.01, ****P<0.0001 vs Hypoxia group.

Fig.5 Effects of HIF-1α inhibitor on hypoxia-induced AC16 cells. A: Western blotting for detecting protein expression levels in AC16 cells. B: Intracellular ROS levels detected by DCFH-DA staining (×40). C: Cell apoptosis detected by TUNEL assay (×40). Data are presented as Mean±SD (n=3). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs Hypoxia group; #P<0.05, ####P<0.0001 vs Hypoxia+H-exo group.

Fig.6 Effect of HIF-1α inhibitor on hypoxia-induced RCFs. A: Western blotting for detecting protein expression levels in RCFs cells. B: Proliferative ability of RCFs detected by EdU assay (×40). Data are presented as Mean±SD (n=3). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs Hypoxia group; #P<0.05, ##P<0.01 vs Hypoxia+H-exo group.

References 31

[1]	Li X, Zhang Q, Nasser MI, et al. Oxygen homeostasis and cardiovascular disease: A role for HIF[J]. Biomed Pharmacother, 2020, 128: 110338. doi：10.1016/j.biopha.2020.110338
[2]	Xie SY, Xing Y, Shi WK, et al. Cardiac fibroblast heat shock protein 47 aggravates cardiac fibrosis post myocardial ischemia-reperfusion injury by encouraging ubiquitin specific peptidase 10 dependent Smad4 deubiquitination[J]. Acta Pharm Sin B, 2022, 12(11): 4138-53. doi：10.1016/j.apsb.2022.07.022
[3]	Bacmeister L, Schwarzl M, Warnke S, et al. Inflammation and fibrosis in murine models of heart failure[J]. Basic Res Cardiol, 2019, 114(3): 19. doi：10.1007/s00395-019-0722-5
[4]	Wang JH, Chen X, Huang W. microRNA-369 attenuates hypoxia-induced cardiomyocyte apoptosis and inflammation via targeting TRPV3[J]. Braz J Med Biol Res, 2021, 54(3): e10550. doi：10.1590/1414-431x202010550
[5]	Baehr A, Umansky KB, Bassat E, et al. Agrin promotes coordinated therapeutic processes leading to improved cardiac repair in pigs[J]. Circulation, 2020, 142(9): 868-81. doi：10.1161/circulationaha.119.045116
[6]	Feng JJ, Zhan JC, Ma SS. LRG1 promotes hypoxia-induced cardiomyocyte apoptosis and autophagy by regulating hypoxia-inducible factor-1α[J]. Bioengineered, 2021, 12(1): 8897-907. doi：10.1080/21655979.2021.1988368
[7]	Han CS, Yang JJ, Sun JC, et al. Extracellular vesicles in cardiovascular disease: Biological functions and therapeutic implications[J]. Pharmacol Ther, 2022, 233: 108025. doi：10.1016/j.pharmthera.2021.108025
[8]	Kalluri R, LeBleu VS. The biology, function, and biomedical applications of exosomes[J]. Science, 2020, 367(6478): eaau6977. doi：10.1126/science.aau6977
[9]	Pan YH, Wu WP, Jiang XX, et al. Mesenchymal stem cell-derived exosomes in cardiovascular and cerebrovascular diseases: From mechanisms to therapy[J]. Biomed Pharmacother, 2023, 163: 114817. doi：10.1016/j.biopha.2023.114817
[10]	Zhang ZB, Zou YM, Song CY, et al. Advances in the study of exosomes in cardiovascular diseases[J]. J Adv Res, 2024, 66: 133-53. doi：10.1016/j.jare.2023.12.014
[11]	Tian Z, Ning H, Wang XY, et al. Endothelial autophagy promotes atheroprotective communication between endothelial and smooth muscle cells via exosome-mediated delivery of miR-204-5p[J]. Arterioscler Thromb Vasc Biol, 2024, 44(8): 1813-32. doi：10.1161/atvbaha.123.319993
[12]	Otani K, Yokoya M, Kodama T, et al. Plasma exosomes regulate systemic blood pressure in rats[J]. Biochem Biophys Res Commun, 2018, 503(2): 776-83. doi：10.1016/j.bbrc.2018.06.075
[13]	Wang C, Li ZL, Liu YN, et al. Exosomes in atherosclerosis: performers, bystanders, biomarkers, and therapeutic targets[J]. Theranostics, 2021, 11(8): 3996-4010. doi：10.7150/thno.56035
[14]	Wang Y, Zhao RZ, Shen CY, et al. Exosomal CircHIPK3 released from hypoxia-induced cardiomyocytes regulates cardiac angiogenesis after myocardial infarction[J]. Oxid Med Cell Longev, 2020, 2020: 8418407. doi：10.1155/2020/8418407
[15]	Liu SJ, Chen J, Shi J, et al. M1-like macrophage-derived exosomes suppress angiogenesis and exacerbate cardiac dysfunction in a myocardial infarction microenvironment[J]. Basic Res Cardiol, 2020, 115(2): 22. doi：10.1007/s00395-020-0781-7
[16]	Zhao YC, Xiong WD, Li CF, et al. Hypoxia-induced signaling in the cardiovascular system: pathogenesis and therapeutic targets[J]. Signal Transduct Target Ther, 2023, 8(1): 431. doi：10.1038/s41392-023-01652-9
[17]	Ding J, Wang BY, Yang YF, et al. Ciprofol ameliorates myocardial ischemia/reperfusion injury by inhibiting ferroptosis through upregulating HIF-1α[J]. Drug Des Devel Ther, 2024, 18: 6115-32. doi：10.2147/dddt.s480514
[18]	Maimaitiaili N, Zeng YX, Ju PN, et al. NLRC3 deficiency promotes hypoxia-induced pulmonary hypertension development via IKK/NF-κB p65/HIF-1α pathway[J]. Exp Cell Res, 2023, 431(2): 113755. doi：10.1016/j.yexcr.2023.113755
[19]	Sun JC, Shen H, Shao LB, et al. HIF-1α overexpression in mesenchymal stem cell-derived exosomes mediates cardiopro-tection in myocardial infarction by enhanced angiogenesis[J]. Stem Cell Res Ther, 2020, 11(1): 373. doi：10.1186/s13287-020-01881-7
[20]	Wen ZZ, Mai Z, Zhu XL, et al. Mesenchymal stem cell-derived exosomes ameliorate cardiomyocyte apoptosis in hypoxic conditions through microRNA144 by targeting the PTEN/AKT pathway[J]. Stem Cell Res Ther, 2020, 11(1): 36. doi：10.1186/s13287-020-1563-8
[21]	Fang TT, Ma CC, Yang BY, et al. Roxadustat improves diabetic myocardial injury by upregulating HIF-1α/UCP2 against oxidative stress[J]. Cardiovasc Diabetol, 2025, 24(1): 67. doi：10.1186/s12933-025-02601-2
[22]	Ikeda M, Ide T, Tadokoro T, et al. Excessive hypoxia-inducible factor-1α expression induces cardiac rupture via p53-dependent apoptosis after myocardial infarction[J]. J Am Heart Assoc, 2021, 10(17): e020895. doi：10.1161/jaha.121.020895
[23]	Yang Z, Huang YJ, Zhu L, et al. SIRT6 promotes angiogenesis and hemorrhage of carotid plaque via regulating HIF-1α and reactive oxygen species[J]. Cell Death Dis, 2021, 12: 77. doi：10.1038/s41419-020-03372-2
[24]	Janbandhu V, Tallapragada V, Patrick R, et al. Hif-1a suppresses ROS-induced proliferation of cardiac fibroblasts following myocardial infarction[J]. Cell Stem Cell, 2022, 29(2): 281-97. e12. doi：10.1016/j.stem.2021.10.009
[25]	Bao QK, Zhang BY, Zhou L, et al. CNP ameliorates macrophage inflammatory response and atherosclerosis[J]. Circ Res, 2024, 134(8): e72-91. doi：10.1161/circresaha.123.324086
[26]	Lei L, Mason S, Liu DG, et al. Hypoxia-inducible factor-dependent degeneration, failure, and malignant transformation of the heart in the absence of the von Hippel-Lindau protein. Mol[J]. Cell Biol, 2008, 28: 3790-803. doi：10.1128/mcb.01580-07
[27]	Lian G, Li XP, Zhang LQ, et al. Macrophage metabolic reprogramming aggravates aortic dissection through the HIF1α-ADAM17 pathway[J]. eBioMedicine, 2019, 49: 291-304. doi：10.1016/j.ebiom.2019.09.041
[28]	Wang J, Wang XJ, Zhang Y, et al. TXNIP knockout improves cardiac function after myocardial infarction by promoting angiogenesis and reducing cardiomyocyte apoptosis[J]. Cardiovasc Diagn Ther, 2022, 12(3): 289-304. doi：10.21037/cdt-21-732
[29]	Sui XZ, Wei HC, Wang DC. Novel mechanism of cardiac protection by valsartan: synergetic roles of TGF-β1 and HIF-1α in Ang II-mediated fibrosis after myocardial infarction[J]. J Cell Mol Med, 2015, 19(8): 1773-82. doi：10.1111/jcmm.12551
[30]	Viswanadha VP, Dhivya V, Beeraka NM, et al. The protective effect of piperine against isoproterenol-induced inflammation in experimental models of myocardial toxicity[J]. Eur J Pharmacol, 2020, 885: 173524. doi：10.1016/j.ejphar.2020.173524
[31]	Refaie MMM, El-Hussieny M, Abdel-Hakeem EA, et al. Phosphodiesterase inhibitor, Vinpocetine, guards against doxorubicin induced cardiotoxicity via modulation of HIF/VEGF and cGMP/cAMP/SIRT signaling pathways[J]. Hum Exp Toxicol, 2022, 41: 09603271221136209. doi：10.1177/09603271221136209