Human umbilical cord mesenchymal stem cell exosomes pretreated with atorvastatin alleviate high glucose-induced injury of human retinal vascular endothelial cells through the protein kinase B/ endothelial nitric oxide synthase pathway_Chinese Journal of Ocular Fundus Diseases

Authors：

Wu Bin , Liu Yangchen , Zhang Wei ,  He Guanghui

Tianjin Eye Hospital, Tianjin Key Lab of Ophthalmology and Vision Science, Tianjin 300020, China;

Corresponding author：

He Guanghui, Email: peterho812@126.com

Keywords：

Atorvastatin; Mesenchymal stem cell; Exosomes; High glucose; Cell experiment

DOI：

10.3760/cma.j.cn511434-20241210-00479

Video：

Export PDF Favorites Scan Get Citation

Abstract Full text Figures/Tables Video References Cited by

Objective To investigate whether exosomes derived from atorvastatin (ATV)-pretreated human umbilical cord mesenchymal stem cells (ATV-MSC-EXO) alleviate high glucose-induced injury in human retinal vascular endothelial cells (HREC) via the protein kinase B (AKT)/endothelial nitric oxide synthase (eNOS) signaling pathway. Methods The optimal pretreatment concentration of atorvastatin (ATV) was determined using the cell counting Kit-8 (CCK-8) assay. Exosomes derived from mesenchymal stem cells (MSC-EXO) and ATV-pretreated MSCs (ATV-MSC-EXO) were isolated and extracted, and their morphology and surface markers were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blotting (WB). The uptake capacity of exosomes by human retinal vascular endothelial cells (HREC) was evaluated using a fluorescence labeling assay. In vitro cultured HREC were divided into the following groups: normal control group (NC group), high glucose group (HG group), high glucose+MSC-EXO group (MSC-EXO group), high glucose+ATV-MSC-EXO group (ATV-MSC-EXO group), high glucose+ATV-MSC-EXO+AKT inhibitor group (ATV-MSC-EXO-MK-2206-2HCL group), and high glucose+ATV-MSC-EXO+eNOS inhibitor group (ATV-MSC-EXO-L-NAME group). Cell proliferation and apoptosis were detected using CCK-8 and flow cytometry, respectively. The protein expression levels of B-cell lymphoma/leukemia-2 (Bcl-2), Bcl-2-associated protein (Bax), and Caspase-3 were measured by WB. In addition, the regulatory effects of ATV-MSC-EXO on the AKT/eNOS signaling pathway and its downstream functional molecules were analyzed by detecting the phosphorylation levels of AKT (P-AKT/AKT) and eNOS (P-eNOS/eNOS) via WB, the mRNA expression levels of AKT and eNOS by quantitative real-time polymerase chain reaction (qPCR), and the concentrations of nitric oxide (NO) and endothelin-1 (ET-1) using commercial NO and ET-1 assay kits. Results The optimal pretreatment concentration of ATV was 1 μmol/L. ATV-MSC-EXO exhibited similar morphology and particle size to MSC-EXO and were efficiently taken up by HREC. Under high glucose conditions, ATV-MSC-EXO significantly enhanced the viability of HREC (F=83.24, P＜0.000 1) and inhibited apoptosis (F=77.39, P＜0.000 1). Western blot analysis further confirmed that ATV-MSC-EXO upregulated the expression of the anti-apoptotic protein Bcl-2 (F=53.17), while downregulating the pro-apoptotic proteins Bax (F=36.49) and Caspase-3 (F=60.75) (P＜0.001). In addition, ATV-MSC-EXO markedly increased the protein levels of P-AKT/AKT (F=107.60) and P-eNOS/eNOS (F=38.59), as well as the relative mRNA expression of AKT, eNOS, P-AKT, and P-eNOS (F= 203.60, 315.00, 234.00, 47.30; P＜0.000 1). Furthermore, ATV-MSC-EXO promoted NO production (F= 407.40) and suppressed the relative expression of ET-1 (F= 49.76) (P＜0.000 1). Conclusion ATV-MSC-EXO enhances the viability and inhibits apoptosis of HREC under high glucose conditions by activating the AKT/eNOS signaling pathway.

1.	Li Y, Liu Y, Liu S, et al. Diabetic vascular diseases: molecular mechanisms and therapeutic strategies[J]. Signal Transduct Target Ther, 2023, 8(1): 152. DOI: 10.1038/s41392-023-01400-z.
2.	Lin F, Yang Y, Wei S, et al. Hydrogen sulfide protects against high glucose-induced human umbilical vein endothelial cell injury through activating PI3K/Akt/eNOS pathway[J]. Drug Des Devel Ther, 2020, 14: 621-633. DOI: 10.2147/DDDT.S242521.
3.	Liu M, Xiang G, Lu J, et al. TRAIL protects against endothelium injury in diabetes via Akt-eNOS signaling[J]. Atherosclerosis, 2014, 237(2): 718-724. DOI: 10.1016/j.atherosclerosis.2014.10.013.
4.	杨敏, 罗向霞, 康莉, 等. 磷脂酰肌醇3-激酶/蛋白激酶B信号通路在糖尿病视网膜病变与动脉粥样硬化发病机制中的作用[J]. 中华糖尿病杂志, 2018, 10(5): 373-376. DOI: 10.3760/cma.j.issn.1674-5809.2018.05.015.Yang M, Luo XX, Kang L, et al. Effects of phosphatidylinositol 3-kinase/protein kinase B signaling pathway on the pathogenesis of diabetic retinopathy and atherosclerosis[J]. Chin J Diabetes Mellitus, 2018, 10(5): 373-376. DOI: 10.3760/cma.j.issn.1674-5809.2018.05.015.
5.	Yan J, Tie G, Park B, et al. Recovery from hind limb ischemia is less effective in type 2 than in type 1 diabetic mice: roles of endothelial nitric oxide synthase and endothelial progenitor cells[J]. J Vasc Surg, 2009, 50(6): 1412-1422. DOI: 10.1016/j.jvs.2009.08.007.
6.	Bai L, Wang Y. Mesenchymal stem cells-derived EXOomes alleviate senescence of retinal pigment epithelial cells by activating PI3K/AKT-Nrf2 signaling pathway in early diabetic retinopathy[J/OL]. Exp Cell Res, 2024, 441(2): 114170[2024-07-15]. https://pubmed.ncbi.nlm.nih.gov/39019426/. DOI: 10.1016/j.yexcr.2024.114170.
7.	Huang P, Wang L, Li Q, et al. Atorvastatin enhances the therapeutic efficacy of mesenchymal stem cells-derived EXOomes in acute myocardial infarction via up-regulating long non-coding RNA H19[J]. Cardiovasc Res, 2020, 116(2): 353-367. DOI: 10.1093/cvr/cvz139.
8.	Yu M, Liu W, Li J, et al. EXOomes derived from atorvastatin-pretreated MSC accelerate diabetic wound repair by enhancing angiogenesis via AKT/eNOS pathway[J/OL]. Stem Cell Res Ther, 2020, 11(1): 350[2020-08-12]. https://pubmed.ncbi.nlm.nih.gov/32787917/. DOI: 10.1186/s13287-020-01824-2.
9.	Yu B, Li XR, Zhang XM. Mesenchymal stem cell-derived extracellular vesicles as a new therapeutic strategy for ocular diseases[J]. World J Stem Cells, 2020, 12(3): 178-187. DOI: 10.4252/wjsc.v12.i3.178.
10.	Moisseiev E, Anderson JD, Oltjen S, et al. Protective effect of intravitreal administration of EXOomes derived from mesenchymal stem cells on retinal ischemia[J]. Curr Eye Res, 2017, 42(10): 1358-1367. DOI: 10.1080/02713683.2017.1319491.
11.	Zhang W, Wang Y, Kong Y. EXOomes derived from mesenchymal stem cells modulate miR-126 to ameliorate hyperglycemia-induced retinal inflammation via targeting HMGB1[J]. Invest Ophthalmol Vis Sci, 2019, 60(1): 294-303. DOI: 10.1167/iovs.18-25617.
12.	Safwat A, Sabry D, Ragiae A, et al. Adipose mesenchymal stem cells-derived EXOomes attenuate retina degeneration of streptozotocin-induced diabetes in rabbits[J/OL]. J Circ Biomark, 2018, 7: 1849454418807827[2018-10-28]. https://pubmed.ncbi.nlm.nih.gov/30397416/. DOI: 10.1177/1849454418807827.
13.	Wu J, Lei MX, Xie XY, et al. Rosiglitazone inhibits high glucose-induced apoptosis in human umbilical vein endothelial cells through the PI3K/Akt/eNOS pathway[J]. Can J Physiol Pharmacol, 2009, 87(7): 549-555. DOI: 10.1139/y09-040.
14.	Liu D, Wu M, Lu Y, et al. Protective effects of 6-Gingerol on vascular endothelial cell injury induced by high glucose via activation of PI3K-AKT-eNOS pathway in human umbilical vein endothelial cells[J]. Biomed Pharmacother, 2017, 93: 788-795. DOI: 10.1016/j.biopha.2017.07.037.
15.	Wang Y, Cui L, Xu H, et al. TRPV1 agonism inhibits endothelial cell inflammation via activation of eNOS/NO pathway[J]. Atherosclerosis, 2017, 260: 13-19. DOI: 10.1016/j.atherosclerosis.2017.03.016.
16.	Duan MX, Zhou H, Wu QQ, et al. Andrographolide protects against hg-induced inflammation, apoptosis, migration, and impairment of angiogenesis via PI3K/AKT-eNOS signalling in HUVECs[J/OL]. Mediators Inflamm, 2019, 2019: 6168340[2019-10-07]. https://pubmed.ncbi.nlm.nih.gov/31686985/. DOI: 10.1155/2019/6168340.
17.	Jeong SJ, Dasgupta A, Jung KJ, et al. PI3K/AKT inhibition induces caspase-dependent apoptosis in HTLV-1-transformed cells[J]. Virology, 2008, 370(2): 264-272. DOI: 10.1016/j.virol.2007.09.003.
18.	Liu Q, Zhang L, Shan Q, et al. Total flavonoids from Astragalus alleviate endothelial dysfunction by activating the Akt/eNOS pathway[J]. J Int Med Res, 2018, 46(6): 2096-2103. DOI: 10.1177/0300060517717358.
19.	Cui J, Zhang B, Gao M, et al. The protective effect of tetrahydroxystilbene glucoside on high glucose-induced injury in human umbilical vein endothelial cells through the PI3K/Akt/eNOS pathway and regulation of Bcl-2/Bax[J]. J Vasc Res, 2021, 58(5): 301-310. DOI: 10.1159/000511035.
20.	Barton M, Yanagisawa M, Vanhoutte PM, et al. Endothelin XII[J]. Life Sci, 2012, 91(13-14): 449-451. DOI: 10.1016/j.lfs.2012.07.016.
21.	Chou JC, Rollins SD, Ye M, et al. Endothelin receptor-A antagonist attenuates retinal vascular and neuroretinal pathology in diabetic mice[J]. Invest Ophthalmol Vis Sci, 2014, 55(4): 2516-2525. DOI: 10.1167/iovs.13-13676.
22.	Singh G, Sharma B, Jaggi AS, et al. Efficacy of bosentan, a dual ETA and ETB endothelin receptor antagonist, in experimental diabetes induced vascular endothelial dysfunction and associated dementia in rats[J]. Pharmacol Biochem Behav, 2014, 124: 27-35. DOI: 10.1016/j.pbb.2014.05.002.

1. Li Y, Liu Y, Liu S, et al. Diabetic vascular diseases: molecular mechanisms and therapeutic strategies[J]. Signal Transduct Target Ther, 2023, 8(1): 152. DOI: 10.1038/s41392-023-01400-z.
2. Lin F, Yang Y, Wei S, et al. Hydrogen sulfide protects against high glucose-induced human umbilical vein endothelial cell injury through activating PI3K/Akt/eNOS pathway[J]. Drug Des Devel Ther, 2020, 14: 621-633. DOI: 10.2147/DDDT.S242521.
3. Liu M, Xiang G, Lu J, et al. TRAIL protects against endothelium injury in diabetes via Akt-eNOS signaling[J]. Atherosclerosis, 2014, 237(2): 718-724. DOI: 10.1016/j.atherosclerosis.2014.10.013.
4. 杨敏, 罗向霞, 康莉, 等. 磷脂酰肌醇3-激酶/蛋白激酶B信号通路在糖尿病视网膜病变与动脉粥样硬化发病机制中的作用[J]. 中华糖尿病杂志, 2018, 10(5): 373-376. DOI: 10.3760/cma.j.issn.1674-5809.2018.05.015.Yang M, Luo XX, Kang L, et al. Effects of phosphatidylinositol 3-kinase/protein kinase B signaling pathway on the pathogenesis of diabetic retinopathy and atherosclerosis[J]. Chin J Diabetes Mellitus, 2018, 10(5): 373-376. DOI: 10.3760/cma.j.issn.1674-5809.2018.05.015.
5. Yan J, Tie G, Park B, et al. Recovery from hind limb ischemia is less effective in type 2 than in type 1 diabetic mice: roles of endothelial nitric oxide synthase and endothelial progenitor cells[J]. J Vasc Surg, 2009, 50(6): 1412-1422. DOI: 10.1016/j.jvs.2009.08.007.
6. Bai L, Wang Y. Mesenchymal stem cells-derived EXOomes alleviate senescence of retinal pigment epithelial cells by activating PI3K/AKT-Nrf2 signaling pathway in early diabetic retinopathy[J/OL]. Exp Cell Res, 2024, 441(2): 114170[2024-07-15]. https://pubmed.ncbi.nlm.nih.gov/39019426/. DOI: 10.1016/j.yexcr.2024.114170.
7. Huang P, Wang L, Li Q, et al. Atorvastatin enhances the therapeutic efficacy of mesenchymal stem cells-derived EXOomes in acute myocardial infarction via up-regulating long non-coding RNA H19[J]. Cardiovasc Res, 2020, 116(2): 353-367. DOI: 10.1093/cvr/cvz139.
8. Yu M, Liu W, Li J, et al. EXOomes derived from atorvastatin-pretreated MSC accelerate diabetic wound repair by enhancing angiogenesis via AKT/eNOS pathway[J/OL]. Stem Cell Res Ther, 2020, 11(1): 350[2020-08-12]. https://pubmed.ncbi.nlm.nih.gov/32787917/. DOI: 10.1186/s13287-020-01824-2.
9. Yu B, Li XR, Zhang XM. Mesenchymal stem cell-derived extracellular vesicles as a new therapeutic strategy for ocular diseases[J]. World J Stem Cells, 2020, 12(3): 178-187. DOI: 10.4252/wjsc.v12.i3.178.
10. Moisseiev E, Anderson JD, Oltjen S, et al. Protective effect of intravitreal administration of EXOomes derived from mesenchymal stem cells on retinal ischemia[J]. Curr Eye Res, 2017, 42(10): 1358-1367. DOI: 10.1080/02713683.2017.1319491.
11. Zhang W, Wang Y, Kong Y. EXOomes derived from mesenchymal stem cells modulate miR-126 to ameliorate hyperglycemia-induced retinal inflammation via targeting HMGB1[J]. Invest Ophthalmol Vis Sci, 2019, 60(1): 294-303. DOI: 10.1167/iovs.18-25617.
12. Safwat A, Sabry D, Ragiae A, et al. Adipose mesenchymal stem cells-derived EXOomes attenuate retina degeneration of streptozotocin-induced diabetes in rabbits[J/OL]. J Circ Biomark, 2018, 7: 1849454418807827[2018-10-28]. https://pubmed.ncbi.nlm.nih.gov/30397416/. DOI: 10.1177/1849454418807827.
13. Wu J, Lei MX, Xie XY, et al. Rosiglitazone inhibits high glucose-induced apoptosis in human umbilical vein endothelial cells through the PI3K/Akt/eNOS pathway[J]. Can J Physiol Pharmacol, 2009, 87(7): 549-555. DOI: 10.1139/y09-040.
14. Liu D, Wu M, Lu Y, et al. Protective effects of 6-Gingerol on vascular endothelial cell injury induced by high glucose via activation of PI3K-AKT-eNOS pathway in human umbilical vein endothelial cells[J]. Biomed Pharmacother, 2017, 93: 788-795. DOI: 10.1016/j.biopha.2017.07.037.
15. Wang Y, Cui L, Xu H, et al. TRPV1 agonism inhibits endothelial cell inflammation via activation of eNOS/NO pathway[J]. Atherosclerosis, 2017, 260: 13-19. DOI: 10.1016/j.atherosclerosis.2017.03.016.
16. Duan MX, Zhou H, Wu QQ, et al. Andrographolide protects against hg-induced inflammation, apoptosis, migration, and impairment of angiogenesis via PI3K/AKT-eNOS signalling in HUVECs[J/OL]. Mediators Inflamm, 2019, 2019: 6168340[2019-10-07]. https://pubmed.ncbi.nlm.nih.gov/31686985/. DOI: 10.1155/2019/6168340.
17. Jeong SJ, Dasgupta A, Jung KJ, et al. PI3K/AKT inhibition induces caspase-dependent apoptosis in HTLV-1-transformed cells[J]. Virology, 2008, 370(2): 264-272. DOI: 10.1016/j.virol.2007.09.003.
18. Liu Q, Zhang L, Shan Q, et al. Total flavonoids from Astragalus alleviate endothelial dysfunction by activating the Akt/eNOS pathway[J]. J Int Med Res, 2018, 46(6): 2096-2103. DOI: 10.1177/0300060517717358.
19. Cui J, Zhang B, Gao M, et al. The protective effect of tetrahydroxystilbene glucoside on high glucose-induced injury in human umbilical vein endothelial cells through the PI3K/Akt/eNOS pathway and regulation of Bcl-2/Bax[J]. J Vasc Res, 2021, 58(5): 301-310. DOI: 10.1159/000511035.
20. Barton M, Yanagisawa M, Vanhoutte PM, et al. Endothelin XII[J]. Life Sci, 2012, 91(13-14): 449-451. DOI: 10.1016/j.lfs.2012.07.016.
21. Chou JC, Rollins SD, Ye M, et al. Endothelin receptor-A antagonist attenuates retinal vascular and neuroretinal pathology in diabetic mice[J]. Invest Ophthalmol Vis Sci, 2014, 55(4): 2516-2525. DOI: 10.1167/iovs.13-13676.
22. Singh G, Sharma B, Jaggi AS, et al. Efficacy of bosentan, a dual ETA and ETB endothelin receptor antagonist, in experimental diabetes induced vascular endothelial dysfunction and associated dementia in rats[J]. Pharmacol Biochem Behav, 2014, 124: 27-35. DOI: 10.1016/j.pbb.2014.05.002.

Chinese Journal of Ocular Fundus Diseases

Latest ArticlesHuman umbilical cord mesenchymal stem cell exosomes pretreated with atorvastatin alleviate high glucose-induced injury of human retinal vascular endothelial cells through the protein kinase B/ endothelial nitric oxide synthase pathway

Abstract Full text Figures/Tables Video References Cited by

Format

Content