Effects of S100A4 overexpression on retinal capillary cells in a retinal ischemia-reperfusion model in rats_Chinese Journal of Ocular Fundus Diseases

Authors：

Pan Yiji , Yang Jiayi , Xing Yiqiao ,  He Tao

Eye Center, Renmin Hospital of Wuhan University, Wuhan 430060, China;

Corresponding author：

He Tao, Email: ht178933175@163.com

Keywords：

Retinal ischemia-reperfusion injury; S100A4; Endothelial cells; Pericyte; Retinal ganglion cells; Animal experiment

DOI：

10.3760/cma.j.cn511434-20230803-00333

Video：

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Objective To observe the effects of overexpression of S100A4 protein on retinal capillary cells and retinal ganglion cells (RGC) after retinal ischemia-reperfusion injury (RIRI). Methods One hundred healthy adult male C57BL/6 mice were randomly divided into normal control group (group C), RIRI group, adeno-associated virus (AAV2)-S100A4 green fluorescent protein (GFP) intravitreal injection group (group S), RIRI+AAV2-GFP intravitreal injection group (group GIR), and RIRI+AAV2-S100A4-GFP intravitreal injection group (group SIR), with 20 mice in each group. The RIRI model was established using the high intraocular pressure anterior chamber method in the RIRI, GIR and SIR groups of mice. Eyes were enucleated 3 days after modelling by over anaesthesia. The number of retinal capillary endothelial cells and pericytes in the retinal capillaries of mice in each group was observed by retinal trypsinised sections and hematoxylin-eosin and periodic acid-Schiff staining; immunofluorescence staining was used to observe endothelial cell, pericyte coverage and RGC survival; The relative expression of Toll-like receptor 4 (TLR4), p38 MAPK and nuclear factor erythroid 2-related factor 2 (NRF2) in retinal tissues was measured by Western blot. One-way analysis of variance was used to compare data between groups. Results Three days after modeling, the endothelial cell to pericyte ratio in group C was compared with group S and SIR, and the difference was not statistically significant (F=106.30, P＞0.05); the SIR group was compared with group RIRI and GIR, and the difference was statistically significant (F=106.30, P＜0.000 1). Comparison of endothelial cell coverage in each group, the difference was not statistically significant (F=3.44, P＞0.05); compared with the pericyte coverage in group C, the RIRI group and the GIR group were significantly lower, and the difference was statistically significant (F=62.69, P＜0.001). Compared with the RGC survival rate in group C, it was significantly lower in RIRI and GIR groups, and the difference was statistically significant (F=171.60, P＜0.000 1); compared with RIRI and GIR groups, the RGC survival rate in SIR group was significantly higher, and the difference was statistically significant (F=171.60, P＜0.000 1). The relative expression levels of TLR4, p38 and NRF2 proteins were statistically significant among all groups (F=42.65, 20.78, 11.55; P＜0.05). Conclusions Pericytes are more sensitive to ischemia than endothelial cells after retinal RIRI in mice, and early vascular cell loss is dominated by pericytes rather than endothelial cells. The overexpression of S100A4 protein protects against loss of pericytes and RGC after RIRI by inhibiting the TLR4/p38/NRF2 signaling pathway.

Citation： Pan Yiji, Yang Jiayi, Xing Yiqiao, He Tao. Effects of S100A4 overexpression on retinal capillary cells in a retinal ischemia-reperfusion model in rats. Chinese Journal of Ocular Fundus Diseases, 2024, 40(4): 296-302. doi: 10.3760/cma.j.cn511434-20230803-00333 Copy

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27.	Thimmulappa RK, Lee H, Rangasamy T, et al. Nrf2 is a critical regulator of the innate immune response and survival during experimental sepsis[J]. J Clin Invest, 2006, 116(4): 984-995. DOI: 10.1172/JCI25790.
28.	Li H, Li J, Zhang G, et al. HMGB1-induced p62 overexpression promotes snail-mediated epithelial-mesenchymal transition in glioblastoma cells via the degradation of GSK-3beta[J]. Theranostics, 2019, 9(7): 1909-1922. DOI: 10.7150/thno.30578.
29.	Liu B, Deng X, Jiang Q, et al. Scoparone improves hepatic inflammation and autophagy in mice with nonalcoholic steatohepatitis by regulating the ROS/P38/Nrf2 axis and PI3K/AKT/mTOR pathway in macrophages[J/OL]. Biomed Pharmacother, 2020, 125: 109895[2020-01-28]. https://pubmed.ncbi.nlm.nih.gov/32000066/. DOI: 10.1016/j.biopha.2020.109895.

1. Huang H. Pericyte-endothelial interactions in the retinal microvasculature[J/OL]. Int J Mol Sci, 2020, 21(19): 7413[2020-10-08]. https://pubmed.ncbi.nlm.nih.gov/33049983/. DOI: 10.3390/ijms21197413.
2. Ornelas S, Berthiaume AA, Bonney SK, et al. Three-dimensional ultrastructure of the brain pericyte-endothelial interface[J]. J Cereb Blood Flow Metab, 2021, 41(9): 2185-2200. DOI: 10.1177/0271678X211012836.
3. Bek T. Inner retinal ischaemia: current understanding and needs for further investigations[J]. Acta Ophthalmol, 2009, 87(4): 362-367. DOI: 10.1111/j.1755-3768.2008.01429.x.
4. Yu H, Kalogeris T, Korthuis RJ. Reactive species-induced microvascular dysfunction in ischemia/reperfusion[J]. Free Radic Biol Med, 2019, 135: 182-197. DOI: 10.1016/j.freeradbiomed.2019.02.031.
5. Nakahara T, Hoshino M, Hoshino S, et al. Structural and functional changes in retinal vasculature induced by retinal ischemia-reperfusion in rats[J]. Exp Eye Res, 2015, 135: 134-145. DOI: 10.1016/j.exer.2015.02.020.
6. Gonzales AL, Klug NR, Moshkforoush A, et al. Contractile pericytes determine the direction of blood flow at capillary junctions[J]. Proc Natl Acad Sci USA, 2020, 117(43): 27022-27033. DOI: 10.1073/pnas.1922755117.
7. Yemisci M, Gursoy-Ozdemir Y, Vural A, et al. Pericyte contraction induced by oxidative-nitrative stress impairs capillary reflow despite successful opening of an occluded cerebral artery[J]. Nat Med, 2009, 15(9): 1031-1037. DOI: 10.1038/nm.2022.
8. Hall CN, Reynell C, Gesslein B, et al. Capillary pericytes regulate cerebral blood flow in health and disease[J]. Nature, 2014, 508(7494): 55-60. DOI: 10.1038/nature13165.
9. Cheng G, Tian K, Zhang L, et al. S100A4 gene silencing in oxygen-induced ischemic retinopathy inhibits retinal neovascularization via down-regulation of CREB expression[J]. Graefe's Arch Clin Exp Ophthalmol, 2016, 254(1): 97-108. DOI: 10.1007/s00417-015-3158-0.
10. Abu EA, Nawaz MI, De Hertogh G, et al. S100A4 is upregulated in proliferative diabetic retinopathy and correlates with markers of angiogenesis and fibrogenesis[J]. Mol Vis, 2014, 20: 1209-1224.
11. Alarcon-Martinez L, Shiga Y, Villafranca-Baughman D, et al. Pericyte dysfunction and loss of interpericyte tunneling nanotubes promote neurovascular deficits in glaucoma[J/OL]. Proc Natl Acad Sci USA, 2022, 119(7): e2110329119[2022-02-15]. https://pubmed.ncbi.nlm.nih.gov/35135877/. DOI: 10.1073/pnas.2110329119.
12. Yang J, Yang N, Luo J, et al. Overexpression of S100A4 protects retinal ganglion cells against retinal ischemia-reperfusion injury in mice[J/OL]. Exp Eye Res, 2020, 201: 108281[2020-10-06]. https://pubmed.ncbi.nlm.nih.gov/33031790/. DOI: 10.1016/j.exer.2020.108281.
13. Roy S, Tonkiss J, Roy S. Aging increases retinal vascular lesions characteristic of early diabetic retinopathy[J]. Biogerontology, 2010, 11(4): 447-455. DOI: 10.1007/s10522-010-9263-x.
14. Cogan DG, Kuwabara T. Comparison of retinal and cerebral vasculature in trypsin digest preparations[J]. Br J Ophthalmol, 1984, 68(1): 10-12. DOI: 10.1136/bjo.68.1.10.
15. Kugler EC, Greenwood J, MacDonald RB. The "Neuro-Glial-Vascular" Unit: the role of glia in neurovascular unit formation and dysfunction[J/OL]. Front Cell Dev Biol, 2021, 9: 732820[2021-09-27]. https://pubmed.ncbi.nlm.nih.gov/34646826/. DOI: 10.3389/fcell.2021.732820.
16. Trost A, Bruckner D, Rivera FJ, et al. Pericytes in the retina[J]. Adv Exp Med Biol, 2019, 1122: 1-26. DOI: 10.1007/978-3-030-11093-2_1.
17. Dalkara T, Alarcon-Martinez L. Cerebral microvascular pericytes and neurogliovascular signaling in health and disease[J]. Brain Res, 2015, 1623: 3-17. DOI: 10.1016/j.brainres.2015.03.047.
18. Motiejūnaite R, Kazlauskas A. Pericytes and ocular diseases[J]. Exp Eye Res, 2008, 86(2): 171-177. DOI: 10.1016/j.exer.2007.10.013.
19. Kisler K, Nelson AR, Rege SV, et al. Pericyte degeneration leads to neurovascular uncoupling and limits oxygen supply to brain[J]. Nat Neurosci, 2017, 20(3): 406-416. DOI: 10.1038/nn.4489.
20. Huang J, Zhao Q, Li M, et al. The effects of endothelium-specific CYP2J2 overexpression on the attenuation of retinal ganglion cell apoptosis in a glaucoma rat model[J]. FASEB J, 2019, 33(10): 11194-11209. DOI: 10.1096/fj.201900756R.
21. Wang M, Liu H, Xia N, et al. Intraocular pressure-induced endothelial dysfunction of retinal blood vessels is persistent, but does not trigger retinal ganglion cell loss[J]. Antioxidants (Basel), 2022, 11(10): 1864. DOI: 10.3390/antiox11101864.
22. Peppiatt CM, Howarth C, Mobbs P, et al. Bidirectional control of CNS capillary diameter by pericytes[J]. Nature, 2006, 443(7112): 700-704. DOI: 10.1038/nature05193.
23. O'Farrell FM, Mastitskaya S, Hammond-Haley M, et al. Capillary pericytes mediate coronary no-reflow after myocardial ischaemia[J/OL]. Elife, 2017, 6: e29280[2017-11-09]. https://pubmed.ncbi.nlm.nih.gov/29120327/. DOI: 10.7554/eLife.29280.
24. Kiryushko D, Novitskaya V, Soroka V, et al. Molecular mechanisms of Ca(2+) signaling in neurons induced by the S100A4 protein[J]. Mol Cell Biol, 2006, 26(9): 3625-3638. DOI: 10.1128/MCB.26.9.3625-3638.2006.
25. Méhes E, Biri-Kovács B, Isai DG, et al. Matrigel patterning reflects multicellular contractility[J/OL]. PLoS Comput Biol, 2019, 15(10): e1007431[2019-10-25]. https://pubmed.ncbi.nlm.nih.gov/31652274/. DOI: 10.1371/journal.pcbi.1007431.
26. Kilic U, Kilic E, Matter CM, et al. TLR-4 deficiency protects against focal cerebral ischemia and axotomy-induced neurodegeneration[J]. Neurobiol Dis, 2008, 31(1): 33-40. DOI: 10.1016/j.nbd.2008.03.002.
27. Thimmulappa RK, Lee H, Rangasamy T, et al. Nrf2 is a critical regulator of the innate immune response and survival during experimental sepsis[J]. J Clin Invest, 2006, 116(4): 984-995. DOI: 10.1172/JCI25790.
28. Li H, Li J, Zhang G, et al. HMGB1-induced p62 overexpression promotes snail-mediated epithelial-mesenchymal transition in glioblastoma cells via the degradation of GSK-3beta[J]. Theranostics, 2019, 9(7): 1909-1922. DOI: 10.7150/thno.30578.
29. Liu B, Deng X, Jiang Q, et al. Scoparone improves hepatic inflammation and autophagy in mice with nonalcoholic steatohepatitis by regulating the ROS/P38/Nrf2 axis and PI3K/AKT/mTOR pathway in macrophages[J/OL]. Biomed Pharmacother, 2020, 125: 109895[2020-01-28]. https://pubmed.ncbi.nlm.nih.gov/32000066/. DOI: 10.1016/j.biopha.2020.109895.

Chinese Journal of Ocular Fundus Diseases

Effects of S100A4 overexpression on retinal capillary cells in a retinal ischemia-reperfusion model in rats

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