Adipocyte-Derived Exosomal LINC00968 Promotes Mouse Retina Microvascular Endothelial Cell Dysfunction in a High-Glucose Environment by Modulating the miR-361–5p/TRAF3 Axis

Abstract

As a major cause of mortality, cardiovascular disease is associated with obesity and diabetes. However, the molecular mechanism by which diabetes-obesity causes cardiovascular complications is largely unknown. In this study, the crosstalk mediated by 3T3-L1 preadipocytes and mouse retina microvascular endothelial cells (mRMECs) was determined after co-culturing performed with a Transwell system or measuring exosome uptake by mRMECs. CCK-8 assays, EdU incorporation assays, TUNEL staining, and ELISAs were used to evaluate the functions of mRMECs. Related protein markers were analyzed by western blotting. Our results showed that LINC00968 levels were significantly elevated in the exosomes derived from H-Glu-induced 3T3-L1 preadipocytes. Both H-Glu treatment and co-culture with 3T3-L1 cells damaged mRMECs, as indicated by lower rates of proliferation and higher rates of apoptosis and cell adhesion molecule expression, as well as by induced inflammation and oxidative stress, which were enhanced by combined H-Glu and co-culture treatment. Furthermore, H-Glu and co-culture treatment increased LINC00968 expression in mRMECs, and the exosomes collected from 3T3-L1 cells had a similar effect. Functionally, LINC00968 inhibition protected mRMECs against the effects of H-Glu and co-culture treatment, while LINC00968 played the opposite role. LINC00968 was found to target miR-361–5p, and TRAF3 was identified as a target gene of miR-361–5p. Finally, miR-361–5p overexpression alleviated the effects of LINC00968 on H-Glu-induced mRMEC dysfunction in vitro. In conclusion, our results indicated that in an H-glu environment, adipocyte exosomes damage microvascular endothelial cells via a LINC00968/miR-361–5p/TRAF3 signaling pathway, which could possibly serve as a target for treating diabetes-obesity-triggered microvascular complications.

Key words

3T3-L1 preadipocytes - exosomes - microvascular endothelial cell injury - LINC00968 - diabetes

• References

• 1 Gonzalez N, Moreno-Villegas Z, Gonzalez-Bris A. et al. Regulation of visceral and epicardial adipose tissue for preventing cardiovascular injuries associated to obesity and diabetes. Cardiovasc Diabetol 2017; 16: 44
• 2 El-Atat F, McFarlane SI, Sowers JR. Diabetes, hypertension, and cardiovascular derangements: pathophysiology and management. Curr Hypertens Rep 2004; 6: 215-223
• 3 King RJ, Ajjan RA. Vascular risk in obesity: Facts, misconceptions and the unknown. Diabetes Vasc Dis Res 2017; 14: 2-13
• 4 Barrett EJ, Liu Z, Khamaisi M. et al. Diabetic microvascular disease: an endocrine society scientific statement. J Clin Endocrinol Metab 2017; 102: 4343-4410
• 5 Meakin PJ, Coull BM, Tuharska Z. et al. Elevated circulating amyloid concentrations in obesity and diabetes promote vascular dysfunction. J Clin Invest 2020; 130: 4104-4117
• 6 Knapp M, Tu X, Wu R. Vascular endothelial dysfunction, a major mediator in diabetic cardiomyopathy. Acta Pharmacol Sin 2019; 40: 1-8
• 7 Bugiardini R, Manfrini O, De Ferrari GM. Unanswered questions for management of acute coronary syndrome: risk stratification of patients with minimal disease or normal findings on coronary angiography. Arch Inter Med 2006; 166: 1391-1395
• 8 Shi Y, Vanhoutte PM. Macro- and microvascular endothelial dysfunction in diabetes. J Diabetes 2017; 9: 434-449
• 9 Gilbert RE. Endothelial loss and repair in the vascular complications of diabetes: pathogenetic mechanisms and therapeutic implications. Circ J 2013; 77: 849-856
• 10 Lawson C, Vicencio JM, Yellon DM. et al. Microvesicles and exosomes: new players in metabolic and cardiovascular disease. J Endocrinol 2016; 228: R57-R71
• 11 Colombo M, Raposo G, Thery C. Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles. Ann Rev Cell. Develop Biol 2014; 30: 255-289
• 12 Gezer U, Ozgur E, Cetinkaya M. et al. Long non-coding RNAs with low expression levels in cells are enriched in secreted exosomes. Cell Biol Int 2014; 38: 1076-1079
• 13 Hu W, Ding Y, Wang S. et al. The construction and analysis of the aberrant lncRNA-miRNA-mRNA network in adipose tissue from type 2 diabetes individuals with obesity. J Diabetes Res 2020; 3980742
• 14 Wang QC, Wang ZY, Xu Q. et al. lncRNA expression profiles and associated ceRNA network analyses in epicardial adipose tissue of patients with coronary artery disease. Sci Rep 2021; 11: 1567
• 15 Wang X, Zhao Z, Zhang W. et al. Long noncoding RNA LINC00968 promotes endothelial cell proliferation and migration via regulating miR-9-3p expression. J Cell Biochem 2018; 120: 8214-8221
• 16 Katsiki N, Anagnostis P, Kotsa K. et al. Obesity, metabolic syndrome and the risk of microvascular complications in patients with diabetes mellitus. Curr Pharm Des 2019; 25: 2051-2059
• 17 Pefanis E, Wang J, Rothschild G. et al. RNA exosome-regulated long non-coding RNA transcription controls super-enhancer activity. Cell 2015; 161: 774-789
• 18 Ai Y, Wei H, Wu S. et al. Exosomal LncRNA LBX1-AS1 derived from RBPJ overexpressed-macrophages inhibits oral squamous cell carcinoma progress via miR-182-5p/FOXO3. Front Oncol 2021; 11: 605884
• 19 Wang X, Gu H, Huang W. et al. Hsp20-mediated activation of exosome biogenesis in cardiomyocytes improves cardiac function and angiogenesis in diabetic mice. Diabetes 2016; 65: 3111-3128
• 20 Ying W, Riopel M, Bandyopadhyay G. et al. Adipose tissue macrophage-derived exosomal miRNAs can modulate in vivo and in vitro insulin sensitivity. Cell 2017; 171: 372-384 e312
• 21 Wang Y, Zhou J, Xu YJ. et al. Long non-coding RNA LINC00968 acts as oncogene in NSCLC by activating the Wnt signaling pathway. J Cell Physiol 2018; 233: 3397-3406
• 22 Li Z, Yu Z, Meng X. et al. LncRNA LINC00968 accelerates the proliferation and fibrosis of diabetic nephropathy by epigenetically repressing p21 via recruiting EZH2. Biochem Biophys Res Commun 2018; 504: 499-504
• 23 Ala U. Competing Endogenous RNAs, Non-Coding RNAs and Diseases: An Intertwined Story. Cells 2020; 9: 1574
• 24 Kar S, Bali KK, Baisantry A. et al. Genome-wide sequencing reveals microRNAs downregulated in cerebral cavernous malformations. J Mol Neurosci 2017; 61: 178-188
• 25 Zhang X, Ye Q, Gong D. et al. Apelin-13 inhibits lipoprotein lipase expression via the APJ/PKCalpha/miR-361-5p signaling pathway in THP-1 macrophage-derived foam cells. Acta Biochim Biophys Sin (Shanghai) 2017; 49: 530-540
• 26 Tao Y, Zhou J, Wang Z. et al. Human bone mesenchymal stem cells-derived exosomal miRNA-361-5p alleviates osteoarthritis by downregulating DDX20 and inactivating the NF-kappaB signaling pathway. Bioorg Chem 2021; 113: 104978
• 27 Chen Z, Canet MJ, Sheng L. et al. Hepatocyte TRAF3 promotes insulin resistance and type 2 diabetes in mice with obesity. Mol Metab 2015; 4: 951-960
• 28 Grunewald ZI, Ramirez-Perez FI, Woodford ML. et al. TRAF3IP2 (TRAF3 interacting protein 2) mediates obesity-associated vascular insulin resistance and dysfunction in male mice. Hypertension 2020; 76: 1319-1329
• 29 Gu H, Yu J, Dong D. et al. The miR-322-TRAF3 circuit mediates the pro-apoptotic effect of high glucose on neural stem cells. Toxicol Sci 2015; 144: 186-196
• 30 Sun Y, Zhou Y, Shi Y. et al. Expression of miRNA-29 in pancreatic β cells promotes inflammation and diabetes via TRAF3. Cell Rep 2021; 34: 108576