Mesenchymal marker expression is elevated in Müller cells exposed to high glucose and in animal models of diabetic retinopathy
Müller cells are retinal glial cells and exhibit a fibroblast-like phenotype and ability to migrate in diabetic retinopathy (DR). However, expression of mesenchymal markers, which promote fibrosis in various organs, has not been characterized in the diabetic retina. We examined changes in the expression of these markers in Müller cells exposed to high glucose and in animal models of diabetic retinopathy. High glucose conditions increased mesenchymal maker expression and migration in Müller cells. Snail, N-cadherin, Vimentin, β-catenin, and α-smooth muscle actin (α-SMA) levels were all dramatically increased in retinas from humans with diabetic retinopathy (DR) and from DR mouse models. In addition, Snail overexpression increased the expression of connective tissue growth factor (CTGF) and fibronectin, while Snail knockdown attenuated high glucose-induced increases in fibronectin and CTGF expression. These results demonstrate for the first time that mesenchymal markers are upregulated in retinas from a diabetic mouse model, and that Snail and N-cadherin levels are also increased in Müller cells exposed to high glucose. This suggests mesenchymal proteins may play a crucial role in the development of DR.Immunostaining revealed that vimentin, N-cadherin, α-SMA, and Snail were expressed in epiretinal membranes from PDR patients. Fibronectin and connective tissue growth factor (CTGF), which are important profibrotic growth factors in DR, were also expressed in epiretinal membranes from PDR patients (Figure
1).L-glucose, D-glucose, and streptozocin were purchased from Sigma (St. Louis, MO, USA). α-SMA (A2547), β-actin (A5411), and Twist (T6451) antibodies were purchased from Sigma-Aldrich (St Louis, MO, USA). Antibodies against Vimentin (550513), N-cadherin (610920), and fibronectin (610077) were obtained from BD (Franklin Lakes, NJ, USA). Antibodies for Snail (3879S), β-catenin (9582S), and Glial Fibrillary Acidic Protein (GFAP, 3670S) were from Cell Signaling Technology (Danvers, MA, USA). CTGF (ab6922) and Glutamine synthase (GS, ab176562) antibodies were purchased from Abcam (Cambridge, MA, USA). Glutamine synthase (GS, MAB302) antibody was from Millipore (Billerica, MA, USA). Goat Anti-mouse(PI-2000) and anti-rabbit (PI-1000) horseradish peroxidase (HRP)-conjugated secondary antibodies were from Vector Laboratories (Burlingame, CA, USA). Alexa Flour 488 goat anti-rabbit/anti-mouse (A21206/A21202) and Alexa Fluor 594 goat anti-rabbit/anti-mouse (A21207/A21203) antibodies and 4’,6-Diamidino-2-phenylindole (DAPI, D1306) were from Life Technology (St. Louis, MO, USA).We thank Zhenzhen Fang for technical assistance and the members of the laboratory for their helpful comments on the manuscript.CONFLICTS OF INTERESTThe authors report no conflicts of interest.GRANT SUPPORTNational Nature Science Foundation of China, grant numbers 81200706, 81272338, 81272515, 81370945, 81471033, 81572342, 81570871, 81570764 and 81600641; National Key Sci-Tech Special Project of China, grant numbers 2013ZX09102-053 and 2015GKS-355; Program for Doctoral Station in University, grant numbers 20120171110053 and 20130171110053; Key Project of Nature Science Foundation of Guangdong Province, China, Grant Number: 2015A030311043 and 2016A030311035; Guangdong Natural Science Fund, grant numbers S2012040006986, 2014A030313073, 2015A030313029, and 2015A030313103; Guandong Science and Technology Project, grant numbers 2014A020212023 and 2015B090903063; Key Sci-tech Research Project of Guangzhou Municipality, China, grant numbers 2014J4100162, 201508020033, and 201510010052; Pearl River Nova Program of Guangzhou Municipality, China, grant number 201610010186; Changjiang Scholars and Innovative Research Team in University, grant number 985 project PCSIRT 0947; Fundermental Research Funds for the Central Universities of China (Youth Program, 13ykpy06, 14ykpy05, 16ykpy24).