Chemerin promotes microangiopathy in diabetic retinopathy via activation of ChemR23 in rat primary microvascular endothelial cells

Abstract

Purpose: The correlation between chemerin and diabetic retinopathy (DR) has been demonstrated previously. We aimed to investigate the potential inflammatory and angiogenic roles of chemerin in DR using rat primary retinal microvascular endothelial cells (RRMECs).

Methods: RRMECs were incubated in low- and high-glucose media, and stable chemerin receptor (ChemR23) knockdown in RRMECs was established by lentiviral infection. Real-time quantitative PCR (RT-qPCR), enzyme-linked immunosorbent assay (ELISA), and western blotting were employed to investigate the mRNA and protein expression of intercellular adhesion molecule-1 (ICAM-1), vascular endothelial growth factor (VEGF), tumor necrosis factor-α (TNF-α), and the interleukin-6 receptor (IL-6R) to explore the inflammatory and angiogenic effects of chemerin. A scratch assay was employed to evaluate the effect of chemerin on RRMEC migration.

Results: Chemerin and TNF-α markedly increased the mRNA and protein expression of ICAM-1 in RRMECs (p<0.001). ChemR23 knockdown may have decreased the ICAM-1 expression under low- and high-glucose conditions (p<0.001). Even in the ChemR23-knockdown group, TNF-α significantly increased the mRNA and protein levels of ICAM-1 under low- and high-glucose conditions (p<0.001). Chemerin promoted VEGF expression under low- and high-glucose conditions. ChemR23 knockdown markedly decreased VEGF levels under low- and high-glucose conditions (p<0.05) and significantly decreased RRMEC migration (p<0.001).

Conclusions: Chemerin promotes the expression of ICAM-1, the secretion of VEGF, and the migration of RRMECs via the activation of ChemR23.

Figure 1

High concentrations of glucose increased chemerin expression, intercellular adhesion molecule-1 (ICAM-1), and tumor necrosis factor-α receptor (TNFR), whereas they had no effect on ChemR23 or interleukin-6 receptor (IL-6R). A,B: Western blot analysis of the expression of ChemR23 under different glucose concentrations. C: Real-time quantitative PCR (RT-qPCR) showed the mRNA expression of ChemR23 under different glucose conditions. D: RT-qPCR showed the mRNA expression of chemerin under different glucose conditions. E-H: Western blot analysis of ICAM-1, TNFR, and IL-6R proteins, normalized to tubulin. I: The expression of ICAM-1 in rat primary retinal microvascular endothelial cells (RRMECs) detected by RT-qPCR. J: The expression of TNFR in RRMECs detected by RT-qPCR. K: The expression of IL-6R in RRMECs detected by RT-qPCR. Data are shown as mean ± standard deviation (SD) of six replicates. **p<0.01, ***p<0.001.

Figure 2

The expression of tumor necrosis factor-α receptor (TNFR) and interleukin-6 receptor (IL-6R) was not affected by ChemR23 knockdown in rat primary retinal microvascular endothelial cells (RRMECs). A and C-E: Western blot analysis of ChemR23, TNFR, and IL-6R proteins, normalized to tubulin under low- (5.5 mM) and high-glucose (30 mM) conditions. B: The expression of ChemR23 in RRMECs detected by real-time quantitative PCR (RT-qPCR). Data are shown as mean ± standard deviation (SD) of six replicates. *p<0.05, **p<0.01, ***p<0.001. Abbreviations: NC, RRMECs transfected with empty vector; shChemR23, RRMECs transfected with shRNA targeting ChemR23.

Figure 3

Chemerin/ChemR23 pathway–mediated intercellular adhesion molecule-1 (ICAM-1) expression. A-D: Western blot analysis of ChemR23, tumor necrosis factor-α receptor (TNFR), and ICAM-1 proteins with/without chemerin stimulation (10 nM for 24 h) or ChemR23 knockdown under low- and high-glucose conditions, normalized to tubulin. E: The expression of ICAM-1 in RRMECs detected by real-time quantitative PCR (RT-qPCR) under low-glucose condition (5.5 mM). F: The expression of ICAM-1 in rat primary retinal microvascular endothelial cells (RRMECs) detected by RT-qPCR under high-glucose conditions (30 mM). Data are shown as mean ± standard deviation (SD) of six replicates. *p<0.05, ***p<0.001. Abbreviations: NC, RRMECs transfected with empty vector; sh, RRMECs transfected with shRNA targeting ChemR23; ch, with chemerin stimulation (10 nM for 24 h); ch/sh, RRMECs transfected with shRNA targeting ChemR23 and chemerin stimulation (10 nM for 24 h).

Figure 4

Chemerin and tumor necrosis factor-α (TNF-α) both increased intercellular adhesion molecule-1 (ICAM-1) expression via the activation of ChemR23 and nuclear factor-κB (NF-κB), respectively. A-C: Western blot analysis of TNF-α receptor (TNFR) and ICAM-1 proteins with/without TNF-α (30 ng/ml for 6 h) stimulation or ChemR23 knockdown under low- and high-glucose conditions, normalized to tubulin. A,D,E,F: Western blot analysis of inhibitor of nuclear factor kappa-B alpha (Iκ-Bα) and intranuclear NF-κB (n-NF-κB) proteins with/without TNF-α stimulation or ChemR23 knockdown under low- and high-glucose conditions. The protein expression of Iκ-Bα and NF-κB was normalized to tubulin, whereas the protein expression of n-NF-κB was normalized to His-H3. Data are shown as mean ± standard deviation (SD) of six replicates. **p<0.01, ***p<0.001. Abbreviations: NC, RRMECs transfected with empty vector; shChemR23, RRMECs transfected with shRNA targeting ChemR23.

Figure 5

Chemerin promotes vascular endothelial growth factor (VEGF) expression via the activation of ChemR23 in RRMECs. A: The expression of VEGF detected by enzyme-linked immunosorbent assay (ELISA) under different glucose conditions (5.5, 20, and 30 mM). B,C: RT-qPCR detected VEGF expression with/without chemerin stimulation (10 nM for 24 h) or ChemR23 knockdown under low- (5.5 mM) and high-glucose (30 mM) glucose conditions. D: VEGF expression detected by ELISA with/without chemerin stimulation (10 nM for 24 h) or ChemR23 knockdown under low- (5.5 mM) and high-glucose (30 mM) conditions. Data were shown as mean ± standard deviation (SD) of six replicates. *p<0.05, **p<0.01, ***p<0.001. Abbreviations: NC, RRMECs transfected with empty vector; sh, RRMECs transfected with shRNA targeting ChemR23; ch, with chemerin stimulation (10 nM for 24 h); ch/sh, RRMECs transfected with shRNA targeting ChemR23 and chemerin stimulation (10 nM for 24 h).

Figure 6

Chemerin promotes the migration of rat primary retinal microvascular endothelial cells (RRMECs) via activation of ChemR23. A,B: The effects of different glucose concentrations (5.5, 20, and 30 mM) on RRMEC migration. C,D: The effect of the chemerin/ChemR23 pathway on RRMEC migration under low-glucose condition (5.5 mM). E,F: The effect of the chemerin/ChemR23 pathway on RRMEC migration under high-glucose condition (30 mM). The representative images were photographed and quantitative analysis of the scraping area was performed at 0 and 24 h after scraping. The experiment was conducted in triplicate. **p<0.01, ***p<0.001. Abbreviations: NC, RRMECs transfected with empty vector; shChemR23, RRMECs transfected with shRNA targeting ChemR23.

Figure 7

Schematic summary of chemerin/ChemR23 pathway in rat primary retinal microvascular endothelial cells (RRMECs). A high concentration of glucose increases the secretion of vascular endothelial growth factor (VEGF) and chemerin in RRMECs. High concentration of glucose increases the expression of tumor necrosis factor-α (TNF-α) receptor (TNFR) but has no effect on the expression of ChemR23. Chemerin increases the mRNA and protein expression of VEGF and intercellular adhesion molecule-1 (ICAM-1) via chemerin/ChemR23 pathway. TNF-α can also increase the mRNA and protein expression of ICAM-1 via activation of TNFR and nuclear translocation of nuclear factor-κB (NF-κB). TNF-α and chemerin both regulate ICAM-1 expression.