Increased adhesion molecules and leukocytes adhesion in human islet endothelial cells by advanced glycation end products

Abstract

Objective To investigate the effect of advanced glycation end products (AGEs) on adhesion molecules expression and leukocyte adhesion on human islet microvascular endothelial cells (HIMVEC), and the related mechanisms. Methods HIMVEC was treated with AGEs (final concentration 200 mg/L). Cell-based enzyme-linked immunosorbent assay(ELISA) and western blotting were used to detect adhesion molecules level of HIMVEC; the leukocyte-HIMVEC adhesion was evaluated after BCECF-labeled human leukocytes incubated with HIMVEC.The expression levels of receptor of AGEs (RAGE), protein kinase C β (PKCβ) and protein kinase A (PKA) were detected by real-time PCR and Western blotting.The adhesion molecules expression and leukocyte adhesion were evaluated after cells treated with PKCβ inhibitor (LY333531) or PKA activator (8-Br-cAMP). t-test were used to compare the difference between groups for statistical analysis. Results Compared to control group, P-selectin, E-selectin and Vascular cell adhesion molecule-1 (VCAM-1) were all up-regulated on HIMVEC by AGEs treatment (1.1±0.13 vs 0.64±0.14, 0.83±0.06 vs 0.47±0.05, 0.87±0.09 vs 0.43±0.07, t=4.93, 9.40, 7.61, all P<0.05), and leukocytes adhesion was increased in AGEs group than control (54±4 vs 23±3, t=12.69, P<0.05). Compared to AGEs group, the expression levels of P-selectin, E-selectin and VCAM-1 were decreased by pre-treated with anti-RAGE antibody (t=5.69, 6.89, 5.43, all P<0.05), and leukocyte adhesion was also inhibited by RAGE antibody (54±4 vs 31±4, t=8.22, P<0.05). Compared to control group, after treated with AGEs for 4 h or 18 h, the mRNA and protein level of RAGE and PKCβ was up-regulated, but PKA was down-regulated (t=10.94, 7.76, 21.82, 5.85, 10.96, 11.47, all P<0.05). Compared to AGEs group, PKCβ inhibitor (LY333531) or PKA activator (8-Br-cAMP) can down-regulate the expression of P-selectin, E-selectin and VCAM-1 (t=7.60, 6.60, 6.25, 11.58, 4.08, 3.47, all P<0.05). And leukocyte adhesion was also reduced in LY333531 or 8-Br-cAMP group than AGEs group (t=7.67, 8.89, both P<0.05). Conclusion AGEs increased adhesion molecules expression and leukocyte adhesion on HIMVEC by up-regulating PKCΒ and down-regulating PKA, which might be one reason for leukocyte infiltration in islets of type 2 diabetes. Key words: Advanced glycation end products; Human islet microvascular endothelial cell; Adhesion molecules; Leukocyte