Background: Mönckeberg’s calcification in diabetes, known as medial artery calcification, is an independent predictor of cardiovascular mortality. However, the mechanism underlying this phenomenon remains to be elucidated. We demonstrate that advanced glycation end products (AGEs) induce calcification of vascular smooth muscle cells through the receptor for AGE (RAGE)/p38 mitogen-activated protein kinase (MAPK) signaling pathway. Methods: We detected vascular calcification by von Kossa staining. Alkaline phosphatase (ALP) activity was determined by measuring p-nitrophenol. Osteocalcin concentrations were measured using ELISA. Western blotting for protein phosphorylation and real-time RT-PCR for expression of mRNA were used. Results: AGEs induced calcification of vascular smooth muscle cells. AGEs also induced the expression of Runx2 mRNA. In addition, AGEs increased ALP activity and osteocalcin secretion. Furthermore, AGEs induced phosphorylation of p38 MAPK, and this phosphorylation was inhibited by the anti-RAGE blocking antibody. Increased ALP activity was inhibited by the p38 MAPK inhibitor or anti-RAGE blocking antibody. Furthermore, the p38 MAPK inhibitor and anti-RAGE blocking antibody both inhibited AGE-induced calcification of vascular smooth muscle cells. Diabetic serum induced calcification of smooth muscle cells and the calcification was inhibited by RAGE blocking. Conclusion: Our findings indicate that AGEs induce calcification of vascular smooth muscle cells by osteoblast-like differentiation of smooth muscle cells through RAGE/p38 MAPK.
a b s t r a c tAlthough increased vascular permeability is known to be a major characteristic of diabetic vasculopathy, the precise mechanisms and relevance of advanced glycation end products (AGE) to hyperpermeability of vessels remains unclear. Here, we studied changes in cytoskeletal configuration and the signaling mechanism induced by AGE in human endothelial cells. AGE-BSA stimulation induced Rho activation, intercellular gap formation, prominent actin stress fiber and cell contraction without changing VE-cadherin, and subsequently transendothelial diffusion of FITC-labeled dextran. These processes induced by AGE-BSA were inhibited by either Rho-kinase inhibitor Y27632 or anti-RAGE antibody. We also showed that RhoA and RAGE spontaneously formed a complex. These findings suggest that activation of RAGE/Rho is involved in AGE-BSA-induced hyperpermeability through gap formation and actin reorganization in diabetes.
We investigated the relationship between HSP27 phosphorylation and collagen-stimulated activation of platelets in patients with diabetes mellitus (DM). Platelet-rich plasma was prepared from blood of type 2 DM patients. The platelet aggregation was analyzed in size of aggregates by an aggregometer using a laser scattering method. The protein phosphorylation was analyzed by Western blotting. Phosphorylated-HSP27 and PDGF-AB released from platelets were measured by ELISA. The phosphorylated-HSP27 levels at Ser-78 and Ser-82 induced by collagen were directly proportional to the platelet aggregation. Total HSP27 levels in platelets were decreased concomitantly with the phosphorylation. The released HSP27 levels were significantly correlated with the phosphorylated levels of HSP27 in the platelets stimulated by 0.3 μg/ml collagen. The low dose collagen-stimulated release of HSP27 was detected but relatively small in healthy donors. The released levels of PDGF-AB were in parallel with the levels of released HSP27. Area under the curve (AUC) of small aggregation (9-25 μm) induced by 0.3 μg/ml collagen was inversely proportional to the levels of released HSP27. AUC of large aggregation (50-70 μm) was directly proportional to the levels of released HSP27. Exogenous recombinant phosphorylated- HSP27 hardly affected the aggregation or the released levels of PDGF-AB induced by collagen. These results strongly suggest that HSP27 is released from human platelets accompanied with its phosphorylation induced by collagen, which is correlated with the acceleration of platelet aggregation in type 2 DM patients.
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