Deletion of AT2 Receptor Prevents SHP-1-Induced VEGF Inhibition and Improves Blood Flow Reperfusion in Diabetic Ischemic Hindlimb.

OBJECTIVE: Ischemia caused by narrowing of femoral artery is a major cause of peripheral arterial disease and morbidity affecting patients with diabetes mellitus. We have previously reported that the inhibition of the angiogenic response to VEGF (vascular endothelial growth factor) in diabetic mice was associated with the increased expression of SHP-1 (SH2 domain-containing phosphatase 1), a protein that can be activated by the AT2 (angiotensin II type 2) receptor. Deletion of AT2 receptor has been shown to promote angiogenesis within the ischemic muscle. However, the relative impact of AT2 receptor in diabetic condition remains unknown. APPROACH AND RESULTS: Nondiabetic and diabetic AT2 null (Atgr2-/Y) mice underwent femoral artery ligation after 2 months of diabetes mellitus. Blood perfusion was measured every week ≤4 weeks post-surgery. Expression of the VEGF, SHP-1, and renin-angiotensin pathways was evaluated. Blood flow in the ischemic muscle of diabetic Atgr2-/Y mice recovered faster and ≤80% after 4 weeks compared with 51% recovery in diabetic control littermates. Diabetic Atgr2-/Y had reduced apoptotic endothelial cells and elevated small vessel formation compared with diabetic Atgr2+/Y mice, as well as increased SHP-1 expression and reduced VEGF receptor activity. In endothelial cells, high glucose levels and AT2 agonist treatment did not change SHP-1, VEGF, and VEGF receptor expression. However, the activity of SHP-1 and its association with the VEGF receptors were increased, causing inhibition of the VEGF action in endothelial cell proliferation and migration. CONCLUSIONS: Our results suggest that the deletion of AT2 receptor reduced SHP-1 activity and restored VEGF actions, leading to an increased blood flow reperfusion after ischemia in diabetes mellitus.

PKC-ß activation inhibits IL-18-binding protein causing endothelial dysfunction and diabetic atherosclerosis.

AIMS: Clinical observations showed a correlation between accelerated atherosclerosis in diabetes and high plasmatic level of IL-18, a pro-inflammatory cytokine. IL-18 enhances the production of inflammatory cytokines and cellular adhesion molecules contributing to atherosclerotic plaque formation and instability. Previous studies indicated that protein kinase C (PKC)-ß inhibition prevented macrophage-induced cytokine expression involved in diabetic (DM) atherosclerotic plaque development. However, the role of PKC-ß activation on IL-18/IL-18-binding protein (IL-18BP) pathway causing endothelial dysfunction and monocyte adhesion in diabetes has never been explored. METHODS AND RESULTS: Apoe(-/-) mice were rendered DM and fed with western diet containing ruboxistaurin (RBX), a PKC-ß inhibitor. After 20 weeks, atherosclerotic plaque composition was quantified. Compared with non-diabetic, DM mice exhibited elevated atherosclerotic plaque formation, cholestoryl ester content and macrophage infiltration, as well as reduced IL-18BP expression in the aorta which was prevented with RBX treatment. Endothelial cells (ECs) and macrophages were exposed to normal or high glucose (HG) levels with or without palmitate and recombinant IL-18 for 24 h. The combined HG and palmitate condition was required to increase IL-18 expression and secretion in macrophages, while it reduced IL-18BP expression in EC causing up-regulation of the vascular cell adhesion molecule (VCAM)-1 and monocyte adhesion. Elevated VCAM-1 expression and monocyte adherence were prevented by siRNA, RBX, and IL-18 neutralizing antibody. CONCLUSION: Our study unrevealed a new mechanism by which PKC-ß activation promotes EC dysfunction caused by the de-regulation of the IL-18/IL-18BP pathway, leading to increased VCAM-1 expression, monocyte/macrophage adhesion, and accelerated atherosclerotic plaque formation in diabetes.