Person:

Rask-Madsen, Christian

To characterize glucagon-like peptide (GLP)-1 signaling and its effect on renal endothelial dysfunction and glomerulopathy. We studied the expression and signaling of GLP-1 receptor (GLP-1R) on glomerular endothelial cells and the novel finding of protein kinase A–dependent phosphorylation of c-Raf at Ser259 and its inhibition of angiotensin II (Ang II) phospho–c-Raf(Ser338) and Erk1/2 phosphorylation. Mice overexpressing protein kinase C (PKC)β2 in endothelial cells (EC-PKCβ2Tg) were established. Ang II and GLP-1 actions in glomerular endothelial cells were analyzed with small interfering RNA of GLP-1R. PKCβ isoform activation induced by diabetes decreased GLP-1R expression and protective action on the renal endothelium by increasing its degradation via ubiquitination and enhancing phospho–c-Raf(Ser338) and Ang II activation of phospho-Erk1/2. EC-PKCβ2Tg mice exhibited decreased GLP-1R expression and increased phospho–c-Raf(Ser338), leading to enhanced effects of Ang II. Diabetic EC-PKCβ2Tg mice exhibited greater loss of endothelial GLP-1R expression and exendin-4–protective actions and exhibited more albuminuria and mesangial expansion than diabetic controls. These results showed that the renal protective effects of GLP-1 were mediated via the inhibition of Ang II actions on cRaf(Ser259) and diminished by diabetes because of PKCβ activation and the increased degradation of GLP-1R in the glomerular endothelial cells.

To determine whether insulin action on endothelial cells promotes or protects against atherosclerosis, we generated apolipoprotein E null mice in which the insulin receptor gene was intact or conditionally deleted in vascular endothelial cells. Insulin sensitivity, glucose tolerance, plasma lipids, and blood pressure were not different between the two groups, but atherosclerotic lesion size was more than 2-fold higher in mice lacking endothelial insulin signaling. Endothelium-dependent vasodilation was impaired and endothelial cell VCAM-1 expression was increased in these animals. Adhesion of mononuclear cells to endothelium in vivo was increased 4-fold compared with controls but reduced to below control values by a VCAM-1-blocking antibody. These results provide definitive evidence that loss of insulin signaling in endothelium, in the absence of competing systemic risk factors, accelerates atherosclerosis. Therefore, improving insulin sensitivity in the endothelium of patients with insulin resistance or type 2 diabetes may prevent cardiovascular complications.

Purpose. Although vascular endothelial growth factor (VEGF) is a key mediator of retinal vascular permeability (RVP), there may be additional humoral contributors. Hepatocyte growth factor (HGF) induces endothelial cell separation, regulates expression of cell adhesion molecules and is increased in the vitreous fluid of patients with proliferative diabetic retinopathy. The purpose of this study was to evaluate the in vivo effects of HGF on RVP and retinal hemodynamics and delineate the signaling pathways. Methods. RVP was assessed by vitreous fluorescein fluorophotometry in rats. Time course and dose-response were determined after intravitreal HGF injection. MAP kinase (MAPK), phosphatidylinositol 3-kinase (PI-3 kinase), and protein kinase C (PKC) involvement were examined by using selective inhibitors. Retinal blood flow (RBF) and mean circulation time (MCT) were evaluated by video fluorescein angiography. Results. HGF increased RVP in a time- and dose-dependent manner. HGF-induced RVP was evident 5 minutes after injection, and reached maximal levels after 25 minutes (+107% versus vehicle, P = 0.002). This effect was comparable to that of maximum VEGF stimulation (134% ± 128% at 25 ng/mL). Selective inhibitors of MAPK (PD98059) and PI-3 kinase (LY294002) suppressed HGF-induced RVP by 86% ± 44% (P = 0.015) and 97% ± 59% (P = 0.021), respectively. Non-isoform-selective inhibition of PKC did not significantly decrease HGF-induced RVP. Although VEGF increases RBF and reduces MCT, HGF did not affect either. Conclusions. HGF increases RVP in a time- and dose-dependent manner at physiologically relevant concentrations with a magnitude and profile similar to that of VEGF, without affecting retinal hemodynamics. Thus, HGF may represent another clinically significant contributor to retinal edema distinct from the actions of VEGF.

The risk of several cancers, including colorectal cancer, is increased in patients with obesity and type 2 diabetes, conditions characterized by hyperinsulinemia and insulin resistance. Because hyperinsulinemia itself is an independent risk factor for cancer development, we examined tissue-specific insulin action in intestinal tumor formation. In vitro, insulin increased proliferation of primary cultures of intestinal tumor epithelial cells from ApcMin/+ mice by over 2-fold. Surprisingly, targeted deletion of insulin receptors in intestinal epithelial cells in ApcMin/+ mice did not change intestinal tumor number or size distribution on either a low or high-fat diet. We therefore asked whether cells in the tumor stroma might explain the association between tumor formation and insulin resistance. To this end, we generated ApcMin/+ mice with loss of insulin receptors in vascular endothelial cells. Strikingly, these mice had 42% more intestinal tumors than controls, no change in tumor angiogenesis, but increased expression of vascular cell adhesion molecule-1 (VCAM-1) in primary culture of tumor endothelial cells. Insulin decreased VCAM-1 expression and leukocyte adhesion in quiescent tumor endothelial cells with intact insulin receptors and partly prevented increases in VCAM-1 and leukocyte adhesion after treatment with tumor necrosis factor-α. Knockout of insulin receptors in endothelial cells also increased leukocyte adhesion in mesenteric venules and increased the frequency of neutrophils in tumors. We conclude that although insulin is mitogenic for intestinal tumor cells in vitro, its action on tumor cells in vivo is via signals from the tumor microenvironment. Insulin resistance in tumor endothelial cells produces an activated, proinflammatory state that promotes tumorigenesis. Improvement of endothelial dysfunction may reduce colorectal cancer risk in patients with obesity and type 2 diabetes.