Background Endothelial dysfunction contributes to the development of atherosclerosis in patients with diabetes mellitus, but the mechanisms of endothelial dysfunction in this setting are incompletely understood. Recent studies have shown altered mitochondrial dynamics in diabetes mellitus with increased mitochondrial fission and production of reactive oxygen species (ROS). We investigated the contribution of altered dynamics to endothelial dysfunction in diabetes. Methods and Results We observed mitochondrial fragmentation (P=0.002) and increased expression of fission-1 protein (Fis1, P<0.0001) in venous endothelial cells freshly isolated from patients with diabetes mellitus (n=10) compared to healthy controls (n=9). In cultured human aortic endothelial cells exposed to 30 mM glucose, we observed a similar loss of mitochondrial networks and increased expression of Fis1 and dynamin-related protein-1 (Drp1), proteins required for mitochondrial fission. Altered mitochondrial dynamics was associated with increased mitochondrial ROS production and a marked impairment of agonist-stimulated activation of endothelial nitric oxide synthase (eNOS) and cGMP production. Silencing Fis1 or DRP1 expression with siRNA blunted high glucose-induced alterations in mitochondrial networks, ROS production, eNOS activation, and cGMP production. An intracellular ROS scavenger provided no additional benefit, suggesting that increased mitochondrial fission may impair endothelial function via increased ROS. Conclusions These findings implicate increased mitochondrial fission as a contributing mechanism for endothelial dysfunction in diabetic states.

Cardiovascular disease is a major complication of diabetes mellitus, and improved strategies for prevention and treatment are needed. Endothelial dysfunction contributes to the pathogenesis and clinical expression of atherosclerosis in diabetes mellitus. This article reviews the evidence linking endothelial dysfunction to human diabetes mellitus and experimental studies that investigated the responsible mechanisms. We then discuss the implications of these studies for current management and for new approaches for the prevention and treatment of cardiovascular disease in patients with diabetes mellitus. KeywordsEndothelium; Nitric oxide; Inflammation; Diabetes mellitus; Mitochondria; Protein kinase C Type 2 diabetes mellitus is a growing public health problem [1] and a major cause of cardiovascular disease in the United States [2]. Type 2 diabetes is associated with systemic insulin resistance, which promotes hyperglycemia and dyslipidemia [3], and it has been proposed that these metabolic abnormalities account for increased cardiovascular risk. Endothelial dysfunction contributes to the pathogenesis and clinical expression of atherosclerosis and has been linked to Type 2 diabetes mellitus and insulin resistance in experimental and clinical studies [4]. This article will review the concept of endothelial dysfunction and the evidence linking it to human diabetes mellitus. We will then review © Springer Science+Business Media, LLC 2010 Correspondence to: Joseph A. Vita. NIH Public Access Author ManuscriptRev Endocr Metab Disord. Author manuscript; available in PMC 2010 June 9. What is endothelial dysfunction?Once thought to be simply a passive lining for blood vessels, it is now recognized that the vascular endothelium is a key determinant of vascular health. Broadly speaking, the term "endothelial dysfunction" refers to an impairment of the ability of the endothelium to properly maintain vascular homeostasis [5]. Although the term is often used in reference to a loss of bioavailable nitric oxide (NO), endothelial dysfunction also reflects increased production of vasoconstrictors and disturbed regulation of inflammation, thrombosis, and cell growth in the vascular wall [5,6]. Numerous studies have linked endothelial dysfunction and resultant atherosclerosis with insulin resistant states such as obesity and diabetes [7][8][9][10].The endothelium plays a key role in the regulation of arterial tone and blood flow. In this regard, the endothelium orchestrates the production of vasodilator molecules such as NO, prostacyclin, and endothelium-derived hyperpolarizing factor (EDHF), and vasoconstrictors, including endothelin-1 (ET-1) and angiotensin II [5]. Stimuli for production of endothelium-dependent vasodilators include physiologically relevant factors such as acetylcholine, thrombin, serotonin, angiotensin II, and alpha adrenergic agonists. In general, these factors also promote vasoconstriction via direct effects on vascular smooth muscle. Endothelium-derived NO and other vasodilators oppose such vasocons...

Background Abnormal endothelial function promotes atherosclerotic vascular disease in diabetes. Experimental studies indicate that disruption of endothelial insulin signaling through the activity of protein kinase C-β (PKCβ) and nuclear factor κB (NFκB) reduces nitric oxide availability. We sought to establish whether similar mechanisms operate in the endothelium in human diabetes mellitus. Methods and Results We measured protein expression and insulin response in freshly isolated endothelial cells from patients with Type 2 diabetes mellitus (n=40) and non-diabetic controls (n=36). Unexpectedly, we observed 1.7-fold higher basal endothelial nitric oxide synthase (eNOS) phosphorylation at serine 1177 in patients with diabetes (P=0.007) without a difference in total eNOS expression. Insulin stimulation increased eNOS phosphorylation in non-diabetic subjects but not in diabetic patients (P=0.003) consistent with endothelial insulin resistance. Nitrotyrosine levels were higher in diabetic patients indicating endothelial oxidative stress. PKCβ expression was higher in diabetic patients and was associated with lower flow-mediated dilation (r=−0.541, P=0.02) Inhibition of PKCβ with LY379196 reduced basal eNOS phosphorylation and improved insulin-mediated eNOS activation in patients with diabetes. Endothelial NFκB activation was higher in diabetes and was reduced with PKCβ inhibition. Conclusions We provide evidence for the presence of altered eNOS activation, reduced insulin action and inflammatory activation in the endothelium of patients with diabetes. Our findings implicate PKCβ activity in endothelial insulin resistance.

Background Non-surgical bleeding (NSB) is the most common adverse event in patients with continuous-flow left ventricular assist devices (LVADs) and is caused by arteriovenous malformations (AVMs). We hypothesized that deregulation of an angiogenic factor, Angiopoietin-2 (Ang-2), in LVAD patients leads to increased angiogenesis and higher NSB. Methods Ang-2 and thrombin levels were measured by ELISA and Western blotting, respectively, in blood samples from 101 patients with heart failure (HF), LVAD, or orthotopic heart transplant (OHT). Ang-2 expression in endothelial biopsy was quantified by immunofluorescence. Angiogenesis was determined by in vitro tube formation using serum from each patient with or without Ang-2-blocking antibody. Ang-2 gene expression was measured by RT-PCR in endothelial cells incubated with plasma from each patient with or without the thrombin receptor blocker Vorapaxar. Results Compared with HF or OHT patients, serum levels and endothelial expression of Ang-2 were higher in LVAD patients (p=0.001 and p<0.001, respectively). This corresponded with increased angiogenic potential of serum from patients with LVADs (p<0.001), which was normalized with Ang-2 blockade. Furthermore, plasma from LVAD patients contained higher amounts of thrombin (p=0.003) which was associated with activation of the contact coagulation system. Plasma from LVAD patients induced more Ang-2 gene expression in endothelial cells (p<0.001) which was reduced with thrombin receptor blockade (p=0.013). LVAD patients with Ang-2 levels above the mean (12.32 ng/mL) had more NSB events compared with patients with Ang-2 levels below the mean (p=0.003). Conclusions Our findings indicate that thrombin-induced Ang-2 expression in LVAD patients leads to increased angiogenesis in vitro and may be associated with higher NSB events. Ang-2 therefore may contribute to AVM formation and subsequent bleeding in LVAD patients.