Linking the beneficial effects of current therapeutic approaches in diabetes to the vascular endothelin system

The rising epidemic of diabetes worldwide is of significant concern. Although the ultimate objective is to prevent the development and find a cure for the disease, prevention and treatment of diabetic complications is very important. Vascular complications in diabetes, or diabetic vasculopathy, include macro- and microvascular dysfunction and represent the principal cause of morbidity and mortality in diabetic patients. Endothelial dysfunction plays a pivotal role in the development and progression of diabetic vasculopathy. Endothelin-1 (ET-1), an endothelial cell-derived peptide, is a potent vasoconstrictor with mitogenic, pro-oxidative and pro-inflammatory properties that are particularly relevant to the pathophysiology of diabetic vasculopathy. Overproduction of ET-1 is reported in patients and animal models of diabetes and the functional effects of ET-1 and its receptors are also greatly altered in diabetic conditions. The current therapeutic approaches in diabetes include glucose lowering, sensitization to insulin, reduction of fatty acids and vasculoprotective therapies. However, whether and how these therapeutic approaches affect the ET-1 system remain poorly understood. Accordingly, in the present review, we will focus on experimental and clinical evidence that indicates a role for ET-1 in diabetic vasculopathy and on the effects of current therapeutic approaches in diabetes on the vascular ET-1 system

Spironolactone treatment attenuates vascular dysfunction in type 2 diabetic mice by decreasing oxidative stress and restoring NO/GC signaling

Type 2 diabetes (DM2) increases the risk of cardiovascular disease. Aldosterone, which has pro-oxidative and pro-inflammatory effects in the cardiovascular system, is positively regulated in DM2. We assessed whether blockade of mineralocorticoid receptors (MR) with spironolactone decreases reactive oxygen species (ROS)-associated vascular dysfunction and improves vascular nitric oxide (NO) signaling in diabetes. Leptin receptor knockout [LepRdb/LepRdb (db/db)] mice, a model of DM2, and their counterpart controls [LepRdb/LepR+, (db/+) mice] received spironolactone (50 mg/kg body weight/day) or vehicle (ethanol 1%) via oral per gavage for 6 weeks. Spironolactone treatment abolished endothelial dysfunction and increased endothelial nitric oxide synthase (eNOS) phosphorylation (Ser1177) in arteries from db/db mice, determined by acetylcholine-induced relaxation and Western Blot analysis, respectively. MR antagonist therapy also abrogated augmented ROS-generation in aorta from diabetic mice, determined by lucigenin luminescence assay. Spironolactone treatment increased superoxide dismutase-1 and catalase expression, improved sodium nitroprusside and BAY 41-2272-induced relaxation, and increased soluble guanylyl cyclase (sGC) β subunit expression in arteries from db/db mice. Our results demonstrate that spironolactone decreases diabetes-associated vascular oxidative stress and prevents vascular dysfunction through processes involving increased expression of antioxidant enzymes and sGC. These findings further elucidate redox-sensitive mechanisms whereby spironolactone protects against vascular injury in diabetes

Isolation and culture of endothelial cells from large vessels

The endothelium, which is at the interface between circulating blood and the vascular wall, comprises a simple squamous layer of cells that lines the inner surface of all blood vessels. Endothelial cells are highly metabolically active and play an important role in many physiological functions, including control of vasomotor tone, blood cell trafficking, vascular permeability, and maintenance of vascular integrity (Mensah, Vascul Pharmacol 46(5):310–314, 2007; Yetik-Anacak and Catravas, Vascul Pharmacol 45(5):268–276, 2006). Endothelial cells are characteristically ‘quiescent’ in that they do not actively proliferate, with the average lifespan of an endothelial cell being >1 year. The endothelium is very sensitive to mechanical stimuli (stretch, shear stress, pressure), humoral agents (angiotensin II (Ang II), endothelin-1 (ET-1), aldosterone, bradykinin, thromoxane) and chemical factors (glucose, reactive oxygen species (ROS)) and responds by releasing endothelial-derived mediators, such as nitric oxide (NO), prostacyclin (PGI2), platelet-activating factor (PAF), C-type atrial natriuretic peptide (ANP), and ET-1 to regulate vascular tone, prevent thrombosis and inflammation, and maintain structural integrity. Primary culture of endothelial cells is an important tool in dissecting the role of the endothelium in many physiological or pathological responses. This chapter describes the explant method for culture of endothelial cells from large vessels. Cells derived by the protocol described here can be used for cell biology and molecular biology studies in hypertension and other cardiovascular diseases where endothelial function may be impaired

Isolation and culture of vascular smooth muscle cells from small and large vessels

Primary culture of vascular smooth muscle cells is an important in vitro model for the dissection of molecular mechanisms related to a specific physiological or pathological response at the cellular level. Cultured cells also provide an excellent model to study cell biology. This chapter describes a user-friendly and practical protocol for isolation of vascular smooth muscle cells from small and large vessels by enzymatic dissociation, which can be applied to vessels from different species, including rodents and humans