These results confirm that adhesion to immobilised TP508 is mediated through v3. Open in a separate window Figure 2 Integrin v3 mediates adhesion to Rilapladib TP508A) Adhesion of HEK-v3 cells to immobilised TP508 is mediated through integrin v3. of NO. Factors influencing decreased production of NO include reduced expression of endothelial nitric oxide synthase (eNOS), failure to activate eNOS and a lack of substrate or eNOS cofactors (2). Reduced vasodilation results in impaired blood flow and response to angiogenic factors that could lead to atherosclerosis, plaque formation and cardiovascular pathology (3). VED is associated with several pathological conditions, including hypercholesterolaemia, hypertension, atherosclerosis, stroke, diabetes mellitus and SOX18 congestive heart failure (4C9). Consequently, disturbed endothelial function is of clinical significance. Proposed pharmaceutical treatments for VED exist, including L-arginine, I-methylnicotinamide, phosphodiesterase type 5 inhibitors and endothelin receptor antagonists, but many have been approved only for specific indications related to VED such as hypertension and erectile dysfunction (10). TP508, a 23-amino acid synthetic peptide representing residues 508 to 530 of human prothrombin, elicits a variety of potential therapeutic effects in a NO-dependent manner. A previous study has shown that TP508 exhibited reversal of VED in porcine ischaemic hearts and human endothelial cells via an upregulation of endothelial NO synthesis (11). Recent studies using normal and hypercholesteraemic pig models show that TP508 treatment results in a significant reduction in infarct size as well as elevated levels of phosphorylated eNOS (peNOS) compared to untreated pigs (12, 13). These studies using isolated coronary arterioles from ischaemic regions show that TP508 significantly enhances vascular dilation in an endothelium-dependent manner (11C13). These results provide evidence supporting eNOS phosphorylation and endothelium-dependent coronary microvascular relaxation by TP508. The effects of TP508 are well documented, but its mechanism of action Rilapladib has not yet been completely elaborated. TP508 was initially selected for its ability to compete with the binding of thrombin to its receptors on fibroblasts (14). However, it lacks the protease domain of thrombin and is unlikely to activate intracellular signalling through protease-activated receptors (PARs) in a manner similar to that observed for thrombin. Interestingly, the region of thrombin that corresponds to TP508 contains an RGD sequence that is buried within Rilapladib the structure of native thrombin and may only be exposed when thrombin is proteolytically cleaved or its structure is altered by high salts or immobilisation onto charged surfaces (15, 16). Thrombin has previously been shown to support endothelial cell adhesion in an RGD-dependent manner when immobilised to a surface (15C17). This interaction was determined to be mediated primarily through integrins v3 and 51 (16, 18C19). Other studies have provided evidence that some thrombin-induced signalling events are mediated through v3 specifically (20C21). Our working hypothesis is that TP508 mitigates VED by inducing NO via the integrin v3. Due to the presence of an RGD sequence in TP508, integrins that specifically recognise this motif, including v3 and 51, were targeted as potential receptors. The involvement of integrins was investigated by determining the ability of endothelial cells and cells over-expressing selective integrins to adhere to immobilised TP508 and the ability of soluble TP508 to compete for the natural ligands fibrinogen or fibronectin. Additionally, surface plasmon resonance (SPR) studies were performed to measure the direct interaction of TP508 and v3. Integrin-mediated signalling, NO production and vasorelaxation were also studied. The role of this interaction in NO induction was determined by using inhibitors of integrin function to block TP508-mediated NO induction. Results from this study indicate that TP508 induction of NO synthesis in endothelial cells is mediated by integrin v3. Materials and methods Cells, peptides and antibodies Human umbilical vein endothelial cells (HUVEC) were obtained from Lonza (Boston, MA, USA). All experiments using Rilapladib HUVEC were performed at passages 2 to 4. Human embryonic kidney Rilapladib 293 (HEK 293) cells were purchased from ATCC (Manassas, VA, USA). The v3-expressing HEK 293 cells (HEK-v3 were constructed as previously described (22), using full-length 3 cDNA provided by Dr. T. OToole (Lerner Research.