Osteoprotegerin: a pancreatic islets dysfunction and vascular injury modulator.

Abstract

Background. Osteoprotegerin (OPG) is a soluble glycoprotein of the tumor necrosis factor (TNF) receptor superfamily, which was initially identified as a key regulator in bone turnover. It acts as a decoy receptor for the receptor activator of nuclear factor kB ligand (RANKL) and for the TNF-related apoptosis-inducing ligand (TRAIL), counterbalancing their biological effects. OPG is produced by a wide range of tissues, including the cardiovascular system, and its levels are particularly high in aortic and renal arteries. Several studies have clearly demonstrated that the serum levels of OPG are elevated in diabetic and nondiabetic patients affected by cardiovascular diseases, and increased levels of OPG represent a risk factor for cardiovascular mortality, especially in diabetic patients. However, in spite of the reported findings, the physiopathological role of elevated serum levels of OPG in vascular biology and in pancreatic islet function are not well understood. Aim of the study. The aims of our studies were: Study 1. Evaluate the potential role of OPG in the pathogenesis of diabetes associated atherosclerosis. Study 2. Investigate OPG effects on pancreatic islet function and its interaction with local pancreatic renin-angiotensin system (RAS). Materials and Methods. Study 1.A. In vivo study: 80 apoE knockout male mice were further randomized into 4 groups (n=20) and followed for 3 months. One group of non diabetic animals received an intraperitoneal (i.p.) injection of vehicle and served as a control; another group of non-diabetic animals received every 3 weeks an i.p. injection of human recombinant OPG (OPG). The other two groups, rendered diabetic by 5 daily i.p. injections of streptozotocin (55mg/Kg/die), received injections of OPG or an equivalent volume of vehicle. At the end of the study, animals were culled, the blood was collected for biochemical analysis, and the entire aorta was excised out to study the total plaques extent and to evaluate the lesion composition and complexity of the aortic plaques. B. In vitro study: Murine vascular smooth muscle cells (VMSC) were treated with different concentrations of OPG, TGFβ and SB431542 (TGFβ- type 1 receptor inhibitor). Subsequently, cellular proliferation and pro-fibrotic markers gene expression were evaluated at different time points. OPG protein release was measured in growth media (ELISA technique). Study 2. 40 male mice C57Bl/6J, aged 10 weeks, were randomized into 4 groups (n=10) and studied for 3 months. Group 1 received every 3 weeks an i.p. injection of vehicle and served as a control. Group 2 received every 3 weeks an i.p. injection of OPG. Group 3 received the ACE inhibitor ramipril at the dose of 10mg/Kg/die in drinking water in co-treatment with i.p. injections of vehicle. Group 4 received ramipril in co-treatment with i.p. injections of OPG. At the end of the study, animals were culled, the blood was collected for biochemical analysis, and the pancreas was dissected out for subsequent quantitative RT-PCR measurements and immunohistochemical analysis. Results. Study 1.A. At the end of the study, diabetic animals injected with OPG presented a significant increase in total plaques extent, with an increase of smooth muscle cells content in aortic plaques. Moreover OPG treated animals showed an increase in the collagen content in aortic media in respect to control mice. B. OPG promoted VSMC proliferation and pro-fibrotic markers gene expression. TGFβ treatment of VSMC induced a dose-dependent increase of OPG gene and protein expression, that was completed prevented by pre-treatment with the SB431542 inhibitor. Study 2. OPG-treated animals showed increased islet monocyte-macrophage infiltration, fibrosis and apoptosis with reduction of islet function. The remodeling of islet architecture was associated with increased pancreatic expression of components of the RAS, growth factor genes (TGFβ and CTGF) and inflammatory molecules (MCP-1 and VCAM-1). Prevention of these changes with improvement of insulin secretion was observed in ramipril treated animals. Conclusion. Study 1.A-B OPG seems to play an important pathogenetic role in the development and progression of diabetic atherosclerosis. Study 2. Our data suggest that OPG might play an important role in promoting beta cell dysfunction and the upregulation of the local RAS represents one possible mechanism responsible for the OPG-induced beta cell dysfunction