A E Canfield

The involvement of matrix glycoproteins in vascular calcification and fibrosis: an immunohistochemical study

Calcification and fibrointimal proliferation are associated with advanced complicated atherosclerosis in large arteries but may also occur in smaller vessels, resulting in ischaemic tissue necrosis. This study investigates whether the mechanisms of calcification and intimal fibrosis are similar in vessels of different sizes. The localization of osteopontin (OPN), matrix Gla protein (MGP), thrombospondin‐1 (TSP‐1), and cartilage oligomeric matrix protein (COMP) was investigated in three types of human vascular lesions: atherosclerosis, chronic vascular rejection (CVR) in renal allografts, and calcific uraemic arteriolopathy (calciphylaxis). These lesions were chosen as they affect different sized blood vessels and they exhibit a fibroproliferative intimal reaction, with or without calcification, resulting in luminal obliteration and ischaemic complications. OPN, MGP, TSP‐1, and COMP were not detected in normal blood vessels. However, OPN and MGP were expressed at sites of calcification within atherosclerotic lesions and in microvessels in calciphylaxis, suggesting that calcification in different sized vessels may occur by a common mechanism. These proteins were not detected in areas of fibrointimal proliferation. In contrast, TSP‐1 was localized primarily within the fibrous tissue of atherosclerotic lesions and was also expressed in the expanded fibrous intima of arteries showing CVR. COMP was localized primarily within the fibrous tissue under the lipid core of the majority of advanced atherosclerotic lesions. TSP‐1 and COMP were also detected in areas of microcalcification in atherosclerotic lesions and TSP‐1 was detected adjacent to areas of calcification in calciphylaxis. However, neither TSP‐1 nor COMP was localized to calcific foci within these lesions. The localization of OPN, MGP, TSP‐1, and COMP to pathological, but not normal arterial intima supports a pathogenetic role for these proteins in the development of vascular fibrosis and calcification. Modulation of their production and activity may offer a novel approach to the therapy of a number of vascular diseases. Copyright

Hepatocyte growth factor and c‐Met expression in pericytes: implications for atherosclerotic plaque development

Intraplaque neovascularization contributes to the progression of atherosclerosis. Our aim is to understand the mobilization of cells and factors involved in this process. We investigated the localization of hepatocyte growth factor (HGF) and its receptor, c‐Met, in human atherosclerotic plaques, together with the effects of HGF on pericyte migration in vitro. Atherosclerotic femoral arterial segments were collected and analysed from 13 subjects who were undergoing lower limb amputation. Pericytes were identified in human lesions using a 3G5 antibody. Immunohistochemical analysis localized HGF mainly around microvessels, in association with some, but not all, CD31‐positive endothelial cells. c‐Met expression was mainly associated with smooth muscle cells and pericytes, around some, but not all, microvessels within the atherosclerotic lesions; no detection was apparent in normal internal mammary arteries. Using RT–PCR, we demonstrated expression of HGF and c‐Met in a rat pericyte cell‐line, TR–PCT1, and in primary pericytes. HGF treatment of TR‐PCT1 cells induced their migration, but not their proliferation, in a dose‐dependent manner (10–100 ng/ml, p < 0.01), an effect mediated by activation of the serine/threonine kinase Akt, shown by western blot analysis. Treating the cells with the PI3K inhibitors Wortmannin (0.1 µM) or LY294002 (10 µM) abolished these effects. This work demonstrates the expression of c‐Met and HGF in human atherosclerotic arteries, in association with SM‐actin‐positive cells and CD‐31‐positive cells, respectively. HGF induces pericyte migration via PI3‐kinase and Akt activation in vitro. HGF and c‐Met may be involved in neovascularization during plaque development, and may recruit pericytes to neovessels. Since pericytes are thought to mechanically stabilize new blood vessels, these factors may function to protect against haemorrhage. Copyright

Contribution of VCAF-positive cells to neovascularization and calcification in atherosclerotic plaque development.

Calcification of the vessel wall is a regulated process with many similarities to osteogenesis. Progenitor cells may play a role in this process. Previously, we identified a novel gene, Vascular Calcification Associated Factor (VCAF), which was shown to be important in pericyte osteogenic differentiation. The aim of this study was to determine the localization and expression pattern of VCAF in human cells and tissues. Immunohistochemical analysis of seven atherosclerotic arteries confirmed VCAF protein expression within calcified lesions. In addition, individual VCAF‐positive cells were detected within the intima and adventitia in areas where sporadic 3G5‐positive pericytes were localized. Furthermore, VCAF‐positive cells were identified in newly formed microvessels in association with CD34‐positive/CD146‐positive/c‐kit‐positive cells as well as in intact CD31‐positive endothelium in internal mammary arteries. Western blot analysis confirmed the presence of VCAF (18 kD) in protein lysates extracted from human smooth muscle cells, endothelial cells, macrophages, and osteoblasts. In fracture callus samples from three patients, VCAF was detected in osteoblasts and microvessels. This study demonstrates the presence of VCAF in neovessels and raises the possibility that VCAF could be a new marker for vascular progenitor cells involved in a number of differentiation pathways. These data may have implications for the prevention or treatment of vascular disease. Copyright