Composition in situ and in vitro of vascular smooth muscle laminin in the rat

Abstract

Vascular smooth muscle cells are surrounded by a basal lamina containing an array of macromolecules: included among these are the laminins, a family of oligomeric glycoproteins composed of subunits encoded by different genes. In this study, we have used monoclonal antibodies to several of these subunits, including S-laminin, laminin B2, and laminin B1, to study these proteins in tail artery, superior mesenteric artery, and aorta of rats. In situ, immunostaining for the B2 and S chains was present in the basal lamina, between the smooth muscle cells, throughout the tunica media. In contrast, B1 chain immunostaining was concentrated around cells in the inner media. To investigate whether smooth muscle cells can produce S-laminin, laminin B2, and laminin B1, smooth muscle cells from the superior mesenteric artery were grown in culture and laminin subunit expression determined. In early culture (4 days), immunostaining showed abundant laminin B2 and less B1 synthesis and incorporation into the matrix. Staining for S-laminin was even less intense than for B1 and was localized to areas where cells were densely packed. The same pattern of S-laminin immunostaining was seen during early culture in cells grown on fibronectin, type IV collagen, or gelatin. Immunoblotting detected S-laminin in the conditioned medium from early cultured cells. In later culture (12 days), S-laminin incorporation into the matrix increased markedly compared to incorporation at 4 days. At this time, cells are much more densely packed and multilayered with extensive matrix accumulation. Cyclical stretching of cells in vitro did not increase immunostaining for S-laminin. Together these data show that S-laminin is a component of the arterial media in situ and that in vitro S-laminin is synthesized by smooth muscle cells. Increased incorporation of S-laminin into the matrix in later culture correlates with the presence of a more extensive matrix, suggesting that matrix organization may be critical to S-laminin incorporation.