MAB1921P Sigma-AldrichAnti-Laminin β1 Antibody, clone 4E10

We show that endothelial cell (EC)-generated vascular guidance tunnels (ie, matrix spaces created during tube formation) serve as conduits for the recruitment and motility of pericytes along EC ablumenal surfaces to facilitate vessel maturation events, including vascular basement membrane matrix assembly and restriction of EC tube diameter. During quail development, pericyte recruitment along microvascular tubes directly correlates with vascular basement membrane matrix deposition. Pericyte recruitment to EC tubes leads to specific induction of fibronectin and nidogen-1 (ie, matrix-bridging proteins that link together basement membrane components) as well as perlecan and laminin isoforms. Coincident with these events, up-regulation of integrins, alpha(5)beta(1), alpha(3)beta(1), alpha(6)beta(1), and alpha(1)beta(1), which bind fibronectin, nidogens, laminin isoforms, and collagen type IV, occurs in EC-pericyte cocultures, but not EC-only cultures. Integrin-blocking antibodies to these receptors, disruption of fibronectin matrix assembly, and small interfering RNA suppression of pericyte tissue inhibitor of metalloproteinase (TIMP)-3 (a known regulator of vascular tube stabilization) all lead to decreased EC basement membrane, resulting in increased vessel lumen diameter, a key indicator of dysfunctional EC-pericyte interactions. Thus, pericyte recruitment to EC-lined tubes during vasculogenesis is a stimulatory event controlling vascular basement membrane matrix assembly, a fundamental maturation step regulating the transition from vascular morphogenesis to stabilization.

Laminin is a basement membrane (BM) glycoprotein composed of three of five subunits, the A, M, B1, B2, and the S chain. Four forms of laminin, A-B1-B2, A-S-B2, M-B1-B2, and M-S-B2, have been identified. Laminin is implicated in various biological processes such as cell adhesion and differentiation. We studied immunohistochemically the expression of the four laminin subunits A, M, B1, B2 as well as of neural cell adhesion molecule (N-CAM, CD56), a marker of regenerating myofibers, in various neuromuscular disorders. In normal muscle, the predominant subunits of myofiber laminin were M, B1, and B2. The A chain was only faintly expressed in myofiber BM. In inflammatory myopathies and dystrophinopathies myofiber laminin A expression was greatly increased. An average of 80% and 63% of laminin A-positive myofibers in inflammatory myopathies and dystrophinopathies, respectively, were additionally CD56 positive. Laminin A and CD56 expression in denervating diseases and mitochondrial myopathies were negligible. Expression of M, B1, and B2 subunits did not seem to be altered in the diseased conditions examined above. The data suggest that laminin A is upregulated in inflammatory myopathies and dystrophinopathies and, most markedly in regenerating myofibers.

A protein with properties of laminin has been isolated from human placental extracts by using monoclonal antibodies. Placental tissue was extracted with 0.5 M NaCl and high molecular weight proteins were isolated from the extract by salt precipitation and gel filtration on Sepharose 6B. The resulting protein fraction which contained material cross-reactive with anti-sera to rat laminin was used as immunogen to prepare hybridomas. Thirteen hybrids produced antibodies which reacted with basement membrane-associated antigens in indirect immunofluorescence of tissues. One of these, 4E10, was characterized in detail. This monoclonal antibody reacted with human laminin as shown by several lines of evidence. Immunoprecipitation from metabolically labeled culture media of a human amniotic epithelial cell line with the 4E10 antibody followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed polypeptides with Mr similar to those of rat laminin. Immunochromatography of placental extracts obtained by limited pepsin digestion yielded material with main polypeptides at 160 and 130 kilodaltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis after reduction. These peptic fragments cross-reacted with rat laminin in immunodiffusion and enzyme immunoassay, and a polyclonal antiserum against the fragments reacted with basement membranes in tissues in a manner identical with the 4E10 antibody. Electron microscopic images of the human peptic fragments showed structures similar to the cross-shaped images of murine laminins, although the short arms were truncated to various degrees or even absent. The isolated peptic fragments also displayed biological activity similar to that of murine laminins in that the outgrowth of neurites by neuronal cells was promoted on plates coated with the fragments.