Grazia Conforti

Production and characterization of two monoclonal antibodies directed against the integrin beta 1 chain.

The production and characterization of two new monoclonal antibodies (MAbs), designated MAR4 and MAR5, raised against the partially purified α5β1 integrin, are described. The reactivity of these 2 MAbs on tumor cell lines indicated that they reacted on all the cells expressing the β1 subunit independently of the α5 expression. Both MAbs were found to immunoprecipitate on 3 cell lines, a protein of 120 KD corresponding to the molecular weight be the β1 chain, in addition to proteins of other MW corresponding to the α subunits differentially expressed by these cells. The cross-competition experiments showed that MAR4 and MAR5 recognize the same epitope. These 2 MAbs seem to be useful reagents for the characterization of the VLA expression in tumor cells.

Biochemical and Functional Characteristics of Fibrinogen Interaction with Endothelial Cells

Several observations indicate that endothelial cells interact with fibrinogen, fibrin, and their derivative products. The main biological function of fibrinogen on endothelial cells so far identified is its ability to promote adhesion, motility, and cytoskeletal organization of these cells. In other words, when fibrinogen is bound to a substratum, it behaves as a matrix protein like fibronectin, collagen, or vitronectin. Fibrinogen specifically binds to endothelial cells with low affinity and with a high number of binding sites. In addition, the putative fibrinogen receptor in endothelial cells appears to be related to the other extracellular matrix protein receptors.

Role of Integrins in Endothelial Cell Function

Endothelial cell interaction with extracellular matrix proteins plays an important role in the maintenance of vascular wall integrity and in the formation of new vessels (Form et al.,1986; Ingberg and Folkman,1989).

RECEPTORS FOR EXTRACELLULAR MATRIX PROTEINS IN ENDOTHELIAL CELLS

The basement membrane of endothelial cells (EC) is organized as a complex network of numerous glycoproteins which include fibronectin (FN), vitronectin (VN), collagens and von Willebrand factor (vW). Beside these substrata which are directly produced by EC, fibrinogen (FG) and fibrin tend to accumulate at sites of vascular injury and may provide a new matrix over which EC can migrate and proliferate (Dejana et al., 1988). The interaction of EC with the protein components of the matrix, causes a series of cell responses as adhesion, spreading, cytoskeletal organization and cell migration. These multiple cellular reactions appear to be mediated by specific receptors which on the outer side of the membrane specifically recognize and bind different components of the extracellular matrix and, on the cytoplasmic side, link a chain of proteins involved in the mechanism of adhesion and cytoskeletal organization (Geiger, 1985).

Endothelial Cell-Matrix Interactions in Health and Disease

Human endothelial cells (EC) adhere, spread and organize their cytoskeleton on a variety of molecules of the extracellular matrix such as fibronectin, vitronectin, laminin and collagen (Form et al,1983; Ingber and Folkman, 1989). EC express receptor molecules which on the outer side of the membrane, recognize and bind different components of the extracellular matrix and, on the cytoplasmic side, transmit intracellular signals and link a chain of proteins of the membrane-microfilament interaction complex involved in the mechanism of adhesion and cytoskeletal organization (Burridge,1986). Most of the EC receptors for extracellular matrix components belong to a recently discovered superfamily of adhesive membrane proteins denominated “integrins” (Ruoslahti and Pierschbacher, 1987; Hynes, 1986). These receptors have several structural and functional homologies so that it is believed that they differentiated from a common ancestral gene. They are all heterodimers of two non-covalently linked subunits. The larger subunit has been termed “α chain” and the smaller subunit “β chain”. The name “integrins” arose because they are integral membrane proteins, i.e. each subunit has a transmembrane segment, a small C-terminal cytoplasmic domain and a large N-terminal extracellular domain (Ruoslahti and Pierschbacher, 1987; Hynes, 1986). Some of these receptors specifically recognize in the ligand proteins a sequence of only three aminoacids (arginine-glycine-aspartic acid, RGD). Many proteins contain this sequence but not all of them are recognized by an integrin receptor; however, this sequence is the cell recognition site of a large number of extracellular matrix and plasma proteins including fibrinogen, vitronectin, fibronectin, thrombospondin and von Willebrand factor (Ruoslahti and Pierschbacher, 1987). Despite the similarities in the cell binding sequences in ligand proteins, the cell can recognize them individually through specific and separate receptors. This suggests that other aminoacids surrounding the RGD sequence, and the RGD steric conformation, confer specificity to the interaction with one integrin receptor or another (Ruoslahti and Pierschbacher, 1987).