Abstract. Microvascular endothelial cells (MEC) use a set of surface receptors to adhere not only to the vascular basement membrane but, during angiogenic stimulation, to the interstitium. We examined how cultured human MEC interact with laminin-rich basement membranes. By using a panel of monoclonal antibodies, we found that MEC cells express a number of integrin-related receptor complexes, including cq/3t, ¢x~, c~3/3,, ots/3~, ¢xd3~, and ¢x,/33. Attachment to laminin, a major adhesive protein in basement membranes, was studied in detail. Blocking monoclonal antibodies specific to different integrin receptor complexes showed that the otd3~ complex was important for MEC adhesion to laminin. In addition, blocking antibody also implicated the vitronectin receptor (o~/33) in laminin adhesion. We used ligand affinity chromatography of detergent-solubilized receptor complexes to further define receptor specificity. On laminin-Sepharose columns, we identified several integrin receptor complexes whose affinity for the ligand was dependent on the type of divalent cation present. Several/3~ complexes, including Otl/31, O~2/31, and otd3t bound strongly to laminin. In agreement with the antibody blocking experiments, c~d33 was found to bind well to laminin. However, unlike binding to its other ligands (e.g., vitronectin, fibrinogen, von Willebrand factor), Otv/33 interaction with larninin did not appear to be Arg-GlyAsp (RGD) sensitive. Finally, immunofluorescent staining demonstrated both/3~ and/33 complexes in vinculin-positive focal adhesion plaques on the basal surface of MEC adhering to laminin-coated substrates. The results indicate that both these subfamilies of integrin heterodimers are involved in promoting MEC adhesion to laminin and the vascular basement membrane.

A novel integrin, a,#,, that specifically binds with high affinity to laminin has been identified on melanoma cells. This complex was purified from both human and murine melanoma cells by laminin-affinity chromatography, and the a7 subunit was recovered after gel electrophoresis. N-terminal amino acid sequence analysis of the a7 subunit from both human and mouse cells verifies that this integrin is distinct from other a chains in the #I family, although strikingly similar to the as subunit. By using specific proteolytically derived fragments of laminin, it was determined that the a7f1 complex binds selectively to the E8 region, which represents part of the long arm of laminin. In contrast, the receptor failed to bind to the P1 fragment, which contains the intersection of the short arms of laminin. Although the a7,,1 complex was commonly expressed in melanoma cells, this integrin was not detected in normal melanocytes, suggesting that a7 expression may be associated with malignant transformation. These results establish the existence of a novel integrin that binds to the E8 domain of laminin and appears to mediate cell adhesion to this ligand.

Microvascular endothelial cells (MEC) must use a set of surface receptors to adhere not only to the vascular basement membrane but, during angiogenic stimulation, to the interstitium. We examined how cultured MEC isolated from human foreskin interact with their subendothelial matrix. MEC were able to attach to diverse extracellular matrix proteins, including fibronectin (Fn), vitronectin (Vn), laminin (Ln), type I and IV collagen, as well as to fibrinogen and gelatin. Adhesion to Fn, but not to laminin or collagens, was specifically blocked in the presence of Arg-Gly-Asp (RGD)-containing peptides. When surface radioiodinated MEC were solubilized and subjected to affinity chromatography on Fn-Sepharose columns, two polypeptides of 150 and 125 kD, corresponding to the integrin heterodimer alpha 5 beta 1, were identified. MEC also express a complex of 150 (alpha) and 95 kD (beta 3) that is related to the Vn receptor. Immunofluorescent staining of MEC cultures with antibodies to the integrin beta 1 subunit demonstrated receptors on the basolateral surface at focal adhesion plaques that co-localized with vinculin and with Fn-positive matrix fibers. Occasionally, antibodies to the Vn receptor stained the vinculin-positive focal adhesion plaques that frequently co-localized with the beta 1 complex. However, in cultures of MEC that were attached to substrates coated with alternating strips of Fn and Vn, the beta 1 complex was preferentially localized to the Fn substrate, while the Vn receptor was concentrated on the Vn substrate. The results indicate that MEC express at least two different heterodimer adhesion receptors that belong to the integrin super-family and appear to have distinct ligand specificities: the Fn receptor and the Vn receptor. These receptors mediate cell adhesion to the extracellular matrix and presumably have an important role in hemostasis and neovascularization.

Invasion of melanoma cells into the underlying interstitial stromal matrix is the initial step for subsequent local and distant metastasis. The invading tumor cell must interact with the extracellular matrix during the early stages of invasion and later during penetration of lymphatic and blood vessels. This interaction with different types of extracellular matrix predicts that the invasive cell must possess surface adhesion receptors with diverse ligand specificities, including the capacity to bind different types of collagens and adhesive glycoproteins. Metastatic melanoma cells do in fact express multiple adhesion receptors, including several of the receptors from the integrin family of heterodimers. The integrin receptors can be either extremely specific for a single ligand or capable of binding multiple ligands. It is likely that the tumor cell's repertoire of adhesion receptors may influence not only its adhesive properties but its metastatic characteristics as well. There is evidence that normal melanocytes have an integrin profile distinct from that of melanoma cells. In particular, melanocytes adhere poorly to laminin while metastatic melanoma cells bind well to this ligand. This difference in adhesion between the two cell types appears to reflect the fact that melanoma cells express a melanoma-specific integrin (alpha 7 beta 1) that binds laminin and is not detectable in normal melanocytes. The presence of increased laminin receptors and enhanced laminin binding in melanoma cells may contribute to the malignant phenotype.

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