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Monkeys with excellent reproductive histories were immunized with the laminin peptides YIGSR, RGD, IKVAV, and YD, a control sequence with no known biological function. Sera from the YIGSR-immunized monkey became toxic, causing neural tube defects in whole rat embryo cultures, and this monkey experienced fetal loss after immunization. Sera from the RGD-immunized monkey also became embryotoxic in culture after immunization, but this monkey appeared to become infertile as she failed to initiate a pregnancy for at least 2 years after immunization. In contrast, embryos cultured on sera from the IKVAV- or YD-immunized monkeys were predominantly normal and both monkeys completed successful pregnancies. Antibody levels to the respective peptides or to laminin were not predictive of embryotoxicity, but antibody binding to homogenized yolk sacs as well as to yolk sacs of cultured embryos was associated with sera embryotoxicity and reproductive outcomes in vivo. These observations suggested that the laminin sequences YIGSR and RGD may play a role in immune-mediated reproductive failure by reacting directly with embryonic tissue and could provide a basis for identifying individuals at risk for both spontaneous abortion and infertility.

We previously characterized a 110-kDa membrane-associated laminin-binding protein (LBP110) from brain which binds the laminin A chain -Ile-Lys-Val-Ala-Val-(IKVAV) site and increases in injury. Here we demonstrate that antisera directed against different epitopes of beta-amyloid precursor protein (APP) recognize LBP110 and that APP is recognized by LBP110 antiserum. APP specifically binds IKVAV and not another biologically active laminin-derived peptide containing the amino acid sequence -Tyr-Ile-Gly-Ser-Arg-. PC-12 cells transfected with antisense APP RNA produce less APP and LBP110, and they form fewer processes when cultured on either laminin or the IKVAV peptide. Thus, LBP110 is a member of the APP family and a function for APP in neurite outgrowth is now defined.

Scatter factor (also known as hepatocyte growth factor) is a glycoprotein secreted by stromal cells that stimulates cell motility and proliferation. In vitro, scatter factor stimulates vascular endothelial cell migration, proliferation, and organization into capillary-like tubes. Using two different in vivo assays, we showed that physiologic quantities of purified native mouse scatter factor and recombinant human hepatocyte growth factor induce angiogenesis (the formation of new blood vessels). The angiogenic activity was blocked by specific anti-scatter factor antibodies. Scatter factor induced cultured microvascular endothelial cells to accumulate and secrete significantly increased quantities of urokinase, an enzyme associated with development of an invasive endothelial phenotype during angiogenesis. We further showed that immunoreactive scatter factor is present surrounding sites of blood vessel formation in psoriatic skin. These findings suggest that scatter factor may act as a paracrine mediator in pathologic angiogenesis associated with human inflammatory disease.

To evaluate the feasibility of using a replication-deficient recombinant adenovirus to transfer human genes to the human endothelium, human umbilical vein endothelial cells were infected in vitro with adenovirus vectors containing the lacZ gene or a human alpha 1-antitrypsin (alpha 1AT) cDNA. After in vitro infection with the lacZ adenovirus vector, cultured endothelial cells expressed beta-galactosidase. In parallel studies with the alpha 1AT adenovirus vector, infected cells expressed human alpha 1AT transcripts, as evidenced by in situ hybridization and Northern analysis, and de novo synthesized and secreted glycosylated, functional alpha 1AT within 6 hr of infection, as shown by [35S]methionine labeling and immunoprecipitation. Quantification of the culture supernatants demonstrated 0.3-0.6 micrograms of human alpha 1AT secreted per 10(6) cells in 24 hr, for at least 14 days after adenovirus vector infection. To demonstrate the feasibility of direct transfer of genes into endothelial cells in human blood vessels, lacZ or alpha 1AT adenovirus vectors were placed in the lumen of intact human umbilical veins ex vivo. Histologic evaluation of the veins after 24 hr demonstrated transfer and expression of the lacZ gene specifically to the endothelium. alpha 1AT adenovirus infection resulted both in expression of alpha 1AT transcripts in the endothelium and in de novo synthesis and secretion of alpha 1AT. Quantification of alpha 1AT in the vein perfusates showed average levels of 13 micrograms/ml after 24 hr. These observations strongly support the feasibility of in vivo human gene transfer to the endothelium mediated by replication-deficient adenovirus vectors.

The ability of matrigel, a reconstituted basement membrane gel, to induce the differentiation of baby mouse kidney cells has been examined in a hormonally defined serum-free medium. Primary cultures of baby mouse kidney cells were observed to form tubules over a time interval of 1-2 weeks in matrigel. Electron microscopic studies showed that tubules with lumens were present, and the tubule morphology was similar to that of the collecting duct. When using matrigel from which the growth factors had been removed, tubule formation no longer occurred, unless the medium was further supplemented with epidermal growth factor (10 ng/ml). Transforming growth factor alpha stimulated tubule formation as effectively as epidermal growth factor, whereas transforming growth factor beta had an inhibitory effect on tubule formation. These data suggest that both an extracellular matrix and specific growth factors may regulate kidney differentiation during development.

Tumor cells attach, degrade, and migrate through basement membranes as they metastasize. Laminin, a major glycoprotein of basement membranes, promotes the metastatic activity of tumor cells by stimulating the attachment and migration of the cells and their secretion of collagenase IV. We have identified a synthetic peptide of 19 amino acids (Cys-Ser-Arg-Ala-Arg-Lys-Gln-Ala-Ala-Ser-Ile-Lys-Val-Ala-Val-Ser-Ala-Asp -Arg) from the sequence of the A chain of laminin that increases experimental metastases of the lungs by murine melanoma cells. The peptide is active when injected either intravenously or intraperitoneally. The peptide increased collagenase IV activity, a key enzyme in the breakdown of basement membranes, to the same extent as laminin. This peptide represents an active site on laminin for promotion of the metastatic phenotype and generates a probe for studying the regulation of malignant activities.

Smooth muscle cells use fibronectin to bind to type I and type III collagens but bind to type V collagen by a trypsin-resistant intrinsic glycoconjugate. The binding site on type V collagen is located in the alpha A chain. By using collagen-coated filters in a modified Boyden chamber assay for chemotaxis, it was observed that the platelet-derived growth factor was chemotactic for smooth muscle cells but that several other growth factors were inactive. We suggest that the migration of smooth muscle cells from the media to the intima of a blood vessel, which leads to the formation of an atherosclerotic plaque, may be the result of a chemotactic migration of cells responsive to the platelet-derived growth factor.

The interaction of a line of human breast carcinoma cells (MCF7) with basement membrane components, particularly laminin, was altered by exposure of the cells to estrogen as well as by transfection of the cells with the v-Ha-ras oncogene. In both cases, the cells show a greater ability to attach to a laminin substrate, to migrate to laminin, to grow in the presence of a basement membrane matrix, and to cross barriers of reconstituted basement membrane. These responses were associated with an increase in the expression of laminin receptors. It is postulated that the increase in the invasive behavior of the cells treated with estrogen or transfected with v-Ha-ras is related to the increased number of laminin receptors and their interaction with laminin. Estrogen had no discernible effect on the v-Ha-ras transfected cells. It appears that in the MCF7 cells, the malignant phenotype is under hormonal control and that this control is bypassed after v-Ha-ras transfection.

Laminin, a basement membrane glycoprotein promotes both cell attachment and neurite outgrowth. Separate domains on laminin elicit these responses, suggesting that distinct receptors occur on the surface of cells. NG108-15 neuroblastoma-glioma cells rapidly extend long processes in the presence of laminin. We report here that 125I-labeled laminin specifically binds to these cells and to three membrane proteins of 67, 110, and 180 kDa. These proteins were isolated by affinity chromatography on laminin-Sepharose. The 67-kDa protein reacted with antibody to the previously characterized receptor for cell attachment to laminin. Antibodies to the 110-kDa and 180-kDa bands demonstrated that the 110-kDa protein was found in a variety of epithelial cell lines and in brain, whereas the 180-kDa protein was neural specific. Antibodies prepared against the 110-kDa and 180-kDa proteins inhibited neurite outgrowth induced by the neurite-promoting domain of laminin, whereas antibodies to the 67-kDa laminin receptor had no effect on neurite outgrowth. We conclude that neuronal cells have multiple cell-surface laminin receptors and that the 110-kDa and 180-kDa proteins are involved in neurite formation.

Fibronectin mediates the adhesion of cells to collagen by first binding to the collagen substrate, followed by attachment of the cells to the fibronectin-collagen complex. Bovine brain gangliosides were found to block fibronectin-mediated cell adhesion to collagen in a concentration-dependent manner. The gangliosides did not block the binding of fibronectin to collagen but did prevent the attachment of the cells to the fibronectin-collagen complex. Of the individual gangliosides tested, GT1 and GD1a were the most effective inhibitors followed by GD1b greater than GM1 greater than GM2; GM3 was not an inhibitor. The inhibition of cell adhesion also was observed with the oligosaccharide portion of the gangliosides, but not with ceramides or with a variety of free sugars or glycosaminoglycans. Mild periodate oxidation of mixed gangliosides or of GD1a modified their sialic acid residues and the oxidized gangliosides were no longer inhibitory; subsequent reduction with NaBH4 did not restore the inhibitory activity of the modified gangliosides. These results suggest that specific gangliosides or related sialic acid-containing glycoconjugates on the cell surface may act as the receptors for fibronectin.

In cartilage, type II collagen is present as thin, short, randomly oriented fibrils. In vitro, however, type II collagen forms fibrils of large diameter, indicating that additional factors may be involved in the regulation of collagen fibril formation. We have examined extracts of a cartilage-producing tumor for the presence of collagen-binding proteins. In addition to fibronectin and link protein, a Mr 54,000 protein was found to bind to collagen fibrils as well as to native and denatured type II collagen. Immunological studies using antibody against the protein indicate that it is a cartilage matrix protein, not present in bone or in several other tissues. In vitro studies show that the Mr 54,000 protein in combination with cartilage proteoglycan decreases the rate of type II fibril formation and causes the fibrils to be of small diameter (24 +/- 8 nm). These studies indicate that complexes between collagen and proteoglycans mediated by this protein may regulate the assembly of cartilage matrix.

The attachment of chondrocytes to collagen substrates is stimulated by serum but not by fibronectin. The active material in serum was partially purified and was shown to be a protein by its sensitivity to trypsin and heat and its chromatographic properties. This factor, which we have named chondronectin, is distinct from fibronectin and does not stimulate fibroblast attachment. Because material with similar attachment-enhancing activity is produced by chondrocytes and is extractable from cartilage, chondronectin may be a chondrocyte-specific attachment protein.

The C1q subunit of complement component C1 is known to bind to immune complexes, which often are deposited in basement membrane. We investigated the possibility that this deposition is a result of binding to laminin, a large basement membrane glycoprotein. C1q showed saturable binding to immobilized laminin; this binding was increased at reduced ionic strength. Intact C1 did not bind laminin. A ternary complex was formed by laminin, C1q, and aggregated IgG. This complex formation was dependent on and proportional to the amount of C1q bound to the aggregated IgG. Binding of laminin to C1q occurred with a Kd of 2 nM and was stronger than the binding of C1q to fibronectin. Preliminary data, including electron micrographs of rotary-shadowed preparations, suggest that laminin binds to the collagen-like tail of C1q. Electron microscopy localized the site of interaction with C1q to a short arm of laminin. Since laminin is found only in basement membranes, the interaction between laminin and C1q could be involved in the deposition and retention of immune complexes in these structures.