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H K Kleinman

Publications and source records attributed to H K Kleinman.

At least 19 recordsLinked to original sources
PubMed1992

Role of the SIKVAV site of laminin in promotion of angiogenesis and tumor growth: an in vivo Matrigel model.

BACKGROUND: Angiogenesis (vascularization) has a critical role in tumor growth and metastasis, and peptides containing the SIKVAV amino acid sequence (Ser-Ile-Lys-Val-Ala-Val) have been shown to stimulate many angiogenic activities in vitro. The use of model systems to identify agents that stimulate or inhibit angiogenesis may lead to the development of new antitumor strategies. PURPOSE: Our purpose was to use an in vivo murine model system to study the angiogenic activity of a synthetic peptide derived from the laminin A protein chain and containing the SIKVAV amino acid sequence. We also examined the ability of the peptide to enhance tumor growth in vivo. METHODS: The SIKVAV-containing peptide was mixed with Matrigel, a reconstituted basement membrane extract used to assay stimulation of angiogenesis. The mixture was subcutaneously injected into C57BL/6 mice. At various times after injection, the Matrigel plug was excised, and angiogenic activity was assessed by histologic examination and immunohistochemical staining with an antibody to the von Willebrand factor (vWF), an endothelium-specific antigen. In other experiments, the mixture of peptide and Matrigel was co-injected with B16F10 murine melanoma cells into C57BL/6 mice, and the resultant tumors were assessed for size and vascularization. RESULTS: When co-injected with Matrigel at doses as low as 10 micrograms, the SIKVAV-containing peptide stimulated angiogenesis fourfold greater than that seen in controls, and maximum angiogenic activity was observed 2 weeks after injection. This peptide was angiogenic in a dose-dependent manner up to a 100-micrograms dose. When co-injected with Matrigel and B16F10 melanoma cells, the peptide enhanced tumor growth by approximately 2.5-fold, and tumor vascularization was significantly increased (P = .027) over that observed after injection with melanoma cells and Matrigel alone. CONCLUSIONS: These data demonstrate that the laminin-derived SIKVAV-containing peptide is angiogenic in a new in vivo model system and can enhance tumor vascularization and growth.

Amino Acid Sequence↗
PubMed1992

Adenovirus-mediated transfer of a recombinant human alpha 1-antitrypsin cDNA to human endothelial cells.

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.

Adenoviruses, Human↗
PubMed1992

Malignant transformation of NIH-3T3 cells after subcutaneous co-injection with a reconstituted basement membrane (matrigel).

NIH-3T3 cells are non-tumorigenic when injected into athymic mice. If these cells are mixed with an extract of basement-membrane proteins (matrigel) and injected s.c., they form locally invasive and highly vascularized tumors. Cells cultured from the NIH-3T3-matrigel-induced tumors showed a transformed phenotype and lacked contact inhibition. When cultured in a gel of matrigel, they proliferated and formed branched and invasive colonies. In contrast, the parental NIH-3T3 cells cultured on matrigel remained as cell aggregates and were not invasive. I.V. injections of the tumor-derived NIH-3T3 cells produced many colonies on the surface of the lungs, whereas the parental NIH-3T3 cells were not metastatic. Zymographic analysis of the conditioned media obtained from both the tumor-derived and parental NIH-3T3 cells demonstrated higher amounts of the 72-kDa gelatinase (type-IV collagenase) enzyme in the tumor-derived cells. Also, tumor-derived NIH-3T3 cells, but not parental NIH-3T3 cells, secreted the 92-kDa type-IV collagenase. These studies suggest that the interaction of pre-malignant NIH-3T3 cells with extracellular matrix components may contribute to the process of tumor progression.

Animals↗
PubMed1992

Stimulation of human prostatic carcinoma tumor growth in athymic mice and control of migration in culture by extracellular matrix.

The tumorigenicity, migration, growth and invasiveness of certain tumor cells is stimulated by basement membranes. Here we have examined the effect of Matrigel, an extract of basement membrane proteins, on the behavior of several prostate cancer cell lines, testing their growth and invasiveness in vitro and in vivo. Cells of the Tsu-prI line were more invasive than PC-3, Du-145, or LNCaP cells. Peptide inhibitors implicated laminin in the migration and invasion of these cells. When these cells were suspended in Matrigel and injected into nude mice, their growth was greatly enhanced, since large tumors formed in athymic nude mice whereas virtually no tumors were observed in the absence of Matrigel. The growth of a slowly growing line, LNCaP, was increased by exogenous basic fibroblast growth factor when injected with Matrigel. A laminin cell adhesion peptide, YIGSR, was a potent inhibitor of Matrigel-stimulated tumor growth implicating cell-laminin interactions in this process. These results suggest that tumor growth of prostate adenocarcinoma cells may be dependent both on cellular growth factors and on cell-matrix interactions mediated by laminin which facilitate the development of transplanted tumors in nude mice.

Animals↗
PubMed1992

Reconstituted basement membrane (Matrigel) promotes the survival and influences the growth of murine tumors.

The effects of reconstituted basement membrane (Matrigel) on in vivo survival and growth of several murine tumors were studied. Survival of tumor cells was enhanced in all experiments which resulted in increased incidence and/or in increased tumor mass. While basement membrane enhanced the in vivo growth of B16F6 melanoma cells, survival of these mice was prolonged. Basement membrane increased the incidence but reduced the growth of Ehrlich ascites tumor. Walker-256 hypercalcemic breast carcinosarcoma growth was enhanced and glandular-like structures were observed when grown on Matrigel. The results indicate that the enhanced survival of tumor cells in the presence of basement membrane is not unequivocally linked with increased malignancy.

Animals↗
PubMed1992

Interaction of endothelial cells with a laminin A chain peptide (SIKVAV) in vitro and induction of angiogenic behavior in vivo.

Endothelial cells are known to bind to laminin, and two peptides derived from the laminin A (CTFALRGDNP) and B1 (CDPGYIGSR) chains block the capillary-like tube formation on a laminin-rich basement membrane matrix, Matrigel. In the present study, we have used various in vitro and in vivo assays to investigate the angiogenic-biologic effects of a third active site in the laminin A chain, CSRARKQAASIKVAVSADR (designated PA22-2) on endothelial cells. The SIKVAV-containing peptide was as active as the YIGSR-containing peptide for endothelial cell attachment but was less active than either the RGD-containing peptide or intact laminin. Endothelial cells seeded on this peptide appeared fibroblastic with many extended processes, unlike the normal cobblestone morphology observed on tissue culture plastic. In addition, in contrast to normal tube formation on Matrigel, short irregular structures formed, some of which penetrated the matrix and sprouting was more apparent. Analysis of endothelial cell conditioned media of cells cultured in the presence of this peptide indicated degradation of the Matrigel and zymograms demonstrated active collagenase IV (gelatinase) at 68 and 62 Kd. A murine in vivo angiogenesis assay and the chick yolk sac/chorioallantoic membrane assays with the peptide demonstrated increased endothelial cell mobilization, capillary branching, and vessel formation. These data suggest that the -SIKVAV-site may play an important role in initiating branching and formation of new capillaries from the parent vessels, a behavior that is observed in vivo in response to tumor growth or in the normal vascular response to injury.

Amino Acid Sequence↗
PubMed1992

Glandular-like morphogenesis of the human submandibular tumor cell line A253 on basement membrane components.

We have studied the interaction of a human tumor cell line, A253, derived from a submandibular gland carcinoma with a differentiation promoting reconstituted basement membrane extract, Matrigel. When cultured on plastic, these cells maintain a flat, cobblestone, epithelial morphology. On Matrigel, A253 cells initially form a honeycomb network of cords of cells which subsequently thickens. With time, these cords of cells become discontinuous and blunted, whereupon multilobular clusters of cells develop. These clusters possess a lumen with polarized, PAS(+) cells containing numerous desmosomes and an abundance of glycogen. Culture of the cells on laminin, the most abundant protein found in Matrigel, also induces this morphologic differentiation. Using synthetic laminin-derived peptides, the biologically active IKVAV-containing site of laminin was most active in attachment assays, as well as in inhibiting glandular-like morphogenesis when added to the media of cells cultured on Matrigel. Antibodies to the cell surface 67- and 32-kDa laminin binding proteins partially inhibited the glandular-like morphogenesis, suggesting that multiple interactions with laminin are likely required for the differentiation process. Our data demonstrate that A253 cells can undergo glandular-like morphogenesis on basement membrane and that laminin appears to be the major initiating factor.

Blotting, Western↗
PubMed1992

Identification of multiple active growth factors in basement membrane Matrigel suggests caution in interpretation of cellular activity related to extracellular matrix components.

We have recently demonstrated the formation of interconnecting canalicular cell processes in bone cells upon contact with basement membrane components. Here we have determined whether growth factors in the reconstituted basement membrane (Matrigel) were active in influencing the cellular network formation. Various growth factors including transforming growth factor beta (TGF-beta), epidermal growth factor (EGF), insulin-like growth factor 1, bovine fibroblast growth factor (bFGF), and platelet-derived growth factor (PDGF) were identified in Matrigel. Exogenous TGF-beta blocked the cellular network formation. Conversely, addition of TGF-beta 1 neutralizing antibodies to Matrigel stimulated the cellular network formation. bFGF, EGF, and PDGF all promoted cellular migration and organization on Matrigel. Addition of bFGF to MC3T3-E1 cells grown on Matrigel overcame the inhibitory effect of TGF-beta. Some TGF-beta remained bound to type IV collagen purified from the Engelbreth-Holm-Swarm tumor matrix. These data demonstrate that reconstituted basement membrane contains growth factors which influence cellular behavior, suggesting caution in the interpretation of experiments on cellular activity related to Matrigel, collagen type IV, and possibly other extracellular matrix components.

Animals↗
PubMed1992

Protein kinase C regulates endothelial cell tube formation on basement membrane matrix, Matrigel.

Human umbilical vein endothelial cells differentiate within 12 h to form capillary-like networks of tube structures when the cells are plated on Matrigel, a mixture of basement membrane proteins. Nothing is known about the intracellular signaling events involved in this differentiation. As a first step to define the process, we investigated the possible role of protein kinase C activation by beta-phorbol 12-myristate 13-acetate (PMA) in regulating the formation of the tube structures. In this model, PMA increased tube formation several-fold in a dose-dependent manner with half-maximum stimulation of tube formation at approximately 5 nM PMA. In the absence of serum, essentially little or no tubes were formed on Matrigel unless PMA was added to the medium. Only active phorbol analogs increased tube formation, while the protein kinase C inhibitor, H-7, blocked tube formation. The protein kinase C activators and inhibitors were effective only when added at or just after plating of the cells and did not affect already formed tubes. This study suggests that protein kinase C is involved in the early events of in vitro endothelial cell tube formation on Matrigel.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1992

Functional domains of cell adhesion molecules.

A number of molecules involved in cell adhesion (e.g. fibronectin, laminin, collagens I and IV, thrombospondin, entactin) have now been identified and the consequent roles that they play in the processes of growth, migration, differentiation and tumor spread have been described. Active sequences of the molecules have been identified using synthetic peptides derived from specific domains. Several adhesive molecules contain multiple active domains with different biological activities.

Amino Acid Sequence↗
PubMed1992

Recombinant human bone morphogenetic protein 2B stimulates PC12 cell differentiation: potentiation and binding to type IV collagen.

Bone morphogenetic protein 2B (BMP 2B, also known as BMP 4) induces cartilage and bone morphogenesis in ectopic extraskeletal sites. BMP 2B is one of several bone morphogenetic proteins which along with activins and inhibins are members of the transforming growth factor-beta (TGF-beta) family. Both BMP 2B and activin A, but not TGF-beta 1, induce rat pheochromocytoma PC12 neuronal cell differentiation and expression of VGF, a nervous system-specific mRNA. PC12 cells exhibited approximately 2,500 receptors per cell for BMP 2B with an apparent dissociation constant of 19 pM. Extracellular matrix components, including fibronectin, laminin, and collagen type IV potentiated the activity of BMP and activin A, with the latter being the most active. Direct experiments demonstrated that radioiodinated BMP 2B bound to collagen type IV better than to either laminin or fibronectin. These data demonstrate a common neurotrophic activity of both BMP 2B and activin A, and suggest that these regulatory molecules alone and in conjunction with extracellular matrix components may play a role in both the development and repair of nervous tissue.

Activins↗
PubMed1992

Alzheimer disease brain extract stimulates branching of laminin-mediated neuronal processes.

Patients with Alzheimer disease (AD) suffer mental deterioration associated with neurofibrillary tangle and senile plaque formation in the brain. Here we have determined the effects of brain extracts from normal and from AD patients on neuronal process formation by a pheochromocytoma (PC-12) and a neuroblastoma x glioma hybrid cell line (NG108-15). PC12 cells show a dose-related stimulation of branching of neuronal processes by AD brain extracts with cells cultured on a laminin substrate. The neurotrophic effects of extracts of AD brains may be related to the abnormal sprouting and neurofibrillary tangle formation observed in the brain in this disorder.

Adrenal Gland Neoplasms↗
PubMed1992

Laminin enhances binding of Toxoplasma gondii tachyzoites to J774 murine macrophage cells.

We investigated the effects of the extracellular matrix proteins laminin and fibronectin on the attachment of tachyzoites of Toxoplasma gondii to the murine macrophage cell line J774. Laminin but not fibronectin increased parasite attachment to J774 cells in a dose-dependent fashion. Cyclic YIGSR, a laminin-derived peptide which inhibits laminin binding to the 32/67-kDa laminin-binding protein on host cells, blocked the laminin-mediated enhancement of parasite attachment. An antiserum to the 32/67-kDa laminin-binding protein also inhibited binding of parasites to J774 cells. These results, in conjunction with our previous observations (G. C. Furtado, F. L. Collins, and K. A. Joiner, submitted for publication), demonstrate that tachyzoites bearing surface laminin bind to multiple laminin receptors in attaching to different target cells.

Animals↗
PubMed1992

Induction of an invasive phenotype in benign tumor cells with a laminin A-chain synthetic peptide.

K-1735 clones 10 and M2 are cell lines cloned from a UV-induced murine melanoma. While both lines are highly tumorigenic, only the M2 cells are highly invasive in vitro and metastatic in vivo. Here we have exposed the clone 10 cells to the synthetic peptide PA22-2, which contains the IKVAV sequence from the A chain of laminin and which, like laminin, induces collagenase IV production and enhances metastasis formation by B16F10 cells. Zymogram analysis of conditioned media from clone 10 cells cultured on the peptide demonstrated a dose-dependent increase in collagenase IV activity. When clone 10 cells were cultured on a reconstituted basement membrane (Matrigel), this peptide caused an invasive phenotype comparable to the M2 cells. The invasive clone 10 cells were, however, unable to form lung colonies in vivo in the presence of this peptide. We conclude that this peptide represents an active site on laminin which is able to stimulate the invasiveness of this tumor cell line, but that this activity is not sufficient to confer metastatic potential.

Amino Acid Sequence↗
PubMed1991

Identification of a 110-kDa nonintegrin cell surface laminin-binding protein which recognizes an A chain neurite-promoting peptide.

Laminin is a potent promoter of neurite outgrowth, and a synthetic peptide of 19 amino acids, PA22-2, from the A chain has been found to promote process formation. Using peptide affinity chromatography, we have identified a 110-kDa, cell surface ligand from both neural cells and brain which binds this sequence. This binding protein does not share immunological identity with the B1 chain of integrin, and reduction does not alter its mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antibody to the 110-kDa protein stained cellular processes in vivo. Sequence analysis of the first 18 amino acids from the amino terminus yielded almost exact sequence identity with nucleolin, a major 110-kDa nucleolar phosphoprotein. Antibody to nucleolin, however, does not interact with the neural-derived, laminin-peptide-binding 110-kDa protein. The 110-kDa protein appears to be a ligand for a specific site on laminin.

Amino Acid Sequence↗
PubMed1991

Distinct immunoreactivity to 110 kDa laminin-binding protein in adult and lesioned rat forebrain.

A phosphorylated, approximately 110 kDa laminin-binding protein (110 kDa LBP) from mouse brain has been previously identified. This protein recognizes a neurite-outgrowth promoting 19-amino acid synthetic peptide (PA 22-2) derived from the laminin A chain. In the present study, an antibody against the 110 kDa LBP was used to localize immunoreactivity in the normal adult rat brain and also following a stab wound and ischemic lesion. Immunoreactive cells were found in layers II/III and V of the cerebral cortex and within apical dendrites of pyramidal neurons. Specific immunoreactivity was also found in the stratum lucidum in the CA3 region of the hippocampus which exhibited densely stained mossy fibers and terminals. Mechanical and ischemic lesions induced intense immunolabeling of reactive glial cells around the lesion site. The distinct and anatomically restricted localization of the immunostain in adult and lesioned rat brain suggests that 110 kDa LBP-like molecules might have an important function in forebrain structures and may be involved in the response to CNS injury.

Animals↗