Biomedical subjects

H K Kleinman

Publications and source records attributed to H K Kleinman.

At least 163 records · Page 9Linked to original sources

PubMed1991

Differential effects of interferon gamma and alpha on in vitro model of angiogenesis.

The formation of blood vessels in vivo (angiogenesis) is an important process and is usually initiated in response to injury, tumor growth, or normal tissue development. We have studied the effect of human interferon (IFN) alpha (alpha) and gamma (gamma) on the capillary-like network formation in an in vitro model of angiogenesis using human umbilical vein endothelial cells (HUVEC). When HUVEC cells are plated on Matrigel (reconstituted basement membrane matrix enriched in laminin), a network of capillary like structures (endotubes) rapidly forms. IFN-alpha enhanced the tube formation in a dose-dependent manner, whereas IFN-gamma significantly inhibited the tube formation. In addition, both the enhancement and inhibition of angiogenesis by IFN-alpha and gamma was found to be greater if the cells were pretreated with IFN for 12 hr before plating on the Matrigel. These results suggest that IFN may play an important role in several vascular processes including early stages of wound healing, recanalization of thrombi, tumor growth, metastasis, normal growth, and development.

Endothelium, Vascular↗

PubMed1991

The RGD containing site of the mouse laminin A chain is active for cell attachment, spreading, migration and neurite outgrowth.

The laminin A chain has been sequenced by cDNA cloning and was found to contain an RGD sequence. Synthetic peptides containing the RGD sequence and flanking amino acids were active in mediating cell adhesion, spreading, migration, and neurite outgrowth. Furthermore, endothelial cell attachment to a laminin substrate was inhibited by an RGD-containing synthetic peptide. Antisera against the integrin (fibronectin) receptor, and monoclonal antibody to the integrin, VLA-6, inhibited cell interaction with laminin, as well as with peptides containing an RGD sequence. These results suggest that the RGD containing site of laminin is active and interacts with the integrin family of receptors in certain cells.

Amino Acid Sequence↗

PubMed1991

cAMP promotes branching of laminin-induced neuronal processes.

Laminin is a potent stimulator of neurite outgrowth. We have examined the signal transduction events involved in the neuronal cell response to laminin. Cyclic nucleotides, calcium, and sodium-proton exchange do not appear to be required for the transduction of the laminin signal during neurite outgrowth. Direct measurement of cAMP and cGMP levels shows no changes in NG108-15 cells when cultured on laminin. Exogenous cAMP alone had no effect on either the rate of process formation or process length, but did alter the morphology of laminin-induced neurites. A four-fold increase in the number of branches per neurite and a two-to-three-fold increase in the number of neurites per cell were observed in both NG108-15 and PC12 cells cultured on laminin when either 8-BrcAMP or forskolin was added. The cAMP-induced branching was also observed when PC12 cells were cultured on a laminin-derived synthetic peptide (PA22-2), which contains the neurite-promoting amino acid sequence IKVAV. By immunofluorescence analysis with axonal or dendritic markers, the PC12 processes on laminin and PA22-2 were axonal, not dendritic, and the cAMP-induced morphological changes were due to axonal branching. These data demonstrate that changes in cAMP are not involved in laminin-mediated neurite outgrowth, but cAMP can modulate the effects of laminin.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1991

Fetal rat septal cells adhere to and extend processes on basement membrane, laminin, and a synthetic peptide from the laminin A chain sequence.

Responses of rat embryonic septal cells to reconstituted basement membrane, laminin, and laminin A chain-derived synthetic peptides were studied in culture. Dissociated fetal E16/17 septal cells were grown for three days on differently coated plastic substrata. Reconstituted basement membrane (Matrigel), laminin, and a 19-amino acid synthetic peptide CSRARKQAASIKVAVSADR-NH2 (PA22-2) from the laminin A chain sequence mediated cell-substratum adhesion and promoted neurite outgrowth. In contrast, cells did not attach to or form processes on uncoated plastic or on plastic substrata coated with synthetic, laminin-derived control peptides. Polyethylenimine (PEI) supported the adhesion and survival of fetal septal cells; however, when laminin was added to the medium during cell plating or 18 hr afterward, a dose-dependent increase was observed in neurite outgrowth of cells attached to this substratum. Cells grown for 6 days on PEI in the presence of laminin showed a determined increase in the number of cholinergic neurons as marked by acetylcholinesterase staining. These data suggest that the subpopulation of cholinergic septal neurons present in the septal cells studied here were also responding to laminin. The results of this in vitro study suggest potential uses for basement membrane, laminin, or synthetic peptides, such as PA22-2, in fetal septal grafts to enhance regeneration in the damaged septo-hippocampal system.

Acetylcholinesterase↗

PubMed1991

Basement membrane and the SIKVAV laminin-derived peptide promote tumor growth and metastases.

Laminin, the major glycoprotein component of basement membrane, promotes the malignant phenotype. Cells which are adherent to laminin are more malignant than the non-adherent cells and in certain tumor cells, the number of laminin receptors is positively correlated with malignancy. Laminin also increases collagenase IV activity, an enzyme demonstrated to be critical for tumor spread. A site on laminin, containing the amino acid sequence SIKVAV, has been identified which when injected intravenously with B16F10 melanoma cells, causes an increase in the number of colonies on the surface of the lungs. This peptide does not affect tumor cell arrest in the vasculature or the immune system. It does promote angiogenesis in various in vitro and in vivo models, thereby facilitating tumor cell survival. When a complex mixture of laminin-enriched basement membrane components (Matrigel) is coinjected with tumor cells subcutaneously, tumor incidence and growth increases. Various tumor cell lines and primary isolates, which previously could not form tumors in mice, can be induced to grow rapidly in the presence of Matrigel. Slowly growing tumors or arrested tumors can also be induced to grow more quickly with additional injections of Matrigel. When an SIKVAV-containing synthetic peptide is coinjected with B16F10 tumor cells and Matrigel subcutaneously in mice, larger tumors are formed than that observed with either Matrigel or cells alone. Such studies define the role of laminin in tumor growth and spread and generate new models for studying therapeutic agents. Of particular interest is the ability to grow primary isolates which generally do not grow in mice.

Amino Acid Sequence↗

PubMed1991

Intracellular mechanisms involved in basement membrane induced blood vessel differentiation in vitro.

The extracellular matrix, particularly basement membranes, plays an important role in angiogenesis (blood vessel formation). Previous work has demonstrated that a basement membranelike substrate (Matrigel) induces human umbilical vein endothelial cells to rapidly form vessel-like tubes (Kubota, et al., 1988; Grant et al., 1989b); however, the precise mechanism of tube formation is unclear. Using this in vitro model, we have investigated morphologic changes occurring during tube formation and the cytoskeletal and protein synthesis requirements of this process. Electron microscopy showed that endothelial cells attach to the Matrigel surface, align, and form cylindrical structures that contain a lumen and polarized cytoplasmic organelles. The cytoskeleton is reorganized into bundles of actin filaments oriented along the axis of the tubes and is located at the periphery of the cells. The addition of colchicine or cytochalasin D blocked tube formation, indicating that both microfilaments and microtubules are involved in this process. Cycloheximide blocked tube formation by 100%, indicating that the process also required protein synthesis. In particular, collagen synthesis seems to be required for tube formation because cis-hydroxyproline inhibited tube formation, whereas either the presence of ascorbic acid or the addition of exogenous collagen IV to the Matrigel increased tube formation. Our results indicate that endothelial cell attachment to Matrigel induces the reorganization of the cytoskeleton and elicits the synthesis of specific proteins required for the differentiated phenotype of the cells.

Ascorbic Acid↗

PubMed1991

Identification and partial characterization of laminin binding proteins in immature rat Sertoli cells.

Laminin, a major component of basement membrane extracellular matrices, promotes differentiation in a number of cell types, including Sertoli cells. We have identified and characterized Sertoli cells. We have identified and characterized Sertoli cell surface molecules which interact with laminin. Using laminin-Sepharose affinity chromatography and [125I]laminin binding to Sertoli cell plasma membranes, binding proteins have been identified with the Mr 110,000, 67,000, 55,000, 45,000, 36,000, and 25,000. In addition, the Mr 110,000 and 67,000 laminin binding proteins were phosphorylated. The 67,000, 45,000, and 36,000 react with antibodies to the previously characterized laminin receptor and these antibodies stain the basolateral surface of Sertoli cells in vivo. Cultured Sertoli cells stain for laminin receptor both on the cell surface and within the cells. Antiserum to the 32,000 and 67,000 laminin binding proteins partially inhibited spreading of Sertoli cells on a laminin-coated culture dish, suggesting a functional importance of those proteins in Sertoli cell differentiation. The 25,000 and 45,000 laminin binding proteins reacted with integrin antibodies, but no high-molecular-weight forms could be detected. Integrin was localized to the cell surface and intracellularly but antibodies did not block Sertoli cell spreading on laminin. This work represents the first identification and characterization of extracellular matrix binding proteins in an endocrine organ and suggests an important role for the nonintegrin 32/67 laminin binding proteins.

Amino Acid Sequence↗

PubMed1991

HIV-1 infection of human T lymphocytes results in enhanced alpha 5 beta 1 integrin expression.

Altered T cell adherence after human immunodeficiency virus 1 (HIV-1) infection may contribute to viral pathogenesis in the acquired immune deficiency syndrome. To address this hypothesis, we assessed mechanisms of T cell adherence to extracellular matrix proteins in vitro. We found that after HIV-1 infection, both chronically infected H9 CD4+ T cells and acutely infected primary peripheral blood lymphocytes acquired the ability to adhere to the extracellular matrix glycoprotein fibronectin, to a lesser extent to type IV collagen and laminin, but not to type I collagen. H9 cells chronically infected with two of the three HIV-1 strains studied showed approximately a sevenfold increase in attachment to fibronectin, while the same cells infected with the human retrovirus HIV-2 did not. Adhesion was accompanied by changes in morphology, including marked spreading and increased filopodia. These alterations were not blocked by the protein kinase C inhibitor H-7, which did inhibit TPA-induced T cell attachment to fibronectin. Monoclonal antibodies against both the alpha 5 and the beta 1 subunits of the classical fibronectin receptor as well as an Arg-Gly-Asp (RGD) peptide inhibited attachment, whereas anti-alpha 4 monoclonal antibodies and the CS1 peptide did not. Binding to collagen IV was also inhibited by the anti-beta 1 monoclonal antibody, but not the other antibodies. Cells metabolically labeled with [35S]methionine and analyzed by immunoprecipitation with polyclonal anti-beta 1 integrin antibody showed a 2.5-fold increase in integrin synthesis in infected cells compared to uninfected controls. This increase in synthesis was associated with an increase in cell surface expression of both alpha 5 and beta 1 integrins by FACS (registered trademark of Becton Dickinson for a fluorescence-activated cell sorter) analysis. Enhanced expression of integrins such as alpha 5 beta 1 may cause T cell adherence to a variety of tissues, where released viral gene products may induce some of the tissue-specific manifestations of HIV-1 infection.

Amino Acid Sequence↗

PubMed1991

Adult and fetal human mesangial cells interact with specific laminin domains.

Mesangial cells are centrally located pericytes in the renal glomerulus. They are surrounded by an extracellular matrix and directly contact the glomerular basement membrane in vivo. Because these interactions are critical for renal development and function, we have studied human mesangial cell interactions with laminin, a major adhesive component of basement membranes present in the extracellular matrix of the mesangium. Human fetal and adult mesangial cell attachment was stimulated by both laminin and the laminin-derived synthetic peptides YIGSR-NH2, CQAGTFALRGDNPQG-NH2, and CIKVAVS-NH2. Furthermore, mesangial cells spread on laminin as well as on both the RGD-containing and CIKVAVS peptides. When added in solution, all three peptides inhibited mesangial cell attachment to laminin, and the latter two peptides inhibited mesangial cell spreading on laminin. Laminin affinity column chromatography demonstrated several low-molecular-mass laminin-binding proteins ranging from between 35 and 42 kDa, which predominated in fetal mesangial cells, whereas a higher molecular mass laminin-binding protein of 65 kDa was predominant in adult mesangial cells. Western blot analysis with an anti-32-kDa laminin-binding protein antibody showed increased expression of both 31- and 42-kDa proteins in fetal mesangial cells when compared with the adult. The antisera to the 32-kDa laminin-binding protein also inhibited fetal mesangial spreading on the CIKVAVS peptide. Western blot analysis with an anti-67-kDa laminin-binding protein antibody revealed a 110-kDa protein in adult mesangial cells that was not present in fetal mesangial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1991

Matrix proteins induce neuroblastoma cell differentiation without altering cell growth.

The maturation of embryonal neural crest cells is thought to be regulated in part by the milieu into which these cells migrate. Neuroblastoma (NB) is a tumor of very early childhood that is thought to arise in association with the arrested differentiation of embryonal neural crest cells. In culture, neuroblastoma tumor cells differentiate in the presence of retinoic acid, which is also known to influence extracellular matrix protein synthesis. We have cultured neuroblastoma cells on laminin (LN) and fibronectin (FN) substrata to examine the role of extracellular matrix in retinoic acid (RA)-induced differentiation of these tumor cells. These proteins caused morphologic changes in NB cells indistinguishable from those caused by RA. Antiserum to each of these proteins blocked the effects induced by the corresponding protein, but neither antiserum affected the action of RA. Despite the induction of a neuronal morphologic change, matrix proteins did not alter the proliferation of NB cells. These results indicate that LN and FN modulate the differentiation of NB cells without inducing growth arrest and that RA-induced differentiation does not require these matrix proteins.

Blotting, Northern↗

PubMed1991

High-affinity binding of interferon-gamma to a basement membrane complex (matrigel).

Recently it was demonstrated that growth factors are bound to the extracellular matrix, and can regulate cell behavior. Using three different types of binding assays, we have examined the interaction of interferon-gamma with a basement membrane produced by the Engelbreth-Holm-Swarm tumor. Basement membrane was found to bind interferon-gamma in both a time- and concentration-dependent manner. Equilibrium binding analysis revealed a high-affinity site with a dissociation constant of 1.5 10(-9) M and a maximum binding capacity of 1.6 10(9) sites/mm2 of basement membrane. Competition studies show that the binding is inhibited by heparan sulfate, suggesting that basement membrane-heparan sulfate proteoglycan could be the binding site. This interaction was clearly confirmed by native polyacrylamide gel electrophoresis and dot-blot analysis with purified basement membrane molecules. Furthermore, the carboxy-terminal part of the interferon-gamma molecule contains an amino acid cluster, very closely related to a consensus sequence, present in more than 20 proteins known to bind sulfated glycosaminoglycans such as heparin. These data demonstrate a possible role of extracellular matrix components in storing cytokines and in modulating the cellular response to such factors.

Antibodies, Monoclonal↗

PubMed1991

Basement membrane increases G-protein levels and follicle-stimulating hormone responsiveness of Sertoli cell adenylyl cyclase activity.

On a basement membrane substrate, Sertoli cells in culture have been shown to assume a phenotype similar to that of the in vivo differentiated cells. Sertoli cells from 10-day-old rats were cultured on plastic and on different extracellular matrix substrates [laminin, a reconstituted basement membrane (Matrigel), and a synthetic laminin peptide containing the arginine-glycine-aspartic acid (RGD) tripeptide sequence] to investigate the effects of the extracellular matrix on FSH responsiveness. Both laminin and Matrigel markedly enhanced the cAMP response to FSH and cholera toxin, indicating modifications at the level of guanine nucleotide-binding regulatory (G) proteins. Furthermore, Sertoli cell grown on either of these two substrates responded to physiological levels of FSH (25-50 ng/ml), whereas pharmacological levels of FSH (500 ng/ml) were required for cells grown on either plastic or on the RGD-containing laminin peptide. Immunoblotting of Sertoli cell plasma membranes with antibodies directed against the alpha-subunit of the stimulatory G-protein (Gs alpha) of adenylyl cyclase indicated that Sertoli cell culture on either laminin or Matrigel increased the amounts of Gs alpha. These results were further confirmed by immunoprecipitating the Gs alpha protein from the particulate fraction of [35S]methionine metabolically labeled Sertoli cells. However, Northern blot analysis using a cDNA probe for Gs alpha did not demonstrate changes in gene expression when Sertoli cells were grown on the various substrates. Immunofluorescent studies revealed that the Gs complex of adenylyl cyclase was preferentially located at the base of the Sertoli cells at the site of contact with the extracellular matrix. These data suggest that culture of epithelial Sertoli cells on basement membrane substrates enhances the Gs complex of adenylyl cyclase and the cAMP response to FSH, consistent with the more differentiated morphology and function of the cells.

Adenylyl Cyclases↗

PubMed1991

Identification of an amino acid sequence in the laminin A chain mediating mast cell attachment and spreading.

PT18 mast cells and mouse bone marrow-derived mast cells have been shown to adhere and spread when in contact with a laminin substratum. Mouse bone marrow cells, however, first require activation with phorbol myristate acetate (PMA), ionophore, or antigen-specific IgE with antigen in order to exhibit these phenomena. Here, we have studied the interaction of these cells with three active synthetic peptides derived from different domains of laminin. PT18 cells and mouse bone marrow mast cells attached and spread on the 19 amino acid synthetic laminin A chain-derived peptide PA22-2, containing the active five amino acid sequence IKVAV, and this attachment did not require prior activation of the mouse bone marrow mast cells with PMA or IgE plus antigen. These cells did not adhere to the B1 chain peptide YIGSR-NH2 or the RGD-containing peptide from the A chain. PT18 cell adherence to laminin was inhibited by soluble peptide PA22-2, but not by either YIGSR-NH2, the RGD-containing, or control peptides. Antisera to the PA22-2 peptide completely abolished adherence to PA22-2, but only partially inhibited mast cell adherence to laminin. Antibody to the 67,000-32,000 MW laminin-binding protein receptor blocked cell adhesion to laminin and to the active A chain peptide. Thus, mast cell adhesion and spreading on laminin may be mediated by an interaction with the IKVAV sequence on the laminin A chain.

Amino Acid Sequence↗

PubMed1990

Differentiation of canalicular cell processes in bone cells by basement membrane matrix components: regulation by discrete domains of laminin.

We have investigated the interaction of rat primary calvarial bone cells and a mouse osteoblast-like cell line MC3T3-E1 with basement membrane components. On a reconstituted gel of basement membrane, both cell types attached and formed isolated clusters that developed long interconnecting cell processes similar to the canalicular network observed in bone. The differentiation of the osteoblastic phenotype was stimulated as determined by increased alkaline phosphatase production and the deposition of mineral. Antibodies to laminin and to a 32/67 kd laminin receptor blocked this differentiation. Cell morphology was altered by the addition of active laminin-derived synthetic peptides, YIGSR-NH2 and CSRARKQAASIKVAVSADR-NH2, but not by an active RGD-containing peptide. When coated directly on plastic, all three peptides promoted cell adhesion, demonstrating that bone cells interact with specific molecular domains of laminin. These data demonstrate that basement membrane plays a key role in formation of a network of cytoplasmic processes resembling the osteocyte canalicular network in bone.

Amino Acid Sequence↗

PubMed1990

Interaction of osteogenin, a heparin binding bone morphogenetic protein, with type IV collagen.

Osteogenin, an extracellular matrix component of bone, is a heparin binding differentiation factor that initiates endochondral bone formation in rats when implanted subcutaneously with an insoluble collagenous matrix. We have examined the interaction of osteogenin with various extracellular matrix components including basement membranes. Osteogenin, purified from bovine bone, binds avidly to type IV collagen and to a lesser extent to both type I and IX collagens. Osteogenin binds equally well to both native and denatured type IV collagen. Both alpha 1 and alpha 2 chains of type IV collagen are recognized by osteogenin. Osteogenin binds to a collagen IV affinity column, and is eluted by 6.0 M urea with 1 M NaCl, pH 7.4, and the eluate contained the osteogenic activity as demonstrated in vivo. Binding of osteogenin to collagen IV is not influenced by either laminin or fibronectin. These results imply that osteogenin binding to extracellular matrix components including collagens I and IV and heparin may have physiological relevance, and such interactions may modulate its local action.

Animals↗

PubMed1990

Laminin-mediated process formation in neuronal cells involves protein dephosphorylation.

Laminin mediates neural adhesion and process formation. A possible signal transduction pathway for laminin was investigated in both NG108-15 and PC12 neuronal cells using radiolabeling studies as well as various stimulators and inhibitors of phosphatases and kinases. Using [32P]-ortho-phosphate, laminin caused a decrease in the TCA-precipitable counts. Further, laminin stimulated dephosphorylation of laminin binding proteins of 110 kDa, 67 kDa, and 45 kDa and this dephosphorylation was blocked by the phosphatase inhibitor, okadaic acid, and the protein kinase C stimulator, TPA. The phosphatase inhibitors okadaic acid and vanadate, as well as the protein kinase C stimulators, TPA and DAG, blocked laminin-mediated process formation. Inhibitors of kinase activity such as H-7, H-8, and H-9 increased laminin-mediated neural process formation. Since phosphate incorporation into laminin-binding proteins is decreased by laminin and because both phosphatase inhibitors and kinase stimulators inhibit laminin-mediated process formation, we conclude that dephosphorylation events promote the neural cell response to laminin.

Adrenal Gland Neoplasms↗

PubMed1990

Laminin promotes formation of cord-like structures by Sertoli cells in vitro.

Basement membranes are thin extracellular matrices which contact epithelial cells and promote their adhesion, migration, differentiation, and morphogenesis. These matrices are composed of collagen IV, heparan sulfate proteoglycan, laminin, and entactin as well as other minor components. Sertoli cells, like most epithelial cells, are in contact at their basal surface with a basement membrane. When cultured within three-dimensional basement membrane gels (Matrigel), Sertoli cells reorganize into cords that resemble testicular seminiferous cords found in the in vivo differentiating testis. Anti-laminin and anti-entactin antisera inhibit this cord morphogenesis by Sertoli cells whereas antisera against type IV and type I collagen, heparan sulfate proteoglycan, fibronectin, and preimmune sera had no effect. The RGD (RGDS-NH2) sequence, found in the cell binding domain of the integrin family of cell adhesion molecules as well as in the A chain of laminin and in entactin, effectively inhibited Sertoli cell cord formation at a concentration of 1.0 mg/ml but was unable to prevent Sertoli cell attachment at concentrations as high as 2.0 mg/ml. A synthetic pentapeptide from a cell-binding domain of the B1 chain of laminin. YIGSR-NH2, inhibited cord formation at a concentration of 0.25 mg/ml, but Sertoli cells were still adherent to the basement membrane matrix. At concentrations greater than 0.50 mg/ml, Sertoli cells detached. Antiserum against the YIGSR-NH2-containing sequence was also effective in inhibiting cord formation by Sertoli cells. Ligand (YIGSR-NH2 peptide) blot analysis of Sertoli cell lysates revealed an interaction with a major band at 60 kDa and with minor bands at 39 and 127 kDa. Furthermore, in Western blot analysis the anti-67-kDa laminin-binding protein antibody recognized a 59- to 60-kDa protein in Sertoli cells. The data indicate that laminin is involved in both Sertoli cell attachment and migration during formation of histotypic cord structures by these cells in culture. Two separate laminin cell-binding domains appear to be involved in Sertoli cell cord morphogenesis in vitro and are likely to participate in the formation of seminiferous cords in vivo.

Amino Acid Sequence↗

PubMed1990

Altered basement membrane protein biosynthesis by primary cultures of cpk/cpk mouse kidney.

Previously, kidneys from three-week-old cpk/cpk C57/B16 mice were found to contain elevated mRNA levels for the basement membrane components collagen IV and laminin [1]. Here primary cultures of kidney epithelial cells derived from cpk/cpk C57/B16 mice were established and the production of these proteins in culture was studied. Primary cultures of cpk/cpk mouse kidney epithelial cells were observed to have a more polygonal, flattened morphology than cells from unaffected littermate kidneys. The rate of collagen IV and laminin biosynthesis was determined by means of [35S] labelling studies followed by immunoprecipitation. Collagen IV and laminin biosynthesis are elevated by approximately twofold or more in primary cultures derived from 20-day-old cpk/cpk mice, as compared with parallel primary cultures derived from their unaffected littermates. Similarly, laminin B1 chain mRNA is elevated in primary cultures derived from 20-day-old cpk/cpk mice. In primary cultures derived from younger (day 11) mice, similar differences in the rates of both collagen and laminin biosynthesis were not observed between the two culture types. These observations are consistent with the previously reported age-dependent differences observed in laminin and in collagen IV gene expression in both cpk/cpk and wild-type mouse kidneys, and suggest that the regulation of overproduction of these proteins is due to an alteration in the kidney cells and not due to systemic factors.

Animals↗