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PubMed2006

The serpin protease nexin-1 regulates vascular smooth muscle cell adhesion, spreading, migration and response to thrombin.

BACKGROUND: Protease nexin-1 (PN-1) is an important physiological regulator of thrombin in the brain. PN-1 is also present in aortic smooth muscle cells and may thus participate in vascular biology. However, little is known about its function in the vessel wall. OBJECTIVES: In this study, we investigated the effect of PN-1 overexpression in smooth muscle cells (SMCs), on their sensitivity to thrombin, and their capacity for adhesion, spreading and migration. RESULTS: Two clones exhibiting a two- to threefold increase in PN-1 expression were selected and compared with untransfected and mock-transfected cells. Overexpression of PN-1 was observed to inhibit thrombin-induced cell responses as indicated by a twofold decrease in induction of PAI-1 expression, a decreased calcium mobilization in response to low thrombin concentrations and a twofold increase in the capacity to inhibit thrombin catalytic activity. Overexpression of PN-1 did not modify adhesion, spreading, and migration of SMCs on type I collagen. In contrast, SMCs overexpressing PN-1 exhibited a 40% reduction in adhesion, a 50% reduction in spreading and a complete absence of migration on vitronectin when compared with control SMCs. CONCLUSIONS: Our studies thus reveal that PN-1 is likely to play a critical role in regulating essential cell functions such as (i) thrombin-induced responses, which are dependent on its antiprotease activity, and (ii) adhesion, spreading, and migration, which are independent of its antiprotease activity and may be related to its interaction with other partners, such as vitronectin in the present case.

Amyloid beta-Protein Precursor↗
PubMed1995

Interaction of osteopontin with osteoclast integrins.

Mammalian osteoclasts express three integrin receptors--alpha v beta 3 (vitronectin receptor), alpha 2 beta 1 and alpha v beta 1. The vitronectin receptor recognizes bone matrix proteins, including bone sialoproteins, in an RGD-dependent manner, whereas adhesion to collagen involves beta 1 integrins. Interference with integrin function, by anti-receptor antibodies or RGD-peptides, blocks bone resorption. Data on the mechanism of osteoclast adhesion to sialoproteins and the differential synthesis of osteopontin and bone sialoprotein by osteoclasts is presented. Thus, osteoclasts adhere to both osteopontin and bone sialoprotein with a characteristic irregular morphology with numerous, peripherally placed, actin-rich podosomes. Adhesion is predominantly RGD and beta 3 dependent, though alpha v beta 1 may also be involved in adhesion to bone sialoprotein. KQAGD and AGDV, but not H12, fibrinogen peptides induce osteoclast 'rounding' on osteopontin suggesting there is an alternative anti-adhesive signal to 'RGD.' However, adhesion is not completely inhibited and is not specific for osteopontin as equivalent effects are seen with adhesion to serum. The role of sialoproteins in osteoclast adhesion in situ in the skeleton is complicated by the finding of endogenous synthesis of osteopontin, but not bone sialoprotein, by osteoclasts. The disposition of osteoclast integrins during resorption and the role of integrins and sialoprotein-derived peptides in osteoclast adhesion and function is also reviewed.

Amino Acid Sequence↗
PubMed1986

Platelet membrane glycoprotein IIb/IIIa: member of a family of Arg-Gly-Asp--specific adhesion receptors.

Adhesive interactions of the platelet surface with plasma proteins such as fibrinogen and fibronectin play an important role in thrombosis and hemostasis. The binding of both of these proteins to platelets is inhibited by synthetic peptides containing the sequence Arg-Gly-Asp, which corresponds to the cell adhesion site in fibronectin and is also present in the alpha chain of fibrinogen. An affinity matrix made of an insolubilized heptapeptide containing the Arg-Gly-Asp sequence selectively binds the platelet membrane glycoprotein IIb/IIIa from detergent extracts of platelets. When incorporated into liposome membranes, the isolated protein confers to the liposomes the ability to bind to surfaces coated with fibrinogen, fibronectin, and vitronectin but not to surfaces coated with thrombospondin or albumin. This platelet receptor is related to the previously identified fibronectin and vitronectin receptors in that it recognizes an Arg-Gly-Asp sequence but differs from the other receptors in its wider specificity toward various adhesive proteins. These results establish the existence of a family of adhesion receptors that recognize the sequence Arg-Gly-Asp.

Amino Acid Sequence↗
PubMed1993

Mycobacterium avium-M. intracellulare binds to the integrin receptor alpha v beta 3 on human monocytes and monocyte-derived macrophages.

Mycobacterium avium-M. intracellulare is an intracellular pathogen responsible for the highest incidence of disseminated bacterial infection in patients with AIDS. Treatment of the infection is difficult and has been of limited efficacy. Attachment of the organism to macrophages is a critical early step in the establishment of the disease. In the present study, we isolated and identified a receptor that mediates attachment of M. avium-M. intracellulare to human peripheral blood monocytes and monocyte-derived macrophages. On Western blotting, (immunoblotting), the receptor was found to cross-react with antibodies against a human vitronectin receptor (alpha v beta 3). The receptor could be purified from monocyte extracts by using monoclonal antibodies (MAbs) against the alpha v subunit of vitronectin receptor coupled to CNBr-Sepharose 4B, as well as with the adhesive tripeptide sequence arginine-glycine-aspartic acid (RGD) coupled to CNBr-Sepharose 4B. Surface-bound MAbs directed against alpha v beta 3 were found to inhibit the attachment of M. avium-M. intracellulare to monocyte-derived macrophages in an in vitro inhibition assay, while MAbs directed against CD14, CD18, alpha 2 beta 1 and platelet glycoprotein gpIIb/IIIa receptors did not inhibit this attachment. These observations suggest that alpha v beta 3 on the surface of human monocytes and monocyte-derived macrophages may function as a receptor for M. avium-M. intracellulare. Identification of a receptor for M. avium-M. intracellulare on macrophages may offer new approaches to the prevention and control of M. avium-M. intracellulare infection at the cellular level.

Amino Acid Sequence↗
PubMed2003

Lung epithelial cells and extracellular matrix components induce expression of Pneumocystis carinii STE20, a gene complementing the mating and pseudohyphal growth defects of STE20 mutant yeast.

Pneumocystis carinii causes severe pneumonia in immunocompromised hosts. The binding of P. carinii to alveolar epithelial cells and extracellular matrix constituents such as fibronectin and vitronectin is a central feature of infection, which initiates proliferation of the organism. Herein, we demonstrate that P. carinii binding to lung cells specifically alters the gene expression of the organism, regulating fungal growth. Subtractive hybridization was performed to isolate P. carinii genes expressed following binding to mammalian extracellular matrix constituents. P. carinii STE20 (PCSTE20), a gene participating in mating and pseudohyphal growth of other fungi, was identified following adherence to the extracellular matrix constituents fibronectin, vitronectin, collagen, and lung epithelial cells. The expression of PCSTE20 and a related P. carinii mitogen-activated protein kinase (MAPK) kinase gene, also implicated in signaling of mating, were both specifically upregulated by binding to matrix protein. The expression of general cyclin-dependent kinases and other MAPKs not involved in mating pathways were not altered by organism binding. PCSTE20 expression was also strongly enhanced following organism attachment to A549 lung epithelial cells. When expressed in a Saccharomyces cerevisiae ste20Delta mutant, PCSTE20 suppressed defects in both mating and pseudohyphal growth. These findings are consistent with the observed proliferation and filopodial extension of Pneumocystis organisms adherent to the epithelium in the lungs of immunocompromised hosts. PCSTE20 expression appears to represent a significant component in the regulation of the life cycle of this intractable opportunistic pathogen.

Amino Acid Sequence↗
PubMed1995

Phenotypic characterization of epidemic versus sporadic strains of methicillin-resistant Staphylococcus aureus.

Forty strains of methicillin-resistant Staphylococcus aureus (MRSA) were divided on the basis of their epidemiologic behavior into two subgroups, sporadic MRSA (SMRSA) and epidemic MRSA (EMRSA) strains. The strains were examined for binding of 125I-labelled fibronectin, vitronectin, collagen, Fc fragments of immunoglobulin G, and fibrinogen. A significant difference between EMRSA and SMRSA strains was found for binding of 125I-labelled fibrinogen and for Fc fragments of immunoglobulin G, (P < 0.05). No significant difference in the binding of 125I-labelled fibronectin and collagen was found between EMRSA and SMRSA strains. The binding of 125I-labelled vitronectin to MRSA strains was found to be aspecific. Capsular serotypes of the strains were determined with monoclonal antibodies against capsular types 5 and 8. Strains could be divided into the following four groups: types 5, 8, and 5/8 and nontypeable. More nontypeable strains were found in the EMRSA group (66.6%). Significantly more EMRSA strains (79%) than SMRSA strains (44%) produced alpha-toxin (P < 0.025). Logistic regression analysis using a combination of the parameters 125I-labelled immunoglobulin G binding, capsular type, and alpha-toxin production predicted the epidemic character with a sensitivity of 83% and a specificity of 75%.

Bacterial Toxins↗
PubMed1999

Cellular entry of hantaviruses which cause hemorrhagic fever with renal syndrome is mediated by beta3 integrins.

Hantaviruses replicate primarily in the vascular endothelium and cause two human diseases, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). In this report, we demonstrate that the cellular entry of HFRS-associated hantaviruses is facilitated by specific integrins expressed on platelets, endothelial cells, and macrophages. Infection of human umbilical vein endothelial cells and Vero E6 cells by the HFRS-causing hantaviruses Hantaan (HTN), Seoul (SEO), and Puumala (PUU) is inhibited by antibodies to alphavbeta3 integrins and by the integrin ligand vitronectin. The cellular entry of HTN, SEO, and PUU viruses, but not the nonpathogenic Prospect Hill (PH) hantavirus (i.e., a virus with no associated human disease), was also mediated by introducting recombinant alphaIIbbeta3 or alphavbeta3 integrins into beta3-integrin-deficient CHO cells. In addition, PH infectivity was not inhibited by alphavbeta3-specific sera or vitronectin but was blocked by alpha5beta1-specific sera and the integrin ligand fibronectin. RGD tripeptides, which are required for many integrin-ligand interactions, are absent from all hantavirus G1 and G2 surface glycoproteins, and GRGDSP peptides did not inhibit hantavirus infectivity. Further, a mouse-human hybrid beta3 integrin-specific Fab fragment, c7E3 (ReoPro), also inhibited the infectivity of HTN, SEO, and PUU as well as HPS-associated hantaviruses, Sin Nombre (SN) and New York-1 (NY-1). These findings indicate that pathogenic HPS- and HFRS-causing hantaviruses enter cells via beta3 integrins, which are present on the surfaces of platelets, endothelial cells, and macrophages. Since beta3 integrins regulate vascular permeability and platelet function, these findings also correlate beta3 integrin usage with common elements of hantavirus pathogenesis.

Abciximab↗
PubMed1995

Human scleral elastic system: an immunoelectron microscopic study.

An immunocytochemical study was conducted on elastic components in the sclera of seven aged human eyes. By conventional electron microscopy, elastic tissue consists of three distinct fibre types--elastic fibres, elaunin fibres, and oxytalan fibres. The distribution of six components associated with the elastic system (elastin, amyloid P component, laminin, fibronectin, gp 115, and vitronectin) were studied by immunogold transmission electron microscopy. The codistribution of amyloid P component and laminin was further studied by double immunolabelling. Both elastic and elaunin fibres contained elastin. The microfibrillar sheaths of elastic fibres labelled for amyloid P component, those of elaunin fibres for amyloid P and laminin, and those of oxytalan fibres for laminin only. No labelling was observed for fibronectin, gp 115, and vitronectin. In terms of the proteins investigated, the biochemical profile of the three fibre types was not completely identical and was manifest as different affinities in the binding of serum amyloid P component and an association with laminin.

Aged↗
PubMed1996

Integrin mediation of type II cell adherence to provisional matrix proteins.

Lung injury causes alveolar type I epithelial cell death, basement membrane denudation, and alveolar flooding with serum fibronectin and fibrinogen. For successful restoration of normal architecture, the epithelium must be regenerated from progenitor type II alveolar cells. Using adhesion assays, we examined whether type II alveolar cells adhere to the provisional matrix proteins fibronectin, fibrinogen, and fibrin, and whether integrins mediate this adherence. Rat type II cells adhered to fibronectin, vitronectin, fibrinogen, and fibrin. Synthetic RGD (arginine-glycine-aspartic acid) peptide blocked this adhesion. Flow cytometry and Western analysis indicated that type II cells expressed beta 1- and alpha v beta 3-integrins. Anti-beta 1-and anti-alpha v beta 3-integrin antibodies blocked type II cell adhesion to fibronectin and to fibronectin and fibrinogen, respectively. In summary, type II cells adhered to fibronectin, fibrinogen, and fibrin, and adhesion was partially mediated by integrins. This study provides the first evidence of type II cell adhesion to fibrin gels and vitronectin, beta 1- and alpha v beta 3-integrin mediation of type II cell adhesion, and the presence of the alpha v beta 3-integrin on type II epithelial cells.

Animals↗
PubMed2000

Vascular expression of the alpha(v)beta(3)-integrin in lung and other organs.

The expression of the alpha(v)beta(3)-integrin in nonproliferating vascular beds remains unclear. To determine possible organ-specific differences, we compared alpha(v)beta(3)-integrin expression in the lung and other organs. Paraffin-embedded tissue sections of lung, liver, brain, muscle and skin obtained from rats were processed for immunohistochemistry with a monoclonal (LM609) and a polyclonal antibody (AB1903) against the alpha(v)beta(3)-integrin. Immunogold electron microscopy was used to localize alpha(v)beta(3)-integrin in rat lung microvasculature. With the use of custom-designed primers, lung sections were subjected to in situ PCR in a thermal cycler to amplify alpha(v) or beta(3) mRNA. To confirm specific amplification, PCR products were further hybridized in situ with an alpha(v) or beta(3) cDNA probe. In the lung, the alpha(v)beta(3)-integrin protein as well as alpha(v) and beta(3) mRNAs was extensively evident in the endothelium of extra-alveolar and alveolar microvessels, in vascular smooth muscle, and in large bronchial epithelium but not in the epithelium of alveolar ducts or alveoli. Ultrastructural immunogold labeling showed the presence of the integrin on the luminal and abluminal faces of the lung microvascular endothelium but not on the apical surface of the alveolar epithelium. Staining for the integrin was generally negative in blood vessels of several systemic organs, although weak staining was evident in branches of the hepatic portal vein. The constitutive presence of the alpha(v) and beta(3) mRNAs and the alpha(v)beta(3)-integrin in the lung microvascular bed suggests that gene transcription for the integrin is ongoing in lung vessels. Because it binds vitronectin, the lung vascular alpha(v)beta(3)-integrin may play a role in ligation of bloodborne, vitronectin-containing macromolecular complexes formed in inflammation.

Animals↗
PubMed1996

Alpha-V/beta-3 and alpha-V/beta-5 integrin distribution in neoplastic kidney.

Integrins are transmembrane glycoproteins that mediate cell-cell and cell-matrix interactions. Altered integrin expression may contribute to tumor progression, invasiveness and metastases. The alpha-V/beta-3 (alpha v beta 3; osteopontin/ vitronectin receptor) has recently been implicated in neovascularization and tumor-induced angiogenesis. alpha v-Subunit also associates with beta 5 to form an alpha v beta 5-complex, another vitronectin receptor. We studied tissue distribution of alpha v beta 3-and alpha v beta 5-integrins, as well as alpha 1- and beta 1-subunits in nephrectomy samples from 7 subjects with localized renal cell carcinoma. Grossly and histologically uninvolved regions ('normal') from the same nephrectomy specimens were used for comparison. Integrin expression was studied with specific monoclonal antibodies and the immunoperoxidase technique. alpha v beta 3 was expressed in the glomerular epithelial cells, Bowman's capsule, vascular endothelium, and weakly in tubular epithelial cells. alpha v beta 5 had a similar distribution except for minimal expression on vascular endothelium. alpha 1-Expression was observed in mesangium and but weakly in Bowman's capsule. beta 1-Expression was seen in glomerular epithelial cells, Bowman's capsule, vascular epithelium and tubular epithelial cells. Unlike in 'normals', neoplastic expression was more heterogeneous alpha v beta 3 was expressed in tumor cells in 4/7 cases, vascular endothelium in 6/6, and in stroma in 4/7. alpha v beta 5 was weakly expressed in tumor cells in 4/5, vascular endothelium in 5/5, and stroma in 4/5 cases. alpha 1-Expression was seen in tumor cells in 3/7, vascular endothelium in 4/7 and in stroma in 7/7 cases. beta 1-Expression was seen in tumor cells in 7/7 cases, vascular endothelium in 7/7, and in stroma in 4/7 cases. This study delineates the pattern of expression of the alpha v beta 3-and alpha v beta 5-integrins in 'normal' and neoplastic human kidney. Variations in alpha v beta 3-and alpha v beta 5-integrin expression may play a role in normal and neoplastic processes of the kidney.

Adult↗
PubMedDate not supplied

Identification of a 60-kD antigen associated with malignant growth of human breast tissue.

Antisera against a novel 60-kD antigen (P60) were developed using immunomasking strategy which shows malignant growth-related expression in human breast cancer tissue. Cell adhesion assay in the presence of P60 antibody clearly indicates a role for P60 directly or indirectly in adhesion of tumor cells to extracellular matrix protein vitronectin. These results indicate that P60 could be a potential marker to distinguish benign from malignant growth of human breast tissue, and that P60 could be an integrin or a non-integrin receptor, or a receptor-related protein to vitronectin.

Animals↗
PubMed1992

The effect of mast cell chymase on extracellular matrix: studies in autoimmune thyroiditis and in cultured thyroid cells.

In the first part of the study we analyzed the morphology of mast cells in autoimmune thyroiditis of BB/W rats. In the early stage of thyroiditis mast cells showed exocytosis of granules into the interstitium; this was associated with disorganization of the extracellular matrix and the appearance of a translucent ground substance in stroma. Mast cells were not seen in the mononuclear infiltrates in the later stages of thyroiditis. In order to further study the effect of mast cells on the extracellular matrix, we evaluated the effect of mast cell lysate and purified chymase on the matrix of cultured thyroid cells. Mast cells were obtained from peritoneal cavity; mast cell chymase was purified by anion exchange chromatography. After exposure to chymase there was a reduction of pericellular fibronectin in cultured thyroid cells, while laminin in matrix remained unchanged. Similarly, as found by gel electrophoresis, soluble fibronectin and vitronectin were digested by chymase in the reaction mixture. Cell attachment on both fibronectin and vitronectin was significantly decreased upon exposure of matrix proteins to chymase. The effects of chymase were abolished by enzyme inhibitor phenylmethane sulfonyl fluoride. These data suggest that mast cells possess proteolytic enzymes capable of digesting different host proteins which may have a role in the thyroid cell interaction with the surrounding matrix.

Animals↗
PubMed2002

PlA polymorphism of integrin beta 3 differentially modulates cellular migration on extracellular matrix proteins.

OBJECTIVE: Cell migration is central to multiple physiological and pathologic processes and involves interactions between integrins on the cell surface and the extracellular matrix. The Leu33Pro (PlA) polymorphism of integrin beta3 has been reported to be associated with a greater rate of restenosis after angioplasty, a process involving endothelial and smooth muscle cell migration. We have addressed the possibility that the Leu33Pro polymorphism could modify the migratory behavior of Chinese hamster ovary (CHO) cells expressing the beta3-containing integrin complexes. METHODS AND RESULTS: Haptotactic migratory responses of CHO alpha(IIb)beta3 cells to fibronectin and vitronectin were not statistically different between the Leu33 and Pro33 cells. However, CHO cells with the Pro33 (PlA2) polymorphism had an enhanced haptotactic migratory response to fibrinogen and von Willebrand Factor. This enhanced migration (1) could be blocked by the alpha(IIb)beta3-complex-specific neutralizing mAb 10E5, by 7E3, a neutralizing mAb specific for the beta3 integrin, and by the alpha(IIb)beta3-blocking peptide Integrilin; (2) was not observed with a CHO cell line expressing an activating beta3 Cys435 to Ala mutation; and (3) was attributable to increased activity of mitogen-activated protein kinase and cyclooxygenase. CHO cell lines expressing the Pro33 isoform of alpha(v)beta3 had an enhanced haptotactic migratory response to vitronectin and osteopontin but not fibrinogen. CONCLUSIONS: The Leu33Pro polymorphism alters the migratory behavior of cells on extracellular matrix substrates, and the alpha subunit influences the substrate specificity of this genetic effect.

Animals↗
PubMed2000

CD9 participates in endothelial cell migration during in vitro wound repair.

CD9, a widely expressed membrane protein of the tetraspanin family, has been implicated in diverse functions, such as signal transduction, cell adhesion, and cell motility. We tested the effects of an anti-CD9 monoclonal antibody (ALMA.1) on the migration and proliferation of human vascular endothelial cells (ECs) during repair of an in vitro mechanical wound mimicking angiogenic processes. ALMA.1 induced dose-dependent inhibition of wound repair with a 35+/-1.5% decrease at 20 microg/mL. Only cell migration was affected, because the rate of proliferation of ECs at the lesion margin was not modified and because the inhibition of repair was also observed for nonproliferating irradiated ECs. Monoclonal antibodies against CD63 tetraspanin (H5C6) and control mouse IgG (MOPC-21) were inactive. CD9, one of the most abundant proteins at the surface of ECs, colocalized with beta(1) or beta(3) integrins on EC membranes in double-labeling immunofluorescence experiments with ALMA.1 and an anti-beta(1) (4B4) or anti-beta(3) (SDF.3) monoclonal antibody. Moreover, ALMA.1 and 4B4 had additive inhibitory effects on lesion repair, whereas 4B4 alone also inhibited EC proliferation. In transmembrane Boyden-type assays, ALMA.1 induced dose-dependent inhibition of EC migration toward fibronectin and vitronectin with 45+/-6% and 31+/-10% inhibition, respectively, at 100 microg/mL. 4B4 inhibited migration toward fibronectin at 10 microg/mL but had no effect in the case of vitronectin. Adhesion of ECs to immobilized anti-CD9 monoclonal antibodies induced tyrosine-phosphorylated protein levels similar to those observed during interactions with beta(1) or beta(3) integrins. These results point to the involvement of CD9 in EC adhesion and migration during lesion repair and angiogenesis, probably through cooperation with integrins. As such, CD9 is a potential target to inhibit angiogenesis in metastatic and atherosclerotic processes.

Antibodies↗
PubMedDate not supplied

Molecular modeling of protein-glycosaminoglycan interactions.

Forty-nine regions in 21 proteins were identified as potential heparin-binding sites based on the sequence organizations of their basic and nonbasic residues. Twelve known heparin-binding sequences in vitronectin, apolipoproteins E and B-100, and platelet factor 4 were used to formulate two search strings for identifying potential heparin-binding regions in other proteins. Consensus sequences for glycosaminoglycan recognition were determined as [-X-B-B-X-B-X-] and [-X-B-B-B-X-X-B-X-] where B is the probability of a basic residue and X is a hydropathic residue. Predictions were then made as to the heparin-binding domains in endothelial cell growth factor, purpurin, and antithrombin-III. Many of the natural sequences conforming to these consensus motifs show prominent amphipathic periodicities having both alpha-helical and beta-strand conformations as determined by predictive algorithms and circular dichroism studies. The heparin-binding domain of vitronectin was modeled and formed a hydrophilic pocket that wrapped around and folded over a heparin octasaccharide, yielding a complementary structure. We suggest that these consensus sequence elements form potential nucleation sites for the recognition of polyanions in proteins and may provide a useful guide in identifying heparin-binding regions in other proteins. The possible relevance of protein-glycosaminoglycans interactions in atherosclerosis is discussed.

Algorithms↗
PubMed2001

Effect of extracellular matrix elements on angiotensin II-induced calcium release in vascular smooth muscle cells from normotensive and hypertensive rats.

The interaction of the vascular smooth muscle cells (VSMCs) with the components of the matrix determines several functions of the cell, such as growth and differentiation. In contrast, an alteration in angiotensin (Ang) II-induced Ca(2+) mechanisms in VSMCs was reported in genetic hypertension. In this study, we wished to assess the effect of different components of the extracellular matrix on the increase of [Ca(2+)](i) induced by Ang II in VSMCs from spontaneously hypertensive rats (SHR) compared with those from normotensive Wistar-Kyoto rats (WKY). Results demonstrate for the first time that elements of the extracellular matrix modulate the Ang II-induced Ca(2+) transport mechanisms. This modulation is different in cells from WKY compared with those from SHR. Thus, growing cells from SHR on collagen I, collagen IV, fibronectin, vitronectin, or Matrigel induced a significant decrease in Ang II-induced Ca(2+) release from internal stores, whereas in cells from WKY, no effect could be observed except for those grown on collagen I, which increased Ca(2+) release. Fibronectin and vitronectin, however, induced a decrease in Ang II-induced Ca(2+) influx in WKY, whereas no effect could be observed in SHR. Conversely, collagen I and collagen IV induced an increase in this influx in SHR but not in WKY, whereas Matrigel increased the influx in both strains. These results suggest a modulation of the Ang II-associated signaling events by the matrix elements via the focal adhesion points. The understanding of these synergies should provide insight into issues such as development of hypertrophy of large vessels in hypertension.

Angiotensin II↗
PubMed2002

Inhibition of protein kinase Calpha prevents endothelial cell migration and vascular tube formation in vitro and myocardial neovascularization in vivo.

Although protein kinase C (PKC) activation is required for endothelial cell (EC) growth, migration, adhesion, and vessel formation, the role of individual PKC isoenzymes in these events is not defined. Because PKCalpha has been previously linked with enhanced EC migration and response to angiogenic growth factors, we characterized a specific phosphorothioate-modified 21-mer antisense PKCalpha (AS-PKCalpha). AS-PKCalpha (500 nmol/L) prevented the expression of PKCalpha protein by 90% in human ECs and did not reduce the expression of any other PKC isoenzyme. AS-PKCalpha reduced human EC migration by 64% compared with its control oligonucleotide in a "scratch" wounding assay, and AS-PKCalpha reduced human EC adhesion to the extracellular matrix protein vitronectin by 18%. Phosphorylation of mitogen-activated protein kinase (extracellular signal-regulated kinase 1/2) induced by vascular endothelial growth factor was inhibited by 30% in human ECs transfected with AS-PKCalpha. Compared with control, AS-PKCalpha also reduced the number of EC tubes formed in a 3D type I collagen gel assay by 37.5%. Finally, using an osmotic minipump, we infused AS-PKCalpha into mice in which myocardial infarction was induced by coronary ligation and found that the oligonucleotide was primarily taken up by intramyocardial blood vessels. Compared with the results with control oligonucleotide, AS-PKCalpha oligonucleotide inhibited the number of anti-PKCalpha-stained blood vessels by 48% and reduced the total vessel number by 72% as well. In conclusion, the expression of PKCalpha is required for full EC migration, adhesion to vitronectin, vascular endothelial growth factor-induced extracellular signal-regulated kinase activation, and tube formation and is likely to be of importance in myocardial angiogenesis in vivo after ischemia.

Animals↗