Differential regulation of phosphoinositide metabolism by alphaVbeta3 and alphaVbeta5 integrins upon smooth muscle cell migration.

Smooth muscle cell migration is a key step of atherosclerosis and angiogenesis. We demonstrate that alpha(V)beta(3) and alpha(V)beta(5) integrins synergistically regulate smooth muscle cell migration onto vitronectin. Using an original haptotactic cell migration assay, we measured a strong stimulation of phosphoinositide metabolism in migrating vascular smooth muscle cells. Phosphatidic acid production and phosphoinositide 3-kinase IA activation were triggered only upon alpha(V)beta(3) engagement. Blockade of alpha(V)beta(3) engagement or phospholipase C activity resulted in a strong inhibition of smooth muscle cell spreading on vitronectin. By contrast, blockade of alpha(V)beta(5) reinforced elongation and polarization of cell shape. Moreover, Pyk2-associated tyrosine kinase and phosphoinositide 4-kinase activities measured in Pyk2 immunoprecipitates were stimulated upon cell migration. Blockade of either alpha(V)beta(3) or alpha(V)beta(5) function, as well as inhibition of phospholipase C activity, decreased both Pyk2-associated activities. We demonstrated that the Pyk2-associated phosphoinositide 4-kinase corresponded to the beta isoform. Our data point to the metabolism of phosphoinositides as a regulatory pathway for the differential roles played by alpha(V)beta(3) and alpha(V)beta(5) upon cell migration and identify the Pyk2-associated phosphoinositide 4-kinase beta as a common target for both integrins.

Prevention of neointima formation by mibefradil after vascular injury in rats: comparison with ACE inhibition.

Cilazapril, an angiotensin-converting enzyme inhibitor, and mibefradil, a selective T-type voltage-operated calcium channel blocker, have been shown to prevent neointima formation after vascular injury. The goal of the present study was to evaluate the mechanism of action of both drugs. For this purpose, the influence of the renin angiotensin system on the effects of mibefradil (30 mg/kg po) and cilazapril (10 mg/kg po) on neointima formation after carotid injury were evaluated in normotensive rats (normal renin angiotensin system) and DOCA hypertensive rats (suppressed renin angiotensin system). In addition, in order to differentiate an effect on cell migration or cell proliferation, both drugs were given either before or after the smooth muscle migration phase. Finally, cilazapril and mibefradil were given in combination. In normotensive rats, mibefradil and cilazapril decreased neointima formation, resulting in neointima/media ratios of 38% (p < 0.05) and 53% (p < 0.01), respectively. However, in DOCA hypertensive rats, mibefradil was active, with a reduction of the neointima/media ratio by 63% (p < 0.001), whereas cilazapril reduced it only slightly (19%) and not significantly. In addition, cilazapril was active only when treatment started before the migration phase (63%, reduction in neointima/media ratio, p < 0.001) but not when started thereafter (13% reduction in neointima/media ratio, n.s.). In contrast, treatment with mibefradil was also active when started after the migration phase (51% reduction in neointima/ media ratio, p < 0.001 when treatment started 1 day before balloon injury and 41%, p < 0.01 when treatment started 5 days after balloon injury). The combination of both drugs was additive (67% reduction in neointima/media ratio, p < 0.001 vs. control). These experiments clearly show that mibefradil and cilazapril have a different mechanism of action after vascular injury. Mibefradil most likely prevents the proliferation of smooth muscle cells. In contrast, cilazapril most likely inhibits the migration of smooth muscle cells. These two different mechanisms of action explain why the effects of both drugs are additive.

Mibefradil prevents neointima formation after vascular injury in rats. Possible role of the blockade of the T-type voltage-operated calcium channel.

Mibefradil is a novel calcium antagonist that is selective for the T-type voltage-operated calcium channel rather than the L type. Because T-type calcium channels are present on rapidly proliferating cells and mediate the increase of intracellular calcium induced by some growth factors, such as platelet-derived growth factor, we hypothesized that the blockade of T channels could prevent the excessive smooth muscle cell proliferation that occurs in conditions such as vascular injury. To test this hypothesis, we evaluated in rats the effects of mibefradil (which blocks both L- and T-type channels) on neointima formation after vascular injury, and we compared them with those of equihypotensive doses of amlodipine and verapamil (which block only L-type channels). Mibefradil (30 mg/kg) decreased the area of neointima formed 14 days after balloon injury by 54% (P < .001). In contrast, neither verapamil nor amlodipine had an effect despite a blood pressure reduction at least equal to that of mibefradil. The in vivo effect of mibefradil was indeed an inhibition of smooth muscle cell proliferation, as shown by thymidine incorporation experiments. This antiproliferative effect of mibefradil was also observed in vitro in smooth muscle cells stimulated by fetal calf serum. In this condition also, verapamil was ineffective. We conclude that in rats mibefradil has a potent antiproliferative effect on smooth muscle cells after vascular injury. This effect might be due to blockade of voltage-operated T channels.

Dimerization of extracellular domains of platelet-derived growth factor receptors. A revised model of receptor-ligand interaction.

The alpha- and beta-subunits of the receptor for platelet-derived growth factor (PDGFR) were found to be autophosphorylated in the absence of ligands at high expression levels which suggests a propensity of PDGFRs to dimerize spontaneously. When the extracellular domains (ED) of both receptors were expressed and purified to homogeneity, they could be dimerized specifically by the different PDGF isoforms. PDGF-BB-induced dimerization was dependent on uncleaved loop I sequences present on both chains. Whereas, in solution, the EDs were weak competitors for PDGF binding to cellular PDGFRs, they formed high and low affinity complexes upon immobilization on solid phase. Cross-competition experiments defined two distinct binding sites on PDGFR alpha-ED. PDGF-AB bound only to the low affinity form of immobilized PDGFR beta-ED and could not dimerize PDGFR beta-ED. Cross-linking studies, however, revealed that both chains of PDGF-AB can interact with a PDGFR beta-ED monomer. Cross-linking of PDGF homodimers with EDs also yielded complexes which contained more than two ligand chains. These results led to a revised model of receptor-ligand interaction and indicate that monomeric PDGF should be able to dimerize PDGF receptors.

Transcriptional regulation of basic fibroblast growth factor gene expression in capillary endothelial cells.

The growth of capillary endothelial cells (BCE) is an important regulatory step in the formation of capillary blood vessels. In vivo, the proliferation of these cells is stringently controlled. In vitro they can be stimulated by polypeptide growth factors, such as acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF). Since bFGF is synthesized and stored by vascular endothelial cells, this mitogen may play an important role in an autocrine growth regulation during angiogenesis. Here, evidence is presented for induction of the mRNA of bFGF by bFGF itself. A similar increase of bFGF mRNA was observed in response to thrombin and after treatment with phorbol ester. These results suggest that an autocrine loop may exist that may serve to modulate the mitogenic response in BCE under various physiological conditions, (e.g., wound healing and new capillary formation).

Expression of acidic and basic fibroblast growth factors in human and bovine vascular smooth muscle cells.

The expression and synthesis of acidic and basic fibroblast growth factors (aFGF and bFGF) in cultures of bovine and human vascular smooth muscle cells (BSMC and HSMC) was studied. BSMC express and synthesize only bFGF, whereas HSMC express and synthesize both bFGF and aFGF. The presence of bFGF in BSMC is shown by the following criteria: (1) the growth factor activity in BSMC lysates binds to a heparin-affinity column and elutes as a single peak at 1.5-1.7 M NaCl, characteristic for bFGF; (2) this extract is mitogenic for smooth muscle cells; (3) Northern blot analysis demonstrates three distinct bFGF mRNAs of 7.0, 4.0, and 1.9 kb; no aFGF mRNA species were detected. Analysis of human umbilical vein endothelial cells yielded similar results: Heparin-affinity chromatography and Northern blot analysis failed to demonstrate the presence of aFGF despite the detection of bFGF by these techniques. In contrast, HSMC synthesize two growth factor activities: First, they bind to an immobilized heparin column and elute as two separate peaks at 1.2 and 1.5-1.7 M NaCl, characteristic for aFGF and bFGF; and second. Northern blot analysis demonstrates the expression of aFGF mRNA of 4.6 kb and bFGF mRNAs of 7.0, 4.0 and 1.9 kb. Furthermore, it is shown that aFGF and bFGF are potent mitogens for smooth muscle cells in vitro.

Species-specific high molecular weight forms of basic fibroblast growth factor.

bFGF was extracted from either mouse, rat and human cell lines or mouse, rat bovine and human brain tissue and partially purified by cation exchange chromatography and heparin-affinity chromatography. When the heparin-affinity purified proteins were probed on Western blots with antisera against either a highly conserved internal bFGF sequence or recombinant 18 kDa bFGF, species-specific forms of bFGF were detected. bFGF proteins from rat and mouse sources were of apparent molecular weight 18,000, 21,500 and 22,000 whereas those from human sources were of 18,000, 22,500 and 24,000. Bovine bFGF proteins were similar to the multiple human bFGFs. The 22.5 kDa and 24 kDa proteins from human cells were also recognized by an antibody specific for the N-terminally extended forms of human bFGF, whereas this antibody failed to detect 18 kDa bFGF. We showed that the differences in molecular weight between human and rat bFGFs are consistent with the predicted ATG (methionine) or alternative CTG (leucine) translational start sites in the 5' upstream sequences of bFGF cDNAs. In addition we show that, irrespective of the species of origin, the larger bFGF proteins may be separated from 18 kDa bFGF by Mono S chromatography.

Purification of 18- and 22-kDa forms of basic fibroblast growth factor from rat cells transformed by the ras oncogene.

Normal Rat-1 fibroblasts and Rat-1 cells transformed by the H-ras oncogene (Rat-1-EJ) were analyzed for cell-associated growth factor activity. The two cell lines grew at the same rate, but at any given stage of growth the Rat-1-EJ cells synthesized two to four times more cell-associated growth factor activity than did the Rat-1 cells. For each cell line, the level of cell-associated growth factor activity was five to eight times greater at confluent densities compared to sparse densities. Heparin affinity chromatography and Western blot analysis demonstrated that the cell-associated growth factor was basic fibroblast growth factor (bFGF). The bFGF synthesized by the Rat-1-EJ cells appeared in two molecular mass forms, about 40% as an 18-kDa form which comigrated with recombinant bFGF and about 60% as a higher molecular mass doublet of about 22 kDa. The two forms of bFGF were biologically active and could be separated on a Mono S cation exchange column. Separation and purification to homogeneity of both the 18-kDa bFGF and the 22-kDa bFGF doublet were achieved by a combination of CM-Sepharose cation exchange, heparin affinity-fast performance liquid chromatography, and C4 reverse phase high performance liquid chromatography.

Nonenzymatic glycosylation of albumin in vivo. Identification of multiple glycosylated sites.

Nonenzymatic glycosylation of albumin in vivo occurs at multiple sites. Glucose gets attached to Lys-199, Lys-281, Lys-439, and Lys-525 as well as to some other lysine residues. The principal glycosylated site is Lys-525. Approximately 33% of the overall glycosylation occurs at this site. This site specificity is remarkable and is postulated to be a consequence of local catalysis of the nonenzymatic glycosylation reaction. It appears that positively charged amino groups in the protein catalyze the Amadori rearrangement at specific sites. The principal glycosylated site, Lys-525, lies in a Lys-Lys sequence; other glycosylated sites lie in a Lys-Lys, Lys-His, and Lys-His-Lys sequence or are near disulfide bridges, which are likely to place amino groups of more remote parts of the protein closer to these sites. The occurrence of nonenzymatic glycosylation at most of the identified sites in albumin from diabetic patients is explained by the concept of local acid-base catalysis of the Amadori rearrangement.

Albumin-directed antibodies in diabetes: demonstration of human serum albumin-directed IgM autoantibodies.

Sera of 406 individuals, 174 Type 1 (insulin-dependent) diabetic patients, 125 non-diabetic family members and 107 unrelated control subjects, were screened for the presence of antibodies against glycated albumin. In none of these sera could such antibodies be detected. However, antibodies directed towards monomeric, unmodified human serum albumin were detected in 13 sera. These albumin autoantibodies were of the IgM class, and occurred in sera from nondiabetic persons (0.9-1.6%) and with a five-fold higher frequency in sera from diabetic patients (5.2%). The presence of albumin antibodies was neither related to the presence of diabetic late complications, islet cell antibodies, HLA-status nor duration of diabetes. The albumin antibodies were also found in sera from persons carrying antibodies against mumps (17%) or Epstein-Barr virus.