Thrombin activates a Y box-binding protein (DNA-binding protein B) in endothelial cells.

Thrombin stimulates the expression of multiple genes in endothelial cells (ECs), but the trans-acting factors responsible for this induction remain undefined. We have previously described a thrombin-inducible nuclear factor (TINF), which binds to an element in the PDGF B promoter and is responsible for the thrombin inducibility of this gene. Inactive cytoplasmic TINF is rapidly activated and translocated to nuclei of ECs upon stimulation with thrombin. We have now purified TINF from thrombin-treated ECs. Amino acid sequencing revealed it to be a member of the Y-box protein family, and the sole Y-box protein-encoding cDNA we detected in human or bovine ECs corresponded to DNA-binding protein B (dbpB). DbpB translocated to the nucleus after thrombin stimulation of ECs as shown by FACS analysis of nuclei from ECs expressing GFP-dbpB fusion proteins. During thrombin activation, dbpB was found to be cleaved, yielding a 30-kDa NH(2)-terminal fragment that recognized the thrombin-response element sequence, but not the Y-box consensus sequence. Preincubation of ECs with protein tyrosine phosphatase inhibitors completely blocked dbpB activation by thrombin and blocked induction of endogenous PDGF B-chain mRNA and promoter activation by thrombin. Y-box proteins are known to act constitutively to regulate the expression of several genes. Activation of this class of transcription factors in response to thrombin or any other agonist represents a novel signaling pathway.

Thrombin regulates PDGF expression in bovine glomerular endothelial cells.

The proteolytic enzyme thrombin is produced during activation of the coagulation pathway. Intraglomerular fibrin deposition and thrombosis are common pathologic features of several glomerular diseases, including transplant rejection. The effect of thrombin on platelet-derived growth factor (PDGF) production and DNA synthesis in well characterized bovine glomerular endothelial cells (G/endo) was studied. DNA synthesis was measured as the amount of [3H]thymidine incorporated into acid-insoluble material. PDGF released in the supernatant was measured by Western blotting and by a radioreceptor assay. PDGF mRNA expression was analyzed by solution hybridization, using human genomic PDGF B-chain (c-sis) and A-chain cDNA probes. G/endo constitutively secrete PDGF activity in serum-free medium. Thrombin stimulates PDGF production and increases the expression of mRNA that hybridizes with labeled B-chain but not A-chain probe, whereas epidermal growth factor and transforming growth factor-alpha stimulate the expression of PDGF A-chain mRNA. In addition, thrombin stimulates DNA synthesis with a peak effect at 24 h. Unlike endothelial cells from other microvascular beds, G/endo did not respond to any of the three PDGF isoforms BB, AB, or AA. These data demonstrate that bovine G/endo produce PDGF and that thrombin stimulates de novo synthesis of PDGF from these cells. Because mesangial, but not bovine, G/endo express PDGF receptors, PDGF released by G/endo is likely to modulate mesangial cell functions such as proliferation and matrix production by means of a paracrine mechanism.

Distinct mechanisms for N-acetylcysteine inhibition of cytokine-induced E-selectin and VCAM-1 expression.

We have examined the effects of N-acetyl-L-cysteine (NAC), a well-characterized, thiol-containing antioxidant, on agonist-induced monocytic cell adhesion to endothelial cells (EC). NAC inhibited interleukin-1 (IL-1 beta)-induced, but not basal, adhesion with 50% inhibition at approximately 20 mM. Monocytic cell adhesion to EC in response to tumor necrosis factor-alpha (TNF-alpha), lipopolysaccharide (LPS), alpha-thrombin, or phorbol 12-myristate 13-acetate (PMA) was similarly inhibited by NAC. Unlike published studies with pyrrolidinedithiocarbamate, which specifically inhibited vascular cell adhesion molecule 1 (VCAM-1), NAC inhibited IL-1 beta-induced mRNA and cell surface expression of both E-selectin and VCAM-1. NAC had no effect on the half-life of E-selectin or VCAM-1 mRNA. Although NAC reduced nuclear factor-kappa B (NF-kappa B) activation in EC as measured by gel-shift assays using an oligonucleotide probe corresponding to the consensus NF-kappa B binding sites of the VCAM-1 gene (VCAM-NF-kappa B), the antioxidant had no appreciable effect when an oligomer corresponding to the consensus NF-kappa B binding site of the E-selectin gene (E-selectin-NF-kappa B) was used. Because NF-kappa B has been reported to be redox sensitive, we studied the effects of NAC on the EC redox environment. NAC caused an expected dramatic increase in the reduced glutathione (GSH) levels in EC. In vitro studies demonstrated that whereas the binding affinity of NF-kappa B to the VCAM-NF-kappa B oligomer peaked at a GSH-to-oxidized glutathione (GSSG) ratio of approximately 200 and decreased at higher ratios, the binding to the E-selectin-NF-kappa B oligomer appeared relatively unaffected even at ratios > 400, i.e., those achieved in EC treated with 40 mM NAC. These results suggest that NF-kappa B binding to its consensus sequences in the VCAM-1 and E-selectin gene exhibits marked differences in redox sensitivity, allowing for differential gene expression regulated by the same transcription factor. Our data also demonstrate that NAC increases the GSH-to-GSSG ratio within the EC suggesting one possible mechanism through which this antioxidant inhibits agonist-induced monocyte adhesion to EC.

Influence of local delivery of the protein tyrosine kinase receptor inhibitor tyrphostin-47 on smooth-muscle cell proliferation in a rat carotid balloon-injury model.

Smooth-muscle cell proliferation in response to arterial injury represents an important etiologic factor in restenosis after angioplasty. Tyrphostin-47, a protein tyrosine kinase inhibitor, inhibits smooth-muscle cell proliferation in vitro. In this study tyrphostin-47 was incorporated into matrixes to determine whether prolonged local delivery would result in a reduction of neointimal proliferation after arterial injury in a rat carotid balloon-injury model. A polymer matrix (polylactic polyglycolic acid copolymer and pluronic gel F-127, mean matrix weight 7.83 +/- 0.39 mg) was loaded with tyrphostin-47 (25% w/w). Release studies demonstrated delivery of 11% of the incorporated drug over a 21-day release period. In cell culture, tyrphostin-47 released from the polymer matrix produced a reduction in smooth-muscle cell proliferation (p < 0.0007). Balloon denudation injury of the left common carotid artery of 34 animals was performed. In 12 animals, polymer matrixes containing tyrphostin-47 were wrapped around the injured arteries to provide prolonged drug delivery (estimated dosage 28 micrograms/kg/24 hr); in 10 animals a polymer matrix without tyrphostin-47 was implanted; and in 12 animals only balloon injury was performed. The mean neointimal cross-sectional areas, luminal areas, and intima/media ratios were not significantly different among animals receiving local treatment with tyrphostin-47, sham polymer after injury, or balloon injury without polymer implantation. We conclude that despite inhibition of smooth-muscle cell proliferation by tyrphostin-47 in vitro, sustained local delivery of this tyrosine kinase inhibitor does not result in a reduction of neointimal proliferation in the rat carotid injury model.

Effects of antisense c-myb oligonucleotides on vascular smooth muscle cell proliferation and response to vessel wall injury.

The process of restenosis after arterial balloon dilatation has been demonstrated to involve smooth muscle cell hyperplasia. Initial reports with antisense oligonucleotides directed against the proto-oncogene c-myb suggest marked in vitro specificity and in vivo efficacy. In the present study, we sought to confirm and extend the hypothesis that antisense to c-myb results in a specific antiproliferative effect with a comprehensive assessment by using different oligonucleotide preparations, different species, and tissue and cellular uptake experiments. Phosphorothioate-protected oligonucleotides representing the appropriate sequence for antisense to c-myb and multiple controls were used to inhibit proliferation of platelet-derived growth factor- and fetal bovine serum-stimulated rat, dog, and human aortic smooth muscle cells in vitro and neointimal proliferation in the rat carotid injury model. In vitro experiments using identical culture conditions in rat, dog, and human aortic smooth muscle cells failed to show specificity as well as consistency in growth inhibitory effects that could be attributed to an antisense mechanism. Proliferation of smooth muscle cell growth in culture was consistently inhibited with oligomers containing a contiguous 4-guanosine residue motif. In vivo, the rat carotid injury neointimal hyperplasia was similar for antisense c-myb (0.095 +/- 0.009 mm2) and sense c-myb (0.090 +/- 0.009 mm2). Fluorescent-labeled oligonucleotides were present in tissue after local delivery via pluronic gel, and their activity rapidly declined over a 72-hour period. Our findings point to the potential nonspecificity and lack of consistency of the antisense oligonucleotide to c-myb in vitro and in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Thrombin receptor-activating peptides differentially stimulate platelet-derived growth factor production, monocytic cell adhesion, and E-selectin expression in human umbilical vein endothelial cells.

Recent studies have shown that the synthetic peptides SFL LRN and SFL LRN PND KYEPF (thrombin receptor-activating peptides (TRAP)) derived from the deduced sequence of the new amino terminus of the cleaved thrombin receptor can mimic thrombin receptor activation, act as full agonists for platelet activation, and induce prostaglandin I2 production as well as cytosolic Ca2+ increase in human umbilical vein endothelial cells (HUVEC). Here, we have compared the ability of these synthetic peptide ligands and thrombin to stimulate platelet-derived growth factor (PDGF) production by, and monocyte adhesion to, HUVEC. Thrombin (50 units/ml) and TRAP (25 microM) maximally stimulated monocyte adhesion. Furthermore, the stimulation of E-selectin cell surface expression and the steady-state E-selectin mRNA levels by thrombin and TRAP were comparable. Thrombin (50 units/ml) stimulated PDGF production 400% above the basal level in 24 h, whereas the 6-mer and 14-mer TRAP, even at 200 microM, did not significantly stimulate PDGF production. Northern analysis, however, revealed that TRAP at 100 microM stimulated PDGF-A and -B chain mRNA expression to a level similar to that induced by thrombin. These results suggest that activation of cell signaling by TRAP can mimic thrombin and is sufficient for the stimulation of monocyte adhesion to HUVEC; however, thrombin-stimulated PDGF production by HUVEC may require mechanisms in addition to the signaling events initiated by TRAP or may require the participation of a novel thrombin receptor.

A cationic region of the platelet-derived growth factor (PDGF) A-chain (Arg159-Lys160-Lys161) is required for receptor binding and mitogenic activity of the PDGF-AA homodimer.

Platelet-derived growth factor-AA and -BB homodimers and -AB heterodimers bind with high affinity to the platelet-derived growth factor (PDGF) alpha-receptor. Basic polypeptides such as polylysine and protamine sulfate compete with PDGF for receptor binding, suggesting a role for ligand positive charge in the binding interaction. A pentapeptide amino acid sequence with a cationic tripeptide core is perfectly conserved between the A- and B-chains (Val158-Arg159-Lys160-Lys161-Pro162) and was therefore considered as a possible alpha-receptor-binding domain. We have investigated the functional importance of positive charge within this region of the PDGF A-chain by using site-directed mutagenesis to convert the cationic core amino acids to the acidic sequence triglutamic acid. cDNAs encoding wild-type (PDGF-AAwt) and charge mutant (PDGF-AAcm) proteins were expressed following stable transfection of Chinese hamster ovary cells. Proper assembly and secretion of PDGF-AAcm was verified by metabolic labeling with [35S]cysteine, immunoprecipitation, and SDS-polyacrylamide gel electrophoresis analysis under nonreducing and reducing conditions. PDGF-AAcm was secreted as two major species of disulfide-linked A-chain homodimers identical in molecular mass to those observed for PDGF-AAwt (32 and 35 kDa). Secreted PDGF-AAwt and PDGF-AAcm proteins were purified to homogeneity and subjected to structural and functional analyses. Compared to purified PDGF-AAwt, PDGF-AAcm displayed a marked reduction in both binding affinity for PDGF alpha-receptors and mitogenic activity in Swiss 3T3 cells. Large reductions were also observed in the ability of semipurified PDGF-AAcm to stimulate calcium influx and the production of inositol phosphates. Measurement of circular dichroism spectra of highly purified PDGF-AAcm and PDGF-AAwt revealed no significant difference in secondary structure. Collectively, these results indicate that the cationic Arg159-Lys160-Lys161 region plays a critical role in the biological activity of PDGF-AA by direct participation in ligand binding to the PDGF alpha-receptor.

Endothelin stimulates PDGF secretion in cultured human mesangial cells.

Endothelin, a 17-DKa peptide originally described as a potent vasoconstrictor, also stimulates the release of important regulators of glomerular hemodynamics such as atrial natriuretic factor and renin. In the present study we investigated the role of endothelin in the release of another potent vasoconstrictor and mitogen of human mesangial cells, the platelet-derived growth factor. Endothelin stimulated PDGF release at 12 hours and the effect was sustained for 36 hours. This effect was associated with the enhanced induction of mRNAs encoding PDGF A- and B-chain. Endothelin also induced mitogenesis in human mesangial cells which was accompanied by activation of phospholipase C with increased inositol phosphate turnover. These data suggest a mechanism by which endothelin may regulate mesangial cell function in disease states.

Lipopolysaccharide induces competence genes JE and KC in Balb/C 3T3 cells.

The expression of the early genes JE and KC has been examined in Balb/C 3T3 cells treated with bacterial lipopolysaccharide (LPS). Previous studies showed that JE and KC mRNAs are induced in murine peritoneal macrophages treated with LPS, suggesting a role for these genes in inflammatory responses. Consistent with this possibility are recently published cDNA sequences which document that both genes are members of a superfamily of inflammation- and/or growth-related cytokines. In the present study, we provide evidence that the mRNAs for JE and KC are specifically induced by LPS treatment of Balb/c 3T3 cells. The LPS-stimulated expression of JE and KC was dose dependent, and exhibited a transient time course; message levels were maximal between 2 and 4 hr and declined by 8 hr. The LPS-augmented accumulation of JE and KC occurred even in the presence of cyclohexamide, which additionally had a superinducing effect on the expression of both genes. Cyclohexamide alone, in the absence of LPS, also induced JE and KC mRNA accumulation. LPS-stimulated JE and KC mRNA expression was dependent upon the stimulation of transcription as determined by nuclear "run-on" studies. Comparative analyses indicated that, under the conditions employed, LPS was a somewhat less effective stimulant of JE expression than PDGF or EGF, and was more effective than PDGF and equivalent to EGF in its ability to augment KC accumulation. Unlike PDGF and EGF, LPS did not stimulate DNA synthesis by Balb/c 3T3 cells at any time over the 72 hr period examined. The ability of the inflammatory, non-mitogenic stimulus LPS to selectively induce JE and KC mRNA expression by fibroblasts may reflect their participation in inflammation and wound healing as secretory cells.

Lipopolysaccharide-inducible macrophage early genes are induced in Balb/c 3T3 cells by platelet-derived growth factor.

We have previously described the isolation and characterization of a set of cDNA clones encoding lipopolysaccharide (LPS)-induced early genes in murine peritoneal macrophages. The treatment of macrophages with LPS also stimulates the expression of four early or competence genes (c-fos, c-myc, JE, and KC) described in platelet-derived growth factor-stimulated Balb/c 3T3 cells. These latter findings led to the hypothesis that long term, adaptive responses such as DNA synthesis in fibroblasts and functional activation of macrophages may share multiple mechanistic pathways. To test this possibility, we have examined the expression of four LPS-inducible macrophage genes in platelet-derived growth factor-stimulated Balb/c 3T3 fibroblasts. The results demonstrate that three of these four genes are expressed in 3T3 cells in a fashion reminiscent of other growth factor-stimulated competence genes. All three mRNAs are expressed even in the presence of cycloheximide and two of the three exhibit superinducibility. The accumulation of these specific mRNA species was dependent upon the stimulation of transcription as determined by nuclear "run-off" studies. The platelet-derived growth factor dose dependence is comparable both for stimulation of DNA synthesis and expression of the three early genes. Furthermore, expression of all three genes preceded the entry of the cells into S phase, suggesting an association with cell cycle entry. Stimulation of 3T3 cells with epidermal growth factor resulted in DNA synthesis but not early gene expression. This latter result indicates that these early gene products are not necessary for 3T3 cell mitogenesis. Nevertheless, the expression of these genes in two different cell types in association with two distinct functional responses suggests that they contribute common functions either in terms of the physiologic response in which these cells participate (e.g. inflammation) or in the regulatory mechanisms which govern such responses.

Production of platelet-derived growth factorlike protein by rat mesangial cells in culture.

Rat mesangial cells (MC) release a factor that competes in a dose-dependent manner with 125I-labeled platelet-derived growth factor (PDGF) for binding to human foreskin fibroblasts (HFF). The competitor activity in mesangial cell conditioned medium (MCCM) is reversible, trypsin sensitive, and inhibited by anti-PDGF IgG. MCCM also expresses potent mitogenic activity to HFF. Anti-PDGF IgG, in concentrations that completely abolished the mitogenic activity of pure PDGF and the competitor activity of MCCM, only partially (33-41%) inhibits this mitogenic activity. The PDGF receptor competing activity as well as the total mitogenic activity, coelutes with labeled pure PDGF on Sephacryl S-200 gel chromatography. Cation exchange chromatography of concentrated MCCM yields a major mitogen peak with little competitor activity and a smaller mitogenic peak with comparable competitor activity, suggestive of the presence of other mitogens in MCCM besides the PDGF-like protein. PDGF is a potent mitogen and may play a role at inflammatory sites. The production of PDGF-like protein by MC may provide insights for understanding the pathogenesis of glomerular diseases.