Search

NO generated by endothelial cells is vasoprotective by antagonizing platelet adhesion and smooth muscle contraction. Since vascular smooth muscle cells (VSMCs) produce NO in response to cytokine stimulation and after arterial injury, we speculated that NO produced by VSMCs could compensate for the loss of endothelium. Using balloon catheter denudation of the rat carotid artery as a model for arterial injury and restenosis, we have evaluated the time course of expression of inducible NO synthase (iNOS) by in situ hybridization and immunohistochemistry and studied the effect of iNOS on platelet adhesion and blood flow of the injured vessel. iNOS mRNA and protein were expressed in the innermost layer of the media from day 1 and were subsequently detected in the neointima, whereas no expression was detectable in the uninjured artery. Systemic administration of N ω -nitro- l -arginine methyl ester (L-NAME) resulted in a twofold to threefold increase in the adhesion of 111 In-labeled platelets to the injured vessel wall. Platelet adhesion was also enhanced threefold by local delivery of L-NAME from a gel surrounding the injured vessel, whereas the stereoisomer, D-NAME, had no effect. Finally, inhibition of NO synthase led to a 24% reduction of the blood flow in the injured carotid artery. These results demonstrate that arterial injury triggers the expression of iNOS in the lesion and that NO produced by iNOS inhibits platelet adhesion and restores blood flow. This could explain the disappearance of platelet thrombi from deendothelialized arterial surfaces within a few days after injury and indicates the importance of NO generated by iNOS for the maintenance of vascular tone. Thus, expression of iNOS in lesions may represent a protective mechanism that compensates for the loss of endothelium.

We measured regional cerebral blood flow (rCBF) in young and old Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats by a minimally invasive microsphere technique. Blood flows to the cerebrum, diencephalon, mesencephalon, cerebellum and pons-medulla during normocapnia were determined. To test the ability of the vessels to dilate, rCBF was also measured during hypercapnia. Reactivity to CO2 was calculated as delta rCBF/delta paCO2. In the old SHR, blood flow to the pons-medulla (88 +/- 8 ml/min/100 g, mean +/- SEM) was markedly lower than that in the young SHR (107 +/- 4 ml/min/100 g, p < 0.05), whereas the difference of those values in the old and young WKY rats was slight (0.05 < p < 0.1). There were no differences in the values of blood flow to the cerebrum, diencephalon, mesencephalon or cerebellum between the young and old rats in both species. Cerebrovascular CO2 reactivity was markedly impaired in the old SHR (p < 0.05) compared to that in the young SHR, but the difference in reactivity in the WKY rats was not significant. The results indicate that blood flow to the pons-medulla is reduced in association with age, particularly in the hypertensive animals, and that the ability of the cerebral vessels to dilate is impaired homogeneously by the combination of chronic hypertension and aging.

We studied the effects of interferon gamma (IFN-gamma), a T-cell lymphokine, on the proliferation and chemotaxis of vascular smooth muscle cells (SMC). Recombinant human IFN-gamma dose-dependently inhibited the proliferation of SMC cultured in the presence of 20% fetal calf-serum. It also inhibited PDGF-induced chemotaxis of SMC. Similar concentrations of IFN-gamma induced DNA-synthesis of SMC cultured in mitogen-depleted medium for 5 days. The inhibition and the stimulation of SMC proliferation were accompanied by concomitant decrease and increase in the number of PDGF receptors. Our study indicated that IFN-gamma is a bidirectional regulator of SMC proliferation.

Proliferation of cultured human vascular endothelial cells may be negatively regulated by the protein kinase C (PKC) pathway, because phorbol 12-myristate, 13-acetate (PMA) inhibits DNA synthesis and cell population doubling in PKC-retaining cells, but not in cells depleted of PKC by a long-term exposure to PMA. We investigated the mechanism through which PKC arrests the cell cycle with regard to cyclin A, which has been reported to play a key role in G1/S progression activating CDK2. Cyclin A mRNA was elevated from late G1 in accordance with the protein expression, which reached the maximal level during the S phase. PMA added at late G1 potently reduced the levels of cyclin A mRNA and the protein in concentration-dependent manners parallel to its effect on the proliferation. However, it failed to inhibit the expression in PKC-depleted cells. The mRNA reduction by PMA was due to inhibition of the transcription. The PMA effects were mimicked by multiple doses of 1,2-dioctanoylglycerol. These findings suggest that PKC inhibits G1/S progression through suppression of cyclin A gene transcription in endothelial cells.

Involvement of the immunological mechanisms in atherogenesis has recently been suggested by immunohistological detection of macrophages and T lymphocytes in atherosclerotic lesions. In the present study, we have investigated the regulatory effect of interferon-gamma (IFN-gamma), a cytokine secreted by activated T cells, on the production and secretion of platelet-derived growth factor (PDGF) from macrophages in culture. The human monocytic leukemia cell line, THP-1, was treated with phorbol 12-myristate 13-acetate (PMA) for 24 h to induce macrophage differentiation and PDGF production, and then various doses of recombinant human IFN-gamma (0-1000 I.U./ml) were added to the culture. After 48 h, the conditioned medium and the cells were harvested and analyzed for PDGF production. PDGF-dependent mitogenic activity in the conditioned medium, estimated by neutralization of mitogenic activity with anti-PDGF antibody, was suppressed by IFN-gamma treatment. Radioimmunoassays for PDGF also revealed a decrease in both PDGF-AA and -BB in the conditioned medium with IFN-gamma treatment, whereas neither total cell DNA as an indication of cell number nor overall protein synthesis based on [3H]leucine incorporation were decreased. Northern analysis of total RNA extracted from the cells demonstrated that IFN-gamma suppressed the level of PDGF mRNA. Analysis of mRNA degradation in the presence of actinomycin D demonstrated that the decrease in PDGF mRNA was not due to enhanced degradation of mRNA. A similar inhibitory effect of IFN-gamma on PDGF mRNA levels was also found in monocyte-derived macrophages cultured in the presence of granulocyte-macrophage colony stimulating factor. These results suggest that IFN-gamma modulates production and secretion of PDGF from macrophages and that the functions of macrophages in atherogenesis may be regulated by the cellular interactions between T cells and macrophages through the action of cytokines such as IFN-gamma.

The scavenger receptor (SR-A) is considered to play a role in host defense by scavenging endotoxins, oxidized proteins, and denatured or otherwise modified self components, which are routed toward degradation in macrophages. Recent data suggest that SR-A also functions as an adhesion molecule. Our previous finding of SR-A expression by high endothelial cells of venules and on follicular dendritic cells in peripheral lymph nodes prompted us to investigate whether SR-A can act as an addressin for lymphocytes. We describe here that activated B cells adhere to CHO cells transfected with either the type I or type II isoform of SR-A. In contrast, resting B cells isolated from peripheral blood did not adhere to SR-A transfected CHO cells. Other types of leukocytes did not bind to SR-A. The adhesive properties of B lymphocytes in different stages of activation were further explored using lymphoma cell lines of the B cell lineage. The in vitro EBV-transformed B cell line IARC171 showed enhanced adhesiveness to SR-A, whereas the Burkitt lymphoma cell lines, BL41, Rael, and Bl16 did not. The SR-A-dependent adhesion of B-lymphoblasts occurred both at 37 and 4 degrees C, suggesting that it was not dependent on cell metabolism. The known polyanionic ligands for SR-A, fucoidan, and acetylated low density lipoprotein, which bind to a positively charged portion of the collagen-like domain of SR-A, did not inhibit adhesion. This finding suggests that SR-A mediates adhesion of activated B lymphocytes through a binding site that differs from the one that binds polyanionic ligands. Together, our data suggest that SR-A plays a role in the recruitment of activated B cells into lymph nodes and inflammatory lesions by acting as an adhesion molecule for such cells.

The effects of protein tyrosine kinase inhibitors on platelet-derived growth factor (PDGF)-induced chemotaxis in cultured rat aortic smooth muscle cells (SMCs) were investigated to elucidate the role of tyrosine phosphorylation in the chemotaxis of vascular SMCs. Two tyrosine kinase inhibitors, methyl 2,5-dihydroxycinnamate and genistein, inhibited PDGF-induced chemotaxis, the IC50 being 5 and 150 mumol/L, respectively. Methyl cinnamate and genistein partly inhibited the adhesion of SMC to collagen-coated dishes. A chemotaxis assay using double-well culture dishes revealed that both agents also inhibited cell migration after adhesion. H-7, a C kinase inhibitor, did not inhibit either chemotaxis or SMC adhesion at 100 mumol/L. Western blot analysis using anti-phosphotyrosine revealed that the tyrosine kinase inhibitors inhibited the tyrosine phosphorylation of at least two proteins of molecular weight 85 and 95 kD under our experimental conditions. An immunocytochemical study revealed that these inhibitors eliminated tyrosine phosphorylation along the cell margins; these agents also inhibited the reorganization of microtubules and stress fibers, both of which are involved in directional cell locomotion. These findings suggest that tyrosine kinases may play an important role in SMC chemotaxis.

Interferon gamma (IFN-gamma) is a multifunctional lymphokine secreted by activated T lymphocytes, which are found in atherosclerotic lesions. IFN-gamma has been reported to suppress the proliferation of vascular smooth muscle cells (SMCs). However, as we report in this paper, IFN-gamma is mitogenic for vascular SMCs under certain circumstances. Recombinant human IFN-gamma (1-100 units/ml), in a dose-dependent fashion, stimulated cell multiplication and [3H]thymidine and 5-bromo-2'-deoxyuridine incorporation into DNA by cultured arterial SMCs that had been growth arrested by culturing in 1% plasma-derived serum for 5 days. IFN-gamma also accentuated the mitogenic activity of platelet-derived growth factor (PDGF)-BB. A time-course study revealed that there was a time lag of 4-6 hours between the G1-->S transition of quiescent SMCs stimulated by IFN-gamma and that of SMCs stimulated by PDGF-BB. A synergistic effect of IFN-gamma on the mitogenicity of PDGF became apparent after a similar time lag, suggesting that the IFN-gamma-related mitogenicity is mediated by a substance(s) secreted by IFN-gamma-treated SMCs. In fact, conditioned medium of IFN-gamma-treated SMCs was mitogenic for SMCs. Mitogenic activity in the conditioned medium was also detected by an assay using Swiss 3T3 cells, which originate from mice and, therefore, are not responsive to human IFN-gamma. The production of the mitogenic factor was blocked by anti-IFN-gamma antibody. Mitogenicity of the conditioned medium was not eliminated by addition of neutralizing antibody against PDGF, indicating that any autocrine growth factor(s) secreted by IFN-gamma-treated SMCs was not PDGF.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of prostaglandins (PGs) A and J, which are antitumor eicosanoids, on the proliferation of cultured vascular smooth muscle cells were investigated. Serum-stimulated DNA synthesis was potently inhibited by PGA1, PGA2, PGJ2, and δ12-PGJ2 in similar dose-dependent fashions. The effects of PGA1 and PGA2 were reversible when they were removed from the culture media, whereas recoveries were only partial in the cells treated with PGJ2 and δ12-PGJ2. PGs were effective even if they were added immediately before entry into S phase. Inhibition of DNA synthesis was sustained when hydroxyurea, which blocks cell cycle at the G 1 S border, was added after the removal of PGA2, and vice versa; PGs blocked DNA synthesis when they were added after the removal of hydroxyurea. Levels of c-myc mRNA formed two peaks during the G1 phase, at 1–2 h and at 8–12 h. The PGs did not affect the first elevation, but enhanced the second and sustained it up to 18–24 h, whereas in controls, c-myc mRNA decreased quickly after entry into S phase. The rate of degradation of c-myc mRNA was much smaller in PG-treated cells than in nontreated cells. We conclude, therefore, that PGA and PGJ inhibit a crucial event(s) in the cell cycle occurring at the G 1 S border, but that this inhibition is not accompanied by the reduction in c-myc gene expression in contrast with some types of tumor cells treated With PGs.