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1. |
The IntimaSoil for Atherosclerosis and Restenosis |
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Circulation Research,
Volume 77,
Issue 3,
1995,
Page 445-465
Stephen M. Schwartz,
Denis deBlois,
Edward R.M. O'Brien,
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ISSN:0009-7330
出版商:OVID
年代:1995
数据来源: OVID
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2. |
Characterization of 5 prime End of Human Thromboxane Receptor Gene; Organizational Analysis and Mapping of Protein Kinase C-Responsive Elements Regulating Expression in Platelets |
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Circulation Research,
Volume 77,
Issue 3,
1995,
Page 466-474
Drew D. D'Angelo,
Michael G. Davis,
William A. Houser,
Jeremy J. Eubank,
Michael E. Ritchie,
Gerald W. Dorn II,
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摘要:
Platelet thromboxane receptors are acutely and reversibly upregulated after acute myocardial infarction. To determine if platelet thromboxane receptors are under transcriptional control, we isolated and characterized human genomic DNA clones containing the 5 prime flanking region of the thromboxane receptor gene. The exon-intron structure of the 5 prime portion of the thromboxane receptor gene was determined initially by comparing the nucleotide sequence of the 5 prime flanking genomic clone with that of a novel human uterine thromboxane receptor cDNA that extended the mRNA 141 bp further upstream than the previously identified human placental cDNA. A major transcription initiation site was located in three human tissues approximate equals 560 bp upstream from the translation initiation codon and 380 bp upstream from any previously identified transcription initiation site. The thromboxane receptor gene has neither a TATA nor a CAAT consensus site. Promoter function of the 5 prime flanking region of the thromboxane receptor gene was evaluated by transfection of thromboxane receptor gene promoter/chloramphenicol acetyltransferase (CAT) chimera plasmids into plateletlike K562 cells. Thromboxane receptor promoter activity, as assessed by CAT expression, was relatively weak but was significantly enhanced by phorbol ester treatment. Functional analysis of 5 prime deletion constructs in transfected K562 cells and gel mobility shift localized the major phorbol ester-responsive motifs in the thromboxane receptor gene promoter to a cluster of activator protein-2 (AP-2) binding consensus sites located approximate equals 1.8 kb 5 prime from the transcription initiation site. These studies are the first to determine the structure and organization of the 5 prime end of the thromboxane receptor gene and demonstrate that thromboxane receptor gene expression can be regulated by activation of protein kinase C via induction of an AP-2-like nuclear factor binding to upstream promoter elements. These findings strongly suggest that the mechanism for previously described upregulation of platelet thromboxane receptors after acute myocardial infarction is increased thromboxane receptor gene transcription in platelet-progenitor cells.(Circ Res. 1995;77:466-474.)
ISSN:0009-7330
出版商:OVID
年代:1995
数据来源: OVID
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3. |
Endothelium-Specific In Vivo Gene Transfer |
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Circulation Research,
Volume 77,
Issue 3,
1995,
Page 475-485
Andrew H. Schulick,
Gang Dong,
Kurt D. Newman,
Renu Virmani,
David A. Dichek,
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摘要:
Targeted expression of genetic material within the vascular endothelium is potentially a powerful tool for the investigation of endothelial cell (EC) biology. We developed, optimized, and characterized an efficient somatic transgenic model of EC-specific gene transfer. Rat carotid arteries were infused with adenovirus expressing a beta-galactosidase (beta-gal) gene. The level and cell-type specificity of recombinant gene expression were measured by assaying beta-gal activity in vessel extracts and by counting transduced cells in histological sections. Toxicity was evaluated by counting total ECs (3 days) and by measuring neointimal formation (14 days). Effects of transduction on the proliferation of vascular cells were measured with bromodeoxyuridine and [sup 3 H]thymidine. Maximum recombinant gene expression resulted from infusion of 1 times 1010to 1 times 1011plaque-forming units (pfu) per milliliter; approximate equals 35% of luminal ECs were transduced. A high degree of EC specificity (90% to 98% of total transduced cells) was maintained over this range of virus concentrations. More highly concentrated virus resulted in loss of beta-gal expression and a large decrease in luminal EC number (97% decrease, P less than .001). Gene transfer at 4 times 1010pfu/mL was efficient, preserved EC integrity, and caused minimal neointimal formation. After gene transfer, there were early (3-day) increases in both EC and smooth muscle cell proliferation. At 14 days, only EC proliferation remained elevated (18% versus 1.4% in vehicle-infused arteries, P equals .005). This animal model permits efficient highly EC-specific gene transfer. Vascular toxicity is minimal, although the EC proliferative index is elevated. This model will be useful in experiments that elucidate the biological role of EC gene products and define pathways of EC gene regulation and signal transduction in vivo.(Circ Res. 1995;77:475-485.)
ISSN:0009-7330
出版商:OVID
年代:1995
数据来源: OVID
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4. |
Contractile Responsiveness of Ventricular Myocytes to Isoproterenol Is Regulated by Induction of Nitric Oxide Synthase Activity in Cardiac Microvascular Endothelial Cells in Heterotypic Primary Culture |
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Circulation Research,
Volume 77,
Issue 3,
1995,
Page 486-493
Dan Ungureanu-Longrois,
Jean-Luc Balligand,
Ikutaro Okada,
William W. Simmons,
Lester Kobzik,
Charles J. Lowenstein,
Steven L. Kunkel,
Thomas Michel,
Ralph A. Kelly,
Thomas W. Smith,
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摘要:
Unlike large-vessel endothelial cells in cell culture, cardiac microvascular endothelial cells (CMEC) isolated from adult rat ventricular muscle exhibit little detectable constitutive nitric oxide (NO) synthase activity after isolation in vitro but respond to specific combinations of inflammatory mediators with an increase in inducible NO synthase (iNOS; type 2 NO synthase) activity. CMEC iNOS is induced by soluble inflammatory mediators in lipopolysaccharide-activated rat alveolar macrophage-conditioned medium at 24 hours, and this induction can be partially prevented by either interleukin-1 (IL-1) receptor antagonist or a polyclonal anti-rat tumor necrosis factor-alpha (TNF-alpha) antiserum. Interferon-gamma (IFN-gamma), which by itself does not induce iNOS in CMEC, potentiates and accelerates iNOS induction by IL-1 beta. Transforming growth factor-beta (TGF-beta) decreases iNOS activity, protein content, and mRNA abundance in IL-1 beta- and IFN-gamma-pretreated CMEC. To determine whether NO released by CMEC would affect myocyte contractile function in vitro, freshly isolated ARVM were allowed to settle onto confluent, serum-starved CMEC that had been pretreated for 24 hours with IL-1 beta, a cytokine that alone does not affect myocyte contractile function in vitro. Baseline contractile amplitude, at 2 Hz and 37 degrees C, of myocytes in heterotypic culture with IL-1 beta-pretreated CMEC was not different from that of myocytes in control, homotypic myocyte cultures. However, cocultured myocytes exhibited decreased contractile responsiveness to 2 nmol/L isoproterenol compared with control cells, and this could be reversed by the addition of 1 mmol/L NG-monomethyl-L-arginine, an inhibitor of NOS. Thus, activation of endothelial cell iNOS by specific cytokines affects the contractile responsiveness to isoproterenol of adjacent cardiac myocytes in vitro. Reciprocal cell-cell signaling leading to TGF-beta release and activation could act to limit the extent of endothelial cell iNOS induction in vivo.(Circ Res. 1995;77:486-493.)
ISSN:0009-7330
出版商:OVID
年代:1995
数据来源: OVID
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5. |
Induction of Nitric Oxide Synthase Activity by Cytokines in Ventricular Myocytes Is Necessary but Not Sufficient to Decrease Contractile Responsiveness to beta-Adrenergic Agonists |
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Circulation Research,
Volume 77,
Issue 3,
1995,
Page 494-502
Dan Ungureanu-Longrois,
Jean-Luc Balligand,
William W. Simmons,
Ikutaro Okada,
Lester Kobzik,
Charles J. Lowenstein,
Steven L. Kunkel,
Thomas Michel,
Ralph A. Kelly,
Thomas W. Smith,
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摘要:
Recent evidence has documented that increased activity of an inducible nitric oxide synthase (iNOS; type 2 NO synthase) in primary isolates of adult rat ventricular myocytes after exposure to soluble mediators in medium conditioned by lipopolysaccharide-activated macrophages is associated with a decrease in their contractile responsiveness to beta-adrenergic agonists. It remained unclear which specific inflammatory cytokines in this medium contribute to the induction of iNOS activity in myocytes and whether induction of iNOS would result in an obligatory decline in contractile function. Interleukin (IL)-1 beta and tumor necrosis factor-alpha (TNF-alpha) were both present in the lipopolysaccharide-activated macrophage-conditioned medium. However, only IL-1 receptor antagonist and not an anti-rat TNF-alpha antiserum diminished the extent of iNOS induction in myocytes exposed to this medium and prevented a decline in contractile responsiveness to isoproterenol. When recombinant cytokines were used, IL-1 beta, TNF-alpha, and IFN-gamma each induced iNOS activity in cardiac myocytes at 24 hours. However, only the combination of IL-1 beta and IFN-gamma reproducibly caused contractile dysfunction in cardiac myocytes. Among the constituents of the defined medium routinely used for maintenance of adult rat ventricular myocytes in primary culture, it was noted that insulin (10minus7 mol/L) was required for NO production, as detected by nitrite release in cytokine-pretreated myocytes, although insulin had no effect on the extent of induction of iNOS mRNA or maximal enzyme activity in myocyte cell lysates. Insulin was also required for the decrease in contractile responsiveness to isoproterenol to be manifest. Thus, induction of iNOS is necessary but not sufficient to cause inflammatory cytokine-induced contractile dysfunction in cardiac myocytes.(Circ Res. 1995;77:494-502.)
ISSN:0009-7330
出版商:OVID
年代:1995
数据来源: OVID
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6. |
Thrombin Regulates Expression of Monocyte Chemoattractant Protein-1 in Vascular Smooth Muscle Cells |
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Circulation Research,
Volume 77,
Issue 3,
1995,
Page 503-509
Ulrich O. Wenzel,
Bruno Fouqueray,
Giuseppe Grandaliano,
Yong-Soo Kim,
Costantinos Karamitsos,
Anthony J. Valente,
Hanna E. Abboud,
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摘要:
Thrombin, a serine protease generated at sites of vascular injury, plays a role in the pathogenesis of atherosclerosis and restenosis after angioplasty. Adherence of monocytes to the endothelium and migration into the subendothelial space is an important early event in the pathogenesis of atherosclerosis. Monocyte chemoattractant protein 1 (MCP-1) may be an important mediator of monocyte recruitment to the tissue in this and other diseases. We have characterized the expression of MCP-1 in vascular smooth muscle cells (VSMCs) isolated from human renal artery and studied its regulation by thrombin. Serum-deprived cells release monocyte chemotactic activity that is neutralized (80%) by an MCP-1 antibody. The antibody recognized a 13- and 15-kD protein in smooth muscle cell-conditioned medium. Thrombin stimulates MCP-1 gene expression in a concentration- and time-dependent manner. An increase over basal levels was observed with concentrations of thrombin as low as 0.05 U/mL. The maximal effect occurred at 5 U/mL. The stimulatory effect was detected within 1 hour, reached a maximum at 3 hours, and was still present at 8 to 24 hours after the addition of thrombin. A concentration- and time-dependent effect of thrombin on MCP-1 gene expression was also found in rat VSMCs. The thrombin protease inhibitor hirudin blocked thrombin-induced MCP-1 expression. Thrombin stimulated the release of MCP-1 protein in conditioned medium of human VSMCs as measured by radioimmunoassay and chemotactic assay. Thrombin also increased monocyte chemotactic activity in short-term organ cultures of rat aortic rings and in first passage cells. The effect of thrombin on MCP-1 was mimicked by a thrombin receptor-activating peptide (NH2-Ser-Phe-Leu-Leu-Arg-Asn-Pro-COOH). These data describe an important biological activity of thrombin in VSMCs and provide a novel mechanism whereby locally released thrombin may contribute to the pathogenesis of atherosclerosis or restenosis after angioplasty.(Circ Res. 1995;77:503-509.)
ISSN:0009-7330
出版商:OVID
年代:1995
数据来源: OVID
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7. |
Native Low-Density Lipoprotein Increases Endothelial Cell Nitric Oxide Synthase Generation of Superoxide Anion |
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Circulation Research,
Volume 77,
Issue 3,
1995,
Page 510-518
Kirkwood A. Pritchard,
Laura Groszek,
David M. Smalley,
William C. Sessa,
Mingdan Wu,
Patricio Villalon,
Michael S. Wolin,
Michael B. Stemerman,
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摘要:
To examine mechanisms by which native lowdensity lipoprotein (n-LDL) perturbs endothelial cell (EC) release of superoxide anion (O2minus) and nitric oxide (NO), ECs were incubated with n-LDL at 240 mg cholesterol per deciliter for 4 days with media changes every 24 hours. n-LDL increases EC release of O2minusby more than fourfold and increases nitrite production by 57%. In the conditioned media from day-4 incubations, n-LDL increases total nitrogen oxides 20 times control EC (C-EC) levels. However, n-LDL did not alter EC NO synthase (eNOS) enzyme activity as measured by the [sup 3 H]citrulline assay. Nomega-Nitro-L-arginine methyl ester, a specific inhibitor of eNOS activity, increases C-EC release of O2minusby more than 300% but decreases LDL-treated EC (LDL-EC) release by more than 95%. L-Arginine inhibits the release of O2minusfrom LDL-ECs by more than 95% but did not effect C-EC release of O2minus. Indomethacin and SKF 525A partially attenuate LDL-induced increases in O2minusproduction by approximate equals 50% and 30%, respectively. Thus, n-LDL increases O2minusand NO production, which increases the likelihood of the formation of peroxynitrite (ONOO sup minus), a potent oxidant. n-LDL increases the levels of nitrotyrosine, a stable oxidation product of ONOOminus, and tyrosine by approximate equals 50%. In spite of this increase in oxidative metabolism, analysis of thiobarbituric acid substances reveals that no significant changes in the oxidation of n-LDL occur during the 24-hour incubations with ECs. These data indicate that n-LDL directly perturbs endothelial oxidative metabolism and uncouples L-arginine metabolism from NO release to increase eNOS generation of O2minus. Such changes may represent one of the earliest EC perturbations in atherogenesis.(Cir Res. 1995;77:510-518.)
ISSN:0009-7330
出版商:OVID
年代:1995
数据来源: OVID
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8. |
Epidermal Growth Factor Receptor-Targeted Cytotoxin Inhibits Neointimal Hyperplasia In VivoResults of Local Versus Systemic Administration |
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Circulation Research,
Volume 77,
Issue 3,
1995,
Page 519-529
Christopher J. Pastore,
Jeffrey M. Isner,
Patricia A. Bacha,
Marianne Kearney,
J. Geoffrey Pickering,
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摘要:
Smooth muscle cell accumulation is a key feature of restenosis that may be inhibited by the delivery of receptortargeted cytotoxins. DAB389EGF is a recombinant fusion protein in which the receptor-binding domain of diphtheria toxin has been replaced by human epidermal growth factor (EGF). We investigated the effectiveness of DAB389EGF to inhibit neointimal hyperplasia in the balloon-injured rat carotid artery. Incubation of rat carotid arteries with125I-labeled EGF revealed extensive EGF binding sites in the neointima of ballooninjured arteries. Sixty rats subsequently received either saline or DAB389EGF (total dose, 0.15 mg) delivered immediately following balloon injury either systemically, via 14-day continuous osmotic pump infusion, or locally, via 30-minute intraluminal incubation. The effect of both treatment strategies was measured 2 weeks after injury by cross-sectional morphometric analysis of intimal area, the ratio of intimal/medial area (I/M), and the percent luminal narrowing (%LN). In addition, proliferative activity was assessed by immunostaining for the presence of the proliferating cell nuclear antigen (PCNA). Compared with controls, systemic delivery of fusion toxin significantly reduced intimal area, I/M, and %LN by 40%, 40%, and 29%, respectively. However, these rats exhibited 2% weight loss, indicating mild systemic toxicity. Local, intraluminal administration of DAB sub 389 EGF yielded a more pronounced reduction in intimal area, I/M, and %LN by 74%, 79%, and 72%, respectively. This inhibitory effect was preserved at 3 weeks postinjury, and PCNA immunostaining of locally treated arteries revealed a virtual absence of proliferative activity in the neointima and media at this timepoint. In contrast to systemically treated rats, rats receiving fusion toxin locally gained weight at a rate similar to controls, indicating avoidance of systemic toxicity. We conclude that DAB389EGF is a potent inhibitor of neointimal hyperplasia in vivo and that whereas an inhibitory effect may be achieved by systemic delivery, local delivery appears to be more potent, avoids systemic toxicity, and thus represents a feasible strategy to preempt restenosis.(Circ Res. 1995;77:519-529.)
ISSN:0009-7330
出版商:OVID
年代:1995
数据来源: OVID
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9. |
Elevated Levels of cAMP Inhibit Protein Kinase C-Independent Mechanisms of Endothelial Platelet-Derived Growth Factor-B Chain and Intercellular Adhesion Molecule-1 Gene Induction by Lysophosphatidylcholine |
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Circulation Research,
Volume 77,
Issue 3,
1995,
Page 530-535
Hiroshi Ochi,
Noriaki Kume,
Eiichiro Nishi,
Toru Kita,
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摘要:
Lysophosphatidylcholine (lyso-PC), a polar phospholipid product increased in atherogenic lipoproteins and atherosclerotic lesions, has been shown to differentially induce functional intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 and mRNA for platelet-derived growth factor (PDGF)-A and -B chains and heparin-binding epidermal growth factor-like growth factor in various cultured endothelial cells. In this study, we have demonstrated increased expression of cell- and matrix-associated forms of PDGF-B chain (PDGF-B) protein elicited by lyso-PC and further characterized potential signal transduction mechanisms responsible for lyso-PC-induced gene expression, focusing on PDGF-B and ICAM-1 genes in cultured human umbilical vein endothelial cell models. Cycloheximide almost completely inhibited PDGF-B but not ICAM-1 mRNA induction elicited by lyso-PC, suggesting that dependence on de novo protein synthesis for PDGF-B is different from that for ICAM-1. Prolonged exposure to phorbol myristate acetate (PMA), which depletes protein kinase C (PKC), or staurosporine, a PKC inhibitor, did not block lyso-PC-induced increases in PDGF-B or ICAM-1 mRNA. Forskolin and dibutyryl cAMP, which elevate intracellular cAMP levels, blocked both PDGF-B and ICAM-1 upregulation elicited by lyso-PC; however, these cAMP-elevating agents did not suppress ICAM-1 upregulation by PMA. Taken together, PDGF-B and ICAM-1 gene induction by lyso-PC may involve different signaling mechanisms; however, both appear to be independent of PMA-regulatable PKC activation but are suppressed by increased levels of intracellular cAMP.(Circ Res. 1995;77:530-535.)
ISSN:0009-7330
出版商:OVID
年代:1995
数据来源: OVID
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10. |
Evidence That Expression of Inducible Nitric Oxide Synthase in Response to Endotoxin Is Augmented in Atherosclerotic Rabbits |
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Circulation Research,
Volume 77,
Issue 3,
1995,
Page 536-543
Christopher G. Sobey,
Robert M. Brooks,
Donald D. Heistad,
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摘要:
Atherosclerotic lesions contain monocytes/macrophages and vascular smooth muscle cells and thus may have an increased capacity for generation of nitric oxide by inducible nitric oxide synthase (NOS). We used three approaches (contractile responses, generation of L-citrulline from L-arginine, and staining with NADPH-diaphorase) to test the hypothesis that after administration of lipopolysaccharide (LPS) in vivo, generation of nitric oxide by inducible NOS is augmented in atherosclerotic arteries. New Zealand White (normal, n equals 18) and Watanabe heritable hyperlipidemic (atherosclerotic, n equals 21) rabbits were anesthetized and injected intravenously with vehicle or LPS. Contractile responsiveness of aortic segments was examined in vitro 4 hours after injection of LPS in vivo. There was a substantial (approximately fivefold) decrease in contractile sensitivity of aortas from LPS-treated atherosclerotic rabbits and a small (approximately twofold) decrease in normal rabbits. Incubation of aortic segments with aminoguanidine, which inhibits inducible NOS, restored contractile responsiveness after LPS treatment. In vitro assay of conversion of [sup 14 C]L-arginine to [sup 14 C]L-citrulline by aortic segments demonstrated marked (approximately fivefold) increase in calcium-independent conversion of [sup 14 C]L-arginine by LPS-treated atherosclerotic, but not normal, aortas. NADPH-diaphorase staining demonstrated positive cells only in the endothelium of normal rabbits and in the lesions and media of the atherosclerotic aortas in both vehicle- and LPS-treated rabbits. The general distribution of these NADPH-diaphorase-positive cells resembled that of smooth muscle cells and not macrophages. Thus, impairment of contractile responses, generation of L-citrulline, and staining with NADPH-diaphorase suggest that atherosclerotic arteries have increased capacity for generation of nitric oxide by inducible NOS.(Circ Res. 1995;77:536-543.)
ISSN:0009-7330
出版商:OVID
年代:1995
数据来源: OVID
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