RESUMO
Human urotensin-II (U-II) is the most potent vasoactive peptide identified to date, and may be involved in hypertension and atherosclerosis. We investigated the effects of the interactions between U-II or other vasoactive agents and mildly oxidized low-density lipoprotein (mox-LDL) or hydrogen peroxide (H2O2) on the induction of vascular smooth muscle cell (VSMC) proliferation. Growth-arrested rabbit VSMCs were incubated with vasoactive agents (U-II, endothelin-1, angiotensin-II, serotonin, or thromboxane-A2) in the presence or absence of mox-LDL or H2O2. [3H]Thymidine incorporation into DNA was measured as an index of VSMC proliferation. On interaction with mox-LDL or H2O2, U-II induced the greatest increase in [3H]thymidine incorporation among these vasoactive agents. A low concentration of U-II (10 nmol/l) enhanced the potential mitogenic effect of low concentrations of mox-LDL (120 to 337%) and H2O2 (177 to 226%). U-II at 50 nmol/l showed the maximal mitogenic effect (161%), which was abolished by G protein inactivator (GDP-beta-S), c-Src tyrosine kinase inhibitor (radicicol), protein kinase C (PKC) inhibitor (Ro31-8220), extracellular signal-regulated kinase (ERK) kinase inhibitor (PD98059), or Rho kinase inhibitor (Y27632). Mox-LDL at 5 microg/ml showed the maximal mitogenic effect (211%), which was inhibited by free radical scavenger (catalase), intracellular and extracellular antioxidants (N-acetylcysteine and probucol), nicotinamide adenine dinucleotide phosphate oxidase inhibitor (diphenylene iodonium), or c-Jun N-terminal kinase (JNK) inhibitor (SP600125). These results suggested that U-II acts in synergy with mox-LDL in inducing VSMC DNA synthesis at the highest rate among these vasoactive agents. Activation of the G protein/c-Src/PKC/ERK and Rho kinase pathways by U-II together with the redox-sensitive JNK pathway by mox-LDL may explain the synergistic interaction between these agents.
Assuntos
Peróxido de Hidrogênio/farmacologia , Lipoproteínas LDL/farmacologia , Mitógenos/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Oxidantes/farmacologia , Urotensinas/farmacologia , Aldeídos/farmacologia , Angiotensina II/farmacologia , Animais , Aorta Torácica/citologia , Células Cultivadas , DNA/biossíntese , Sinergismo Farmacológico , Endotelina-1/farmacologia , Humanos , Lipoproteínas LDL/antagonistas & inibidores , Lisofosfatidilcolinas/metabolismo , Lisofosfatidilcolinas/farmacologia , Masculino , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Coelhos , Serotonina/farmacologia , Serotoninérgicos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Tromboxano A2/farmacologia , Vasoconstritores/farmacologiaRESUMO
OBJECTIVES: We sought to compare the neurohormonal responses and clinical effects of long-term, high-dose versus low-dose enalapril in patients with chronic heart failure (CHF). BACKGROUND: Examination of neurohormonal and clinical responses in patients receiving different doses of angiotensin-converting enzyme (ACE) inhibitors may provide insight into the potential for additional suppression with angiotensin II (AT-II) or aldosterone antagonists. METHODS: Seventy-five patients with CHF were randomized to receive either high-dose (40 mg/day, n = 37) or low-dose (5 mg/day, n = 38) enalapril over six months. The results from exercise testing, echocardiography, tissue-specific ACE activity and monthly pre- and post-enalapril neurohormonal levels were compared. RESULTS: Despite greater intra-group improvements in plasma renin activity and serum aldosterone levels in the high-dose group, no statistically significant differences were observed between the two groups in all variables, except for serum ACE activity at the end of study. Elevated serum aldosterone and plasma AT-II levels were observed in 35% and 85% of patients, respectively, at 34 weeks, an inter-group difference that was not statistically significant. A trend toward higher levels of tissue-specific ACE activity in the high-dose group compared with the low-dose group at the end of study was observed (p = 0.054). A predefined composite end point of clinical events had a trend toward better improvement in the high-dose group. CONCLUSIONS: This study could not demonstrate a difference between high- and low-dose enalapril in terms of serum aldosterone and plasma AT-II suppression, despite a dose-dependent reduction in serum ACE activity. Even at maximal doses of enalapril, elevated serum aldosterone and plasma AT-II levels were frequently observed.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/administração & dosagem , Enalapril/administração & dosagem , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/fisiopatologia , Adulto , Aldosterona/sangue , Angiotensina II/sangue , Inibidores da Enzima Conversora de Angiotensina/sangue , Relação Dose-Resposta a Droga , Método Duplo-Cego , Enalapril/sangue , Epinefrina/sangue , Feminino , Insuficiência Cardíaca/sangue , Insuficiência Cardíaca/diagnóstico por imagem , Humanos , Masculino , Pessoa de Meia-Idade , Norepinefrina/sangue , Estudos Prospectivos , UltrassonografiaRESUMO
Mildly oxidized LDL (mox-LDL) has been shown to induce monocyte-endothelial interactions and vascular smooth muscle cell (VSMC) proliferation, key events in the formation of the atherosclerotic lesion. Growth factors and vasoactive peptides are also thought to play a major role in atherogenesis. We examined the interaction between mox-LDL and well-known vasoactive agents such as serotonin (5-HT), angiotensin II (Ang-II), endothelin-1 (ET-1), or urotensin II (U-II) in inducing DNA synthesis in VSMCs. Growth-arrested VSMCs were incubated with different concentrations of native LDL, mox-LDL, or highly oxidized LDL (ox-LDL) with 5-HT, Ang-II, ET-1, or U-II in the absence or presence of N-acetylcysteine (NAC), an intracellular free radical scavenger. DNA synthesis in VSMCs was examined by [3H]thymidine incorporation into cellular DNA. Mox-LDL and ox-LDL stimulated [3H]thymidine incorporation with a maximal effect at 5 microg/ml (211%, 154%), which values were significantly greater than that for native LDL (128%). 5-HT, Ang-II, ET-1, or U-II also stimulated [3H]thymidine incorporation in a dose-dependent manner. 5-HT had a maximal stimulatory effect at a concentration of 50 micromol/l (205%), Ang-II at 1.75 micromol/l (202%), ET-1 at 0.1 micromol/l (205%), and U-II at 0.05 micromol/l (161%). When added together, mox-LDL (100 ng/ml)-induced [3H]thymidine incorporation was potentiated by low concentrations of 5-HT (1 micromol/l), Ang-II (0.5 micromol/l), ET-1 (1 nmol/l), or U-II (10 nmol/l) (114% to 330%, 325%, 338%, or 345%, respectively). Synergistic interactions of mox-LDL with 5-HT, Ang-II, ET-1, or U-II were significantly inhibited by NAC (400 micromol/l). Our results suggest that mild oxidation of LDL may enhance its atherogenic potential and exert a synergistic interaction with vasoactive agents in inducing DNA synthesis via the generation of reactive oxygen species in VSMCs.
Assuntos
Acetilcisteína/farmacologia , Antioxidantes/farmacologia , Fármacos Cardiovasculares/farmacologia , Lipoproteínas LDL/farmacologia , Mitógenos/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Animais , Células Cultivadas , DNA/biossíntese , Humanos , Lipoproteínas LDL/antagonistas & inibidores , Masculino , Mitógenos/antagonistas & inibidores , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , CoelhosRESUMO
OBJECTIVE: Niacin has been used for more than 50 years to treat dyslipidemia, yet the mechanisms underlying its lipid-modifying effects remain unknown, a situation stemming at least in part from a lack of validated animal models. The objective of this study was to determine if the dyslipidemic hamster could serve as such a model. MATERIALS/METHODS: Dyslipidemia was induced in Golden Syrian hamsters by feeding them a high-fat, high-cholesterol, and high-fructose (HF/HF) diet. The effect of high-dose niacin treatment for 18 days and 28 days on plasma lipid levels and gene expression was measured. RESULTS: Niacin treatment produced significant decreases in plasma total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), triglycerides (TG), and free fatty acids (FFA), but had no measureable effect on high-density lipoprotein cholesterol (HDL-C) in the dyslipidemic hamster. Niacin treatment also produced significant increases in hepatic adenosine ATP-Binding Cassette A1 (ABCA1) mRNA, ABCA1 protein, apolipoprotein A-I (Apo A-I) mRNA, and adipose adiponectin mRNA in these animals. CONCLUSIONS: With the exception of HDL-C, the lipid effects of niacin treatment in the dyslipidemic hamster closely parallel those observed in humans. Moreover, the effects of niacin treatment on gene expression of hepatic proteins related to HDL metabolism are similar to those observed in human cells in culture. The HF/HF-fed hamster could therefore serve as an animal model for niacin's lowering of proatherogenic lipids and mechanisms of action relative to lipid metabolism.
Assuntos
Dieta Hiperlipídica/efeitos adversos , Frutose/efeitos adversos , Hipolipemiantes/farmacologia , Niacina/farmacologia , Niacina/fisiologia , Transportador 1 de Cassete de Ligação de ATP/biossíntese , Transportador 1 de Cassete de Ligação de ATP/genética , Adiponectina/biossíntese , Adiponectina/genética , Animais , Apolipoproteínas E/metabolismo , Western Blotting , Proteínas de Transferência de Ésteres de Colesterol/metabolismo , HDL-Colesterol/sangue , LDL-Colesterol/sangue , Cricetinae , Dieta , Ácidos Graxos não Esterificados/sangue , Expressão Gênica/efeitos dos fármacos , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipoproteínas/metabolismo , Masculino , Mesocricetus , Receptores de LDL/metabolismo , Triglicerídeos/sangueRESUMO
Considerable attention has been focused on both highly oxidized low-density lipoprotein (ox-LDL) and mildly oxidized LDL (mox-LDL) as important risk factors for cardiovascular disease. Further, 5-hydroxytryptamine (5-HT) appears to play a crucial role in the development of atherosclerotic plaque. We assessed the interaction of oxidatively modified LDL and its major oxidative components, ie, hydrogen peroxide (H2O2), lysophosphatidylcholine (LPC), and 4-hydroxy-2-nonenal (HNE) with 5-HT on DNA synthesis in vascular smooth muscle cells (VSMCs). Growth-arrested rabbit VSMCs were incubated in serum-free medium with native LDL, mox-LDL, ox-LDL (all 50 ng/mL), H2O2 (0.5 microM), LPC (1 microM), or HNE (0.1 microM) for 24 hours followed by 5-HT (5 microM) for another 24 hours. DNA synthesis in VSMCs was measured by [3H]thymidine incorporation. Significant effects on [3H]thymidine incorporation were observed in VSMCs incubated with mox-LDL (129%), ox-LDL (129%), H2O2 (119%), LPC (115%), HNE (127%), or 5-HT (183%) in contrast with native LDL (113%). The mitogenic effect of 5-HT was potentiated by mox-LDL, ox-LDL, H2O2, LPC, or HNE (183 to 365%, 274%, 304%, 339%, or 273%, respectively) but not by native LDL (240%). The mitogen-activated protein kinase (MAPK) kinase inhibitor PD98059 (10 microM) significantly inhibited the mitogenic effect of 5-HT but did not influence the effects of mox-LDL, ox-LDL, H2O2, LPC, or HNE. The intracellular antioxidant N-acetylcysteine (400 microM) significantly inhibited the mitogenic effects of mox-LDL, ox-LDL, H2O2, LPC, and HNE but not that of 5-HT. Our results suggest that mox-LDL, ox-LDL, and their major components H2O2, LPC, and HNE act synergistically with 5-HT in inducing VSMC DNA synthesis via MAPK and redox-sensitive pathways, contributing to the development of atherosclerotic plaque.
Assuntos
Lipoproteínas LDL/farmacologia , Mitógenos/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Serotonina/farmacologia , Animais , Arteriosclerose/etiologia , Divisão Celular/efeitos dos fármacos , Flavonoides/farmacologia , Humanos , Masculino , Músculo Liso Vascular/citologia , Coelhos , Transdução de SinaisRESUMO
Endothelin-1 (ET-1) and oxidized low-density lipoprotein (ox-LDL) are associated with atherosclerosis and essential hypertension. We assessed the effect of mildly oxidized LDL (mox-LDL) and ox-LDL and their major oxidative components, i.e., reactive oxygen species (ROS), lysophosphatidylcholine (LPC), and 4-hydroxy-2-nonenal (HNE) and their interaction with ET-1 on vascular smooth muscle cell (VSMC) proliferation. Growth-arrested VSMCs isolated from the rabbit aorta were incubated with different concentrations of LDL, mox-LDL, ox-LDL, hydrogen peroxide (H(2)O(2)) (a donor of ROS), LPC, or HNE with or without ET-1. DNA synthesis in VSMCs was measured by [(3)H] thymidine incorporation. Mox-LDL, ox-LDL, H(2)O(2), LPC, HNE, or ET-1 stimulated DNA synthesis in a dose-dependent manner. Maximal effect was observed at 5 microg/ml for mox-LDL (162%) or ox-LDL (154%), 15 microM LPC (156%), 5 microM H2O2 (177%), 1 microM HNE (144%), and 0.1 microM ET-1 (195%). By contrast, LDL was without any significant effect. When added together, there was no synergistic effect of LDL, H2O2, or HNE with ET-1 on DNA synthesis. However, the effect of mox-LDL (0.1 microg/ml), ox-LDL (0.5 microg/ml), or LPC (10 microM) was potentiated by ET-1 (114%-338%, 133%-425%, 118%-333%, respectively). The mitogenic effect of mox-LDL, ox-LDL, or LPC and their interaction with ET-1 were inhibited by defatted albumin (10 microg/ml), antioxidant N-acetylcysteine (400 microM), the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor diphenylene iodonium (1 microM). The ET(A/B) receptor antagonist TAK044 (1 microM) or the MAPK kinase inhibitor PD098059 (10 microM) inhibited the mitogenic effect of ET-1 and its interaction with mox-LDL, ox-LDL, or LPC. The synergistic interaction of mox-LDL, ox-LDL, or LPC with ET-1 was completely reversed by the combined use of N-acetylcysteine and TAK044. Our results suggest that mox-LDL, ox-LDL, and their major phospholipid component LPC act synergistically with ET-1 in inducing VSMC proliferation by way of the activation of redox-sensitive and MAPK pathways.
Assuntos
Endotelina-1/fisiologia , Lipoproteínas LDL/fisiologia , Lisofosfatidilcolinas/farmacologia , Músculo Liso Vascular/metabolismo , Acetilcisteína/farmacologia , Aldeídos/farmacologia , Animais , Aorta Torácica/citologia , Aorta Torácica/metabolismo , Contagem de Células , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Células Cultivadas , DNA/biossíntese , Antagonistas dos Receptores de Endotelina , Flavonoides/farmacologia , Sequestradores de Radicais Livres/farmacologia , Inibidores do Crescimento/farmacologia , Peróxido de Hidrogênio/farmacologia , Masculino , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Músculo Liso Vascular/citologia , NADPH Oxidases/antagonistas & inibidores , Oniocompostos/farmacologia , Oxidantes/farmacologia , Peptídeos Cíclicos/farmacologia , Coelhos , Espécies Reativas de Oxigênio/metabolismoRESUMO
INTRODUCTION: Cell-cell interaction is an essential component of atherosclerotic plaque development. Activated monocytes appear to play a central role in the development of atherosclerosis, not only through foam cell formation but also via the production of various growth factors that induce proliferation of different cell types that are involved in the plaque development. Using serum free co-culture method, we determined the effect of monocytes on endothelial cell proliferation. METHODS: Endothelial cell proliferation is determined by the amount of [3H]thymidine incorporated in to the DNA. Basic fibroblast growth factor (b-FGF), vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) levels in the conditioned medium were determined by ELISA. RESULTS: Conditioned medium from unactivated monocytes partially inhibited endothelial cell proliferation, whereas conditioned medium from activated monocytes promoted endothelial cell proliferation. The mitogenic effect of conditioned medium derived from activated monocytes is due to the presence of b-FGF, VEGF and IL-8. Neutralizing antibodies against b-FGF, VEGF and IL-8 partially reversed the mitogenic effect of conditioned medium derived from activated monocytes. When b-FGF, VEGF and IL-8 were immunoprecipitated from conditioned medium derived from activated monocytes, it is less mitogenic to endothelial cells. CONCLUSION: Activated monocytes may play an important role in the development of atherosclerotic plaque by producing endothelial cell growth factors.
Assuntos
Indutores da Angiogênese/farmacologia , Arteriosclerose/fisiopatologia , Comunicação Celular/efeitos dos fármacos , Comunicação Celular/fisiologia , Fenômenos Fisiológicos Celulares/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiopatologia , Monócitos/efeitos dos fármacos , Monócitos/fisiologia , Indutores da Angiogênese/análise , Arteriosclerose/etiologia , Meios de Cultura Livres de Soro/química , Meios de Cultura Livres de Soro/farmacologia , Fatores de Crescimento Endotelial/análise , Endotélio Vascular/química , Fator 2 de Crescimento de Fibroblastos/análise , Humanos , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intercelular/análise , Interleucina-8/análise , Linfocinas/análise , Monócitos/química , Timidina/análise , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
We examined the mechanism of action of lysophosphatidylcholine (lyso-PC), which is suggested to be involved in the pathogenesis of atherosclerosis and inflamatory disorders, and its interaction with well-known vasoactive compounds such as hydrogen peroxide (H2O2), thromboxane A2 (TX-A2), serotonin (5-HT), angiotensin II (Ang-II), endothelin-1 (ET-1), or urotensin II (U-II) on VSMC proliferation. Growth-arrested rabbit VSMCs were incubated with given concentrations of lyso-PC with H202, TX-A2, 5-HT, Ang-II, ET-1, or U-II. [3H]Thymidine incorporation into DNA was measured as an index of VSMC proliferation. Lyso-PC induced a maximal effect on [3H]thymidine incorporation at a concentration of 15 microM (156%), and its effect was significantly inhibited by the phospholipase C inhibitor U73122 (10 microM), the intracellular antioxidant NAC (400 microM), and the NADPH oxidase inhibitor diphenylene iodonium (1 microM), but not by the MAPK kinase inhibitor (10 microM). H2O2, TX-A2, 5-HT, Ang-II, ET-1, or U-II also stimulated [3H]thymidine incorporation in a dose-dependent manner. A non-mitogenic concentration of lyso-PC (5 microM) significantly potentiated the effect of low concentrations of H2O2 (0.1 microM, 110 to 222%), TX-A2 (5 microM, 120 to 202%), 5-HT (5 microM, 182 to 259%), Ang-II (0.5 microM, 167 to 304%), ET-1 (0.01 microM, 139 to 297%), or U-II (0.025 microM, 120 to 332%) on [3H]thymidine incorporation. The results suggest that lyso-PC acts synergistically with the vasoactive compounds H2O2, TX-A2, 5-HT, Ang-II, ET-1, or U-II in inducing VSMC proliferation, which may play an important role in the progression of atherosclerosis.