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1.
Int J Mol Sci ; 22(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34445519

RESUMO

Cardiovascular disease is the leading cause of morbidity and mortality in diabetes. Recent clinical studies indicate that sodium-glucose co-transporter 2 (SGLT2) inhibitors improve cardiovascular outcomes in patients with diabetes. The mechanism underlying the beneficial effect of SGLT2 inhibitors is not completely clear but may involve direct actions on vascular cells. SGLT2 inhibitors increase the bioavailability of endothelium-derived nitric oxide and thereby restore endothelium-dependent vasodilation in diabetes. In addition, SGLT2 inhibitors favorably regulate the proliferation, migration, differentiation, survival, and senescence of endothelial cells (ECs). Moreover, they exert potent antioxidant and anti-inflammatory effects in ECs. SGLT2 inhibitors also inhibit the contraction of vascular smooth muscle cells and block the proliferation and migration of these cells. Furthermore, studies demonstrate that SGLT2 inhibitors prevent postangioplasty restenosis, maladaptive remodeling of the vasculature in pulmonary arterial hypertension, the formation of abdominal aortic aneurysms, and the acceleration of arterial stiffness in diabetes. However, the role of SGLT2 in mediating the vascular actions of these drugs remains to be established as important off-target effects of SGLT2 inhibitors have been identified. Future studies distinguishing drug- versus class-specific effects may optimize the selection of specific SGLT2 inhibitors in patients with distinct cardiovascular pathologies.


Assuntos
Complicações do Diabetes/prevenção & controle , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Remodelação Vascular/efeitos dos fármacos , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Complicações do Diabetes/metabolismo , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Humanos , Óxido Nítrico/metabolismo , Inibidores do Transportador 2 de Sódio-Glicose/uso terapêutico
2.
Redox Biol ; 32: 101527, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32278282

RESUMO

Recent cardiovascular outcome trials found that sodium-glucose cotransporter-2 (SGLT2) inhibitors reduce cardiovascular disease and mortality in type 2 diabetic patients; however, the underlying mechanisms are not fully known. Since the proliferation and migration of vascular smooth muscle cells (SMCs) contributes to the development of arterial lesions, we hypothesized that SGLT2 inhibitors may exert their beneficial cardiovascular effects by inhibiting the growth and movement of vascular SMCs. Treatment of rat or human aortic SMCs with clinically relevant concentrations of canagliflozin, but not empagliflozin or dapagliflozin, inhibited cell proliferation and migration. The inhibition of SMC growth by canagliflozin occurred in the absence of cell death, and was associated with the arrest of SMCs in the G0/G1 phase of the cell cycle and diminished DNA synthesis. Canagliflozin also resulted in the induction of heme oxygenase-1 (HO-1) expression, and a rise in HO activity in vascular SMCs, whereas, empagliflozin or dapagliflozin had no effect on HO activity. Canagliflozin also activated the HO-1 promoter and this was abrogated by mutating the antioxidant responsive element or by overexpressing dominant-negative NF-E2-related factor-2 (Nrf2). The induction of HO-1 by canagliflozin relied on reactive oxygen species (ROS) formation and was negated by antioxidants. Finally, silencing HO-1 expression partially rescued the proliferative and migratory response of canagliflozin-treated SMCs, and this was reversed by carbon monoxide and bilirubin. In conclusion, the present study identifies canagliflozin as a novel inhibitor of vascular SMC proliferation and migration. Moreover, it demonstrates that canagliflozin stimulates the expression of HO-1 in vascular SMCs via the ROS-Nrf2 pathway, and that the induction of HO-1 contributes to the cellular actions of canagliflozin. The ability of canagliflozin to exert these pleiotropic effects may contribute to the favorable clinical actions of the drug and suggest an extra potential benefit of canagliflozin relative to other SGLT2 inhibitors.


Assuntos
Heme Oxigenase-1 , Músculo Liso Vascular , Animais , Canagliflozina/farmacologia , Proliferação de Células , Células Cultivadas , Heme Oxigenase (Desciclizante) , Heme Oxigenase-1/genética , Humanos , Miócitos de Músculo Liso , Ratos
3.
Front Pharmacol ; 10: 362, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31057401

RESUMO

Recent clinical trials revealed that sodium-glucose co-transporter 2 (SGLT2) inhibitors significantly reduce cardiovascular events in type 2 diabetic patients, however, canagliflozin increased limb amputations, an effect not seen with other SGLT2 inhibitors. Since endothelial cell (EC) dysfunction promotes diabetes-associated vascular disease and limb ischemia, we hypothesized that canagliflozin, but not other SGLT2 inhibitors, impairs EC proliferation, migration, and angiogenesis. Treatment of human umbilical vein ECs (HUVECs) with clinically relevant concentrations of canagliflozin, but not empagliflozin or dapagliflozin, inhibited cell proliferation. In particular, 10 µM canagliflozin reduced EC proliferation by approximately 45%. The inhibition of EC growth by canagliflozin occurred in the absence of cell death and was associated with diminished DNA synthesis, cell cycle arrest, and a striking decrease in cyclin A expression. Restoration of cyclin A expression via adenoviral-mediated gene transfer partially rescued the proliferative response of HUVECs treated with canagliflozin. A high concentration of canagliflozin (50 µM) modestly inhibited HUVEC migration by 20%, but markedly attenuated their tube formation by 65% and EC sprouting from mouse aortas by 80%. A moderate 20% reduction in HUVEC migration was also observed with a high concentration of empagliflozin (50 µM), while neither empagliflozin nor dapagliflozin affected tube formation by HUVECs. The present study identified canagliflozin as a robust inhibitor of human EC proliferation and tube formation. The anti-proliferative action of canagliflozin occurs in the absence of cell death and is due, in part, to the blockade of cyclin A expression. Notably, these actions are not seen with empagliflozin or dapagliflozin. The ability of canagliflozin to exert these pleiotropic effects on ECs may contribute to the clinical actions of this drug.

4.
Biochem Pharmacol ; 156: 204-214, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30144404

RESUMO

Glutaminase-1 (GLS1) is a mitochondrial enzyme found in endothelial cells (ECs) that metabolizes glutamine to glutamate and ammonia. Although glutaminolysis modulates the function of human umbilical vein ECs, it is not known whether these findings extend to human ECs beyond the fetal circulation. Furthermore, the molecular mechanism by which GLS1 regulates EC function is not defined. In this study, we show that the absence of glutamine in the culture media or the inhibition of GLS1 activity or expression blocked the proliferation and migration of ECs derived from the human umbilical vein, the human aorta, and the human microvasculature. GLS1 inhibition arrested ECs in the G0/G1 phase of the cell cycle and this was associated with a significant decline in cyclin A expression. Restoration of cyclin A expression via adenoviral-mediated gene transfer improved the proliferative, but not the migratory, response of GLS1-inhibited ECs. Glutamine deprivation or GLS1 inhibition also stimulated the production of reactive oxygen species and this was associated with a marked decline in heme oxygenase-1 (HO-1) expression. GLS1 inhibition also sensitized ECs to the cytotoxic effect of hydrogen peroxide and this was prevented by the overexpression of HO-1. In conclusion, the metabolism of glutamine by GLS1 promotes human EC proliferation, migration, and survival irrespective of the vascular source. While cyclin A contributes to the proliferative action of GLS1, HO-1 mediates its pro-survival effect. These results identify GLS1 as a promising therapeutic target in treating diseases associated with aberrant EC proliferation, migration, and viability.


Assuntos
Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Células Endoteliais/enzimologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glutaminase/metabolismo , Glutamina/farmacologia , Aorta/citologia , Benzenoacetamidas/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Ciclina A/genética , Ciclina A/metabolismo , Diazo-Oxo-Norleucina/farmacologia , Células Endoteliais/efeitos dos fármacos , Glutaminase/antagonistas & inibidores , Glutaminase/genética , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Humanos , Interferência de RNA , Tiadiazóis/farmacologia , Veias/citologia
5.
Amino Acids ; 50(6): 747-754, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29700652

RESUMO

This study investigated the temporal activation of arginase in obese Zucker rats (ZR) and determined if arginase inhibition prevents the development of hypertension and improves insulin resistance in these animals. Arginase activity, plasma arginine and nitric oxide (NO) concentration, blood pressure, and insulin resistance were measured in lean and obese animals. There was a chronological increase in vascular and plasma arginase activity in obese ZR beginning at 8 weeks of age. The increase in arginase activity in obese animals was associated with a decrease in insulin sensitivity and circulating levels of arginine and NO. The rise in arginase activity also preceded the increase in blood pressure in obese ZR detected at 12 weeks of age. Chronic treatment of 8-week-old obese animals with an arginase inhibitor or L-arginine for 4 weeks prevented the development of hypertension and improved plasma concentrations of arginine and NO. Arginase inhibition also improved insulin sensitivity in obese ZR while L-arginine supplementation had no effect. In conclusion, arginase inhibition prevents the development of hypertension and improves insulin sensitivity while L-arginine administration only mitigates hypertension in obese animals. Arginase represents a promising therapeutic target in ameliorating obesity-associated vascular and metabolic dysfunction.


Assuntos
Arginase/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Hipertensão/tratamento farmacológico , Resistência à Insulina , Obesidade/tratamento farmacológico , Animais , Arginase/metabolismo , Arginina/sangue , Hipertensão/sangue , Masculino , Óxido Nítrico/sangue , Obesidade/sangue , Ratos , Ratos Zucker
6.
Free Radic Biol Med ; 102: 37-46, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27867098

RESUMO

Although endothelial cells produce substantial quantities of ammonia during cell metabolism, the physiologic role of this gas in these cells is not known. In this study, we investigated if ammonia regulates the expression of heme oxygenase-1 (HO-1), and if this enzyme influences the biological actions of ammonia on endothelial cells. Exogenously administered ammonia, given as ammonium chloride or ammonium hydroxide, or endogenously generated ammonia stimulated HO-1 protein expression in cultured human and murine endothelial cells. Dietary supplementation of ammonia also induced HO-1 protein expression in murine arteries. The increase in HO-1 protein by ammonia in endothelial cells was first detected 4h after ammonia exposure and was associated with the induction of HO-1 mRNA, enhanced production of reactive oxygen species (ROS), and increased expression and activity of NF-E2-related factor-2 (Nrf2). Ammonia also activated the HO-1 promoter and this was blocked by mutating the antioxidant responsive element or by overexpressing dominant-negative Nrf2. The induction of HO-1 expression by ammonia was dependent on ROS formation and prevented by N-acetylcysteine or rotenone. Finally, prior treatment of endothelial cells with ammonia inhibited tumor necrosis factor-α-stimulated cell death. However, silencing HO-1 expression abrogated the protective action of ammonia and this was reversed by the administration of carbon monoxide but not bilirubin or iron. In conclusion, this study demonstrates that ammonia stimulates the expression of HO-1 in endothelial cells via the ROS-Nrf2 pathway, and that the induction of HO-1 contributes to the cytoprotective action of ammonia by generating carbon monoxide. Moreover, it identifies ammonia as a potentially important signaling gas in the vasculature that promotes endothelial cell survival.


Assuntos
Amônia/metabolismo , Células Endoteliais/metabolismo , Heme Oxigenase-1/genética , Fator 2 Relacionado a NF-E2/genética , Acetilcisteína/administração & dosagem , Amônia/administração & dosagem , Cloreto de Amônio/administração & dosagem , Animais , Artérias/efeitos dos fármacos , Artérias/metabolismo , Monóxido de Carbono/administração & dosagem , Sobrevivência Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Heme Oxigenase-1/biossíntese , Humanos , Camundongos , Fator 2 Relacionado a NF-E2/metabolismo , Regiões Promotoras Genéticas/genética , Espécies Reativas de Oxigênio/metabolismo , Rotenona/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/genética
7.
Free Radic Biol Med ; 94: 218-29, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26968795

RESUMO

The use of HIV protease inhibitors (PIs) has extended the duration and quality of life for HIV-positive individuals. However there is increasing concern that this antiviral therapy may promote premature cardiovascular disease by impairing endothelial cell (EC) function. In the present study, we investigated the effect of HIV PIs on EC function and determined if the enzyme heme oxygenase (HO-1) influences the biological action of these drugs. We found that three distinct PIs, including ritonavir, atazanavir, and lopinavir, stimulated the expression of HO-1 protein and mRNA. The induction of HO-1 was associated with an increase in NF-E2-related factor-2 (Nrf2) activity and reactive oxygen species (ROS). PIs also stimulated HO-1 promoter activity and this was prevented by mutating the antioxidant responsive element or by overexpressing dominant-negative Nrf2. In addition, the PI-mediated induction of HO-1 was abolished by N-acetyl-l-cysteine and rotenone. Furthermore, PIs blocked EC proliferation and migration and stimulated the expression of intercellular adhesion molecule-1 and the adhesion of monocytes on ECs. Inhibition of HO-1 activity or expression potentiated the anti-proliferative and inflammatory actions of PIs which was reversed by bilirubin but not carbon monoxide. Alternatively, adenovirus-mediated overexpression of HO-1 attenuated the growth-inhibitory and inflammatory effect of PIs. In contrast, blocking HO-1 activity failed to modify the anti-migratory effect of the PIs. Thus, induction of HO-1 via the ROS-Nrf2 pathway in human ECs counteracts the anti-proliferative and inflammatory actions of PIs by generating bilirubin. Therapeutic approaches targeting HO-1 may provide a novel approach in preventing EC dysfunction and vascular disease in HIV-infected patients undergoing antiretroviral therapy.


Assuntos
Bilirrubina/metabolismo , Doenças Cardiovasculares/tratamento farmacológico , Infecções por HIV/tratamento farmacológico , Heme Oxigenase-1/genética , Fator 2 Relacionado a NF-E2/genética , Acetilcisteína/administração & dosagem , Sulfato de Atazanavir/administração & dosagem , Sulfato de Atazanavir/efeitos adversos , Doenças Cardiovasculares/induzido quimicamente , Doenças Cardiovasculares/virologia , Proliferação de Células/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Infecções por HIV/virologia , Inibidores da Protease de HIV/administração & dosagem , Inibidores da Protease de HIV/efeitos adversos , Heme Oxigenase-1/antagonistas & inibidores , Heme Oxigenase-1/metabolismo , Humanos , Lopinavir/administração & dosagem , Lopinavir/efeitos adversos , Regiões Promotoras Genéticas/genética , Espécies Reativas de Oxigênio/metabolismo , Ritonavir/administração & dosagem , Ritonavir/efeitos adversos , Rotenona/administração & dosagem
8.
Front Biosci (Elite Ed) ; 8: 205-12, 2016 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-26709656

RESUMO

The vascular endothelium is continuously exposed to cyclic mechanical strain due to the periodic change in vessel diameter as a result of pulsatile blood flow. Since emerging evidence indicates the cyclic strain plays an integral role in regulating endothelial cell function, the present study determined whether application of a physiologic regimen of cyclic strain (6% at 1 hertz) influences the proliferation of human arterial endothelial cells. Prolonged exposure of human dermal microvascular or human aortic endothelial cells to cyclic strain for up to 7 days resulted in a marked decrease in cell growth. The strain-mediated anti-proliferative effect was associated with the arrest of endothelial cells in the G2/M phase of the cell cycle, did not involve cell detachment or cytotoxicity, and was due to the induction of p21. Interestingly, the inhibition in endothelial cell growth was independent of the strain regimen since prolonged application of constant or intermittent 6% strain was also able to block endothelial cell proliferation. The ability of chronic physiologic cyclic strain to inhibit endothelial cell growth represents a previously unrecognized mechanism by which hemodynamic forces maintain these cells in a quiescent, non-proliferative state.


Assuntos
Proliferação de Células , Endotélio Vascular/citologia , Estresse Mecânico , Células Cultivadas , Endotélio Vascular/enzimologia , Indução Enzimática , Humanos , Quinases Ativadas por p21/biossíntese
9.
Obesity (Silver Spring) ; 23(2): 383-90, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25557182

RESUMO

OBJECTIVE: This study investigated whether arginase contributes to endothelial dysfunction and hypertension in obese rats. METHODS: Endothelial function and arginase expression were examined in skeletal muscle arterioles from lean and obese Zucker rats (ZRs). Arginase activity, arginine bioavailability, and blood pressure were measured in lean and obese animals. RESULTS: Arginase activity and expression was increased while global arginine bioavailability decreased in obese ZRs. Acetylcholine or luminal flow caused dilation of isolated skeletal muscle arterioles, but this was reduced or absent in vessels from obese ZRs. Treatment of arterioles with a nitric oxide synthase inhibitor blocked dilation in lean arterioles and eliminated differences among lean and obese vessels. In contrast, arginase inhibitors or l-arginine enhanced vasodilation in obese ZRs and abolished differences between lean and obese animals, while d-arginine had no effect. Finally, mean arterial blood pressure was significantly increased in obese ZRs. However, administration of l-arginine or arginase inhibitors lowered blood pressure in obese but not lean animals, and this was associated with an improvement in systemic arginine bioavailability. CONCLUSIONS: Arginase promotes endothelial dysfunction and hypertension in obesity by reducing arginine bioavailability. Therapeutic approaches targeting arginase represent a promising approach in treating obesity-related vascular disease.


Assuntos
Arginase/genética , Endotélio Vascular/fisiopatologia , Regulação da Expressão Gênica , Hipertensão/genética , Obesidade/complicações , RNA/genética , Vasodilatação/fisiologia , Animais , Arginase/biossíntese , Arteríolas/enzimologia , Arteríolas/fisiopatologia , Pressão Sanguínea/fisiologia , Modelos Animais de Doenças , Endotélio Vascular/enzimologia , Hipertensão/enzimologia , Hipertensão/fisiopatologia , Masculino , Obesidade/enzimologia , Obesidade/genética , Ratos , Ratos Zucker , Reação em Cadeia da Polimerase em Tempo Real
10.
Biochem Pharmacol ; 87(2): 303-11, 2014 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-24239896

RESUMO

Endothelial cell (EC) dysfunction is involved in the pathogenesis of contrast-induced acute kidney injury, which is a major adverse event following coronary angiography. In this study, we evaluated the effect of contrast media (CM) on human EC proliferation, migration, and inflammation, and determined if heme oxygenase-1 (HO-1) influences the biological actions of CM. We found that three distinct CM, including high-osmolar (diatrizoate), low-osmolar (iopamidol), and iso-osmolar (iodixanol), stimulated the expression of HO-1 protein and mRNA. The induction of HO-1 was associated with an increase in NF-E2-related factor-2 (Nrf2) activity and reactive oxygen species (ROS). CM also stimulated HO-1 promoter activity and this was prevented by mutating the antioxidant responsive element or by overexpressing dominant-negative Nrf2. In addition, the CM-mediated induction of HO-1 and activation of Nrf2 was abolished by acetylcysteine. Finally, CM inhibited the proliferation and migration of ECs and stimulated the expression of intercellular adhesion molecule-1 and the adhesion of monocytes on ECs. Inhibition or silencing of HO-1 exacerbated the anti-proliferative and inflammatory actions of CM but had no effect on the anti-migratory effect. Thus, induction of HO-1 via the ROS-Nrf2 pathway counteracts the anti-proliferative and inflammatory actions of CM. Therapeutic approaches targeting HO-1 may provide a novel approach in preventing CM-induced endothelial and organ dysfunction.


Assuntos
Meios de Contraste/toxicidade , Células Endoteliais/enzimologia , Células Endoteliais/patologia , Heme Oxigenase-1/fisiologia , Proliferação de Células/efeitos dos fármacos , Angiografia Coronária/efeitos adversos , Angiografia Coronária/métodos , Células Endoteliais/efeitos dos fármacos , Heme Oxigenase-1/farmacologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Inflamação/induzido quimicamente , Inflamação/metabolismo , Inflamação/patologia , Células U937
11.
Front Immunol ; 4: 119, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23730303

RESUMO

Endothelial dysfunction is a characteristic feature in diabetes that contributes to the development of vascular disease. Recently, arginase has been implicated in triggering endothelial dysfunction in diabetic patients and animals by competing with endothelial nitric oxide synthase for substrate l-arginine. While most studies have focused on the coronary circulation and large conduit blood vessels, the role of arginase in mediating diabetic endothelial dysfunction in other vascular beds has not been fully investigated. In the present study, we determined whether arginase contributes to endothelial dysfunction in skeletal muscle arterioles of diabetic rats. Diabetes was induced in male Sprague Dawley rats by streptozotocin injection. Four weeks after streptozotocin administration, blood glucose, glycated hemoglobin, and vascular arginase activity were significantly increased. In addition, a significant increase in arginase I and II mRNA expression was detected in gracilis muscle arterioles of diabetic rats compared to age-matched, vehicle control animals. To examine endothelial function, first-order gracilis muscle arterioles were isolated, cannulated in a pressure myograph system, exposed to graded levels of luminal flow, and internal vessel diameter measured. Increases in luminal flow (0-50 µL/min) caused progressive vasodilation in arterioles isolated from control, normoglycemic animals. However, flow-induced vasodilation was absent in arterioles obtained from streptozotocin-treated rats. Acute in vitro pretreatment of blood vessels with the arginase inhibitors N (ω)-hydroxy-nor-l-arginine or S-(2-boronoethyl)-l-cysteine restored flow-induced responses in arterioles from diabetic rats and abolished differences between diabetic and control animals. Similarly, acute in vitro pretreatment with l-arginine returned flow-mediated vasodilation in vessels from diabetic animals to that of control rats. In contrast, d-arginine failed to restore flow-induced dilation in arterioles isolated from diabetic animals. Administration of sodium nitroprusside resulted in a similar degree of dilation in arterioles isolated from control or diabetic rats. In conclusion, the present study identifies arginase as an essential mediator of skeletal muscle arteriolar endothelial dysfunction in diabetes. The ability of arginase to induce endothelial dysfunction in skeletal muscle arterioles may further compromise glucose utilization and facilitate the development of hypertension in diabetes.

12.
Am J Physiol Heart Circ Physiol ; 304(12): H1634-43, 2013 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-23604711

RESUMO

Endothelial cells (ECs) are constantly subjected to cyclic strain that arises from periodic change in vessel wall diameter as a result of pulsatile blood flow. Application of physiological levels of cyclic strain inhibits EC apoptosis; however, the underlying mechanism is not known. Since heme oxygenase-1 (HO-1) is a potent inhibitor of apoptosis, the present study investigated whether HO-1 contributes to the antiapoptotic action of cyclic strain. Administration of physiological cyclic strain (6% at 1 Hz) to human aortic ECs stimulated an increase in HO-1 activity, protein, and mRNA expression. The induction of HO-1 was preceded by a rise in reactive oxygen species (ROS) and Nrf2 protein expression. Cyclic strain also stimulated an increase in HO-1 promoter activity that was prevented by mutating the antioxidant responsive element in the promoter or by overexpressing dominant-negative Nrf2. In addition, the strain-mediated induction of HO-1 and activation of Nrf2 was abolished by the antioxidant N-acetyl-l-cysteine. Finally, application of cyclic strain blocked inflammatory cytokine-mediated EC death and apoptosis. However, the protective action of cyclic strain was reversed by the HO inhibitor tin protoporphyrin-IX and was absent in ECs isolated from HO-1-deficient mice. In conclusion, the present study demonstrates that a hemodynamically relevant level of cyclic strain stimulates HO-1 gene expression in ECs via the ROS-Nrf2 signaling pathway to inhibit EC death. The ability of cyclic strain to induce HO-1 expression may provide an important mechanism by which hemodynamic forces promote EC survival and vascular homeostasis.


Assuntos
Apoptose , Células Endoteliais/enzimologia , Endotélio Vascular/enzimologia , Heme Oxigenase-1/metabolismo , Estresse Mecânico , Estresse Fisiológico , Acetilcisteína/farmacologia , Elementos de Resposta Antioxidante , Aorta/citologia , Sobrevivência Celular , Células Endoteliais/metabolismo , Células Endoteliais/fisiologia , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiologia , Inibidores Enzimáticos/farmacologia , Sequestradores de Radicais Livres/farmacologia , Fator de Transcrição de Proteínas de Ligação GA/genética , Fator de Transcrição de Proteínas de Ligação GA/metabolismo , Heme Oxigenase-1/antagonistas & inibidores , Heme Oxigenase-1/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Metaloporfirinas/farmacologia , Protoporfirinas/farmacologia , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Transcrição Genética , Ativação Transcricional
13.
Biochem Pharmacol ; 84(8): 1045-54, 2012 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-22864061

RESUMO

Sildenafil is a cGMP-specific phosphodiesterase type 5 inhibitor that augments cGMP accumulation following the activation of soluble guanylate cyclase (sGC). In this study, we investigated whether sildenafil promotes the production of the sGC-stimulatory gases, carbon monoxide and nitric oxide, by stimulating the expression of the inducible isoforms of heme oxygenase (HO-1) and nitric oxide synthase (iNOS) in vascular smooth muscle cells (SMCs). Sildenafil increased HO-1 expression and potentiated cytokine-mediated expression of iNOS and NO synthesis by SMCs. The induction of HO-1 was unaffected by the sGC inhibitor 1H-(1,2,4)oxadiazolo[4,3-α]quinozalin-1-one (ODQ) or the protein kinase G inhibitor (8R,9S,11S)-(-)-2-methyl-9-methoxyl-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11a-triazadibenzo(a,g)cyclocta9(cde)trinen-1-one (KT 5823). However, the sildenafil-mediated increase in HO-1 promoter activity was abolished by mutating the antioxidant responsive elements in the promoter or by overexpressing a dominant-negative mutant of NF-E2-related factor-2 (Nrf2). Furthermore, the induction of HO-1 by sildenafil was accompanied by an increase in reactive oxygen species (ROS) and blocked by N-acetyl-L-cysteine and rotenone. In contrast, the enhancement of cytokine-stimulated NO synthesis by sildenafil was prevented by ODQ and the protein kinase A inhibitor (9S,10S,12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo(1,2,3-fg:3',2',1'-kl)pyrrolo(3,4-i)(1,6)benzodiazocine-10-carboxylic acid hexyl ester (KT 5720) and duplicated by lipophilic analogs of cGMP. In conclusion, these studies demonstrate that sildenafil stimulates the expression of HO-1 and iNOS via the ROS-Nrf2 and sGC-cGMP pathway, respectively. The ability of sildenafil to block the catabolism of cGMP while stimulating the synthesis of sGC-stimulatory gaseous monoxides through the induction of HO-1 and iNOS provides a potent mechanism by which cGMP-dependent vascular actions of this drug are amplified.


Assuntos
Monóxido de Carbono/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Óxido Nítrico/biossíntese , Inibidores da Fosfodiesterase 5/farmacologia , Piperazinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Sulfonas/farmacologia , Vasodilatadores/farmacologia , Animais , Northern Blotting , Western Blotting , Células Cultivadas , Heme Oxigenase (Desciclizante)/genética , Músculo Liso Vascular/enzimologia , Regiões Promotoras Genéticas , Purinas/farmacologia , Ratos , Espécies Reativas de Oxigênio/metabolismo , Citrato de Sildenafila
14.
J Pharmacol Exp Ther ; 342(3): 827-34, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22700432

RESUMO

AMP-activated protein kinase (AMPK) is an evolutionary conserved energy-sensing enzyme that regulates cell metabolism. Emerging evidence indicates that AMPK also plays an important role in modulating endothelial cell function. In the present study, we investigated whether AMPK modulates endothelial cell growth. Treatment of cultured human umbilical vein endothelial cells with the AMPK activators 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside (AICAR), 6,7-dihydro-4-hydroxy-3-(2'-hydroxy[1,1'-biphenyl]-4-yl)-6-oxo-thieno[2,3-b]pyridine-5-carbonitrile (A-769662), or metformin inhibited cell proliferation and DNA synthesis. The antiproliferative action of AICAR was largely prevented by the adenosine kinase inhibitor 5'-iodotubercidin and mimicked by infecting endothelial cells with an adenovirus expressing constitutively active AMPK. In contrast, pharmacological blockade of endothelial nitric oxide synthase or heme oxygenase-1 activity failed to reverse the inhibition of endothelial cell growth by AICAR. Flow cytometry experiments revealed that pharmacological activation of AMPK arrested endothelial cells in the G0/G1 phase of the cell cycle, and this was associated with increases in p53 phosphorylation and p53, p21, and p27 protein expression and decreases in cyclin A protein expression and retinoblastoma protein phosphorylation. In addition, silencing p21 and p27 expression partially restored the mitogenic response of AMPK-activated cells. Finally, activation of AMPK by AICAR blocked the migration of endothelial cells after scrape injury and stimulated tube formation by endothelial cells plated onto Matrigel-coated plates. In conclusion, these studies demonstrate that AMPK activation inhibits endothelial cell proliferation by elevating p21 and p27 expression. In addition, they show that AMPK regulates endothelial cell migration and differentiation and identify AMPK as an attractive therapeutic target in treating diseases associated with aberrant endothelial cell growth.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Células Endoteliais da Veia Umbilical Humana/enzimologia , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Ciclina A/metabolismo , Ativação Enzimática , Fase G1/efeitos dos fármacos , Heme Oxigenase-1/metabolismo , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Óxido Nítrico Sintase Tipo III/metabolismo , Fosforilação/efeitos dos fármacos , Pironas/farmacologia , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Proteína do Retinoblastoma/metabolismo , Ribonucleotídeos/farmacologia , Tiofenos/farmacologia
15.
Front Pharmacol ; 3: 48, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22470341

RESUMO

Bilirubin is a heme metabolite generated by the concerted action of the enzymes heme oxygenase and biliverdin reductase. Although long considered a toxic byproduct of heme catabolism, recent preclinical, and clinical studies indicate the bilirubin exerts beneficial effects in the circulation. In the present study, we determined whether local administration of bilirubin attenuates neointima formation following injury of rat carotid arteries. In addition, the ability of bilirubin to regulate the proliferation and migration of human arterial smooth muscle cells (SMCs) was investigated. Local perivascular administration of bilirubin immediately following balloon injury of rat carotid arteries significantly attenuated neointima formation. Bilirubin-mediated inhibition of neointimal thickening was associated with a significant decrease in ERK activity and cyclin D1 and A protein expression, and an increase in p21 and p53 protein expression in injured blood vessels. Treatment of human aortic SMCs with bilirubin inhibited proliferation and migration in a concentration-dependent manner without affecting cell viability. In addition, bilirubin resulted in a concentration-dependent increase in the percentage of cells in the G(0)/G(1) phase of the cell cycle and this was paralleled by a decrease in the fraction of cells in the S and G(2)M phases of the cell cycle. Finally, bilirubin had no effect on mitochondrial function and ATP content of vascular SMCs. In conclusion, these studies demonstrate that bilirubin inhibits neointima formation after arterial injury and this is associated with alterations in the expression of cell cycle regulatory proteins. Furthermore, bilirubin blocks proliferation and migration of human arterial SMCs and arrests SMCs in the G(0)/G(1) phase of the cell cycle. Bilirubin represents an attractive therapeutic agent in treating occlusive vascular disease.

16.
Biochem Pharmacol ; 82(4): 371-9, 2011 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-21635873

RESUMO

We recently identified adenosine monophosphate-activated protein kinase (AMPK) as a novel inducer of heme oxygenase-1 (HO-1) and surprisingly found that compound C (6-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-3-pyridin-4-yl-pyrazolo[1,5-a] pyrimidine), a cell-permeable inhibitor of AMPK, could also elevate HO-1 suggesting other AMPK-independent actions for this agent. In this study, we investigated the biochemical mechanism by which compound C stimulates HO-1 expression in human endothelial cells (ECs) and determined the biological significance of the induction of HO-1 by compound C in these cells. Compound C stimulated a concentration- and time-dependent increase in HO-1 expression and an increase in HO-1 promoter activity that was abrogated by mutating the antioxidant responsive elements (AREs) in the HO-1 promoter or by overexpressing a dominant negative mutant of NF-E2-related factor 2 (Nrf2). Compound C also stimulated Nrf2 expression this was associated with an increase in the production of reactive oxygen species and with a decline in intracellular glutathione levels. Interestingly, the glutathione donor N-acetyl-l-cysteine or the NADPH oxidase inhibitor apocynin blocked the induction of HO-1 by compound C. Finally, compound C stimulated EC death and this was potentiated by silencing HO-1 expression and reversed by the administration of CO, biliverdin, or bilirubin. In conclusion, this study demonstrates that compound C stimulates HO-1 gene expression in human vascular endothelium via the activation of the Nrf2/ARE signaling pathway to counteract compound C-mediated cell death. The ability of compound C to induce HO-1 expression may contribute to the pleiotropic actions of this agent and suggest caution when using compound C to probe for AMPK functions.


Assuntos
Endotélio Vascular/citologia , Endotélio Vascular/enzimologia , Regulação Enzimológica da Expressão Gênica , Heme Oxigenase-1/biossíntese , Fator 2 Relacionado a NF-E2/biossíntese , Pirazóis/farmacologia , Pirimidinas/farmacologia , Elementos de Resposta/fisiologia , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/metabolismo , Antioxidantes/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Endotélio Vascular/efeitos dos fármacos , Humanos , Elementos de Resposta/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
17.
Am J Physiol Heart Circ Physiol ; 301(3): H888-94, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21666111

RESUMO

We recently demonstrated that preconditioning with an exogenous hydrogen sulfide donor (NaHS-PC) 24 h before ischemia and reperfusion (I/R) causes postcapillary venules to shift to an anti-inflammatory phenotype in C57BL/6J wild-type (WT) mice such that these vessels fail to support increases in postischemic leukocyte rolling (LR) and leukocyte adhesion (LA). The objective of the present study was to determine whether heme oxygenase-1 (HO-1) is a mediator of these anti-inflammatory effects noted during I/R in mice preconditioned with NaHS. Intravital fluorescence microscopy was used to visualize LR and LA in single postcapillary venules of the murine small intestine. I/R induced marked increases in LR and LA, effects that were prevented by NaHS-PC. Treatment with the HO inhibitor tin protoporphyrin IX, but not the inactive protoporphyrin CuPPIX, just before reperfusion prevented the anti-inflammatory effects of antecedent NaHS. The anti-inflammatory effects of NaHS-PC were mimicked by preconditioning with hemin, an agent that induces HO-1 expression. We then evaluated the effect of NaHS as a preconditioning stimulus in mice that were genetically deficient in HO-1 (HO-1(-/-) on an H129 background with appropriate WT strain controls). NaHS-PC was ineffective in HO-1(-/-) mice. Our work indicates that HO-1 serves as an effector of the anti-inflammatory effects of NaHS-PC during I/R 24 h later.


Assuntos
Anti-Inflamatórios/farmacologia , Células Endoteliais/efeitos dos fármacos , Heme Oxigenase-1/metabolismo , Sulfeto de Hidrogênio/metabolismo , Intestino Delgado/irrigação sanguínea , Proteínas de Membrana/metabolismo , Traumatismo por Reperfusão/prevenção & controle , Sulfetos/farmacologia , Análise de Variância , Animais , Anti-Inflamatórios/metabolismo , Adesão Celular/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Células Endoteliais/enzimologia , Células Endoteliais/imunologia , Inibidores Enzimáticos/farmacologia , Heme Oxigenase-1/antagonistas & inibidores , Heme Oxigenase-1/deficiência , Heme Oxigenase-1/genética , Migração e Rolagem de Leucócitos/efeitos dos fármacos , Masculino , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Metaloporfirinas/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Fluorescência , Fenótipo , Protoporfirinas/farmacologia , Traumatismo por Reperfusão/enzimologia , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/imunologia , Sulfetos/metabolismo , Fatores de Tempo , Vênulas/efeitos dos fármacos , Vênulas/enzimologia , Vênulas/imunologia
18.
J Pharmacol Exp Ther ; 338(2): 476-84, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21566210

RESUMO

6-[4-(2-Piperidin-1-yl-ethoxy)-phenyl]-3-pyridin-4-yl-pyrazolo[1,5-a] pyrimidine (compound C) is a cell-permeable pyrrazolopyrimidine derivative that acts as a potent inhibitor of AMP-activated protein kinase (AMPK). Although compound C is often used to determine the role of AMPK in various physiological processes, it also evokes AMPK-independent actions. In the present study, we investigated whether compound C influences vascular smooth muscle cell (SMC) function through the AMPK pathway. Treatment of rat aortic SMCs with compound C (0.02-10 µM) inhibited vascular SMC proliferation and migration in a concentration-dependent fashion. These actions of compound C were not mimicked or affected by silencing AMPKα expression or infecting SMCs with an adenovirus expressing a dominant-negative mutant of AMPK. In contrast, the pharmacological activator of AMPK 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside inhibited the proliferation and migration of SMCs in a manner that was strictly dependent on AMPK activity. Flow cytometry experiments revealed that compound C arrested SMCs in the G(0)/G(1) phase of the cell cycle, and this was associated with a decrease in cyclin D1 and cyclin A protein expression and retinoblastoma protein phosphorylation and an increase in p21 protein expression. Finally, local perivascular delivery of compound C immediately after balloon injury of rat carotid arteries markedly attenuated neointima formation. These studies identify compound C as a novel AMPK-independent regulator of vascular SMC function that exerts inhibitory effects on SMC proliferation and migration and neointima formation after arterial injury. Compound C represents a potentially new therapeutic agent in treating and preventing occlusive vascular disease.


Assuntos
Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Inibição de Migração Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/enzimologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Artérias Carótidas/efeitos dos fármacos , Artérias Carótidas/enzimologia , Inibição de Migração Celular/fisiologia , Células Cultivadas , Masculino , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley
19.
Am J Physiol Heart Circ Physiol ; 300(1): H84-93, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21037234

RESUMO

The present study determined whether AMP-activated protein kinase (AMPK) regulates heme oxygenase (HO)-1 gene expression in endothelial cells (ECs) and if HO-1 contributes to the biological actions of this kinase. Treatment of human ECs with the AMPK activator 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAR) stimulated a concentration- and time-dependent increase in HO-1 protein and mRNA expression that was associated with a prominent increase in nuclear factor-erythroid 2-related factor 2 (Nrf2) protein. Induction of HO-1 was also observed in rat carotid arteries after the in vivo application of AICAR. Induction of HO-1 by AICAR was blocked by the AMPK inhibitor compound C, the adenosine kinase inhibitor 5'-iodotubercidin, and by silencing AMPK-α(1/2) and was mimicked by the AMPK activator A-769662 and by infecting ECs with an adenovirus expressing constitutively active AMPK-α(1). AICAR also induced a significant rise in HO-1 promoter activity that was abolished by mutating the antioxidant responsive elements of the HO-1 promoter or by the overexpression of dominant negative Nrf2. Finally, activation of AMPK inhibited cytokine-mediated EC death, and this was prevented by the HO inhibitor tin protoporphyrin-IX or by silencing HO-1 expression. In conclusion, AMPK stimulates HO-1 gene expression in human ECs via the Nrf2/antioxidant responsive element signaling pathway. The induction of HO-1 mediates the antiapoptotic effect of AMPK, and this may provide an important adaptive response to preserve EC viability during periods of metabolic stress.


Assuntos
Adenilato Quinase/metabolismo , Células Endoteliais/fisiologia , Heme Oxigenase-1/genética , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Análise de Variância , Animais , Northern Blotting , Western Blotting , Caspase 3/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Heme Oxigenase-1/metabolismo , Humanos , Regiões Promotoras Genéticas , Pirazóis/farmacologia , Pirimidinas/farmacologia , RNA Interferente Pequeno , Ratos , Ratos Sprague-Dawley , Ribonucleotídeos/farmacologia , Fatores de Tempo
20.
J Trauma ; 69(2): 384-91, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20699748

RESUMO

BACKGROUND: Hemorrhagic shock causes hypoperfusion of peripheral tissues and promotes endothelial dysfunction, which may lead to further tissue injury. Trauma increases extrahepatic activity of arginase, an enzyme which competes for l-arginine with nitric oxide synthase, and plays a key role in the development of endothelial dysfunction during aging, hypertension, and diabetes. However, the role of arginase in hemorrhage-induced endothelial dysfunction has not been studied. This study tests the hypothesis that arginase inhibition improves endothelial function after hemorrhage. METHODS: Male Sprague-Dawley rats were implanted with indwelling arterial catheters for blood pressure measurements and blood removal. Awake animals were subjected to a 45% fixed volume controlled hemorrhage and blood pressure was monitored. Unbled rats served as controls. Skeletal muscle arterioles were isolated 24 hours after hemorrhage and cannulated in a pressure myograph system. To study endothelial function, arterioles were exposed to constant midpoint, but altered endpoint pressures, to establish graded levels of luminal flow and internal diameter was measured. RESULTS: Hemorrhage lowered mean arterial pressure that spontaneously recovered to 78% and 88% of baseline in 2 hours and 20 hours, respectively. Vascular arginase II and blood glucose levels were elevated, whereas hemoglobin and insulin levels were decreased 24 hours after blood loss. In posthemorrhage arterioles, flow-induced dilation was abolished. Acute in vitro treatment with an inhibitor of arginase, N-hydroxy-nor-l-arginine, restored flow-induced dilation to unbled control levels. Similarly, the arginase and nitric oxide synthase substrate, l-arginine, but not the inactive isomer, d-arginine, restored flow-induced dilation. CONCLUSIONS: These results indicate that arginase contributes to endothelial dysfunction in resistance vessels after significant hemorrhage.


Assuntos
Arginase/metabolismo , Endotélio Vascular/enzimologia , Óxido Nítrico Sintase/metabolismo , Choque Hemorrágico/enzimologia , Animais , Arginase/antagonistas & inibidores , Arginina/metabolismo , Velocidade do Fluxo Sanguíneo , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Masculino , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Fatores de Risco , Resistência Vascular/efeitos dos fármacos , Resistência Vascular/fisiologia
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