RESUMO
Heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme degradation, is a cytoprotective enzyme upregulated in the vasculature by increased flow and inflammatory stimuli. Human genetic data suggest that a diminished HO-1 expression may predispose one to abdominal aortic aneurysm (AAA) development. In addition, heme is known to strongly induce HO-1 expression. Utilizing the porcine pancreatic elastase (PPE) model of AAA induction in HO-1 heterozygous (HO-1+/-, HO-1 Het) mice, we found that a deficiency in HO-1 leads to augmented AAA development. Peritoneal macrophages from HO-1+/- mice showed increased gene expression of pro-inflammatory cytokines, including MCP-1, TNF-alpha, IL-1-beta, and IL-6, but decreased expression of anti-inflammatory cytokines IL-10 and TGF-beta. Furthermore, treatment with heme returned AAA progression in HO-1 Het mice to a wild-type profile. Using a second murine AAA model (Ang II-ApoE-/-), we showed that low doses of the HMG-CoA reductase inhibitor rosuvastatin can induce HO-1 expression in aortic tissue and suppress AAA progression in the absence of lipid lowering. Our results support those studies that suggest that pleiotropic statin effects might be beneficial in AAA, possibly through the upregulation of HO-1. Specific targeted therapies designed to induce HO-1 could become an adjunctive therapeutic strategy for the prevention of AAA disease.
Assuntos
Aneurisma da Aorta Abdominal/genética , Aneurisma da Aorta Abdominal/patologia , Expressão Gênica , Heme Oxigenase-1/genética , Animais , Aneurisma da Aorta Abdominal/tratamento farmacológico , Aneurisma da Aorta Abdominal/metabolismo , Peso Corporal , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Ativação Enzimática , Genes Reporter , Heme/metabolismo , Heme/farmacologia , Heme Oxigenase-1/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Mediadores da Inflamação/metabolismo , Lipídeos/sangue , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Knockout , Regiões Promotoras Genéticas , SuínosRESUMO
Identification and treatment of abdominal aortic aneurysm (AAA) remain among the most prominent challenges in vascular medicine. MicroRNAs (miRNAs) are crucial regulators of cardiovascular pathology and represent intriguing targets to limit AAA expansion. Here we show, by using two established murine models of AAA disease along with human aortic tissue and plasma analysis, that miR-24 is a key regulator of vascular inflammation and AAA pathology. In vivo and in vitro studies reveal chitinase 3-like 1 (Chi3l1) to be a major target and effector under the control of miR-24, regulating cytokine synthesis in macrophages as well as their survival, promoting aortic smooth muscle cell migration and cytokine production, and stimulating adhesion molecule expression in vascular endothelial cells. We further show that modulation of miR-24 alters AAA progression in animal models, and that miR-24 and CHI3L1 represent novel plasma biomarkers of AAA disease progression in humans.
Assuntos
Aneurisma da Aorta Abdominal/metabolismo , Células Endoteliais/metabolismo , Macrófagos/metabolismo , MicroRNAs/metabolismo , Miócitos de Músculo Liso/metabolismo , Animais , Aorta/metabolismo , Aneurisma da Aorta Abdominal/etiologia , Biomarcadores/sangue , Células Cultivadas , Proteína 1 Semelhante à Quitinase-3 , Modelos Animais de Doenças , Progressão da Doença , Glicoproteínas/metabolismo , Humanos , Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Senescence of cells is associated with shortened or damaged telomeres and is characterized by permanent exit from the cell cycle and altered function. Cellular senescence is caused by repeated cell division, and also conditions of stress including inflammation and reactive oxygen species can lead to the development of premature senescence. At the cellular level, proliferative and oxidative-stress induced cell senescence related to a pro-inflammatory state might strongly contribute to age-associated impaired tissue and organ functions. Vascular cells (endothelial cells, vascular smooth muscle cells) and bone marrow-derived endothelial progenitor cells have been repeatedly shown to have pivotal role in the maintenance and regeneration of cardiovascular tissue. Therefore, the molecular mechanisms of vascular cell senescence have been extensively studied. However, therapeutic approaches to prevent cellular senescence in cardiovascular disease (CVD) are still limited. Hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), and fibroblast growth factor (FGF) are all potent angiogenic growth factors in animal models of ischemia, but their therapeutic effects are not the same in animal experiments and clinical trials. A multicenter, double-blind, placebo-controlled phase III clinical trial in Japan and a US phase II clinical trial of HGF gene therapy for critical limb ischemia (CLI) demonstrated a significant improvement in primary end points and an increase in transcutaneous partial pressure of oxygen even after one year compared with placebo, whereas effectiveness of VEGF and FGF treatment for CLI has not yet been shown. Moreover, our recent publication and another researcher demonstrated that HGF acts as an anti-inflammatory cytokine, while VEGF and FGF act as pro-inflammatory cytokine. This review overviews the outcomes of clinical trials using angiogenic growth factors, which have shown a dramatic effect in several animal studies. Additionally, interventions with HGF aimed at improving the regenerative capacity of stem/progenitor cells and vascular cells by preventing cellular senescence are discussed.
Assuntos
Células Progenitoras Endoteliais/metabolismo , Animais , Doenças Cardiovasculares/metabolismo , Senescência Celular/fisiologia , Células Progenitoras Endoteliais/citologia , Fatores de Crescimento de Fibroblastos/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The effectiveness of angioplasty and stenting in intracranial atherosclerotic diseases is controversial due to high rates of delayed restenosis and hemorrhage compared with extracranial arteries. However, the mechanisms underlying these differences are still unclear, because their pathophysiology is yet to be examined. To address this issue, we established a novel vascular injury model in the intracranial internal carotid arteries (IICAs) in mice, and analyzed the remodeling process in comparison to that of the femoral arteries (FAs). In IICAs, neointimal hyperplasia was observed from day 14 and grew until day 56. Although smooth muscle cells (SMCs) emerged in the neointima from day 28, SMCs in the injured media were continuously lost with eventual extinction of the media. Re-endothelialization was started from day 7 and completed on day 28. Accumulation of macrophages was continued in the adventitia until day 56. Compared with FAs, the following points are unique in IICAs: (1) delayed continuous formation of neointima; (2) accumulation of macrophages in the media on day 14; (3) continuous loss of SMCs in the media followed by extinction of the media itself; and (4) continuously growing adventitia. These pathophysiologic differences might be associated with unfavorable outcomes in percutaneous transluminal angioplasty and stenting in intracranial arteries.
Assuntos
Artérias Cerebrais/patologia , Transtornos Cerebrovasculares/patologia , Animais , Antibacterianos/administração & dosagem , Antibacterianos/farmacologia , Artéria Carótida Interna/patologia , Circulação Cerebrovascular , Citocinas/líquido cefalorraquidiano , Artéria Femoral/patologia , Marcação In Situ das Extremidades Cortadas , Fluxometria por Laser-Doppler , Macrófagos/patologia , Masculino , Camundongos , Neointima/patologia , Neointima/prevenção & controle , Sirolimo/administração & dosagem , Sirolimo/farmacologia , Fixação de TecidosRESUMO
OBJECTIVE: Lipopolysaccharide (LPS) triggers sepsis and systemic inflammatory response syndrome, which results in multiple organ failure. Our recent reports demonstrated that hepatocyte growth factor (HGF) attenuated angiotensin II-induced oxidative stress via epithelial growth factor receptor (EGFR) degradation in vascular smooth muscle cells. Here, we examined whether HGF can protect against systemic inflammatory response syndrome induced by LPS and investigated the mechanism. METHODS AND RESULTS: HGF inhibited the increase in the expression of vascular cell adhesion molecule-1 and EGFR by LPS in vitro. HGF inhibited colocalization of EGFR and Src homology domain 2-containing inositol 5'-phosphatase 2. Furthermore, HGF inhibited reactive oxygen species production. We also injected LPS into HGF transgenic mice with increased HGF serum concentration and their littermates. HGF transgenic mice reduced LPS-induced vascular cell adhesion molecule-1 and reactive oxygen species compared with control, accompanied by significant EGFR degradation. Furthermore, HGF transgenic mice significantly improved survival in the LPS injection model. CONCLUSIONS: The present study revealed inhibition of LPS-induced vascular cell adhesion molecule-1 expression by HGF via the degradation of EGFR. We demonstrated that HGF regulated Src homology domain 2-containing inositol 5'-phosphatase 2 recruitment to EGFR and inhibited LPS-induced inflammation via EGFR degradation. This effect of HGF may be useful for the treatment of inflammatory disease.
Assuntos
Receptores ErbB/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Lipopolissacarídeos , Estresse Oxidativo , Síndrome de Resposta Inflamatória Sistêmica/metabolismo , Animais , Aorta/efeitos dos fármacos , Aorta/metabolismo , Permeabilidade Capilar , Células Cultivadas , Modelos Animais de Doenças , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Cloridrato de Erlotinib , Fator de Crescimento de Hepatócito/genética , Humanos , Inositol Polifosfato 5-Fosfatases , Rim/efeitos dos fármacos , Rim/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Monoéster Fosfórico Hidrolases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , Interferência de RNA , Espécies Reativas de Oxigênio/metabolismo , Síndrome de Resposta Inflamatória Sistêmica/induzido quimicamente , Síndrome de Resposta Inflamatória Sistêmica/prevenção & controle , Transfecção , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
OBJECTIVE: As angiogenic growth factors can stimulate the development of collateral arteries, a concept called therapeutic angiogenesis, we performed a phase I/IIa open-label clinical trial using intramuscular injection of naked plasmid DNA encoding hepatocyte growth factor (HGF). We reported long-term evaluation of 2 years after HGF gene therapy in 22 patients with severe peripheral arterial disease. METHODS AND RESULTS: Twenty-two patients with peripheral arterial disease or Buerger disease staged by Fontaine IIb (n=7), III (n=4), and IV (n=11) were treated with HGF plasmid, either 2 mg or 4 mg ×2. Increase in ankle-branchial pressure index >0.1 was observed in 11 of 14 patients (79 %) at 2 years after gene therapy and in 11 of the 17 patients (65%) at 2 months. Reduction in rest pain (>2 cm in visual analog scale) was observed in 9 of 9 patients (100%) at 2 years and in 8 of 13 (62%) patients at 2 months. At 2 years, 9 of 10 (90%) ischemic ulcers reduced by >25%, accompanied by a reduction in the size of ulcer. Severe complications and adverse effects caused by gene transfer were not detected in any patient throughout the period up to 2 years. CONCLUSIONS: Overall, the present study demonstrated long-term efficacy of HGF gene therapy up to 2 years. These findings may be cautiously interpreted to indicate that intramuscular injection of naked HGF plasmid is safe, feasible, and can achieve successful improvement of ischemic limbs as sole therapy.
Assuntos
Terapia Genética/métodos , Fator de Crescimento de Hepatócito/genética , Doença Arterial Periférica/mortalidade , Doença Arterial Periférica/terapia , Plasmídeos/uso terapêutico , Adulto , Idoso , Amputação Cirúrgica/estatística & dados numéricos , Feminino , Seguimentos , Humanos , Incidência , Injeções Intramusculares , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Medição da Dor , Plasmídeos/administração & dosagem , Plasmídeos/genética , Taxa de Sobrevida , Tromboangiite Obliterante/mortalidade , Tromboangiite Obliterante/terapia , Resultado do TratamentoRESUMO
BACKGROUND: Lipoprotein (a) (Lp(a)) is one of the risk factors for peripheral artery disease (PAD). Our previous report demonstrated that hepatocyte growth factor (HGF) gene therapy attenuated the impairment of collateral formation in Lp(a) transgenic mice. Since risk factors for atherosclerosis accelerate endothelial senescence and impair angiogenesis, we examined the role of Lp(a) in dysfunction and senescence of endothelial progenitor cells (EPC) and endothelial cells. METHODS: In vitro and in vivo incorporation assays were performed using ex-vivo expanded DiI-labeled human EPC. Senescence of cultured endothelial cells, production of oxidative stress and angiogenesis function were evaluated by SA-ß-galactosidase staining, dihydroethidium (DHE) staining and Matrigel assay, respectively. RESULTS: EPC transplantation significantly stimulated recovery of ischemic limb perfusion, while EPC pre-treated with Lp(a) did not increase ischemic limb perfusion. Impairment of angiogenesis by EPC with Lp(a) was associated with a significant decrease in CD31-positive capillaries and DiI-labeled EPC. Importantly, Lp(a) significantly accelerated the onset of senescence and production of reactive oxygen species (ROS) in human aortic endothelial cells, accompanied by a significant increase in the protein expression of p53 and p21. On the other hand, HGF significantly attenuated EPC dysfunction, senescence, ROS production, and p53 and p21 expression induced by Lp(a). CONCLUSION: Lp(a) might affect atherosclerosis via acceleration of senescence, ROS production, and functional impairment of the endothelial cell lineage. HGF might have inhibitory effects on these atherogenic actions of Lp(a).
Assuntos
Células Endoteliais/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Lipoproteína(a)/antagonistas & inibidores , Neovascularização Fisiológica , Células-Tronco/metabolismo , Animais , Aterosclerose/metabolismo , Aterosclerose/terapia , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Fator de Crescimento de Hepatócito/farmacologia , Membro Posterior/irrigação sanguínea , Humanos , Isquemia/metabolismo , Isquemia/terapia , Lipoproteína(a)/metabolismo , Lipoproteína(a)/farmacologia , Camundongos , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Transplante de Células-Tronco , Células-Tronco/efeitos dos fármacos , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismoRESUMO
Identification and treatment of abdominal aortic aneurysm (AAA) remains among the most prominent challenges in vascular medicine. MicroRNAs are crucial regulators of cardiovascular pathology and represent possible targets for the inhibition of AAA expansion. We identified microRNA-21 (miR-21) as a key modulator of proliferation and apoptosis of vascular wall smooth muscle cells during development of AAA in two established murine models. In both models (AAA induced by porcine pancreatic elastase or infusion of angiotensin II), miR-21 expression increased as AAA developed. Lentiviral overexpression of miR-21 induced cell proliferation and decreased apoptosis in the aortic wall, with protective effects on aneurysm expansion. miR-21 overexpression substantially decreased expression of the phosphatase and tensin homolog (PTEN) protein, leading to increased phosphorylation and activation of AKT, a component of a pro-proliferative and antiapoptotic pathway. Systemic injection of a locked nucleic acid-modified antagomir targeting miR-21 diminished the pro-proliferative impact of down-regulated PTEN, leading to a marked increase in the size of AAA. Similar results were seen in mice with AAA augmented by nicotine and in human aortic tissue samples from patients undergoing surgical repair of AAA (with more pronounced effects observed in smokers). Modulation of miR-21 expression shows potential as a new therapeutic option to limit AAA expansion and vascular disease progression.
Assuntos
Aneurisma da Aorta Abdominal/genética , Aneurisma da Aorta Abdominal/patologia , MicroRNAs/metabolismo , Nicotina/farmacologia , Angiotensina II/farmacologia , Animais , Aneurisma da Aorta Abdominal/complicações , Aneurisma da Aorta Abdominal/enzimologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Proliferação de Células/efeitos dos fármacos , Perfilação da Expressão Gênica , Humanos , Inflamação/complicações , Inflamação/genética , Inflamação/patologia , Interleucina-6/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , PTEN Fosfo-Hidrolase/metabolismo , Elastase Pancreática , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sus scrofa , Regulação para Cima/efeitos dos fármacosRESUMO
Serotonin (5-hydroxytryptamine, 5-HT) plays a crucial role in peripheral artery disease (PAD) and diabetes mellitus (DM). In these conditions, the balance between the 5-HT2A receptor in smooth muscle cells and the 5-HT1B receptor in endothelial cells (ECs) regulates vascular tonus. In the present study, we focused on the role of 5-HT in endothelial dysfunction using a selective 5-HT2A receptor blocker, sarpogrelate. In human EC, 5-HT markedly stimulated eNOS expression and the phosphorylation of eNOS, Akt and ERK1/2. In addition, a dose-dependent increase in tubule-formation on Matrigel was observed after 5-HT treatment. In contrast, high glucose significantly inhibited tubule formation and eNOS expression through inactivation of Akt, while 5-HT significantly attenuated these actions of high glucose (P<0.01). These results indicate that 5-HT stimulated angiogenesis through activation of Akt in ECs. However, in clinical situations, 5-HT seems to act as the "devil". To examine the role of 5-HT in diabetic PAD, a hindlimb ischemia model was created in diabetic mice. The blood flow ratio of the ischemic to non-ischemic limb was significantly lower in DM mice than in normal mice, while sarpogrelate significantly attenuated the decrease in the blood flow ratio compared to control (P<0.01). Consistently, the decrease in eNOS expression and Akt activity in DM mice was significantly attenuated by sarpogrelate. Overall, the present study demonstrated that selective inhibition of 5-HT2A by sarpogrelate significantly restored ischemic limb blood perfusion in a severe diabetic mouse model through stimulation of the eNOS/Akt pathway via the endothelial 5-HT1B receptor. Enhancement of vasodilation and angiogenesis by sarpogrelate might provide a unique treatment for PAD and DM patients.
Assuntos
Indutores da Angiogênese/farmacologia , Diabetes Mellitus Experimental/tratamento farmacológico , Angiopatias Diabéticas/tratamento farmacológico , Isquemia/tratamento farmacológico , Músculo Esquelético/irrigação sanguínea , Neovascularização Fisiológica/efeitos dos fármacos , Óxido Nítrico Sintase Tipo III/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor 5-HT1B de Serotonina/efeitos dos fármacos , Antagonistas da Serotonina/farmacologia , Serotonina/metabolismo , Succinatos/farmacologia , Animais , Células Cultivadas , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Experimental/fisiopatologia , Angiopatias Diabéticas/enzimologia , Angiopatias Diabéticas/patologia , Angiopatias Diabéticas/fisiopatologia , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Células Endoteliais/patologia , Ativação Enzimática , Membro Posterior , Humanos , Isquemia/enzimologia , Isquemia/patologia , Isquemia/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Receptor 5-HT1B de Serotonina/metabolismo , Receptor 5-HT2A de Serotonina/efeitos dos fármacos , Receptor 5-HT2A de Serotonina/metabolismo , Fluxo Sanguíneo Regional/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Vasodilatação/efeitos dos fármacosRESUMO
Gene therapy offers a novel approach for the prevention and treatment of a variety of diseases, but it is not yet a common option in the real world because of various problems. Viral vectors show high efficiency of gene transfer, but they have some problems with toxicity and immunity. On the other hand, plasmid DNA-based gene transfer is very safe, but its efficiency is relatively low. Especially, plasmid DNA gene therapy is used for cardiovascular disease because plasmid DNA transfer is possible for cardiac or skeletal muscle. Clinical angiogenic gene therapy using plasmid DNA gene transfer has been attempted in patients with peripheral artery disease, but a Phase III clinical trial did not show sufficient efficiency. Recently, a Phase III clinical trial of hepatocyte growth factor gene therapy in peripheral artery disease (PAD) showed improvement of ischemic ulcers, but it could not salvage limbs from amputation. In addition, a Phase I/II clinical study of fibroblast growth factor gene therapy in PAD extended amputation-free survival, but it seemed to fail in Phase III. In this situation, we and others have developed plasmid DNA-based gene transfer using ultrasound with microbubbles to enhance its efficiency while maintaining safety. Ultrasound-mediated gene transfer has been reported to augment the gene transfer efficiency and select the target organ using cationic microbubble phospholipids which bind negatively charged DNA. Ultrasound with microbubblesis likely to create new therapeutic options inavariety of diseases.
Assuntos
Técnicas de Transferência de Genes , Terapia Genética/métodos , Microbolhas , Plasmídeos/administração & dosagem , Ensaios Clínicos Fase II como Assunto , Ensaios Clínicos Fase III como Assunto , Humanos , Doença Arterial Periférica/terapia , Plasmídeos/genética , Transfecção , Ultrassonografia , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Both angiotensin II (Ang II) and transforming growth factor (TGF)-ß1 are thought to be involved in the progression of chronic kidney disease. In contrast, hepatocyte growth factor (HGF) counteracts the actions of Ang II and TGF-ß1. Therefore, in this study, we investigated the molecular mechanisms of how HGF antagonizes the Ang II-TGF-ß axis in renal cells. In cultured human mesangial cells, TGF-ß1 increased angiotensin type 1 receptor (AT(1)R) mRNA, mainly dependent on the Akt/phosphatidylinositol 3-kinase signaling pathway. Furthermore, TGF-ß1 decreased the expression and phosphatase activity of phosphatase and tensin homolog, deleted on chromosome 10 (PTEN), a negative regulator of the phosphatidylinositol 3-kinase/Akt pathway. These data revealed positive feedback of the Ang II-TGF-ß pathway, because Ang II increased TGF-ß expression. In contrast, HGF significantly attenuated the increase in AT(1)R gene expression, and inhibited the decrease in PTEN induced by TGF-ß1. Of importance, a PTEN-specific inhibitor significantly attenuated the reduction in TGF-ß1-induced AT(1)R expression by HGF. These data suggest that HGF attenuated TGF-ß1-induced AT(1)R expression through the PTEN/Akt pathway. To investigate this hypothesis, we performed in vivo experiments in mice with increased circulating levels of HGF produced by transgenically expressing HGF under control of a cardiac-specific transgene (HGF-Tg). In HGF-Tg mice, renal injury and fibrosis were significantly decreased, associated with reduction in AT(1)R expression and increase in PTEN after Ang II infusion, as compared with control mice. Moreover, these renal protective effects were abrogated by a neutralizing antibody against HGF. Thus, the present study demonstrated that HGF counteracts the vicious cycle of Ang II-TGF-ß1-AT(1)R, mediating the inhibition of PTEN.
Assuntos
Angiotensina II/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Células Mesangiais/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Células Cultivadas , Fator de Crescimento de Hepatócito/farmacologia , Humanos , Células Mesangiais/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/farmacologiaRESUMO
The release of free fatty acids (FFAs) from adipocytes (i.e. lipolysis) is increased in obesity and is a contributory factor to the development of insulin resistance. A recently identified adipokine, apelin, is up-regulated in states of obesity. Although apelin is secreted by adipocytes, its functions in them remain largely unknown. To determine whether apelin affects lipolysis, FFA, glycerol, and leptin levels, as well as abdominal adiposity, were measured at baseline and after reintroduction of exogenous apelin in apelin-null mice. To examine apelin's effects in vitro, isoproterenol-induced FFA/glycerol release, and hormone-sensitive lipase (HSL) and acetyl CoA carboxylase phosphorylation were investigated in 3T3-L1 cells and isolated wild-type adipocytes. Serum FFA, glycerol, and leptin concentrations, as well as abdominal adiposity, were significantly increased in apelin-null vs. wild-type mice; these changes were ameliorated in response to exogenous apelin. Apelin also reduced isoproterenol-induced FFA release in adipocytes isolated from wild-type but not APJ-null mice. In 3T3-L1 cells and isolated adipocytes, apelin attenuated isoproterenol-induced FFA/glycerol release. Apelin's inhibition was reversed by pertussis toxin, the G(q) inhibitor glycoprotein antagonist 2A, and the AMP-activated protein kinase inhibitors compound C and dorsomorphin. Apelin increased HSL phosphorylation at Ser-565 and also abrogated isoproterenol-induced HSL phosphorylation at Ser-563. Notably, apelin increased acetyl CoA carboxylase phosphorylation, suggesting AMPK activation. In conclusion, apelin negatively regulates lipolysis. Its actions may be mediated by pathways involving G(q), G(i), and AMP-activated protein kinase.
Assuntos
Adenilato Quinase/metabolismo , Proteínas de Transporte/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Lipólise/fisiologia , Células 3T3-L1 , Adenilato Quinase/genética , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Adipocinas , Agonistas Adrenérgicos beta/farmacologia , Animais , Apelina , Proteínas de Transporte/genética , AMP Cíclico , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Ácidos Graxos não Esterificados , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Glicerol , Inositol 1,4,5-Trifosfato/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Isoproterenol/farmacologia , Leptina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutAssuntos
Indutores da Angiogênese/uso terapêutico , Isquemia Miocárdica/terapia , Fator 2 de Crescimento de Fibroblastos/uso terapêutico , Fator 4 de Crescimento de Fibroblastos/genética , Terapia Genética/métodos , Humanos , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/uso terapêutico , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/uso terapêuticoRESUMO
OBJECTIVE: To evaluate the safety and feasibility of intramuscular gene transfer using naked plasmid DNA-encoding hepatocyte growth factor (HGF) and to assess its potential therapeutic benefit in patients with critical limb ischemia. METHODS AND RESULTS: Gene transfer was performed in 22 patients with critical limb ischemia by intramuscular injection of HGF plasmid, either 2 or 4 mg, 2 times. Safety, ankle-brachial index, resting pain on a 10-cm visual analog scale, wound healing, and walking distance were evaluated before treatment and at 2 months after injection. No serious adverse event caused by gene transfer was detected over a follow-up of 6 months. Of particular importance, no peripheral edema, in contrast to that seen after treatment with vascular endothelial growth factor, was observed. In addition, the systemic HGF protein level did not increase during the study. At 2 months after gene transfer, the mean ± SD ankle-brachial index increased from 0.46 ± 0.08 to 0.59 ± 0.13 (P<0.001), the mean ± SD size of the largest ischemic ulcers decreased from 3.08 ± 1.54 to 2.32 ± 1.88 cm (P=0.007), and the mean ± SD visual analog scale score decreased from 5.92 ± 1.67 to 3.04 ± 2.50 cm (P=0.001). An increase in ankle-brachial index by >0.1, a reduction in ulcer size by >25%, and a reduction in visual analog scale score by >2 cm at 2 months after gene transfer were observed in 11 (64.7%) of 17 limbs, 18 (72%) of 25 ulcers, and 8 (61.5%) of 13 limbs, respectively. CONCLUSIONS: Intramuscular injection of naked HGF plasmid is safe and feasible and can achieve successful improvement of ischemic limbs as sole therapy.
Assuntos
Terapia Genética , Fator de Crescimento de Hepatócito/biossíntese , Isquemia/terapia , Extremidade Inferior/irrigação sanguínea , Neovascularização Fisiológica , Idoso , Índice Tornozelo-Braço , Estado Terminal , Feminino , Terapia Genética/efeitos adversos , Hemodinâmica , Fator de Crescimento de Hepatócito/genética , Humanos , Injeções Intramusculares , Isquemia/genética , Isquemia/metabolismo , Isquemia/fisiopatologia , Japão , Masculino , Pessoa de Meia-Idade , Neovascularização Fisiológica/genética , Dor/genética , Dor/metabolismo , Dor/fisiopatologia , Dor/prevenção & controle , Medição da Dor , Estudos Prospectivos , Recuperação de Função Fisiológica , Fatores de Tempo , Resultado do Tratamento , Caminhada , CicatrizaçãoRESUMO
Thrombosis, the localized clotting of blood, occurs in both the arterial and venous circulation, and has a major impact on health outcomes. The primary etiology of myocardial infarctions, and approximately 80% of strokes, is acute arterial thrombosis. In combination this represents the most common cause of death in the Western world, while the third leading cause of cardiovascular-associated death is venous thromboembolism. An understanding of the pathogenic changes in the vessel wall and the blood that result in thrombosis is crucial for developing safer and more effective antithrombotic drugs. Dabigatran etexilate belongs to a new class of direct thrombin inhibitors. Following oral administration, dabigatran reaches peak plasma concentrations within 2 hours, shows linear pharmacokinetics, and a limited (but important) amount of direct drug interactions. Given once daily at 150 mg or 220 mg, it has proven to be competitive with enoxaparin in the prevention of venous thromboembolism after major orthopedic surgery, with a comparable safety profile. For stroke prevention in patients suffering from atrial fibrillation, dabigatran administered at a dose of 110 mg twice daily was associated with rates of stroke and systemic embolism that were similar to those associated with warfarin, as well as lower rates of hemorrhage. Dabigatran given at a dose of 150 mg twice daily, as compared with warfarin, was associated with lower rates of stroke and systemic embolism but similar rates of major hemorrhage. Oral bioavailability of dabigatran, together with a rapid onset and offset of action and predictable anticoagulation response, makes this newly available antithrombotic drug an attractive alternative to traditional anticoagulant therapies for numerous thrombosis-related indications.
Assuntos
Anticoagulantes/uso terapêutico , Benzimidazóis/uso terapêutico , Coagulação Sanguínea/efeitos dos fármacos , Doenças Cardiovasculares/tratamento farmacológico , Fibrinolíticos/uso terapêutico , Piridinas/uso terapêutico , Trombina/antagonistas & inibidores , Administração Oral , Animais , Anticoagulantes/administração & dosagem , Anticoagulantes/efeitos adversos , Anticoagulantes/farmacocinética , Benzimidazóis/administração & dosagem , Benzimidazóis/efeitos adversos , Benzimidazóis/farmacocinética , Doenças Cardiovasculares/sangue , Dabigatrana , Esquema de Medicação , Interações Medicamentosas , Fibrinolíticos/administração & dosagem , Fibrinolíticos/efeitos adversos , Fibrinolíticos/farmacocinética , Hemorragia/induzido quimicamente , Humanos , Piridinas/administração & dosagem , Piridinas/efeitos adversos , Piridinas/farmacocinética , Resultado do TratamentoRESUMO
BACKGROUND: Extracellular matrix degradation is a sentinel pathologic feature of abdominal aortic aneurysm (AAA) disease. Diabetes mellitus, a negative risk factor for AAA, may impair aneurysm progression through its influence on the fibrinolytic system. We hypothesize that hyperglycemia limits AAA progression through effects on endogenous plasminogen activator inhibitor-1 (PAI-1) levels and subsequent reductions in plasmin generation. METHODS: Experimental AAAs were induced in diabetic and control mice via the intra-aortic elastase infusion method. Serial transabdominal high-frequency ultrasound examinations were performed to monitor aortic diameter following elastase infusion. Circulating PAI-1 and plasmin alpha2-antiplasmin (PAP) complex concentrations were determined by ELISA and local expression of PAI-1 levels was examined by RT-PCR and immunohistochemistry. RESULTS: Hyperglycemia was associated with reduced AAA diameter, increased plasma PAI-1 concentration and reduced plasmin generation. Aneurysmal aortic PAI-1 gene expression increased in parallel with plasma concentration, with peak expression occurring early after aneurysm initiation. CONCLUSION: Hyperglycemia increases PAI-1 expression and attenuates AAA diameter in experimental AAA disease. These results emphasize the role of the fibrinolytic pathway in AAA pathophysiology, and suggest a candidate mechanism for hyperglycemic inhibition of AAA disease.
Assuntos
Aneurisma da Aorta Abdominal/sangue , Aneurisma da Aorta Abdominal/complicações , Hiperglicemia/complicações , Serpinas/sangue , Animais , Aorta Abdominal/metabolismo , Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/prevenção & controle , Modelos Animais de Doenças , Fibrinolisina/metabolismo , Fibrinólise , Galectina 3/metabolismo , Expressão Gênica , Humanos , Hiperglicemia/sangue , Imuno-Histoquímica , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Risco , Serpina E2 , Serpinas/genética , alfa 2-Antiplasmina/metabolismoRESUMO
The recently discovered peptide apelin is known to be involved in the maintenance of insulin sensitivity. However, questions persist regarding its precise role in the chronic setting. Fasting glucose, insulin, and adiponectin levels were determined on mice with generalized deficiency of apelin (APKO). Additionally, insulin (ITT) and glucose tolerance tests (GTT) were performed. To assess the impact of exogenously delivered apelin on insulin sensitivity, osmotic pumps containing pyroglutamated apelin-13 or saline were implanted in APKO mice for 4 wk. Following the infusion, ITT/GTTs were repeated and the animals euthanized. Soleus muscles were harvested and homogenized in lysis buffer, and insulin-induced Akt phosphorylation was determined by Western blotting. Apelin-13 infusion and ITTs/GTTs were also performed in obese diabetic db/db mice. To probe the underlying mechanism for apelin's effects, apelin-13 was also delivered to cultured C2C12 myotubes. 2-[3H]deoxyglucose uptake and Akt phosphorylation were assessed in the presence of various inhibitors. APKO mice had diminished insulin sensitivity, were hyperinsulinemic, and had decreased adiponectin levels. Soleus lysates had decreased insulin-induced Akt phosphorylation. Administration of apelin to APKO and db/db mice resulted in improved insulin sensitivity. In C2C12 myotubes, apelin increased glucose uptake and Akt phosphorylation. These events were fully abrogated by pertussis toxin, compound C, and siRNA knockdown of AMPKalpha1 but only partially diminished by LY-294002 and not at all by L-NAME. We conclude that apelin is necessary for the maintenance of insulin sensitivity in vivo. Apelin's effects on glucose uptake and Akt phosphorylation are in part mediated by a G(i) and AMPK-dependent pathway.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Diabetes Mellitus Tipo 2/fisiopatologia , Resistência à Insulina/fisiologia , Obesidade/fisiopatologia , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Adipocinas , Animais , Apelina , Células Cultivadas , Cromonas/farmacologia , Desoxiglucose/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Sacarose Alimentar/farmacologia , Inibidores Enzimáticos/farmacologia , Hiperinsulinismo/metabolismo , Hiperinsulinismo/fisiopatologia , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Morfolinas/farmacologia , Músculo Esquelético/metabolismo , Mioblastos/citologia , Mioblastos/metabolismo , NG-Nitroarginina Metil Éster/farmacologia , Obesidade/metabolismo , Fosforilação/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores para Leptina/genética , Receptores para Leptina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , TrítioRESUMO
RATIONALE: Neointimal hyperplasia contributes to atherosclerosis and restenosis after percutaneous coronary intervention. Vascular injury in each of these conditions results in the release of mitogenic growth factors and hormones that contribute to pathological vascular smooth muscle cell growth and inflammation. Hepatocyte growth factor (HGF) is known as an antiinflammatory growth factor, although it is downregulated in injured tissue. However, the precise mechanism how HGF reduces inflammation is unclear. OBJECTIVE: To elucidate the mechanism how HGF and its receptor c-Met reduces angiotensin II (Ang II)-induced inflammation. METHODS AND RESULTS: HGF reduced Ang II-induced vascular smooth muscle cell growth and inflammation by controlling translocation of SHIP2 (Src homology domain 2-containing inositol 5'-phosphatase 2), which led to Ang II-dependent degradation of epithelial growth factor receptor. Moreover, the present study also revealed a preventive effect of HGF on atherosclerotic change in an Ang II infusion and cuff HGF transgenic mouse model. CONCLUSIONS: These data suggest that the HGF/c-Met system might regulate extrinsic factor signaling that maintains the homeostasis of organs.
Assuntos
Angiotensina II/metabolismo , Aterosclerose/metabolismo , Receptores ErbB/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Inflamação/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Transdução de Sinais , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Animais , Aterosclerose/patologia , Proliferação de Células , Células Cultivadas , Receptores ErbB/genética , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Fator de Crescimento de Hepatócito/genética , Humanos , Hiperplasia , Inflamação/patologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/patologia , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Monoéster Fosfórico Hidrolases/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional , Transporte Proteico , Proteínas Proto-Oncogênicas c-met , Interferência de RNA , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Superóxido Dismutase/metabolismo , Fatores de Tempo , Transfecção , UbiquitinaçãoRESUMO
Although both hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) are potent angiogenic growth factors in animal models of ischemia, their characteristics are not the same in animal experiments and clinical trials. To elucidate the discrepancy between HGF and VEGF, we compared the effects of HGF and VEGF on endothelial progenitor cells under angiotensin II stimulation, which is a well-known risk factor for atherosclerosis. Here, we demonstrated that HGF, but not VEGF, attenuated angiotensin II-induced senescence of endothelial progenitor cells through a reduction of oxidative stress by inhibition of the phosphatidylinositol-3,4,5-triphosphate/rac1 pathway. Potent induction of neovascularization of endothelial progenitor cells by HGF, but not VEGF, under angiotensin II was also confirmed by in vivo experiments using several models, including HGF transgenic mice.