RESUMO
OBJECTIVE: Mitochondria consistently change their morphology in a process regulated by proteins, including Drp1 (dynamin-related protein 1), a protein promoting mitochondrial fission. Drp1 is involved in the mechanisms underlying various cardiovascular diseases, such as myocardial ischemia/reperfusion injury, heart failure, and pulmonary arterial hypertension. However, its role in macrophages, which promote various vascular diseases, is poorly understood. We therefore tested our hypothesis that macrophage Drp1 promotes vascular remodeling after injury. METHOD AND RESULTS: To explore the selective role of macrophage Drp1, we created macrophage-selective Drp1-deficient mice and performed femoral arterial wire injury. In these mice, intimal thickening and negative remodeling were attenuated at 4 weeks after injury when compared with control mice. Deletion of macrophage Drp1 also attenuated the macrophage accumulation and cell proliferation in the injured arteries. Gain- and loss-of-function experiments using cultured macrophages indicated that Drp1 induces the expression of molecules associated with inflammatory macrophages. Morphologically, mitochondrial fission was induced in inflammatory macrophages, whereas mitochondrial fusion was induced in less inflammatory/reparative macrophages. Pharmacological inhibition or knockdown of Drp1 decreased the mitochondrial reactive oxygen species and chemotactic activity in cultured macrophages. Co-culture experiments of macrophages with vascular smooth muscle cells indicated that deletion of macrophage Drp1 suppresses growth and migration of vascular smooth muscle cells induced by macrophage-derived soluble factors. CONCLUSIONS: Macrophage Drp1 accelerates intimal thickening after vascular injury by promoting macrophage-mediated inflammation. Macrophage Drp1 may be a potential therapeutic target of vascular diseases.
Assuntos
Dinaminas/metabolismo , Artéria Femoral/metabolismo , Macrófagos Peritoneais/metabolismo , Mitocôndrias/metabolismo , Neointima , Remodelação Vascular , Lesões do Sistema Vascular/metabolismo , Animais , Proliferação de Células , Quimiotaxia , Técnicas de Cocultura , Modelos Animais de Doenças , Dinaminas/deficiência , Dinaminas/genética , Artéria Femoral/lesões , Artéria Femoral/patologia , Artéria Femoral/fisiopatologia , Ativação de Macrófagos , Macrófagos Peritoneais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/patologia , Dinâmica Mitocondrial , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Músculo Liso Vascular/fisiopatologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Células RAW 264.7 , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Fatores de Tempo , Lesões do Sistema Vascular/genética , Lesões do Sistema Vascular/patologia , Lesões do Sistema Vascular/fisiopatologiaRESUMO
OBJECTIVE: Plaque erosion is increasing its importance as one of the mechanisms of acute coronary syndromes in this statin era. However, the clinical efficacy of currently used lipid-lowering agents in the prevention of thrombotic complications associated with plaque erosion has not been clarified. Therefore, we examined the therapeutic effects of ezetimibe or rosuvastatin monotherapy on spontaneous atherothrombotic occlusion. APPROACH AND RESULTS: Femoral arteries of Japanese white rabbits, fed a high-cholesterol diet, were injured by balloon catheter, and then angiotensin II was continuously administrated. In 94% of these arteries, spontaneous thrombotic occlusions were observed after 5 weeks (median) of balloon injury. Histochemical analyses indicated that the injured arteries had similar pathological features to human plaque erosions; (1) spontaneous thrombotic occlusion, (2) lack of endothelial cells, and (3) tissue factor expression in vascular smooth muscle cells. Ezetimibe (1.0 mg/kg per day), but not rosuvastatin (0.6 mg/kg per day), significantly decreased thrombotic occlusion of arteries accompanied with accelerated re-endothelialization and the decreases of serum oxysterols despite the comparable on-treatment serum cholesterol levels. The 7-ketocholesterol inhibited the migration of human umbilical vein endothelial cells. Both 7-ketocholesterol and 27-hydroxycholesterol increased tissue factor expression in cultured rat vascular smooth muscle cells. Tissue factor expression was also induced by serum from vehicle- or rosuvastatin-treated rabbits, but the induction was attenuated with serum from ezetimibe-treated rabbits. CONCLUSIONS: We have established a novel rabbit model of spontaneous atherothromobotic occlusion without plaque rupture that is feasible to test the therapeutic effects of various pharmacotherapies. Ezetimibe may decrease atherothrombotic complications after superficial plaque erosion by reducing serum oxysterols.
Assuntos
Anticolesterolemiantes/farmacologia , Arteriopatias Oclusivas/prevenção & controle , Aterosclerose/tratamento farmacológico , Ezetimiba/farmacologia , Artéria Femoral/efeitos dos fármacos , Oxisteróis/sangue , Placa Aterosclerótica , Trombose/prevenção & controle , Lesões do Sistema Vascular/tratamento farmacológico , Angiotensina II , Animais , Arteriopatias Oclusivas/sangue , Arteriopatias Oclusivas/patologia , Aterosclerose/sangue , Aterosclerose/patologia , Biomarcadores/sangue , Células Cultivadas , Colesterol na Dieta , Dieta Hiperlipídica , Modelos Animais de Doenças , Regulação para Baixo , Artéria Femoral/metabolismo , Artéria Femoral/patologia , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Masculino , Coelhos , Ratos , Rosuvastatina Cálcica/farmacologia , Transdução de Sinais/efeitos dos fármacos , Trombose/sangue , Trombose/patologia , Lesões do Sistema Vascular/sangue , Lesões do Sistema Vascular/patologiaRESUMO
Statins are known to improve pulmonary arterial hypertension (PAH) by their anti-inflammatory and anti-proliferative effects in animal models. However, recent clinical studies have reported that clinically approved statin doses failed to improve clinical outcomes in patients with PAH. We therefore hypothesized that nanoparticle (NP) -mediated targeting of pitavastatin could attenuate the progression of established PAH.We induced PAH by subcutaneously injecting monocrotaline (MCT) in Sprague-Dawley rats. On day 14 after the MCT injection, animals that displayed established PAH on echocardiography were included. On day 17, they were randomly assigned to the following 5 groups: daily intravenous administration of (1) vehicle, (2) fluorescein-isothiocyanate-NP, (3) pitavastatin, (4) pitavastatin-NP, or (5) oral sildenafil. Intravenous NP was selectively delivered to small pulmonary arteries and circulating CD11b-positive leukocytes. On day 21, pitavastatin-NP attenuated the progression of PAH at lower doses than pitavastatin alone. This was associated with the inhibition of monocyte-mediated inflammation, proliferation, and remodeling of the pulmonary arteries. Interestingly, sildenafil attenuated the development of PAH, but had no effects on inflammation or remodeling of the pulmonary arteries. In separate experiments, only treatment with pitavastatin-NP reduced the mortality rate at day 35.NP-mediated targeting of pitavastatin to small pulmonary arteries and leukocytes attenuated the progression of established MCT-induced PAH and improved survival. Therapeutically, pitavastatin-NP was associated with anti-inflammatory and anti-proliferative effects on small pulmonary arteries, which was completely distinct from the vasodilatory effect of sildenafil. Pitavastatin-NP can be a novel therapeutic modality for PAH.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Inibidores de Hidroximetilglutaril-CoA Redutases/administração & dosagem , Hipertensão Pulmonar/tratamento farmacológico , Nanopartículas/administração & dosagem , Quinolinas/administração & dosagem , Administração Intravenosa , Animais , Progressão da Doença , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Hipertensão Pulmonar/induzido quimicamente , Leucócitos , Masculino , Monocrotalina , Artéria Pulmonar , Quinolinas/uso terapêutico , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Resultado do TratamentoRESUMO
Pulmonary hypertension (PH) is a disease with poor prognosis, caused by the obstruction/stenosis of small pulmonary arteries. Statin is known to have vasodilating and anti-inflammatory property and is considered to be a candidate of therapeutic agents for the treatment of PH, but its efficacy has not been verified in clinical trials. We have formulated pitavastatin incorporating nanoparticles composed of poly (lactic-co-glycolic acid) (NK-104-NP) to improve drug delivery to the pulmonary arteries and evaluated their safety and pharmacokinetics in healthy volunteers. To accomplish this purpose, phase I clinical trials were conducted. In the single intravenous administration regimen, 40 healthy subjects were enrolled and PK (pharmacokinetic) parameters in 4 groups (1, 2, 4, and 8 mg as pitavastatin calcium) were as follows: 1.00 hour after the administration, the plasma concentration of pitavastatin reached Cmax (the maximum drug concentration) in all groups. Cmax, AUC0-t (area under the curve from time 0 to the last measurable concentration) and AUC0-∞ (area under the curve from time 0 extrapolated to infinite time) were increased in a dose-dependent manner. Population pharmacokinetic analysis based on these results indicated no accumulation of pitavastatin after repeated administration of NK-104-NP for 7 days. In this 7-day administration trial, the mean Cmax and AUC0-∞ of pitavastatin were not significantly different between days 1 and 7, suggesting that pitavastatin is unlikely to accumulate after repeated administration. In these trials, three adverse events (AEs) were reported, but they were resolved without any complications and judged to have no causal relationships with NK-104-NP. These results indicate that the innovative nanotechnology-based medicine NK-104-NP exhibited dose-dependent pharmacokinetics and was well tolerated with no significant AEs in healthy volunteers.
Assuntos
Inibidores de Hidroximetilglutaril-CoA Redutases/farmacocinética , Hipertensão Pulmonar/tratamento farmacológico , Pulmão/irrigação sanguínea , Quinolinas/farmacocinética , Administração Intravenosa , Adulto , Sistemas de Liberação de Medicamentos , Voluntários Saudáveis , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/administração & dosagem , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Hipertensão Pulmonar/mortalidade , Hipertensão Pulmonar/patologia , Japão/epidemiologia , Pulmão/efeitos dos fármacos , Pulmão/patologia , Masculino , Nanotecnologia/métodos , Artéria Pulmonar/efeitos dos fármacos , Quinolinas/administração & dosagem , Quinolinas/sangue , Quinolinas/uso terapêuticoRESUMO
The Notch signaling pathway regulates the development of various cell types and organs, and also contributes to disease mechanisms in adults. Accumulating evidence suggests its role in cardiovascular and metabolic diseases. Notch signaling components also control the phenotype of immune cells. Delta-like ligand 4 (Dll4) of the Notch pathway promotes proinflammatory activation of macrophages in vitro and in vivo. Dll4 blockade attenuates chronic atherosclerosis, vein graft disease, vascular calcification, insulin resistance, and fatty liver in mice. The Dll4-Notch axis may, thus, participate in the shared mechanisms for cardiometabolic disorders, serving as a potential therapeutic target for ameliorating these global health problems.
Assuntos
Doenças Cardiovasculares/metabolismo , Sistema Cardiovascular/metabolismo , Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Ativação de Macrófagos , Macrófagos/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Ligação ao Cálcio , Doenças Cardiovasculares/imunologia , Doenças Cardiovasculares/patologia , Sistema Cardiovascular/imunologia , Sistema Cardiovascular/patologia , Humanos , Inflamação/imunologia , Inflamação/patologia , Macrófagos/imunologia , Macrófagos/patologia , MorfogêneseRESUMO
OBJECTIVE: Inflammatory monocytes/macrophages produce various proteinases, including matrix metalloproteinases, and degradation of the extracellular matrix by these activated proteinases weakens the mechanical strength of atherosclerotic plaques, which results in a rupture of the plaque. Peroxisome proliferator-activated receptor-γ induces a polarity shift of monocytes/macrophages toward less inflammatory phenotypes and has the potential to prevent atherosclerotic plaque ruptures. Therefore, we hypothesized that nanoparticle-mediated targeted delivery of the peroxisome proliferator-activated receptor-γ agonist pioglitazone into circulating monocytes could effectively inhibit plaque ruptures in a mouse model. APPROACH AND RESULTS: We prepared bioabsorbable poly(lactic-co-glycolic-acid) nanoparticles containing pioglitazone (pioglitazone-NPs). Intravenously administered poly(lactic-co-glycolic-acid) nanoparticles incorporated with fluorescein isothiocyanate were found in circulating monocytes and aortic macrophages by flow cytometric analysis. Weekly intravenous administration of pioglitazone-NPs (7 mg/kg per week) for 4 weeks decreased buried fibrous caps, a surrogate marker of plaque rupture, in the brachiocephalic arteries of ApoE(-/-) mice fed a high-fat diet and infused with angiotensin II. In contrast, administration of control-NPs or an equivalent dose of oral pioglitazone treatment produced no effects. Pioglitazone-NPs inhibited the activity of matrix metalloproteinases and cathepsins in the brachiocephalic arteries. Pioglitazone-NPs regulated inflammatory cytokine expression and also suppressed the expression of extracellular matrix metalloproteinase inducer in bone marrow-derived macrophages. CONCLUSIONS: Nanoparticle-mediated delivery of pioglitazone inhibited macrophage activation and atherosclerotic plaque ruptures in hyperlipidemic ApoE(-/-) mice. These results demonstrate a promising strategy with a favorable safety profile to prevent atherosclerotic plaque ruptures.
Assuntos
Aterosclerose/tratamento farmacológico , Fármacos Cardiovasculares/farmacologia , Diferenciação Celular/efeitos dos fármacos , Portadores de Fármacos , Ácido Láctico/química , Macrófagos Peritoneais/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Nanopartículas , Placa Aterosclerótica , Ácido Poliglicólico/química , Tiazolidinedionas/farmacologia , Administração Intravenosa , Angiotensina II , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/induzido quimicamente , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Tronco Braquiocefálico/efeitos dos fármacos , Tronco Braquiocefálico/metabolismo , Tronco Braquiocefálico/patologia , Fármacos Cardiovasculares/administração & dosagem , Fármacos Cardiovasculares/química , Catepsinas/metabolismo , Células Cultivadas , Química Farmacêutica , Citocinas/metabolismo , Dieta Hiperlipídica , Modelos Animais de Doenças , Progressão da Doença , Mediadores da Inflamação/metabolismo , Macrófagos Peritoneais/metabolismo , Macrófagos Peritoneais/patologia , Masculino , Metaloproteinases da Matriz/metabolismo , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/metabolismo , Monócitos/patologia , Fenótipo , Pioglitazona , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Ruptura Espontânea , Tiazolidinedionas/administração & dosagem , Tiazolidinedionas/químicaRESUMO
Left ventricular (LV) remodeling after myocardial infarction (MI) causes heart failure. Although medical therapies including angiotensin converting enzyme inhibitors show inhibitory effects on post-infarct LV remodeling, the prognosis of patients with post-infarct heart failure is still poor. Accumulating evidence suggests that an inflammatory response is implicated in the process of post-infarct LV remodeling. Therefore, we hypothesized that anti-inflammatory therapy by nanoparticle-mediated monocyte/macrophage-targeting delivery of pitavastatin may protect the heart from post-infarct LV remodeling.Male C57BL/6 mice were subjected to permanent coronary ligation and pitavastatin-incorporating nanoparticles (Pitavastatin-NPs) were intravenously injected for 3 to 5 consecutive days. Pitavastatin-NPs were delivered to CD11b+ monocytes/macrophages, but not to cardiomyocytes. Treatment with Pitavastatin-NPs after establishment of MI attenuated post-infarct LV remodeling accompanied by a reduction of monocytes/macrophages in the heart, whereas pitavastatin solution treatment did not. Pitavastatin-NPs inhibited mobilization of monocytes from the spleen after MI. In mice after splenectomy, Pitavastatin-NPs still decreased the number of monocytes/macrophages in the infarcted heart and inhibited post-infarct LV remodeling.Nanoparticle-mediated delivery of pitavastatin to monocytes/macrophages may be a novel therapeutic strategy to protect the heart from post-infarct LV remodeling. Inhibition of monocyte mobilization from the bone marrow is one of the major mechanisms by which Pitavastatin-NPs attenuated post-infarct LV remodeling.
Assuntos
Sistemas de Liberação de Medicamentos , Insuficiência Cardíaca/prevenção & controle , Inflamação/tratamento farmacológico , Infarto do Miocárdio/fisiopatologia , Nanopartículas/administração & dosagem , Quinolinas/administração & dosagem , Remodelação Ventricular/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/fisiopatologia , Inibidores de Hidroximetilglutaril-CoA Redutases/administração & dosagem , Injeções Intravenosas , Macrófagos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monócitos , Infarto do Miocárdio/complicaçõesRESUMO
OBJECTIVE: Despite its large clinical impact, the underlying mechanisms for vein graft failure remain obscure and no effective therapeutic solutions are available. We tested the hypothesis that Notch signaling promotes vein graft disease. APPROACH AND RESULTS: We used 2 biotherapeutics for Delta-like ligand 4 (Dll4), a Notch ligand: (1) blocking antibody and (2) macrophage- or endothelial cell (EC)-targeted small-interfering RNA. Dll4 antibody administration for 28 days inhibited vein graft lesion development in low-density lipoprotein (LDL) receptor-deficient (Ldlr(-/-)) mice, and suppressed macrophage accumulation and macrophage expression of proinflammatory M1 genes. Dll4 antibody treatment for 7 days after grafting also reduced macrophage burden at day 28. Dll4 silencing via macrophage-targeted lipid nanoparticles reduced lesion development and macrophage accumulation, whereas EC-targeted Dll4 small-interfering RNA produced no effects. Gain-of-function and loss-of-function studies suggested in vitro that Dll4 induces proinflammatory molecules in macrophages. Macrophage Dll4 also stimulated smooth muscle cell proliferation and migration and suppressed their differentiation. CONCLUSIONS: These results suggest that macrophage Dll4 promotes lesion development in vein grafts via macrophage activation and crosstalk between macrophages and smooth muscle cells, supporting the Dll4-Notch axis as a novel therapeutic target.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Macrófagos/metabolismo , Proteínas de Membrana/metabolismo , Neointima , Veia Safena/transplante , Veia Cava Inferior/transplante , Proteínas Adaptadoras de Transdução de Sinal , Animais , Anticorpos/farmacologia , Proteínas de Ligação ao Cálcio , Artérias Carótidas/cirurgia , Comunicação Celular , Diferenciação Celular , Movimento Celular , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Macrófagos/imunologia , Masculino , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Interferência de RNA , Receptores de LDL/deficiência , Receptores de LDL/genética , Veia Safena/metabolismo , Veia Safena/patologia , Transdução de Sinais , Fatores de Tempo , Transfecção , Remodelação Vascular , Veia Cava Inferior/imunologia , Veia Cava Inferior/metabolismo , Veia Cava Inferior/patologiaRESUMO
BACKGROUND: Preventing atherosclerotic plaque destabilization and rupture is the most reasonable therapeutic strategy for acute myocardial infarction. Therefore, we tested the hypotheses that (1) inflammatory monocytes play a causative role in plaque destabilization and rupture and (2) the nanoparticle-mediated delivery of pitavastatin into circulating inflammatory monocytes inhibits plaque destabilization and rupture. METHODS AND RESULTS: We used a model of plaque destabilization and rupture in the brachiocephalic arteries of apolipoprotein E-deficient (ApoE(-/-)) mice fed a high-fat diet and infused with angiotensin II. The adoptive transfer of CCR2(+/+)Ly-6C(high) inflammatory macrophages, but not CCR2(-/-) leukocytes, accelerated plaque destabilization associated with increased serum monocyte chemoattractant protein-1 (MCP-1), monocyte-colony stimulating factor, and matrix metalloproteinase-9. We prepared poly(lactic-co-glycolic) acid nanoparticles that were incorporated by Ly-6G(-)CD11b(+) monocytes and delivered into atherosclerotic plaques after intravenous administration. Intravenous treatment with pitavastatin-incorporated nanoparticles, but not with control nanoparticles or pitavastatin alone, inhibited plaque destabilization and rupture associated with decreased monocyte infiltration and gelatinase activity in the plaque. Pitavastatin-incorporated nanoparticles inhibited MCP-1-induced monocyte chemotaxis and the secretion of MCP-1 and matrix metalloproteinase-9 from cultured macrophages. Furthermore, the nanoparticle-mediated anti-MCP-1 gene therapy reduced the incidence of plaque destabilization and rupture. CONCLUSIONS: The recruitment of inflammatory monocytes is critical in the pathogenesis of plaque destabilization and rupture, and nanoparticle-mediated pitavastatin delivery is a promising therapeutic strategy to inhibit plaque destabilization and rupture by regulating MCP-1/CCR2-dependent monocyte recruitment in this model.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Monócitos/efeitos dos fármacos , Nanopartículas/metabolismo , Placa Aterosclerótica/tratamento farmacológico , Quinolinas/farmacocinética , Transferência Adotiva , Animais , Apolipoproteínas E/genética , Tronco Braquiocefálico/efeitos dos fármacos , Tronco Braquiocefálico/imunologia , Tronco Braquiocefálico/patologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/imunologia , Quimiocina CCL2/antagonistas & inibidores , Quimiocina CCL2/metabolismo , Modelos Animais de Doenças , Terapia Genética/métodos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacocinética , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/imunologia , Monócitos/patologia , Nanopartículas/uso terapêutico , Placa Aterosclerótica/imunologia , Placa Aterosclerótica/patologia , Receptores CCR2/genéticaRESUMO
Vascular calcification, which is a major complication of diabetes mellitus, is an independent risk factor for cardiovascular disease. Osteogenic differentiation of vascular smooth muscle cells (VSMCs) is one of the key mechanisms underlying vascular calcification. Emerging evidence suggests that macrophage-derived extracellular vesicles (EVs) may be involved in calcification within atherosclerotic plaques in patients with diabetes mellitus. However, the role of macrophage-derived EVs in the progression of vascular calcification is largely unknown. In this study, we investigated whether macrophage-derived EVs contribute to the osteogenic differentiation of VSMCs under high glucose conditions. We isolated EVs that were secreted by murine peritoneal macrophages under normal glucose (EVs-NG) or high glucose (EVs-HG) conditions. miRNA array analysis in EVs from murine macrophages showed that miR-17-5p was significantly increased in EVs-HG compared with EVs-NG. Prediction analysis with miRbase identified transforming growth factor ß receptor type II (TGF-ß RII) as a potential target of miR-17-5p. EVs-HG as well as miR-17-5p overexpression with lipid nanoparticles inhibited the gene expression of Runx2, and TGF-ß RII. Furthermore, we demonstrated that VSMCs transfected with miR-17-5p mimic inhibited calcium deposition. Our findings reveal a novel role of macrophage-derived EVs in the negative regulation of osteogenic differentiation in VSMCs under high glucose conditions.
Assuntos
Diferenciação Celular , Vesículas Extracelulares , Glucose , MicroRNAs , Músculo Liso Vascular , Miócitos de Músculo Liso , Osteogênese , Transdução de Sinais , Fator de Crescimento Transformador beta , MicroRNAs/genética , MicroRNAs/metabolismo , Animais , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/citologia , Glucose/farmacologia , Glucose/metabolismo , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Fator de Crescimento Transformador beta/metabolismo , Camundongos , Miócitos de Músculo Liso/metabolismo , Vesículas Extracelulares/metabolismo , Calcificação Vascular/metabolismo , Calcificação Vascular/genética , Calcificação Vascular/patologia , Receptor do Fator de Crescimento Transformador beta Tipo II/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II/genética , Masculino , Camundongos Endogâmicos C57BL , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genéticaRESUMO
BACKGROUND AND AIMS: In advanced atherosclerotic lesions, macrophage deaths result in necrotic core formation and plaque vulnerability. Cyclophilin D (CypD) is a mitochondria-specific cyclophilin involved in the process of cell death after organ ischemia-reperfusion. However, the role of CypD in atherosclerosis, especially in necrotic core formation, is unknown. Therefore, this experiment aims to clarify the role of CypD in necrotic core formation. METHODS: To clarify the specific role of CypD, encoded by Ppif in mice, apolipoprotein-E/CypD-double knockout (Apoe-/-Ppif-/-) mice were generated. These mice were fed a high-fat diet containing 0.15 % cholesterol for 24 weeks to accelerate atherosclerotic lesion development. RESULTS: Deletion of CypD decreased the necrotic core size, accompanied by a reduction of macrophage apoptosis compared to control Apoe-/- mice. In RAW264.7 cells, siRNA-mediated knockdown of CypD attenuated the release of cytochrome c from the mitochondria to the cytosol induced by endoplasmic reticulum stress inducer thapsigargin. In addition, necroptosis, induced by TNF-α and caspase inhibitor, was attenuated by knockdown of CypD. Ly-6Chigh inflammatory monocytes in peripheral blood leukocytes and mRNA expression of Il1b in the aorta were decreased by deletion of CypD. In contrast, siRNA-mediated knockdown of CypD did not significantly decrease Il1b nor Ccl2 mRNA expression in RAW264.7 cells treated with LPS and IFN-γ, suggesting that inhibition of inflammation in vivo is likely due to decreased cell death in the atherosclerotic lesions rather than a direct action of CypD deletion on the macrophage. CONCLUSIONS: These results indicate that CypD induces macrophage death and mediates necrotic core formation in advanced atherosclerotic lesions. CypD could be a novel therapeutic target for treating atherosclerotic vascular diseases.
Assuntos
Aterosclerose , Macrófagos , Mitocôndrias , Necrose , Peptidil-Prolil Isomerase F , Placa Aterosclerótica , Animais , Peptidil-Prolil Isomerase F/metabolismo , Peptidil-Prolil Isomerase F/genética , Macrófagos/metabolismo , Aterosclerose/patologia , Aterosclerose/metabolismo , Aterosclerose/genética , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Células RAW 264.7 , Modelos Animais de Doenças , Apoptose , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Necroptose , Masculino , Camundongos Knockout , Apolipoproteínas E/genética , Apolipoproteínas E/deficiência , Ciclofilinas/metabolismo , Ciclofilinas/genética , Ciclofilinas/deficiência , Dieta Hiperlipídica , Interleucina-1beta/metabolismo , Antígenos LyRESUMO
Despite widespread adoption of tissue clearing techniques in recent years, poor access to suitable light-sheet fluorescence microscopes remains a major obstacle for biomedical end-users. Here, we present descSPIM (desktop-equipped SPIM for cleared specimens), a low-cost ($20,000-50,000), low-expertise (one-day installation by a non-expert), yet practical do-it-yourself light-sheet microscope as a solution for this bottleneck. Even the most fundamental configuration of descSPIM enables multi-color imaging of whole mouse brains and a cancer cell line-derived xenograft tumor mass for the visualization of neurocircuitry, assessment of drug distribution, and pathological examination by false-colored hematoxylin and eosin staining in a three-dimensional manner. Academically open-sourced ( https://github.com/dbsb-juntendo/descSPIM ), descSPIM allows routine three-dimensional imaging of cleared samples in minutes. Thus, the dissemination of descSPIM will accelerate biomedical discoveries driven by tissue clearing technologies.
Assuntos
Encéfalo , Imageamento Tridimensional , Microscopia de Fluorescência , Animais , Camundongos , Encéfalo/diagnóstico por imagem , Humanos , Microscopia de Fluorescência/métodos , Microscopia de Fluorescência/instrumentação , Imageamento Tridimensional/métodos , Linhagem Celular TumoralRESUMO
OBJECTIVE: Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is pathologically characterized by focal fibrinoid necrosis, in which ANCA-mediated neutrophil extracellular trap (NET) formation and subsequent endothelial cell necrosis occur. Cyclophilin D (CypD) plays an important role in mediation of cell necrosis and inflammation via the opening of mitochondrial permeability transition pores. This study was undertaken to examine the role of CypD in AAV pathogenesis. METHODS: We assessed the role and mechanism of CypD in ANCA-stimulated neutrophils in vitro by immunostaining and electron microscopy observation. We performed a comprehensive RNA-sequencing analysis on ANCA-treated murine neutrophils. To investigate the role of CypD in vivo, we assessed disease features in CypD-knockout mice and wild-type mice using 2 different murine AAV models: anti-myeloperoxidase IgG transfer-induced AAV and spontaneous AAV. RESULTS: In vitro experiments showed that pharmacologic and genetic inhibition of CypD suppressed ANCA-induced NET formation via the suppression of reactive oxygen species and cytochrome c release from the mitochondria. RNA-sequencing analyses in ANCA-treated murine neutrophils revealed the involvement of inflammatory responses, with CypD deficiency reducing ANCA-induced alterations in gene expression. Furthermore, analyses of upstream regulators revealed the relevance of intracellular calcium (CypD activator) and cyclosporin (CypD inhibitor) in ANCA stimulation, indicating that the CypD-dependent opening of mitochondrial permeability transition pores is associated with ANCA-induced neutrophil activation and NETosis. In both AAV mouse models, the genetic deletion of CypD ameliorated crescentic glomerulonephritis via the inhibition of CypD-dependent neutrophil and endothelial necrosis. CONCLUSION: CypD targeting is a novel and specific therapeutic strategy for AAV via the resolution of necrotizing vasculitis.
Assuntos
Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos , Anticorpos Anticitoplasma de Neutrófilos , Peptidil-Prolil Isomerase F , Animais , Camundongos , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/tratamento farmacológico , Inflamação , Necrose , Neutrófilos/metabolismo , RNARESUMO
AIM: Abdominal aortic aneurysm (AAA) is a lethal and multifactorial disease. To prevent a rupture and dissection of enlarged AAA, prophylactic surgery and stenting are currently available. There are, however, no medical therapies preventing these complications of AAA. Statin is one of the candidates, but its efficacy on AAA formation/progression remains controversial. We have previously demonstrated that nanoparticles (NPs) incorporating pitavastatin (Pitava-NPs)-clinical trials using these nanoparticles have been already conducted-suppressed progression of atherosclerosis in apolipoprotein E-deficient ( Apoe-/-) mice. Therefore, we have tested a hypothesis that monocytes/macrophages-targeting delivery of pitavastatin prevents the progression of AAA. METHODS: Angiotensin II was intraperitoneally injected by osmotic mini-pumps to induce AAA formation in Apoe-/- mice. NPs consisting of poly(lactic-co-glycolic acid) were used for in vivo delivery of pitavastatin to monocytes/macrophages. RESULTS: Intravenously administered Pitava-NPs (containing 0.012 mg/kg/week pitavastatin) inhibited AAA formation accompanied with reduction of macrophage accumulation and monocyte chemoattractant protein-1 (MCP-1) expression. Ex vivo molecular imaging revealed that Pitava-NPs not only reduced macrophage accumulation but also attenuated matrix metalloproteinase activity in the abdominal aorta, which was underpinned by attenuated elastin degradation. CONCLUSION: These results suggest that Pitava-NPs inhibit AAA formation associated with reduced macrophage accumulation and MCP-1 expression. This clinically feasible nanomedicine could be an innovative therapeutic strategy that prevents devastating complications of AAA.
Assuntos
Aneurisma da Aorta Abdominal/prevenção & controle , Inibidores de Hidroximetilglutaril-CoA Redutases/administração & dosagem , Macrófagos/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Nanopartículas , Quinolinas/administração & dosagem , Angiotensina II , Animais , Aneurisma da Aorta Abdominal/sangue , Aneurisma da Aorta Abdominal/patologia , Apolipoproteínas E , Quimiocina CCL2/sangue , Modelos Animais de Doenças , Masculino , Metaloproteinases da Matriz/sangue , Camundongos , Camundongos Endogâmicos C57BL , Copolímero de Ácido Poliláctico e Ácido PoliglicólicoRESUMO
AIM: To assess the results of a phase I/IIa open-label dose-escalation clinical trial of 5-day repeated intramuscular administration of pitavastatin-incorporated poly (lactic-co-glycolic acid) nanoparticles (NK-104-NP) in patients with chronic limb threatening ischemia (CLTI). METHODS: NK-104-NP was formulated using an emulsion solvent diffusion method. NK-104-NP at four doses (nanoparticles containing 0.5, 1, 2, and 4 mg of pitavastatin calcium, n=4 patients per dose) was investigated in a dose-escalation manner and administered intramuscularly into the ischemic limbs of 16 patients with CLTI. The safety and therapeutic efficacy of treatment were investigated over a 26-week follow-up period. RESULTS: No cardiovascular or other serious adverse events caused by NK-104-NP were detected during the follow-up period. Improvements in Fontaine and Rutherford classifications were noted in five patients (one, three, and one in the 1-, 2-, and 4-mg dose groups, respectively). Pharmacokinetic parameters including the maximum serum concentration and the area under the blood concentration-time curve increased with pitavastatin treatment in a dose-dependent manner. The area under the curve was slightly increased at day 5 compared with that at day 1 of treatment, although the difference was not statistically significant. CONCLUSIONS: This is the first clinical trial of pitavastatin-incorporated nanoparticles in patients with CLTI. Intramuscular administration of NK-104-NP to the ischemic limbs of patients with CLTI was safe and well tolerated and resulted in improvements in limb function.
Assuntos
Isquemia Crônica Crítica de Membro , Nanopartículas , Humanos , Quinolinas/uso terapêuticoRESUMO
Emerging evidence suggests that 7-ketocholesterol (7-KC), one of the most abundant dietary oxysterols, causes inflammation and cardiovascular diseases. Here we show the deteriorating effects of dietary 7-KC on myocardial ischemia-reperfusion (IR) injury and detailed the molecular mechanisms. A high-fat high-cholesterol diet containing 7-KC (7KWD) for 3 weeks increased the plasma 7-KC level compared with high-fat high-cholesterol diet in mice. In wild-type mice but not in CCR2-/- mice, dietary 7-KC increased the myocardial infarct size after IR. Flow cytometry revealed that the ratio of Ly-6Chigh inflammatory monocytes to total monocytes was increased in the 7KWD group. Unbiased RNA sequencing using murine primary macrophages revealed that 7-KC regulated the expression of transcripts related to inflammation and cholesterol biosynthesis. We further validated that in vitro, 7-KC induced endoplasmic reticulum stress, mitochondrial reactive oxygen species production, and nuclear factor-kappa B activation, which are associated with increased mRNA levels of proinflammatory cytokines. Administration of N-acetyl-L-cysteine or siRNA-mediated knockdown of PKR-like endoplasmic reticulum kinase or endoplasmic reticulum oxidase 1α suppressed the levels of 7-KC-induced inflammation. Dietary 7-KC exacerbates myocardial IR injury through monocyte/macrophage-mediated inflammation. Endoplasmic reticulum stress and oxidative stress are involved in the 7-KC-induced proinflammatory response in macrophages.
Assuntos
Traumatismo por Reperfusão Miocárdica , Traumatismo por Reperfusão , Animais , Dieta , Estresse do Retículo Endoplasmático , Inflamação/metabolismo , Cetocolesteróis , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão/metabolismoRESUMO
Background The opening of mitochondrial permeability transition pore and inflammation cooperatively progress myocardial ischemia-reperfusion (IR) injury, which hampers therapeutic effects of primary reperfusion therapy for acute myocardial infarction. We examined the therapeutic effects of nanoparticle-mediated medicine that simultaneously targets mitochondrial permeability transition pore and inflammation during IR injury. Methods and Results We used mice lacking cyclophilin D (CypD, a key molecule for mitochondrial permeability transition pore opening) and C-C chemokine receptor 2 and found that CypD contributes to the progression of myocardial IR injury at early time point (30-45 minutes) after reperfusion, whereas C-C chemokine receptor 2 contributes to IR injury at later time point (45-60 minutes) after reperfusion. Double deficiency of CypD and C-C chemokine receptor 2 enhanced cardioprotection compared with single deficiency regardless of the durations of ischemia. Deletion of C-C chemokine receptor 2, but not deletion of CypD, decreased the recruitment of Ly-6Chigh monocytes after myocardial IR injury. In CypD-knockout mice, administration of interleukin-1ß blocking antibody reduced the recruitment of these monocytes. Combined administration of polymeric nanoparticles composed of poly-lactic/glycolic acid and encapsulating nanoparticles containing cyclosporine A or pitavastatin, which inhibit mitochondrial permeability transition pore opening and monocyte-mediated inflammation, respectively, augmented the cardioprotection as compared with single administration of nanoparticles containing cyclosporine A or pitavastatin after myocardial IR injury. Conclusions Nanoparticle-mediated simultaneous targeting of mitochondrial injury and inflammation could be a novel therapeutic strategy for the treatment of myocardial IR injury.
Assuntos
Anti-Inflamatórios/farmacologia , Ciclosporina/farmacologia , Portadores de Fármacos , Mitocôndrias Cardíacas/efeitos dos fármacos , Poro de Transição de Permeabilidade Mitocondrial/antagonistas & inibidores , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miócitos Cardíacos/efeitos dos fármacos , Nanopartículas , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Quinolinas/farmacologia , Animais , Anti-Inflamatórios/química , Peptidil-Prolil Isomerase F/genética , Peptidil-Prolil Isomerase F/metabolismo , Ciclosporina/química , Modelos Animais de Doenças , Combinação de Medicamentos , Composição de Medicamentos , Mediadores da Inflamação/metabolismo , Interleucina-1beta/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/patologia , Poro de Transição de Permeabilidade Mitocondrial/metabolismo , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Quinolinas/química , Receptores CCR2/genética , Receptores CCR2/metabolismo , Fatores de TempoRESUMO
OBJECTIVE: We have demonstrated that vascular endothelial growth factor (VEGF) expression is upregulated in injured vascular wall, and blockade of VEGF inhibited monocyte infiltration and neointima formation in several animal models. In the present study, we aimed to clarify relative role of two VEGF receptors, flt-1 versus flk-1/KDR, in neointima formation after injury using flt-1 tyrosine kinase-deficient (Flt-1 TK(-/-)) mice and soluble Flt-1(sFlt-1) gene transfer. METHODS AND RESULTS: Neointima formation was comparable between wild-type and Flt-1 TK(-/-) mice 28 days after intraluminal wire injury in femoral arteries. By contrast, neointima formation was significantly suppressed by sFlt-1 gene transfer into Flt-1 TK(-/-) mice that blocks VEGF action on flk-1 (intima/media ratio: 2.8+/-0.4 versus 1.4+/-0.4, P<0.05). The inhibition of neointima formation was preceded by significant reduction of monocyte chemoattractant protein (MCP-1) expression in vascular smooth muscle cells (VSMCs) and monocyte infiltration 7 days after injury. Gene transfer of sFlt-1 or treatment of flk-1-specific antibody significantly inhibited VEGF-induced MCP-1 expression determined by RT-PCR in cultured aortic tissue and VSMCs. MCP-1-induced chemotaxis was equivalent between wild-type and Flt-1 TK(-/-) mice. CONCLUSIONS: These results suggest that endogenous VEGF accelerates neointima formation through flk-1 by regulating MCP-1 expression in VSMCs and macrophage-mediated inflammation in injured vascular wall in murine model of wire injury.
Assuntos
Artéria Femoral/enzimologia , Técnicas de Transferência de Genes , Músculo Liso Vascular/enzimologia , Túnica Íntima/enzimologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Quimiocina CCL2/metabolismo , Quimiotaxia de Leucócito , Constrição Patológica , Modelos Animais de Doenças , Eletroporação , Artéria Femoral/lesões , Artéria Femoral/patologia , Hiperplasia , Macrófagos Peritoneais/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/lesões , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/enzimologia , Neovascularização Fisiológica , Técnicas de Cultura de Tecidos , Túnica Íntima/lesões , Túnica Íntima/patologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/deficiência , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
OBJECTIVE: Recent clinical studies of therapeutic neovascularization using angiogenic growth factors demonstrated smaller therapeutic effects than those reported in animal experiments. We hypothesized that nanoparticle (NP)-mediated cell-selective delivery of statins to vascular endothelium would more effectively and integratively induce therapeutic neovascularization. METHODS AND RESULTS: In a murine hindlimb ischemia model, intramuscular injection of biodegradable polymeric NP resulted in cell-selective delivery of NP into the capillary and arteriolar endothelium of ischemic muscles for up to 2 weeks postinjection. NP-mediated statin delivery significantly enhanced recovery of blood perfusion to the ischemic limb, increased angiogenesis and arteriogenesis, and promoted expression of the protein kinase Akt, endothelial nitric oxide synthase (eNOS), and angiogenic growth factors. These effects were blocked in mice administered a nitric oxide synthase inhibitor, or in eNOS-deficient mice. CONCLUSIONS: NP-mediated cell-selective statin delivery may be a more effective and integrative strategy for therapeutic neovascularization in patients with severe organ ischemia.
Assuntos
Indutores da Angiogênese/administração & dosagem , Portadores de Fármacos , Endotélio Vascular/efeitos dos fármacos , Isquemia/tratamento farmacológico , Músculo Esquelético/irrigação sanguínea , Nanopartículas , Neovascularização Fisiológica/efeitos dos fármacos , Quinolinas/administração & dosagem , Indutores da Angiogênese/sangue , Proteínas Angiogênicas/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Endotélio Vascular/enzimologia , Endotélio Vascular/fisiopatologia , Inibidores Enzimáticos/farmacologia , Membro Posterior , Humanos , Injeções Intramusculares , Isquemia/enzimologia , Isquemia/fisiopatologia , Ácido Láctico/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Óxido Nítrico Sintase Tipo III/deficiência , Óxido Nítrico Sintase Tipo III/genética , Ácido Poliglicólico/metabolismo , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quinolinas/sangue , Fluxo Sanguíneo Regional/efeitos dos fármacos , Fatores de TempoRESUMO
Two decades have passed since therapeutic angiogenesis was proposed to promote reparative collateral growth as an alternative therapy for ischemic diseases in patients for whom neither surgical revascularization nor endovascular therapy was suitable. When therapeutic angiogenesis first began, local administration was conducted using recombinant growth factor proteins or gene-encoding growth factors for endothelial cells. Since then, autologous stem cells and endothelial progenitor cell transplantation therapy have been developed. Although many clinical trials have been performed on patients, most therapies have not yet become standard treatments. We have developed a nanoparticle (NP)-mediated, drug-targeting delivery system using bioabsorbable poly-lactic/glycolic acid (PLGA) NPs. In several animal models, pitavastatin-incorporated (Pitava)-NPs showed significant therapeutic effects on critical limb ischemia. Because PLGA NPs are delivered selectively to vascular endothelial cells after intramuscular administration, it is suggested that therapeutic angiogenesis/arteriogenesis plays an important role in the mechanism by which Pitava-NPs exert beneficial therapeutic effects. To translate this to clinical medicine, we have performed studies and produced Pitava-NPs in compliance with good laboratory practice/good manufacturing practice regulations, and completed a phase I/II clinical trial, reporting the safety and efficacy of Pitava-NP intramuscular injection for patients with critical limb ischemia. This review will focus on therapeutic angiogenesis/arteriogenesis for peripheral arterial disease induced by Pitava-NPs.