RESUMO
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
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
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
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
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.
RESUMO
AIMS: Myocardial ischaemia-reperfusion (IR) injury hampers the therapeutic effect of revascularization in patients with acute myocardial infarction (AMI). Innate immunity for damage-associated protein patterns promotes the process of IR injury; however, the blockade of Toll-like receptor 4 (TLR4) in myocardial IR injury has not been translated into clinical practice. Therefore, we aimed to examine whether the nanoparticle-mediated administration of TAK-242, a chemical inhibitor of TLR4, attenuates myocardial IR injury in a clinically feasible protocol in a mouse model. METHODS AND RESULTS: We have prepared poly-(lactic-co-glycolic acid) nanoparticles containing TAK-242 (TAK-242-NP). TAK-242-NP significantly enhanced the drug delivery to monocytes/macrophages in the spleen, blood, and the heart in mice. Intravenous administration of TAK-242-NP (containing 1.0 or 3.0 mg/kg TAK-242) at the time of reperfusion decreased the infarct size, but the TAK-242 solution did not even when administered at a dosage of 10.0 mg/kg. TAK-242-NP inhibited the recruitment of Ly-6Chigh monocytes to the heart, which was accompanied by decreased circulating HMGB1, and NF-κB activation and cytokine expressions in the heart. TAK-242-NP did not decrease the infarct size further in TLR4-deficient mice, confirming the TLR4-specific mechanism in the effects of TAK-242-NP. Furthermore, TAK-242-NP did not decrease the infarct size further in CCR2-deficient mice, suggesting that monocyte/macrophage-mediated inflammation is the primary therapeutic target of TAK-242-NP. CONCLUSION: The nanoparticle-mediated delivery of TAK-242-NP represent a novel and clinical feasible strategy in patients undergone coronary revascularization for AMI by regulating TLR4-dependent monocytes/macrophages-mediated inflammation.
Assuntos
Anti-Inflamatórios/administração & dosagem , Portadores de Fármacos , Macrófagos/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Infarto do Miocárdio/prevenção & controle , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miocárdio/metabolismo , Nanopartículas , Sulfonamidas/administração & dosagem , Receptor 4 Toll-Like/antagonistas & inibidores , Remodelação Ventricular/efeitos dos fármacos , Animais , Anti-Inflamatórios/química , Modelos Animais de Doenças , Composição de Medicamentos , Proteína HMGB1/metabolismo , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/metabolismo , Monócitos/patologia , Infarto do Miocárdio/genética , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/patologia , NF-kappa B/metabolismo , Nanomedicina , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Receptores CCR2/genética , Receptores CCR2/metabolismo , Transdução de Sinais , Sulfonamidas/química , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Função Ventricular Esquerda/efeitos dos fármacosRESUMO
Aims: Monocyte-mediated inflammation is a major mechanism underlying myocardial ischaemia-reperfusion (IR) injury and the healing process after acute myocardial infarction (AMI). However, no definitive anti-inflammatory therapies have been developed for clinical use. Pioglitazone, a peroxisome proliferator-activated receptor-gamma (PPARγ) agonist, has unique anti-inflammatory effects on monocytes/macrophages. Here, we tested the hypothesis that nanoparticle (NP)-mediated targeting of pioglitazone to monocytes/macrophages ameliorates IR injury and cardiac remodelling in preclinical animal models. Methods and results: We formulated poly (lactic acid/glycolic acid) NPs containing pioglitazone (pioglitazone-NPs). In a mouse IR model, these NPs were delivered predominantly to circulating monocytes and macrophages in the IR heart. Intravenous treatment with pioglitazone-NPs at the time of reperfusion attenuated IR injury. This effect was abrogated by pre-treatment with the PPARγ antagonist GW9662. In contrast, treatment with a pioglitazone solution had no therapeutic effects on IR injury. Pioglitazone-NPs inhibited Ly6Chigh inflammatory monocyte recruitment as well as inflammatory gene expression in the IR hearts. In a mouse myocardial infarction model, intravenous treatment with pioglitazone-NPs for three consecutive days, starting 6 h after left anterior descending artery ligation, attenuated cardiac remodelling by reducing macrophage recruitment and polarizing macrophages towards the pro-healing M2 phenotype. Furthermore, pioglitazone-NPs significantly decreased mortality after MI. Finally, in a conscious porcine model of myocardial IR, pioglitazone-NPs induced cardioprotection from reperfused infarction, thus providing pre-clinical proof of concept. Conclusion: NP-mediated targeting of pioglitazone to inflammatory monocytes protected the heart from IR injury and cardiac remodelling by antagonizing monocyte/macrophage-mediated acute inflammation and promoting cardiac healing after AMI.
Assuntos
Anti-Inflamatórios/farmacologia , Portadores de Fármacos , Macrófagos/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Infarto do Miocárdio/tratamento farmacológico , Miocárdio/metabolismo , Nanopartículas , PPAR gama/agonistas , Pioglitazona/farmacologia , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Cicatrização/efeitos dos fármacos , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/química , Modelos Animais de Doenças , Injeções Intravenosas , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/metabolismo , Monócitos/patologia , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Miocárdio/patologia , PPAR gama/metabolismo , Pioglitazona/administração & dosagem , Pioglitazona/química , Receptores CCR2/genética , Receptores CCR2/metabolismo , Transdução de Sinais , Suínos , Porco MiniaturaRESUMO
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
BACKGROUND: There is an unmet need to develop an innovative cardioprotective modality for acute myocardial infarction, for which interventional reperfusion therapy is hampered by ischemia-reperfusion (IR) injury. We recently reported that bioabsorbable poly(lactic acid/glycolic acid) (PLGA) nanoparticle-mediated treatment with pitavastatin (pitavastatin-NP) exerts a cardioprotective effect in a rat IR injury model by activating the PI3K-Akt pathway and inhibiting inflammation. To obtain preclinical proof-of-concept evidence, in this study, we examined the effect of pitavastatin-NP on myocardial IR injury in conscious and anesthetized pig models. METHODS AND RESULTS: Eighty-four Bama mini-pigs were surgically implanted with a pneumatic cuff occluder at the left circumflex coronary artery (LCx) and telemetry transmitters to continuously monitor electrocardiogram as well as to monitor arterial blood pressure and heart rate. The LCx was occluded for 60 minutes, followed by 24 hours of reperfusion under conscious conditions. Intravenous administration of pitavastatin-NP containing ≥ 8 mg/body of pitavastatin 5 minutes before reperfusion significantly reduced infarct size; by contrast, pitavastatin alone (8 mg/body) showed no therapeutic effects. Pitavastatin-NP produced anti-apoptotic effects on cultured cardiomyocytes in vitro. Cardiac magnetic resonance imaging performed 4 weeks after IR injury revealed that pitavastatin-NP reduced the extent of left ventricle remodeling. Importantly, pitavastatin-NP exerted no significant effects on blood pressure, heart rate, or serum biochemistry. Exploratory examinations in anesthetized pigs showed pharmacokinetic analysis and the effects of pitavastatin-NP on no-reflow phenomenon. CONCLUSIONS: NP-mediated delivery of pitavastatin to IR-injured myocardium exerts cardioprotective effects on IR injury without apparent adverse side effects in a preclinical conscious pig model. Thus, pitavastatin-NP represents a novel therapeutic modality for IR injury in acute myocardial infarction.
Assuntos
Infarto do Miocárdio/tratamento farmacológico , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Reperfusão Miocárdica , Miocárdio/patologia , Nanopartículas/química , Quinolinas/administração & dosagem , Quinolinas/uso terapêutico , Pesquisa Translacional Biomédica , Anestesia , Animais , Pressão Sanguínea , Cardiotônicos/administração & dosagem , Cardiotônicos/uso terapêutico , Morte Celular , Células Cultivadas , Estado de Consciência , Modelos Animais de Doenças , Frequência Cardíaca , Testes de Função Renal , Ácido Láctico/química , Testes de Função Hepática , Imageamento por Ressonância Magnética , Infarto do Miocárdio/sangue , Infarto do Miocárdio/complicações , Infarto do Miocárdio/fisiopatologia , Traumatismo por Reperfusão Miocárdica/sangue , Traumatismo por Reperfusão Miocárdica/complicações , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miócitos Cardíacos/patologia , Fenômeno de não Refluxo , Fosforilação , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quinolinas/sangue , Quinolinas/farmacocinética , Volume Sistólico , Suínos , Porco Miniatura , Remodelação VentricularRESUMO
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
Recent technical innovation has enabled chemical modifications of small materials and various kinds of nanoparticles have been created. In clinical settings, nanoparticle-mediated drug delivery systems have been used in the field of cancer care to deliver therapeutic agents specifically to cancer tissues and to enhance the efficacy of drugs by gradually releasing their contents. In addition, nanotechnology has enabled the visualization of various molecular processes by targeting proteinases or inflammation. Nanoparticles that consist of poly (lactic-co-glycolic) acid (PLGA) deliver therapeutic agents to monocytes/macrophages and function as anti-inflammatory nanoparticles in combination with statins, angiotensin receptor antagonists, or agonists of peroxisome proliferator-activated receptor-γ (PPARγ). PLGA nanoparticle-mediated delivery of pitavastatin has been shown to prevent inflammation and ameliorated features associated with plaque ruptures in hyperlipidemic mice. PLGA nanoparticles were also delivered to tissues with increased vascular permeability and nanoparticles incorporating pitavastatin, injected intramuscularly, were retained in ischemic tissues and induced therapeutic arteriogenesis. This resulted in attenuation of hind limb ischemia. Ex vivo treatment of vein grafts with imatinib nanoparticles before graft implantation has been demonstrated to inhibit lesion development. These results suggest that nanoparticle-mediated drug delivery system can be a promising strategy as a next generation therapy for atherosclerotic vascular diseases.
Assuntos
Anti-Inflamatórios/administração & dosagem , Aterosclerose/prevenção & controle , Nanopartículas/administração & dosagem , Doenças Vasculares/prevenção & controle , Animais , Humanos , CamundongosRESUMO
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
Emerging evidence suggests that Notch signaling not only regulates biological processes during development but also participates in the pathogenesis of various diseases in adults, including tumor angiogenesis, hematopoietic malignancies, and cardiometabolic syndromes. Notch signaling involves several ligands and receptors that have unique and overlapping functions. Therefore, blocking function of a ligand or receptor with a neutralizing antibody is a useful approach to examine the specific role of each Notch component. In addition, administration of Notch signaling blocking antibodies in experimental animals offers important insights into clinical translation of Notch biology. In this chapter, we describe examples of in vitro and in vivo loss-of-function experiments with blockade of Notch ligands, particularly Delta-like ligand 4 (Dll4).
Assuntos
Anticorpos Neutralizantes/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Proteínas de Membrana/antagonistas & inibidores , Receptores Notch/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Anticorpos Neutralizantes/imunologia , Técnicas de Cultura de Células/métodos , Linhagem Celular , Ensaios Enzimáticos/métodos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Luciferases/metabolismo , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , CamundongosRESUMO
Macrophages and smooth muscle cells (SMCs) represent major players in the pathogenesis of atherosclerotic vascular diseases. SMCs often reside in close proximity to macrophage clusters. Activated macrophages may promote pro-atherogenic functions of SMCs. Addressing macrophage-dependent mechanisms of SMC activation may provide new insight into atherogenesis and new therapies for various vascular diseases. Direct evidence for such interplay between atherosclerosis-associated cell types, however, remains scant. While SMC-derived macrophage foam cells have long been reported, recent evidence has also identified SMC-like cells of monocyte origin, suggesting dynamic interchangeability of these cell types. Future efforts may help to understand the interplay between key cell types and offer new paradigms in vascular medicine and pharmacology.
Assuntos
Aterosclerose/metabolismo , Aterosclerose/patologia , Comunicação Celular/fisiologia , Macrófagos/metabolismo , Macrófagos/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Animais , Humanos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologiaRESUMO
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
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
The angiotensin II (Ang II) type 1a (AT1a) receptor is expressed on multiple cell types in atherosclerotic lesions, including bone marrow-derived cells and vascular wall cells, and mediates inflammatory and proliferative responses. Indeed, Ang II infusion accelerates atherogenesis in hyperlipidemic mice by recruiting monocytes and by activating vascular wall cells. Here, we investigated the relative roles of AT1a receptors in the bone marrow vs. the vascular wall in Ang II-induced atherogenesis. Apolipoprotein E-knockout (ApoE(-/-)) mice with or without bone marrow AT1a receptor were generated by experimental bone marrow transplantation using AT1a(+/+) or AT1a(-/-) recipients. In these mice, 28-d Ang II infusion induced significant atherosclerosis in the aorta, and the severity of plaque formation was not affected by the absence of bone marrow AT1a receptor. We then generated AT1a(-/-)ApoE(-/-) mice with or without bone marrow AT1a receptor. Ang II-induced plaque formation was blunted irrespective of the presence of bone marrow AT1a receptor. Host AT1a receptor deficiency was found to suppress Ang II-induced reactive oxygen species production. In addition, AT1a receptor deficiency also impaired monocyte chemoattractant protein-1 (MCP-1) and vascular cell adhesion molecule-1 (VCAM-1) expression in the arterial wall 7 d after Ang II initiation. These molecules normally initiate later macrophage-mediated inflammation in the vascular wall. By contrast, AT1a receptor deficiency in the bone marrow did not affect MCP-1-induced monocyte chemotaxis in vitro. In conclusion, AT1a receptors in the host vascular wall, but not in the bone marrow, are essential in Ang II-induced atherogenesis.
Assuntos
Artérias/patologia , Aterosclerose/patologia , Aterosclerose/fisiopatologia , Medula Óssea/patologia , Receptor Tipo 1 de Angiotensina/genética , Angiotensina II/farmacologia , Animais , Apolipoproteínas E/genética , Artérias/metabolismo , Artérias/fisiopatologia , Aterosclerose/metabolismo , Medula Óssea/metabolismo , Medula Óssea/fisiopatologia , Transplante de Medula Óssea , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Quimiotaxia de Leucócito/fisiologia , Quimera , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Receptor Tipo 1 de Angiotensina/metabolismo , Receptores CCR2/genética , Vasoconstritores/farmacologiaRESUMO
OBJECTIVE: We have previously shown that the intramuscular transfer of the anti-monocyte chemoattractant protein-1 (MCP-1) gene (called 7ND) is able to prevent experimental restenosis. The aim of this study was to determine the in vivo efficacy and safety of local delivery of 7ND gene via the gene-eluting stent in reducing in-stent neointima formation in rabbits and in cynomolgus monkeys. METHODS AND RESULTS: We here found that in vitro, 7ND effectively inhibited the chemotaxis of mononuclear leukocytes and also inhibited the proliferation/migration of vascular smooth muscle cells. We then coated stents with a biocompatible polymer containing a plasmid bearing the 7ND gene, and deployed these stents in the iliac arteries of rabbits and monkeys. 7ND gene-eluting stents attenuated stent-associated monocyte infiltration and neointima formation after one month in rabbits, and showed long-term inhibitory effects on neointima formation when assessments were carried out at 1, 3, and 6 months in monkeys. CONCLUSIONS: Strategy of inhibiting the action of MCP-1 with a 7ND gene-eluting stent reduced in-stent neointima formation with no evidence of adverse effects in rabbits and monkeys. The 7ND gene-eluting stent could be a promising therapy for treatment of restenosis in humans.
Assuntos
Arteriopatias Oclusivas/prevenção & controle , Quimiocina CCL2/metabolismo , Técnicas de Transferência de Genes , Terapia Genética/métodos , Artéria Ilíaca/cirurgia , Stents , Procedimentos Cirúrgicos Vasculares/instrumentação , Animais , Arteriopatias Oclusivas/etiologia , Arteriopatias Oclusivas/genética , Arteriopatias Oclusivas/metabolismo , Arteriopatias Oclusivas/patologia , Materiais Biocompatíveis , Células Cultivadas , Quimiocina CCL2/genética , Quimiotaxia de Leucócito , Constrição Patológica/prevenção & controle , Modelos Animais de Doenças , Estudos de Viabilidade , Humanos , Artéria Ilíaca/patologia , Cinética , Leucócitos Mononucleares/patologia , Macaca fascicularis , Macrófagos/patologia , Masculino , Metais , Mutação , Plasmídeos/metabolismo , Desenho de Prótese , Coelhos , Fatores de Tempo , Túnica Íntima/patologia , Procedimentos Cirúrgicos Vasculares/efeitos adversosRESUMO
Cholesterol embolization (CE) is a potentially serious complication associated with invasive arterial maneuvers, in which standard therapy has not been established. We experienced two cases of CE in patients with severe atherosclerosis whose renal function deteriorated within a few months after invasive arterial maneuvers. CE was confirmed either by renal biopsy (case 1) or skin biopsy (case 2). Oral administration of prednisolone at a daily dose of 30 mg (0.4 mg/kg) was effective to improve their renal function. Our observation suggests that corticosteroid therapy may be beneficial in some patients with CE.