RESUMO
Respiratory complex I is an efficient driver for oxidative phosphorylation in mammalian mitochondria, but its uncontrolled catalysis under challenging conditions leads to oxidative stress and cellular damage. Ischemic conditions switch complex I from rapid, reversible catalysis into a dormant state that protects upon reoxygenation, but the molecular basis for the switch is unknown. We combined precise biochemical definition of complex I catalysis with high-resolution cryo-electron microscopy structures in the phospholipid bilayer of coupled vesicles to reveal the mechanism of the transition into the dormant state, modulated by membrane interactions. By implementing a versatile membrane system to unite structure and function, attributing catalytic and regulatory properties to specific structural states, we define how a conformational switch in complex I controls its physiological roles.
Assuntos
Complexo I de Transporte de Elétrons , Isquemia , Animais , Microscopia Crioeletrônica , Complexo I de Transporte de Elétrons/metabolismo , Complexo I de Transporte de Elétrons/química , Isquemia/enzimologia , Bicamadas Lipídicas/metabolismo , Mitocôndrias/enzimologia , Fosforilação Oxidativa , Bovinos , Ubiquinona/química , Ubiquinona/metabolismo , Conformação Proteica em alfa-HéliceRESUMO
The post-translational modification of intracellular proteins by O-linked ß-GlcNAc (O-GlcNAc) has emerged as a critical regulator of cardiac function. Enhanced O-GlcNAcylation activates cytoprotective pathways in cardiac models of ischemia-reperfusion (I/R) injury; however, the mechanisms underpinning O-GlcNAc cycling in response to I/R injury have not been comprehensively assessed. The cycling of O-GlcNAc is regulated by the collective efforts of two enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which catalyze the addition and hydrolysis of O-GlcNAc, respectively. It has previously been shown that baseline heart physiology and pathophysiology are impacted by sex. Here, we hypothesized that sex differences in molecular signaling may target protein O-GlcNAcylation both basally and in ischemic hearts. To address this question, we subjected male and female WT murine hearts to ex vivo ischemia or I/R injury. We assessed hearts for protein O-GlcNAcylation, abundance of OGT, OGA, and glutamine:fructose-6-phosphate aminotransferase (GFAT2), activity of OGT and OGA, and UDP-GlcNAc levels. Our data demonstrate elevated O-GlcNAcylation in female hearts both basally and during ischemia. We show that OGT activity was enhanced in female hearts in all treatments, suggesting a mechanism for these observations. Furthermore, we found that ischemia led to reduced O-GlcNAcylation and OGT-specific activity. Our findings provide a foundation for understanding molecular mechanisms that regulate O-GlcNAcylation in the heart and highlight the importance of sex as a significant factor when assessing key regulatory events that control O-GlcNAc cycling. These data suggest the intriguing possibility that elevated O-GlcNAcylation in females contributes to reduced ischemic susceptibility.
Assuntos
Acetilglucosamina , Coração , Miocárdio , N-Acetilglucosaminiltransferases , Caracteres Sexuais , Transdução de Sinais , Animais , Feminino , Masculino , Camundongos , Acetilglucosamina/metabolismo , Coração/fisiologia , Isquemia/enzimologia , Isquemia/metabolismo , Miocárdio/enzimologia , Miocárdio/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Processamento de Proteína Pós-TraducionalRESUMO
Objective: The dominant driver of arteriogenesis is elevated shear stress sensed by the endothelial glycocalyx thereby promoting arterial outward remodeling. Hyaluronan, a critical component of the endothelial glycocalyx, is synthesized by 3 HAS isoenzymes (hyaluronan synthases 1-3) at the plasma membrane. Considering further the importance of HAS3 for smooth muscle cell and immune cell functions we aimed to evaluate its role in collateral artery growth. Approach and Results: Male Has3-deficient (Has3-KO) mice were subjected to hindlimb ischemia. Blood perfusion was monitored by laser Doppler perfusion imaging and endothelial function was assessed by measurement of flow-mediated dilation in vivo. Collateral remodeling was monitored by high resolution magnetic resonance angiography. A neutralizing antibody against CD44 (clone KM201) was injected intraperitoneally to analyze hyaluronan signaling in vivo. After hindlimb ischemia, Has3-KO mice showed a reduced arteriogenic response with decreased collateral remodeling and impaired perfusion recovery. While postischemic leukocyte infiltration was unaffected, a diminished flow-mediated dilation pointed towards an impaired endothelial cell function. Indeed, endothelial AKT (protein kinase B)-dependent eNOS (endothelial nitric oxide synthase) phosphorylation at Ser1177 was substantially reduced in Has3-KO thigh muscles. Endothelial-specific Has3-KO mice mimicked the hindlimb ischemia-induced phenotype of impaired perfusion recovery as observed in global Has3-deficiency. Mechanistically, blocking selectively the hyaluronan binding site of CD44 reduced flow-mediated dilation, thereby suggesting hyaluronan signaling through CD44 as the underlying signaling pathway. Conclusions: In summary, HAS3 contributes to arteriogenesis in hindlimb ischemia by hyaluronan/CD44-mediated stimulation of eNOS phosphorylation at Ser1177. Thus, strategies augmenting endothelial HAS3 or CD44 could be envisioned to enhance vascularization under pathological conditions.
Assuntos
Células Endoteliais/enzimologia , Membro Posterior/irrigação sanguínea , Receptores de Hialuronatos/metabolismo , Hialuronan Sintases/metabolismo , Isquemia/enzimologia , Neovascularização Fisiológica , Óxido Nítrico Sintase Tipo III/metabolismo , Animais , Circulação Colateral , Modelos Animais de Doenças , Humanos , Hialuronan Sintases/genética , Isquemia/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Óxido Nítrico Sintase Tipo III/genética , Fosforilação , Fluxo Sanguíneo Regional , Transdução de Sinais , Fatores de TempoRESUMO
Objective: Critical limb ischemia is a major complication of diabetes characterized by insufficient collateral vessel development and proper growth factor signaling unresponsiveness. Although mainly deactivated by hypoxia, phosphatases are important players in the deregulation of proangiogenetic pathways. Previously, SHP-1 (Scr homology 2-containing phosphatase-1) was found to be associated with the downregulation of growth factor actions in the diabetic muscle. Thus, we aimed to gain further understanding of the impact of SHP-1 on smooth muscle cell (SMC) function under hypoxic and diabetic conditions. Approach and Results: Despite being inactivated under hypoxic conditions, high glucose level exposure sustained SHP-1 phosphatase activity in SMC and increased its interaction with PDGFR (platelet-derived growth factor receptor)-ß, thus reducing PDGF proangiogenic actions. Overexpression of an inactive form of SHP-1 fully restored PDGF-induced proliferation, migration, and signaling pathways in SMC exposed to high glucose and hypoxia. Nondiabetic and diabetic mice with deletion of SHP-1 specifically in SMC were generated. Ligation of the femoral artery was performed, and blood flow was measured for 4 weeks. Blood flow reperfusion, vascular density and maturation, and limb survival were all improved while vascular apoptosis was attenuated in diabetic SMC-specific SHP-1 null mice as compared to diabetic mice. Conclusions: Diabetes and high glucose level exposure maintained SHP-1 activity preventing hypoxia-induced PDGF actions in SMC. Specific deletion of SHP-1 in SMC partially restored blood flow reperfusion in the diabetic ischemic limb. Therefore, local modulation of SHP-1 activity in SMC could represent a potential therapeutic avenue to improve the proangiogenic properties of SMC under ischemia and diabetes.
Assuntos
Indutores da Angiogênese/farmacologia , Diabetes Mellitus Experimental/enzimologia , Angiopatias Diabéticas/enzimologia , Membro Posterior/irrigação sanguínea , Isquemia/enzimologia , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Animais , Glicemia/metabolismo , Estudos de Casos e Controles , Bovinos , Hipóxia Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/fisiopatologia , Angiopatias Diabéticas/genética , Angiopatias Diabéticas/fisiopatologia , Ativação Enzimática , Humanos , Isquemia/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/enzimologia , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/enzimologia , Miócitos de Músculo Liso/patologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Transdução de SinaisRESUMO
[Figure: see text].
Assuntos
Membro Posterior/irrigação sanguínea , Células Endoteliais da Veia Umbilical Humana/enzimologia , Isquemia/enzimologia , Metiltransferases/metabolismo , MicroRNAs/metabolismo , Infarto do Miocárdio/enzimologia , Neovascularização Fisiológica , Adenosina/análogos & derivados , Adenosina/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Humanos , Isquemia/genética , Isquemia/fisiopatologia , Masculino , Metiltransferases/genética , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Infarto do Miocárdio/genética , Infarto do Miocárdio/fisiopatologia , Transdução de Sinais , Trombospondina 1/genética , Trombospondina 1/metabolismoRESUMO
[Figure: see text].
Assuntos
5'-Nucleotidase/administração & dosagem , Adenosina/metabolismo , Portadores de Fármacos , Membro Posterior/irrigação sanguínea , Isquemia/tratamento farmacológico , Maleimidas/química , Doença Arterial Periférica/tratamento farmacológico , Polietilenoglicóis/química , 5'-Nucleotidase/química , 5'-Nucleotidase/metabolismo , Animais , Células Cultivadas , Preparações de Ação Retardada , Modelos Animais de Doenças , Composição de Medicamentos , Feminino , Humanos , Hidrogéis , Isquemia/enzimologia , Isquemia/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Ativação de Neutrófilo/efeitos dos fármacos , Doença Arterial Periférica/enzimologia , Doença Arterial Periférica/fisiopatologia , Fluxo Sanguíneo Regional , Regulação para CimaRESUMO
[Figure: see text].
Assuntos
Heme Oxigenase-1/metabolismo , Inflamassomos/metabolismo , Isquemia/enzimologia , Lisossomos/enzimologia , Macrófagos/enzimologia , Proteínas de Membrana/metabolismo , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/enzimologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Animais , Células Cultivadas , Bases de Dados Genéticas , Modelos Animais de Doenças , Heme Oxigenase-1/genética , Membro Posterior , Humanos , Inflamassomos/genética , Mediadores da Inflamação/metabolismo , Isquemia/genética , Isquemia/fisiopatologia , Masculino , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético/fisiopatologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Neovascularização Fisiológica , Proteólise , Recuperação de Função Fisiológica , Fluxo Sanguíneo RegionalRESUMO
ABSTRACT: Promoting angiogenesis is a critical treatment strategy for ischemic cardiovascular diseases. Shexiang Baoxin Pill (SBP), a traditional Chinese medicine, has been reported to be capable of relieving angina and improve heart function by promoting angiogenesis. The aim of this study was to determine the role of mitochondrial aldehyde dehydrogenase 2 (ALDH2) in SBP-induced angiogenesis. Left femoral artery ligation was performed in wild-type mice (WT) and ALDH2 knockout mice, which were administrated with SBP (20 mg/kg/d) or equal volume saline per day by gastric gavage for 2 weeks. Perfusion recovery, angiogenesis in chronic hind limb ischemia, was significantly improved in the WT + SBP group than in the WT group. However, these beneficial effects were absent in ALDH2 knockout mice. In vitro, hypoxia impaired the ability of proliferation, migration and tube formation, sprouting angiogenesis, and promoted apoptosis in cardiovascular microvascular endothelial cells, whereas the hypoxia damage was restored by SBP. The protective effect of SBP was remarkably weakened by ALDH2 knockdown. Furthermore, SBP suppressed hypoxia-induced ALDH2/protein kinase B (AKT)/mammalian target of rapamycin pathways. In conclusion, this study demonstrated that SBP protected lower limb from ischemia injury through the ALDH2-dependent pathway. The protective mechanism of SBP in cardiovascular microvascular endothelial cells was partly mediated through ALDH2/AKT/mammalian target of rapamycin pathways.
Assuntos
Aldeído-Desidrogenase Mitocondrial/metabolismo , Indutores da Angiogênese/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Células Endoteliais/efeitos dos fármacos , Membro Posterior/irrigação sanguínea , Isquemia/tratamento farmacológico , Neovascularização Fisiológica/efeitos dos fármacos , Aldeído-Desidrogenase Mitocondrial/genética , Animais , Hipóxia Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/enzimologia , Ativação Enzimática , Isquemia/enzimologia , Isquemia/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Sprague-Dawley , Fluxo Sanguíneo Regional , Transdução de SinaisRESUMO
AIMS: Peripheral artery disease is a complication of diabetes leading to critical hindlimb ischemia. Diabetes-induced inhibition of VEGF actions is associated with the activation of protein kinase Cδ (PKCδ). We aim to specifically investigate the role of PKCδ in endothelial cell (EC) function and VEGF signaling. METHODS: Nondiabetic and diabetic mice, with (ec-Prkcd-/-) or without (ec-Prkcdf/f) endothelial deletion of PKCδ, underwent femoral artery ligation. Blood flow reperfusion was assessed up to 4 weeks post-surgery. Capillary density, EC apoptosis and VEGF signaling were evaluated in the ischemic muscle. Src homology region 2 domain-containing phosphatase-1 (SHP-1) phosphatase activity was assessed in vitro using primary ECs. RESULTS: Ischemic muscle of diabetic ec-Prkcdf/f mice exhibited reduced blood flow reperfusion and capillary density while apoptosis increased as compared to nondiabetic ec-Prkcdf/f mice. In contrast, blood flow reperfusion and capillary density were significantly improved in diabetic ec-Prkcd-/- mice. VEGF signaling pathway was restored in diabetic ec-Prkcd-/- mice. The deletion of PKCδ in ECs prevented diabetes-induced VEGF unresponsiveness through a reduction of SHP-1 phosphatase activity. CONCLUSIONS: Our data provide new highlights in mechanisms by which PKCδ activation in EC contributed to poor collateral vessel formation, thus, offering novel therapeutic targets to improve angiogenesis in the diabetic limb.
Assuntos
Diabetes Mellitus Experimental/enzimologia , Células Endoteliais/enzimologia , Membro Posterior/irrigação sanguínea , Isquemia/enzimologia , Neovascularização Fisiológica , Proteína Quinase C-delta/deficiência , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Apoptose , Bovinos , Células Cultivadas , Circulação Colateral , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/fisiopatologia , Células Endoteliais/patologia , Isquemia/genética , Isquemia/fisiopatologia , Camundongos Knockout , Densidade Microvascular , Proteína Quinase C-delta/genética , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Fluxo Sanguíneo Regional , Transdução de SinaisRESUMO
Heat shock protein 32 (Hsp32), also known as heme oxygenase1 (HO1), is an enzyme that exists in microsomes. HO1 can be induced by a variety of stimuli, including heavy metals, heat shock, inflammatory stimuli, heme and its derivatives, stress, hypoxia, and biological hormones. HO1 is the ratelimiting enzyme of heme catabolism, which splits heme into biliverdin, carbon monoxide (CO) and iron. The metabolites of HO1 have antiinflammatory and antioxidant effects, and provide protection to the cardiovascular system and transplanted organs. This review summarizes the biological characteristics of HO1 and the functional significance of its products, and specifically elaborates on its protective effect on skin flaps. HO1 improves the survival rate of ischemic skin flaps through antiinflammatory, antioxidant and vasodilatory effects of enzymatic reaction products. In particular, this review focuses on the role of carbon monoxide (CO), one of the primary metabolites of HO1, in flap survival and discusses the feasibility and existing challenges of HO1 in flap surgery.
Assuntos
Heme Oxigenase-1/metabolismo , Isquemia/enzimologia , Transplante de Pele , Pele/enzimologia , Retalhos Cirúrgicos , Animais , Humanos , Isquemia/patologia , Pele/patologiaRESUMO
OBJECTIVE: Hmox1 (heme oxygenase-1) is a stress-induced enzyme that catalyzes the degradation of heme to carbon monoxide, iron, and biliverdin. Induction of Hmox1 and its products protect against cardiovascular disease, including ischemic injury. Hmox1 is also a downstream target of the transcription factor HIF-1α (hypoxia-inducible factor-1α), a key regulator of the body's response to hypoxia. However, the mechanisms by which Hmox1 confers protection against ischemia-mediated injury remain to be fully understood. Approach and Results: Hmox1 deficient (Hmox1-/-) mice had impaired blood flow recovery with severe tissue necrosis and autoamputation following unilateral hindlimb ischemia. Autoamputation preceded the return of blood flow, and bone marrow transfer from littermate wild-type mice failed to prevent tissue injury and autoamputation. In wild-type mice, ischemia-induced expression of Hmox1 in skeletal muscle occurred before stabilization of HIF-1α. Moreover, HIF-1α stabilization and glucose utilization were impaired in Hmox1-/- mice compared with wild-type mice. Experiments exposing dermal fibroblasts to hypoxia (1% O2) recapitulated these key findings. Metabolomics analyses indicated a failure of Hmox1-/- mice to adapt cellular energy reprogramming in response to ischemia. Prolyl-4-hydroxylase inhibition stabilized HIF-1α in Hmox1-/- fibroblasts and ischemic skeletal muscle, decreased tissue necrosis and autoamputation, and restored cellular metabolism to that of wild-type mice. Mechanistic studies showed that carbon monoxide stabilized HIF-1α in Hmox1-/- fibroblasts in response to hypoxia. CONCLUSIONS: Our findings suggest that Hmox1 acts both downstream and upstream of HIF-1α, and that stabilization of HIF-1α contributes to Hmox1's protection against ischemic injury independent of neovascularization.
Assuntos
Heme Oxigenase-1/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Isquemia/enzimologia , Proteínas de Membrana/metabolismo , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/enzimologia , Traumatismo por Reperfusão/prevenção & controle , Animais , Hipóxia Celular , Células Cultivadas , Modelos Animais de Doenças , Metabolismo Energético , Feminino , Fibroblastos/enzimologia , Fibroblastos/patologia , Glucose/metabolismo , Heme Oxigenase-1/deficiência , Heme Oxigenase-1/genética , Membro Posterior , Isquemia/genética , Isquemia/patologia , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos Endogâmicos BALB C , Camundongos Knockout , Músculo Esquelético/patologia , Necrose , Estabilidade Proteica , Fluxo Sanguíneo Regional , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologiaRESUMO
Proteotoxicity from insufficient clearance of misfolded/damaged proteins underlies many diseases. Carboxyl terminus of Hsc70-interacting protein (CHIP) is an important regulator of proteostasis in many cells, having E3-ligase and chaperone functions and often directing damaged proteins towards proteasome recycling. While enhancing CHIP functionality has broad therapeutic potential, prior efforts have all relied on genetic upregulation. Here we report that CHIP-mediated protein turnover is markedly post-translationally enhanced by direct protein kinase G (PKG) phosphorylation at S20 (mouse, S19 human). This increases CHIP binding affinity to Hsc70, CHIP protein half-life, and consequent clearance of stress-induced ubiquitinated-insoluble proteins. PKG-mediated CHIP-pS20 or expressing CHIP-S20E (phosphomimetic) reduces ischemic proteo- and cytotoxicity, whereas a phospho-silenced CHIP-S20A amplifies both. In vivo, depressing PKG activity lowers CHIP-S20 phosphorylation and protein, exacerbating proteotoxicity and heart dysfunction after ischemic injury. CHIP-S20E knock-in mice better clear ubiquitinated proteins and are cardio-protected. PKG activation provides post-translational enhancement of protein quality control via CHIP.
Assuntos
Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Isquemia/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Motivos de Aminoácidos , Animais , Proteínas Quinases Dependentes de GMP Cíclico/genética , Feminino , Coração/fisiopatologia , Humanos , Isquemia/enzimologia , Isquemia/genética , Isquemia/fisiopatologia , Masculino , Camundongos , Miocárdio/metabolismo , Fosforilação , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genéticaRESUMO
Flow-limiting atherosclerotic lesions of arteries supplying the limbs are a cause of symptoms in patients with peripheral artery disease (PAD). Musculoskeletal metabolic factors also contribute to the pathophysiology of claudication, which is manifest as leg discomfort that impairs walking capacity. Accordingly, we conducted a case-control study to determine whether skeletal muscle metabolic gene expression is altered in PAD. Calf skeletal muscle gene expression of patients with PAD and healthy subjects was analyzed using microarrays. The top-ranking gene differentially expressed between PAD and controls (FDR < 0.001) was PLA2G16, which encodes adipose-specific phospholipase A2 (AdPLA) and is implicated in the maintenance of insulin sensitivity and regulation of lipid metabolism. Differential expression was confirmed by qRT-PCR; PLA2G16 was downregulated by 68% in patients with PAD (p < 0.001). Expression of Pla2g16 was then measured in control (db/+) and diabetic (db/db) mice that underwent unilateral femoral artery ligation. There was significantly reduced expression of Pla2g16 in the ischemic leg of both control and diabetic mice (by 51%), with significantly greater magnitude of reduction in the diabetic mice (by 79%). We conclude that AdPLA is downregulated in humans with PAD and in mice with hindlimb ischemia. Reduced AdPLA may contribute to impaired walking capacity in patients with PAD via its effects on skeletal muscle metabolism. Further studies are needed to fully characterize the role of AdPLA in PAD and to investigate its potential as a therapeutic target for alleviating symptoms of claudication.
Assuntos
Claudicação Intermitente/enzimologia , Isquemia/enzimologia , Músculo Esquelético/enzimologia , Doença Arterial Periférica/enzimologia , Fosfolipases A2 Independentes de Cálcio/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Idoso , Animais , Estudos de Casos e Controles , Modelos Animais de Doenças , Feminino , Regulação Enzimológica da Expressão Gênica , Humanos , Resistência à Insulina , Claudicação Intermitente/genética , Claudicação Intermitente/fisiopatologia , Isquemia/genética , Isquemia/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Músculo Esquelético/fisiopatologia , Doença Arterial Periférica/genética , Doença Arterial Periférica/fisiopatologia , Fosfolipases A2 Independentes de Cálcio/genética , Proteínas Supressoras de Tumor/genética , CaminhadaRESUMO
BACKGROUND: NT5Egenetic mutations are known to result in calcification of joints and arteries (CALJA), and worldwide, 14 patients from 7 families have been reported.MethodsâandâResults:A total of 5 patients from 2 independent families with CALJA were found in Japan. Of them, 3 complained of intermittent claudication (IC), and 1 suffered from bilateral chronic limb-threatening ischemia (CLTI). Whole-exome sequencing analysis revealed an identical mutation pattern (c.G3C on the exon 1 start codon) that was unique compared withNT5Emutations reported in other countries. CONCLUSIONS: Vascular specialists need to recognize CALJA as a rare cause of ischemic IC and CLTI.
Assuntos
5'-Nucleotidase/genética , Calcinose/genética , Claudicação Intermitente/genética , Isquemia/genética , Artropatias/genética , Mutação , Calcificação Vascular/genética , Doenças Vasculares/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Calcinose/diagnóstico , Calcinose/enzimologia , Doença Crônica , Éxons , Feminino , Proteínas Ligadas por GPI/genética , Predisposição Genética para Doença , Humanos , Claudicação Intermitente/diagnóstico , Claudicação Intermitente/enzimologia , Isquemia/diagnóstico , Isquemia/enzimologia , Artropatias/diagnóstico , Artropatias/enzimologia , Masculino , Pessoa de Meia-Idade , Fenótipo , Calcificação Vascular/diagnóstico por imagem , Calcificação Vascular/enzimologia , Doenças Vasculares/diagnóstico , Doenças Vasculares/enzimologia , Sequenciamento do ExomaRESUMO
SET domain with lysine methyltransferase 7/9 (Set7/9), a histone lysine methyltransferase (HMT), recently suggested to exert a critical role among kidney disorders, whereas its role in diabetes associated IRI co-morbidity remains complete elusive. The present study aimed to understand the role of SET7/9 and histone methylation in regulation of inflammatory signaling under IRI in diabetes mellitus and non-diabetic rats. Our results demonstrated that IRI caused renal dysfunction via increased blood urea nitrogen (BUN) levels in ND and DM rats. The NF-κB mediated inflammatory cascade like increased p-NF-κB, reduced IκBα levels followed by enhanced leukocyte infiltration as shown by increased MCP-1 expressions. IRI results in increased histone H3 methylation at lysine 4 and 36 (H3K4Me2, H3K36Me2), and decreased histone H3 methylation at lysine 9. Additionally, IRI increased the protein and mRNA expression of H3K4Me2 specific histone methyltransferase-SET7/9 in DM and ND rats. The abovementioned results remain prominent in DM rats compared to ND rats followed by IRI. Further, treatment with a novel SET7/9 inhibitor; cyproheptadine, significantly improved renal functioning via reducing the BUN levels in ND and DM rats. Hence, this study demonstrated the role of SET7/9 in mediating active transcription via H3K4Me2, ultimately regulated the NFκB-mediated inflammatory cascade. Therefore, SET7/9 can be explored as novel target for drug development against IRI under DM and ND conditions.
Assuntos
Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Experimental/patologia , Progressão da Doença , Histona-Lisina N-Metiltransferase/metabolismo , Isquemia/enzimologia , Isquemia/patologia , Rim/patologia , Animais , Biomarcadores/metabolismo , Ciproeptadina/farmacologia , Ciproeptadina/uso terapêutico , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/fisiopatologia , Histona-Lisina N-Metiltransferase/genética , Histonas/metabolismo , Hiperglicemia/patologia , Inflamação/patologia , Isquemia/tratamento farmacológico , Isquemia/fisiopatologia , Rim/enzimologia , Rim/fisiopatologia , Túbulos Renais Proximais/enzimologia , Túbulos Renais Proximais/patologia , Túbulos Renais Proximais/fisiopatologia , Masculino , Metilação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos WistarRESUMO
OBJECTIVE: Diabetes mellitus is a significant risk factor for peripheral artery disease. Diabetes mellitus induces chronic states of oxidative stress and vascular inflammation that increase neutrophil activation and release of myeloperoxidase. The goal of this study is to determine whether inhibiting myeloperoxidase reduces oxidative stress and neutrophil infiltration, increases vascularization, and improves blood flow in a diabetic murine model of hindlimb ischaemia. METHODS: Leptin receptor-deficient (db/db) mice were subjected to hindlimb ischaemia. Ischaemic mice were treated with N-acetyl-lysyltyrosylcysteine-amide (KYC) to inhibit myeloperoxidase. After ligating the femoral artery, effects of treatments were determined with respect to hindlimb blood flow, neutrophil infiltration, oxidative damage, and the capability of hindlimb extracellular matrix to support human endothelial cell proliferation and migration. RESULTS: KYC treatment improved hindlimb blood flow at 7 and 14 days in db/db mice; decreased the formation of advanced glycation end products, 4-hydroxynonenal, and 3-chlorotyrosine; reduced neutrophil infiltration into the hindlimbs; and improved the ability of hindlimb extracellular matrix from db/db mice to support endothelial cell proliferation and migration. CONCLUSION: These results demonstrate that inhibiting myeloperoxidase reduces oxidative stress in ischaemic hindlimbs of db/db mice, which improves blood flow and reduces neutrophil infiltration such that hindlimb extracellular matrix from db/db mice supports endothelial cell proliferation and migration.
Assuntos
Indutores da Angiogênese/farmacologia , Diabetes Mellitus/metabolismo , Inibidores Enzimáticos/farmacologia , Isquemia/tratamento farmacológico , Músculo Esquelético/irrigação sanguínea , Neovascularização Fisiológica/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Oligopeptídeos/farmacologia , Peroxidase/antagonistas & inibidores , Animais , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Diabetes Mellitus/genética , Diabetes Mellitus/fisiopatologia , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Membro Posterior , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Isquemia/enzimologia , Isquemia/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infiltração de Neutrófilos/efeitos dos fármacos , Neutrófilos/enzimologia , Estresse Oxidativo/efeitos dos fármacos , Peroxidase/metabolismo , Receptores para Leptina/deficiência , Receptores para Leptina/genética , Fluxo Sanguíneo Regional , Transdução de SinaisRESUMO
Hemodialyzed patients (HD) have high prevalence of peripheral arterial disease. In the general population, lipoprotein-associated phospholipase A2 (Lp-PLA2 ) is associated with peripheral arterial disease but no data are available for renal subjects. The aim of this study was to evaluate the relationship between Lp-PLA2 and lower limb ischemia among dialyzed patients. One hundred and two dialyzed subjects, with median (IQR) age of 71 (59-78) years, enrolled in June 2013 and followed until June 2018, were investigated for Lp-PLA2 activity and the occurrence of peripheral arterial disease and lower limb ischemia. The median (IQR) levels of Lp-PLA2 were 184 nmol/min/mL (156.5-214.5). The 43 HD patients with abnormal Lp-PLA2 activity (>194 nmol/min/mL) had higher levels of total and LDL-cholesterol, ApoB/A1 ratio, and higher occurrence of lower limb ischemia during the follow up (44% vs 17%, P = .003). In multivariate analysis, Lp-PLA2 activity (P = .018) and diabetes (P < .001) were independently associated with time to lower limb ischemia, and when the presence of previous PAD was added to the multivariate model, only presence of previous PAD (P < .001) and Lp-PLA2 (P = .003) remained associated. Lp-PLA2 is an independent predictor of lower limb ischemia in dialyzed patients.
Assuntos
1-Alquil-2-acetilglicerofosfocolina Esterase/sangue , Doença Arterial Periférica/sangue , Doença Arterial Periférica/epidemiologia , Diálise Renal , Idoso , Feminino , Humanos , Isquemia/sangue , Isquemia/enzimologia , Perna (Membro)/irrigação sanguínea , Masculino , Pessoa de Meia-Idade , Doença Arterial Periférica/enzimologia , Prevalência , Medição de RiscoRESUMO
AIMS: Cord blood-derived endothelial colony-forming cells (CB-ECFCs) are a defined progenitor population with established roles in vascular homeostasis and angiogenesis, which possess low immunogenicity and high potential for allogeneic therapy and are highly sensitive to regulation by reactive oxygen species (ROS). The aim of this study was to define the precise role of the major ROS-producing enzyme, NOX4 NADPH oxidase, in CB-ECFC vasoreparative function. METHODS AND RESULTS: In vitro CB-ECFC migration (scratch-wound assay) and tubulogenesis (tube length, branch number) was enhanced by phorbol 12-myristate 13-acetate (PMA)-induced superoxide in a NOX-dependent manner. CB-ECFCs highly-expressed NOX4, which was further induced by PMA, whilst NOX4 siRNA and plasmid overexpression reduced and potentiated in vitro function, respectively. Increased ROS generation in NOX4-overexpressing CB-ECFCs (DCF fluorescence, flow cytometry) was specifically reduced by superoxide dismutase, highlighting induction of ROS-specific signalling. Laser Doppler imaging of mouse ischaemic hindlimbs at 7 days indicated that NOX4-knockdown CB-ECFCs inhibited blood flow recovery, which was enhanced by NOX4-overexpressing CB-ECFCs. Tissue analysis at 14 days revealed consistent alterations in vascular density (lectin expression) and eNOS protein despite clearance of injected CB-ECFCs, suggesting NOX4-mediated modulation of host tissue. Indeed, proteome array analysis indicated that NOX4-knockdown CB-ECFCs largely suppressed tissue angiogenesis, whilst NOX4-overexpressing CB-ECFCs up-regulated a number of pro-angiogenic factors specifically-linked with eNOS signalling, in parallel with equivalent modulation of NOX-dependent ROS generation, suggesting that CB-ECFC NOX4 signalling may promote host vascular repair. CONCLUSION: Taken together, these findings indicate a key role for NOX4 in CB-ECFCs, thereby highlighting its potential as a target for enhancing their reparative function through therapeutic priming to support creation of a pro-reparative microenvironment and effective post-ischaemic revascularization.
Assuntos
Células Progenitoras Endoteliais/transplante , Isquemia/cirurgia , Músculo Esquelético/irrigação sanguínea , NADPH Oxidase 4/metabolismo , Neovascularização Fisiológica , Animais , Movimento Celular , Células Cultivadas , Microambiente Celular , Modelos Animais de Doenças , Células Progenitoras Endoteliais/enzimologia , Sangue Fetal/citologia , Membro Posterior , Humanos , Isquemia/enzimologia , Isquemia/genética , Isquemia/fisiopatologia , Camundongos Endogâmicos NOD , NADPH Oxidase 4/genética , Espécies Reativas de Oxigênio/metabolismo , Recuperação de Função Fisiológica , Transdução de SinaisRESUMO
Peripheral artery disease is a severe complication of diabetes. We have reported that the deletion of angiotensin type 2 receptor in diabetic mice promoted vascular angiogenesis in the ischaemic muscle 4 weeks following ischaemia. However, the angiotensin type 2 receptor deletion beneficial effects occurred 2 weeks post surgery suggesting that angiotensin type 2 receptor may regulate other pro-angiogenic signalling pathways during the early phases of ischaemia. Nondiabetic and diabetic angiotensin type 2 receptor-deficient mice (Agtr2-/Y) underwent femoral artery ligation after 2 months of diabetes. Blood perfusion was measured every week up to 2 weeks post surgery. Expression of vascular endothelial growth factor, vascular endothelial growth factor receptor and endothelial nitric oxide synthase expression and activity were evaluated. Blood flow reperfusion in the ischaemic muscle of diabetic Agtr2+/Y mice was recovered at 35% as compared to a 68% recovery in diabetic Agtr2-/Y mice. The expression of vascular endothelial growth factor and its receptors was diminished in diabetic Agtr2+/Y mice, an observation not seen in diabetic Agtr2-/Y mice. Interestingly, Agtr2-/Y mice were protected from diabetes-induced glutathionylation, nitration and decreased endothelial nitric oxide synthase expression, which correlated with reduced endothelial cell death and enhanced vascular density in diabetic ischaemic muscle. In conclusion, our results suggest that the deletion of angiotensin type 2 receptor promotes blood flow reperfusion in diabetes by favouring endothelial cell survival and function.
Assuntos
Diabetes Mellitus/enzimologia , Células Endoteliais/enzimologia , Glutationa/metabolismo , Isquemia/enzimologia , Músculo Esquelético/irrigação sanguínea , Neovascularização Fisiológica , Nitratos/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Doença Arterial Periférica/enzimologia , Receptor Tipo 2 de Angiotensina/deficiência , Animais , Apoptose , Velocidade do Fluxo Sanguíneo , Bovinos , Células Cultivadas , Diabetes Mellitus/genética , Modelos Animais de Doenças , Células Endoteliais/patologia , Membro Posterior , Isquemia/genética , Isquemia/fisiopatologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Doença Arterial Periférica/genética , Doença Arterial Periférica/fisiopatologia , Processamento de Proteína Pós-Traducional , Receptor Tipo 2 de Angiotensina/genética , Recuperação de Função Fisiológica , Fluxo Sanguíneo RegionalRESUMO
Neprilysin (NEP) is an endogenously induced peptidase for modulating production and degradation of various peptides in humans. It is most abundantly present in kidney and regulates the intrinsic renal homeostatic mechanism. Recently, drugs inhibiting NEP have been approved for the use in heart failure. In the context of increased prevalence of ischemia associated renal failure, NEP could be an attractive target for treating kidney failure. In the kidney, targeting NEP may possess potential benefits as well as adverse consequences. The unfavorable outcomes of NEP are mainly attributed to the degradation of the natriuretic peptides (NPs). NPs are involved in the inhibition of the renin-angiotensin-aldosterone system (RAAS) and activation of the sympathetic system contributing to the tubular and glomerular injury. In contrary, NEP exerts the beneficial effect by converting angiotensin-1 (Ang I) to angiotensin-(1-7) (Ang-(1-7)), thus activating MAS-related G-protein coupled receptor. MAS receptor antagonizes angiotensin type I receptor (AT-1R), reduces reactive oxygen species (ROS) and inflammation, thus ameliorating renal injury. However, the association of NEP with complex cascades of renal ischemia remains vague. Therefore, there is a need to evaluate the putative mechanism of NEP and its overlap with other signaling cascades in conditions of renal ischemia.