RESUMEN
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.
Asunto(s)
Complejo I de Transporte de Electrón , Isquemia , Animales , Microscopía por Crioelectrón , Complejo I de Transporte de Electrón/metabolismo , Complejo I de Transporte de Electrón/química , Isquemia/enzimología , Membrana Dobles de Lípidos/metabolismo , Mitocondrias/enzimología , Fosforilación Oxidativa , Bovinos , Ubiquinona/química , Ubiquinona/metabolismo , Conformación Proteica en Hélice alfaRESUMEN
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.
Asunto(s)
Acetilglucosamina , Corazón , Miocardio , N-Acetilglucosaminiltransferasas , Caracteres Sexuales , Transducción de Señal , Animales , Femenino , Masculino , Ratones , Acetilglucosamina/metabolismo , Corazón/fisiología , Isquemia/enzimología , Isquemia/metabolismo , Miocardio/enzimología , Miocardio/metabolismo , N-Acetilglucosaminiltransferasas/metabolismo , Procesamiento Proteico-PostraduccionalRESUMEN
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.
Asunto(s)
Células Endoteliales/enzimología , Miembro Posterior/irrigación sanguínea , Receptores de Hialuranos/metabolismo , Hialuronano Sintasas/metabolismo , Isquemia/enzimología , Neovascularización Fisiológica , Óxido Nítrico Sintasa de Tipo III/metabolismo , Animales , Circulación Colateral , Modelos Animales de Enfermedad , Humanos , Hialuronano Sintasas/genética , Isquemia/fisiopatología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados para ApoE , Óxido Nítrico Sintasa de Tipo III/genética , Fosforilación , Flujo Sanguíneo Regional , Transducción de Señal , Factores de TiempoRESUMEN
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.
Asunto(s)
Inductores de la Angiogénesis/farmacología , Diabetes Mellitus Experimental/enzimología , Angiopatías Diabéticas/enzimología , Miembro Posterior/irrigación sanguínea , Isquemia/enzimología , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Neovascularización Fisiológica/efectos de los fármacos , Factor de Crecimiento Derivado de Plaquetas/farmacología , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Animales , Glucemia/metabolismo , Estudios de Casos y Controles , Bovinos , Hipoxia de la Célula , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/fisiopatología , Angiopatías Diabéticas/genética , Angiopatías Diabéticas/fisiopatología , Activación Enzimática , Humanos , Isquemia/fisiopatología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/enzimología , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/enzimología , Miocitos del Músculo Liso/patología , Proteína Tirosina Fosfatasa no Receptora Tipo 6/genética , Transducción de SeñalRESUMEN
[Figure: see text].
Asunto(s)
Miembro Posterior/irrigación sanguínea , Células Endoteliales de la Vena Umbilical Humana/enzimología , Isquemia/enzimología , Metiltransferasas/metabolismo , MicroARNs/metabolismo , Infarto del Miocardio/enzimología , Neovascularización Fisiológica , Adenosina/análogos & derivados , Adenosina/metabolismo , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica , Humanos , Isquemia/genética , Isquemia/fisiopatología , Masculino , Metiltransferasas/genética , Ratones Endogámicos C57BL , MicroARNs/genética , Infarto del Miocardio/genética , Infarto del Miocardio/fisiopatología , Transducción de Señal , Trombospondina 1/genética , Trombospondina 1/metabolismoRESUMEN
[Figure: see text].
Asunto(s)
5'-Nucleotidasa/administración & dosificación , Adenosina/metabolismo , Portadores de Fármacos , Miembro Posterior/irrigación sanguínea , Isquemia/tratamiento farmacológico , Maleimidas/química , Enfermedad Arterial Periférica/tratamiento farmacológico , Polietilenglicoles/química , 5'-Nucleotidasa/química , 5'-Nucleotidasa/metabolismo , Animales , Células Cultivadas , Preparaciones de Acción Retardada , Modelos Animales de Enfermedad , Composición de Medicamentos , Femenino , Humanos , Hidrogeles , Isquemia/enzimología , Isquemia/fisiopatología , Masculino , Ratones Endogámicos C57BL , Activación Neutrófila/efectos de los fármacos , Enfermedad Arterial Periférica/enzimología , Enfermedad Arterial Periférica/fisiopatología , Flujo Sanguíneo Regional , Regulación hacia ArribaRESUMEN
[Figure: see text].
Asunto(s)
Hemo-Oxigenasa 1/metabolismo , Inflamasomas/metabolismo , Isquemia/enzimología , Lisosomas/enzimología , Macrófagos/enzimología , Proteínas de la Membrana/metabolismo , Músculo Esquelético/irrigación sanguínea , Músculo Esquelético/enzimología , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Animales , Células Cultivadas , Bases de Datos Genéticas , Modelos Animales de Enfermedad , Hemo-Oxigenasa 1/genética , Miembro Posterior , Humanos , Inflamasomas/genética , Mediadores de Inflamación/metabolismo , Isquemia/genética , Isquemia/fisiopatología , Masculino , Proteínas de la Membrana/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Esquelético/fisiopatología , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Neovascularización Fisiológica , Proteolisis , Recuperación de la Función , Flujo Sanguíneo RegionalRESUMEN
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.
Asunto(s)
Aldehído Deshidrogenasa Mitocondrial/metabolismo , Inductores de la Angiogénesis/farmacología , Medicamentos Herbarios Chinos/farmacología , Células Endoteliales/efectos de los fármacos , Miembro Posterior/irrigación sanguínea , Isquemia/tratamiento farmacológico , Neovascularización Fisiológica/efectos de los fármacos , Aldehído Deshidrogenasa Mitocondrial/genética , Animales , Hipoxia de la Célula , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Células Endoteliales/enzimología , Activación Enzimática , Isquemia/enzimología , Isquemia/fisiopatología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas Sprague-Dawley , Flujo Sanguíneo Regional , Transducción de SeñalRESUMEN
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.
Asunto(s)
Diabetes Mellitus Experimental/enzimología , Células Endoteliales/enzimología , Miembro Posterior/irrigación sanguínea , Isquemia/enzimología , Neovascularización Fisiológica , Proteína Quinasa C-delta/deficiencia , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Apoptosis , Bovinos , Células Cultivadas , Circulación Colateral , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/fisiopatología , Células Endoteliales/patología , Isquemia/genética , Isquemia/fisiopatología , Ratones Noqueados , Densidad Microvascular , Proteína Quinasa C-delta/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 6/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Flujo Sanguíneo Regional , Transducción de SeñalRESUMEN
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.
Asunto(s)
Hemo-Oxigenasa 1/metabolismo , Isquemia/enzimología , Trasplante de Piel , Piel/enzimología , Colgajos Quirúrgicos , Animales , Humanos , Isquemia/patología , Piel/patologíaRESUMEN
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.
Asunto(s)
Hemo-Oxigenasa 1/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Isquemia/enzimología , Proteínas de la Membrana/metabolismo , Músculo Esquelético/irrigación sanguínea , Músculo Esquelético/enzimología , Daño por Reperfusión/prevención & control , Animales , Hipoxia de la Célula , Células Cultivadas , Modelos Animales de Enfermedad , Metabolismo Energético , Femenino , Fibroblastos/enzimología , Fibroblastos/patología , Glucosa/metabolismo , Hemo-Oxigenasa 1/deficiencia , Hemo-Oxigenasa 1/genética , Miembro Posterior , Isquemia/genética , Isquemia/patología , Masculino , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones Endogámicos BALB C , Ratones Noqueados , Músculo Esquelético/patología , Necrosis , Estabilidad Proteica , Flujo Sanguíneo Regional , Daño por Reperfusión/genética , Daño por Reperfusión/patologíaRESUMEN
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.
Asunto(s)
Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Isquemia/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Secuencias de Aminoácidos , Animales , Proteínas Quinasas Dependientes de GMP Cíclico/genética , Femenino , Corazón/fisiopatología , Humanos , Isquemia/enzimología , Isquemia/genética , Isquemia/fisiopatología , Masculino , Ratones , Miocardio/metabolismo , Fosforilación , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
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.
Asunto(s)
Claudicación Intermitente/enzimología , Isquemia/enzimología , Músculo Esquelético/enzimología , Enfermedad Arterial Periférica/enzimología , Fosfolipasas A2 Calcio-Independiente/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Anciano , Animales , Estudios de Casos y Controles , Modelos Animales de Enfermedad , Femenino , Regulación Enzimológica de la Expresión Génica , Humanos , Resistencia a la Insulina , Claudicación Intermitente/genética , Claudicación Intermitente/fisiopatología , Isquemia/genética , Isquemia/fisiopatología , Masculino , Ratones Endogámicos C57BL , Persona de Mediana Edad , Músculo Esquelético/fisiopatología , Enfermedad Arterial Periférica/genética , Enfermedad Arterial Periférica/fisiopatología , Fosfolipasas A2 Calcio-Independiente/genética , Proteínas Supresoras de Tumor/genética , CaminataRESUMEN
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.
Asunto(s)
5'-Nucleotidasa/genética , Calcinosis/genética , Claudicación Intermitente/genética , Isquemia/genética , Artropatías/genética , Mutación , Calcificación Vascular/genética , Enfermedades Vasculares/genética , Adulto , Anciano , Anciano de 80 o más Años , Calcinosis/diagnóstico , Calcinosis/enzimología , Enfermedad Crónica , Exones , Femenino , Proteínas Ligadas a GPI/genética , Predisposición Genética a la Enfermedad , Humanos , Claudicación Intermitente/diagnóstico , Claudicación Intermitente/enzimología , Isquemia/diagnóstico , Isquemia/enzimología , Artropatías/diagnóstico , Artropatías/enzimología , Masculino , Persona de Mediana Edad , Fenotipo , Calcificación Vascular/diagnóstico por imagen , Calcificación Vascular/enzimología , Enfermedades Vasculares/diagnóstico , Enfermedades Vasculares/enzimología , Secuenciación del ExomaRESUMEN
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.
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Diabetes Mellitus Experimental/enzimología , Diabetes Mellitus Experimental/patología , Progresión de la Enfermedad , N-Metiltransferasa de Histona-Lisina/metabolismo , Isquemia/enzimología , Isquemia/patología , Riñón/patología , Animales , Biomarcadores/metabolismo , Ciproheptadina/farmacología , Ciproheptadina/uso terapéutico , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/fisiopatología , N-Metiltransferasa de Histona-Lisina/genética , Histonas/metabolismo , Hiperglucemia/patología , Inflamación/patología , Isquemia/tratamiento farmacológico , Isquemia/fisiopatología , Riñón/enzimología , Riñón/fisiopatología , Túbulos Renales Proximales/enzimología , Túbulos Renales Proximales/patología , Túbulos Renales Proximales/fisiopatología , Masculino , Metilación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas WistarRESUMEN
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.
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Inductores de la Angiogénesis/farmacología , Diabetes Mellitus/metabolismo , Inhibidores Enzimáticos/farmacología , Isquemia/tratamiento farmacológico , Músculo Esquelético/irrigación sanguínea , Neovascularización Fisiológica/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Oligopéptidos/farmacología , Peroxidasa/antagonistas & inhibidores , Animales , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Diabetes Mellitus/genética , Diabetes Mellitus/fisiopatología , Modelos Animales de Enfermedad , Matriz Extracelular/metabolismo , Miembro Posterior , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Isquemia/enzimología , Isquemia/fisiopatología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Infiltración Neutrófila/efectos de los fármacos , Neutrófilos/enzimología , Estrés Oxidativo/efectos de los fármacos , Peroxidasa/metabolismo , Receptores de Leptina/deficiencia , Receptores de Leptina/genética , Flujo Sanguíneo Regional , Transducción de SeñalRESUMEN
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.
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1-Alquil-2-acetilglicerofosfocolina Esterasa/sangre , Enfermedad Arterial Periférica/sangre , Enfermedad Arterial Periférica/epidemiología , Diálisis Renal , Anciano , Femenino , Humanos , Isquemia/sangre , Isquemia/enzimología , Pierna/irrigación sanguínea , Masculino , Persona de Mediana Edad , Enfermedad Arterial Periférica/enzimología , Prevalencia , Medición de RiesgoRESUMEN
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.
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Células Progenitoras Endoteliales/trasplante , Isquemia/cirugía , Músculo Esquelético/irrigación sanguínea , NADPH Oxidasa 4/metabolismo , Neovascularización Fisiológica , Animales , Movimiento Celular , Células Cultivadas , Microambiente Celular , Modelos Animales de Enfermedad , Células Progenitoras Endoteliales/enzimología , Sangre Fetal/citología , Miembro Posterior , Humanos , Isquemia/enzimología , Isquemia/genética , Isquemia/fisiopatología , Ratones Endogámicos NOD , NADPH Oxidasa 4/genética , Especies Reactivas de Oxígeno/metabolismo , Recuperación de la Función , Transducción de SeñalRESUMEN
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.
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Diabetes Mellitus/enzimología , Células Endoteliales/enzimología , Glutatión/metabolismo , Isquemia/enzimología , Músculo Esquelético/irrigación sanguínea , Neovascularización Fisiológica , Nitratos/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Enfermedad Arterial Periférica/enzimología , Receptor de Angiotensina Tipo 2/deficiencia , Animales , Apoptosis , Velocidad del Flujo Sanguíneo , Bovinos , Células Cultivadas , Diabetes Mellitus/genética , Modelos Animales de Enfermedad , Células Endoteliales/patología , Miembro Posterior , Isquemia/genética , Isquemia/fisiopatología , Ratones Endogámicos C57BL , Ratones Noqueados , Enfermedad Arterial Periférica/genética , Enfermedad Arterial Periférica/fisiopatología , Procesamiento Proteico-Postraduccional , Receptor de Angiotensina Tipo 2/genética , Recuperación de la Función , Flujo Sanguíneo RegionalRESUMEN
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.