RESUMEN
Diabetic nephropathy is characterized by excessive accumulation of extracellular matrix (ECM) leading to renal fibrosis, progressive deterioration of renal function, and eventually to end stage renal disease. Matrix metalloproteinases (MMPs) are known to regulate synthesis and degradation of the ECM. Earlier, we demonstrated that imbalanced MMPs promote adverse ECM remodeling leading to renal fibrosis in type-1 diabetes. Moreover, elevated macrophage infiltration, pro-inflammatory cytokines and epithelialâmesenchymal transition (EMT) are known to contribute to the renal fibrosis. Various bioactive compounds derived from the medicinal plant, Azadirachta indica (neem) are shown to regulate inflammation and ECM proteins in different diseases. Nimbidiol is a neem-derived diterpenoid that is considered as a potential anti-diabetic compound due to its glucosidase inhibitory properties. We investigated whether Nimbidiol mitigates adverse ECM accumulation and renal fibrosis to improve kidney function in type-1 diabetes and the underlying mechanism. Wild-type (C57BL/6J) and type-1 diabetic (C57BL/6-Ins2Akita/J) mice were treated either with saline or with Nimbidiol (0.40 mg kg-1 d-1) for eight weeks. Diabetic kidney showed increased accumulation of M1 macrophages, elevated pro-inflammatory cytokines and EMT. In addition, upregulated MMP-9 and MMP-13, excessive collagen deposition in the glomerular and tubulointerstitial regions, and degradation of vascular elastin resulted to renal fibrosis in the Akita mice. These pathological changes in the diabetic mice were associated with functional impairments that include elevated resistive index and reduced blood flow in the renal cortex, and decreased glomerular filtration rate. Furthermore, TGF-ß1, p-Smad2/3, p-P38, p-ERK1/2 and p-JNK were upregulated in diabetic kidney compared to WT mice. Treatment with Nimbidiol reversed the changes to alleviate inflammation, ECM accumulation and fibrosis and thus, improved renal function in Akita mice. Together, our results suggest that Nimbidiol attenuates inflammation and ECM accumulation and thereby, protects kidney from fibrosis and dysfunction possibly by inhibiting TGF-ß/Smad and MAPK signaling pathways in type-1 diabetes.
Asunto(s)
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1 , Nefropatías Diabéticas , Diterpenos , Ratones , Animales , Nefropatías Diabéticas/patología , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Ratones Endogámicos C57BL , Fibrosis , Factor de Crecimiento Transformador beta1/metabolismo , Riñón/metabolismo , Diterpenos/metabolismo , Inflamación/patología , GlucosidasasRESUMEN
Hydrogen sulfide (H(2)S) has recently been identified as a regulator of various physiological events, including vasodilation, angiogenesis, antiapoptotic, and cellular signaling. Endogenously, H(2)S is produced as a metabolite of homocysteine (Hcy) by cystathionine ß-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3MST). Although Hcy is recognized as vascular risk factor at an elevated level [hyperhomocysteinemia (HHcy)] and contributes to vascular injury leading to renovascular dysfunction, the exact mechanism is unclear. The goal of the current study was to investigate whether conversion of Hcy to H(2)S improves renovascular function. Ex vivo renal artery culture with CBS, CSE, and 3MST triple gene therapy generated more H(2)S in the presence of Hcy, and these arteries were more responsive to endothelial-dependent vasodilation compared with nontransfected arteries treated with high Hcy. Cross section of triple gene-delivered renal arteries immunostaining suggested increased expression of CD31 and VEGF and diminished expression of the antiangiogenic factor endostatin. In vitro endothelial cell culture demonstrated increased mitophagy during high levels of Hcy and was mitigated by triple gene delivery. Also, dephosphorylated Akt and phosphorylated FoxO3 in HHcy were reversed by H(2)S or triple gene delivery. Upregulated matrix metalloproteinases-13 and downregulated tissue inhibitor of metalloproteinase-1 in HHcy were normalized by overexpression of triple genes. Together, these results suggest that H(2)S plays a key role in renovasculopathy during HHcy and is mediated through Akt/FoxO3 pathways. We conclude that conversion of Hcy to H(2)S by CBS, CSE, or 3MST triple gene therapy improves renovascular function in HHcy.
Asunto(s)
Cistationina betasintasa/genética , Cistationina gamma-Liasa/genética , Terapia Genética , Sulfuro de Hidrógeno/metabolismo , Hiperhomocisteinemia/terapia , Sulfurtransferasas/genética , Animales , Células Cultivadas , Cistationina betasintasa/metabolismo , Cistationina gamma-Liasa/metabolismo , Endostatinas/biosíntesis , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/metabolismo , Homocisteína/metabolismo , Hiperhomocisteinemia/genética , Hiperhomocisteinemia/metabolismo , Hipertensión Renovascular/genética , Hipertensión Renovascular/terapia , Metaloproteinasa 13 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Técnicas de Cultivo de Órganos , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/biosíntesis , Proteínas Proto-Oncogénicas c-akt/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Arteria Renal/metabolismo , Sulfurtransferasas/metabolismo , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Lesiones del Sistema VascularRESUMEN
Accumulating evidences suggest that homocysteine, a non-protein amino acid, is involved in vessel remodeling and blood flow at elevated level, although the exact mechanism is unclear. Here we hypothesized that homocysteine affects vein in such a way that vein develops arterial phenotype. We tested our hypothesis employing wild type (WT, C57BL/6J) and CBS+/- (cystathionine ß-synthase heterozygote, a genetic model of hyperhomocysteinemia) supplemented with or without folic acid (FA, a homocysteine lowering agent). Vena cava blood flow was measured by ultrasound transonic flow probe. Tissue collagen and elastin were detected by histochemistry. Super oxide was detected by dihydroethidium (DHE) staining. Expressions of MMP-2, -9, -12, TIMP -2,-4, were measured by Western blot. MMP-13, TIMP-1, -3, and vein and aortic markers, EphB4 and EphrinB2, respectively were measured by RT-PCR. The results indicated relatively low blood flow and significant increase of collagen/elastin ratio in the CBS+/- mice compared to WT. Although FA treatment did not alter blood flow in CBS+/- mice, the collagen/elastin ratio was normalized. A relatively increased content of super oxide and gelatinase activity was observed in CBS+/- vena cava vs WT and normalized by FA treatment. Western blot analyses showed significant increase in MMP-9,-12 and decrease in TIMP-2, -4 expressions. Expressions of MMP-13, TIMP-1 and -3, Ephrin B2 were increased, whereas EphB4 was decreased with reverse change in FA treatment, with no change in MMP-13 and TIMP-1. We conclude that chronic HHcy causes vascular remodeling that expresses arterial phenotype in vein.
RESUMEN
Myocardial infarction (MI) results in significant metabolic derangement, causing accumulation of metabolic by product, such as homocysteine (Hcy). Hcy is a nonprotein amino acid generated during nucleic acid methylation and demethylation of methionine. Folic acid (FA) decreases Hcy levels by remethylating the Hcy to methionine, by 5-methylene tetrahydrofolate reductase (5-MTHFR). Although clinical trials were inconclusive regarding the role of Hcy in MI, in animal models, the levels of 5-MTHFR were decreased, and FA mitigated the MI injury. We hypothesized that FA mitigated MI-induced injury, in part, by mitigating cardiac remodeling during chronic heart failure. Thus, MI was induced in 12-wk-old male C57BL/J mice by ligating the left anterior descending artery, and FA (0.03 g/l in drinking water) was administered for 4 wk after the surgery. Cardiac function was assessed by echocardiography and by a Millar pressure-volume catheter. The levels of Hcy-metabolizing enzymes, cystathionine ß-synthase (CBS), cystathionine γ-lyase (CSE), and 5-MTHFR, were estimated by Western blot analyses. The results suggest that FA administered post-MI significantly improved cardiac ejection fraction and induced tissue inhibitor of metalloproteinase, CBS, CSE, and 5-MTHFR. We showed that FA supplementation resulted in significant improvement of myocardial function after MI. The study eluted the importance of homocysteine (Hcy) metabolism and FA supplementation in cardiovascular disease.
Asunto(s)
Ácido Fólico/farmacología , Corazón/efectos de los fármacos , Hematínicos/farmacología , Homocisteína/metabolismo , Metaloproteinasas de la Matriz/metabolismo , Infarto del Miocardio/metabolismo , Inhibidores Tisulares de Metaloproteinasas/metabolismo , Animales , Suplementos Dietéticos , Ácido Fólico/administración & dosificación , Ácido Fólico/uso terapéutico , Corazón/fisiopatología , Hematínicos/administración & dosificación , Hematínicos/uso terapéutico , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Infarto del Miocardio/tratamiento farmacológico , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/fisiología , Neovascularización Fisiológica/efectos de los fármacos , Neovascularización Fisiológica/fisiología , Resultado del Tratamiento , Función Ventricular Izquierda/efectos de los fármacos , Función Ventricular Izquierda/fisiologíaRESUMEN
BACKGROUND/AIMS: Sodium thiosulfate (STS) has been shown to be an antioxidant and calcium solubilizer, but the possible role of STS in dysfunctional ventricles remains unknown. Here, we assessed the effects of STS in the failing heart. METHODS: Heart failure was created by an arteriovenous fistula (AVF). Mice were divided into 4 groups: sham, AVF, sham + STS, and AVF + STS. STS (3 mg/ml) was supplemented with drinking water for 6 weeks in the appropriate surgery groups after surgery. RESULTS: M-mode echocardiograms showed ventricular contractile dysfunction with reduced aortic blood flow in AVF mice, whereas STS treatment prevented the decline in cardiac function. Ventricular collagen, MMP-2 and -9, and TIMP-1 were robustly increased with a decreasing trend in adenylate cyclase VI expression; however, STS supplementation reversed these effects in AVF mice. Among 2 enzymes that produce endogenous hydrogen sulfide (H(2)S), cystathionine-gamma-lyase (CSE) expression was attenuated in AVF mice with no changes in cystathionine-beta-synthase (CBS) expression. In addition, reduced production of H(2)S in AVF ventricular tissue was normalized with STS supplementation. Moreover, cardiac tissues were more responsive to H(2)S when AVF mice were supplemented with STS compared to AVF alone. CONCLUSIONS: These results suggested that STS modulated cardiac dysfunction and the extracellular matrix, in part, by increasing ventricular H(2)S generation.
Asunto(s)
Cardiotónicos/farmacología , Insuficiencia Cardíaca/tratamiento farmacológico , Sulfuro de Hidrógeno/metabolismo , Tiosulfatos/farmacología , Adenilil Ciclasas/genética , Adenilil Ciclasas/metabolismo , Animales , Aorta/fisiopatología , Fístula Arteriovenosa , Enfermedad Crónica , Colágeno/efectos de los fármacos , Colágeno/metabolismo , Cistationina gamma-Liasa/efectos de los fármacos , Cistationina gamma-Liasa/metabolismo , Ecocardiografía , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Insuficiencia Cardíaca/fisiopatología , Masculino , Metaloproteinasa 2 de la Matriz/efectos de los fármacos , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/efectos de los fármacos , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Contracción Miocárdica/efectos de los fármacos , Inhibidor Tisular de Metaloproteinasa-1/efectos de los fármacos , Inhibidor Tisular de Metaloproteinasa-1/metabolismoRESUMEN
Elevated levels of homocysteine (Hcy) (known as hyperhomocysteinemia HHcy) are involved in dilated cardiomyopathy. Hcy chelates copper and impairs copper-dependent enzymes. Copper deficiency has been linked to cardiovascular disease. We tested the hypothesis that copper supplement regresses left ventricular hypertrophy (LVH), fibrosis and endothelial dysfunction in pressure overload DCM mice hearts. The mice were grouped as sham, sham + Cu, aortic constriction (AC), and AC + Cu. Aortic constriction was performed by transverse aortic constriction. The mice were treated with or without 20 mg/kg copper supplement in the diet for 12 weeks. The cardiac function was assessed by echocardiography and electrocardiography. The matrix remodeling was assessed by measuring matrix metalloproteinase (MMP), tissue inhibitor of metalloproteinases (TIMPs), and lysyl oxidase (LOX) by Western blot analyses. The results suggest that in AC mice, cardiac function was improved with copper supplement. TIMP-1 levels decreased in AC and were normalized in AC + Cu. Although MMP-9, TIMP-3, and LOX activity increased in AC and returned to baseline value in AC + Cu, copper supplement showed no significant effect on TIMP-4 activity after pressure overload. In conclusion, our data suggest that copper supplement helps improve cardiac function in a pressure overload dilated cardiomyopathic heart.
Asunto(s)
Cardiomiopatía Dilatada/tratamiento farmacológico , Cobre/administración & dosificación , Suplementos Dietéticos , Insuficiencia Cardíaca/tratamiento farmacológico , Hemodinámica/efectos de los fármacos , Homocisteína/metabolismo , Hipertrofia Ventricular Izquierda/tratamiento farmacológico , Miocardio/metabolismo , Animales , Aorta/cirugía , Presión Sanguínea , Western Blotting , Cardiomiopatía Dilatada/metabolismo , Cardiomiopatía Dilatada/fisiopatología , Constricción , Cobre/metabolismo , Modelos Animales de Enfermedad , Ecocardiografía , Electrocardiografía , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Endotelio Vascular/fisiopatología , Femenino , Fibrosis , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/fisiopatología , Homocisteína/sangre , Hipertrofia Ventricular Izquierda/metabolismo , Hipertrofia Ventricular Izquierda/fisiopatología , Masculino , Metaloproteinasas de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Miocardio/enzimología , Miocardio/patología , Proteína-Lisina 6-Oxidasa/metabolismo , Inhibidores Tisulares de Metaloproteinasas/metabolismo , Remodelación Ventricular/efectos de los fármacosRESUMEN
Hyperhomocysteinemia (HHcy) is associated with atherosclerotic events involving the modulation of arachidonic acid (AA) metabolism and the activation of matrix metalloproteinase-9 (MMP-9). Cytochrome P450 (CYP) epoxygenase-2J2 (CYP2J2) is abundant in the heart endothelium, and its AA metabolites epoxyeicosatrienoic acids (EETs) mitigates inflammation through NF-kappabeta. However, the underlying molecular mechanisms for MMP-9 regulation by CYP2J2 in HHcy remain obscure. We sought to determine the molecular mechanisms by which P450 epoxygenase gene transfection or EETs supplementation attenuate homocysteine (Hcy)-induced MMP-9 activation. CYP2J2 was over-expressed in mouse aortic endothelial cells (MAECs) by transfection with the pcDNA3.1/CYP2J2 vector. The effects of P450 epoxygenase transfection or exogenous supplementation of EETs on NF-kappabeta-mediated MMP-9 regulation were evaluated using Western blot, in-gel gelatin zymography, electromobility shift assay, immunocytochemistry. The result suggested that Hcy downregulated CYP2J2 protein expression and dephosphorylated PI3K-dependent AKT signal. Hcy induced the nuclear translocation of NF-kappabeta via downregulation of IKbetaalpha (endogenous cytoplasmic inhibitor of NF-kappabeta). Hcy induced MMP-9 activation by increasing NF-kappabeta-DNA binding. Moreover, P450 epoxygenase transfection or exogenous addition of 8,9-EET phosphorylated the AKT and attenuated Hcy-induced MMP-9 activation. This occurred, in part, by the inhibition of NF-kappabeta nuclear translocation, NF-kappabeta-DNA binding and activation of IKbetaalpha. The study unequivocally suggested the pivotal role of EETs in the modulation of Hcy/MMP-9 signal.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Hiperhomocisteinemia/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Oxigenasas/genética , Oxigenasas/metabolismo , Factor de Transcripción ReIA/antagonistas & inhibidores , Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido 8,11,14-Eicosatrienoico/farmacología , Animales , Células Cultivadas , Citocromo P-450 CYP2J2 , Activación Enzimática/efectos de los fármacos , Inducción Enzimática/efectos de los fármacos , Homocisteína/farmacología , Hiperhomocisteinemia/enzimología , Proteínas I-kappa B/metabolismo , Metaloproteinasa 9 de la Matriz/biosíntesis , Ratones , Inhibidor NF-kappaB alfa , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factor de Transcripción ReIA/metabolismo , TransfecciónRESUMEN
Ectodomain shedding has emerged as an important regulatory step in the function of transmembrane proteins. Intercellular adhesion molecule-1 (ICAM-1), an adhesion receptor that mediates inflammatory and immune responses, undergoes shedding in the presence of inflammatory mediators and phorbol 12-myristate 13-acetate (PMA). The shedding of ICAM-1 in ICAM-1-transfected 293 cells upon PMA stimulation and in endothelial cells upon tumor necrosis factor-alpha stimulation was blocked by metalloproteinase inhibitors, whereas serine protease inhibitors were ineffective. p-Aminophenylmercuric acetate, a mercuric compound that is known to activate matrix metalloproteinases, up-regulated ICAM-1 shedding. TIMP-3 (but not TIMP-1 or -2) effectively blocked cleavage. This profile suggests the involvement of the ADAM family of proteases in the cleavage of ICAM-1. The introduction of enzymatically active tumor necrosis factor-alpha-converting enzyme (TACE) into ICAM-1-expressing cells up-regulated cleavage. Small interfering RNA directed against TACE blocked ICAM-1 cleavage. ICAM-1 transfected into TACE-/- fibroblasts did not show increased shedding over constitutive levels in the presence of PMA, whereas cleavage did occur in ICAM-1-transfected TACE+/+ cells. These results indicate that ICAM-1 shedding is mediated by TACE. Blocking the shedding of ICAM-1 altered the cell adhesive function, as ICAM-1-mediated cell adhesion was up-regulated in the presence of TACE small interfering RNA and TIMP-3, but not TIMP-1. However, cleavage was found to occur at multiple sites within the stalk domain of ICAM-1, and numerous point mutations within the region did not affect cleavage, indicating that TACE-mediated cleavage of ICAM-1 may not be sequence-specific.