RESUMEN
Impaired regulation of renin in Dahl salt-sensitive rats (SS/JRHsdMcwi, SS) contributes to attenuated angiogenesis in this strain. This study examined angiogenic function and genomic structure of regions surrounding the renin gene using subcongenic strains of the SS and BN/NHsdMcwi (BN) rat to identify important genomic variations between SS and BN involved in angiogenesis. Three candidate regions on Chr 13 were studied: two congenic strains containing 0.89 and 2.62 Mb portions of BN Chr 13 that excluded the BN renin allele and a third strain that contained a 2.02 Mb overlapping region that included the BN renin allele. Angiogenesis induced by electrical stimulation of the tibialis anterior muscle was attenuated in the SS compared with the BN. Congenics carrying the SS renin allele had impaired angiogenesis, while strains carrying the BN renin allele had angiogenesis restored. The exception was a congenic including a region of BN genome 0.4 Mb distal to renin that restored both renin regulation and angiogenesis. This suggests that there is a distant regulatory element in the BN capable of restoring normal regulation of the SS renin allele. The importance of ANG II in the restored angiogenic response was demonstrated by blocking with losartan. Sequencing of the 4.05 Mb candidate region in SS and BN revealed a total of 8,850 SNPs and other sequence variants. An analysis of the genes and their variants in the region suggested a number of pathways that may explain the impaired regulation of renin and angiogenesis in the SS rat.
Asunto(s)
Genoma/genética , Neovascularización Fisiológica/genética , Renina/genética , Animales , Animales Congénicos , Peso Corporal/genética , Cromosomas de los Mamíferos/genética , Estimulación Eléctrica , Exones/genética , Regulación de la Expresión Génica , Redes Reguladoras de Genes/genética , Inmunohistoquímica , Músculo Esquelético/anatomía & histología , Músculo Esquelético/citología , Tamaño de los Órganos/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas Dahl , Renina/metabolismoRESUMEN
Autologous bone marrow cell (BMC) transplantation has been shown as a potential approach to treat various ischemic diseases. However, under many conditions BMC dysfunction has been reported, leading to poor cell engraftment and a failure of tissue revascularization. We have previously shown that skeletal muscle angiogenesis induced by electrical stimulation (ES) is impaired in the SS/Mcwi rats and that this effect is related to a dysregulation of the renin angiotensin system (RAS) that is normalized by the replacement of chromosome 13 derived from the Brown Norway rat (SS-13(BN)/Mcwi consomic rats). The present study explored bone marrow-derived endothelial cell (BM-EC) function in the SS/Mcwi rat and its impact on skeletal muscle angiogenesis induced by ES. SS/Mcwi rats were randomized to receive BMC from: SS/Mcwi; SS-13(BN)/Mcwi; SS/Mcwi rats infused with saline or ANG II (3 ng kg(-1) min(-1)). BMC were injected in the stimulated tibialis anterior muscle of SS/Mcwi rats. Vessel density was evaluated in unstimulated and stimulated muscles after 7 days of ES. BMC isolated from SS/Mcwi or SS/Mcwi rats infused with saline failed to restore angiogenesis induced by ES. However, BMC isolated from SS-13(BN)/Mcwi and SS/Mcwi rats infused with ANG II effectively restored the angiogenesis response in the SS/Mcwi recipient. Furthermore, ANG II infusion increased the capacity of BM-EC to induce endothelial cell tube formation in vitro and slightly increased VEGF protein expression. This study suggests that dysregulation of the RAS in the SS/Mcwi rat contributes to impaired BM-EC function and could impact the angiogenic therapeutic potential of BMC.
Asunto(s)
Células de la Médula Ósea/fisiología , Músculo Esquelético/irrigación sanguínea , Neovascularización Fisiológica/fisiología , Sistema Renina-Angiotensina/fisiología , Angiotensina II/farmacología , Animales , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Trasplante de Médula Ósea , Células Cultivadas , Estimulación Eléctrica , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/fisiología , Humanos , Modelos Biológicos , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiología , Neovascularización Fisiológica/efectos de los fármacos , Ratas , Ratas Endogámicas BN , Ratas Endogámicas Dahl , Sistema Renina-Angiotensina/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
We have previously shown that skeletal muscle angiogenesis induced by electrical stimulation is significantly attenuated when SS-13BN/Mcwi rats are fed a high-salt diet. This effect was associated with a large increase in endothelial cell (EC) apoptosis. We hypothesized that the low levels of ANG II during high-salt diet would increase EC apoptosis and consequently diminish the angiogenic response. To test this hypothesis, a series of in vitro and in vivo studies was performed. EC apoptosis and viability were evaluated after incubation with ANG II under serum-free conditions. After 24 h of incubation, ANG II increased EC viability and Bcl-2-to-Bax ratio along with a dose-dependent decrease in EC apoptosis. This effect was blocked by the ANG II type 1 receptor antagonist losartan. To confirm our in vitro results, ANG II (3 ng.kg(-1).min(-1)) was chronically infused in rats fed a high-salt diet (4% NaCl). ANG II decreased EC apoptosis and produced a significant increase (40%) in skeletal muscle angiogenesis after electrical stimulation. These in vivo results were in agreement with our in vitro results and demonstrate that the attenuation of ANG II levels during a high-salt diet may induce EC apoptosis and consequently block the angiogenic response induced by electrical stimulation. Furthermore, under normal conditions, ANG II increases EC viability and protects EC from apoptosis possibly by inactivation of the mitochondrial apoptotic pathway.
Asunto(s)
Angiotensina II/metabolismo , Apoptosis , Células Endoteliales/metabolismo , Músculo Esquelético/irrigación sanguínea , Neovascularización Fisiológica , Angiotensina II/administración & dosificación , Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Animales , Apoptosis/efectos de los fármacos , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Estimulación Eléctrica , Células Endoteliales/efectos de los fármacos , Células Endoteliales/patología , Infusiones Intravenosas , Losartán/farmacología , Mitocondrias/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Ratas , Ratas Endogámicas , Cloruro de Sodio Dietético/administración & dosificación , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Previous studies have indicated the importance of angiotensin II (ANG II) in skeletal muscle angiogenesis. The present study explored the effect of regulation of the renin gene on angiogenesis induced by electrical stimulation with the use of physiological, pharmacological, and genetic manipulations of the renin-angiotensin system (RAS). Transfer of the entire chromosome 13, containing the physiologically regulated renin gene, from the normotensive inbred Brown Norway (BN) rat into the background of an inbred substrain of the Dahl salt-sensitive (SS/Mcwi) rat restored renin levels and the angiogenic response after electrical stimulation. This restored response was significantly attenuated when SS-13(BN)/Mcwi consomic rats were treated with lisinopril or high-salt diet. The role of ANG II on this effect was confirmed by the complete restoration of skeletal muscle angiogenesis in SS/Mcwi rats infused with subpressor doses of ANG II. Congenic strains derived from the SS-13(BN)/Mcwi consomic were used to further verify the role of the renin gene in this response. Microvessel density was markedly increased after stimulation in congenic strains that contained the renin gene from the BN rat (congenic lines A and D). This angiogenic response was suppressed in control strains that carried regions of the BN genome just above (congenic line C) or just below (congenic line B) the renin gene. The present study emphasizes the importance of maintaining normal renin regulation as well as ANG II levels during the angiogenesis process with a combination of physiological, genetic, and pharmacological manipulation of the RAS.
Asunto(s)
Estimulación Eléctrica , Músculo Esquelético/irrigación sanguínea , Neovascularización Fisiológica/genética , Renina/genética , Renina/fisiología , Animales , Animales Congénicos , Antihipertensivos/farmacología , Presión Sanguínea/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Cruzamientos Genéticos , Dieta Hiposódica , Hipertensión/tratamiento farmacológico , Lisinopril/farmacología , Lisinopril/uso terapéutico , Masculino , Músculo Esquelético/anatomía & histología , Tamaño de los Órganos/efectos de los fármacos , Ratas , Ratas Endogámicas BN , Ratas Endogámicas Dahl , Sistema Renina-Angiotensina/efectos de los fármacos , Sistema Renina-Angiotensina/fisiología , Sodio en la Dieta/farmacologíaRESUMEN
1. This study was performed to evaluate the effect of chronic high salt intake on local cardiac and renal components of the renin-angiotensin system (RAS) and its impact on cardiac remodelling and function after myocardial infarction (MI). 2. Rats submitted to coronary artery ligation to produce MI or sham operation (SO) were randomized to receive 1% NaCl solution or tap water as drinking water for 4 weeks. Plasma renin activity (PRA) and angiotensin-converting enzyme (ACE) activity were quantified. Tissue angiotensin (Ang) II and ACE activity were determined by ELISA and a fluorimetric assay, respectively. Renal and cardiac AT(1) and AT(2) receptor protein levels were quantified by western blot. 3. Independent of the lower PRA levels, MI promoted a significant increase in the left ventricular/bodyweight ratio and impaired cardiac function. The cardiac RAS was activated after MI with a significant increase in ACE activity, AngII and AT(1) receptor levels. The RAS was slightly attenuated under high-salt conditions. 4. Interestingly, high salt intake increased the expression of the AT(2) receptor by approximately twofold in the kidney of MI rats compared with the SO control group. Because of its natriuretic effect, the AT(2) receptor may counterbalance the salt overload and prevent the additional impairment of cardiac function. 5. The present study indicates that 4 weeks after MI, high salt intake did not further increase cardiac hypertrophy or further impair cardiac function in MI rats. A chronic increase in salt intake significantly suppressed PRA, but did not prevent activation of the local RAS or the progression of cardiac remodelling and left ventricular dysfunction caused by MI. 6. The present results show that inhibition of systemic renin production with salt overload does not affect ventricular remodelling after MI in rats. This suggests that local activation of the RAS in the heart, which was not suppressed by salt overload, exerts a predominant role for local adaptations of the heart after MI.
Asunto(s)
Infarto del Miocardio/complicaciones , Sistema Renina-Angiotensina/efectos de los fármacos , Cloruro de Sodio Dietético/farmacología , Disfunción Ventricular/fisiopatología , Angiotensina II/metabolismo , Animales , Western Blotting , Peso Corporal/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Corazón/efectos de los fármacos , Corazón/fisiopatología , Masculino , Contracción Miocárdica/efectos de los fármacos , Peptidil-Dipeptidasa A/sangre , Peptidil-Dipeptidasa A/metabolismo , Ratas , Ratas Wistar , Receptor de Angiotensina Tipo 1/metabolismo , Receptor de Angiotensina Tipo 2/metabolismo , Renina/sangre , Cloruro de Sodio Dietético/administración & dosificación , Factores de Tiempo , Disfunción Ventricular/etiología , Función Ventricular Izquierda/efectos de los fármacos , Presión Ventricular/efectos de los fármacos , Remodelación Ventricular/efectos de los fármacosRESUMEN
Angiogenesis, under normal conditions, is a tightly regulated balance between pro- and antiangiogenic factors. The goal of this study was to investigate the mechanisms involved in the control of the skeletal muscle angiogenic response induced by electrical stimulation during the suppression of plasma renin activity (PRA) with a high-salt diet. Rats fed 0.4% or 4% salt diets were exposed to electrical stimulation for 7 days. The tibialis anterior (TA) muscles from stimulated and unstimulated hindlimbs were removed and prepared for gene expression analysis, CD31-terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) double-staining assay, and Bcl-2 and Bax protein expression by Western blot. Rats fed a low-salt diet showed a dramatic angiogenesis response in the stimulated limb compared with the unstimulated limb. This angiogenesis response was significantly attenuated when rats were placed on a high-salt diet. Microarray analysis showed that in the stimulated limb of rats fed a low-salt diet many genes related to angiogenesis were upregulated. In contrast, in rats fed a high-salt diet most of the genes upregulated in the stimulated limb function in apoptosis and cell cycle arrest. Endothelial cell apoptosis, as analyzed by CD31-TUNEL staining, increased by fourfold in the stimulated limb compared with the unstimulated limb. There was also a 48% decrease in the Bcl-2-to-Bax ratio in stimulated compared with unstimulated limbs of rats fed a high-salt diet, confirming severe apoptosis. This study suggests that the increase in endothelial cell apoptosis in TA muscle might contribute to the attenuation of angiogenesis response observed in rats fed a high-salt diet.