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Introduccion: Los diureticos forman parte del tratamiento antihipertensivo actual con efectividad e impacto clinico demostrados. Sin embargo, los efectos de estos farmacos sobre el remodelado de la pared arterial en la hipertension arterial (HTA) han sido poco evaluados. Objetivos: Determinar y comparar el efecto de Hi-droclorotiazida (HCTZ) y de Espironolactona (ESP) en la hipertrofia de la pared aortica en la HTA experimental. Metodologia: Estudio comparativo en 4 grupos experimentales. Se utilizaron ratas Sprague Dawley macho de 150 ± 10 grs. unifrectomizadas tratadas con desoxicorticosterona Acetato (DOCA, 100 mg/Kg/sem sbc) por 6 semanas. Como controles (Sham) se usaron ratas unifrectomizadas. A partir de la tercera semana con DOCA se administro diureticos en dos grupos adicionales durante 3 semanas. Uno recibio HCTZ (6 mg/ kg/dia) y otro ESP (100 mg/Kg/dia), via gavage. Al finalizar los tratamientos se determino la presion arterial sistolica (PAS), masa corporal (MC), peso del corazon (PC) y masa cardiaca relativa (MCR). El grado de hipertrofia de la pared aortica se determino midiendo el grosor de la tunica media (GTM), area de la tunica media (ATM), area luminal (AL) y la relacion ATM/AL en cortes tenidos con hematoxilina-eosina. Resultados: En las ratas DOCA no tratadas hubo un aumento significativo de PAS (51%), MCR (79%), ATM (44%), GTM (57%), y de la razon ATM/AL (43%) respecto al grupo Sham. Ambos tratamientos (Hctz y Esp) redujeron en forma muy importante y significativamente la PAS, MCR, ATM, GTM y la razon ATM/AL en magnitudes similares y tambien por cada mm de Hg de descenso de la PAS logrado. Conclusion: Ademas del efecto antihipertensivo, tanto hidroclorotiazida como espironolactona previenen y/o revierten en magnitud similar el desarrollo de hipertrofia de la pared aortica en este modelo de HTA experimental.

Introduction. Isoproterenol treatment of Brown Norway and Lewis rats (high and low plasma angiotensin-I-converting enzyme activity, respectively) results in similar cardiac hypertrophy but higher cardiac fibrosis in Brown Norway rats. Materials and methods. Rats were infused in vivo with isoproterenol for two or 10 days. Cardiac fibrosis and inflammation were evaluated histochemically. We measured the mRNAs of pro-fibrotic factors (transforming growth factor β1, endothelin-1) and pro-inflammatory factors (monocyte chemoattractant protein-1). In studies with cardiac fibroblasts incubated with isoproterenol in vitro , we measured cell proliferation, angiotensin-I-converting enzyme and matrix metalloprotease 2 activities and deposition of collagen type I and fibronectin. Results. After treatment with isoproterenol for two days, there were large areas of myocardial injury and numerous inflammatory foci in the left ventricle, these being greater in Brown-Norway than in Lewis rats. After treatment with isoproterenol for 10 days, there were large areas of damage with extensive collagen deposition only in the left ventricle; both strains exhibited this damage which was, however, more severe in Brown-Norway than in Lewis rats. After treatment with isoproterenol for two, but not 10, days, greater amounts of monocyte chemoattractant protein-1 mRNA were found in Brown Norway than in Lewis rats. Cell proliferation, activities of angiotensin-I-converting enzyme and matrix metalloprotease 2, amounts of collagen type I and fibronectin were similar in cardiac fibroblasts from both strains; changes after isoproterenol (10 µM) were also similar in both strains. Conclusion. We conclude that the greater cardiac fibrosis in Brown Norway rats treated with isoproterenol correlates with the early and higher expression of proinflammatory factors.

Changes in cardiac osmolarity occur in myocardial infarction. Osmoregulatory mechanisms may, therefore, play a crucial role in cardiomyocyte survival. Tonicity-responsive enhancer binding protein (TonEBP) is a key transcription factor participating in the adaptation of cells to increases in tonicity. However, it is unknown whether cardiac TonEBP is activated by tonicity. Hypertonicity activated transcriptional activity of TonEBP, increased the amounts of both TonEBP mRNA and protein, and induced both the mRNA and protein of TonEBP target genes (aldose reductase and heat shock protein-70). Hypotonicity decreased the amount of TonEBP protein indicating bidirectional osmoregulation of this transcription factor. Adenoviral expression of a dominant negative TonEBP suppressed the hypertonicity-dependent increase of aldose reductase protein. These results indicated that TonEBP controls osmoregulatory mechanisms in cardiomyocytes.

Pulmonary arterial hypertension (PAH) is a rare and progressive disease. Long-term survival remains poor despite of advances in specific vasodilator therapy.To describe the survival rate in a cohort of PAH patients in two referral centers in Chile.One hundred fifteen patients aged 43 ± 15.6 years (85% females) with PAH qualified for this study. Their median pulmonary artery pressure was 55.4 ± 14 mmHg and their six minutes walking capacity was 368 ± 119 m. They were followed for 58 ± 0.4 months and their actual survival rates were compared with the estimated survival using the equation proposed by the French registry of PAH.One, two and three year survival rates were 97, 94 and 89%, respectively. The observed survival rates were greater than the estimated survival.The improvement in survival rates observed in this cohort of patients is similar to what has been described in literature.

Insulin-like growth factor-1, angiotensin-(1-7) and angiotensin-(1-9) have been proposed to be important mediators in cardioprotection. A large body of evidence indicates that insulin like growth factor-1 has pleotropic actions in the heart (i.e., contractility, metabolism, hypertrophy, autophagy, senescence and cell death) and, conversely, its deficiency is associated with impaired cardiac function. Recently, we reported that insulin like growth factor-1 receptor is also located in plasma membrane invaginations with perinuclear localization, highlighting the role of nuclear Ca(2+) signaling in the heart. In parallel, angiotensin-(1-7) and angiotensin (1-9) acting through Mas receptor and angiotensin type 2 receptor have emerged as a novel anti-hypertensive molecules promoting vasodilatation and preventing heart hypertrophy. In this review we discuss the scientific evidence available regarding insulin-like growth factor-1, angiotensin-(1-7) and angiotensin-(1-9) in cardioprotection and its potential application as novel therapeutic targets for treating cardiac diseases.

La hipertrofia cardiaca constituye una de las principales formas de respuesta del cardiomiocito a estimulos mecanicos y neurohormonales y permite al miocito generar mayor trabajo, con aumento de la funcion de la bomba cardiaca. Esta accion compensadora, sin embargo, se ve en algun momento sobrepasada por el estres biomecanico, lo que da lugar al cuadro de insuficiencia cardiaca, que causa una gran morbilidad y mortalidad. En los complejos procesos moleculares que llevan al crecimiento del miocito cardiaco intervienen receptors de membrana, segundos mensajeros y factores de transcripcion. La via final comun en que convergen estos agentes intracelulares es la expresion genica, cuyos cambios estan siendo caracterizados cada vez con mas detalle. Esta modificacion genica se caracteriza por la reexpresion de genes fetales, evento considerado como el marcador molecular de hipertrofia patologica, ausente en condiciones de crecimiento ventricular fisiologico. La posibilidad de detener o revertir la hipertrofia patologica y, asi, detener la evolucion hacia insuficiencia cardiaca, ha generado un considerable interes y mucha informacion al respecto. El objetivo de la presente revision es mostrar esquematicamente el conocimiento actual de la patogenia molecular de la hipertrofia patologica del cardiomiocito, con enfasis en los nuevos interrogantes y lineas de investigacion.

To determine circulating angiotensin-(1-7) [Ang-(1,7)] levels in rats with different angiotensin converting enzyme (ACE) genotypes and to evaluate the effect of hypertension on levels of this heptapeptide, plasma levels of angiotensin II (Ang II) and Ang-(1-7) were determined by HPLC and radioimmunoassay in (a) normotensive F0 and F2 homozygous Brown Norway (BN; with high ACE) or Lewis (with low ACE) rats and (b) in hypertensive F2 homozygous male rats (Goldblatt model). Genotypes were characterized by PCR and plasma ACE activity measured by fluorimetry. Plasma ACE activity was 2-fold higher (p < 0.05) in homozygous BN compared to homozygous Lewis groups. In the Goldblatt groups, a similar degree of hypertension and left ventricular hypertrophy was observed in rats with both genotypes. Plasma Ang II levels were between 300-400% higher (p < 0.05) in the BN than in the Lewis rats, without increment in the hypertensive animals. Plasma Ang-(1-7) levels were 75-87% lower in the BN rats (p < 0.05) and they were significantly higher (p < 0.05) in the hypertensive rats from both genotypes. Plasma levels of Ang II and Ang-(1-7) levels were inversely correlated in the normotensive rats (r = -0.64; p < 0.001), but not in the hypertensive animals. We conclude that there is an inverse relationship between circulating levels of Ang II and Ang-(1-7) in rats determined by the ACE gene polymorphism. This inverse relation is due to genetically determined higher ACE activity. Besides, plasma levels of Ang-(1-7) increase in renovascular hypertension.

In humans and rats, angiotensin I–converting enzyme activity is significantly determined by a gene polymorphism. Homozygous Brown Norway rats have higher plasma angiotensin I–converting enzyme activity and circulating angiotensin II (Ang II) levels than Lewis rats. Because Ang II induces NAD(P)H oxidase activation, we hypothesized here that Brown Norway rats have higher vascular NAD(P)H oxidase activity and superoxide anion production than Lewis rats. Homozygous Brown Norway (n=15) and Lewis (n=13) male rats were used. Plasma angiotensin I–converting enzyme activity (by fluorimetry), Ang II levels (by high-performance liquid chromatography and radioimmunoassay), and aortic NAD(P)H oxidase activity, as well as superoxide anion production (by chemiluminescence with lucigenin) were measured. Plasma angiotensin I–converting enzyme activity and Ang II levels were 100% higher in Brown Norway rats than in Lewis rats ( P P P

The small guanosine triphosphatase Rho and its target, Rho kinase, play important roles in both blood pressure regulation and vascular smooth muscle contraction. Rho is activated by agonists of receptors coupled to cell membrane G protein, such as angiotensin II and phenylephrine. Once Rho is activated, it translocates to the cell membrane where it, in turn, activates Rho kinase. Activated Rho kinase phosphorylates myosin light chain phosphatase, which is then inhibited. This sequence stimulates vascular smooth muscle contraction, stress fiber formation,and cell migration. In this way, Rho and Rho kinase activation have important effects on several cardiovascular diseases. Currently available substances that specifically inhibit this signaling pathway could offer clinical benefits in several cardiovascular, as well as noncardiovascular diseases, such as arterial hypertension, pulmonary hypertension, cerebral or coronary spasm, post-angioplasty restenosis, and erectile dysfunction.

La guanosina trifosfatasa pequena Rho y su blanco Rho-cinasa desempenan un papel importante en la regulacion de la presion arterial y en la contraccion de la musculatura lisa vascular. Diversos agonistas de los receptores acoplados a la proteina G de la membrana celular como, por ejemplo, la angiotensina II y la fenilefrina, activan Rho, que se transloca asi hacia la membrana plasmatica donde activa Rho-cinasa. Al activarse Rho-cinasa por RhoA se fosforila la fosfatasa de la cadena ligera de miosina, con lo cual esta fosfatasa es inhibida, y se favorece de esta manera la contraccion de las celulas musculares lisas vasculares, la formacion de fibras de estres y la migracion celular. Asi, la activacion de Rho y de Rho-cinasa tiene efectos importantes en diversas enfermedades cardiovasculares. Al disponer en la actualidad de inhibidores de esta via de senalizacion, diversas enfermedades cardiovasculares (y tambien otras no cardiovasculares) se podrian beneficiar clinicamente, entre ellas la hipertension arterial, la hipertension pulmonar, el espasmo cerebral y coronario, la reestenosis postangioplastia y la disfuncion erectil.

In humans, the effect of angiotensin-converting enzyme (ACE) gene polymorphisms in cardiovascular disease is still controversial. In the rat, a microsatellite marker in the ACE gene allows differentiation of the ACE gene polymorphism among strains with different ACE levels. We tested the hypothesis that this ACE gene polymorphism determines the extent of cardiac fibrosis induced by isoproterenol (Iso) in the rat. We used a male F2generation (homozygous LL and BB ACE genotypes determined by polymerase chain reaction) derived from two rat strains [Brown-Norway (BB) and Lewis (LL)] that differ with respect to their plasma ACE activities. For induction of left ventricular (LV) hypertrophy (LVH) and cardiac fibrosis, rats were infused with Iso (5 mg·kg–1·day–1) or saline (control) for 10 days and euthanized at day 1 after the last injection. The interstitial collagen volumetric fraction (ICVF), collagen I, and fibronectin content, but not collagen III content, were significantly higher in the homozygous BB rats than in homozygous LL rats. Differences in metalloprotease (MMP)-9, but not in MMP-2 activities as well as in cardiac cell proliferation, were also detected between LL and BB rats treated with Iso. LV ACE activity was higher in BB rats than LL rats and correlated with ICVF ( r = 0.61, P < 0.002). No changes were observed in plasma ACE activities, ANG II plasma or LV levels, plasma renin activity, and ACE and ANG II type 1 receptor (AT1R) mRNA levels in the LV of rats with the two different ACE polymorphisms. Iso induced a similar degree of LVH [assessed by an increase in LV weight 100 per body weight, LV-to-right ventricle (RV) ratio, and LV protein content] in LL and BB rats. We concluded that rats in the F2generation with high plasma ACE activity developed more fibrosis but to a similar degree of LVH compared with rats with low plasma ACE activity.

Summary: This study investigated whether long-term administration of isoproterenol (ISO) induces differential expression of angiotensin-converting enzyme (ACE) in lung, plasma, and left ventricle (LV) during development of left ventricular hypertrophy (LVH) and myocardial fibrosis. Male Sprague-Dawley rats (n = 7–9 per group) were treated with isoproterenol (ISO) 5 mg/kg per day for 10 days or saline and examined at 1, 15, and 33 days after the last injection. ISO stimulated the development of left ventricular hypertrophy (LVH); relative LV weight (mg LV 100/body weight), LV protein content, and LV β-myosin heavy chain levels increased at day 1. LVH regressed at days 15 and 33. ISO also increased myocardial fibrosis (assessed by hydroxyproline content and morphometry) at days 15 and 33. There no were changes in arterial blood pressure. Long-term β-adrenergic stimulation with ISO increased ACE expression in lung, LV, and plasma during development of LVH and myocardial fibrosis. However, time courses were markedly different. ISO stimulated a sustained increase in lung and plasma ACE activities, whereas ISO induced a high LV ACE. Plasma ACE activity paralleled lung ACE activity. LV ACE activity correlated with ACE mRNA levels and paralleled development of LVH. Our data suggest long-term β-adrenergic stimulation induced a differential temporal expression of LV, lung, and plasma ACE in rat during development of LVH and myocardial fibrosis.

Antecedentes: Angiotensina [Ang-(1-9)] disminuye la presion arterial (PA) y el remodel amiento cardiaco en la hipertension arterial experimental independiente de renina. No hay antecedentes sobre el efecto de Ang-(1-9) en la progresion de dano renal de ratas hipertensas por expansion de volumen (con renina baja). Objetivo: Determinar la participacion de Ang-(1-9) en la progresion del dano renal en ratas hipertensas (DOCA-sal). Metodos: Se utilizaron ratas Sprague Dawley macho de 150 ± 10 g uninefrectomizadas tratadas con DOCA (60mg/Kg/2 veces sem, im) por 4 semanas. Como controles (Sham) se usaron ratas uninefrectomizadas. Desde la 2a semana las ratas DOCA, con presion arterial sistolica (PAS) > 140 mmHg, recibieron vehiculo o Ang-(1-9) [602 ng/Kg min] por 2 semanas (minibomba Alzet). Se evaluo la respuesta inflamatoria y el dano renal profibrotico por la presencia de macrofagos infiltrativos y de mio-fibroblastos intersticiales. Se determino, ademas, la presion arterial sistolica (PAS), masa corporal (MC), masa del rinon derecho (MR) y masa renal relativa (MRR). Resultados: Se observo una disminucion del dano renal en las ratas DOCA-sal, cuando recibieron Ang-(1-9), respecto a aquellas que no la recibieron, evidenciado por una significativa disminucion de macrofagos infiltrativos y miofibroblastos en el intersticio renal. El bloqueo de los receptores Mas y AT2, no tuvieron efectos adicionales. Conclusion: En este modelo experimental, Ang-(1-9) disminuyo la hipertension y redujo significativamente la infiltracion de macrofagos y la aparicion de miofibroblastos en el intersticio renal. Estos resultados son la primera evidencia de que Ang-(1-9) reduce la fibrosis tubulo-intersticial renal y el dano renal hipertensivo.

Abstract — — Angiotensin I is a substrate for both ACE and for neutral endopeptidase 24.11 (NEP). We hypothesized that high ACE expression is related to low NEP activity. Accordingly, circulating and tissue NEP and ACE activities were measured by fluorometry in homozygous rats (F 0 and F 2 ) for the Lewis microsatellite allele (LL, low ACE) and for the Brown Norway microsatellite allele (BB, high ACE). Plasma, lung, and aortic ACE activities in F 0 and F 2 were higher in BB rats than in LL rats ( P 0 and F 2 homozygous ( P r =−0.6 and −0.598 in F 0 and F 2 , respectively; P r =−0.77 in F 0 and r =−0.59 in F 2 , P r =−0.696 and −0.584 in F 0 and F 2 , respectively; P

Introduccion Las concentraciones de angiotensina II pueden inhibirse parcialmente durante la administracion cronica de inhibidores de la enzima conversiva de la angiotensina (ECA), limitando desde el punto de vista clinico su eficacia en el tratamiento de la hipertension arterial. Existen pocos estudios que relacionan directamente la actividad de la ECA y la prevencion precoz de hipertrofia ventricular izquierda (HVI) secundaria a hipertension arterial durante la administracion de un inhibidor de la ECA (IECA). Objetivo Evaluar los efectos de la inhibicion precoz de la ECA con perindopril sobre el desarrollo de hipertension, HVI y concentraciones de angiotensina II plasmatica y en el ventriculo izquierdo en el modelo Goldblatt en la rata (Gb; 2 rinones-un pinzado) a las 2 semanas de la cirugia. Resultados La presion arterial sistolica y la masa ventricular izquierda relativa aumentaron un 42 y un 20%, respectivamente, en el grupo Gb (p Conclusiones En este modelo experimental de HVI hipertensiva existe una activacion temprana de la ECA plasmatica y cardiaca. La administracion precoz de un IECA previene el desarrollo de hipertension e HVI al inhibir el aumento de angiontensina II en el plasma y el ventriculo izquierdo.

Combined rho kinase and renin–angiotensin system inhibition: a new therapeutic perspective for renal and cardiovascular remodeling

Background: Little is known about the biological effects of angiotensin-(1-9), but available evidence shows that angiotensin-(1-9) has beneficial effects in preventing/ameliorating cardiovascular remodeling. Objective: In this study, we evaluated whether angiotensin-(1-9) decreases hypertension and reverses experimental cardiovascular damage in the rat. Methods and results: Angiotensin-(1-9) (600 ng/kg per min for 2 weeks) reduced already-established hypertension in rats with early high blood pressure induced by angiotensin II infusion or renal artery clipping. Angiotensin-(1-9) also improved cardiac (assessed by echocardiography) and endothelial function in small-diameter mesenteric arteries, cardiac and aortic wall hypertrophy, fibrosis, oxidative stress, collagen and transforming growth factor type beta - 1 protein expression (assessed by western blot). The beneficial effect of angiotensin-(1-9) was blunted by coadministration of the angiotensin type 2(AT2) receptor blocker PD123319 (36 ng/kg per min) but not by coadministration of the Mas receptor blocker A779 (100 ng/kg per min). Angiotensin-(1-9) treatment also decreased circulating levels of Ang II, angiotensin-converting enzyme activity and oxidative stress in aorta and left ventricle. Whereas, Ang-(1-9) increased endothelial nitric oxide synthase mRNA levels in aorta as well as plasma nitrate levels. Conclusion: Angiotensin-(1-9) reduces hypertension, ameliorates structural alterations (hypertrophy and fibrosis), oxidative stress in the heart and aorta and improves cardiac and endothelial function in hypertensive rats. These effects were mediated by the AT2 receptor but not by the angiotensin-(1-7)/Mas receptor axis.

Chronic RAS (renin–angiotensin system) activation by both AngII (angiotensin II) and aldosterone leads to hypertension and perpetuates a cascade of pro-hypertrophic, pro-inflammatory, pro-thrombotic and atherogenic effects associated with cardiovascular damage. In 2000, a new pathway consisting of ACE2 (angiotensin-converting enzyme2), Ang-(1–9) [angiotensin-(1–9)], Ang-(1–7) [angiotensin-(1–7)] and the Mas receptor was discovered. Activation of this novel pathway stimulates vasodilation, anti-hypertrophy and anti-hyperplasia. For some time, studies have focused mainly on ACE2, Ang-(1–7) and the Mas receptor, and their biological properties that counterbalance the ACE/AngII/AT1R (angiotensin type 1 receptor) axis. No previous information about Ang-(1–9) suggested that this peptide had biological properties. However, recent data suggest that Ang-(1–9) protects the heart and blood vessels (and possibly the kidney) from adverse cardiovascular remodelling in patients with hypertension and/or heart failure. These beneficial effects are not modified by the Mas receptor antagonist A779 [an Ang-(1–7) receptor blocker], but they are abolished by the AT2R (angiotensin type 2 receptor) antagonist PD123319. Current information suggests that the beneficial effects of Ang-(1–9) are mediated via the AT2R. In the present review, we summarize the biological effects of the novel vasoactive peptide Ang-(1–9), providing new evidence of its cardiovascular-protective activity. We also discuss the potential mechanism by which this peptide prevents and ameliorates the cardiovascular damage induced by RAS activation.

The relation between gizzard erosion-black vomit (GE-BV) and gastric secretion is not completely understood. A pharmacological approach to reduce the presence of GE-BV in chicks due to fish meal in diets is also unknown. In this study the use of omeprazole, a H+/K+ ATPase inhibitor, and fish meals of different biotoxicological characteristics, showed that: 1) Omeprazole decreased total gastric acid content, GE scores and severe GE (SGE) cases, in a dose-dependent manner. This reduction was significant at levels higher than 20 mg omeprazole/Kg body weight (BW)/day (p < 0.01). The addition of 50 mg omeprazole/kg BW/day almost completely prevented the incidence of SGE cases and reduced in 50% GE score in chicks (p < 0.01). 2) A significant reduction in specific mortality, near 90%, was also seen with all toxic fish meals when omeprazole (50 mg/Kg BW/day) was added to experimental diets in comparison to control groups. However, no mortality was observed when omeprazole was added to diets containing non-toxic fish meals. 3) In chicks fed with toxic fish meals, addition of different amounts of omeprazole to diets changed the relative weight of proventriculus (p < 0.01) and gizzard (p < 0.05). Maximum effect was obtained with omeprazole concentration higher than 50 mg/Kg BW/day. 4) Omeprazole did not change feed intake in chicks fed with toxic fish meal. However, in some fish meal a reduction on weight gain was observed with the addition of omeprazole.

Introduccion: El modelo experimental de ligadura de la arteria coronaria descendente anterior izquierda (ADA) en ratas, ha sido de gran ayuda para el estudio de la cardiopatia isquemica. Sin embargo, es un procedimiento dificil de realizar y con alta mortalidad operatoria. Existe poca informacion de sus aspectos tecnicos. El objetivo de nuestro trabajo fue describir en detalle la tecnica quirurgica de la ligadura de la ADA en ratas. Material y Metodo: Se utilizaron 51 ratas machos Sprague-Dawley 230 ± 20 gr de peso anestesiadas y conectadas a ventilacion mecanica. A traves de una toracotomia izquierda se ligo la ADA, 2 mm debajo de la interseccion entre la arteria pulmonar y la orejuela izquierda. Al dia siguiente se realizo una ecocardiografia transtoracica para confirmar la presencia de hipocinesia. Resultados: La mortalidad operatoria fue de 7,8% (4 de 51). La principal causa de muerte fue la hemorragia en el intraoperatorio durante la curva de aprendizaje. El tiempo operatorio promedio fue de 45 ± 5 min. Se confirmo la presencia de una hipocinesia de la pared anterolateral en el 100% de las ratas supervivientes. Discusion. Esta tecnica es un procedimiento reproducible y seguro, con una mortalidad menor al 10%.

Estrogen regulation of myocardial chymase and chymase effects on cardiac remodeling are unknown. To test the hypothesis that estrogen prevents pressure overload-induced adverse cardiac remodeling by inhibiting mast cell chymase release, transverse aortic constriction or sham surgery was performed in 7-week-old intact and ovariectomized rats. Three days prior to creating the constriction, additional groups of ovariectomized rats began receiving 17β-Estradiol, a chymase inhibitor, or a mast cell stabilizer. Left ventricular function, cardiomyocyte size, collagen volume fraction, mast cell density and degranulation, and myocardial and plasma chymase levels were assessed 18 days post-surgery. Aortic constriction resulted in ventricular hypertrophy in intact and ovariectomized groups while collagen volume fraction was increased only in ovariectomized rats. Chymase protein content was increased by aortic constriction in the intact and ovariectomized groups with the magnitude of the increase being greater in ovariectomized rats. Mast cell density and degranulation, plasma chymase levels and myocardial active transforming growth factor- 1 levels were increased by aortic constriction only in ovariectomized rats. Estrogen replacement markedly attenuated the constriction-increased myocardial chymase, mast cell density and degranulation, plasma chymase and myocardial active transforming growth factor- 1 as well as prevented ventricular hypertrophy and increased collagen volume fraction. Chymostatin attenuated the aortic constriction induced ventricular hypertrophy and collagen volume fraction in the ovariectomized rats similar to that achieved by estrogen replacement. Nedocromil yielded similar effects except for the reduction of chymase content. We conclude that the estrogen-inhibited release of mast cell chymase is responsible for the cardioprotection against transverse aortic constriction-induced adverse cardiac remodeling.

Despite reduction in cardiovascular (CV) events and end-organ damage with the current pharmacologic strategies, CV disease remains the primary cause of death in the world. Pharmacological therapies based on the renin angiotensin system (RAS) blockade are used extensively for the treatment of hypertension, heart failure, and CV remodeling but in spite of their success the prevalence of end-organ damage and residual risk remain still high. Novel approaches must be discovered for a more effective treatment of residual CV remodeling and risk. The ACE2/Ang-(1–9) axis is a new and important target to counterbalance the vasoconstrictive/proliferative RAS axis. Ang-(1–9) is hydrolyzed slower than Ang-(1–7) and is able to bind the Ang II type 2 receptor. We review here the current experimental evidence suggesting that activation of the ACE2/Ang-(1–9) axis protects the heart and vessels (and possibly the kidney) from adverse cardiovascular remodeling in hypertension as well as in heart failure.

Introducción: La vía intracellular de RhoA/Rho kinasa es activada por agonistas de receptores acoplados a proteínas G pequeñas unidas a membrana. Su activación está relacionada al remodelado cardiovascular patológico. Previamente hemos observado aumento de actividad de Rho kinasa (ROCK) en pacientes con hipertensión arterial (HT) e hipertrofia ventricular izquierda como daño de órgano blanco. Pero su activación en relación a la diabetes no ha sido explorada en estos pacientes. Objetivo: Evaluar activación de Rho kinasa y parámetros de estrés oxidativo en pacientes hipertensos con diabetes tipo II (DMII). Métodos: Estudio comparativo entre pacientes con HT sin tratamiento, HT con DMII y hemoglobina glicosi-lada Alc > 7,5% y un grupo control normotenso. Se realizó ecocardiograma de superficie. Se midió activación de ROCK en leucocitos circulantes midiendo MYPT1 fosforilado/total (p/t) por Western blot y la velocidad de pulso carotídeo-femoral (PWV) para estimar distensibilidad arterial. El stress oxidativo se estimó midiendo ma-londialdehído (MDA) y 8-isoprostano (8-ISO) en suero. Resultados: Se incluyeron 21 pacientes hipertensos con DMII, 38 pacientes hipertensos sin DMII y 34 controles normotensos. La edad promedio fue 51 ± 0,9; 48 ± 0,9 y 52 (p: NS) ± 1,1 y el 47%, 50% y 52% (p: NS) eran mujeres respectivamente. Los pacientes HT con DMII presentaron MYPTl p/t (5,6 ± 1,3; 3,6 ± 0,4; 2,1 ± 0,1 p< 0,01), MDA (1,8 ± 0,4/ 7.5% and normotensi-ve control subjects was performed. LVH was assessed by echocardiography. ROCK activity was measured in peripheral leukocytes by Western blot determination of phosphorilated / total MYPT1 ratio. Arterial compliance was determined by the relationship of carotid and femoral velocity signals (PWV) Oxidative stress was estimated by serum levels of malondialdehyde (MDA) and 8-isoprostane (8-ISO). Results: Hypertensives with DMII (n=21) had a mean age of 51 ± 0.9 years, and 47% were females. Corresponding figures for 38 hypertensive patients without DM and 34 control patients were 48 ± 0,9 and 52 ± 1,1 (NS) and 50% and 52% females, respectively (NS). The MYPT1 p/t ratio was 5,6 ± 1,3; 3,6 ± 0,4; 2,1 ± 0,1 (p

La actividad de Rho kinasa (ROCK) cardíaca en la hipertensión arterial (HTA) y el efecto del tratamiento antihipertensivo conjunto han sido poco estudiados. Hemos planteado que la adición de un inhibidor de ROCK al tratamiento antihipertensivo convencional podría tener efectos preventivos adicionales al uso aislado del antihipertensivo. Objetivo: Determinar la actividad de ROCK ventricular y parámetros de remodelamiento cardíaco en ratas hipertensas con y sin tratamiento antihipertensivo, adicionando un inhibidor directo de ROCK. Métodos. Se usaron ratas Sprague Dawley de 150 grs. ( n = 12 - 13/grupo) unifrectomizadas tratadas con desoxicorticosterona (DOCA, 100 mg/Kg/sem sbc) durante 6 semanas. Como controles se usaron ratas unifrectomizadas. Otros 3 grupos recibieron DOCA y además el antagonista del receptor de angiotensina n, candesartán (10 mg/kg/día) o el inhibidor de la vía ROCK fasudil (50 mg/Kg/dia), o la combinación de ambos (5 y 25 mg/Kg/dia, respectivamente), vía gavage desde la tercera semana post cirugía, durante 3 semanas. Al finalizar los tratamientos se determinó la masa corporal (MC), presión arterial sistólica (PAS) y la masa cardíaca relativa (MCR). Además se midió en el ventrículo izquierdo la fosforilación de la fosfatasa de la miosina (MYPT-1) como índice de activación de ROCK, la infiltración de macrófagos/ monocitos (células ED1 positivas), la expresión proteica de colágeno I (por Western blot) y la expresión génica de la subunidad gp91 de NADPH oxidasa y eNOS por RTPCR. Resultados: Con respecto de las ratas sham, en las ratas hipertensas se observó hipertrofia cardiaca de 63% (p < 0,05), junto a aumento significativo (p < 0,05) de MYPT-1 fosforilado/total (300%), de colágeno miocárdico I (en 14 veces), de células ED1 en 270% y de la expresión génica de la subunidad gp91 de NADPH oxidasa en miocardio en 75%. Además se observó disminución en la expresión génica de eNOS en corazón de 37% (p< 0.05). En las ratas hipertensas DOCA tratadas durante 3 semanas con candesartán, con fasudil o con ambos fármacos en combinación, hubo normalización de la PAS y de MYPT-1 fosforilado/ total, regresión significativa de la hipertrofia cardíaca, normalización del colágeno miocárdico I, del número de células ED1 en la pared cardíaca y de la expresión génica de la subunidad gp91 de NADPH oxidasa. Conclusión: En este modelo experimental de HTA, el uso combinado de un inhibidor de ROCK y de un antagonista del receptor de angiotensina II en dosis bajas fue tan efectivo como cualquiera de ambos fármacos por separado en dosis plena en prevenir el desarrollo de HVI y el remodelado cardíaco hipertensivo. (Fondecyt 1085208).

Uno de los objetivos del tratamiento antihipertensivo, más allá de normalizar las cifras tensionales, es disminuir/regresar el remodelado patológico hipertensivo cardiovascular y renal, de manera de reducir eficientemente el riesgo dado por esta patología. Al respecto, la vía de señalización intracelular de la Rho kinasa (ROCK) es un mecanismo de vasoconstricción y de promoción de remodelado que podría ser un blanco atractivo en el tratamiento antihipertensivo. Objetivo: Evaluar a nivel vascular los niveles de TGF beta, la expresión génica vascular de NADPH oxidasa (fuente de stress oxidativo vascular), y el grado de inflamación parietal y su dependencia de ROCK en la hipertensión arterial (HTA) experimental. Métodos: Se compararon 5 grupos experimentales. Se usó el modelo de HTA por administración de deoxicorticosterona (DOCA, 100mg/Kg, sc una vez/semana) + sal en ratas Sprague Dawley uninefrectomizadas. Como controles, se usaron ratas uninefrectomizadas (Sham). Las ratas DOCA se randomizaron para recibir el inhibidor específico de ROCK fasudil (Fas, 50 mg/kg/d, por gavage, durante 3 semanas), desde la semana 3 post cirugía, o candesartán (Cand, 10 mg/kg/d, por gavage, durante 3 semanas), o fasudil (25 mg/kg/d) + candesartan (5 mg/ kg/d por gavage, 3 semanas ). Al finalizar los experimentos se midieron en la aorta los niveles de MYPT1 fosforilada/total, blanco de ROCK y estimador de su activación (por Western blot), de TGF beta (por Western blot), los niveles de mRNA de las subunidades p22 Phox y gp 91 de la NADPH oxidasa (por RT PCR) y el número de células infamatorias ED1 en anillos aórticos (por inmunohistoquímica). Resultados: La presión arterial sistólica aumentó en las ratas DOCA a 172 ± 7 mm Hg (p < 0,05) y fue normalizada después de 3 semanas de tratamiento con candesartán, fasudil y de candesartán + fasudil. La actividad de ROCK en aorta aumentó en 4 veces en las ratas hipertensas (p < 0,05) y se normalizó con los 3 tratamientos antihipertensivos. En las ratas hipertensas la expresión génica en aorta de las subunidades de NADPH oxidasa p22 Phox y gp91 aumentó significativamente (p < 0.05) en 80 y 100%, respectivamente y se redujo significativamente a valores normales en las ratas que recibieron fasudil y candesartán + fasudil. La expresión génica en la aorta de TGF beta aumentó en las ratas hipertensas 4 veces y se normalizó en los 3 grupos tratados. El número de células ED1 en la pared aórtica aumentó 6 veces en las ratas hipertensas y disminuyó significativamente con los 3 tratamientos. Conclusiones: En este modelo de HTA experimental hay un importante aumento de la actividad de ROCK en la pared aórtica. El tratamiento antihipertensivo previene la inflamación y la sobrexpresión génica de TGF beta en la pared vascular. La sobrexpresión génica de NADPH oxidasa en la pared aórtica en las ratas hipertensas se normaliza solamente al inhibir en forma directa ROCK con un inhibidor específico. Es posible la inhibición directa de ROCK sea una forma más completa de tratamiento antihipertensivo, al prevenir la expresión de las subunidades de NADPH oxidasa p22 Phox y gp91 en la pared aórtica, hecho que no se observó utilizando un antihipertensivo de última generación.

The small guanosine triphosphatase Rho and its target Rho-kinase have significant roles in experimental remodeling and ventricular dysfunction, but no data are available on Rho-kinase activation in patients with heart failure (HF). We hypothesized that, in patients with chronic HF, Rho-kinase in circulating leukocytes is activated and related to left ventricular (LV) remodeling and dysfunction.Accordingly, Rho-kinase activity, assessed by the levels of phosphorylated to total myosin light chain phosphatase 1 (MYPT1-P/T) in circulating leukocytes, and echocardiographic LV function data were compared between patients with HF New York Heart Association functional class II or III due to systolic dysfunction (n = 17), healthy controls (n = 17), and hypertensive patients without HF (n = 17).In the control subjects, mean MYPT1-P/T ratio was 1.2 ± 0.2 (it was similar in the hypertensive patients without HF), whereas in patients with HF, it was significantly increased by100-fold (P.001). Both MYPT1-P/T and log MYPT1-P/T ratios were inversely correlated with ejection fraction (r = -0.54, P.03 and r = -0.86, P.001, respectively). Furthermore, in patients with HF with LV end-diastolic diameter60 mm, MYPT1-P/T ratio was 35.8 ± 18.1, whereas it was significantly higher in patients with LV diameter ≥60 mm (P.05).Rho-Kinase activity is markedly increased in patients with stable chronic HF under optimal medical treatment, and it is associated with pathologic LV remodeling and systolic dysfunction. Mechanisms of Rho-kinase activation in patients with HF, its role in the progression of the disease, and the direct effect of Rho-kinase inhibition need further investigation.

Background Angiotensin-(1-9) is present in human and rat plasma and its circulating levels increased early after myocardial infarction or in animals treated with angiotensin-converting enzyme inhibitor. However, the cardiovascular effects of this peptide are unknown. Objective To determine whether angiotensin-(1-9) is a novel anti-cardiac hypertrophy factor in vitro and in vivo and whether this peptide is involved in the pharmacological effects of cardiovascular drugs acting on the renin-angiotensin system. Methods and results The administration of angiotensin-(1-9) to myocardial infarcted rats by osmotic minipumps (450 ng/kg per min, n = 6) vs. vehicle (n = 8) for 2 weeks decreased plasma angiotensin II levels, inhibited angiotensin-converting enzyme activity and also prevented cardiac myocyte hypertrophy. However, cardiac myocyte hypertrophy attenuation triggered by angiotensin-(1-9) was not modified with the simultaneous administration of the angiotensin-(1-7) receptor antagonist A779 (100 ng/kg per min, n = 6). In experiments in vitro with cultured cardiac myocytes incubated with norepinephrine (10 micromol/l) or with insulin-like growth factor-1 (10 nmol/l), angiotensin-(1-9) also prevented hypertrophy. In other experimental setting, myocardial infarcted rats (n = 37) were randomized to receive either vehicle (n = 12), enalapril (10 mg/kg per day, n = 12) or angiotensin II receptor blocker candesartan (10 mg/kg per day, n = 13) for 8 weeks. Both drugs prevented left ventricle hypertrophy and increased plasma angiotensin-(1-9) levels by several folds. Angiotensin-(1-9) levels correlated negatively with different left ventricular hypertrophy markers even after adjustment for blood pressure reduction. Conclusion Angiotensin-(1-9) is an effective and a novel anti-cardiac hypertrophy agent not acting via the Mas receptor.

BACKGROUND Patients with primary aldosteronism experience greater left ventricular hypertrophy and a higher frequency of cardiovascular events than do essential hypertensive patients with comparable blood pressure levels. Aldosterone has been correlated with increased oxidative stress, endothelial inflammation, and fibrosis, particularly in patients with heart disease. AIM To evaluate oxidative stress, subclinical endothelial inflammation, and myocardial fibrosis markers in patients with primary aldosteronism and essential hypertension. DESIGN AND INDIVIDUALS: We studied 30 primary aldosteronism patients and 70 control essential hypertensive patients, matched by age, sex and median blood pressure. For all patients, we measured the serum levels of aldosterone, plasma renin activity, malondialdehyde (MDA), xanthine oxidase, metalloproteinase-9, ultrasensitive C-reactive protein and amino terminal propeptides of type I (PINP), and type III procollagen. We also evaluated the effect of PA treatment in 19 PA individuals. RESULTS PA patients showed elevated levels of MDA (1.70 ± 0.53 versus 0.94 ± 0.65 μmol/l, P

Antecedentes: Recientemente hemos propuesto en un modelo experimental de infarto al miocardio una significativa interregulación entre los niveles de la enzima convertidora de angiotensina I (ECA) y su homologa (ECA-2), junto con que angiotensina (Ang)-(1-9) más que Ang-(1-7) actuaría como un contrarregulador de Ang II. Sin embargo tal relación no se ha investigado en el remodelado aórtico hipertensivo. Objetivo: Determinar la expresion de ECA y ECA-2, los niveles de Angs I, II, (1-7) y (1-9) y los parámetros de remodelado de la pared aórtica de ratas hipertensas. Métodos: Ratas normotensas Lewis (n=18) fueron randomizadas a hipertension (HTA) por sobrecarga de presion (modelo Goldblatt, GB, 2 riñones-1 pinzado, n=9). Ratas pseudo-operadas se usaron como controles (S, n=9). A las 6 semanas post cirugía, se determinó la masa cardíaca relativa (MCR) y la presion arterial sistólica (PAS). En la aorta torácica se determinó el grosor de la túnica media (GTM), área de la TM (ATM), niveles de mRNA de ECA y ECA-2, factor de crecimiento transformante tipo ß (TGF-ß), inhibidor del activador de plasminógeno (PAI-1) y de la proteína quimioatractante de monocitos (MCP-1) por RT-PCR. La actividad y niveles proteicos de ECA y ECA-2 por fluorimetría y Western blot y los niveles de Angs I, II, (1-7) y (1-9) por HPLC y radioinmunoensayo. Resultados: La MCR y la PAS aumentaron significativamente (p), MCP-1 (53%,) junto con mayor actividad (89%,), niveles proteicos de ECA (130%,) y Ang II (48%,). Esos efectos se asociaron a una significativa disminución del mRNA, los niveles proteicos y actividad de ECA-2 (- 55%, -41%, y 54%, respectivamente) y a menores niveles aórticos (-25%,) de Ang- (1-9), sin diferencias en los niveles de ang-(1-7). Conclusion: Estos resultados fuertemente sugieren que en la hipertension arterial experimental, el remodelado de la pared aórtica está asociado a una interacción entre ECA y ECA-2 y los niveles de Ang II y Ang-(1-9), pero no de Ang-(1-7).

Antecedentes: La sobreexpresion génica de la enzima convertidora de angiotensina I homologa (ECA2) se asocia con prevención de la hipertrofia y fibrosis cardiaca dependiente de angiotensina (Ang) II. Sin embargo se desconoce si su sobreexpresion reduce la hipertensión arterial (HTA) y revierte el consecuente remodelado mio-cárdico (RM) dependiente de Ang II. Objetivo: Determinar si la sobreexpresion adenoviral (Ad) del gen de la ECA2 en el miocardio disminuye la HTA y RM experimental en ratas con niveles genéticamente determinados de ECA y Ang II. Métodos: Ratas homocigotas normotensas Lewis (LL) y Brown Borway (BN), con menores y mayores niveles circulantes de ECA y Ang II, respectivamente, se hicieron hipertensas por el procedimiento Goldblatt (GB). Como controles se usaron ratas seudo-operadas (sham). A la semana 5 post cirugía y con HTA establecida > 140 mmHg, las ratas se randomizaron a inyección intra-miocárdica con un AdECA2 o Ad proteína fluorescente verde (GFP) como controles de infección. A la semana post infección adenoviral, los ratas se sacrificaron y se determinaron peso corporal (PC, g), masa cardiaca (MC, mg), presión arterial sistólica (RAS, mmHg), área (AC, um2) y perímetro (PERC, um) de cardiomiocitos y contenido de colágeno (%) miocárdico (CM), sub-endocárdico (CS)ytotal(CT). Resultados: La HTA aumentó significativamente la MC, MCR, AC, PERC como también el CM, CS y CT en las ratas GB vs Sham, sin diferencias en el PC ni por efecto del polimorfismo de la ECA. La sobreexpresion de ECA2 disminuyó significativamente la RAS (15% y 27%), AC (25% y 25% ) y PERC (17 % y 18%) en las ratas LL y BN vs ratas hipertensas, respectivamente. Estos resultados se asociaron a una disminución significativa del CS (LL = 37%, BN = 39%), CM (LL = 54%) y CT (LL = 42%, BN = 22%) respecto a las ratas GB. Conclusión: En ratas con HTA establecida, la sobre-expresión miocárdica de ECA2 disminuyó la HTA y el desarrollo de hipertrofia y fibrosis cardíaca hipertensiva experimental en ratas con diferentes niveles de ECA y Ang II. FONDECYT 1070662.

Beta adrenergic receptors (AR) are highly polymorphic and important regulators of cardiovascular homeostasis. Among these, beta1 and beta2 AR regulate cardiac contractility and frequency and are important pharmacological targets.To evaluate genotype and gene-gene interaction between beta1-AR Arg389Gly and beta2-AR Arg16Gly, Gln27Glu and Thr164Ile polymorphisms, as risk factors for HF.Eighty chronic HF patients and eighty-eight controls matched by age and sex were genotyped for beta1-AR Arg389Gly, beta2-AR Arg16Gly, Gln27Glu and Thr164Ile polymorphisms.The presence of beta2-AR Glu allele was a risk predictor for HF (odds ratio (OR) = 2.81; 95% confidence intervals (CI) = 1.49-5.31). Interactions that increased the risk for HF were found in patients carrying at least one of the beta2-AR Glu and beta2-AR Gly allele (OR = 3.81; 95% CI = 1.50-0.70) and beta2-AR Glu and beta1-AR Gly allele combination (OR = 5.51; 95% CI = 2.19-13.86). Furthermore, the frequency of beta2-AR Glu allele was higher among patients with a history of acute myocardial infarction (with infarction: 0.534, without: 0.313, p = 0.01).Beta2-AR Glu allele could be a risk predictorfor HF. This risk could be enhanced by the additional presence of beta2-AR Gly16 or beta1-AR Arg389 alleles. The frequency of beta2-AR Gln27 Glu allele was higher among patients with a history of myocardial infarction.

Background: ß adrenergic receptors (AR) are highly polymorphic and important regulators of cardiovascular homeostasis. Among these, ß1 and ß2 AR regulate cardiac contractility and frequency and are important pharmacological targets. Aim: To evaluate genotype and gene-gene interaction between ß1-AR Arg389Gly and ß2-AR ArglSGly GlnZ7Gly and Thrl 64Ile polymorphisms, as risk factors for HF. Material and methods: Eighty chronic HF patients and eighty-eight controls matched by age and sex were genotyped for ß1 -AR Arg389Gly ß2-AR ArgWGly, GlnZ7Glu and Thr164Ile polymorphisms. Results: The presence of ß2-AR Glu afiele was a risk predictor for HF (odds ratio (OR) =2.81; 95% confidence intervals (CI) =1.49-5.31). Interactions that increased the risk for HF were found in patients carrying at least one of the ß2-AR Glu and ß2-AR Gly allele (OR =3.81; 95% CI =1.50-0.70) and ß2-AR Glu and ß1 -AR Gly allele combination (OR =5.51; 95% CI =2.19-13.86). Furthermore, the frequency of ß2-AR Glu allele was higher among patients with a history ofacute myocardial infarction (with infarction: 0.534, without: 0.313, p =0.01). Conclusions: ß2-AR Glu allele could be a risk predictor for HF. This risk could be enhanced by the additional presence of ß2-AR GlyW or ß1-AR Arg389 alleles. The frequency of ß2-AR Gln27 Glu allele was higher among patients with a history of myocardial infarction.

The RhoA/Rho kinase (ROCK) pathway is a new mechanism of remodeling and vasoconstriction. Few data are available regarding ROCK activation when angiotensin I–converting enzyme is high and blood pressure is normal. We hypothesized that ROCK is activated in the vascular wall in normotensive rats with genetically high angiotensin I–converting enzyme levels, and it causes increased vascular expression of genes promoting vascular remodeling and also oxidative stress. Aortic ROCK activation, mRNA and protein levels (of monocyte chemoattractant protein-1, transforming growth factor [TGF]-β 1 , and plasminogen activator inhibitor-1 [PAI-1]), NADPH oxidase activity, and O 2 ·− production were measured in normotensive rats with genetically high (Brown Norway [BN]) and low (Lewis) angiotensin-I–converting enzyme levels and in BN rats treated with the ROCK antagonist fasudil (100 mg/kg per day) for 7 days. ROCK activation was 12-fold higher in BN versus Lewis rats ( P P P P . O 2 − production were increased by 88% and 300%, respectively, in BN rats ( P

Cardiac hypertrophy is one of the main ways in which cardiomyocytes respond to mechanical and neurohormonal stimuli. It enables myocytes to increase their work output, which improves cardiac pump function. However, this compensatory mechanism can become overwhelmed by biomechanical stress, thereby resulting in heart failure, which is associated with high morbidity and mortality. The complex molecular processes that lead to cardiomyocyte growth involve membrane receptors, second messengers, and transcription factors. The common final pathway of all these intracellular substances is gene expression, whose variations are being revealed in increasing detail. The genetic response is characterized by the re-expression of fetal genes, an event which is regarded as the molecular marker of pathologic cardiac hypertrophy, and which is absent in normal physiologic cardiac growth. The possibility of stopping or reversing pathologic cardiac hypertrophy and, thereby, slowing the development of heart failure is a topic of considerable clinical interest and a large amount of relevant data has accumulated. The purpose of this review was to provide a schematic overview of current knowledge about the molecular pathogenesis of cardiomyocyte hypertrophy, with special emphasis on new research topics and investigations.

Cardiac hypertrophy is one of the main ways in which cardiomyocytes respond to mechanical and neurohormonal stimuli. It enables myocytes to increase their work output, which improves cardiac pump function. However, this compensatory mechanism can become overwhelmed by biomechanical stress, thereby resulting in heart failure, which is associated with high morbidity and mortality. The complex molecular processes that lead to cardiomyocyte growth involve membrane receptors, second messengers, and transcription factors. The common final pathway of all these intracellular substances is gene expression, whose variations are being revealed in increasing detail. The genetic response is characterized by the re-expression of fetal genes, an event which is regarded as the molecular marker of pathologic cardiac hypertrophy, and which is absent in normal physiologic cardiac growth. The possibility of stopping or reversing pathologic cardiac hypertrophy and, thereby, slowing the development of heart failure is a topic of considerable clinical interest and a large amount of relevant data has accumulated. The purpose of this review was to provide a schematic overview of current knowledge about the molecular pathogenesis of cardiomyocyte hypertrophy, with special emphasis on new research topics and investigations.

The small guanosine triphosphatase Rho and its target, Rho kinase, play important roles in both blood pressure regulation and vascular smooth muscle contraction. Rho is activated by agonists of receptors coupled to cell membrane G protein, such as angiotensin II and phenylephrine. Once Rho is activated, it translocates to the cell membrane where it, in turn, activates Rho kinase. Activated Rho kinase phosphorylates myosin light chain phosphatase, which is then inhibited. This sequence stimulates vascular smooth muscle contraction, stress fiber formation,and cell migration. In this way, Rho and Rho kinase activation have important effects on several cardiovascular diseases. Currently available substances that specifically inhibit this signaling pathway could offer clinical benefits in several cardiovascular, as well as noncardiovascular diseases, such as arterial hypertension, pulmonary hypertension, cerebral or coronary spasm, post-angioplasty restenosis, and erectile dysfunction.

Neutral endopeptidase (NEP) hydrolyses angiotensins (Ang) I and II and generates angiotensin-(1-7) [Ang-(1-7)]. In humans, the insertion/deletion (I/D) angiotensin-I converting enzyme (ACE) gene polymorphism determined plasma ACE levels by 40%. In rats, a similar polymorphism determines ACE levels which are inversely associated to NEP activity. The objective of this study is to evaluate the relationship between ACE expression and plasma NEP activity in normotensive subjects and in hypertensive patients. In total, 58 consecutive patients with hypertension, evaluated in our Hypertension Clinic, were compared according to their ACE I/D genotypes with 54 control subjects in terms of both plasma ACE activity and NEP activities. Plasma ACE activity was elevated 51 and 70% in both DD ACE groups (normotensives and hypertensives) compared with their respective ID and II ACE groups (P

In patients with hypertension who have the DD-ACE genotype (higher angiotensin-converting enzyme [ACE] activity), plasma levels of angiotensin-(1-7) are 4 times lower than in patients with the II-ACE genotype (lower ACE levels). Angiotensin II levels are similar in both groups.

BACKGROUND AND OBJECTIVE In humans, the insertion/deletion polymorphism in the angiotensin (Ang) I converting enzyme (ACE) gene significantly determines ACE activity. The deletion allele induces higher ACE levels and is associated with hypertension in men. In the rat, a microsatellite marker in the ACE gene allows differentiation of the ACE alleles among strains with different ACE levels. We evaluated the effect of genetically determined ACE expression on the development of renovascular hypertension in the rat. METHODS AND RESULTS Systolic BP (SBP), ACE and angiotensin II (Ang II) levels were measured using the Goldblatt (Gb) model (two kidneys, one clip) in homozygous males of two inbred strains (F2) of Lewis x Brown-Norway (BN) rats. SBP was significantly higher in the BN-Gb rats compared to the Lewis-Gb rats throughout the study (F = 239.6, P < 0.001). An interaction was observed between SBP and strain (F = 2.92, P < 0.01). Plasma ACE activity was 100% higher in the BN-Gb than in the Lewis-Gb rats (P < 0.05). Ang II plasma levels were higher in the BN-sham than in the Lewis-sham rats (255 +/- 22 versus 161 +/- 16 pg/ml, P < 0.05), increased in both Gb groups and correlated significantly with SBP (r = 0.58, P < 0.01). CONCLUSIONS Genetically determined ACE expression in male rats enhances the chronic hypertensive response after the induction of renovascular hypertension. A relationship between circulating Ang II and the development of hypertension was also observed in this experimental model of genetically modulated hypertension.

Despite the major physiologic role of angiotensin-converting enzyme (ACE), few studies have evaluated the ideal conditions for measuring human plasma ACE activity, specifically when using Z-phenylalanine-histidyl-leucine as substrate. This study, performed in volunteer patients, assessed the reproducibility of human plasma ACE activity measured by fluorimetry with Z-phenyl-histidyl-leucine as the substrate. After blood centrifugation, plasma was stored under different conditions until processing. The following sources of variability were evaluated: (1) the interval to centrifugation of blood after collection, (2) the temperature and (3) safe time for storing the plasma after cold centrifugation, (4) the effect of fasting. Plasma ACE activity was 20.6+/-7.7 U/mL, 20.9+/-8 U/mL, and 20.5+/-7.9 U/mL (n = 25) when samples were centrifuged immediately, after 1 hour of blood sampling, and after 3 hours of blood sampling, respectively (not significant). In plasma kept at -20 degrees C, ACE activity was not different after 1 week (17.4+/-4.3 U/mL) nor after 1 month (17.9+/-4 U/mL), whereas baseline ACE was 16.7+/-4.3 U/mL (n = 10). In plasma stored at -80 degrees C, ACE activity was 15.5+/-5.7 U/mL after 1 month (baseline 15+/-5.3 U/mL; not significant; n = 12). No evidence for hydrolysis of the reaction product of ACE (his-leu dipeptide) was observed in plasma samples kept for 1 month at -20 degrees C or at -80 degrees C (by high-performance liquid chromatography analysis). In plasma obtained before breakfast, ACE activity was 12.8+/-7.1 U/mL, and it was 12.3+/-7.5 U/mL 2 hours afterwards (not significant; n = 12). Thus, to determine human plasma ACE activity by fluorimetry with reliability, with Z-phenylalanine-histidyl-leucine used as a substrate, there is a safe interval of at least 3 hours before blood centrifugation at -4 degrees C. Plasma may be kept at -20 degrees C or at -80 degrees C for at least 4 weeks before final processing. Fasting does not influence its enzymatic activity.

Abstract 2177 Background. Cocaine abuse is associated with an increased risk of cardiac and cerebrovascular events, such as myocardial infarction, sudden cardiac death, and ischemic stroke. The underlying mechanisms leading to these complications are not fully understood although intravascular thrombus formation and accelerated atherosclerosis are prominent findings. We have recently demonstrated that chronic cocaine use is associated with endothelial dysfunction (Sáez et al. Thromb Res 2011; 128: 18), a key event in the onset and progression of atherosclerosis. There is growing evidence that the RhoA/Rho kinase (ROCK) pathway has an important pathophysiological role in vascular endothelial dysfunction. Accordingly, we hypothesized that cocaine use induces activation of RhoA/ROCK pathway. Objectives. The main aim of this work was to investigate the activation of RhoA/ROCK pathway through ex vivo, in vivo and in vitro studies. Methods. Ex vivo studies. We studied 13 cocaine dependent individuals (aged 19–52 years mean age 37 years) who met DSM-IV criteria for cocaine dependence, seeking treatment for cocaine abuse and age, sex-matched healthy controls (aged 20–49 years, mean age 35 years). Samples were obtained at admission, within 72 hours of drug exposure. Endothelial cell damage was determined by enumerating circulating endothelial cells (CECs). Rho-kinase activity was assessed by the levels of phosphorylated to total myosin light chain phosphatase 1 (MYPT1-P/T) in circulating leukocytes. In vivo studies. Adult male Sprague-Dawley rats were randomly assigned to receive either cocaine (30mg/kg, provided by NIDA, USA) or saline intraperitoneally once daily for 21 days. The levels of aortic phosphorylated MYPT1 (phospho-MYPT1) were assessed by western blot in aorta extracts. In vitro experiments. Human umbilical vein endothelial cells (HUVECs) were cultured under standard conditions and supplemented for 5 hours with plasma from chronic cocaine users, normal plasma, cocaine (10μM) or vehicle. After media removal, HUVECs were either lysed for determination of ROCK activity or co-cultured with resting platelets and immunostained for von Willebrand factor (FVW). Platelet adhesion was evaluated by immunofluorescence microsocopy. Experiments were conducted in the presence or absence of ROCK inhibitors, Y-27632 (10 μM) or atorvastatin (10μM). Results. Cocaine users showed significantly elevated number of CECs compared to the controls (65 ± 6.6 vs 14 ± 3.4 cells/mL, p: 0.0002). In the control subjects, leukocyte mean MYPT1-P/T ratio was 2.2 ± 0.8 whereas in cocaine addicts were significantly increased (9.8 ± 2.8; p 0.015). ROCK activity was higher by 100% (p: 0.019) in the aortic wall of the cocaine-treated rats compared to sham animals. HUVECs supplemented with plasma from cocaine users showed an increase in ROCK activity by 25% (p: 0.039), released significantly higher amount of FVW (p0.001).ROCK inhibitors, atorvastatin and Y-27632 reduced the release of FVW by HUVECs exposed to plasma from cocaine users by 65% (p: 0.004) and strongly inhibited platelet adhesion (by 75% in plasma-treated cells and by 90% in HUVECs exposed to cocaine, p:< 0.006). Conclusions. We found an increase in Rho kinase activity in peripheral leukocytes of cocaine abusers, in the vessel wall of rats exposed to cocaine and a marked positive effect of ROCK inhibitors on the cellular injury induced by cocaine or plasma from cocaine consumers on endothelial cells. Collectively, these data suggest that activation of RhoA/ROCK pathway plays a key role in cocaine-induced endothelial dysfunction. Inhibition of ROCK may provide therapeutic benefits in a comprehensive treatment for cocaine addiction. Disclosures: No relevant conflicts of interest to declare.