Azorella compacta Organic Extracts Exacerbate Metabolic Dysfunction-Associated Fatty Liver Disease in Mice Fed a High-Fat Diet.

Azorella compacta (A. compacta) is a shrub of the Andean Altiplano of Bolivia, Chile and Peru, consumed by local communities as a traditional medicine for several maladies such as diabetes, hepatic and inflammatory diseases. A. compacta is rich in mulinane- and azorellane-type diterpenoids. For two of these, acute hypoglycemic effects have been described, but the impact of A. compacta diterpenoids on fatty liver disease has not been investigated. Therefore, A. compacta organic fractions were prepared using petroleum ether, dichloromethane and methanol. Their content was characterized by UHPLC/MS, revealing the presence of ten diterpenoids, mainly mulinic acid, azorellanol and mulin-11,13-diene. Next, mice fed with a high-fat diet (HFD), a model of metabolic dysfunction-associated fatty liver disease (MAFLD), received one of the fractions in drinking water for two weeks. After this treatment, hepatic parameters were evaluated. The A. compacta fractions did not reduce hyperglycemia or body weight in the HFD-fed mice but increased the serum levels of hepatic transaminases (AST and ALT), reduced albumin and increased bilirubin, indicating hepatic damage, while histopathological alterations such as steatosis, inflammation and necrosis generated by the HFD were, overall, not ameliorated by the fractions. These results suggest that organic A. compacta extracts may generate hepatic complications in patients with MAFLD.

Tetrahydrobiopterin (BH4) Supplementation Prevents the Cardiorenal Effects of Diabetes in Mice by Reducing Oxidative Stress, Inflammation and Fibrosis.

Background: The effects of diabetes on the cardiovascular system as well as in the kidney are profound, which include hypertrophy and fibrosis. Diabetes also induces oxidative stress, at least in part due to the uncoupling of nitric oxide synthase (NOS); this is a shift in NO production toward superoxide production due to reduced levels of the NOS cofactor tetrahydrobiopterin (BH4). With this in mind, we tested the hypothesis that BH4 supplementation may prevent the development of diabetic cardiomyopathy and nephropathy. Methods: Diabetes was induced in Balb/c mice with streptozotocin. Then, diabetic mice were divided into two groups: one group provided with BH4 (sapropterin) in drinking water (daily doses of 15 mg/kg/day, during eight weeks) and the other that received only water. A third group of normoglycemic mice that received only water were used as the control. Results: Cardiac levels of BH4 were increased in mice treated with BH4 (p = 0.0019). Diabetes induced cardiac hypertrophy, which was prevented in the group that received BH4 (p < 0.05). In addition, hypertrophy was evaluated as cardiomyocyte cross-sectional area. This was reduced in diabetic mice that received BH4 (p = 0.0012). Diabetes induced cardiac interstitial fibrosis that was reduced in mice that received BH4 treatment (p < 0.05). We also evaluated in the kidney the impact of BH4 treatment on glomerular morphology. Diabetes induced glomerular hypertrophy compared with normoglycemic mice and was prevented by BH4 treatment. In addition, diabetic mice presented glomerular fibrosis, which was prevented in mice that received BH4. Conclusions: These results suggest that chronic treatment with BH4 in mice ameliorates the cardiorenal effects of diabetes,, probably by restoring the nitroso−redox balance. This offers a possible new alternative to explore a BH4-based treatment for the organ damage caused by diabetes.

Hydrochlorothiazide Reduces Cardiac Hypertrophy, Fibrosis and Rho-Kinase Activation in DOCA-Salt Induced Hypertension.

BACKGROUND: Thiazides are one of the most common antihypertensive drugs used for hypertension treatment and hydrochlorothiazide (HCTZ) is the most frequently used diuretic for hypertension treatment. The Rho/Rho-kinase (ROCK) path plays a key function in cardiovascular remodeling. We hypothesized that in preclinical hypertension HCTZ reduces myocardial ROCK activation and consequent myocardial remodeling. METHODS: The preclinical model of deoxycorticosterone (DOCA)-salt hypertension was used (Sprague-Dawley male rats). After 3 weeks, in 3 different groups: HCTZ, the ROCK inhibitor fasudil or spironolactone was added (3 weeks). After 6 weeks myocardial hypertrophy and fibrosis, cardiac levels of profibrotic proteins, mRNA levels (RT PCR) of pro remodeling and pro oxidative molecules and ROCK activity were determined. RESULTS: Blood pressure, myocardial hypertrophy and fibrosis were reduced significantly by HCTZ, fasudil and spironolactone. In the heart, increased levels of the pro-fibrotic proteins Col-I, Col-III and TGF-ß1 and gene expression of pro-remodeling molecules TGF-ß1, CTGF, MCP-1 and PAI-1 and the pro-oxidative molecules gp91phox and p22phox were significantly reduced by HCTZ, fasudil and spironolactone. ROCK activity in the myocardium was increased by 54% (P < 0.05) as related to the sham group and HCTZ, spironolactone and fasudil, reduced ROCK activation to control levels. CONCLUSIONS: HCTZ reduced pathologic LVH by controlling blood pressure, hypertrophy and myocardial fibrosis and by decreasing myocardial ROCK activation, expression of pro remodeling, pro fibrotic and pro oxidative genes. In hypertension, the observed effects of HCTZ on the myocardium might explain preventive outcomes of thiazides in hypertension, specifically on LVH regression and incident heart failure.

Angiotensin-(1-9) prevents vascular remodeling by decreasing vascular smooth muscle cell dedifferentiation through a FoxO1-dependent mechanism.

The renin-angiotensin system, one of the main regulators of vascular function, controls vasoconstriction, inflammation and vascular remodeling. Antagonistic actions of the counter-regulatory renin-angiotensin system, which include vasodilation, anti-proliferative, anti-inflammatory and anti-remodeling effects, have also been described. However, little is known about the direct effects of angiotensin-(1-9), a peptide of the counter-regulatory renin-angiotensin system, on vascular smooth muscle cells. Here, we studied the anti-vascular remodeling effects of angiotensin-(1-9), with special focus on the control of vascular smooth muscle cell phenotype. Angiotensin-(1-9) decreased blood pressure and aorta media thickness in spontaneously hypertensive rats. Reduction of media thickness was associated with decreased vascular smooth muscle cell proliferation. In the A7r5 VSMC cell line and in primary cultures of rat aorta smooth muscle cells, angiotensin-(1-9) did not modify basal proliferation. However, angiotensin-(1-9) inhibited proliferation, migration and contractile protein decrease induced by platelet derived growth factor-BB. Moreover, angiotensin-(1-9) reduced Akt and FoxO1 phosphorylation at 30 min, followed by an increase of total FoxO1 protein content. Angiotensin-(1-9) effects were blocked by the AT2R antagonist PD123319, Akt-Myr overexpression and FoxO1 siRNA. These data suggest that angiotensin-(1-9) inhibits vascular smooth muscle cell dedifferentiation by an AT2R/Akt/FoxO1-dependent mechanism.

Effect of Early Normotension with Olmesartan on Rho-kinase Activity in Hypertensive Patients.

BACKGROUND: Angiotensin II is a potent activator of the Rho-kinase (ROCK) pathway, through which it exerts some of its adverse vasoconstrictor effects. Clinical evidence on the effects of blocking the angiotensin II receptor 1 on ROCK activity in hypertensive patients is scarce. OBJECTIVE: To demonstrate that ROCK activity in peripheral blood mononuclear cells (PMBCs) in patients with essential hypertension is reduced earlier than previously observed, along with blood pressure (BP) lowering on treatment with olmesartan. METHODS: Prospective pilot open study; 17 hypertensive patients were treated with progressive olmesartan doses starting with 20 mg qd. BP was measured at 3, 6 and 9 weeks after treatment initiation. If treatment failed to normalize BP after 3 weeks, olmesartan dose was increased to 40 mg qd, and if still hypertensive after 6 weeks, 12.5 mg of hydrochlorothiazide qd was added. ROCK activity was measured at baseline and 9 weeks after treatment as myosin phosphatase target subunit 1 phosphorylation (MYPT1-p/T ratio) in PBMC. RESULTS: Mean baseline BP was 162 ± 4.9/101 ± 2.4 mmHg. After 9 weeks of treatment, both systolic and diastolic BP were reduced by 41 and 22 mmHg, respectively (p<0.05). Mean pretreatment MYPT1- p/T ratio in PMBCs was significantly reduced by 80% after 9 weeks with olmesartan (p<0.01). CONCLUSION: Normotension achieved after 9 weeks in 82% of the patients treated with olmesartan was associated with a significant reduction of ROCK activity in PBMC.

Angiotensin-(1-9) reduces cardiovascular and renal inflammation in experimental renin-independent hypertension.

Hypertension-induced cardiovascular and renal damage can be mediated by activation of the renin-angiotensin-aldosterone system. There are different factors beyond renin-angiotensin-aldosterone system involved in hypertension and renal damage. Inflammation has emerged as an important mediator of hypertension and cardiovascular and kidney damage. Angiotensin-(1-9), a peptide of the renin-angiotensin system, counter-regulates both the physiological and pathological actions of angiotensin II. Recent data has shown that angiotensin-(1-9) protects the heart and blood vessels from adverse cardiovascular remodeling in experimental models of hypertension and/or heart failure and reduces cardiac fibrosis in stroke-prone, spontaneously hypertensive rats. These effects are mediated by the angiotensin II type 2 receptor (AT2R). However, it remains unknown whether angiotensin-(1-9) also has an anti-inflammatory effect. In the present study, we investigate whether angiotensin-(1-9) reduces inflammation and fibrosis in the heart, arteries, and kidney in a DOCA-salt hypertensive model and explore the mechanisms underlying the amelioration of end-organ damage. DOCA-salt hypertensive rats received: a) vehicle, b) angiotensin-(1-9), c) PD123319 (AT2R blocker), d) angiotensin-(1-9) plus A779 (a Mas receptor blocker) or e) angiotensin-(1-9) plus PD123319, and sham rats were used as a control. Our results showed that angiotensin-(1-9) decreased hypertension and increased vasodilation in DOCA-salt hypertensive rats. These actions were partially inhibited by PD123319. Moreover, angiotensin-(1-9) decreased diuresis, fibrosis, and inflammation. These beneficial effects were not mediated by Mas or AT2R blockers. We concluded that angiotensin-(1-9) protects against volume overload-induced hypertensive cardiovascular and kidney damage by decreasing inflammation in the heart, aortic wall, and kidney, through mechanisms independent of the Mas or AT2R.

High-Intensity Exercise Reduces Cardiac Fibrosis and Hypertrophy but Does Not Restore the Nitroso-Redox Imbalance in Diabetic Cardiomyopathy.

Diabetic cardiomyopathy refers to the manifestations in the heart as a result of altered glucose homeostasis, reflected as fibrosis, cellular hypertrophy, increased oxidative stress, and apoptosis, leading to ventricular dysfunction. Since physical exercise has been indicated as cardioprotective, we tested the hypothesis that high-intensity exercise training could reverse the cardiac maladaptations produced by diabetes. For this, diabetes was induced in rats by a single dose of alloxan. Diabetic rats were randomly assigned to a sedentary group or submitted to a program of exercise on a treadmill for 4 weeks at 80% of maximal performance. Another group of normoglycemic rats was used as control. Diabetic rat hearts presented cardiomyocyte hypertrophy and interstitial fibrosis. Chronic exercise reduced both parameters but increased apoptosis. Diabetes increased the myocardial levels of the mRNA and proteins of NADPH oxidases NOX2 and NOX4. These altered levels were not reduced by exercise. Diabetes also increased the level of uncoupled endothelial nitric oxide synthase (eNOS) that was not reversed by exercise. Finally, diabetic rats showed a lower degree of phosphorylated phospholamban and reduced levels of SERCA2 that were not restored by high-intensity exercise. These results suggest that high-intensity chronic exercise was able to reverse remodeling in the diabetic heart but was unable to restore the nitroso-redox imbalance imposed by diabetes.

Diuretics prevent Rho-kinase activation and expression of profibrotic/oxidative genes in the hypertensive aortic wall.

BACKGROUND: Diuretics are current antihypertensive drugs since they reduce blood pressure and cardiovascular risk. Increased vascular tone is modulated in a relevant way by the RhoA/Rho-kinase (ROCK) pathway, by acting on vascular smooth muscle cell contraction. This pathway has also proremodeling vascular effects. There are few data on the role of diuretics on both vascular ROCK activation and on proremodeling effects. We assessed the effects of hydrochlorothiazide (HCTZ) and spironolactone (spiro) alone and in combination with the ROCK inhibitor fasudil (FAS) on ROCK activation, gene expression of proremodeling markers and on hypertrophy in the aortic wall of hypertensive rats. METHODS: Deoxycorticosterone acetate (DOCA)-salt hypertensive rats (male, Sprague-Dawley) were randomized to the specific ROCK inhibitor FAS, HCTZ, spiro or the combinations of FAS/HCTZ or FAS/spiro for 3 weeks. At the end of the study, ROCK activation (by western blot), gene expression of proremodeling markers (by reverse transcription polymerase chain reaction, RT-PCR) and vascular hypertrophy (by morphometry) were determined in the aortic wall. RESULTS: All treatments significantly reduced blood pressure. In the DOCA rats the p-myosin phosphatase target protein-1 (MYPT1)/t-MYPT1 ratio, index of ROCK activation was higher by 2.8 fold (p < 0.05) compared with control rats. All treatments reduced ROCK activation in the aortic wall to control levels (p < 0.05). Besides, significantly increased protein levels of transforming growth factor ß1 (TGF-ß1), gene expression of TGF-ß1, connective tissue growth factor (CTGF), p22 phox and gp91 phox subunits of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, as well as increased media thickness and aortic media area/lumen area (AM/LA) in the untreated hypertensive rats were significantly reduced (p < 0.05) to control levels by all treatments. Similar effects were observed using both diuretics alone or in combination with FAS. CONCLUSIONS: In the aortic wall, both HCTZ and spiro in antihypertensive doses reduce ROCK activation, subsequent expression of genes that promote vascular remodeling and hypertrophy in this experimental model of hypertension. These effects could explain some of their clinical benefits in hypertensive patients.

Simultaneous Rho kinase inhibition in circulating leukocytes and in cardiovascular tissue in rats with high angiotensin converting enzyme levels.

BACKGROUND: The small guanosine triphosphatase RhoA and its direct target Rho kinase (ROCK) play important roles in cardiovascular pathophysiology. Activated ROCK phosphorylates intracellular proteins with detrimental effects on cardiovascular remodeling. Increased ROCK activity in circulating leukocytes is observed in hypertension and in heart failure, but its relationship with ROCK activation in the myocardium and vessels is unknown. We hypothesized that ROCK activation and phosphorylation/activation of some of its key downstream molecules in the heart and arterial wall are reflected in circulating leukocytes. METHODS: Phosphorylation of MYPT1, ERM and p38-MAPK and levels of p65-NF-κB were determined in the left ventricle (LV), aortic wall and circulating leukocytes in rats with high (Brown Norway, BN) and low (Lewis) angiotensin converting enzyme. A group of BN rats received the ROCK inhibitor fasudil (7days). RESULTS: Compared to Lewis rats, in the BN group phosphorylated levels of MYPT1, ERM and p38-MAPK and levels of p65-NF-κB were increased (P<0.05) in the LV (67%, 92%, 52% and 98%, respectively); in the aortic wall (57%, 51%, 68% and 66%, respectively) and in circulating leukocytes (61%, 72%, 49% and 105%, respectively). Fasudil reduced all these levels to those observed in Lewis rats. Phosphorylated MYPT1, ERM, and p38-MAPK and levels of p65-NF-κB in circulating leukocytes were significantly correlated with their respective LV and aortic wall levels (excepting p65-NF-κB in aorta). CONCLUSION: ROCK activity in circulating leukocytes reflects activation of this signaling pathway in the myocardium and aortic wall in this model, and supports its value as a potential cardiovascular remodeling marker.

El efecto anti-hipertensivo de angiotensina-(1-9) es mediado por aumento temprano de la diuresis y natriuresis / Cardiac arrest associated to intramural hematoma of the anterior descending coronary artery

Introducción: Angiotensina (Ang)-(1-9) posee propiedades anti-hipertensivas y efecto protector a nivel cardiovascular en ratas hipertensas. Sin embargo, se desconoce si estos efectos están asociados a un mecanismo de desbalance de sodio a nivel renal. Objetivo: Determinar si el efecto anti-hipertensivo de Ang-(1-9) está asociado a un mecanismo diurético-na-triurético. Método: Ratas macho Sprague Dawley (200 ± 10g) fueron aleatorizadas para recibir Ang II (400 ng/kgmin) vía bomba osmótica. Como control se utilizaron ratas con operación sham (n=18). Después de 2 semanas desde la instalación de bomba, las ratas Sham e hipertensas fueron randomizadas para recibir vehículo (n=10), Ang-(1-9) (602 ng/kg/min, n=17) o una co-administración de Ang-(1-9) y A779 (100 ng kg-1min-1, n=7 bloqueador del receptor MAS) por 2 semanas. Resultados: Se determinó la presión arterial sistólica (PAS), masa ventricular relativa (MVR), área y perímetro de los cardiomiocitos (AC y PC) y la fracción volumétrica de colágeno total (FVCT). Para evaluar la diuresis y natriuresis se utilizaron ratas normotensas que fueron randomizadas para recibir vehículo (n=8) o Ang-(1-9) (600 ngKg-1min-1, n=8) por 6 días. Se observó un incremento significativo(p<0.05) de PAS (33%), MVR (17%), AC (64%), PC (20%), FVCT (46%). La administración crónica de Ang-(1-9) disminuyó PAS (20%), MVR (13 %), AC (35%), PC (20%) y FVCT (20%). Estos efectos no fueron mediados por el receptor MAS. Al comparar las ratas normotensas tratadas con vehículo o Ang-(1-9), se observó un aumento significativo de la diuresis y natriuresis en los días 2 y 3 en los animales con infusión de Ang-(1-9). Conclusión: Ang-(1-9) reduce la hipertensión y el remodelamiento cardíaco en ratas hipertensas. En animales normotensos se demostró que el tratamiento con Ang-(1-9)-induce diuresis y natriuresis. Este es el primer reporte que señala que el efecto de Ang-(1-9) está asociado a una regulación del sodio a nivel renal.

Background: Angiotensin-(1-9) has anti-hypertensive properties and protective cardiovascular effect in hypertensive rats. However, it is unknown whether its effects are related to a kidney mechanism to balance sodium. Aim: To determine if the anti-hypertensive effect of Ang-(1-9) is associated to a diuretic-natriuretic mechanism. Method: Sprague Dawley male rats (200±10 grs) were randomized to receive Angiotensin II by osmotic pump (400 ng/kg/min). Sham operated rats were utilized as control (n=18). Two weeks after pump setting, Sham rats with hypertension were randomized to receive placebo (n=10), Ang-(1-9)(602 ng/kg/min, n=17) or Ang-(1-9) plus A779 (Ang-(1-7) Receptor Mas blocker, 100ng/kg-1min-1, n=7) co-administration for two weeks. Arterial systolic pressure (PAS), ventricular relative mass (MVR), cardiomyocytes area and perimeter (AC and PC) and total collagen volume fraction (FVCT) were measured. Normotensive rats were utilized to evaluate diuresis and natriuresis which were randomized to receive placebo (n=8) or Ang-(1-9) (600ng/kg-1/min-1, n=8) for six days. Results: It was observed a significant rise (p<0.05) of PAS (33%), MVR (17%), AC (64%), PC (26%), FVCT (46%) was observed. Chronic administration of Ang-(1-9) reduced PAS (20%), MVR (13%), AC (35%), PC (20%) and FCVT (20%). All those effects were not mediated by Mas receptor. A significant raise was observed of diuresis and natriuresis at the second and third day of treatment in rats receiving Ang-(1-9) in comparison with normotensive rats treated with placebo. Conclusion: Ang-(1-9) reduces hypertension and cardiac remodeling in hypertensive rats. Ang-(1-9) induces natriuresis and diuresis in normotensive rats. This is the first report showing that Ang-(1-9) is associated to sodium balance in the kidney.

Angiotensina-(1-9) disminuye el daño renal en la hipertensión arterial renina independiente / Angiotensin (1-9) decreases renal damage in hypertension with low renin

Antecedentes: Angiotensina [Ang-(1-9)] disminuye la presión arterial (PA) y el remodel amiento cardíaco en la hipertensión arterial experimental independiente de renina. No hay antecedentes sobre el efecto de Ang-(1-9) en la progresión de daño renal de ratas hipertensas por expansión de volumen (con renina baja). Objetivo: Determinar la participación de Ang-(1-9) en la progresión del daño renal en ratas hipertensas (DOCA-sal). Métodos: 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 presión arterial sistólica (PAS) > 140 mmHg, recibieron vehículo o Ang-(1-9) [602 ng/Kg min] por 2 semanas (minibomba Alzet). Se evaluó la respuesta inflamatoria y el daño renal profibrótico por la presencia de macrófagos infiltrativos y de mio-fibroblastos intersticiales. Se determinó, además, la presión arterial sistólica (PAS), masa corporal (MC), masa del riñón derecho (MR) y masa renal relativa (MRR). Resultados: Se observó una disminución del daño renal en las ratas DOCA-sal, cuando recibieron Ang-(1-9), respecto a aquellas que no la recibieron, evidenciado por una significativa disminución de macrófagos 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) disminuyó la hipertensión y redujo significativamente la infiltración de macrófagos y la aparición de miofibroblastos en el intersticio renal. Estos resultados son la primera evidencia de que Ang-(1-9) reduce la fibrosis túbulo-intersticial renal y el daño renal hipertensivo.

Angiotensin [Ang] - (1-9) decreases blood pressure (BP) and cardiac remodeling in renin independent hypertension. There are not studies about the effect of Ang- (1-9) in the progression of hypertensive renal damage by volume overload (low-renin). The aim of this study was to determine the effect of Ang- (1-9) on renal damage in volume overload hypertensive rats by (DOCA-salt rats). Methods: Male Sprague Dawley rats, 150 +/- 10 g, were uni-nephrectomized and treated with DOCA (60mg/Kg) 2 times per week, for 4 weeks. Uninephrec-tomized rats were used as controls (Sham). From the 2nd week on DOCA rats with systolic blood pressure (SBP)> 140 mmHg received vehicle or Ang- (1-9) [602 ng / kg min] for 2 weeks (Alzet minipump). Inflammatory and profibrotic renal damage was evaluated by the presence of infiltrating macrophages and interstitial myofibroblasts. Results: Compared to rats receiving vehicle renal damage in DOCA-salt rats decreased when they received Ang- (1-9), as evidenced by a significant decrease in infiltrating macrophages and myofibroblasts in the renal interstitium. Mas and AT2 receptor blockade had no additional effect. The SBP, body mass (BM), mass of the right kidney (MR) and relative renal mass (MRR) were also determined. Conclusion: in this experimental model, Ang-(1-9) decreased hypertension and significantly reduced macrophage infiltration and the appearance of myofibroblasts in the renal interstitium. These results are the first evidence that Ang-(1-9) reduces renal tubulointerstitial fibrosis and hypertensive renal damage.

Angiotensin-(1-9) reverses experimental hypertension and cardiovascular damage by inhibition of the angiotensin converting enzyme/Ang II axis.

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 ß - 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.

Increased rho-kinase activity in hypertensive patients with left ventricular hypertrophy.

BACKGROUND: There is experimental evidence on the role of Rho-kinase (ROCK) activation in cardiac hypertrophy but no information on its role in human hypertension and left ventricular hypertrophy (LVH). We hypothesized that ROCK activity is higher in hypertensive patients with LVH compared with hypertensive patients without LVH. METHODS: We conducted a cross-sectional study comparing untreated hypertensive patients with (n = 41) and without LVH (n = 46) determined by echocardiography with a healthy normotensive control group (n = 51). Measurements included LV mass, dimensions, and function and ROCK activity determined in circulating leukocytes by measuring Western blot levels of phosphorylated to total myosin light chain phosphatase 1 (MYPT1-p/t). RESULTS: Compared with normotensive subjects, MYPT1-p/t was significantly increased by 4.5-fold in the hypertensive patients without LVH and by 9-fold in the hypertensive patients with LVH. Compared with the hypertension without LVH group, MYPT1-p/t was significantly increased by 2-fold in the hypertension with LVH gorup. In patients with eccentric LVH, the mean MYPT1-p/t ratio was significantly higher by 4-fold compared with hypertensive patients without eccentric LVH. Patients with an E/e' ratio ≥15 (n = 6) showed a higher MYPT1-p/t ratio (by 26%) compared with patients with a lower E/e' ratio (P ≤ 0.01). CONCLUSIONS: ROCK activity is higher in hypertensive patients with LVH compared with hypertensive patients without LVH, and it is further increased when eccentric LVH is present. Thus, in hypertension, ROCK activation is related to pathological cardiac remodeling and might have a role as an LVH marker.

Angiotensina-(1-9) disminuye el remodelamiento cardiovascular hipertensivo independiente de los niveles de ECA y de angiotensina II / Angiotensin-(1-) reduces hypertensive cardiovascular remodeling independent of ACE and Ang II levels

Resumen: La enzima convertidora de angiotensina I (ECA2) a través de Angiotensina (Ang)-(1-9) más que Ang-(1-7) contrarresta los efectos deletéreos de ECA y Ang II. Se desconoce si Ang-(1-9) es efectiva en el tratamiento del remodelamiento cardiovascular (RMCV) hipertensivo, en ratas con polimorfismo del gen de la ECA. Objetivo: Determinar el efecto de Ang-(1-9) en el tratamiento del RMCV hipertensivo en ratas con niveles genéticamente determinados de ECA y Ang II. Métodos: Ratas normotensas homocigotas, Lewis (LL) y Brown Norway (BN), se les indujo HTA a través del modelo Goldblatt (GB, 2 riñones-1 pinzado). Después de 4 semanas, las ratas hipertensas se rando-mizaron para recibir Ang-(1-9) (602 ng/Kg min) o una coadministración de Ang-(1-9)+A779 (100 ng/Kg min, antagonista del receptor MAS de Ang-(1-7)) durante 14 días mediante una minibomba. Como controles se usaron ratas sometidas a operación ficticia (Sham). Se determinó masa corporal (MC), presión arterial sistólica (PAS), masa ventricular (MV), área de cardiomiocitos (AC), área y grosor de la túnica media (ATM, GTM), fracción volumétrica de colágeno total (FVCT) en el ventrículo izquierdo (VI), niveles proteicos de colágeno tipo I (Col I) en la aorta (Ao) y la infiltración de macrófagos en Ao y VI, por medio de su molécula especifica ED1 (ED1-Ao, ED1-VI). Resultados: La administración de Ang-(1-9) disminuyó significativamente PAS, MV, AC, FVCT, Col I, ATM, GTM, ED1-Ao (-) y ED1-VI, en las ratas hipertensas LL y BN respecto a las ratas GB sin tratamiento, respectivamente. Este efecto no fue inhibido por el antagonista A779. El polimorfismo de la ECA no modificó la respuesta al tratamiento. Conclusión: Ang-(1-9) redujo eficazmente la HTA y el RMCV secundario, independiente al polimorfismo en el gen de la ECA. Este efecto posiblemente es directo ya que no fue mediado por Ang-(1-7). Fondecyt 1100874.

Background: The angiotensin I converting enzyme 2 (ACE2) counteracts the deleterious effects of ACE and Ang II through angiotensin (Ang) -(1-9) rather than Ang-(1-7). In addition, it is not clear whether Ang-(1-9) is effective in the reversal of hypertensive cardiovascular remodeling (CVRM) in rats with ACE gene polymorphism. Objective: To determine the effect of Ang-(1-9) in the prevention of hypertensive CVRM in rats with genetically determined levels of ACE and Ang II. Methods: In normotensive homozygous Lewis (LL) and Brown Norway (BN) rats hypertension was induced by the Goldblatt 2 kidney-1 pinch model. After 4 weeks, rats were randomized to receive Ang- (1-9) (602 ng / Kg min) or the co administration of Ang- (19) + A779 (100 ng / kg min, a MAS receptor antagonist of Ang- (1-7)) for 14 days. Sham operated rats were used as controls. We determined body mass (BM), systolic blood pressure (SBP), ventricular mass (VM), cardiomyocyte area (CA), area and thickness of the aortic media (ATM, TTM), LV total collagen volume fraction (FVCT), type I collagen protein levels (Col I) in the aorta (Ao) and macrophage infiltration in LV and Ao, through its specific molecule ED1 (ED1-Ao, ED1-VI). Results: Continuous administration of Ang- (1-9) significantly decreased SBP, VM, CA, TCVF, Col I, TTM, and ED1 in the aorta and left ventricle of hypertensive rats. This effect was not inhibited by the antagonist A779. ACE polymorphism did not modify the response to treatment. Conclusion: Ang- (1-9) effectively reduced hypertension induced CVRM independent of ACE gene polymorphism. This effect was not mediated by Ang- (1-7).

Mayores niveles de ECA y angiotensina II determinados genéticamente, se asocian a menor actividad del eje ECA2/angiotensina-(1-9) y mayor remodelamiento de la pared aórtica de ratas hipertensas / Increased levels of ACE and angiotensin II genetilaccy determined, are associated with lower ACE2/Angiotensin-(1-9) activity axis and aortic wall increased remodeling in hypertensive rats

Introducción: El polimorfismo del gen de la enzima convertidora de angiotensina I (ECA) determina mayor actividad de la ECA y mayores niveles de angioten-sina (Ang) II. Un polimorfismo similar ha sido descrito en humanos. La ECA2, a través de Ang-(1-9) más que Ang-(1-7), contrarresta los efectos deletéreos de Ang II. Se desconoce si el polimorfismo de la ECA frente a un estímulo hipertensivo modifica el eje ECA2/Ang-(1-9) y determina mayor remodelamiento de la pared aórtica de ratas hipertensas. Objetivo: Determinar el efecto del polimorfismo del gen de la ECA en la actividad del eje ECA2/Ang-(1-9) y su efecto en el remodelamiento de la pared aórtica secundaria a la hipertensión arterial (HTA) experimental. Métodos: Se usaron ratas macho homocigotas de 150 gr BN y LL. Se indujo HTA por el procedimiento Goldblatt (GB, 2 K-1clip). Ratas pseudo-operadas se usaron como controles (Sham). A las 6 semanas post cirugía se determinaron en la aorta las actividades de ECA y ECA2, los niveles de Ang II/Ang-(1-9), colágeno tipo I, células positivas para el marcador de inflamación ED-1, área y grosor de la túnica media (ATM, GTM). Resultados: El polimorfismo de la ECA con mayores niveles de ECA y Ang II determinó una mayor disminución de la actividad de ECA2, menores niveles de Ang-(1-9) y mayor remodelamiento de la pared aórtica tanto en animales normotensos como hipertensos. Conclusión: El polimorfismo de la ECA con mayor actividad de ECA y AngII determina una interregu-lación de los ejes ECA/AngII y ECA2/Ang-(1-9) lo que se asocia a mayor remodelamiento de la pared aórtica. Fondecyt 1100874.

background: The angiotensin converting enzyme (ACE) gene polymorphism determines increased ACE activity and angiotensin (Ang) II levels in Brown Norway rats (BN), compared to Lewis rats (LL). Similar polymorphism has been described in humans. ACE2 through Ang-(1-9) rather than Ang-(1-7) counteracts the deleterious effects of Ang II. It is unknown whether the ACE polymorphism counteracts the ECA2/Ang1-9 axis and determines increased remodeling of the aortic wall in hypertensive rats. Objective: To determine the effects of ACE gene polymorphism in the ECA2/Ang1-9 axis activity and its impact on the aortic wall remodeling secondary to hypertension (HT). Methods: Male homozygous rats BN and LL were used. Hypertension was induced by the Goldblatt procedure (GB, 2 K-1clip). Pseudo-operated rats were used as controls (Sham). At 6 weeks after surgery, we determined the body weight (BW) and systolic blood pressure (SBP). In aorta, we determined the ACE and ACE2 activities, Ang II/Ang1-9 levels, protein expression of collagen type I, positive cells for ED-1 inflammatory cells and medial thickness (MT) and area (MA) of aortic wall. Results: ACE polymorphism with higher levels of ACE and Ang II determined a significant decrease of ACE2 activity, Ang-(1-9) levels and aortic wall remodeling in normotensives and hypertensives rats. Conclusion: ACE polymorphism with increased ACE activity and AngII levels determines a significant inter-regulation between ACE/AngII and ACE2/Ang-(1-9) axis which is associated with increased remodeling of the aortic wall. Fondecyt 1100874.

Markedly increased Rho-kinase activity in circulating leukocytes in patients with chronic heart failure.

BACKGROUND: 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. METHODS: 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). RESULTS: 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 by >100-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 diameter <60 mm, MYPT1-P/T ratio was 35.8 ± 18.1, whereas it was significantly higher in patients with LV diameter ≥60 mm (P < .05). CONCLUSIONS: 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.

Rho kinase inhibition activates the homologous angiotensin-converting enzyme-angiotensin-(1-9) axis in experimental hypertension.

BACKGROUND: Angiotensin II (Ang II) levels depend on renin, angiotensin-converting enzyme (ACE), and on the homologous angiotensin-converting enzyme (ACE2). Increased ACE and Ang II levels are associated with higher Rho kinase activity. However, the relationship between Rho kinase activation and ACE2 in hypertension is unknown. OBJECTIVE: The role of the Rho kinase signaling pathway in both enzymatic activity and aortic gene expression of ACE2 in deoxycorticosterone acetate (DOCA) hypertensive rats was assessed in the present study. METHODS AND RESULTS: Compared with sham animals, Rho kinase activity was higher by 400% (P<0.05) in the aortic wall of the DOCA hypertensive rats. In addition to blood pressure reduction, the specific Rho kinase inhibitor fasudil reduced aortic Rho kinase activity to levels observed in the sham control group and increased ACE2 enzymatic activity (by 83% in plasma and by 52% in the aortic wall, P<0.05), ACE2, and endothelial nitric oxide synthase (eNOS) aortic mRNA levels (by 340 and 40%, respectively, P<0.05) with respect to the untreated hypertensive DOCA rats. Fasudil also increased significantly plasma levels of Ang-(1-9) in normotensive and in the hypertensive rats. Aortic mRNA and protein levels of transforming growth factor-ß1 (TGF-ß1), plasminogen activator inhibitor 1 (PAI-1), and monocyte chemoattractant protein 1 (MCP-1) were significantly (P<0.05) higher in the untreated DOCA rats and were normalized by fasudil administration. CONCLUSION: In experimental hypertension, Rho-associated, coiled-coil containing protein kinase (ROCK) inhibition reduces blood pressure and increases ACE2 levels and activity. At the same time, ROCK inhibition reduces angiotensin II and increases Ang-(1-9) plasma levels. Fasudil also increases vascular eNOS mRNA levels and reduces aortic overexpression of the remodeling promotion proteins TGF-ß1, PAI-1, and MCP-1. This effect might additionally contribute to the antihypertensive and antiremodeling effects of ROCK inhibition in hypertension.

Mayor actividad de rho kinasa en leucocitos circulantes se asocia a estrés oxidativo y rigidez arterial en hipertensos diabéticos / Increased Rho-kinase activity in peripheral leukocytes is associated to oxidative stress and decreased compliance of the arterial wall in diabetic hypertensive patients

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 por ciento 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 por ciento, 50 por ciento y 52 por ciento (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/

Background: Rho/Rho-kinase intracellular pathway is activated by membrane bound small G-proteins. Activation of Rho/Rho-kinase pathway is related to pathologic cardiac remodeling. We have previously observed this activation (ROCK) in hypertensive patients with left ventricular hypertrophy. The influence of diabetes in this relationship has not been explored. Aim: to evaluate the activation of Rho-kinase and oxi-dative stress in hypertensive patients with type II diabetes (DMII). Methods: A comparative study between patients with untreated hypertension (HT), hypertensive patients with diabetes and hemoglobin A1c > 7.5 percent 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 percent 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 percent and 52 percent females, respectively (NS). The MYPT1 p/t ratio was 5,6 +/- 1,3; 3,6 +/- 0,4; 2,1 +/- 0,1 (p<0.01) in the 3 groups, respectively. MDA for the 3 groups was 1,8 +/- 0,4/

Hidroclorotiazida y espironolactona reducen la hipertrofia de la pared aórtica en la hipertensión arterial experimental / Diuretics are important and effective antihypertensive agents: however, there are a few studies assessing its role on the arterial wall remodelling in hypertension

Introducción: Los diuréticos forman parte del tratamiento antihipertensivo actual con efectividad e impacto clínico demostrados. Sin embargo, los efectos de estos fármacos sobre el remodelado de la pared arterial en la hipertensión arterial (HTA) han sido poco evaluados. Objetivos: Determinar y comparar el efecto de Hidroclorotiazida (HCTZ) y de Espironolactona (ESP) en la hipertrofia de la pared aórtica en la HTA experimental. Metodología: 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 administró diuréticos en dos grupos adicionales durante 3 semanas. Uno recibió HCTZ (6 mg/ kg/día) y otro ESP (100 mg/Kg/dia), vía gavage. Al finalizar los tratamientos se determinó la presión arterial sistólica (PAS), masa corporal (MC), peso del corazón (PC) y masa cardiaca relativa (MCR). El grado de hipertrofia de la pared aórtica se determinó midiendo el grosor de la túnica media (GTM), área de la túnica media (ATM), área luminal (AL) y la relación ATM/AL en cortes teñidos con hematoxilina-eosina. Resultados: En las ratas DOCA no tratadas hubo un aumento significativo de PAS (51 por ciento), MCR (79 por ciento), ATM (44 por ciento), GTM (57 por ciento), y de la razón ATM/AL (43 por ciento) respecto al grupo Sham. Ambos tratamientos (Hctz y Esp) redujeron en forma muy importante y significativamente la PAS, MCR, ATM, GTM y la razón ATM/AL en magnitudes similares y también por cada mm de Hg de descenso de la PAS logrado. Conclusión: Además del efecto antihipertensivo, tanto hidroclorotiazida como espironolactona previenen y/o revierten en magnitud similar el desarrollo de hipertrofia de la pared aórtica en este modelo de HTA experimental.

Aims: To determine and compare the effects of hydrochlorotiazide (Hctz) and spironolactone (Esp) on hypertrophy of the aortic wall in experimental hypertension. Methods: This was a comparative study with 4 experimental groups. We used male. uninephrectomized Sprague Dawley rats (150 +/- 10 grs) treated with des-oxycorticosterone acetate (DOCA, 100 mg/Kg/week sbc) during 6 weeks. As controls uninephrectomized rats (Sham) were used. Starting from the third week with DOCA, two groups recived diuretics by gavage during 3 weeks. One group received Hctz (6 mg/kg/ day) and other group received Sp (100 mg/kg/day). At the end of the study, systolic blood pressure (SBP), body weight, heart weight and relative cardiac weight were measured. Hypertrohy in the aortic wall was assessed by measuring the media thickness (MT), media area (MA), lumen area (LA) and by the AM/LA ratio on hematoxyline-eosine stained cross sections. Results: Compared to the Sham group, in the untreated hypertensive DOCA group, SBP and relative cardiac weight increased significantly (by 51 percent and 79 percent, respectively), MA increased by 44 percent, as well as MT (57 percent) and the AM/LA ratio (43 percent). Both treatments (Hctz and Sp) reduced importantly and significantly SBP, relative cardiac mass as well as MT, MA and the AM/LA ratio at a similar extent and by mm Hg. Conclusion: Besides the antihypertensive effect, both hydrochlorotiazide and spironolactone prevent and/or regress aortic wall hypertrophy in this experimental model of hypertension.

Efectividad del uso combinado de un inhibidor de Rho Kinasa y de un antagonista del receptor de angiotensina II en la prevención de hipertrofia ventricular en ratas hipertensas / Effectiveness of the combined use of a Rho-kinase inhibitor and an Angiotensin II receptor antagonist in the prevention of left ventricular hypertrophy in hypertensive rats

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 por ciento (p < 0,05), junto a aumento significativo (p < 0,05) de MYPT-1 fosforilado/total (300 por ciento), de colágeno miocárdico I (en 14 veces), de células ED1 en 270 por ciento y de la expresión génica de la subunidad gp91 de NADPH...

Background: The effect of cardiac Rho-kinase (ROCK) on hypertension (HT) and cardiac hypertrophy prevention and also the combined anti-hypertensive treatment have been scarcely studied. We hypothesized that the addition of a ROCK inhibitor to conventional anti-hypertensive treatment may have additional beneficial effects. Ainv to determine ventricular ROCK activity and ventricular remodeling in hypertensive rats treated with Angiotensin II inhibition with the addition of a ROCK inhibitor. Methods: Sprague-Dawley rats weighing 150 grams had one kidney removed and received deoxycortisterone acétate (DOCA, 100 mg/kg/week, during 6 weeks). Unilaterally nephrectomized rats were used as controls. The other 3 groups received DOCA along with the Angiotensin II receptor blocker candesartan (10 mg/kg/day) or the combination of both agents (5 and 25 mg/kg/day, respectively) and ROCK inhibitor fasudil (50 mg/kg/day) for 3 weeks starting 3 weeks after surgery. Body mass (BM), systolic blood pressure (SBP) and relative cardiac mass (RCM) were measured. In addition, myosin phosphatase (MYPT-1) phosphorylation was measured as an indicator of ROCK activation. Cardiac infiltration of macrophages/monocytes (ED1 positive cells), collagen I protein contení (by Western Blot) and also cardiac gene expression of NADPH oxydase GP91 subunit and eNOS were determined by RT-PCR. Results: In hypertensive rats we observed cardiac hypertrophy by 63 percent (p < 0.05), a 300 percent increase in cardiac MYPT-1 phosphorylation (p< 0.05), 14 times increase in myocardial collagen type 1,270 percent increase in ED1 cells, a 75 percent increased gene expression of NADPH oxydase GP91 subunit and a 37 percent reduction (p< 0.05) in the gene expression of cardiac eNOS. In hypertensive DOCA rats treated during 3 week with candesartan, fasudil or the combination of both, we observed a significant reduction in cardiac hypertrophy and normalization of SBP, MYPT-1 phosphorylation, ...