Caloric restriction ameliorates angiotensin II-induced mitochondrial remodeling and cardiac hypertrophy.

Angiotensin II-induced cardiac damage is associated with oxidative stress-dependent mitochondrial dysfunction. Caloric restriction (CR), a dietary regimen that increases mitochondrial activity and cellular stress resistance, could provide protection. We tested that hypothesis in double transgenic rats harboring human renin and angiotensinogen genes (dTGRs). CR (60% of energy intake for 4 weeks) decreased mortality in dTGRs. CR ameliorated angiotensin II-induced cardiomyocyte hypertrophy, vascular inflammation, cardiac damage and fibrosis, cardiomyocyte apoptosis, and cardiac atrial natriuretic peptide mRNA overexpression. The effects were blood pressure independent and were linked to increased endoplasmic reticulum stress, autophagy, serum adiponectin level, and 5' AMP-activated protein kinase phosphorylation. CR decreased cardiac p38 phosphorylation, nitrotyrosine expression, and serum insulin-like growth factor 1 levels. Mitochondria from dTGR hearts showed clustered mitochondrial patterns, decreased numbers, and volume fractions but increased trans-sectional areas. All of these effects were reduced in CR dTGRs. Mitochondrial proteomic profiling identified 43 dTGR proteins and 42 Sprague-Dawley proteins, of which 29 proteins were in common in response to CR. We identified 7 proteins in CR dTGRs that were not found in control dTGRs. In contrast, 6 mitochondrial proteins were identified from dTGRs that were not detected in any other group. Gene ontology annotations with the Panther protein classification system revealed downregulation of cytoskeletal proteins and enzyme modulators and upregulation of oxidoreductase activity in dTGRs. CR provides powerful, blood pressure-independent, protection against angiotensin II-induced mitochondrial remodeling and cardiac hypertrophy. The findings support the notion of modulating cardiac bioenergetics to ameliorate angiotensin II-induced cardiovascular complications.

Effects of levosimendan on cardiac gene expression profile and post-infarct cardiac remodelling in diabetic Goto-Kakizaki rats.

The calcium sensitizer levosimendan has shown beneficial effects on cardiac remodelling in spontaneously diabetic Goto-Kakizaki (GK) rats 12 weeks after experimental myocardial infarction (MI). However, the short-term effects and the cellular mechanisms remain partially unresolved. The aim was to study the effects of oral levosimendan treatment on the myocardial gene expression profile in diabetic GK rats 4 weeks after MI/sham operation. MI was induced to diabetic GK rats. Twenty-four hours after surgery, rats were randomized into four groups: MI, MI +levosimendan (1 mg/kg/day), sham-operated and sham-operated +levosimendan. Cardiac function and histology were examined 1, 4 and 12 weeks after MI. The effects of levosimendan on cardiac gene expression profile were investigated by microarray analysis. Levosimendan ameliorated post-infarct heart failure and cardiac remodelling. Levosimendan altered the expression of 264 of MI and sham rats, respectively; these changes were associated with alterations in two Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Levosimendan up-regulated 3 genes in the renin-angiotensin system pathway [angiotensin receptor 1 (Agtr1), chymase 1 (Cma1) and thimet oligopeptidase 1 (Thop1)] and down-regulated 3 genes in the glycerolipid metabolism pathway [diacylglycerol kinase gamma (Dgkg), carboxyl ester lipase (Cel) and Diacylglycerol kinase iota]. Levosimendan induced opposite effects on the gene expression of pleckstrin homology (PH) domain containing family f (Plekhf1), carboxymethylenebutenolidase homologue (Cmbl) (up-regulation) and hydroxyprostaglandin dehydrogenase 15 (Hpgd) (down-regulation) as compared with MI. MI versus sham affected 420 genes and was associated with alterations in 12 KEGG pathways. The beneficial effects of levosimendan on cardiac hypertrophy in sham-operated GK rats was associated with altered expression in 522 genes and associated with three KEGG pathways including purine metabolism, cell cycle pathway and pathways in cancer. Levosimendan protects against post-infarct heart failure and cardiac remodelling. Analysis of the cardiac transcriptome revealed several genes that are regulated by levosimendan. These genes may represent novel drug targets for heart failure and diabetic cardiomyopathy.

Skeletal muscle gene expression profile is modified by dietary protein source and calcium during energy restriction.

BACKGROUND/AIMS: The potential of whey protein and calcium to modify skeletal muscle gene expression during energy restriction (ER) was investigated in a model of diet-induced obesity. METHODS: Obese C57BL/6J mice received casein (calcium 0.4%) and two different high-calcium (1.8%) whey protein-based [whey protein isolate (WPI)+Ca and α-lactalbumin+Ca] diets for ER. RESULTS: Compared to casein, WPI and α-lactalbumin-based diets altered 208 and 287 genes, respectively, of which 186 genes were common to WPI and α-lactalbumin diets. These genes represented 31 KEGG pathways. The Wnt signaling was the most enriched pathway among the 101 genes regulated by α-lactalbumin only, whereas the 22 genes regulated by WPI only were not associated with KEGG pathways. Unlike casein, WPI and α-lactalbumin diets decreased Aldh1a7, Fasn, leptin, Nr4a3 and Scd1 mRNA expression, indicating dietary protein source-dependent alterations in muscle lipid and fatty acid metabolism. Muscle weight or lean body mass maintenance did not differ between groups although modest changes in hypertrophy/atrophy signaling were found. CONCLUSION: The skeletal muscle gene expression profile is modified by the dietary protein source and calcium during ER which may explain, at least in part, the greater anti-obesity effect of whey proteins and calcium compared to casein.

Levosimendan improves cardiac function and survival in rats with angiotensin II-induced hypertensive heart failure.

Calcium-sensitizing agents improve cardiac function in acute heart failure; however, their long-term effects on cardiovascular mortality are unknown. We tested the hypothesis that levosimendan, an inodilator that acts through calcium sensitization, opening of ATP-dependent potassium channels and phosphodiesterase III inhibition, improves cardiac function and survival in double transgenic rats harboring human renin and angiotensinogen genes (dTGRs), a model of angiotensin II (Ang II)-induced hypertensive heart failure. Levosimendan (1 mg kg(-1)) was administered orally to 4-week-old dTGRs and normotensive Sprague-Dawley rats for 4 weeks. Untreated dTGRs developed severe hypertension, cardiac hypertrophy, heart failure with impaired diastolic relaxation, and exhibited a high mortality rate at the age of 8 weeks. Levosimendan did not decrease blood pressure and did not prevent cardiac hypertrophy. However, levosimendan improved systolic function, decreased cardiac atrial natriuretic peptide mRNA expression, ameliorated Ang II-induced cardiac damage and decreased mortality. Levosimendan did not correct Ang II-induced diastolic dysfunction and did not influence heart rate. In a separate survival study, levosimendan increased dTGR survival by 58% and median survival time by 27% (P=0.004). Our findings suggest that levosimendan ameliorates Ang II-induced hypertensive heart failure and reduces mortality. The results also support the notion that the effects of levosimendan in dTGRs are mediated by blood pressure-independent mechanisms and include improved systolic function and amelioration of Ang II-induced coronary and cardiomyocyte damage.

Resveratrol induces mitochondrial biogenesis and ameliorates Ang II-induced cardiac remodeling in transgenic rats harboring human renin and angiotensinogen genes.

There is compelling evidence to indicate an important role for increased local renin-angiotensin system activity in the pathogenesis of cardiac hypertrophy and heart failure. Resveratrol is a natural polyphenol that activates SIRT1, a novel cardioprotective and longevity factor having NAD(+)-dependent histone deacetylase activity. We tested the hypothesis whether resveratrol could prevent from angiotensin II (Ang II)-induced cardiovascular damage. Four-week-old double transgenic rats harboring human renin and human angiotensinogen genes (dTGR) were treated for 4 weeks either with SIRT1 activator resveratrol or SIRT1 inhibitor nicotinamide. Untreated dTGR and their normotensive Sprague-Dawley control rats (SD) received vehicle. Untreated dTGR developed severe hypertension as well as cardiac hypertrophy, and showed pronounced cardiovascular mortality compared with normotensive SD rats. Resveratrol slightly but significantly decreased blood pressure, ameliorated cardiac hypertrophy and prevented completely Ang II-induced mortality, whereas nicotinamide increased blood pressure without significantly influencing cardiac hypertrophy or survival. Resveratrol decreased cardiac ANP mRNA expression and induced cardiac mRNA expressions of mitochondrial biogenesis markers peroxisome proliferator-activated receptor-gamma coactivator (PGC-1alpha), mitochondrial transcription factor (Tfam), nuclear respiratory factor 1 (NRF-1) and cytochrome c oxidase subunit 4 (cox4). Resveratrol dose-dependently increased SIRT1 activity in vitro. Our findings suggest that the beneficial effects of SIRT1 activator resveratrol on Ang II-induced cardiac remodeling are mediated by blood pressure-dependent pathways and are linked to increased mitochondrial biogenesis.

Effects of the calcium sensitizer OR-1896, a metabolite of levosimendan, on post-infarct heart failure and cardiac remodelling in diabetic Goto-Kakizaki rats.

BACKGROUND AND PURPOSE: Levosimendan is a novel, short half-life calcium sensitizer used as pharmacological inotropic support in acute decompensated heart failure. After oral administration, levosimendan is metabolized to OR-1855, which, in rats, is further metabolized into OR-1896. OR-1896 is a long-lasting metabolite of levosimendan sharing the pharmacological properties of the parent compound. EXPERIMENTAL APPROACH: Effects of oral OR-1896 treatment on post-infarct heart failure and cardiac remodelling were assessed in diabetic Goto-Kakizaki (GK) rats, an animal model of type II diabetes. Myocardial infarction (MI) was produced to GK rats by coronary ligation. Twenty-four hours after MI or sham operation, the rats were randomized into four groups: (i) MI; (ii) MI + OR-1896 treatment; (iii) sham; and (iv) sham + OR-1896. Cardiac function and markers of cardiac remodelling were assessed 1, 4 and 12 weeks after MI. KEY RESULTS: OR-1896 increased ejection fraction and fractional shortening in GK rats with MI. OR-1896 ameliorated post-infarct cardiac hypertrophy, and prevented the MI-induced increase in cardiac mRNA for atrial natriuretic peptide, monocyte chemoattractant protein-1 and connective tissue growth factor, markers of pressure/volume overload, inflammation and fibrosis respectively. OR-1896 also suppressed mRNA for senescence-associated p16(INK4A) and p19(ARF). The beneficial effects of OR-1896 were more prominent at week 12 than at week 4. OR-1896 did not influence systolic blood pressure, blood glucose level, myocardial infarct size or cardiovascular mortality. CONCLUSIONS AND IMPLICATIONS: Oral treatment with calcium sensitizer OR-1896 protects against post-infarct heart failure and cardiac remodelling in experimental model of type II diabetes.

Sirtuin1-p53, forkhead box O3a, p38 and post-infarct cardiac remodeling in the spontaneously diabetic Goto-Kakizaki rat.

BACKGROUND: Diabetes is associated with changes in myocardial stress-response pathways and is recognized as an independent risk factor for cardiac remodeling. Using spontaneously diabetic Goto Kakizaki rats as a model of type 2 DM we investigated whether post-translational modifications in the Akt - FOXO3a pathway, Sirt1 - p53 pathway and the mitogen activated protein kinase p38 regulator are involved in post-infarct cardiac remodeling METHODS: Experimental myocardial infarction (MI) was induced by left anterior descending coronary artery ligation in spontaneously diabetic Goto-Kakizaki rats and non-diabetic Wistar controls. Cardiac function was studied by echocardiography. Myocardial hypertrophy, cardiomyocyte apoptosis and cardiac fibrosis were determined histologically 12 weeks post MI or Sham operation. Western blotting was used to study Caspase-3, Bax, Sirt1, acetylation of p53 and phosphorylation of p38, Akt and FOXO3a. Electrophoretic mobility shift assay was used to assess FOXO3a activity and its nuclear localization. RESULTS: Post-infarct heart failure in diabetic GK rats was associated with pronounced cardiomyocyte hypertrophy, increased interstitial fibrosis and sustained cardiomyocyte apoptosis as compared with their non-diabetic Wistar controls. In the GK rat myocardium, Akt- and FOXO3a-phosphorylation was decreased and nuclear localization of FOXO3a was increased concomitantly with increased PTEN protein expression. Furthermore, increased Sirt1 protein expression was associated with decreased p53 acetylation, and phosphorylation of p38 was increased in diabetic rats with MI. CONCLUSIONS: Post-infarct heart failure in diabetic GK rats was associated with more pronounced cardiac hypertrophy, interstitial fibrosis and sustained cardiomyocyte apoptosis as compared to their non-diabetic controls. The present study suggests important roles for Akt-FOXO3a, Sirt1 - p53 and p38 MAPK in the regulation of post-infarct cardiac remodeling in type 2 diabetes.

Metabolomics in angiotensin II-induced cardiac hypertrophy.

Angiotensin II (Ang II) induces mitochondrial dysfunction. We tested whether Ang II alters the "metabolomic" profile. We harvested hearts from 8-week-old double transgenic rats harboring human renin and angiotensinogen genes (dTGRs) and controls (Sprague-Dawley), all with or without Ang II type 1 receptor (valsartan) blockade. We used gas chromatography coupled with time-of-flight mass spectrometry to detect 247 intermediary metabolites. We used a partial least-squares discriminate analysis and identified 112 metabolites that differed significantly after corrections (false discovery rate q <0.05). We found great differences in the use of fatty acids as an energy source, namely, decreased levels of octanoic, oleic, and linoleic acids in dTGR (all P<0.01). The increase in cardiac hypoxanthine levels in dTGRs suggested an increase in purine degradation, whereas other changes supported an increased ketogenic amino acid tyrosine level, causing energy production failure. The metabolomic profile of valsartan-treated dTGRs more closely resembled Sprague-Dawley rats than untreated dTGRs. Mitochondrial respiratory chain activity of cytochrome C oxidase was decreased in dTGRs, whereas complex I and complex II were unaltered. Mitochondria from dTGR hearts showed morphological alterations suggesting increased mitochondrial fusion. Cardiac expression of the redox-sensitive and the cardioprotective metabolic sensor sirtuin 1 was increased in dTGRs. Interestingly, valsartan changed the level of 33 metabolites and induced mitochondrial biogenesis in Sprague-Dawley rats. Thus, distinct patterns of cardiac substrate use in Ang II-induced cardiac hypertrophy are associated with mitochondrial dysfunction. The finding underscores the importance of Ang II in the regulation of mitochondrial biogenesis and cardiac metabolomics, even in healthy hearts.

Oral levosimendan prevents postinfarct heart failure and cardiac remodeling in diabetic Goto-Kakizaki rats.

BACKGROUND: Diabetes increases the risk for fatal myocardial infarction and development of heart failure. Levosimendan, an inodilator acting both via calcium sensitization and opening of ATP-dependent potassium channels, is used intravenously for acute decompensated heart failure. The long-term effects of oral levosimendan on postinfarct heart failure are largely unknown. OBJECTIVE: To examine whether oral treatment with levosimendan could improve cardiac functions and prevent cardiac remodeling after myocardial infarction in a rodent model of type 2 diabetes, the Goto-Kakizaki rat. METHODS: Myocardial infarction (MI) was induced to diabetic Goto-Kakizaki and nondiabetic Wistar rats by coronary ligation. Twenty-four hours after surgery, Goto-Kakizaki and Wistar rats were randomized into four groups: MI group without treatment, MI group with levosimendan for 12 weeks (1 mg/kg per day), sham-operated group, sham-operated group with levosimendan. Blood pressure, cardiac functions as wells as markers of cardiac remodeling were determined. RESULTS: In Goto-Kakizaki rats, MI induced systolic heart failure, pronounced cardiac hypertrophy in the remote area, and sustained cardiomyocyte apoptosis. Postinfarct cardiac remodeling was associated with increased atrial natriuretic peptide, interleukin-6 and connective tissue growth factor mRNA expressions, as well as three-fold increased cardiomyocyte senescence, measured as cardiac p16 mRNA expression. Levosimendan improved cardiac function and prevented postinfarct cardiomyocyte hypertrophy, cardiomyocyte apoptosis, and cellular senescence. Levosimendan also ameliorated MI-induced atrial natriuretic peptide, IL-6, and connective tissue growth factor overexpression as well as MI-induced disturbances in calcium-handling proteins (SERCA2, Na-Ca exchanger) without changes in diabetic status or systemic blood pressure. In nondiabetic Wistar rats, MI induced systolic heart failure; however, the postinfarct cardiac remodeling was associated with less pronounced cardiac hypertrophy, cardiomyocyte apoptosis, inflammatory reaction, and induction of cellular senescence. Levosimendan only partially prevented postinfarct heart failure and cardiac remodeling in Wistar rats. CONCLUSION: Our findings suggest a therapeutic role for oral levosimendan in prevention of postinfarct heart failure and cardiac remodeling in type 2 diabetes and underscore the importance of sustained cardiomyocyte apoptosis and induction of cellular senescence in the pathogenesis.

Forkhead class O transcription factor 3a activation and Sirtuin1 overexpression in the hypertrophied myocardium of the diabetic Goto-Kakizaki rat.

BACKGROUND: Ventricular remodeling in type 2 diabetes predisposes to fatal coronary heart disease. The proapoptotic forkhead class O transcription factor 3a (FOXO3a) and its modulator, the cardioprotective longevity factor and class III histone deacetylase Sirtuin1 (Sirt1), have been implicated in the regulation of the cardiomyocyte lifespan and hypertrophy. OBJECTIVE: To examine whether FOXO3a-Sirt1 activation is involved in diabetes-induced cardiomyocyte apoptosis and ventricular hypertrophy. METHODS: The blood pressure, cardiac functions, cardiomyocyte size, neurohumoral markers, cardiomyocyte apoptosis, nuclear binding of FOXO3a, and Sirt1 expression were determined for 12-week-old spontaneously diabetic Goto-Kakizaki rats and the nondiabetic Wistar control rats. RESULTS: Goto-Kakizaki rats showed a modest increase in blood pressure, pronounced cardiac hypertrophy, impaired systolic function, and increased plasma brain natriuretic peptide level without changes in plasma renin activity, serum aldosterone or urinary noradrenaline excretion. The cardiomyocyte cross-sectional area was increased by 22%. Phosphorylation of FOXO3a was decreased with a concomitant increase in its nuclear translocation. The myocardial expression of the antiapoptotic FOXO3a modulator Sirt1 was increased two-fold. Acetylation of p53 at the Sirt1-specific lysine 373/382 site was markedly decreased. Myocardial caspase-3 and Bax expression were increased, indicating increased apoptotic signaling; however, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling staining did not reveal any significant increase in cardiomyocyte apoptosis. CONCLUSIONS: Diabetes-induced cardiac remodeling in Goto-Kakizaki rats is associated with cardiac hypertrophy, systolic dysfunction, increased apoptotic signaling and activation of the FOXO3a pathway. The present study also suggests that antiapoptotic Sirt1 protects against cardiomyocyte apoptosis and acts as a novel regulator of cardiomyocyte growth.

Effect of dietary calcium and dairy proteins on the adipose tissue gene expression profile in diet-induced obesity.

BACKGROUND/AIMS: Calcium and dairy proteins have been postulated to explain why the intake of dairy products correlates inversely with body mass index in several populations. We have shown that a high-calcium diet with whey protein attenuates weight gain and now we describe the effects of this diet on adipose tissue gene expression. METHODS: Nine-week-old C57Bl/6J mice were divided into two groups (n = 10/group). The control diet was a standard high-fat diet (60% of energy) low in calcium (0.4%). The whey protein diet was a high-calcium (1.8%), high-fat diet with whey protein. After the 21-week treatment, adipose tissue transcript profiling (2 mice/group) was performed using Affymetrix Mouse Genome 430 2.0. RESULTS: The high-calcium diet with whey protein altered the expression of 129 genes (+/- 1.2 fold). Quantitative RT-PCR analysis confirmed the significant up-regulation of Adrb3 (p = 0.002) and leptin (p = 0.0019) in the high-calcium whey group. Insulin and adipocytokine signaling pathways were enriched among the up-regulated genes and the fatty acid metabolism pathway among the down-regulated genes. CONCLUSIONS: High-calcium diet with whey protein significantly modifies adipose tissue gene expression. These preliminary findings reveal that targets of a high-calcium diet with whey protein include genes for Adrb3 and leptin, and help to explain how the intake of dairy products might attenuate obesity.

Lipoic acid supplementation prevents cyclosporine-induced hypertension and nephrotoxicity in spontaneously hypertensive rats.

BACKGROUND: Cyclosporine (CsA) has significantly improved long-term survival after organ transplantations. Hypertension and nephrotoxicity are common side effects during CsA treatment and are aggravated by high salt intake. OBJECTIVE: To examine whether lipoic acid (LA), a natural antioxidant that scavenges reactive oxygen species and regenerates/recycles endogenous antioxidants, could prevent CsA-induced hypertension and nephrotoxicity. METHODS: Six-week-old spontaneously hypertensive rats (SHR) on a high-sodium diet (NaCl 6%) received CsA [5 mg/kg subcutaneously (s.c.)] alone or in combination with LA (0.5% w/w) for 6 weeks. Blood pressure, arterial functions, and tissue morphology were determined. Immunohistochemistry, quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and high-pressure liquid chromatography were used for kidney and heart samples. RESULTS: CsA induced severe hypertension, cardiac hypertrophy, endothelial dysfunction, and pronounced albuminuria. Histologically, the kidneys showed severe thickening of the media of the afferent arteries with fibrinoid necrosis, perivascular monocyte/macrophage infiltration and nitrotyrosine overexpression. CsA induced the expression of fibrogenic connective tissue growth factor both in the heart and kidneys. The detrimental effects of CsA were associated with upregulation of myocardial atrial natriuretic peptide (ANP) mRNA expression, paradoxical activation of the renin-angiotensin system (RAS), induction of renal reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase, and overexpression of oxidative stress-induced transcription factor NRF2. LA lowered blood pressure, ameliorated cardiac hypertrophy and endothelial dysfunction, and totally normalized albuminuria. In LA-treated rats, renal and cardiac morphologies were indistinguishable from those of SHR controls. CsA-induced myocardial ANP and connective tissue growth factor (CTGF) mRNA overexpression, RAS activation, NADPH oxidase induction, and NRF2 overexpression were prevented by LA. LA induced the mRNA expression of gamma-glutamylcysteine ligase, the rate-limiting enzyme in glutathione synthesis, and markedly increased hepatic cysteine and glutathione concentrations. CONCLUSIONS: Our findings suggest a salutary role for lipoic acid supplementation in the prevention of CsA-induced hypertension and nephrotoxicity, and underscore the importance of increased oxidative stress in the pathogenesis of CsA toxicity.

Vascular and renal effects of vasopeptidase inhibition and angiotensin-converting enzyme blockade in spontaneously diabetic Goto-Kakizaki rats.

BACKGROUND: The renin-angiotensin system plays an important role in the pathogenesis of diabetes-induced vascular and renal complications. Vasopeptidase inhibitors simultaneously inhibit angiotensin-converting enzyme (ACE) and neutral endopeptidase. OBJECTIVE: To compare the effectiveness of vasopeptidase inhibition and ACE inhibition in preventing hypertension, endothelial dysfunction and diabetic nephropathy in spontaneously diabetic Goto-Kakizaki (GK) rats. METHODS: Eight-week-old GK rats received omapatrilat (40 mg/kg) or enalapril (30 mg/kg) for 12 weeks, either during a normal-sodium or high-sodium diet (7% w/w). Blood pressure, arterial functions and renal morphology were determined. RESULTS: Blood pressure and albuminuria were increased in GK rats compared to non-diabetic Wistar controls. Endothelium-dependent vascular relaxation in response to acetylcholine (ACh) and endothelium-independent vascular relaxation in response to sodium nitroprusside (SNP) were impaired in GK rats. Experiments with N-nitro-L-arginine methyl ester (L-NAME), diclofenac, and L-NAME + diclofenac suggested that cyclooxygenase and endothelium-derived hyperpolarizing factor components of endothelium-dependent vascular relaxation were also impaired. A high-sodium diet aggravated hypertension and diabetes-induced vascular and renal complications. Omapatrilat and enalapril normalized blood pressure and albuminuria during the normal-sodium diet, and effectively ameliorated diabetes-induced renal complications also during the high-sodium diet. However, omapatrilat improved endothelium-dependent relaxation to ACh to a greater extent (85 +/- 5%) than enalapril (68 +/- 6%, P < 0.05). Diclofenac pre-incubation eliminated this difference between omapatrilat and enalapril in ACh-induced vascular relaxation, suggesting that it was mediated, at least in part, via the cyclooxygenase pathway. CONCLUSIONS: Despite comparable blood pressure-lowering and renoprotective properties, omapatrilat may be more effective in preventing vascular dysfunction during diabetes compared to enalapril in GK rats.

Magnesium supplementation prevents angiotensin II-induced myocardial damage and CTGF overexpression.

OBJECTIVES AND DESIGN: Magnesium deficiency promotes vasoconstriction and myocardial damage. Recent studies provide evidence that Ang II mobilizes intracellular Mg through AT1 receptor-mediated pathways. We tested the hypothesis of whether magnesium supplementation prevents Ang II-induced myocardial damage and induction of the profibrotic connective tissue growth factor (CTGF). METHODS: Four-week-old double transgenic rats harboring human renin and angiotensinogen genes (dTGR) were given dietary magnesium supplementation (0.6%) for 3 weeks. Control dTGR and normotensive Sprague-Dawley (SD) rats received normal diet (Mg 0.2%). Histopathological, immunohistochemical and mRNA analysis were used to detect the treatment-related effects of dietary magnesium in dTGR. RESULTS: Magnesium (Mg) supplementation decreased blood pressure, ameliorated cardiac hypertrophy, protected against the development of Ang II-induced myocardial damage and increased serum ionized Mg2+ concentration (all variables P < 0.05). There was no difference in serum ionized Mg2+ concentration between dTGR and SD rats. Myocardial connective tissue growth factor (CTGF) mRNA and protein expressions were increased by 300% in dTGR (P < 0.05), especially in areas with myocardial infarctions and vascular inflammation. Magnesium supplementation prevented Ang II-induced myocardial CTGF overexpression (P < 0.05). Magnesium supplementation also improved the therapeutic effects of the calcineurin inhibitor tacrolimus, which produced marked hypomagnesemia when given as monotherapy. CONCLUSION: Our findings suggest a salutary effect for magnesium supplementation in the treatment of Ang II-induced myocardial complications.

High sodium intake increases vascular superoxide formation and promotes atherosclerosis in apolipoprotein E-deficient mice.

Hypertension is a major risk factor for atherosclerosis. We tested the hypothesis whether high salt intake aggravates endothelial dysfunction and promotes atherosclerosis in apolipoprotein E-deficient mice (ApoE(-)/(-) mice) and their littermate controls (C57Bl/6 mice). The role of increased oxidative stress was also examined. A high-salt diet (NaCl 7%) for 12 weeks increased blood pressure and induced cardiac hypertrophy and albuminuria more pronouncedly in ApoE(-)/(-) mice compared with C57Bl/6. Endothelium-dependent vascular relaxation in response to acetylcholine was almost maximally impaired in ApoE(-)/(-) mice during a normal sodium diet. A high-salt diet did not further impair NO-mediated vascular relaxation. A high-salt diet also markedly attenuated endothelium-dependent relaxation in C57Bl/6 mice. Preincubation with the superoxide scavenger Tiron normalized endothelial function almost completely in both mice strains indicating the central role of increased oxidative stress in the pathogenesis. Aortic superoxide production and the extent of atherosclerotic lesions were greater in ApoE(-)/(-) mice on a normal-salt diet compared with C57Bl/6. The high-salt diet increased vascular superoxide formation and promoted atherosclerosis in ApoE(-)/(-) mice. Changes in dietary salt intake did not influence serum lipids in either mouse strains. Our findings suggest a detrimental role for high salt intake in the development of atherosclerosis and underscore the importance of increased oxidative stress in the pathogenesis salt-induced vascular damage.

Entacapone protects from angiotensin II-induced inflammation and renal injury.

OBJECTIVES AND DESIGN: Angiotensin II (Ang II)-induced renal damage is associated with perivascular inflammation and increased oxidative stress. We tested the hypothesis whether entacapone, a catechol-O-methyltransferase (COMT) inhibitor exerting antioxidative and anti-inflammatory properties, protects against the Ang II-induced inflammatory response and end-organ damage. METHODS: Samples from double-transgenic rats harbouring human renin and human angiotensinogen genes (dTGR) and normotensive Sprague-Dawley rats (SD) were assessed by light microscopy, immunohistochemistry, reverse transcriptase-polymerase chain reaction (RT-PCR), and high pressure liquid chromatography. The effects of entacapone treatment for 3 weeks were examined in dTGR and SD. RESULTS: Entacapone completely prevented cardiovascular mortality and decreased albuminuria by 85% in dTGR. Entacapone ameliorated Ang II-induced vascular and glomerular damage, leucocyte infiltration, and intercellular adhesion molecule-1 (ICAM-1) overexpression in the kidneys. Serum 8-isoprostane concentration, as well as renal nitrotyrosine and 8-hydroxydeoxyguanosine expressions, all markers of oxidative stress, were markedly increased in dTGR and normalized by entacapone. Entacapone also decreased p22phox mRNA expression in the kidney. COMT expression was increased by 500% locally in the renal vascular wall in dTGR; however, COMT activity in the whole kidney remained unchanged. Urinary dopamine excretion, a marker of renal dopaminergic tone, was decreased by 50% in untreated dTGR. Even though entacapone decreased renal COMT activity by 40%, the renal dopaminergic tone remained unchanged in entacapone-treated dTGR. CONCLUSION: Our findings suggest that entacapone provides protection against Ang II-induced renal damage through antioxidative and anti-inflammatory mechanisms, rather than by COMT inhibition-induced changes in renal dopaminergic tone.

Angiotensin II induces connective tissue growth factor gene expression via calcineurin-dependent pathways.

Connective tissue growth factor (CTGF) is a polypeptide implicated in the extracellular matrix synthesis. Previous studies have provided evidence that angiotensin II (Ang II) promotes collagen synthesis and regulates collagen degradation. We investigated whether or not CTGF mediates the profibrotic effects of Ang II in the heart and kidneys and the role of calcineurin-dependent pathways in CTGF gene regulation. In transgenic rats harboring human renin and angiotensinogen genes, Ang II induced an age-dependent increase in myocardial CTGF expression, which was 3.5-fold greater compared to normotensive Sprague Dawley (SD) rats. CTGF overexpression correlated closely with the Ang II-induced rise in blood pressure. CTGF mRNA and protein were located predominantly in areas with leukocyte infiltration, myocardial, and vascular lesions and co-localized with TGFbeta(1), collagen I, and collagen III mRNA expressions. Ang II induced CTGF mRNA and protein to a lesser extent in the kidneys, predominantly in glomeruli, arterioles, and in the interstitium with ample inflammation. However, no expression was found in the right ventricle or pulmonary arteries. Blockade of calcineurin activity by cyclosporine A completely normalized Ang II-induced CTGF overexpression in heart and kidney, suppressed the inflammatory response, and mitigated Ang II-induced cell proliferation and apoptosis. In contrast, blockade of mTOR (target of rapamycin) pathway by everolimus, further increased the expression of CTGF even though everolimus ameliorated cell proliferation and T-cell-mediated inflammation. Our findings provide evidence that CTGF mediates Ang II-induced fibrosis in the heart and kidneys via blood pressure and calcineurin-dependent pathways.

Cardiovascular and renal effects of cyclooxygenase inhibition in transgenic rats harboring mouse renin-2 gene (TGR[mREN2]27).

The present study examined the role of cyclooxygenase-synthetized prostanoids in the pathogenesis of angiotensin-II-induced inflammatory response and vascular injury in transgenic rats harboring mouse renin-2 gene (mREN2 rats). Five- to six-week-old, heterozygous mREN2 rats received the following drug regimens for 8 weeks: (1) controls; (2) cyclooxygenase-2 inhibitor (MF-tricyclic [3-(3,4-difluorophenyl)-4-(4-(methylsulfonyl) phenyl)-2(5H)-furanone], 14 mg kg(-1) p.o.); (3) cyclooxygenase-1/cyclooxygenase-2 inhibitor (sulindac, 14 mg kg(-1) p.o.); (4) angiotensin II receptor antagonist (losartan 40 mg kg(-1) p.o.); (5) MF-tricyclic + losartan; (6) sulindac + losartan. Normotensive Sprague-Dawley rats served as controls. mREN2 rats developed pronounced hypertension, cardiac hypertrophy, and albuminuria as compared to normotensive Sprague-Dawley controls. mREN2 rats showed pronounced perivascular inflammation and morphological damage in the kidneys and the heart. Both MF-tricyclic and sulindac further increased blood pressure and albuminuria in mREN2 rats. Neither MF-tricyclic nor sulindac were able to prevent angiotensin-II-induced perivascular inflammation and morphological changes in the heart or in the kidneys. Myocardial and renal cyclooxygenase-2 mRNA expressions were decreased in mREN2 rats, whereas no difference was found in cyclooxygenase-1 mRNA expressions. Sulindac increased both cyclooxygenase-1 and cyclooxygenase-2 gene expressions, whereas MF-tricyclic increased only cyclooxygenase-2 gene expressions. Losartan normalized blood pressure, cardiac hypertrophy, albuminuria, inflammatory response and morphological changes in mREN2 rats, both in the presence and absence of cyclooxygenase inhibitors. Our findings indicate that cyclooxygenase does not play a central role in the pathogenesis of angiotensin-II-induced inflammatory response and vascular injury in mREN2 rats.