Background: Placebo-controlled trials have found that angiotensin-converting enzyme inhibitors (ACEIs) decrease proteinuria and slow the progression of nondiabetic nephropathies. However, head-to-head comparisons of ACEIs and calcium channel blockers (CCBs) have shown conflicting results. Indeed, a recent meta-analysis concluded that there is still uncertainty about the greater renoprotection seen with ACEIs or angiotensin II receptor blockers in nondiabetic patients with renal disease, particularly when using true glomerular filtration rate (GFR) as the primary outcome.Objective: The objective of this 3-year, randomized, multicenter, double-blind, placebo-controlled study was to compare true GFR decline (measured by yearly Cr-51-EDTA blood clearance) in nondiabetic, non-nephrotic adult hypertensive patients with estimated creatinine clearance of 20 to 60 mL/min . 1.73 m(2),when randomized to a CCB (amlodipine, 5-10 mg/d) or an ACEI (enalapril, 5-20 mg/d).Methods: Patients (aged 18-80 years) entered a 4-week placebo run-in washout period and previous antihypertensive drugs were tapered off over 2 weeks. Add-on treatments were atenolol (50-100 mg/d), loop diuretics (furosemide, 20-500 mg/d or torsemide, 5-200 mg/d), alpha-blockers (prazosin, 2.5-5 mg/d or doxazosin, 1-16 mg/d), and centrally acting drugs (rilmenidine, 1-2 mg/d or methyldopa, 250-500 mg/d). The primary end point was true GFR measured by yearly Cr-51-EDTA blood clearance. Secondary end points included a clinical composite of renal events and tolerability collected by a full clinical and laboratory evaluation at each study visit. Post hoc analyses for the change in GFR, proteinuria, and time to clinical events were also planned on baseline proteinuria subgroups (< 1 and >= 1g/d) before unblinding the database.Results: Three hundred eighteen patients entered the run-in period and 263 patients (156 men/107 women; mean age, 58 years) were randomized to receive either amlodipine (5 mg/d, n = 132) or enalapril (5 mg/d, n = 131). Blood pressure declined from 165/102 mm Hg to 138/84 mm Hg and 138/85 mm Hg with amlodipine and enalapril, respectively (no between-group significance). Only 20.8% of the patients randomized to ACEI treatment received diuretics at the last observation. No statistically significant difference was found between amlodipine and enalapril in GFR decline (-4.92 and -3.98 mL/min . 1.73 m(2), respectively, at last observation) and composite secondary end point after a median follow-up of 2.9 years, including in the subgroup of patients with proteinuria > 1 g/d at baseline. Protein excretion rate decreased significantly from baseline in patients taking enalapril plus diuretics (median -270 mg/d; P < 0.001) but not in patients taking amlodipine plus diuretics (-25 mg/d at last observation).Conclusion: In this cohort of nondiabetic, nonnephrotic hypertensive patients, no statistically significant difference in true GFR decline was found over 3 years between amlodipine-treated patients and enalapril-treated patients with main add-on treatment with beta-blockers, including in the subgroup of patients with proteinuria > 1 g/d.

Objective The transcription factor nuclear factor-kappaB (NF-kappa B) has been implicated in cardiomyocyte hypertrophy in vitro as well as in vivo; however, it is unknown if activation of NF-kappa B plays a mandatory role in the hypertrophic process. Here we characterize the importance of NF-kappa B signaling in moderate and severe left ventricular (LV) hypertrophy in rats with chronic pressure overload induced by angiotensin II (Ang II) infusion.Methods and results Electrophoretic mobility shift assay analysis revealed that Ang 11 infusion (2.5 mu g/kg per min) for 6 days increased LV NF-kappa B/DNA-binding activity in a biphasic manner in Sprague-Dawley rats. Pyrrolidine dithiocarbamate (PDTC) (1100 mg/kg per day), an NF-kappa B inhibitor, abolished Ang U-induced NF-kappa B activation and concomitant increase in tumor necrosis factor-alpha gene expression, while activator protein-1/DNA binding was not affected. Inhibition of NF-kappa B signaling for 6 days significantly attenuated Ang II-induced increases in LV/body weight ratio, LV mean wall thickness and cardiomyocyte cross-sectional area, without compromising LV systolic function. Moreover, PDTC abolished Ang II-induced cardiomyocyte apoptosis and interstitial fibrosis, and attenuated the gene expression of type I collagen. In contrast, a moderate LV hypertrophy induced by Ang II at a lower dose (0.5 mu g/kg per min) was not associated with a significant activation of NF-kappa B, and PDTC treatment had no effect on the hypertrophic indices.Conclusion Our in-vivo data indicate a critical role of NF-kappa B signaling in the advanced stage of the remodeling process, whereas development of moderate LV hypertrophy is not dependent on NF-kappa B activation.

The Na(+)/K(+)-ATPase inhibitor ouabain has been shown to trigger hypertrophic growth of cultured cardiomyocytes; however, the significance of endogenous ouabain-like compound (OLC) in the hypertrophic process in vivo is unknown. Here we characterized the involvement of OLC in left ventricular (LV) hypertrophy induced by norepinephrine (NE) and angiotensin II (Ang II) infusions in rats. Administration of NE (300 microg/kg/h) via subcutanously implanted osmotic minipumps for 72 h resulted in a significant increase in left ventricular weight to body weight (LVW/BW) ratio (P<0.001) and a substantial up-regulation of atrial natriuretic peptide (ANP) gene expression (13.2-fold, P<0.001). NE infusion induced a transient increase in plasma OLC levels at 12 h (P<0.05), which returned to control levels by 72 h. Adrenalectomy markedly reduced both basal and NE-induced increase in plasma OLC levels. LVW/BW ratio was not modulated by adrenalectomy; however, ANP gene expression was blunted by 44% (P<0.01) and 47% (P<0.05) at 12 and 72 h, respectively. In agreement, adrenalectomy reduced up-regulation of ANP without affecting LV mass in rats infused with Ang II (33 microg/kg/h). Administration of exogenous ouabain (1 nM to 100 microM) for 24 h had no effect on ANP gene expression in cultured neonatal rat ventricular myocytes. However, the up-regulation of ANP mRNA levels induced by the alpha-adrenergic agonist phenylephrine (1 microM) was markedly enhanced by ouabain (100 microM) (5.6-fold vs. 9.6-fold, P<0.01). These data show that OLC as an adrenal-derived factor may be required for the induction LV ANP gene expression during the hypertrophic process.

The transcription factor signal transducer and activator of transcription 3 (STAT3) has been implicated in the hypertrophic response of cultured cardiomyocytes; however, its precise function in the...

In left ventricular hypertrophy increased ventricular expression of several genes, including atrial- and B-type natriuretic peptide (ANP, BNP) and adrenomedullin (AM) has been reported . However it remained to be clarified if altered expression of these genes is a consistent marker of LVH. Using three different acute in vivo models of LVH, we assessed the relationship between gene inductions, hemodynamics and the development of LVH. Conscious male SD (n=330) rats were treated by NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME 100mg/kg/day in drinking water) or infused subcutaneously by angiotensin II (Ang II, 33 m g/kg/h), b -agonist, isoproterenol (ISO, 100 m g/kg/h) or saline for 6, 12, 72 hours, 1 week or 2 weeks. Examination by in vivo telemetry, L-NAME and Ang II markedly elevated mean arterial pressure, meanwhile it was not altered by ISO. Ratio of LV weight to body weight was significantly elevated by L-NAME (+11.8 %, 2 weeks), Ang II (+15.4 %, 2 weeks) and ISO (25.7 %, 72 hours) (P<0.001). In parallel with the development of LVH, ANP mRNA levels were increased 8.9-, 20.4- and 4.8-fold (P<0.001) by L-NAME, Ang II and ISO, respectively. BNP mRNA expression was increased throughout the whole experiment with L-NAME, while Ang II and ISO caused only transiently elevation: 5.2- and 3.2-fold (6 hours, P<0.001) respectively. Ang II produced a rapid, transient increase in ventricular AM mRNA levels (1.4-fold, 12 hours, P<0.05). In contrast, ISO resulted in a marked decline in AM mRNA levels (0.4-fold, 6 and 12 hours, P<0.001), but L-NAME did not have effect on AM geneexpression. Changes in ANP, BNP mRNA levels were followed by an increase in ventricular and plasma peptide levels. The present results show that in all three models alteration in ventricular gene expression of ANP is a sensitive indicator in the acute phase of LVH. However BNP gene expression is early, transiently upregulated at Ang II and ISO treatment, while constantly increased at L-NAME treatment during the 2 weeks of treatment. Meanwhile the gene expression of AM is differentially regulated by various forms of hypertrophic stimuli.

The orphan receptor APJ and its recently identified endogenous ligand, apelin, are expressed in the heart. However, their importance in the human cardiovascular system is not known. This study shows that apelin-like immunoreactivity is abundantly present in healthy human heart and plasma. Gel filtration HPLC analysis revealed that atrial and plasma levels of high molecular weight apelin, possibly proapelin, were markedly higher than those of mature apelin-36 itself. As assessed by quantitative RT-PCR analysis, left ventricular apelin mRNA levels were increased 4.7-fold in chronic heart failure (CHF) due to coronary heart disease (p<0.01) and 3.3-fold due to idiopathic dilated cardiomyopathy (p<0.05), whereas atrial apelin mRNA levels were unchanged. Atrial and plasma apelin-like immunoreactivity as well as atrial and ventricular APJ receptor mRNA levels were significantly decreased in CHF. Our results suggest that a new cardiac regulatory peptide, apelin, and APJ receptor may contribute to the pathophysiology of human CHF.

Our knowledge about precise function of beta 3 receptor (β3R) in the mammalian heart in vivo is poorly characterized. The present study aimed to investigate the role of β3R in cardiac hypertrophy. Combined β1 or β2 antagonist metoprolol (0.5mg/kg/h), the β3R agonist BRL37344 (30mg/kg/h), or both, or vehicle were infused via subcutaneously implanted osmotic mini pumps in 44 male SD-rats for 72 hours. Left ventricular weight/body weight (LV/BW) ratio was elevated in BRL37344 and BRL37344 + metoprolol treated groups. (10.3±0.9% (p<0,01); and 9,5±0.8% (p<0,05) vs. sham respectively). We measured the gene expression of substances whith antihypertrophic or antifibrotic effect, like: atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), and the embryonic contractile protein skeletal α-actin mRNA levels, in the left ventricles by Northern-blotting. BRL37344 elevated ANP expression with 64±18.9% meanwhile the combined therapy increased it with 55±19,6%, (p<0,01) vs. sham), BNP gene expression elevated 74±23.2% (p<0,01) and 123±24.9% vs. sham respectively). None of the above mentioned therapy affect the gene expression of skeletal α-actin at 72 hours. The investigated parameters (LV/BW, ANP, BNP and skeletal α-actin mRNA) did not differ between the BRL37344 or BRL37344 + metoprolol treated group. We conclude that BRL37344 infusion rapidly increased left ventricular mass, and induced the expression of representative genes in cardiac remodeling or hypertrophy. We conclude that BRL37344 treatment in vivo induce left ventricular hypertrophy which is the result of the selective stimulation β3R.

BACKGROUND:The precise function of angiotensin II type 2 receptor (AT2-R) in the mammalian heart in vivo is unknown. Here, we investigated the role of AT2-R in cardiac pressure overload. METHODS AND RESULTS:Rats were infused with vehicle, angiotensin II (Ang II), PD123319 (an AT2-R antagonist), or the combination of Ang II and PD123319 via subcutaneously implanted osmotic minipumps for 12 or 72 hours. Ang II-induced increases in mean arterial pressure, left ventricular weight/body weight ratio, and elevation of skeletal alpha-actin and beta-myosin heavy chain mRNA levels were not altered by PD123319. In contrast, AT2-R blockade resulted in a marked increase in the gene expression of c-fos, endothelin-1, and insulin-like growth factor-1 in Ang II-induced hypertension. In parallel, Ang II-stimulated mRNA and protein expression of atrial natriuretic peptide were significantly augmented by AT2-R blockade. Moreover, PD123319 markedly increased the synthesis of B-type natriuretic peptide. Furthermore, the expression of vascular endothelial growth factor and fibroblast growth factor-1 was downregulated by Ang II only in the presence of AT2-R blockade. CONCLUSIONS:Our results provide evidence that AT2-R plays a functional role in the cardiac hypertrophic process in vivo by selectively regulating the expression of growth-promoting and growth-inhibiting factors.

Recent studies suggest, that chronic nitric oxide synthase inhibition induces hypertension, however it is not accompanied by compensatory left ventricular (LV) hypertrophy. Our aim was to investigate the effect of subacute administration of N(G)-nitro-L-arginine methyl ester (L-NAME) on development of LV hypertrophy and on embrional gene programming. 87 male Sprague Dawley rats received L-NAME (100 mg/kg/die in drinking water) for 1/4, 1/2, 3, 7 and 14 days. To determine ventricular mRNA level we used guanidine isothyocianate, CsCl tissue extraction followed by Northern blotting. RNA were labeled by 32P-dCTP. Results were corrected for 18S mRNA expression. Plasma level of B-type natriuretic peptide (BNP) and pro-atrial natriuretic peptide was measured by radioimmunoassay after Sep Pack C18 extraction. Left ventricular weight/body weight ratios (LV/BW) were elevated throughout the whole experiment and reached maximum at 2 weeks (2.378±0.03 vs. 2.13±0.03, p<0.05; average±SD), while right ventricular weight/body weight ratios were unchanged. The L-NAME treatment raised gene expression of embryonic isoform of contractile proteins (skeletal alpha-actin and beta myosin heavy chain) in the left ventricle (LV) from 6 hours to 2 weeks (1,3-1,5 and 1,6-2,6-fold changes to control, p<0.05; relative densitometric values versus control), while adult isoformes, cardiac alpha-actin and alpha myosin heavy chain mRNA level remained constant. The atrial natriuretic peptide (ANP) gene expression followed the changes of LV/BW ratio, peaked at 2 weeks (8.94±1.52, p<0.001). LV gene expression of BNP was significantly elevated at every time point, being highest at 1 week (1.61±0.13, p<0.01). AM mRNA levels were not altered during the 2 weeks period. Right ventricular expression of ANP and BNP increased only at 2 weeks (3,29±2,0 p<0.05 and 2,92±1,11 p<0.01). The plasma level of N-terminal pro-atrial natriuretic peptide parallel changed to the LV gene expression. The BNP plasma level increased at 6 hours and 2 weeks by 1.8-fold vs sham-operated rats. In vivo subacute L-NAME treatment induces LV hypertrophy in rats and reactivates the embryonic gene program, including contractile protein genes, ANP and BNP.

Increased ventricular expression of several genes, including a newly identified vasorelaxant peptide, adrenomedullin (AM), atrial- and B-type natriuretic peptide (ANP, BNP) has been documented in experimental models of cardiac hypertrophy (CH). Yet, it remained to be clarified if altered expression of these genes is a consistent marker of CH. Using three acute in vivo models of CH as a tool, we assessed the relationship between the induction patterns and the development of CH. Conscious male SD (n=319) rats were infused subcutaneously by angiotensin II (Ang II, 33 ug/kg/h), noradrenaline (NA, 300 ug/kg/h), b-agonist, isoproterenol (ISO, 100 ug/kg/h) or saline for 1/4, 1/2, 3, 7 or 14 days. As proven by in vivo telemetry, Ang II and NA markedly elevated mean arterial pressure, meanwhile it was not altered by ISO. Ratio of LV weight to body weight was elevated as a maximum (P<0.001) by NA (+12.7%, 72 h), ISO (25.7%, 72 h) and Ang II (+15.4%, 2 weeks). Ang II induced a rapid, transient increase (1.4-fold, 12 h, P<0.05), in contrast, ISO resulted in a marked decline in AM mRNA levels (0.4-fold, 6-12 h, P<0.001). NA has left AM gene expression unchanged. BNP mRNA expression was transiently elevated 5.2-, 3.9 and 3.2- fold (6h, P<0.001) by Ang II, NA and ISO, respectively. In parallel with the development of CH, ANP mRNA levels were increased 20.4-, 11.4- and 4.8- fold (P<0.001) by Ang II, NA and ISO, respectively. The present results show that AM is differentially regulated by various forms of hypertrophic stimuli. BNP expression is early, transiently upregulated, meanwhile alteration in the ventricular expression of ANP gene is a sensitive indicator of CH and may result from a number of different stimuli.

To investigate the molecular events during the development of cardiac hypertrophy (CH) combined with diabetes mellitus (DM) male Wistar rats (n=9-13/group) were treated with iv. injections of streptozotocin (60 mg/kg) to induce DM. CH was initiated by subcutaneous infusion of angiotensin II (AII) (33mg/kg/day) for 24 hours, either 2.5 or 7 weeks (early and late phase of experimental DM) after the induction of DM. At both time points 4 groups (n=9-13) were investigated: control, diabetic, AII treated, AII treated and diabetic. AII elevated blood pressure by 25-30 mmHg as indicated by in vivo on-line telemetry and increased the left ventricular weight/body weight (LV/BW) ratio (2.02+0.14 mg/g vs 1.90+0.09 average+SD). DM at 2.5 weeks did not alter LV/BW but increased it at 7 weeks (1.92+0.10; 2.24+0.23, respectively). AII superimposed on DM increased LV/BW at 2.5 weeks but could not further increase it at 7 weeks (2.11+0.13 vs 1.92+0.10 and 2.24+0.13 vs 2.24+0.23 respectively). Atrial natriuretic peptide (ANP) expression in the LV was the best molecular marker of the hypertrophic process, changes in B-type natriuretic peptide (BNP) were similar but smaller, whereas adrenomedullin (AM) expression in the LV did not change markedly under these experimental conditions. DM increased LV ANP expression (2.5 weeks: 3.5+0.7; 7 weeks:2.2+0.2, Northern blotting, relativ denzitometric data normalized to 18S expression). AII alone increased LV ANP similarly at both time points (4.8+0.8 and 3.2+0.4). When AII was superimposed on DM, LV ANP expression was increased further (2.5 weeks: 11.1+0.8 vs 3.5+0.7; 7 weeks: 7.3+0.9 vs 2.2+0.2). According to the results DM induced hypertrophic changes are present 2.5 weeks after initiating experimental DM as indicated by LV ANP expression, but this can only be observed at 7 weeks in the LV/BW ratio. AII treatment in DM rats further increases LV/BW at 2.5 weeks but not at 7 weeks, however tissue ANP and BNP expression is further elevated in both cases. The understanding of the early molecular changes during the development of AII induced cardiac hypertrophy combined with DM needs further investigations.

To investigate the cardiac effects of continous beta receptor stimulation on cardiac mass and left ventricular expression of atrial natriuretic peptide, B-type natriuretic peptide and adrenomedullin, 121 male SD rats were given isoproterenol (100 ug/kg/day) or saline via subcutaneously implanted osmotic minipumps. Isoproterenol treatment elevated heart rate by 32% but blood pressure was unchanged as proved by in vivo on line telemetry. Rats were sacrificed at 0.5 and 3 days of isoproterenol treatment. Left ventricular weight/body weight ratio increased significantly (sham: 2.41+0.05 (mg/g), 0.5 day: 2.80+0.05, p<0.05; 3days: 3.08+0.06, p<0.05). Total RNA was isolated from the tissues and specific mRNA content was measured by Northern blotting. Atrial natriuretic peptide gene expression was elevated (0.5 day: 247+38%, p<0.0001; 3days: 481+55%, p<0.0001), densitometric values corrected for 18s expression and given as % of sham treated animals). B-type natriuretic peptide gene expression peaked at 0.5 days(571%+35%, p<0.0001), whereas left ventricular tissue levels of adrenomedullin mRNA - on the contrary - were markedly attenuated by isoproterenol treatment (0.5 day: 42+2%, p<0.0001; 3days: 69+1%, p<0.05 vs. sham). Concomittant treatment by the beta receptor blocker propranolol abolished the effects of isoproterenol. Propranolol alone transiently increased adrenomedullin and B-type natriuretic peptide gene expression, whereas atrial natriuretic peptide expression was not affected. We conclude that in the early phase of isoproterenol induced cardiac hypertrophy, synthesis of diuretic substances are differentially regulated. Both atrial natriuretic peptide and B-type natriuretic peptide genes are upregulated although in a slightly different time pattern, however adrenomedullin gene expression is suppressed in the left ventricles.

The mechanisms mediating the activation of cardiac gene expression during pressure overload are not fully understood. We examined whether angiotensin II-induced activation of ventricular gene expression is related to blood pressure and ventricular mass or requires other factors by infusing angiotensin II in sham-operated and adrenalectomized rats. In sham-operated rats, angiotensin II (33 microg/kg x h, sc) produced a significant increase in mean arterial pressure (measured by telemetry) within 3 h. Mean arterial pressure (up to 45 h) and the increase in left ventricular hypertrophy in adrenalectomized rats during angiotensin II infusion were similar to those in sham-operated rats. Angiotensin II produced 3.6-fold (P < 0.01) and 20.4-fold (P < 0.001) increases in ventricular atrial natriuretic peptide mRNA levels at 12 and 72 h, respectively. Angiotensin II infusion for 12 h also significantly increased the ventricular mRNA levels of B-type natriuretic peptide (5.2-fold) and adrenomedullin (1.4-fold). Adrenalectomy either abolished (atrial natriuretic peptide and adrenomedullin) or blunted (B-type natriuretic peptide) the early activation of ventricular gene expression by angiotensin II. The baseline synthesis of atrial natriuretic peptide, B-type natriuretic peptide, and adrenomedullin in the ventricle remained unchanged in adrenalectomized rats. In conclusion, our results indicate that factors derived from the adrenals are required for angiotensin II-induced early activation of cardiac gene expression.