Cathepsin A inhibition attenuates myocardial infarction-induced heart failure on the functional and proteomic levels.

BACKGROUND: Myocardial infarction (MI) is a major cause of heart failure. The carboxypeptidase cathepsin A is a novel target in the treatment of cardiac failure. We aim to show that recently developed inhibitors of the protease cathepsin A attenuate post-MI heart failure. METHODS: Mice were subjected to permanent left anterior descending artery (LAD) ligation or sham operation. 24 h post-surgery, LAD-ligated animals were treated with daily doses of the cathepsin A inhibitor SAR1 or placebo. After 4 weeks, the three groups (sham, MI-placebo, MI-SAR1) were evaluated. RESULTS: Compared to sham-operated animals, placebo-treated mice showed significantly impaired cardiac function and increased plasma BNP levels. Cathepsin A inhibition prevented the increase of plasma BNP levels and displayed a trend towards improved cardiac functionality. Proteomic profiling was performed for the three groups (sham, MI-placebo, MI-SAR1). More than 100 proteins were significantly altered in placebo-treated LAD ligation compared to the sham operation, including known markers of cardiac failure as well as extracellular/matricellular proteins. This ensemble constitutes a proteome fingerprint of myocardial infarction induced by LAD ligation in mice. Cathepsin A inhibitor treatment normalized the marked increase of the muscle stress marker CA3 as well as of Igγ 2b and fatty acid synthase. For numerous further proteins, cathepsin A inhibition partially dampened the LAD ligation-induced proteome alterations. CONCLUSIONS: Our proteomic and functional data suggest that cathepsin A inhibition has cardioprotective properties and support a beneficial effect of cathepsin A inhibition in the treatment of heart failure after myocardial infarction.

Cardiac remodeling and myocardial dysfunction in obese spontaneously hypertensive rats.

BACKGROUND: The additive effects of obesity and metabolic syndrome on left ventricular (LV) maladaptive remodeling and function in hypertension are not characterized. METHODS: We compared an obese spontaneously hypertensive rat model (SHR-ob) with lean spontaneously hypertensive rats (SHR-lean) and normotensive controls (Ctr). LV-function was investigated by cardiac magnetic resonance imaging and invasive LV-pressure measurements. LV-interstitial fibrosis was quantified and protein levels of phospholamban (PLB), Serca2a and glucose transporters (GLUT1 and GLUT4) were determined by immunohistochemistry. RESULTS: Systolic blood pressure was similar in SHR-lean and SHR-ob (252 ± 7 vs. 242 ± 7 mmHg, p = 0.398) but was higher when compared to Ctr (155 ± 2 mmHg, p < 0.01 for both). Compared to SHR-lean and Ctr, SHR-ob showed impaired glucose tolerance and increased body-weight. In SHR-ob, LV-ejection fraction was impaired vs. Ctr (46.2 ± 1.1 vs. 59.6 ± 1.9%, p = 0.007). LV-enddiastolic pressure was more increased in SHR-ob than in SHR-lean (21.5 ± 4.1 vs. 5.9 ± 0.81 mmHg, p = 0.0002) when compared to Ctr (4.3 ± 1.1 mmHg, p < 0.0001 for both), respectively. Increased LV-fibrosis together with increased myocyte diameters and ANF gene expression in SHR-ob were associated with increased GLUT1-protein levels in SHR-ob suggestive for an upregulation of the GLUT1/ANF-axis. Serca2a-protein levels were decreased in SHR-lean but not altered in SHR-ob compared to Ctr. PLB-phosphorylation was not altered. CONCLUSION: In addition to hypertension alone, metabolic syndrome and obesity adds to the myocardial phenotype by aggravating diastolic dysfunction and a progression towards systolic dysfunction. SHR-ob may be a useful model to develop new interventional and pharmacological treatment strategies for hypertensive heart disease and metabolic disorders.

AVE8134, a novel potent PPARα agonist, improves lipid profile and glucose metabolism in dyslipidemic mice and type 2 diabetic rats.

AIM: AVE8134 is a structurally novel potent PPARα agonist. The aim of this study is to investigate the efficacy of AVE8134 on lipid profile and glucose metabolism in dyslipidemic mice and type 2 diabetic rats. METHODS: A cell based PPAR Gal4 transactivation assay was constructed for testing the activities of AVE8134 at 3 different PPAR isoforms in vitro. Transgenic human Apo A1 (hApo A1) mice and insulin-resistant ZDF rats were used to evaluate the effects of AVE8134 in vivo. RESULTS: AVE8134 was a full PPARα dominated PPAR agonist (the values of EC(50) for human and rodent PPARα receptor were 0.01 and 0.3 µmol/L, respectively). AVE8134 was not active at PPARδ receptor. In female hApo A1 mice, AVE8134 (1-30 mg·kg(-1)·d(-1), po for 12 d) dose-dependently lowered the plasma triglycerides, and increased the serum HDL-cholesterol, hApo A1 and mouse Apo E levels. In female ZDF rats, AVE8134 (3-30 mg·kg(-1)·d(-1) for 2 weeks) improved insulin-sensitivity index. In pre-diabetic male ZDF rats (at the age of 7 weeks), AVE8134 (10 mg·kg(-1)·d(-1) for 8 weeks) produced an anti-diabetic action comparable to rosiglitazone, without the PPARγ mediated adverse effects on body weight and heart weight. In male ZDF rats (at the age of 6 weeks), AVE8134 (20 mg·kg(-1)·d(-1) for 12 weeks) increased mRNA levels of the target genes LPL and PDK4 about 20 fold in the liver, and there was no relevant effect with rosiglitazone. CONCLUSION: AVE8134 improves lipid profile and glucose metabolism in dyslipidemic mice and type 2 diabetic rats.

The phospholipid-binding protein SESTD1 is a novel regulator of the transient receptor potential channels TRPC4 and TRPC5.

TRPC4 and TRPC5 are two closely related members of the mammalian transient receptor potential cation channel family that have been implicated in important physiological functions, such as growth cone guidance and smooth muscle contraction. To further unravel the role of TRPC4 and TRPC5 in these processes in vivo, detailed information about the molecular composition of native channel complexes and their association with cellular signaling networks is needed. We therefore searched a human aortic cDNA library for novel TRPC4-interacting proteins using a modified yeast two-hybrid assay. This screen identified SESTD1, a previously uncharacterized protein containing a lipid-binding SEC14-like domain as well as spectrin-type cytoskeleton interaction domains. SESTD1 was found to associate with TRPC4 and TRPC5 via the channel's calmodulin- and inositol 1,4,5-trisphosphate receptor-binding domain. In functional studies, we demonstrate that SESTD1 binds several phospholipid species in vitro and is essential for efficient receptor-mediated activation of TRPC5. Notably, phospholipid binding to SESTD1 was Ca(2+)-dependent. Because TRPC4 and -5 conduct Ca(2+), SESTD1-channel signaling may be bidirectional and also couple TRPC activity to lipid signaling through SESTD1. The modulation of TRPC channel function by specific lipid-binding proteins, such as SESTD1, adds another facet to the complex regulation of these channels complementary to the previously described effects of direct channel-phospholipid interaction.

Mice lacking calsarcin-1 are sensitized to calcineurin signaling and show accelerated cardiomyopathy in response to pathological biomechanical stress.

Signaling by the calcium-dependent phosphatase calcineurin profoundly influences the growth and gene expression of cardiac and skeletal muscle. Calcineurin binds to calsarcins, a family of muscle-specific proteins of the sarcomeric Z-disc, a focal point in the pathogenesis of human cardiomyopathies. We show that calsarcin-1 negatively modulates the functions of calcineurin, such that calcineurin signaling was enhanced in striated muscles of mice that do not express calsarcin-1. As a consequence of inappropriate calcineurin activation, mice with a null mutation in calsarcin-1 showed an excess of slow skeletal muscle fibers. The absence of calsarcin-1 also activated a hypertrophic gene program, despite the absence of hypertrophy, and enhanced the cardiac growth response to pressure overload. In contrast, cardiac adaptation to other hypertrophic stimuli, such as chronic catecholamine stimulation or exercise, was not affected. These findings show important roles for calsarcins as modulators of calcineurin signaling and the transmission of a specific subset of stress signals leading to cardiac remodeling in vivo.

The peroxisome proliferator-activated receptor-alpha (PPAR-alpha) agonist, AVE8134, attenuates the progression of heart failure and increases survival in rats.

AIM: To investigate the efficacy of the peroxisome proliferator-activated receptor-alpha (PPARalpha) agonist, AVE8134, in cellular and experimental models of cardiac dysfunction and heart failure. METHODS: In Sprague Dawley rats with permanent ligation of the left coronary artery (post-MI), AVE8134 was compared to the PPARgamma agonist rosiglitazone and in a second study to the ACE inhibitor ramipril. In DOCA-salt sensitive rats, efficacy of AVE8134 on cardiac hypertrophy and fibrosis was investigated. Finally, AVE8134 was administered to old spontaneously hypertensive rats (SHR) at a non-blood pressure lowering dose with survival as endpoint. In cellular models, we studied AVE8134 on hypertrophy in rat cardiomyocytes, nitric oxide signaling in human endothelial cells (HUVEC) and LDL-uptake in human MonoMac-6 cells. RESULTS: In post-MI rats, AVE8134 dose-dependently improved cardiac output, myocardial contractility and relaxation and reduced lung and left ventricular weight and fibrosis. In contrast, rosiglitazone exacerbated cardiac dysfunction. Treatment at AVE8134 decreased plasma proBNP and arginine and increased plasma citrulline and urinary NOx/creatinine ratio. In DOCA rats, AVE8134 prevented development of high blood pressure, myocardial hypertrophy and cardiac fibrosis, and ameliorated endothelial dysfunction. Compound treatment increased cardiac protein expression and phosphorylation of eNOS. In old SHR, treatment with a low dose of AVE8134 improved cardiac and vascular function and increased life expectancy without lowering blood pressure. AVE8134 reduced phenylephrine-induced hypertrophy in adult rat cardiomyocytes. In HUVEC, Ser-1177-eNOS phosphorylation but not eNOS expression was increased. In monocytes, AVE8134 increased the expression of CD36 and the macrophage scavenger receptor 1, resulting in enhanced uptake of oxidized LDL. CONCLUSION: The PPARalpha agonist AVE8134 prevents post-MI myocardial hypertrophy, fibrosis and cardiac dysfunction. AVE8134 has beneficial effects against hypertension-induced organ damages, resulting in decreased mortality. The compound exerts its protective properties by a direct effect on cardiomyocyte hypertrophy, but also indirectly via monocyte signaling and increased endothelial NO production.Acta Pharmacologica Sinica (2009) 30: 935-946; doi: 10.1038/aps.2009.58; published online 8 June 2009.

Effect of atrial electrical remodeling on the efficacy of antiarrhythmic drugs: comparison of amiodarone with I(Kr)- and I(to)/IKur-blockade in vivo.

INTRODUCTION: Amiodarone is the gold standard in the prevention of recurrence of atrial fibrillation (AF), but the causes for its superior clinical efficacy are not understood. We hypothesized that atrial electrical remodeling increases the atrial efficacy of amiodarone. METHODS AND RESULTS: We investigated the effect of an acute intravenous dose of amiodarone on atrial refractory periods (AERP) in sinus rhythm (SR) and after 5, 24, and 72 hours of atrial tachypacing in comparison with the I(Kr) blocker dofetilide and the I(to)/IKur blockers AVE1231 and AVE0118 in five instrumented goats. Electrical remodeling progressively increased the AERP-prolonging effect of 3 mg/kg of AVE1231 and AVE0118 (2-fold increase in AERP at 72 hours vs SR, P < 0.01), but strongly decreased that of 10 mug/kg dofetilide (<0.5-fold, P < 0.05, at 300 and 400 ms basic cycle length). After 5 and 24 hours of tachypacing, the effect of 3 mg/kg amiodarone strongly increased (2-fold, P < 0.01 after 24 hours vs SR). This early gain in AERP prolongation was confirmed in anesthetized pigs with 3.5 hours of atrial tachypacing (2.4-fold increase, P < 0.01). At 72 hours of atrial tachypacing in the goat, however, the early gain was lost and the effect of amiodarone was similar again to that in SR. CONCLUSION: Atrial electrical remodeling changed the efficacy of the antiarrhythmic agents in a different way. The favorable efficacy profile of amiodarone during electrical remodeling, particularly the marked increase in AERP prolongation in early electrical remodeling, may explain its superior clinical efficacy over existing antiarrhythmic drugs.

Blood pressure-independent effect of long-term treatment with the soluble heme-independent guanylyl cyclase activator HMR1766 on progression in a model of noninflammatory chronic renal damage.

Nitric oxide formation is impaired in chronic renal failure. The renoprotective effects of a nonhypotensive dose of HMR1766, a direct activator of the heme enzyme soluble guanylyl cyclase was studied in comparison to an ACE-i in the remnant kidney model. Male Sprague-Dawley rats were subtotally nephrectomized (SNX) or sham operated (sham) and left untreated or started on treatment with HMR1766 or ACE-i in non-hypotensive doses. BP, albumin excretion and parameters of renal damage were analyzed. After a 12-week study, urinary albumin excretion was significantly higher in untreated SNX than in sham; this increase was prevented by ACE-i and ameliorated by HMR1766. Relative kidney and left ventricular weight were significantly higher in untreated SNX compared to sham; these changes were completely prevented by HMR1766. In untreated SNX, glomerulosclerosis (1.02 +/- 0.13) was significantly higher than in sham (0.12 +/- 0.04), SNX+HMR1766 (0.27 +/- 0.04) and SNX+ACE-i (0.46 +/- 0.06). Tubulointerstitial changes went in parallel. Increased glomerular cell number after SNX (71.5 +/- 14 vs. 60 +/- 7.3 in sham) was prevented by HMR1766 (55.7 +/- 7.3), but not by ACE-i (66.6 +/- 9). The results document beneficial BP-independent HMR1766 effects on kidney structure and urinary albumin excretion in a noninflammatory model of renal failure and may argue for a novel therapeutic principle.

Reduction of the aortic inflammatory response in spontaneous atherosclerosis by blockade of macrophage migration inhibitory factor (MIF).

Atherosclerosis is an inflammatory response of the arterial wall to "injury", which is prominently driven by cytokines. The inflammatory mediator macrophage migration inhibitory factor (MIF) is a unique cytokine that was recently associated with atherogenesis. Here, we have investigated whether MIF has a role in spontaneous atherosclerosis by studying apolipoprotein E-deficient (ApoE(-/-)) mice treated with neutralizing anti-MIF monoclonal antibody and comparison with isotype IgG-treated controls. After 14 weeks, the aortas and heart valves were analyzed for inflammatory status, macrophage content and plaque areas. MIF expression in the aortic wall was elevated upon spontaneous atherogenesis, with foam cells representing a major source. Of note, MIF blockade led to a marked reduction in intimal Mac-1-positive macrophages. Similarly, treatment with anti-MIF antibody led to a reduction of a variety of inflammatory mediators typically associated with atherosclerosis including the circulating levels of fibrinogen, MIF and IL-6. Importantly, the local aortic expression of ICAM-1, MMP-2, TNF, IL-12, and CD40L was reduced by MIF blockade, as were the levels of the phospho-c-Jun and C/EBPbeta transcription factors. The observed strong reduction of inflammatory parameters by anti-MIF treatment was associated with a small, yet non-significant, reduction in aortic plaque area. Thus, although MIF's role is not directly linked to plaque volume expansion, in this mouse model of spontaneous atherogenesis, MIF plays an important role in intimal inflammation.

Impaired left ventricular relaxation in type 2 diabetic rats is related to myocardial accumulation of N(epsilon)-(carboxymethyl) lysine.

Myocardial dysfunction in the absence of myocardial ischemia is frequent in patients with diabetes mellitus but the underlying pathomechanism is unclear. We investigated whether accumulation of advanced glycation end products (AGEs) in the diabetic myocardium is related to its functional abnormalities. In 11 male homozygous Zucker diabetic fatty rats (ZDF/Gmi-fa/fa) aged 37 weeks (OBESE) and 11 non-obese, non-diabetic littermates (LEAN), we measured left ventricular function (pressure-volume catheter) and levels of N(epsilon)-(carboxymethyl) lysine (CML), a prototypical AGE, in serum and the left ventricle (competitive enzyme linked immuno-assay). Overt diabetes mellitus (HbA1c > 9%) was present in all OBESE animals but not in LEAN. Systolic left ventricular function was not different between the groups, but the markers of left ventricular relaxation, dP/dt(min) and the relaxation constant tau, were impaired in OBESE. In parallel, CML levels were increased in serum (273 +/- 15 vs. 197 +/- 10 ng/ml, p<0.05) and in the left ventricle (18.4 +/- 1.1 vs. 12.5 +/- 2.0 ng/mg protein, p < 0.05) in OBESE compared to LEAN. There was a linear correlation between tau and the left ventricular CML levels (r = 0.65; p < 0.05). We conclude that type 2 diabetes is associated with predominant left ventricular diastolic dysfunction. Myocardial accumulation of advanced glycation end products may contribute to relaxation abnormalities in type 2 diabetes.

Vasopeptidase inhibition prevents target organ damage and improves survival in spontaneously hypertensive rats.

BACKGROUND: Vasopeptidase inhibition has been shown to be an effective antihypertensive principle but its long-term effects on hypertensive target organ damage are not known. We investigated the myocardial, vascular and renal effects of chronic vasopeptidase inhibition in arterial hypertension. METHODS AND RESULTS: One hundred and thirty-nine male spontaneously hypertensive rats aged 15 months were treated chronically with either the pure angiotensin-converting enzyme (ACE) inhibitor, ramipril (1 mg/kg/d in drinking water, n=46), or the vasopeptidase inhibitor AVE7688 (30 mg/kg/d in chow, n=46), or placebo (n=47) and followed up until they died. After six months, both ramipril and AVE7688 had markedly reduced plasma ACE activity, normalised blood pressure (BP), reduced left ventricular mass and improved systolic function to similar extents. Acetylcholine mediated relaxation of aortic rings was improved by both ramipril and AVE7688. There was substantial albuminuria in the placebo group (albumin-to-creatinine ratio 107+/-54 microg/mg), which was significantly reduced by ramipril to 57+/-34 microg/mg, and practically abolished in the AVE7688 group (22+/-12 microg/mg, p<0.05 vs. placebo and ramipril). Tubulo-interstitial damage (semi-quantitative score) was significantly reduced by AVE7688 and ramipril. Overall mortality was markedly reduced in the ramipril and AVE7688 groups (13% and 16% at six months, respectively), both p<0.05 vs. placebo (71%). CONCLUSIONS: Vasopeptidase inhibition effectively controls BP and reduces myocardial, vascular and renal target organ damage, resulting in a markedly prolonged survival. At similar degrees of plasma ACE inhibition, AVE7688 compared to ramipril offers superior protection against hypertensive kidney damage.

Long term Rho-kinase inhibition ameliorates endothelial dysfunction in LDL-Receptor deficient mice.

Chronic inhibition of Rho-kinase has been recently implicated in retardation of atherogenesis induced by high-fat diet in low-density lipoprotein receptor deficient (LDLR-/-) mice. However, it remains to be examined whether long-term Rho-kinase inhibition will reduce vascular dysfunction in this model. LDLR-/- mice on a high-fat diet were treated either with saline (LDLR-/-) or with the Rho-kinase inhibitor Fasudil (HA1077, 5-Isoquinolinesulfonyl homopiperazine, 100 mg/kg/day by gavage, LDLR-/- +Fasudil) for 10 weeks. Fasudil-treatment normalized endothelial function (measured by means of endothelium-dependent vasorelaxation) in LDLR-/- +Fasudil, to the level of controls (C57BL/6J). No tolerance toward Rho-kinase inhibition has been detected in Fasudil-treated animals. We conclude that long-term Rho-kinase inhibition normalizes endothelial function without development of tolerance.

Vasopeptidase inhibition prevents nephropathy in Zucker diabetic fatty rats.

BACKGROUND: Blocking the renin-angiotensin system is an established therapeutic principle in diabetic nephropathy. We investigated whether inhibition of both neutral endopeptidase and ACE (vasopeptidase inhibition) can prevent functional and morphological features of nephropathy in the Zucker diabetic fatty (ZDF) rat, an animal model of type II diabetes. METHODS: Homozygous (fa/fa) ZDF rats (each n=15) aged 10 weeks were treated with placebo, ramipril (1 mg/kg/day in drinking water), or the vasopeptidase inhibitor AVE7688 (45 mg/kg/day in chow). Metabolic parameters and renal function (metabolic cages) were assessed at baseline (age 10 weeks), and at age 17, 27, and 37 weeks. Twenty heterozygous animals (fa/-) served as lean, nondiabetic controls. At age 37 weeks, the animals were sacrificed and the kidneys analyzed histopathologically. RESULTS: Overt diabetes mellitus (blood glucose >20 mmol/l) was established at age 17 weeks in all homozygous ZDF rats. In the placebo group, urinary protein excretion increased progressively from 8+/-1 (baseline) to 342+/-56 mg/kg/day (week 37) whereas diabetes and proteinuria were absent in the lean control group. Ramipril tended to reduce albuminuria and morphological damage (p=ns) but AVE7688 virtually prevented albuminuria (33+/-12 mg/kg/day, p<0.05 vs. ZDF placebo) and drastically reduced the incidence and severity of glomerulosclerosis and tubulointerstitial damage. CONCLUSIONS: In ZDF rats, development of diabetes mellitus is accompanied by functional and morphological kidney damage that resembles human diabetic nephropathy. Diabetic nephropathy can be prevented by chronic vasopeptidase inhibition.

Progression of kidney injury and cardiac remodeling in obese spontaneously hypertensive rats: the role of renal sympathetic innervation.

BACKGROUND: Hypertension and metabolic syndrome (MetS) are associated with increased sympathetic activation possibly contributing to the progression of renal damage and cardiac remodeling. Renal sympathetic denervation (RDN) decreases sympathetic renal efferent and afferent nerve activity. METHODS: Obese spontaneously hypertensive rats (SHRs-ob) were subjected to RDN at the age of 34 weeks (SHRs-ob + RDN) and were compared with sham-operated SHRs-ob and their normotensive lean controls (Ctrs). Blood pressure was measured by telemetry. Kidney and heart function were determined by magnetic resonance imaging (MRI). Renal and cardiac remodeling were characterized by immunohistochemical analyses. Animals were killed at the age of 48 weeks. RESULTS: In SHRs-ob, RDN attenuated the progressive increase in blood pressure and preserved a mean blood pressure of 156±7mm Hg compared with 220±8mm Hg in sham-operated SHRs-ob at 100 days after RDN, whereas heart rate, body weight, and metabolic parameters remained unchanged. Renal catecholamine and tyrosine hydroxylase levels were significantly reduced after RDN, suggesting effective renal denervation. Progression of renal dysfunction as characterized by increased urinary albumin/creatinine ratio and reduced glomerular filtration rate were attenuated by RDN. In SHRs-ob, renal perfusion was significantly reduced and normalized by RDN. Cardiac fibrosis and cardiac diastolic dysfunction measured by MRI and invasive pressure measurements were significantly attenuated by RDN. CONCLUSIONS: In SHRs-ob, progressive increase in blood pressure and progression of renal injury and cardiac remodelling are mediated by renal sympathetic activation as they were attenuated by RDN.

Regression of ventricular and vascular hypertrophy: are there differences between structurally different angiotensin-converting enzyme inhibitors?

OBJECTIVES: It is well established that angiotensin-converting enzyme (ACE) inhibitors (ACEI) reduce blood pressure (BP) and hypertrophy of the left ventricle and vessels. The aim of our study was to compare chemically different ACEIs regarding their ability to modulate left ventricular and media hypertrophy, ACE activity and plasma endothelin-1 concentrations in spontaneously hypertensive rats (SHRs). DESIGN: After establishing equi-effective dose regimes, SHRs were treated (3 months) with captopril, enalapril, fosinopril or ramipril (2 x 25, 10, 20 or 1 mg/kg per day or corresponding 1% doses for studying blood pressure-independent effects). METHODS AND RESULTS: Systolic blood pressure was reduced in SHRs receiving high doses of captopril, enalapril, fosinopril or ramipril (-61, -54, -35 and -47 mmHg), whereas low doses were ineffective. Left ventricular weight was decreased in animals treated with high doses (captopril/enalapril/fosinopril/ramipril: -17/-19/-17/-19%), but not low doses of agents. Media thickness of thoracal aorta was reduced by administering high doses (captopril/enalapril/fosinopril/ramipril: -31/-32/-27/-26%) and low doses (-16/-22/-22/-19%) of agents. ACE activity was reduced in heart, aorta and kidney of rats treated with high and low doses of all ACE inhibitors, whereby high doses showed more pronounced effects. Plasma endothelin-1 concentrations were not altered. A blood-pressure-ineffective treatment with an AT -antagonist revealed similar effects on cardiovascular hypertrophy. CONCLUSIONS: ACEIs reduce cardiovascular hypertrophy uniformly via an AT -receptor- mediated mechanism, reinforcing the opinion that ACEI effects are indeed class effects. The significance of local renin-angiotensin systems was confirmed by antihypertrophic effects in the aorta that were apparent in the absence of any blood pressure reduction.

Effects of lixisenatide once daily on gastric emptying in type 2 diabetes--relationship to postprandial glycemia.

OBJECTIVES: To determine the effects of lixisenatide, a new once-daily (QD) glucagon-like peptide-1 receptor agonist, on postprandial glucose (PPG) and gastric emptying, and the relationship between these effects in patients with type 2 diabetes mellitus (T2DM). METHODS: Data were obtained from a randomized, double-blind, placebo-controlled, parallel-group study with treatment duration of 28 days in patients with T2DM receiving ≤2 oral antidiabetic drugs. Lixisenatide was injected subcutaneously using an ascending dose range (5-20 µg) increased every fifth day in increments of 2.5 µg. Blood glucose was determined before and after three standardized meals (breakfast, lunch, and dinner). Gastric emptying of the standardized breakfast was determined by a (13)C-octanoic acid breath test at baseline (Day-1) and at Day 28. RESULTS: A total of 21 and 22 patients were randomized to lixisenatide 20 µg QD and placebo, respectively. With lixisenatide 20 µg QD, there was a reduction in PPG when compared with placebo after breakfast (p<0.0001), lunch (p<0.001) and dinner (p<0.05). Hence, lixisenatide 20 µg administered in the morning exhibited a pharmacodynamic effect on blood glucose throughout the day. Gastric emptying (50% emptying time) increased substantially from baseline with lixisenatide 20 µg QD, but not with placebo (change from baseline ± SD: -24.1 ± 133.1 min for placebo and 211.5 ± 278.5 min for lixisenatide; p<0.01). There was an inverse relationship between PPG area under the curve after breakfast and gastric emptying with lixisenatide 20 µg QD (n=17, r(2)=0.51, p<0.05), but not with placebo. CONCLUSIONS: In this study, lixisenatide at a dose of 20 µg QD reduced postprandial glycemic excursions in patients with T2DM, possibly as a result of sustained slowing of gastric emptying.

Inhibition of diacylglycerol-sensitive TRPC channels by synthetic and natural steroids.

TRPC channels are a family of nonselective cation channels that regulate ion homeostasis and intracellular Ca(2+) signaling in numerous cell types. Important physiological functions such as vasoregulation, neuronal growth, and pheromone recognition have been assigned to this class of ion channels. Despite their physiological relevance, few selective pharmacological tools are available to study TRPC channel function. We, therefore, screened a selection of pharmacologically active compounds for TRPC modulating activity. We found that the synthetic gestagen norgestimate inhibited diacylglycerol-sensitive TRPC3 and TRPC6 with IC(50)s of 3-5 µM, while half-maximal inhibition of TRPC5 required significantly higher compound concentrations (>10 µM). Norgestimate blocked TRPC-mediated vasopressin-induced cation currents in A7r5 smooth muscle cells and caused vasorelaxation of isolated rat aorta, indicating that norgestimate could be an interesting tool for the investigation of TRP channel function in native cells and tissues. The steroid hormone progesterone, which is structurally related to norgestimate, also inhibited TRPC channel activity with IC(50)s ranging from 6 to 18 µM but showed little subtype selectivity. Thus, TRPC channel inhibition by high gestational levels of progesterone may contribute to the physiological decrease of uterine contractility and immunosuppression during pregnancy.

Long-term severe diabetes only leads to mild cardiac diastolic dysfunction in Zucker diabetic fatty rats.

AIMS: Type 2 diabetes mellitus (DM) leads to cardiac dysfunction irrespective of hypertension and coronary artery disease; this is called diabetic cardiomyopathy. Here, we investigated the severity of diabetic cardiomyopathy and myocardial remodelling in aged Zucker diabetic fatty (ZDF) rats. METHODS AND RESULTS: Body weight, blood glucose and glycated haemoglobin (Hb(A1c)) levels, and urinary albumin excretion were monitored regularly in ZDF rats (n = 19) and control littermates (n = 19) up to age 45 weeks. ZDF rats were severely diabetic during the entire study period and demonstrated decreased body and heart weights at sacrifice. Left ventricular (LV) function was determined using magnetic resonance imaging (MRI) at age 44 weeks and revealed similar LV ejection fraction and cardiac output index in control and ZDF rats, indicating preserved systolic function. LV pressure characteristics assessed at age 45 weeks showed significant, but mild elevations of LV end-diastolic pressure (+45%) and relaxation time constant Tau (+54%) in ZDF rats, indicating diastolic dysfunction. Histological analyses revealed a significantly increased LV collagen content (+50%), but no cardiomyocyte hypertrophy in ZDF rats. CONCLUSION: The present study clearly shows that long term, severe DM in 45-week-old ZDF rats resulted in relatively mild impairment of diastolic LV function, whereas systolic function was well preserved. These data do not support the notion that diabetes per se is a critical factor in the induction of a clinically relevant degree of cardiac dysfunction. Co-morbidities such as hypertension and coronary artery disease probably have larger impacts on myocardial function in diabetic individuals.

Novel ß-amino acid derivatives as inhibitors of cathepsin A.

Cathepsin A (CatA) is a serine carboxypeptidase distributed between lysosomes, cell membrane, and extracellular space. Several peptide hormones including bradykinin and angiotensin I have been described as substrates. Therefore, the inhibition of CatA has the potential for beneficial effects in cardiovascular diseases. Pharmacological inhibition of CatA by the natural product ebelactone B increased renal bradykinin levels and prevented the development of salt-induced hypertension. However, so far no small molecule inhibitors of CatA with oral bioavailability have been described to allow further pharmacological profiling. In our work we identified novel ß-amino acid derivatives as inhibitors of CatA after a HTS analysis based on a project adapted fragment approach. The new inhibitors showed beneficial ADME and pharmacokinetic profiles, and their binding modes were established by X-ray crystallography. Further investigations led to the identification of a hitherto unknown pathophysiological role of CatA in cardiac hypertrophy. One of our inhibitors is currently undergoing phase I clinical trials.