Thomas Hübschle

Antiatherosclerotic Effects of Small-Molecular-Weight Compounds Enhancing Endothelial Nitric-Oxide Synthase (eNOS) Expression and Preventing eNOS Uncoupling

Many cardiovascular diseases are associated with reduced levels of bioactive nitric oxide (NO) and an uncoupling of oxygen reduction from NO synthesis in endothelial NO synthase (eNOS uncoupling). In human endothelial EA.hy 926 cells, two small-molecular-weight compounds with related structures, 4-fluoro-N-indan-2-yl-benzamide (CAS no. 291756-32-6; empirical formula C16H14FNO; AVE9488) and 2,2-difluoro-benzo[1,3]dioxole-5-carboxylic acid indan-2-ylamide (CAS no. 450348-85-3; empirical formula C17H13F2NO3; AVE3085), enhanced eNOS promoter activity in a concentration-dependent manner; with the responsible cis-element localized within the proximal 263 base pairs of the promoter region. RNA interference-mediated knockdown of the transcription factor Sp1 significantly reduced the basal activity of eNOS promoter, but it did not prevent the transcription activation by the compounds. Enhanced transcription of eNOS by AVE9488 in primary human umbilical vein endothelial cells was associated with increased levels of eNOS mRNA and protein expression, as well as increased bradykinin-stimulated NO production. In both wild-type C57BL/6J mice and apolipoprotein E-knockout (apoE-KO) mice, treatment with AVE9488 resulted in enhanced vascular eNOS expression. In apoE-KO mice, but not in eNOS-knockout mice, treatment with AVE9488 reduced cuff-induced neointima formation. A 12-week treatment with AVE9488 or AVE3085 reduced atherosclerotic plaque formation in apoE-KO mice, but not in apoE/eNOS-double knockout mice. Aortas from apoE-KO mice showed a significant generation of reactive oxygen species. This was partly prevented by nitric-oxide inhibitor Nω-nitro-l-arginine methyl ester, indicating eNOS uncoupling. Treatment of mice with AVE9488 enhanced vascular content of the essential eNOS cofactor (6R)-5,6,7,8-tetrahydro-l-biopterin and reversed eNOS uncoupling. The combination of an up-regulated eNOS expression and a reversal of eNOS uncoupling is probably responsible for the observed vasoprotective properties of this new type of compounds.

Cardioprotective effects of lixisenatide in rat myocardial ischemia-reperfusion injury studies

BackgroundLixisenatide is a glucagon-like peptide-1 analog which stimulates insulin secretion and inhibits glucagon secretion and gastric emptying. We investigated cardioprotective effects of lixisenatide in rodent models reflecting the clinical situation.MethodsThe acute cardiac effects of lixisenatide were investigated in isolated rat hearts subjected to brief ischemia and reperfusion. Effects of chronic treatment with lixisenatide on cardiac function were assessed in a modified rat heart failure model after only transient coronary occlusion followed by long-term reperfusion. Freshly isolated cardiomyocytes were used to investigate cell-type specific mechanisms of lixisenatide action.ResultsIn the acute setting of ischemia-reperfusion, lixisenatide reduced the infarct-size/area at risk by 36% ratio without changes on coronary flow, left-ventricular pressure and heart rate. Treatment with lixisenatide for 10 weeks, starting after cardiac ischemia and reperfusion, improved left ventricular end-diastolic pressure and relaxation time and prevented lung congestion in comparison to placebo. No anti-fibrotic effect was observed. Gene expression analysis revealed a change in remodeling genes comparable to the ACE inhibitor ramipril. In isolated cardiomyocytes lixisenatide reduced apoptosis and increased fractional shortening. Glucagon-like peptide-1 receptor (GLP1R) mRNA expression could not be detected in rat heart samples or isolated cardiomyocytes. Surprisingly, cardiomyocytes isolated from GLP-1 receptor knockout mice still responded to lixisenatide.ConclusionsIn rodent models, lixisenatide reduced in an acute setting infarct-size and improved cardiac function when administered long-term after ischemia-reperfusion injury. GLP-1 receptor independent mechanisms contribute to the described cardioprotective effect of lixisenatide. Based in part on these preclinical findings patients with cardiac dysfunction are currently being recruited for a randomized, double-blind, placebo-controlled, multicenter study with lixisenatide.Trial registration(ELIXA, ClinicalTrials.gov Identifier: NCT01147250)

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.

Novel β-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 β-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.

Cardiac remodeling and myocardial dysfunction in obese spontaneously hypertensive rats

BackgroundThe additive effects of obesity and metabolic syndrome on left ventricular (LV) maladaptive remodeling and function in hypertension are not characterized.MethodsWe 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.ResultsSystolic 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.ConclusionIn 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.

Animal Models of Diabetic Macrovascular Complications: Key Players in the Development of New Therapeutic Approaches

Diabetes mellitus is a lifelong, incapacitating metabolic disease associated with chronic macrovascular complications (coronary heart disease, stroke, and peripheral vascular disease) and microvascular disorders leading to damage of the kidneys (nephropathy) and eyes (retinopathy). Based on the current trends, the rising prevalence of diabetes worldwide will lead to increased cardiovascular morbidity and mortality. Therefore, novel means to prevent and treat these complications are needed. Under the auspices of the IMI (Innovative Medicines Initiative), the SUMMIT (SUrrogate markers for Micro- and Macrovascular hard end points for Innovative diabetes Tools) consortium is working on the development of novel animal models that better replicate vascular complications of diabetes and on the characterization of the available models. In the past years, with the high level of genomic information available and more advanced molecular tools, a very large number of models has been created. Selecting the right model for a specific study is not a trivial task and will have an impact on the study results and their interpretation. This review gathers information on the available experimental animal models of diabetic macrovascular complications and evaluates their pros and cons for research purposes as well as for drug development.

Inhibition of CatA: an emerging strategy for the treatment of heart failure

The lysosomal serine carboxypeptidase CatA has a very important and well-known structural function as well as a, so far, less explored catalytic function. A complete loss of the CatA protein results in the lysosomal storage disease galactosialidosis caused by intralysosomal degradation of β-galactosidase and neuraminidase 1. However, mice with a catalytically inactive CatA enzyme show no signs of this disease. This observation establishes a clear distinction between structural and catalytic functions of the CatA enzyme. Recently, several classes of orally bioavailable synthetic inhibitors of CatA have been identified. Pharmacological studies in rodents indicate a remarkable influence of CatA inhibition on cardiovascular disease progression and identify CatA as a promising novel target for the treatment of heart failure.

Guanylin and uroguanylin mRNA expression is increased following Roux-en-Y gastric bypass, but guanylins do not play a significant role in body weight regulation and glycemic control

HighlightsGut expression of guanylin peptides is elevated in obese patients upon gastric bypass.Guanylin and uroguanylin show no body weight or glucoregulatory effects in mice.Uroguanylin has no effect on glucose‐stimulated insulin secretion in rat pancreas.Adenoviral gene delivery of uroguanylin is weight‐neutral in diet‐induced obese rats.Guanylin peptides do not show promise as novel targets in diabetes and obesity. Aim: To determine whether intestinal expression of guanylate cyclase activator 2A (GUCA2A) and guanylate cyclase activator 2B (GUCA2B) genes is regulated in obese humans following Roux‐en‐Y gastric bypass (RYGB), and to evaluate the corresponding guanylin (GN) and uroguanylin (UGN) peptides for potentially contributing to the beneficial metabolic effects of RYGB. Methods: Enteroendocrine cells were harvested peri‐ and post‐RYGB, and GUCA2A/GUCA2B mRNA expression was compared. GN, UGN and their prohormones (proGN, proUGN) were administered subcutaneously in normal‐weight mice to evaluate effects on food intake and glucose regulation. The effect of pro‐UGN or UGN overexpression, using adeno‐associated virus (AAV) vectors, was assessed in diet‐induced obese (DIO) mice. Intracerebroventricular administration of GN and UGN was performed in rats for assessment of putative centrally mediated effects on food intake. GN and UGN, as well as their prohormones, were evaluated for effects on glucose‐stimulated insulin secretion (GSIS) in rat pancreatic islets and perfused rat pancreas. Results: GUCA2A and GUCA2B mRNA expression was significantly upregulated in enteroendocrine cells after RYGB. Peripheral administration of guanylins or prohormones did not influence food intake, oral glucose tolerance, and GSIS. Central administration of GN and UGN did not affect food intake in rats. Chronic AVV‐mediated overexpression of UGN and proUGN had no effect on body weight or glucose homeostasis in DIO mice. Conclusion: GN and UGN, as well as their prohormones, do not seem to play a significant role in body weight regulation and glycemic control, suggesting that guanylin‐family peptides do not show promise as targets for the treatment of obesity or diabetes.