Thiemo Pfab

The novel DPP-4 inhibitors linagliptin and BI 14361 reduce infarct size after myocardial ischemia/reperfusion in rats

BACKGROUND Dipeptidylpeptidase-4 inhibition is reported to have beneficial effects on myocardial ischemia. Mechanisms might include a reduced degradation of stromal cell-derived factor-1 alpha with subsequent increased recruitment of circulating stem cells and/or incretin receptor-dependent pathways. This study evaluated the novel xanthine-based dipeptidylpeptidase-4 inhibitors linagliptin (BI 1356) and BI 14361 in cardiac ischemia. METHODS Male Wistar rats were pretreated with linagliptin or BI 14361 and subjected to ligation of the left anterior descending coronary artery for 30 min. RESULTS Dipeptidylpeptidase-4 inhibition significantly reduced the infarct size after 7 days (-27.7%, p<0.05) and 8 weeks (-18.0%, p<0.05). There was a significantly improved maximum rate of left ventricular pressure decline (dP/dt min) in linagliptin-treated animals 8 weeks after ischemia/reperfusion. Apart from that, treatment did not improve cardiac function as determined by echocardiography and cardiac catheterization. Immunohistological staining revealed an increased number of cells positive for stromal cell-derived factor-1 alpha, CXCR-4 and CD34 within and around the infarcted area of BI 14361-treated animals. CONCLUSIONS Linagliptin and BI 14361 are able to reduce infarct size after myocardial ischemia. The immunohistological findings support the hypothesis that dipeptidylpeptidase-4 inhibition via reduced cleavage of stromal cell-derived factor-1 alpha might lead to an enhanced recruitment of CXCR-4+ circulating progenitor cells.

Loss of insulin-induced activation of TRPM6 magnesium channels results in impaired glucose tolerance during pregnancy

Hypomagnesemia affects insulin resistance and is a risk factor for diabetes mellitus type 2 (DM2) and gestational diabetes mellitus (GDM). Two single nucleotide polymorphisms (SNPs) in the epithelial magnesium channel TRPM6 (V1393I, K1584E) were predicted to confer susceptibility for DM2. Here, we show using patch clamp analysis and total internal reflection fluorescence microscopy, that insulin stimulates TRPM6 activity via a phosphoinositide 3-kinase and Rac1-mediated elevation of cell surface expression of TRPM6. Interestingly, insulin failed to activate the genetic variants TRPM6(V1393I) and TRPM6(K1584E), which is likely due to the inability of the insulin signaling pathway to phosphorylate TRPM6(T1391) and TRPM6(S1583). Moreover, by measuring total glycosylated hemoglobin (TGH) in 997 pregnant women as a measure of glucose control, we demonstrate that TRPM6(V1393I) and TRPM6(K1584E) are associated with higher TGH and confer a higher likelihood of developing GDM. The impaired response of TRPM6(V1393I) and TRPM6(K1584E) to insulin represents a unique molecular pathway leading to GDM where the defect is located in TRPM6.

Lack of endothelial nitric oxide synthase promotes endothelin-induced hypertension: lessons from endothelin-1 transgenic/endothelial nitric oxide synthase knockout mice.

Endothelin-1 (ET-1) is one of the most potent biologic vasoconstrictors. Nevertheless, transgenic mice that overexpress ET-1 exhibit normal BP. It was hypothesized that vascular effects of ET-1 may be antagonized by an increase of the endothelial counterpart of ET-1, nitric oxide (NO), which is produced by the endothelial NO synthase (eNOS). Therefore, cross-bred animals of ET transgenic mice (ET+/+) and eNOS knockout (eNOS-/-) mice and were generated, and BP and endothelial function were evaluated in these animals. Endothelium-dependent and -independent vascular function was assessed as relaxation/contraction of isolated preconstricted aortic rings. The tissue ET and NO system was determined in aortic rings by quantitative real-time PCR and Western blotting. Systolic BP was similar in ET+/+ and wild-type (WT) mice but was significantly elevated in eNOS-/- mice (117 +/- 4 mmHg versus 94 +/- 6 mmHg in WT mice; P < 0.001) and even more elevated in ET+/+ eNOS-/- cross-bred mice (130 +/- 4 mmHg; P < 0.05 versus eNOS-/-). Maximum endothelium-dependent relaxation was enhanced in ET+/+ mice (103 +/- 6 versus 87 +/- 4% of preconstriction in WT littermates; P < 0.05) and was completely blunted in eNOS-/- (-3 +/- 4%) and ET+/+ eNOS-/- mice (-4 +/- 4%), respectively. Endothelium-independent relaxation was comparable among all groups. Quantitative real-time PCR as well as Western blotting revealed an upregulation of the aortic ET(A) and ET(B) receptors in ET+/+ eNOS-/-, whereas eNOS was absent in aortic rings of eNOS-/- and ET+/+ eNOS-/- mice. ET-1 aortic tissue concentrations were similar in WT mice and ET+/+ eNOS-/- mice most probably as a result of an enhanced clearance of ET-1 by the upregulated ET(B) receptor. These data show for the first time that in transgenic mice that overexpress human ET-1, additional knockout of eNOS results in a further enhancement of BP as compared with eNOS-/- mice. The human ET+/+ eNOS-/- mice therefore represent a novel model of hypertension as a result of an imbalance between the vascular ET-1 and NO systems.

Low Birth Weight, a Risk Factor for Cardiovascular Diseases in Later Life, Is Already Associated With Elevated Fetal Glycosylated Hemoglobin at Birth

Background— It remains unclear whether the association between low birth weight and insulin resistance in adulthood has its origin in utero or whether it develops later in life depending on predisposition and exogenous factors. Methods and Results— Total glycosylated hemoglobin (TGH) was quantified at delivery in 1295 mother/child pairs serving as a surrogate of maternal and fetal glycemia. Multivariable regression analysis considering gestational age at delivery, the child’s sex, maternal body mass index, and smoking during pregnancy revealed that an increase in TGH by 1% in the child was significantly associated with a mean birth weight reduction of 135 g (P<0.0001), whereas the same increase in the mother was associated with a mean birth weight increase of 88 g (P<0.0001). The ratio of fetal/maternal TGH suggests that lighter newborns have a higher percentage of TGH than would be expected from maternal TGH. Conclusions— The study demonstrates for the first time in a large population that there is an inverse association between TGH of a newborn and its birth weight. This might be due to increased insulin resistance in newborns with lower birth weight. Our data suggest that the pathophysiological mechanisms linking prenatal growth and postnatal sensitivity to insulin are present as early as before birth.

Endothelin-Converting Enzyme/Neutral Endopeptidase Inhibitor SLV338 Prevents Hypertensive Cardiac Remodeling in a Blood Pressure–Independent Manner

Hypertensive heart disease is a major contributor to cardiovascular mortality. Endothelin is a potent vasoconstrictive and profibrotic mediator produced by the endothelin-converting enzyme (ECE), whereas natriuretic peptides, degraded by the neutral endopeptidase (NEP), have diuretic, vasodilatory, and antifibrotic properties. Thus, combined ECE/NEP inhibition may halt hypertensive cardiac remodeling. This study examined effects of SLV338, a novel ECE/NEP inhibitor, on cardiac protection in experimental renovascular hypertension (2-kidney, 1-clip [2K1C]). Male rats were allocated to 5 groups: sham-operated rats, untreated animals with 2K1C, 2K1C animals treated with oral SLV338 (30 and 100 mg/kg per day), and 2K1C animals treated with oral losartan (20 mg/kg per day). Treatment duration was 12 weeks. Blood pressure was assessed every 4 weeks. At study end, hearts were taken for histology/computer-aided histomorphometry/immunohistochemistry. Pharmacological properties of SLV338 are described. SLV338 is a dual ECE/NEP inhibitor, as demonstrated both in vitro and in vivo. In the 2K1C study, losartan lowered blood pressure by ⩽46 mm Hg, whereas both dosages of SLV338 had no effect. However, SLV338 (both dosages) completely normalized cardiac interstitial fibrosis, perivascular fibrosis, myocyte diameter, and media:lumen ratio of cardiac arteries, as did losartan. Cardiac transforming growth factor-&bgr;1 expression was significantly enhanced in untreated 2K1C rats versus controls, whereas treatment with SLV338 and losartan prevented this effect. Taken together, dual ECE/NEP inhibitor SLV338 prevents cardiac remodeling to the same extent as losartan, but in a blood pressure–independent manner, in a rat model of renovascular hypertension. This effect is at least partially mediated via suppression of cardiac transforming growth factor-&bgr;1 expression.

Diabetic endothelin B receptor–deficient rats develop severe hypertension and progressive renal failure

The endothelin (ET) system has been implicated in the pathogenesis of diabetic nephropathy. The role of the ET-B receptor (ETBR) is still unclear. The effect of ETBR deficiency on the progression of diabetic nephropathy in a streptozotocin model was analyzed in four groups: (1) Homozygous ETBR-deficient (ETBRd) diabetic rats, (2) ETBRd rats, (3) diabetic controls, and (4) wild-type controls. BP and kidney function were measured for 10 wk, followed by biochemical and histologic analysis of the kidneys. The study demonstrates that ETBRd diabetic rats on a normal-sodium diet develop severe hypertension, albuminuria, and a mild reduction of creatinine clearance. The strong BP rise seems not to be caused by activation of the renin-angiotensin-aldosterone system or by suppression of the nitric oxide system. Elevated plasma ET-1, possibly reflecting a reduced ETBR-dependent clearance, seems to cause the severe hypertension via the ETA receptor. The results do not support the hypothesis that a reduction of ETBR activity inhibits the progression of diabetic nephropathy. The study demonstrates for the first time that the combination of diabetes and ETBR deficiency causes severe low-renin hypertension with progressive renal failure.

Fetal sex determines the impact of maternal PROGINS progesterone receptor polymorphism on maternal physiology during pregnancy.

Background Recent evidence from very rare human diseases suggests that variation in the fetal genome can modify maternal physiology during pregnancy. Here, we tested the hypothesis that fetal sex as a major genetic variant of the fetal genome may affect maternal physiology during pregnancy in genetically susceptible pregnant women. Methods We analyzed the impact of fetal sex on maternal physiology during pregnancy in relationship with the maternal PROGINS progesterone receptor gene polymorphism. Two thousand and eighty-nine (2089) Caucasian women without preexisting diabetes and preexisting hypertension with singleton pregnancies delivering consecutively at the Charité obstetrics department participated in this study. Results The maternal PROGINS progesterone receptor polymorphism on its own had no effect on blood pressure, new onset of proteinuria, and total glycated hemoglobin at delivery. However, by considering the offsprings sex, the AA variant of the PROGINS progesterone receptor polymorphism was associated with profound cardiovascular/metabolic effects; mothers carrying both A alleles (AA genotype) delivering a boy had significantly lower systolic blood pressure during the first trimester of pregnancy versus AA mothers delivering girls (107.9±10.2 vs. 116.6±15.1 mmHg, P = 0.044). Diastolic blood pressure was similarly lower during the first trimester of pregnant AA women delivering boys in comparison with AA women delivering girls (63.4±5.7 vs. 68.2±10.9 mmHg, P = 0.032). Total glycated hemoglobin at delivery was significantly (P = 0.002) higher in AA mothers delivering boys (6.6±0.7%) versus AA mothers delivering girls (5.9±0.6%). Conclusion Our study indicates that fetal sex may substantially affect maternal blood pressure as well as glycemic control during pregnancy in genetically susceptible mothers.

Endothelin B receptor-deficient mice develop endothelial dysfunction independently of salt loading.

Background Rodents without a functional endothelin B (ETB) receptor develop salt-sensitive hypertension. The underlying mechanisms, however, are so far unknown. The ETB receptor is involved in endothelial function by modulating the activity of the endothelial nitric oxide synthesis as well as contributing to the control of endothelial prostacyclin synthesis. In the present study, we analysed whether salt alters endothelial function in rescued ETB receptor-deficient mice. We used mice with a rescue of the lethal phenotype of an ETB knockout. These mice were generated by crossbreeding ETB–/– mice with dopamine-hydroxylase ETB transgenic mice. Methods Adult rescued ETB-deficient mice were kept in parallel with wild-type control animals for 15 days on standard (0.2% NaCl) or salt-enriched (4% NaCl) chow, respectively. Systolic blood pressure was measured by the tail cuff method and endothelium-dependent and endothelium-independent vascular function was assessed in isolated aortic rings under isometric conditions. Results Systolic blood pressure increased on salt-enriched chow in ETB receptor-deficient mice (166 ± 12 mmHg), but neither in wild-type mice on high-salt diet (128 ± 11 mmHg; P < 0.05) nor in ETB receptor-deficient mice on standard chow. The heart rate was similar in all groups at any point of time. Endothelium-dependent relaxation was impaired in ETB receptor-deficient mice (74 ± 3 versus 96 ± 5% of preconstriction for wild-type mice; P < 0.05) and was not significantly affected by a salt-enriched diet. Endothelium-independent relaxation was similar among all groups. Contractions to endothelin-1 were not significantly influenced by preincubation with the ETB receptor antagonist BQ-788, but were completely blunted by preincubation with the ETA receptor antagonist BQ-123 in all animals. Conclusion Rescued ETB receptor-deficient mice develop salt-sensitive hypertension. Nevertheless, in this animal model of ETB receptor deficiency, endothelial function is impaired independent of salt-enriched diet or hypertension. This indicates that, in this model, salt-induced hypertension is not mediated by endothelial dysfunction.

Effects of Aliskiren on Stroke in Rats Expressing Human Renin and Angiotensinogen Genes

Objective Pre-treatment with angiotensin receptor blockers is known to improve neurological outcome after stroke. This study investigated for the first time, whether the renin inhibitor aliskiren has similar neuroprotective effects. Methods Since aliskiren specifically blocks human renin, double transgenic rats expressing human renin and angiotensinogen genes were used. To achieve a systolic blood pressure of 150 or 130 mmHg animals were treated with aliskiren (7.5 or 12.5 mg/kg*d) or candesartan (1.5 or 10 mg/kg*d) via osmotic minipump starting five days before middle cerebral artery occlusion with reperfusion. Infarct size was determined by magnetic resonance imaging. mRNA of inflammatory marker genes was studied in different brain regions. Results The mortality of 33.3% (7 of 21 animals) in the vehicle group was reduced to below 10% by treatment with candesartan or aliskiren (p<0.05). Aliskiren-treated animals had a better neurological outcome 7 days post-ischemia, compared to candesartan (Garcia scale: 9.9±0.7 vs. 7.3±0.7; p<0.05). The reduction of infarct size in the aliskiren group did not reach statistical significance compared to candesartan and vehicle (24 h post-ischemia: 314±81 vs. 377±70 and 403±70 mm3 respectively). Only aliskiren was able to significantly reduce stroke-induced gene expression of CXC chemokine ligand 1, interleukin-6 and tumor necrosis factor-alpha in the ischemic core. Conclusions Head-to-head comparison suggests that treatment with aliskiren before and during cerebral ischemia is at least as effective as candesartan in double transgenic rats. The improved neurological outcome in the aliskiren group was blood pressure independent. Whether this effect is due to primary anti-inflammatory mechanisms has to be investigated further.

Pathophysiology of the endothelin system - lessons from genetically manipulated animal models

Shortly after discovery of ET-1 in 1988, the entire endothelin system was characterized. The endothelin system consists of the three peptides ET-1, ET-2 and ET-3, their G-protein-coupled receptors endothelin receptor A and B (ETRA and ETRB) and the two endothelin-converting enzymes (ECE-1 and ECE-2). Genetically modified animal models are an important tool in biomedical research. Here we describe the key findings obtained from genetically modified animal models either over-expressing compounds of the ET system or lacking these compounds (knockout mice). Results from the different transgenic and knockout models disclose that the ET system plays a major role in embryonic development. Two ET system-dependent neural crest-driven developmental pathways become obvious: one of them being an ET-1/ETAR axis, responsible for cardio-renal function and development as well as cranial development; the other seems to be an ET-3/ETBR mediated signalling pathway. Mutations within this axis are associated with disruptions in epidermal melanocytes and enteric neurons. These findings led to the discovery of similar findings in humans with Hirschsprung disease. In adult life the ET system is most important in the cardiovascular system and plays a role in fibrotic remodelling of the heart, lung and kidney as well as in the regulation of water and salt excretion.