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Hans Michael Piper

Publications and source records attributed to Hans Michael Piper.

At least 19 recordsLinked to original sources
PubMed2006

Impaired release of bioactive parathyroid hormone-related peptide in patients with pulmonary hypertension and endothelial dysfunction.

BACKGROUND: Parathyroid hormone-related protein (PTHrP) is an endothelial-derived vasoactive peptide. This study investigated whether bioactive PTHrP is locally released in a pressure-dependent way. METHODS: A PTHrP antibody directed against the midregional part of PTHrP was used to analyze PTHrP in plasma samples. The biological activity of this PTHrP-like peptide was investigated in vitro. Plasma values were determined in samples from the left pulmonary artery and the arteria femoralis, taken under basal conditions and after the application of oxygen or iloprost to lower the pulmonary pressure. Twenty young patients (mean age 6.5 years), who were catheterized for an analysis of the reactivity of the pulmonary bed, were investigated. Endothelial function was investigated by acetylcholine responsiveness. RESULTS: The antibody recognized a 30-kDa protein with in vitro PTHrP-like activity. In 11 patients (responders) with intact endothelial function, the PTHrP values determined in the left pulmonary artery were higher than those in the arteria femoralis. The local increase in the PTHrP concentration was reduced when either oxygen or iloprost lowered the pressure. Nine patients with endothelial dysfunction did not show any concentration gradients at any time (nonresponders). CONCLUSIONS: The local concentration of bioactive PTHrP is increased in patients with pulmonary hypertension and normal endothelial function.

Acetylcholine↗
PubMed2006

Contractile performance of adult ventricular rat cardiomyocytes is not directly jeopardized by NO/cGMP-dependent induction of pro-apoptotic pathways.

The activation of NO/cGMP pathways can induce pro-apoptotic pathways in cardiomyocytes although only a small number of cardiomyocytes fulfill the criteria of apoptosis. The same pathways reduce the contractile performance of cardiomyocytes. In the present study, we tested the hypothesis that exposure of cells to NO/cGMP for 24 h decrease their contractile performance due to an activation of pro-apoptotic pathways. Experiments were performed on freshly isolated and cultured adult ventricular rat cardiomyocytes. Cells were incubated with 8-bromo-cyclo-GMP (100 nmol/L-1 micromol/L), the NO donor SNAP (1 nmol/L-100 micromol/L), or the guanylyl cyclase activator YC-1 (3 micromol/L). Cell shortening, contraction and relaxation velocities, and diastolic cell lengths were determined at beating frequencies of 0.5, 1, and 2 Hz 24 h later. The activation of pro-apoptotic pathways was determined by staining of cardiomyocytes with an antibody directed against active caspase-3 and quantification of the number of apoptotic cells (annexin staining). Caspase-3 activation and an increase in the number of apoptotic cells was observed, but only at the highest concentrations tested (8-bromo-cyclo-GMP: 1-10 mmol/L; SNAP: 1-100 micromol/L). At these concentrations, none of the drugs decreased the mean cell shortening of cardiomyocytes. However, at concentrations lower than those required for induction of apoptotic cell death, the diastolic cell lengths and sarcomere lengths increased but cell shortening decreased. In conclusion, low concentrations of either NO or cGMP cause a desensitization of myofibrils, as indicated by elongated cell shapes, increased sarcomere lengths and reduced load-free cell shortening. High concentrations of NO/cGMP induce caspase-3 activation and increase the number of cells fulfilling the criteria of apoptotic cell death but did not impair cell function. Therefore, induction of apoptotic cell death per se seems not to contribute to the loss of contractile efficiency on the cellular level.

Animals↗
PubMed2006

Ischemic acidosis causes apoptosis in coronary endothelial cells through activation of caspase-12.

OBJECTIVE: Myocardial ischemia has been shown to induce apoptosis of endothelial cells (EC). However, the mechanism of this endothelial injury is still poorly understood. To analyse the signaling pathway of ischemia-induced EC apoptosis was the aim of the present study. METHODS: The primary culture of rat coronary EC was exposed to simulated ischemia (glucose-free anoxia at pH(o) 6.4). Apoptosis was defined by staining of nuclei with Hoechst-33342 and TUNEL. Cytosolic Ca2+ and pH were measured with Fura-2 and BCECF, respectively. RESULTS: Apoptosis (29.2+/-1.7% of cells) induced by exposure to simulated ischemia for 2 h was accompanied by cytosolic Ca2+ overload (1090+/-52 nmol/l) and acidosis (pHi = 6.52+/-0.13). Simulated ischemia had no significant effect on caspase-8 cleavage, but induced cleavage of caspase-3 and caspase-12 and led to a slight release of cytochrome C. Prevention of cytosolic acidosis (anoxia at pH(o) 7.4) had no effect on cytochrome C release, but significantly reduced apoptosis, attenuated cytosolic Ca2+ overload, and prevented cleavage of caspase-12. A similar effect was achieved by inhibition of Ca2+ release channels in the endoplasmic reticulum with ryanodine and xestospongin C. Knock-down of caspase-12 with small interfering RNA suppressed caspase-3 activation and reduced apoptotic cell number by about 70%. CONCLUSION: Acidosis, rather than anoxia, is an important trigger of apoptosis in EC under simulated ischemia. The main pathway of the simulated ischemia-induced apoptosis consists of the Ca2+ leak from the ER followed by activation of caspase-12 and caspase-3.

Acidosis↗
PubMed2006

Accumulation of extracellular ATP protects against acute reperfusion injury in rat heart endothelial cells.

OBJECTIVE: Ischemia-reperfusion provokes barrier failure of the coronary microvasculature, leading to myocardial edema development that jeopardizes functional recovery of the heart during reperfusion. Here, we tested whether adenosine 5'-triphosphate (ATP), either exogenously applied or spontaneously released during reperfusion, protects the endothelial barrier against an imminent reperfusion injury and whether interventions preventing ATP breakdown augment this protective ATP effect. METHODS: Cultured microvascular coronary endothelial monolayers and isolated-perfused hearts of rat were used. RESULTS: After ischemic conditions were induced, reperfusion of endothelial monolayers activated the endothelial contractile machinery and caused intercellular gap formation. It also led to the release of ATP. When its breakdown was inhibited by 6-N,N-diethyl-beta,gamma-dibromomethylene-D-ATP (ARL 67156; 100 microM), a selective ectonucleotidase inhibitor, contractile activation and gap formation were significantly reduced. Reperfusion in the presence of exogenously added ATP (10 microM) plus ARL caused an additional reduction of both aforementioned effects. In contrast, elevation of ATP degradation by apyrase (1 U/ml), a soluble ectonucleotidase, or addition of adenosine (10 microM) provoked an increase in gap formation during reperfusion that could be completely inhibited by 8-phenyltheophylline (8-PT; 10 microM), an adenosine receptor antagonist. In Langendorff-perfused rat hearts, the reperfusion-induced increase in water content was significantly reduced by ARL plus ATP. Under conditions favouring ATP degradation, an increase in myocardial edema was observed that could be blocked by 8-PT. CONCLUSION: ATP, either released from cells or exogenously applied, protects against reperfusion-induced failure of the coronary endothelial barrier. Inhibition of ATP degradation enhances the stabilizing effect of ATP on barrier function.

Adenosine↗
PubMed2006

Contribution of PI 3-kinase isoforms to angiotensin II- and alpha-adrenoceptor-mediated signalling pathways in cardiomyocytes.

OBJECTIVE: Angiotensin II stimulation increases the formation of reactive oxygen species (ROS), the phosphorylation of p38 mitogen-activated protein kinase (MAPK), and the expression of transforming growth factor beta (TGFbeta) in adult cardiomyocytes. The aim of this study was to determine the involvement of PI 3-kinase and to specify the participation of different isoforms in the angiotensin II-induced formation of ROS in comparison to the hypertrophic pathway triggered by alpha-adrenoceptor stimulation. METHODS: Freshly isolated myocytes were used to examine formation of ROS via H(2)DCF fluorescence. p38 MAPK phosphorylation, p70(S6)-kinase phosphorylation, PI 3-kinase, and TGFbeta expression were measured by Western blotting. Sense and antisense oligonucleotides were used to down-regulate diverse PI 3-kinase isoforms. Hypertrophy was measured by (14)C-phenylalanine incorporation and cell volume. RESULTS: Inhibition of PI 3-kinase by Ly294002 or wortmannin, two inhibitors, decreased formation of ROS, phosphorylation of p38 MAPK, and TGFbeta expression. Down-regulation of the p110beta isoform by antisense oligonucleotides inhibited the angiotensin II-induced signalling pathway but not the alpha-adrenoceptor-mediated hypertrophic growth of cardiomyocytes. In contrast, down-regulation of the p110alpha isoform decreased the alpha-adrenoceptor-mediated hypertrophic growth of cardiomyocytes but did not affect the angiotensin II-mediated signalling pathway. CONCLUSION: Thus, our study identifies an involvement of PI 3-kinase in the angiotensin II-induced formation of ROS and provides a biochemical basis for ligand-specific responses for angiotensin II and alpha-adrenoceptor stimulation as relates to hypertrophy.

Angiotensin II↗
PubMed2005

PKC-independent signal transduction pathways increase SERCA2 expression in adult rat cardiomyocytes.

Catecholamines seem to play a major role in the initial response of the heart to pressure overload. The mechanisms by which alpha(1A)-adrenoceptor stimulation increases protein synthesis and subsequently cell size have been worked out in the past. However, little is known about the functional consequence of this type of hypertrophy. Recent transgenic work seems to indicate an adaptive character of this response, but mechanistic insights have yet to be established. The present study investigates whether chronic (overnight) exposure of cardiomyocytes to phenylephrine, an alpha-adrenoceptor agonist, modifies the expression of calcium-handling proteins and identifies key elements of signal transduction pathways leading to such alterations. Cardiomyocytes exposed to phenylephrine had elevated expression of SR-calcium ATPase (SERCA), but not of the sodium-calcium exchanger (NCX). SERCA induction persisted in the presence of protein kinase C (PKC) inhibitors, but required an increase in diastolic cell calcium levels via activation of the sodium-proton exchanger (NHE) and the reverse mode of the NCX. Downstream of an increase in resting cell calcium concentrations an activation of the calcineurin/NFAT pathway was found to be responsible for SERCA2 induction. Transfection of cardiomyocytes with decoys directed against NFAT activity inhibited the increase in SERCA2 expression. Decoys did not inhibit the concomitant PKC-dependent increase in hypertrophic growth. In the absence of SERCA up-regulation, hypertrophied cardiomyocytes were unable to maintain normal, load-free cell shortening. In conclusion, our data give mechanistic insights into the adaptional process during alpha-adrenoceptor-dependent myocardial hypertrophy.

Adrenergic alpha-Agonists↗
PubMed2005

Margatoxin inhibits VEGF-induced hyperpolarization, proliferation and nitric oxide production of human endothelial cells.

BACKGROUND: Vascular endothelial growth factor (VEGF) induces proliferation of endothelial cells (EC) in vitro and angiogenesis in vivo. Furthermore, a role of VEGF in K(+) channel, nitric oxide (NO) and Ca(2+) signaling was reported. We examined whether the K(+) channel blocker margatoxin (MTX) influences VEGF-induced signaling in human EC. METHODS: Fluorescence imaging was used to analyze changes in the membrane potential (DiBAC), intracellular Ca(2+) (FURA-2) and NO (DAF) levels in cultured human EC derived from human umbilical vein EC (HUVEC). Proliferation of HUVEC was examined by cell counts (CC) and [(3)H]-thymidine incorporation (TI). RESULTS: VEGF (5--50 ng/ml) caused a dose-dependent hyperpolarization of EC, with a maximum at 30 ng/ml (n=30, p<0.05). This effect was completely blocked by MTX (5 micromol/l). VEGF caused an increase in transmembrane Ca(2+) influx (n=30, p<0.05) that was sensitive to MTX and the blocker of transmembrane Ca(2+) entry 2-aminoethoxydiphenyl borate (APB, 100 micromol/l). VEGF-induced NO production was significantly reduced by MTX, APB and a reduction in extracellular Ca(2+) (n=30, p<0.05). HUVEC proliferation, examined by CC and TI, was significantly increased by VEGF and inhibited by MTX (CC: -58%, TI --121%); APB (CC --99%, TI--187%); N-monomethyl-L-arginine (300 micromol/l: CC: -86%, TI --164%). CONCLUSIONS: VEGF caused an MTX-sensitive hyperpolarization which results in an increased transmembrane Ca(2+) entry that is responsible for the effects on endothelial proliferation and NO production.

Calcium↗
PubMed2005

SMAD proteins are involved in apoptosis induction in ventricular cardiomyocytes.

OBJECTIVE: The transcription factor AP-1 is a mediator of hypertrophic growth and apoptosis in cardiomyocytes. This puts AP-1 in the center of two important processes found in the failing heart and implies that variations (i) in the AP-1 composition itself or (ii) in additional, interacting transcription factors are responsible for the diverse actions of AP-1. To test this hypothesis, we performed studies on isolated ventricular cardiomyocytes of rat under hypertrophy- or apoptosis-inducing conditions. METHODS AND RESULTS: The NO donor SNAP (100 microM), which is a pro-apoptotic stimulus in cardiomyocytes, activated AP-1 within 2 h. c-Jun, JunB and FosB are identified as the main components of this AP-1 complex. This complex formation is identical to the composition of AP-1 found under hypertrophic growth stimulation by phenylephrine (PE, 10 muM). Analysis of other transcription factors able to interact with AP-1 revealed activation of SMAD activity only during stimulation with SNAP to 131+/-9.6% (p < 0.05 vs. control, n = 9). The SMAD complex is formed from SMAD4 and 3. Intracellular scavenging of SMAD proteins by transformation of cardiomyocytes with SMAD decoy oligonucleotides or inhibition of SMAD4 synthesis using SMAD4 antisense oligonucleotides reduced the number of apoptotic cells under stimulation with SNAP from 13.3 +/- 1.2% to control levels (8 +/- 1%, p < 0.05, n = 6). TGFbeta, which is a known stimulator of SMAD proteins, is also shown to stimulate apoptosis in cardiomyocytes. Again, simultaneous activation of AP-1 and SMAD is needed for this apoptosis induction. CONCLUSIONS: In conclusion, AP-1/SMAD signaling has been identified as a common pathway in cardiomyocyte apoptosis. In contrast, SMAD proteins are dispensable for AP-1-mediated hypertrophic growth. This finding characterizes SMAD proteins as potential candidates for proteins that shift AP-1 signaling from hypertrophy to apoptosis.

Adrenergic alpha-Agonists↗
PubMed2005

p38 MAP-kinase in cultured adult rat ventricular cardiomyocytes: expression and involvement in hypertrophic signalling.

Both alpha-adrenoceptor- and beta-adrenoceptor-stimulation lead to hypertrophic growth of the myocardium. But only beta-adrenoceptor-stimulation requires the pre-cultivation of cells with active TGF-beta. In order to define signalling molecules that are specifically involved in beta-adrenoceptor-dependent hypertrophy, changes in expression and hypertrophic responsiveness during pre-cultivation with TGF-beta were investigated. Isolated adult ventricular cardiomyocytes from rats were either cultured in 20% (v/v) foetal calf serum (FCS) to activate autocrine released TGF-beta or used without pre-treatment. Protein synthesis was analysed by (14)C-phenylalanine incorporation. Expression of signalling molecules was determined by immunoblotting. During cultivation of cardiomyocytes with active TGF-beta only the expression of p38 MAP-kinase increased. Subsequent stimulation of beta-adrenoceptors induced protein synthesis in a p38 MAP-kinase-dependent way. However, stimulation of beta-adrenoceptors activated p38 MAP-kinase irrespective of pre-treatment with TGF-beta. In the absence of this cytokine, hyperosmolarity or reconstitution of mechanical activity increased protein synthesis via p38 MAP-kinase activation in freshly isolated cells. In conclusion, activation of p38 MAP-kinase is a newly identified necessary signalling step required for beta-adrenoceptor induced hypertrophic growth. Like activation of adenyl cyclase, activation of p38 MAP-kinase is up-stream of the TGF-beta-induced coupling to the regulation of protein synthesis. Reconstitution of mechanical activity mimics the co-activation required and induced by TGF-beta.

Animals↗
PubMed2004

Inhibition of Ca2+-dependent PKC isoforms unmasks ERK-dependent hypertrophic growth evoked by phenylephrine in adult ventricular cardiomyocytes.

OBJECTIVE: The duration of extracellular signal-regulated kinase (ERK) activation and the ERK-dependency of hypertrophic growth differ between stimulation of alpha-adrenoceptors or angiotensin II receptors. As both receptor systems activate different protein kinase C (PKC) isoforms, we hypothesized that PKC isoforms contribute to the specific effect of alpha-adrenoceptor stimulation. METHODS: Isolated adult ventricular cardiomyocytes from rats were used. Different PKC isoforms were inhibited either pharmacologically by six different PKC inhibitors or specifically downregulated by antisense oligonucleotides. ERK activation was determined by phosphorylation relative to total ERK. The rate of protein synthesis was determined by 14C-phenylalanine incorporation. RESULTS: The hypertrophic response of phenylephrine was inhibited in a concentration-dependent fashion by three different inhibitors of Ca2+-independent PKC isoforms (Gö6983, rottlerin, Gö6850), but not by three distinct PKC inhibitors directed preferentially against Ca2+-dependent PKC isoforms (Ro32-0432, HBDDE, Gö6976). Antisense oligonucleotides directed against PKC-alpha, -delta, or -epsilon downregulated their specific isoforms. Their corresponding sense oligonucleotides did not affect PKC isoform expression. The phenylephrine-induced increase in protein synthesis was blocked by antisense oligonucleotides directed against PKC-delta or PKC-epsilon but not PKC-alpha, confirming the pharmacological experiments. Inhibition of Ca2+-dependent PKC isoforms by HBDDE or Gö6976 converted a transient activation of ERK by phenylephrine into a sustained response. Under these conditions, phenylephrine increased protein synthesis in an ERK-dependent way. CONCLUSION: Inhibition of Ca2+-dependent PKC isoforms converts the ERK-independent effect of phenylephrine on protein synthesis into an ERK-dependent induction of protein synthesis. We conclude that co-activation of Ca2+-dependent PKC isoforms by phenylephrine contributes to the specific effect on adult ventricular cardiomyocytes from rat.

Acetophenones↗
PubMed2004

The K+-channel opener NS1619 increases endothelial NO-synthesis involving p42/p44 MAP-kinase.

Ca(2+)-activated K(+) channels with large conductance (BK(Ca)) have been shown to play an important role in the regulation of vascular tone. We examined the role of the p42/p44 MAP-kinase (p42/p44(MAPK)) on nitric oxide (NO) production in human endothelial cells induced by the BK(Ca)-opener NS1619. Using DiBAC-fluorescence imaging a concentration-dependent (2.5-12.5 microM) hyperpolarization induced by NS1619 was observed. A significant increase of intracellular Ca(2+)-concentration by NS1619 was seen using Fura-2-fluorescence-imaging, which was blocked by 2-APB, or reduction of extracellular Ca(2+) (n=30; p<0.05). A cGMP-radioimmunoassay was used to examine NO synthesis. NS1619 significantly increased cGMP levels which was inhibited by LNMMA, iberiotoxin, BAPTA, 2-APB, reduction of extracellular Ca(2+), PD 98059, or U0126 (cGMP (pmol/mg protein): NS1619 3.25 +/- 0.85; NS1619 + L-NMMA 0.86 +/- 0.02; NS1619 + iberiotoxin 0.99 +/- 0.09; NS1619 + BAPTA 0.93 +/- 0.29; NS1619 + 2-APB 0.99 +/- 0.31; NS1619 + Ca(2+)-reduction 1.17 +/- 0.06; NS1619 + PD98059 1.06 +/- 0.49; NS1619 + U0126 1.10 +/- 0.24; n=10; p<0.05). The phosphorylation of eNOS and p42/p44(MAPK) was examined by immunocytochemistry. Phosphorylation of p42/p44(MAPK) was significantly increased after 10 minutes of NS1619 stimulation, whereas eNOS phosphorylation was not changed over a period of 1 to 30 minutes. NS1619-induced hyperpolarization was not affected by treatment with PD 98059 or U0126. Additionally, NS1619 inhibited endothelial proliferation involving a NO-dependent mechanism. Our data demonstrate that NS1619 causes a transmembrane Ca(2+)-influx leading to an increased NO production involving p42/p44(MAPK). This rise of NO formation is responsible for the NS1619 induced reduction of endothelial cell growth.

Benzimidazoles↗
PubMed2003

ATP antagonism of thrombin-induced endothelial barrier permeability.

OBJECTIVES: Thrombin induces endothelial barrier failure by activating the contractile machinery of endothelial cells. Contractile activation is due to an increase in myosin light chain (MLC) phosphorylation. Here, it was investigated whether stimulation of endothelial cells with ATP can interrupt this thrombin-induced pathomechanism. METHODS: In cultured human umbilical vein endothelial cells, cytosolic calcium [Ca(2+)](i) (Fura 2 method), phosphorylation of MLC, isometric tension and permeability for albumin were studied. RESULTS: Thrombin (0.2 U/ml) increased [Ca(2+)](i) from a basal level of 78+/-8 to 570+/-63 nM (mean+/-S.D., n=5, P<0.05), MLC phosphorylation from 71+/-7 to 163+/-18%, isometric tension from 157+/-17 to 232+/-26 microN, and permeability from 2.8+/-0.4 to 11.6+/-1 x 10(-6) cm/s. Co-presence of ATP (10 microM) and thrombin did not alter the [Ca(2+)](i) rise, but reduced MLC phosphorylation to 59.8+/-10%, isometric tension to 174+/-14 microN, and permeability to 5.4+/-0.6 x 10(-6) cm/s. The thrombin-induced rise in MLC phosphorylation was sensitive to reduction of [Ca(2+)](i) It was accompanied by an increase in Rho activation, and was inhibited by Y-27632 (10 microM), a Rho-kinase blocker. The ATP-induced decrease in MLC phosphorylation was not sensitive to [Ca(2+)](i). It was not accompanied by changes in RhoA activation, and could not by suppressed by Y-27632. CONCLUSIONS: ATP antagonizes the Ca(2+)- and Rho-dependent effects of thrombin on MLC phosphorylation most likely by a Ca(2+)- and Rho-independent activation of MLC phosphatase. It thereby functionally antagonizes the thrombin-induced increase in monolayer tension and permeability.

Adenosine Triphosphate↗