Hagen Maxeiner

New insights into paracrine mechanisms of human cardiac progenitor cells.

Cardiac progenitor cells (CPCs) have been shown to promote cardiac regeneration in vivo. Understanding the function of CPCs is essential for further implementation of these cells in the treatment of cardiac diseases. The present study tested the hypothesis that adult CPC exert paracrine effects that lead to an improvement in the functional characteristics of cardiomyocytes. This study also investigated whether aging (we included patients aged between 4 months and 81 years) has any effect on the paracrine mechanisms of CPC.

Pretreatment with PKC activator protects cardiomyocytes against reoxygenation-induced hypercontracture independently of Ca2+ overload

OBJECTIVE Although several studies have shown that activation of protein kinase C (PKC) plays an important role in protection through ischemic preconditioning, little is known about the effects of direct PKC activation on the course of ischemia-reperfusion injury. The aim of this study was to analyse the effects of a pretreatment with the PKC activator 1,2-dioctanoyl-sn-glycerol (1,2DOG). METHODS Isolated adult Wistar rat cardiomyocytes were exposed to 80 min of simulated ischemia (anoxia, pHo 6.4) and 20 min of reoxygenation (pHo 7.4). Cytosolic Ca2+ (fura-2), cytosolic pH (BCECF), Mg2+ (Mg-fura-2), lactate and cell length were measured and compared between control cells and cells treated with 20 mumol/l 1,2DOG before anoxia (10 min treatment and 10 min wash out). RESULTS 1,2DOG-pretreatment delayed the time to extreme ATP depletion, but had no effect on lactate production and cytosolic pH. The accumulation of cytosolic Ca2+ was markedly accelerated in pretreated cells that developed rigor shortening, but reoxygenation-induced hypercontracture was significantly reduced. 1,2DOG, therefore, completely abolished Ca(2+)-dependence of hypercontracture. The effects of pretreatment were fully abolished with 1 mumol/l bisindolylmalcimide (PKC inhibitor). We conclude that PKC preactivation leads to (1) reduction of energy demand, (2) acceleration of Ca2+ overload during anoxia and (3) prevention of reoxygenation-induced hypercontracture independent of anoxic changes in cytosolic Ca2+ and pH.

Interleukin-6 Contributes to the Paracrine Effects of Cardiospheres Cultured from Human, Murine and Rat Hearts

Cardiosphere‐derived cells (CDCs) were cultured from human, murine, and rat hearts. Diluted supernatant (conditioned‐medium) of the cultures improved the contractile behavior of isolated rat cardiomyocytes (CMCs). This effect is mediated by the paracrine release of cytokines. The present study tested the hypothesis, that the cardiovascular state of the donor’s heart influences this effect on CMCs and tries to identify the responsible factors. CDCs were cultured from human tissue samples of cardiac surgery and from murine and rat hearts. The supernatants of cultured CDCs from hypertensive humans and rats showed a higher improvement of the contractile behavior of CMCs compared to CDCs of normotensive origin. Subsequently, the cytokine profile of the supernatants was analyzed. Among the cytokines elevated in supernatants originating from hypertensive humans or rats was Interleukin‑6. CDCs were also generated from Interleukin‑6−/−‐mice and their wildtype littermates. The supernatant of the cultured Interleukin‑6−/−‐CDCs had no effect on the contractile behavior, whereas the supernatant of the Interleukin‑6+/+‐CDCs showed a positive effect. To confirm the hypothesis that Interleukin‑6 contributes to the paracrine effects, CMCs were incubated with Interleukin‑6. It improved the contractile function in a concentration dependent way. Finally, the effect of the supernatant of cultured CDCs derived from a hypertensive human sample could be abolished by simultaneous incubation with a specific Interleukin‑6 antibody. CDCs release cytokines that improve the contractile behavior of CMCs. This effect is more intense in CDCs from hypertensive donors. Interleukin‑6 is involved in this phenomenon. J. Cell. Physiol. 229: 1681–1689, 2014.

TGF-β1 improves cardiac performance via up-regulation of laminin receptor 37/67 in adult ventricular cardiomyocytes

TGF-β1 plays an important role in cardiac fibrosis, apoptosis, induction of hypertrophy and contractile dysfunction. This study investigates whether TGF-β1 plays a role in laminin receptor 37/67 (37/67 LR)-dependent regulation of cardiac performance. Therefore, isolated adult cardiomyocytes were stimulated with TGF-β1, the expression of the 37/67 LR was determined and cell shortening was investigated on cells attached to a non-specific, serum-based attachment substrate or to specific, laminin-coated dishes. The role of the MAP kinases in TGF-β1-dependent induction of the 37/67 LR was examined by addition of PD98059, SB202190 and SP600125. Finally, the expression of receptor mRNA was investigated in transgenic mice constitutively over-expressing TGF-β1 and the relationship to distress score and lung wet weight-to-body weight was analysed. TGF-β1 induced a significant increase of the 37/67 LR mRNA and protein expression. The cytokine induced p38 MAP kinase and JNK, but not ERK. Inhibition of either p38 MAP kinase or JNK attenuated the TGF-β1-dependent increase in 37/67 LR expression. TGF-β1 induced a loss of cell shortening in cells attached to a non-specific substrate, but not in cells on a pre-coated laminin matrix. Inhibition of JNK attenuated the protective effect of laminin receptor up-regulation on cardiac performance. Inhibition of p38 MAP kinase attenuated the depressive effect of TGF-β1 on basal cell shortening. In transgenic mice over-expressing TGF-β1 a strong induction of laminin receptor expression attenuated the severeness of the mice’ symptoms. This study shows a new and protective role of TGF-β1-dependent up-regulation of the 37/67 LR in cardiomyocytes in cardiac remodelling with increased laminin expression.

CYP1B1 mRNA inducibility due to benzo(a)pyrene is modified by the CYP1B1 L432V gene polymorphism

Benzo(a)pyrene (BaP), a primary component of tobacco smoke, is activated by cytochrome P450 1B1 (CYP1B1). Smokers homozygous for the C-allele (*1/*1) at the CYP1B1 Leu432Val polymorphism have shown increased CYP1B1 expression, compared to smokers homozygous for the G-allele *3/*3. Since no difference has been shown in CYP1B1 expression between both genotypes in non-smokers, we assumed that the genetic impact is produced in combination with an exogenous induction (e.g. BaP). To confirm this theory and to quantify the effect, we induced human leucocytes with increasing BaP concentrations and determined CYP1B1 mRNA expression with real-time polymerase chain reaction (PCR). We incubated human leucocytes from 27 healthy donors with BaP concentrations ranging from 2.5 to 250 µM. We identified the CYP1B1 genotypes by melting curve analysis and assessed relative CYP1B1 mRNA expression using real-time PCR. Expression was related to β-2-microglobulin with the 2(-ΔΔCT) method. Inducibility of CYP1B1 mRNA by BaP was higher in leucocytes carrying the CYP1B1*1/*1 genotype than in leucocytes carrying the CYP1B1*3/*3 genotype (P = 0.012). We revealed significant differences, with BaP concentrations of 2.5 µM (P = 0.0094), 5 µM (P = 0.027), 10 µM (P = 0.0006), 25 µM (P = 0.0007) and 50 µM (P = 0.017). Homozygous carriers of the C-allele (*1/*1) at the CYP1B1 Leu432Val polymorphism show a higher response to environmental factors, such as carcinogenic BaP, than homozygous carriers of the G-allele *3/*3.

Effects of corticotropin-releasing hormone on proopiomelanocortin derivatives and monocytic HLA-DR expression in patients with septic shock

Little is known about interactions between immune and neuro-endocrine systems in patients with septic shock. We therefore evaluated whether the corticotropin-releasing hormone (CRH) and/or proopiomelanocortin (POMC) derivatives [ACTH, β-endorphin (β-END), β-lipotropin (β-LPH), α-melanocyte stimulating hormone (α-MSH) or N-acetyl-β-END (Nac-β-END)] have any influences on monocyte deactivation as a major factor of immunosuppression under septic shock conditions. Sixteen patients with septic shock were enrolled in a double-blind, cross-over and placebo controlled clinical study; 0.5μg/(kgbodyweighth) CRH (or placebo) were intravenously administered for 24h. Using flow cytometry we investigated the immunosuppression in patients as far as related to the loss of leukocyte surface antigen-DR expression on circulating monocytes (mHLA-DR). ACTH, β-END immunoreacive material (IRM), β-LPH IRM, α-MSH and Nac-β-END IRM as well as TNF-α and mHLA-DR expression were determined before, during and after treatment with CRH (or placebo). A significant correlation between plasma concentration of α-MSH and mHLA-DR expression and an inverse correlation between mHLA-DR expression and TNF-α plasma level were found. Additionally, a significant increase of mHLA-DR expression was observed 16h after starting the CRH infusion; 8h later, the mHLA-DR expression had decreased again. Our results indicate that the up-regulation of mHLA-DR expression after CRH infusion is not dependent on the release of POMC derivatives. From the correlation between plasma concentration of α-MSH and mHLA-DR expression, we conclude that in patients with septic shock the down-regulation of mHAL-DR expression is accompanied by the loss of monocytic release of α-MSH into the cardiovascular compartment.

Effects of cerivastatin on adrenergic pathways, hypertrophic growth and TGFbeta expression in adult ventricular cardiomyocytes.

The effects of statin treatment in the setting of heart failure have already been shown. Nevertheless, there is little knowledge about its influence on adrenergic pathways in cardiomyocytes. Therefore, this study investigated the impact of cerivastatin on adrenoceptor-mediated signalling pathways in isolated adult ventricular cardiomyocytes. It focused on two endpoints: hypertrophic growth and TGFbeta expression. Cultured cardiomyocytes were used to study rac activation (analysed by its translocation into the membrane fraction), ROS formation (H(2)DCF fluorescence) and hypertrophic growth ((14)C-phenylalanine incorporation). Alpha- and beta-adrenoceptor stimulation showed significant differences regarding rac activation, ROS formation, and p38 MAP kinase activation. Both alpha- and beta-adrenoceptor stimulation induced TGFbeta expression. Upon activation of alpha-adrenergic signalling - although ROS formation was not influenced by cerivastatin - TGFbeta expression decreased. Following beta stimulation, TGFbeta expression as well as rac and p38 MAP kinase activation were reduced after pre-treatment with cerivastatin. Statin treatment did not show any influence on hypertrophic growth. In summary, this study clearly demonstrates the ability of adrenoceptor stimulation to increase TGFbeta expression. One component of the beneficial effects of statin therapy on heart failure might therefore be due to a dominant reduction and inhibition of TGFbeta, which is involved in many pathophysiological processes in cardiomyocytes.