Klara Brixius

Basic Fibroblast Growth Factor Controls Migration in Human Mesenchymal Stem Cells

Little is known about the migration of mesenchymal stem cells (MSCs). Some therapeutic approaches had demonstrated that MSCs were able to regenerate injured tissues when applied from different sites of application. This implies that MSCs are not only able to migrate but also that the direction of migration is controlled. Factors that are involved in the control of the migration of MSCs are widely unknown. The migratory ability of isolated MSCs was tested in different conditions. The migratory capability was examined using Boyden chamber assay in the presence or absence of basic fibroblast growth factor (bFGF), erythropoietin, interleukin‐6, stromal cell‐derived factor‐β, and vascular endothelial growth factor. bFGF in particular was able to increase the migratory activity of MSCs through activation of the Akt/protein kinase B (PKB) pathway. The results were supported by analyzing the orientation of the cytoskeleton. In the presence of a bFGF gradient, the actin filaments developed a parallelized pattern that was strongly related to the gradient. Surprisingly, the influence of bFGF was not only an attraction but also routing of MSCs. The bFGF gradient experiment showed that low concentrations of bFGF lead to an attraction of the cells, whereas higher concentrations resulted in repulsion. This ambivalent effect of bFGF provides the possibility to a purposeful routing of MSCs.

Nebivolol, bucindolol, metoprolol and carvedilol are devoid of intrinsic sympathomimetic activity in human myocardium

The present study investigated whether or not there may be differences in the direct cardiac actions of the novel, highly β1‐selective adrenoceptor antagonist nebivolol (NEB) in comparison to metoprolol (MET), bisoprolol (BIS), carvedilol (CAR) and bucindolol (BUC) in human myocardium (n=9). The rank order of β1‐selectivity as judged by competition experiments to 3H‐CGP 12.1777 in the presence of CGP 207.12 A (300u2003nmolu2003l−1, Kiβ2) or ICI 118.551 (50u2003nmolu2003l−1, Kiβ1) were NEB(Kiβ2/Kiβ1: 40.7)>BIS(15.6)>MET(4.23)>CAR(0.73)>BUC(0.49). The rank order of the negative inotropic potency of the β‐adrenoceptor antagonists measured in left ventricular trabeculae (dilated cardiomyopathy, DCM) as judged by the concentration needed to induce a 50% decrease in isoprenaline (1u2003μmolu2003l−1)‐stimulated force (IC50) was: MET (0.6u2003μmolu2003l−1)>CAR (4.1u2003μmolu2003l−1)>NEB (7.0u2003μmolu2003l−1). NEB, BUC, MET and CAR did not not exert an intrinsic sympathomimetic activity (ISA) as determined by measurements of force development in forskolin (0.3u2003μmolu2003l−1) pre‐treated left ventricular trabeculae, nor by measuring adenylate cyclase activity in forskolin (0.3u2003μmolu2003l−1)‐stimulated assays (crude membranes). This also holds true for radioligand binding assays with or without guanine nucleotide guanyl‐5′‐yl imidodiphosphate (Gpp(NH)p). Although all studied β‐adrenoceptor antagonists lack intrinsic sympathomimetic activity (ISA), they differ in the β1‐selectivity as well as in their direct negative inotropic action. These differences as well as the mode of extracardiac action may have an impact on outcome of patients treated with β‐adrenoceptor antagonists.

Modulation of SERCA: implications for the failing human heart.

Abstract Human heart failure is characterized by distinct alterations in the intracellular homeostasis and key regulators of the sarcoplasmic reticulum Ca2+ sequestration mechanisms. Systolic peak Ca2+ is reduced, diastolic Ca2+ levels are increased and diastolic Ca2+ decay is prolonged. Recently specific changes in the expression, function and modulation of SR Ca2+-ATPase (SERCA) have been elucidated. As such, in a variety of studies SERCA expression appeared to be decreased in the failing human heart, although these findings have been discussed controversially depending on the studied tissue, especially with respect to the non-failing samples and regional variation in the obtained samples. However, consistent findings of a diminished Ca2+ dependent SERCA activation were found. Increasing evidence has been provided that one of the underlying mechanisms for a decreased activation of SERCA is its altered regulation. With respect to this, the modulations through phospholamban and Ca2+-dependent protein kinase II (CaMK II) play a detrimental role in regulating SERCA function. Phospholamban phosphorylation of SERCA at the serine-16 and threonine-17 site is diminished in human heart failure resulting in decreases in the apparent affinity for Ca2+ of the SR Ca2+ uptake rates. In contrast, activation of CaMK II leads to an increased maximal velocity of SR Ca2+ sequestration that may enhance SR Ca2+-load. Additional regulation has been recently elucidated by changes in the apparent coupling ratio of Ca2+ transported per ATP hydrolysed. This review summarizes recent advances in the understanding how SERCA is modulated under physiological and pathophysiological conditions.

Crataegus Special Extract WS® 1442 Induces an Endothelium-Dependent, NO-mediated Vasorelaxation via eNOS-Phosphorylation at Serine 1177

PurposeThis study investigates the influence of WS® 1442, a special extract of Crataegus leaves with flowers, on the relaxation of rat aorta and human mammarian artery (coronary bypass patients).MethodsExperiments were performed in the presence and absence (mechanical disruption) of endothelium. In addition, we investigated three fractions of WS® 1442 (fraction A: lipophilic, containing flavonoids and oligomeric procyanidins (OPC), fraction B: hydrophilic, containing flavonoids and low molecular weight OPC, fraction C: hydrophilic, essentially flavonoid-free and rich in high molecular weight OPC).ResultsWS® 1442 induced a concentration-dependent vasodilation in isolated vessel rings that had been precontracted by 10xa0μM phenylephrine (concentration for halfmaximal relaxation (IC50): rat: 15.1 ± 0.6xa0μg/ml (n = 7), human: 19.3 ± 3.4xa0μg/ml (n = 6)). The maximal vasorelaxation induced after application of 100xa0μg of WS® 1442 was 75.0 ± 5.7% (rat) and 79.2 ± 5.8% (human) of the papaverine (0.1xa0mM)-induced vasodilation. If the experiments were performed in the presence of l-nitroarginine methylester (10xa0μM, eNOS-inhibition) or after mechanical disruption of the endothelium, no vasorelaxation was observed in the presence of WS® 1442. The vasorelaxant properties of WS® 1442 were mediated by fraction C. WS® 1442 induced an NO-liberation from human coronary artery endothelial cells as measured by diaminofluorescein. WS® 1442 induced eNOS-activation was due to a phosphorylation at serine 1177. No eNOS-translocation or phosphorylation at serine 114 or threonine 495 was observed after application of WS® 1442.ConclusionsIt is concluded that WS® 1442, induces an endothelium-dependent, NO-mediated vasorelaxation via eNOS phosphorylation at serine 1177.

Mechanisms of β3-adrenoceptor-induced eNOS activation in right atrial and left ventricular human myocardium

β‐adrenoceptors are important modulators of cardiac function. The present study investigated β3‐adrenergic eNOS activation in human myocardium. We measured nitric oxide (NO) liberation (diaminofluorescence) and signal transduction (immunohistochemistry, phosphorylation of eNOSSer1177, eNOSThr495, eNOSSer114, Akt/protein kinase B (Akt/PKB), and eNOS translocation) in human right atrial (RA, aortocoronary‐bypass OP) and left ventricular nonfailing (LV, rejected donor hearts) myocardium after application of BRL 37344 (BRL), a preferential β3‐adrenoceptor agonist. In both RA and LV, BRL (10 μl) induced a liberation of NO. An eNOS activation via translocation was only observed in RA after application of BRL (10 μM). Yet, the NO liberation in both LV and RA was accompanied by phosphorylation of eNOSSer1177 and Akt/PKB. BRL‐induced eNOS phosphorylation was abolished by LY292004, a blocker of PI‐3 kinase. eNOS‐Ser114 phosphorylation was unchanged in RA, but decreased in LV after β3‐adrenergic stimulation. BRL did not alter phosphorylation of eNOSThr495. In conclusion, receptor‐dependent eNOS activation is differentially regulated in the human heart. In the left ventricle, eNOS activation via phosphorylation seems to be of major importance, whereas in human atrial myocardium eNOS translocation is the predominant mechanism induced by β3‐adrenergic activation.

The preferential β3‐adrenoceptor agonist BRL 37344 increases force viaβ1‐/β2‐adrenoceptors and induces endothelial nitric oxide synthase viaβ3‐adrenoceptors in human atrial myocardium

The present study investigated the effects of the preferential β3‐AR agonist BRL 37344 (BRL) on force of contraction (FOC), Ca2+‐transient and eNOS‐activity in human right atrial myocardium. BRL concentration‐dependently caused an increase in FOC that was paralleled by an increase in Ca2+‐transient and a shortening of time to half peak relaxation (T0.5T). These effects were abolished in the presence of propranolol (0.3 μM). BRL acted as a competitive antagonist towards isoprenaline and in binding experiments it was shown to have a distinct affinity towards β1/2‐AR. In immunohistochemical experiments BRL (10 μM) increased detection of activated eNOS. This effect remained constant in the presence of propranolol (0.3 μM). BRL increased directly detected NO in DAF‐staining experiments. This increase was significantly smaller in the presence of the NO‐inhibitor L‐NAME. The inotropic effects of BRL were not changed in the presence of L‐NMA. These results suggest that the inotropic effects of BRL in human atrium are mediated via β1/2‐AR, whereas the increase of atrial eNOS‐activity is due to β3‐ adrenergic stimulation. This increase in eNOS‐activity did not influence atrial myocardial contractility. In conclusion, this study shows that β3‐adrenergic stimulation is present in human atrium, but may not be functionally as significant as in the left ventricle.

Isometric force kinetics upon rapid activation and relaxation of mouse, guinea pig and human heart muscle studied on the subcellular myofibrillar level.

Abstract The kinetics of force development and relaxation upon rapid application and removal of Ca2+ was measured in bundles of few myofibrils isolated from triton X-100 skinned left ventricular trabeculae of mice (M), guinea pigs (G) and humans (H). Upon rapidly switching from relaxing solution (pCa 7.5) to activating solution (pCa 4.5) at 10 °C, force rose by a single exponential with a rate constant κact of 5.2 s−1 (M), 1.7 s−1 (G) and 0.3 s−1 (H) to a plateau of 0.14 μN/μm2 (M), 0.16 μN/μm2 (G) and 0.15 μN/μm2 (H). A rapid release followed by a rapid restretch to the original length applied during steady-state Ca2+ activation at pCa 4.5 induced an exponential force redevelopment with a rate constant κredev that was indistinguishable from κact, indicating that κact is limited by cross-bridge turnover kinetics rather than by the Ca2+-induced activation of the regulatory system. Upon rapidly switching from pCa 4.5 to pCa 7.5, force decayed in a pronounced biphasic manner. Thus a slow initial, almost linear decay with a rate constant κlin of 1.8 s−1 (M), 0.6 s−1 (G) and 0.15 s−1 (H) and a duration tlin of 0.06 s (M), 0.11 s (G) and 0.3 s (H) was followed by a rapid exponential decay with a rate constant κrel of 18 s−1 (M), 11 s−1 (G) and 4.6 s−1 (H). The pronounced biphasic shapes of the force decays determined here for the first time in cardiac myofibrils differs from the force decays that had been reported for multicellular skinned trabeculae in which relaxation was induced by rapid removal of Ca2+ by flash photolysis of caged Ca2+ chelators. In the skinned trabeculae, no pronounced initial slow phase was observed. The force decays shown here are much more similar to those reported for single skeletal myofibrils. The kinetics of isometric relaxation of skinned trabeculae (i.e., multicellular preparations), therefore, do not reflect the kinetics of force relaxation at the cardiac myofibrillar level.

Mechanisms underlying nebivolol-induced endothelial nitric oxide synthase activation in human umbilical vein endothelial cells.

1 Nebivolol (NEB) has been shown to be a selective blocker of β1‐adrenoceptors with additional vasodilating properties that are mediated, at least in part, by an endothelial‐dependent liberation of nitric oxide (NO). In the present study, we investigated the underlying mechanisms of NEB‐induced vasodilation. 2 Immunohistochemical staining of endothelial nitric oxide synthase (eNOS) was performed in the absence and presence of NEB in human umbilical vein endothelial cells (HUVEC). In addition, we measured the release of nitric oxide (NO) using diaminofluorescein. Metoprolol (MET) was used for comparison. 3 Nebivolol, but not MET (each at 10 µmol/L), caused a time‐dependent increase in NO release from HUVEC, as demonstrated by an increase in DAF fluorescence at 0 versus 10 min (+234 ± 7 and 55 ± 22% basal, respectively). Blockade of β3‐adrenoceptors by SR 59230A (1 µmol/L) partially reduced the NEB‐induced increase in DAF fluorescence. Complete inhibition of NEB‐induced NO liberation was achieved by the simultaneous blockade of β3‐adrenoceptors and oestrogen receptors (with 1 µmol/L ICI 182,780). 4 Application of NEB significantly increased eNOS translocation and serine 1177 phosphorylation of eNOS. However, NEB did not alter eNOS‐phosphorylation at threonine 495 and at serine 114. 5 In conclusion, the endothelium‐dependent NO liberation induced by NEB is due to stimulation of β3‐adrenoceptors and oestrogen receptors and coincides with eNOS translocation and a phosphorylation at eNOS‐serine 1177. These characteristics of NEB may be beneficial not only when treating patients suffering from cardiovascular disease, but may also prevent further deterioration of endothelial dysfunction.

Mesenchymal Stem Cells Induce Endothelial Activation via Paracine Mechanisms

Mesenchymal stem cells (MSCs) are bone marrow-derived, pluripotent cells that possess the ability to transdifferentiate into various mesenchymal tissues such as bone, endothelium, and (heart) muscle. Therefore, these cells may provide a therapeutic tool, especially for the treatment of myocardial infarction. The interaction of the MSCs with the endothelial barrier and their ability to ultimately leave blood vessels after application are crucial in this context. In this study, the authors focused on the soluble factors produced by MSCs and their effect on the intracellular signal transduction of endothelial cells. The authors performed immunohistochemical measurements on human umbilical vein endothelial cells (HUVECs) treated with conditioned stem cell medium and took measurements of the intracellular nitric oxide (NO) levels and calcium changes. After application of conditioned stem cell medium, the authors detected an increase in endothelial NO synthase (eNOS) activity by translocation (Ca(2+)) and by phosphorylation (increase of pAKT and peNOS1177). Additionally, the authors observed an upregulation of pERK within the same time. The phosphorylated eNOS forms are linked to these findings and the increase of intracellular NO in the DAF measurements. Moreover, conditioned medium also increased intracellular calcium levels in endothelial cells. Concluding, the authors postulate that MSCs emit soluble factors that alter the NO and calcium levels of endothelial cells and may be important for facilitate crossing the endothelial barrier.

Unchanged protein expression of sarcoplasmic reticulum Ca2+-ATPase, phospholamban, and calsequestrin in terminally failing human myocardium

Abstract The enhanced diastolic Ca2+ levels observed in cardiac myocytes from patients with idiopathic dilated cardiomyopathy (DCM) may be either a consequence of functional impairment of sarcoplasmic reticulum calcium- ATPase (SERCAxa02) and its regulator protein phospholamban or due to a reduction in the number of SERCAxa02 proteins. As different myocardial membrane preparations may lead to different accumulation of proteins, the present study evaluated two different membrane preparations, in human failing and nonfailing myocardium for comparison of SERCAxa02 activity and the protein expression of SERCAxa02 and phospholamban. Crude membranes and tissue homo-genates without any centrifugation steps were prepared from human nonfailing hearts (donor hearts, NF, n=18) and terminally failing hearts (heart transplant, DCM, n=18). Calsequestrin protein expression was used as an internal control for overall protein expression. In both crude membranes and homogenates maximal SERCAxa02 activity (Vmax) was significantly reduced in failing heart preparations (NF crude membranes, 130±8; DCM crude membranes, 102±5xa0nmol ATP/mg protein per minute). In contrast, the protein expression of SERCAxa02 (NF crude membranes, 488±35; DCM crude membranes, 494±42; P=0.92), phospholamban (NF crude membranes, 497±51; DCM crude membranes, 496±45; P=0.98) and calsequestrin (NF crude membranes, 109±06; DCM crude membranes, 107±08; P=0.84) was unchanged in NF and DCM hearts in both preparation methods. This was also the case when the protein expression was normalized to calsequestrin protein levels. Preparation of sarcoplasmic reticulum in crude membranes led to enhanced purification and consequently higher SERCAxa02, phospholamban, and calsequestrin protein levels in crude membranes than in the homogenates, which was paralleled by an increase in SERCAxa02 enzyme activity. In conclusion, the altered Ca2+ handling in DCM may be a consequence of reduced SERCAxa02 enzyme activity and not the result of differences in protein expression of the Ca2+ regulating proteins SERCAxa02, phospholamban, and calsequestrin in human myocardium. The present study emphasizes the importance of different myocardial membrane preparations with respect to quantitative investigations of protein expression and function.