Biomedical subjects

Wilhelm Bloch

Publications and source records attributed to Wilhelm Bloch.

At least 19 recordsLinked to original sources

PubMed2006

Loss of beta1D-integrin function in human ischemic cardiomyopathy.

Integrins play a pivotal role in cardiomyocyte survival and function, with integrin loss leading to myocyte apoptosis and heart failure. The aim of this study was to characterize whether regulation of integrins may contribute to cardiac remodeling in human ischemic cardiomyopathy (ICM). Myocardial tissues of the left ventricle were obtained from patients with ICM (n = 8) undergoing cardiac transplantation and from unused donor hearts (NF, n = 8). In addition, tissue samples from patients with dilated cardiomyopathy (DCM, n = 5) were analyzed. Expression of integrins beta(1)D and beta(3), the effector proteins focal adhesion kinase (FAK) and melusin, and FAK phosphorylation were examined by Western blotting, real-time-PCR and immunofluorescence analysis, respectively. Beta(1)D-integrin protein was decreased in ICM vs. NF by 36%. Beta(1)D-integrin mRNA levels and beta(1)D-integrin shedding were unchanged. Corresponding to beta(1)D-integrin regulation, FAK and phosphorylated FAK were decreased in ICM vs. NF by 54% and 49%, respectively. beta(3)-integrin and melusin were not altered in ICM. As a mediator of integrin effects, AKT kinase activity was examined. In parallel to beta(1)D-integrin and FAK, AKT activity was decreased in ICM by 44%. In contrast, none of the proteins were significantly altered in DCM compared to NF. Integrins and integrin signaling are regulated differentially in ICM and DCM with a decrease of beta(1)D-integrin and FAK in ICM. The loss of the beta(1)Dintegrin-FAK-complex in ICM was paralleled by a reduced AKT activity supporting in vitro data which demonstrate the pivotal role of intact integrin function in anti-apoptotic signaling and cell survival.

Blotting, Western↗

PubMed2006

Functional expression of the Na+/Ca2+ exchanger in the embryonic mouse heart.

The Na(+)/Ca(2+) exchanger (NCX) is one of the earliest functional genes and is currently assumed to compensate at least in part for the rudimentary sarcoplasmic reticulum in the developing mouse heart. However, to date little is known about the functional expression of NCX during development. This prompted us to investigate the NCX current (I(NCX)) in very early (embryonic day E8.5-E9.5 post coitum), early (E10.5-E11.5), middle (E13.5) and late (E16.5) stage mouse embryonic cardiomyocytes. For standard I(NCX) measurements, [Ca(2+)](i) was buffered to 150 nmol/l and voltage ramps were applied from +60 mV to -120 mV. At very early stages of development, we observed a prominent role of the I(NCX) Ca(2+) inward mode in elevating the cytosolic Ca(2+) concentration ([Ca(2+)](i)). Accordingly, a high I(NCX) density was observed (+60 mV: 4.6+/-0.7 pA/pF, n=14). Likewise, we found a strong Ca(2+) outward mode of I(NCX) (-120 mV: -3.9+/-0.7 pA/pF, n=14). At later stages, however, I(NCX) Ca(2+) inward mode was reduced by 54+/-6% (n=15, p<0.0001) in ventricular and 68+/-10% (n=9, p<0.0006) in atrial cells. For the outward mode, a reduction by 43+/-10% (n=15, p<0.01) in ventricular and 62+/-11% (n=9, p<0.004) in atrial cardiomyocytes was observed. By contrast, NCX isoform expression and the reversal potential did not significantly change during development. Thus, NCX displays a prominent Ca(2+) inward and outward mode during early embryonic heart development pointing to its important contribution to maintain [Ca(2+)](i) homeostasis. The functional and protein expression of NCX declines during further development.

Alternative Splicing↗

PubMed2006

Induction of cardioplegic arrest immediately activates the myocardial apoptosis signal pathway.

Myocardial ischemia-reperfusion, including cardioplegic arrest (CA), has been associated with cardiac apoptosis induction. However, the time course of apoptosis activation and the trigger mechanisms are still unclear. Because apoptosis inhibition may represent a novel therapeutic strategy for long-term myocardial preservation, we sought to investigate the time course of apoptosis signal-pathway induction during CA. As to method, Sprague-Dawley rats (300-350 g) were anesthetized, intubated, and mechanically ventilated. CA was initiated by infusion of ice-cold crystalloid solution (Custodiol, 10 ml/kg) into the aortic root, and hearts were rapidly excised and stored for 0, 30, 60, and 120 min in 0.9% sodium chloride solution (28 degrees C). In controls, no CA was initiated before removal and storage at 28 degrees C. In another group, calcium-rich cardioplegia was used, and an additional group received a caspase-8 inhibitor before CA induction. Left ventricular cytosolic extracts were isolated and investigated for the activity of caspase-3 and -6 (effector caspases) and caspase-8 and -9 (involved in extrinsic and intrinsic pathways of apoptosis induction). Fluorometric activity assays were performed by using specific substrates. As a result, activities of all tested caspases were significantly increased immediately after CA induction compared with controls. Administration of the caspase-8 inhibitor significantly reduced activities of all caspases. With calcium-rich cardioplegia, caspase activities were significantly lower compared with low-calcium CA. Control hearts also showed an increase of caspase activities during cold-storage ischemia without CA but had significantly different time courses compared with hearts with CA. In conclusion, our data show rapid apoptosis signal-pathway induction immediately following CA exposure. Thus apoptosis signal-pathway inhibition as a potential strategy for improved myocardial preservation would have the greatest effect when applied before CA exposure.

Animals↗

PubMed2006

Altered tension cost in (TG(mREN-2)27) rats overexpressing the mouse renin gene.

The present study aimed to characterize cardiac hypertrophy induced by activation of the renin-angiotensin system in terms of functional alterations on the level of the contractile proteins, employing transgenic rats harboring the mouse renin gene (TGR(mREN2)27). Ca2+-dependent tension and myosin ATPase activity were measured in skinned fiber preparations obtained from TGR(mREN2)27 and from age-matched Sprague-Dawley rats (SPDR). Western blots for troponin I (TnI) and troponin T (TnT) were performed and the phosphorylation status of TnI were evaluated in myocardial preparations. TnT and myosin heavy chain (MHC) isoforms were analyzed by RT-PCR. The pCa/tension relationship was shifted to the right in TGR(mREN2)27 compared to SPDR as indicated by increased Ca2+-concentrations required for half maximal activation of tension (SPDR 5.80, 95% confidence limits 5.77-5.82 vs. TGR(mREN2)27 5.69, 95% confidence limits 5.67-5.72, pCa units), while maximal developed tension was unaltered. Even more pronounced was the shift in the relationship between pCa and myosin-ATPase (SPDR 6.01, 95% confidence limits 5.99-6.03 vs. TGR(mREN2)27 5.77, 95% confidence limits 5.73-5.79, pCa units). The maximal myosin-ATPase activity was reduced in TGR(mREN2)27 compared to SPDR, respectively (211.0 +/- 28.77 micromol ADP/s vs. 271.6 +/- 43.66 micromol ADP/s, P < 0.05). Tension cost (ATPase activity/tension) was significantly reduced in TGR(mREN2)27. The beta-MHC expression was significantly increased in TGR(mREN2)27. There was no isoform shift for TnT (protein and mRNA), as well as TnI, and no alteration of the phosphorylation of TnI in TGR(mREN2)27 compared to SPRD. The present study demonstrates that cardiac hypertrophy, induced by an activation of the renin-angiotensin system, leads to adapting alterations on the level of the contractile filaments, which reduce tension cost.

Animals↗

PubMed2006

Acute BDNF and cortisol response to low intensity exercise and following ramp incremental exercise to exhaustion in humans.

The effect of short-term aerobic exercise and a following ramp incremental cycle ergometry to exhaustion on the acute response of the serum concentrations of brain derived neurotrophic factor (BDNF) and cortisol (COR) was examined in 8 healthy male athletes. Venous and capillary blood samples were drawn at rest, immediately after a 10 min warm-up period with aerobic exercise and after a ramp test to exhaustion, as well as 3, 6, 10 and 15 min post exercise. Capillary blood lactate (LA) concentration and blood gases as well as serum BDNF and COR concentrations did not change during the warm-up period. LA was increased (p<0.05) at the end of the ramp test and during recovery period while bicarbonate concentration, carbon dioxide pressure, pH and base excess were decreased (p<0.05) during this period. Serum BDNF was increased at the point of exhaustion (p<0.05) while no significant differences were found between values at rest and those during recovery period. At 10 and 15 min post incremental exercise, COR concentrations were increased (p<0.05) compared to rest. The present study is the first to demonstrate in humans that in contrast to short duration aerobic exercise immediately after a following short duration high-intensity exercise to exhaustion, there is a transient augmentation of serum BDNF concentration. Short-term response of serum BDNF and COR concentrations differs as BDNF returns to baseline level faster than COR.

Adult↗

PubMed2006

Force measurements of human embryonic stem cell-derived cardiomyocytes in an in vitro transplantation model.

Human embryonic stem cell (hESC)-derived cardiomyocytes have been suggested for cardiac cell replacement therapy. However, there are no data on loaded contractions developed by these cells and the regulation thereof. We developed a novel in vitro transplantation model in which beating cardiomyocytes derived from hESCs (line H1) were isolated and transplanted onto noncontractile, ischemically damaged ventricular slices of murine hearts. After 2-3 days, transplanted cells started to integrate mechanically into the existing matrix, resulting in spontaneous movements of the whole preparation. Preparations showed a length-dependent increase of active tension. In transplanted early beating hESC-derived cardiomyocytes, frequency modulation by field stimulation was limited to a small range around their spontaneous beating rate. Our data demonstrate that this novel in vitro transplantation model is well suited to assess the mechanical properties and functional integration of cells suggested for cardiac replacement strategies.

Animals↗

PubMed2006

Engraftment of engineered ES cell-derived cardiomyocytes but not BM cells restores contractile function to the infarcted myocardium.

Cellular cardiomyoplasty is an attractive option for the treatment of severe heart failure. It is, however, still unclear and controversial which is the most promising cell source. Therefore, we investigated and examined the fate and functional impact of bone marrow (BM) cells and embryonic stem cell (ES cell)-derived cardiomyocytes after transplantation into the infarcted mouse heart. This proved particularly challenging for the ES cells, as their enrichment into cardiomyocytes and their long-term engraftment and tumorigenicity are still poorly understood. We generated transgenic ES cells expressing puromycin resistance and enhanced green fluorescent protein cassettes under control of a cardiac-specific promoter. Puromycin selection resulted in a highly purified (>99%) cardiomyocyte population, and the yield of cardiomyocytes increased 6-10-fold because of induction of proliferation on purification. Long-term engraftment (4-5 months) was observed when co-transplanting selected ES cell-derived cardiomyocytes and fibroblasts into the injured heart of syngeneic mice, and no teratoma formation was found (n = 60). Although transplantation of ES cell-derived cardiomyocytes improved heart function, BM cells had no positive effects. Furthermore, no contribution of BM cells to cardiac, endothelial, or smooth muscle neogenesis was detected. Hence, our results demonstrate that ES-based cell therapy is a promising approach for the treatment of impaired myocardial function and provides better results than BM-derived cells.

Animals↗

PubMed2006

Reduced cardiac L-type Ca2+ current in Ca(V)beta2-/- embryos impairs cardiac development and contraction with secondary defects in vascular maturation.

Cardiac myocyte contraction depends on transmembrane L-type Ca2+ currents and the ensuing release of Ca2+ from the sarcoplasmic reticulum. Here we show that these L-type Ca2+ currents are essential for cardiac pump function in the mouse at developmental stages where the functional significance of the heart becomes imperative to blood flow and to the continuing growth and survival of the embryo. Disruption of the Ca(V)beta2 gene, which encodes for the predominant ancillary beta subunit of cardiac Ca2+ channels, resulted in diminished L-type Ca2+ currents in cardiomyocytes of embryonic day 9.5 (E9.5). This led to a functionally compromised heart, causing defective remodeling of intra- and extraembryonic blood vessels and embryonic death following E10.5. The defects in vascular remodeling were also observed when the Ca(V)beta2 gene was selectively targeted in cardiomyocytes, demonstrating that they are secondary to cardiac failure rather than a result of the lack of Ca(V)beta2 proteins in the vasculature. Partial rescue of the Ca2+ channel currents by a Ca2+ channel agonist significantly postponed embryonic death in Ca(V)beta2-/- mice. Taken together, these data strongly support the essential role of L-type Ca2+ channel activity in cardiomyocytes for normal heart development and function and that this is a prerequisite for proper maturation of the vasculature.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed2006

Terminal B cell differentiation is skewed by deregulated interleukin-6 secretion in beta2 integrin-deficient mice.

Absence of the common beta chain (CD18) of beta(2) integrins leads to leukocyte-adhesion deficiency type-1 (LAD1) in humans. Mice with a CD18 null mutation suffer from recurrent bacterial infections, impaired wound healing, and skin ulcers, closely resembling human LAD1. Previous findings in CD18(-/-) mice demonstrated a skewed terminal B cell differentiation with plasmacytosis and elevated serum immunoglobulin G (IgG). As interleukin-6 (IL-6) is a potent enhancer of plasma cell formation and Ig secretion, we assessed IL-6 serum levels of CD18(-/-) and wild-type (WT) mice kept under a conventional or barrier facility or specific pathogen-free (SPF) conditions. We detected an up to 20-fold increase in IL-6 in serum of CD18(-/-) mice compared with WT controls when kept under conventional or barrier facility conditions, respectively. Under SPF conditions, no significant differences in terms of IL-6 serum levels were found between CD18(-/-) and WT mice. However, histological alterations of secondary lymphoid tissues, plasmacytosis, abnormal plasmacytoid cells (Mott cells), and hypergammaglobulinemia persisted. To further analyze the role of IL-6 in these pathological alterations, we established a CD18(-/-) IL-6(-/-) double-deficient mouse mutant. In these mice, serum IgG levels were normal, and the altered plasma cell phenotype, including Mott cells, was no longer detectable. The CD18(-/-) IL-6(-/-) double-deficient mouse model thus demonstrated that IL-6 is responsible for parts of the phenotype seen in the CD18(-/-) mouse mutants. It may be of interest to examine human leukocyte-adhesion deficiency type-1 patients closer and search for pathological changes possibly induced via overproduction of IL-6.

Animals↗

PubMed2006

Mesenchymal stem cells transmigrate over the endothelial barrier.

Mesenchymal stem cells (MSCs) seem to be a useful tool for cellular therapy in injured tissues, e.g. myocardial infarction or cardiomyopathies resulting in heart failure. For therapeutic approaches it is crucial that MSCs cross the endothelial barrier especially in intravascular or rather intracoronary application. Until today little is known about MSCs transmigrating across the endothelium. We performed co-culture experiments of MSCs on an endothelial monolayer to analyse direct interactions. An increasing flattened morphology of the MSCs was followed by a total integration into the monolayer after 2h. We repeated these experiments in isolated heart perfusions with gold-labelled MSCs. Using electron microscopy we detected MSCs exhibited direct cell-cell contacts. Tight junctions between the endothelial cells became abolished resulting in a distinct split between the cells. MSCs developed tight cell-cell contacts and became integrated into the endothelial wall of the capillary vessel. Finally, using confocal laser scanning microscopy, we assessed the ability of the MSCs to fully pass the endothelial barrier. Within the first 30 min, 30+/-8% of MSCs transmigrated, increasing to about half at 60 min (50+/-8%), whereas after 120 min the rate remained nearly unchanged (53+/-10%). This work demonstrates the capability of MSCs for transendothelial migration. Moreover we showed that the vast majority of MSCs migrated within 30 min, an important finding for the exposure times in clinical settings.

Animals↗

PubMed2006

Human bone marrow stroma cells display certain neural characteristics and integrate in the subventricular compartment after injection into the liquor system.

Because the neural differentiation capacity of bone marrow stromal cells (BMSCs) is still a matter of controversial debate, we performed a thorough investigation into the differentiation capacity of human BMSCs and examined their therapeutic potency. BMSCs were isolated from the femur and kept in cell cultures with various cultivation protocols being applied. In standard culture conditions using a fetal calf serum-enriched medium, while not exhibiting a neural phenotype, the majority of cells expressed a variety of neuronal marker proteins as well as the astrocyte marker GFAP. Only a minority of stem cells expressed nestin, a marker for neural precursor cells. Cultivation in serum-free medium supplemented with specific growth factors resulted in a markedly higher percentage of nestin-positive cells. To establish the therapeutic potency of bone marrow-derived cells, the synthesis of neurotrophic factors such as NGF, BDNF and GDNF was analyzed under non-stimulating standard culture conditions as well as after a neural selection procedure. The therapeutic potency of BMSCs was further examined with regard to their migratory potential in vitro and after transplantation in vivo. After stereotactic engraftment into the lateral ventricle of adult rats, mesenchymal stem cells were seen to adhere to the ependymocytes and cells of the choroids plexus. Afterwards grafted cells passed through the ependymal barrier, locating in the subventricular space. Their BMSCs took up a close host graft interaction without any degenerative influence on the host cells. Furthermore, there was morphological as well as immunohistochemical evidence for a transdifferentiation within the host tissue. In addition, BMSCs could be efficiently transduced using a third-generation adenoviral vector, indicating their potential feasibility for a gene-therapeutic option.

Adenoviridae↗

PubMed2006

Endostatin influences endothelial morphology via the activated ERK1/2-kinase endothelial morphology and signal transduction.

Endostatin, the proteolytic fragment of collagen XVIII, is known to be a potent inhibitor of angiogenesis. However, to date, only limited knowledge exists with regard to the effects of endostatin on vessel morphology and the underlying signaling pathway. The aim of the present work was therefore to determine the impact of endostatin and its collagen XV analogue restin on vessel development during wound healing and embryonic angio- and vasculogenesis. Time lapse experiments and electron microscopy demonstrate similar morphological changes evoked by endostatin and the ERK1/2-kinase inhibitor PD98059. Furthermore, we show that ERK1/2 phosphorylation, a crucial signaling event in vascular morphogenesis, is regulated by endostatin via the protein phosphatase 2A PP2A. These findings provide new insight into a key signaling pathway of vascular remodeling evoked by a matrix-derived factor.

Angiogenesis Inhibitors↗

PubMed2006

ENOS is not activated by nebivolol in human failing myocardium.

Nebivolol is a highly selective beta(1)-adrenoceptor blocker with additional vasodilatory properties, which may be due to an endothelial-dependent beta(3)-adrenergic activation of the endothelial nitric oxide synthase (eNOS). beta(3)-adrenergic eNOS activation has been described in human myocardium and is increased in human heart failure. Therefore, this study investigated whether nebivolol may induce an eNOS activation in cardiac tissue. Immunohistochemical stainings were performed using specific antibodies against eNOS translocation and eNOS serine(1177) phosphorylation in rat isolated cardiomyocytes, human right atrial tissue (coronary bypass-operation), left ventricular non-failing (donor hearts) and failing myocardium after application of the beta-adrenoceptor blockers nebivolol, metoprolol and carvedilol, as well as after application of BRL 37344, a specific beta(3)-adrenoceptor agonist. BRL 37344 (10 microM) significantly increased eNOS activity in all investigated tissues (either via translocation or phosphorylation or both). None of the beta-blockers (each 10 microM), including nebivolol, increased either translocation or phosphorylation in any of the investigated tissues. In human failing myocardium, nebivolol (10 microM) decreased eNOS activity. In conclusion, nebivolol shows a tissue-specific eNOS activation. Nebivolol does not activate the endothelial eNOS in end-stage human heart failure and may thus reduce inhibitory effects of NO on myocardial contractility and on oxidative stress formation. This mode of action may be of advantage when treating heart failure patients.

Adrenergic beta-Antagonists↗

PubMed2006

First year changes of myocardial lymphatic endothelial markers in heart transplant recipients.

OBJECTIVE: The lymphatic system plays an important role in interstitial fluid balance, lipid metabolism, and immune response. The recent introduction of specific lymphatic endothelial cell markers has made the investigation of lymphangiogenesis under various conditions and from small tissue samples feasible. It was the purpose of the study to investigate the changes of myocardial lymphatic endothelial markers during the first 12 months after heart transplantation and to analyze if a correlation between lymphatic markers and rejection can be found. METHODS: Right ventricular endomyocardial biopsies taken for routine rejection monitoring from 26 heart transplant recipients were investigated. Selected time points were 0.5, 1, 1.5, 6, and 12 months after human heart transplantation (HTX). Immunohistostaining was performed for VEGFR-3, the receptor for lymphangiogenic vascular endothelial growth factors C and D, for LYVE-1, a novel hyaluronan receptor, restricted to lymphatic vessels, and PROX-1, a homeobox gene product, which plays a key role in lymph vessel development and differentiation. RESULTS: Density of VEGFR-3 positive lymphatics did not change during the first 12 months after transplantation. However, in comparison to the 0.5-month biopsy, density of LYVE-1 and PROX-1 positive lymphatics was significantly decreased at 1 month after transplantation (p<0.03) and at the subsequent time points (p<0.01). Patients with only moderate rejection during the first 12 months (ISHLT<IIIa) had a significantly higher density of VEGFR-3 at 0.5 month in comparison to patients with at least one episode of clinically relevant rejection (ISHLT IIIa or worse, p<0.03). CONCLUSION: Myocardial lymphatics show a significant change of endothelial phenotype after transplantation, as demonstrated by significant quantitative changes of lymphatic endothelial marker density. Patients with at least one rejection of ISHLT IIIa or higher had a significantly lower density of VEGFR-3 at 0.5 month after transplantation. The results of this study warrant further investigation on the impact of transplantation on the lymphatic endothelium. The cause-effect relation between rejection and lymphatic endothelium remains to be investigated.

Adolescent↗

PubMed2006

Skin lesion development in a mouse model of incontinentia pigmenti is triggered by NEMO deficiency in epidermal keratinocytes and requires TNF signaling.

NF-kappaB essential modulator (NEMO), the regulatory subunit of the IkappaB kinase, is essential for NF-kappaB activation. Mutations disrupting the X-linked NEMO gene cause incontinentia pigmenti (IP), a human genetic disease characterized by male embryonic lethality and by a complex pathology affecting primarily the skin in heterozygous females. The cellular and molecular mechanisms leading to skin lesion pathogenesis in IP patients remain elusive. Here we used epidermis-specific deletion of NEMO in mice to investigate the mechanisms causing the skin pathology in IP. NEMO deletion completely inhibited NF-kappaB activation and sensitized keratinocytes to tumor necrosis factor (TNF)-induced death but did not affect epidermal development. Keratinocyte-restricted NEMO deletion, either constitutive or induced in adult skin, caused inflammatory skin lesions, identifying the NEMO-deficient keratinocyte as the initiating cell type that triggers the skin pathology in IP. Furthermore, genetic ablation of tumor necrosis factor receptor 1 (TNFRI) rescued the skin phenotype demonstrating that TNF signaling is essential for skin lesion pathogenesis in IP. These results identify the NEMO-deficient keratinocyte as a potent initiator of skin inflammation and provide novel insights into the mechanism leading to the pathogenesis of IP.

Animals↗

PubMed2006

[On the function of beta3-adrenoceptors in the human heart: signal transduction, inotropic effect and therapeutic prospects].

Beta-adrenergic stimulation is an important regulatory mechanism of cardiac function. Next to beta1- and beta2-adrenoceptors, the expression of a third beta-adrenoceptor population, the beta3-adrenoceptor, has recently been evidenced in the human heart. Stimulation of cardiac beta3-adrenoceptors leads to a decrease in contractility via a release of nitric oxide (NO). In this context, different molecular mechanisms of endothelial nitric oxide synthase (eNOS) activation have been uncovered to occur as a consequence of beta3-adrenergic stimulation. In both nonfailing and failing myocardium, beta3-adrenergic stimulation may have a protective effect against excessive chatecolaminergic stimulation as it occurs during somatic and mental stress and during heart failure. For this reason, the beta3-adrenoceptor is discussed as a possible target for the pharmacological therapy of heart failure.

Adrenergic beta-3 Receptor Agonists↗

PubMed2006

Crataegus special extract WS 1442 induces an endothelium-dependent, NO-mediated vasorelaxation via eNOS-phosphorylation at serine 1177.

PURPOSE: This study investigates the influence of WS(R) 1442, a special extract of Crataegus leaves with flowers, on the relaxation of rat aorta and human mammarian artery (coronary bypass patients). METHODS: Experiments were performed in the presence and absence (mechanical disruption) of endothelium. In addition, we investigated three fractions of WS(R) 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). RESULTS: WS 1442 induced a concentration-dependent vasodilation in isolated vessel rings that had been precontracted by 10 microM phenylephrine (concentration for halfmaximal relaxation (IC(50)): rat: 15.1 +/- 0.6 microg/ml (n = 7), human: 19.3 +/- 3.4 microg/ml (n = 6)). The maximal vasorelaxation induced after application of 100 microg of WS 1442 was 75.0 +/- 5.7% (rat) and 79.2 +/- 5.8% (human) of the papaverine (0.1 mM)-induced vasodilation. If the experiments were performed in the presence of L-nitroarginine methylester (10 microM, 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. CONCLUSIONS: It is concluded that WS 1442, induces an endothelium-dependent, NO-mediated vasorelaxation via eNOS phosphorylation at serine 1177.

Adult↗

PubMed2006

Effect of thawing on nitric oxide synthase III and apoptotic markers in cryopreserved human allografts.

BACKGROUND: Previous investigations suggested apoptosis as a contributing factor to early failure of allograft heart valves. As myocardial apoptosis may be induced by nitric oxide (NO) release, this study investigated NO synthase [NOS-III] activation and apoptosis induction in human cryopreserved allografts during the thawing process. METHODS: Frozen myocardial tissue from ten human allograft heart valves, unsuitable for implantation, was submitted to the following conditions: (1) thawing in paraformaldehyde (Control), thawing according to the standard clinical protocol (Standard), standard-thawing with addition of the NOS-inhibitor N-omega-nitro-l-arginine (L-NA; Standard-LNA), and standard thawing with the NOS-stimulator angiotensin II (Standard-AT-II). Cryo-thin sections were investigated by immunostaining for activated NOS-III, cyclic guanosine monophosphate (cGMP), activated caspase-3, and poly-ADP-ribose polymerase (PARP). Quantitative analyses was performed by television densitometry. RESULTS: For activated NOS-III, gray unit values were significantly higher in the Standard and Standard-AT-II group than in the Control and Standard-LNA groups (p < 0.001). Gray unit values for cGMP, a downstream NO-signal-pathway molecule, showed results grossly corresponding to NOS-III activation. Activated caspase-3 and PARP showed high levels of expression in all groups with no significant differences. CONCLUSIONS: Significant activation of NOS-III and subsequent NO-cGMP signal pathway occurs in human cryopreserved allografts during the thawing process and can be significantly reduced by a NOS-III inhibitor administered during thawing. Activation of the apoptosis pathway is also present after thawing, which was not correlated to NOS-III activation. Further experimental investigation focused on the time course and mechanisms of apoptosis and NOS-III activation are required to evaluate NOS and(or) apoptosis inhibitors as therapeutic strategies for improved allograft preservation.

Apoptosis↗