Biomedical subjects

G Schönfelder

Publications and source records attributed to G Schönfelder.

At least 19 recordsLinked to original sources

PubMed2004

Preeclampsia is associated with loss of neuronal nitric oxide synthase expression in vascular smooth muscle cells of the human umbilical cord.

AIMS: Umbilical blood vessels are not innervated and regulation of blood flow to the placenta must depend on structural changes and the effect of vasoactive factors. Failure to achieve these adaptations may result in reduced fetoplacental perfusion. The purpose of this study was to determine whether neuronal nitric oxide synthase (nNOS) is expressed in human vascular smooth muscle cells (VSMCs) of the fetoplacental circulation. nNOS has been described as a non-endothelial NOS counterregulating vasoconstriction only in the VSMCs of animal models. Therefore, we investigated nNOS expression in the fetoplacental unit from preeclamptic and healthy pregnancies. METHODS AND RESULTS: We investigated nNOS regulation by immunohistochemistry, Western blotting and reverse transcriptase-polymerase chain reaction analysis. nNOS activity was determined by measuring the conversion of L-3H-arginine to L-3H-citrulline. nNOS expression was revealed only in VSMCs of the human umbilical veins, but not in umbilical arteries. A more direct assessment of nNOS activity showed that a small, but consistent amount of nNOS is present in the denuded media of the umbilical vein. In VSMCs of the umbilical veins during preeclampsia a total loss of nNOS protein expression and a significant decrease in mRNA expression were seen. CONCLUSIONS: Loss of nNOS expression is associated with preeclampsia. It may alter the regulation of blood flow in the fetal and maternal placental vasculature in preeclampsia. However, the impact of NO produced by nNOS on the vascular tone of umbilical veins remains to be elucidated.

Adult↗

PubMed2001

*NO and oxyradical metabolism in new cell lines of rat brain capillary endothelial cells forming the blood-brain barrier.

To investigate the relevance of *NO and oxyradicals in the blood-brain barrier (BBB), differentiated and well-proliferating brain capillary endothelial cells (BCEC) are required. Therefore, rat BCEC (rBCEC) were transfected with immortalizing genes. The resulting lines exhibited endothelial characteristics (factor VIII, angiotensin-converting enzyme, high prostacyclin/thromboxane release rates) and BBB markers (gamma-glutamyl transpeptidase, alkaline phosphatase). The control line rBCEC2 (mock transfected) revealed fibroblastoid morphology, less factor VIII, reduced gamma-glutamyl transpeptidase, weak radical defence, low prostanoid metabolism, and limited proliferation. Lines transfected with immortalizing genes (especially rBCEC4, polyoma virus large T antigen) conserved primary properties: epitheloid morphology, subcultivation with high proliferation rate under pure culture conditions, and powerful defence against reactive oxygen species (Mn-, Cu/Zn-superoxide dismutase, catalase, glutathione peroxidase, glutathione) effectively controlling radical metabolism. Only 100 microM H2O2 overcame this defence and stimulated the formation of eicosanoids similarly as in primary cells. Some BBB markers were expressed to a lower degree; however, cocultivation with astrocytes intensified these markers (e.g., alkaline phosphatase) and paraendothelial tightness, indicating induction of BBB properties. Inducible NO synthase was induced by a cytokine plus lipopolysaccharide mixture in all lines and primary cells, resulting in *NO release. Comparing the cell lines obtained, rBCEC4 are stable immortalized and reveal the best conservation of properties from primary cells, including enzymes producing or decomposing reactive species. These cells can be subcultivated in large amounts and, hence, they are suitable to study the role of radical metabolism in the BBB and in the cerebral microvasculature.

Animals↗

PubMed1999

Cerebral endothelial cells release TNF-alpha after stimulation with cell walls of Streptococcus pneumoniae and regulate inducible nitric oxide synthase and ICAM-1 expression via autocrine loops.

TNF-alpha, inducible NO synthase (iNOS), and ICAM-1 are considered to be key proteins in the inflammatory response of most tissues. We tested the hypothesis that cell walls of Streptococcus pneumoniae (PCW), the most common cause of adult bacterial meningitis, induce TNF-alpha, iNOS, and ICAM-1 expression in rat primary brain microvascular endothelial cell cultures. We detected TNF-alpha mRNA by RT-PCR already 1 h after stimulation with PCW, while TNF-alpha protein peaked at 4 h (9.4 +/- 3.6 vs 0.1 +/- 0.1 pg/microgram protein). PCW induced iNOS mRNA 2 h after stimulation, followed by an increase of the NO degradation product nitrite (18.1 +/- 4 vs 5.8 +/- 1.8 at 12 h; 18.1 +/- 4 vs 5.8 +/- 1.8 pmol/microgram protein at 72 h). The addition of TNF-alpha Ab significantly reduced nitrite production to 62.2 +/- 14.4% compared with PCW-stimulated brain microvascular endothelial cells (100%). PCW induced the expression of ICAM-1 (measured by FACS), which was completely blocked by TNF-alpha Ab (142 +/- 18.6 vs 97.5 +/- 12.4%; 100% unstimulated brain microvascular endothelial cells). Cerebral endothelial cells express TNF-alpha mRNA as well as iNOS mRNA and release the bioactive proteins in response to PCW. PCW-induced NO production is mediated in part by an autocrine pathway involving TNF-alpha, whereas ICAM-1 expression is completely mediated by this autocrine loop. By these mechanisms, cerebral endothelial cells may regulate critical steps in inflammatory blood-brain-barrier disruption of bacterial meningitis.

Animals↗

PubMed1999

Phosphorylation of vasodilator-stimulated phosphoprotein: a consequence of nitric oxide- and cGMP-mediated signal transduction in brain capillary endothelial cells and astrocytes.

There is contradictory information on the relevance of nitric oxide (NO) and cGMP for the function of brain capillary endothelial cells (BCEC) forming the blood-brain barrier (BBB). Therefore, NO/cGMP-mediated signal transduction was investigated in cell cultures of BCEC and of astrocytes (AC) inducing BBB properties in BCEC. Constitutive, Ca2+-activated isoforms of NO synthase (NOS) were found in BCEC (endothelial NOS: eNOS) and in AC (neuronal NOS: nNOS), leading to increased NO release after incubation with the Ca2+-ionophore A23187. Both cell types expressed inducible NOS (iNOS) after incubation with cytokines. Soluble guanylate cyclase (sGC) was detected in both cell types. NO-dependent cGMP formation were observed in BCEC and, less pronounced, in AC. Furthermore, both cell types formed cGMP independently of NO via stimulation of particulate guanylate cyclase (pGC). cGMP-dependent protein kinase (PKG) type Ibeta, but not type II, was expressed in BCEC and AC. In BCEC, vasodilator-stimulated phosphoprotein (VASP) was detected, an established substrate of PKG and associated with microfilaments and cell-cell contacts. Phosphorylation of VASP was intensified by increased intracellular cGMP concentrations. The results indicate that BCEC and, to a smaller degree, AC can form NO and cGMP in response to different stimuli. In BCEC, NO/cGMP-dependent phosphorylation of VASP is demonstrated, thus providing a possibility of influencing cell-cell contacts.

Animals↗

PubMed1999

Transgenic rats expressing the human ET-2 gene: a model for the study of endothelin actions in vivo.

Previous studies have characterized the endothelin peptides (ET-1, ET-2, ET-3) as strong vasoconstrictors which are possibly involved in the pathogenesis of cardiovascular disease. Whereas ET-1 and ET-3 have been characterized using a number of approaches, little is known about the function of ET-2. The aim of this study was to define the role of ET-2 in physiology and pathophysiology using a transgenic approach. Transgenic rats expressing a genomic construct of the human ET-2 gene were generated by microinjection of fertilized oocytes from Sprague-Dawley rats. Two transgenic lines were generated, and one line was further characterized in detail. Studies on mRNA expression demonstrated that the transgene is expressed predominantly in kidney, gastrointestinal tract, adrenal gland, lung, and brain. Plasma endothelin levels were elevated 2-fold, and big-endothelin levels were elevated 2.5-fold. Despite these alterations blood pressure in transgenic rats remained normal. Further analysis of transgenic animals revealed that endothelin receptors were not downregulated, and that infusion of exogenous human ET-2 results in an enhanced blood pressure response. These observations suggest the presence of counterregulatory mechanisms influencing the effects of endothelin on blood pressure. One of these mechanisms may involve the nitric oxide system since infusion of an inhibitor of nitric oxide synthase resulted in a greater blood pressure response than in non-transgenic littermates. Despite unchanged blood pressure, alterations were observed in organ development and function, namely of hearts and kidneys, indicating an interference between transgene expression and growth processes. Male rats seem to be more susceptible to endothelin actions. These data show that the elevation in endothelin-2 expression in this transgenic model does not induce hypertension but leads to changes at the end-organ level. Normotension is most likely due to compensatory mechanisms such as increased nitric oxide formation.

Animals↗

PubMed1998

High expression of inducible nitric oxide synthase correlates with intestinal inflammation of interleukin-2-deficient mice.

Severely inflamed colonic sections of IL-2 (-/-) mice showed up to 19-fold increased iNOS mRNA levels. The level of iNOS protein expression corresponded to the increased iNOS mRNA levels as detected by means of Western blot analysis. There was a clear, positive relationship between the level of iNOS expression and the degree of inflammation in the colonic tissue of IL-2 (-/-) and wild-type mice. Our data suggest that iNOS may play a key role in the pathogenesis of ulcerative colitis-like disease in IL-2 (-/-) mice. Further investigation should elucidate the impact of NO on the regulation of the inflammatory process in this model and might contribute to a better understanding of the role iNOS expression in human immune-mediated diseases.

Animals↗

PubMed1998

Stretch-activated cation channel in human umbilical vein endothelium in normal pregnancy and in preeclampsia.

OBJECTIVE: To determine whether stretch-activated cation channels (SAC) are present in intact human umbilical vein endothelium (HUVE) and in an endothelial cell line (EA.hy) and whether they act as endothelial mechanosensors, and to determine whether endothelial SAC in HUVE from women with pregnancies complicated by preeclampsia undergo functional changes compared with those in HUVE from women with normotensive pregnancies. METHODS AND RESULTS: By use of the patch-clamp technique we identified a SAC in intact HUVE and in an endothelial cell line. The SAC had mean conductances of 29+/-5 pS (n = 38) for K+ and 12+/-2 pS (n = 4) for Ca2+. Administration of 50 micromol/I gadolinium, a blocker of mechanosensitive ion channels, completely blocked activity of this channel. We found from single-channel recordings that influx of Ca2+ through SAC directly activated high-conductance Ca2+-dependent potassium channels, proving that a significant influx of Ca2+ through SAC occurs at physiologic concentrations of Ca2+. In a comparative study, apparent channel density of SAC (percentage of patches with SAC activity) in HUVE from women with pregnancies complicated by preeclampsia (36.2 +/- 4.3%) was twofold higher than that in HUVE from women with normal pregnancies (17.9+/-2.9%, P< 0.01). Channel conductance and sensitivity to stretching of SAC were not altered by preeclampsia. CONCLUSIONS: Since SAC are capable of acting as endothelial mechanosensors, the greater than normal density of SAC associated with preeclampsia might reflect an alteration of mechanotransduction.

Adult↗

PubMed1996

Expression of inducible nitric oxide synthase in placenta of women with gestational diabetes.

Gestational diabetes is one of the most prevalent medical complications of pregnancy and causes increased fetal wastage. Investigation of placentas from diabetic mothers indicate chronic disturbances in intervillous, circulation, dilatation of capillaries, and a relatively immature villous structure. Abnormal levels of nitric oxide (NO) may contribute to maternal disorders such as the pathogenesis of diabetic vascular complications. In the normal placenta NO is generated only by endothelial NOS, which apparently serves to regulate vascular tone in the fetoplacental circulation. In contrast, studies have reported the absence of inducible nitric oxide synthase (iNOS) in human placentas under normal conditions. The aim of our study was to investigate whether iNOS is expressed in placentas from patients with gestational diabetes. Reverse transcription-polymerase chain reaction and Western blot analysis demonstrated iNOS mRNA and protein expression in placental tissue only from patients with gestational diabetes. Immunohistochemistry localized iNOS staining to endothelial cells and trophoblasts. We conclude that iNOS can be expressed in human placenta. Its expression might play an important role in placental pathophysiology.

Base Sequence↗

PubMed1995

Role of the cardiac renin-angiotensin system in human heart failure.

The local effects of angiotensin II (ANG II) on the heart may play an important role for the pathophysiology of cardiovascular disease. Numerous in vitro studies have demonstrated that angiotensin II has distinctive cellular effects in the cardiovascular system which are independent from its effects on blood pressure. These have led to the hypothesis that activation of the angiotensin system in the heart could be of functional relevance for the adaptive processes in several cardiovascular disorders such as cardiac hypertrophy heart failure. This concept has been further supported by clinical studies showing the beneficial effects of angiotensin-converting enzyme inhibitors in these circumstances. In order to study the gene regulation of renin-angiotensin system components in cardiac disorders we investigated the gene expression of angiotensin converting enzyme in human heart failure. Results showed that the enzyme is activated locally in this condition, supporting previous studies in animals. Taken together with recent evidence from genetic studies linking the enzyme to myocardial infarction and cardiac hypertrophy, our findings are in support of the notion that angiotensin converting enzyme plays a central role in cardiovascular physiology and pathophysiology.

Angiotensin II↗

PubMed1995

The perivascular contractile sheath of human placental stem villi: its isolation and characterization.

Former studies have shown that fetal blood vessels of stem villi in the human placenta are enclosed by sheaths of presumed contractile cells. Efforts to prove the contractility of these sheaths by isometric and biochemical investigations still suffer from the difficulty of differentiating between extravascular and vascular (media) contractile cells. The present study describes a method for the selective dissection of perivascular tissue sheaths in stem villi of approximately 2-4 mm thickness, which contained abundant alpha-actin immunoreactive cells and were free of adherent vascular smooth muscle cells. These extravascular contractile cells are part of the 'fibrous paravascular sheath', which, in placental pathology, is used as an index of maturity. To emphasize the high number of contractile cells and their location within this sheath, we propose the common term perivascular contractile sheath (PVCS). The isolation method offers the possibility of selectively investigating the contractile forces of the PVCS and of obtaining more insight into its functional role.

Actins↗

PubMed1995

Regulation of the endothelin system in transgenic rats expressing the human endothelin-2 gene.

We have established a transgenic rat model for the expression of the human endothelin-2 (ET-2). These animals exhibit overexpression of the transgene in tissues as well as in plasma. Despite these changes, blood pressure remains normal. To understand the regulatory mechanisms for normotension in the presence of increased ET-2 levels, we have investigated the ET system in more detail. We used competitive reverse transcription-polymerase chain reaction (RT-PCR) to evaluate possible overexpression or downregulation of endothelin A and B receptors at the mRNA level. PCR analyses revealed no significant differences of ETA and ETB receptor expression. In conclusion, the expression of human ET-2 in transgenic rats does not result in hypertension. Normotension in the transgenic animals is independent of ET receptor regulation. The reason for this may be counterregulation by other vasoactive systems, such as the NO system. Future studies will take this into account and will also concentrate on possible histomorphologic alterations caused by mitogenic properties of the endothelins.

Animals↗

PubMed1994

The extravascular contractile system in the human placenta. Morphological and immunocytochemical investigations.

In the human placenta, besides the fetal blood vessel system a second extravascular contractile system exists. It is localized in the chorionic plate and runs in a longitudinal direction and adjacent to fetal blood vessels into the stem villi, where it forms perivascular contractile sheaths. Characteristically, cells of the extravascular contractile system are extremely long and spindle-shaped and give rise to fine cell processes, by which they obviously contact each other or insert into the basement membrane of the trophoblast. They show immunoreactivity with desmin, vimentin, alpha-actin, myosin, nitric oxide synthase type I (brain form) and dipeptidyl peptidase IV. The ultrastructure suggests that cells of the extravascular contractile system are related to smooth muscle cells, including subpopulations with myofibroblastic features. In stem villi a few cells are nitric oxide synthase type I immunoreactive. These cells are thought to be specialized smooth-muscle-like cells of the extravascular contractile system or cells of the extravascular contractile system related to paraneurons that generate nitric oxide, which, in turn, may modulate the tone of perivascular contractile sheaths. The high dipeptidyl peptidase IV activity suggests that modulation of the extravascular contractile system may also occur by substance P.

Actins↗

PubMed1994

Distribution of constitutive nitric oxide synthase immunoreactivity and NADPH-diaphorase activity in murine telogen and anagen skin.

The freely diffusible radical nitric oxide is generated by nitric oxide synthase, and is a pleiotropic, bioregulatory molecule that regulates, e.g., the vascular tone, functions as a major neurotransmitter, and is involved in macrophage-mediated cytotoxicity and platelet aggregation. Constitutive nitric oxide synthase exhibits NADPH-diaphorase activity that can be demonstrated histochemically. To study whether this enzyme is present in mammalian skin during distinct phases of the murine hair cycle, we have examined cryosections of C 57 BL-6 mouse skin in telogen and depilation-induced anagen VI. Histochemical analysis of NADPH-diaphorase activity was complemented by immunohistology, using two specific rabbit antisera against constitutive neuronal nitric oxide synthase. Epidermis and the outer root sheath showed both immunoreactivity for the enzyme and NADPH-diaphorase activity, whereas dermal papilla and sebaceous glands displayed only strong NADPH-diaphorase activity, suggesting that this enzyme histochemical test measures additional enzymes besides nitric oxide synthase. Intrinsic nitric oxide synthase immunoreactivity was also detected by immunoblot in mouse skin homogenates, staining proteins of an apparent 160-kDa molecular weight. Compared to telogen skin, these immunoreactive proteins were quantitatively increased in anagen VI skin. Thus, our study suggests that defined epithelial compartments of normal murine skin are capable of synthesizing nitric oxide and that the molecule may be involved in skin physiology, growth, and remodeling.

Amino Acid Oxidoreductases↗

PubMed1994

[The extravascular contractile system of the human placenta: a new aspect for function of the organ?].

In the chorionic plate and stem villi of the human placenta besides the fetal blood vessel system a second extravascular contractile system (EVCS) exists. The cells of this system contain contractile and intermediate filaments and are dipeptidyl peptidase-IV- and NO-synthase-type-I-(NOS)-immunoreactive. Therefore it can be assumed that these cells cleave the vasodilator Substance P (by DPP IV) and produce NO (by NOS) and may contribute to a modulation of the EVCS.

Amino Acid Oxidoreductases↗

PubMed1989

Etiology and incidence of acute pancreatitis: a 20-year study in a single institution.

Reports from various countries raise the question of changes in the incidence and etiology of acute pancreatitis. This study covers patients admitted with acute pancreatitis to a single university department of medicine between 1965 and 1970 and between 1980 and 1985. The absolute number of patients admitted with acute pancreatitis doubled, whereas the percentage of pancreatitic patients in the total population of admitted patients decreased by 38%. No firm conclusion can be made about an increasing incidence of acute pancreatitis from 1970 to 1985. However, the dominating etiological factor was no longer biliary tract disease but significantly shifted to alcoholism, whereas changes in the severity of the disease were not significant. There was a downward trend in mortality with mortality rates of 13 and 8.6% for the first and second period, respectively; however, because of the small number of patients this change did not reach the level of significance.

Acute Disease↗

PubMed1983

[Hemodynamic changes after application of vasoactive substances in cats with unilateral renal artery constriction in addition to sinoaortal baroreceptor deafferentation].

The relationship between total peripheral resistance (TPR), cardiac output (CO) and venous return (VR) and their role in the response of mean arterial pressure (MAP) on intravenous adrenalin (10 micrograms/kg) and angiotensin II (0.5 micrograms/kg), resp., were investigated in normal cats (A), cats with unilateral renal artery stenosis (B) and cats with sinoaortal baroreceptor deafferentation (C). In the maximum MAP increase in group A there participated both TPR and VR through altered CO. In group B the MAP increased after angiotensin II only in consequence of an enhanced TPR, whereas the VR response was dampened; only after adrenalin the VR contributed a little to MAP increase through CO. In group C, the MAP increase after both substances was due exclusively to increased TPR; at a restricted ejection capacity of the heart the VR cannot act on the level of blood pressure.

Angiotensin II↗

PubMed1983

[Mineralocorticoid-induced changes of vascular reactivity to noradrenaline and angiotensin II in baboons].

In experiments on normotensive baboons of both sex the vascular reactivity on angiotensin II and noradrenaline was studied after an 18-week administration of DOCA (2 mg/b.w./week i. m.) and of common sal (5 g/day with meals). Dosage and duration of DOCA administration was chosen to prevent hypertensive haemodynamic alterations. 40 to 240 seconds after an angiotensin II injection (0.5 micrograms/kg b.w.) the pressor, especially diastolic response in both narcotized and non-narcotized animals intensified markedly. Infusion of noradrenaline (1.0 micrograms/kg b.w./min for 5 min) called forth after 120 min a significant rise in diastolic BP. Described abnormal pressor response is considered to represent a suitable model of a vascular predisposition to hypertension.

Angiotensin II↗

PubMed1982

[Arterial hypertensive dysregulations on the basis of a cerebro-visceral stimulus constellation in baboons].

Basing on the hypothesis that disturbances of cerebral information processing on the basis of acute or chronic stress situations or profound neurotic alterations are being directed to the cardiovascular system only by predisposition to hyperreactivity, the influence of a psycho-nervous-humoral-hormonal stepped load schedule upon central nervous and vegetative functions was studied in baboons. Stochastic interventions into the natural day-night rhythm and application of NaCl and DOCA doses not per se causing a blood pressure rise, either single or in combination for altogether 3 years were used as disturbing factors. It has been revealed that experimental disturbance of the light-dark phases led to lasting deviations of the conditional-reflectory activity in the sense of a predominance of irritation processes. With motor response time, initially unchanged but from the second year of experiment significantly shortened by 35%, the failure rates at differentiation increased, on the average, from 6 to 45% and the intersignal responses by 100%. Even after exposure for several months, no disorders of the cardiovascular system occurred. It was only the coupling with an experimentally induced disturbance of electrolyte distribution that provoked a significant increase in mean arterial pressure, on the average, by 24% of the pre-control value with moderate increase of the circulating blood volume. The increases in free fatty acids and blood glucose concentrations by 14 and 23%, respectively, can be interpreted as additional hypertension-favouring factors. Despite an application of mineral corticoids for more than 1 year, it has been impossible to alter the contraction behaviour of the vascular smooth muscle cell in the sense of an experimentally induced predisposition to arteriolar hyperreactivity outlasting the discontinuation of disturbing factors. With higher nervous activity being clearly disturbed as before, the pressure got back to normal; testing the vascular reactivity to noradrenaline (1.0 microgram/kg b.w/min i.v., for 5 min) or angiotensin II (0.5 microgram/kg b.w. i.v.) at the end of the investigation period gave no enhanced pressure responses. By contrast, animals exposed exclusively to the described combination load for 18 weeks, showed a still normal system pressure and sensitivity to the applied noradrenaline and angiotensin II increased by 75-120% of the pre-control response. A liability of the cardiovascular system at acute stress situations (multiple partial immobilization) in long-term neurotically predamaged monkeys in the 24-h experiment was impressive by a cardiodepression during the nightly regeneration phase, reduced on the average by 35 beats/min against the control group. Thus, our results support the hypothesis of a cerebro-visceral pathoconstellation as the etiological principle of certain forms of the inhomogeneous clinical picture of primary hypertension.

Animals↗