Biomedical subjects

J F Schmedtje

Publications and source records attributed to J F Schmedtje.

At least 19 recordsLinked to original sources

PubMed2000

Sp1 increases expression of cyclooxygenase-2 in hypoxic vascular endothelium. Implications for the mechanisms of aortic aneurysm and heart failure.

Cyclooxygenase-2 (COX-2) catalyzes prostaglandin synthesis from arachidonic acid and is expressed locally in aortic aneurysm and heart failure. Cellular hypoxia is also found in these conditions. We have previously shown that cox-2 is transcriptionally regulated by hypoxia in human umbilical vein endothelial cells (HUVEC) in culture via the transactivation factor NF-kappaB p65, leading to increased production of prostaglandin E(2), an inhibitor of vascular smooth muscle cell proliferation. Sp1 is a transactivation factor known to be important in the regulation of cytokine expression in association with NF-kappaB. We hypothesized that Sp1 is involved in the induction of cox-2 in hypoxic HUVEC. Electrophoretic mobility shift assays with hypoxic HUVEC nuclear protein showed that both Sp1 and the related protein Sp3 specifically bound to the cox-2 promoter. Immunoblotting demonstrated that hypoxia increased the nuclear localization of Sp1 but did not change the Sp3 content in HUVEC. Overexpression of Sp1 through transfection of HUVEC enhanced cox-2 promoter activity as measured by reporter gene expression and by the production of COX-2. The specificity of the results was confirmed by mutation of the Sp1-binding site in the cox-2 promoter construct and by reproducibility in an Sp-deficient Drosophila SL2 cell line. The regulatory role of Sp1 discovered in this work supports the concept that a mechanistic link exists between vascular cellular hypoxia and mediators of inflammation associated with aortic aneurysm and heart failure.

Base Sequence↗

PubMed1998

Hypoxia induces high-mobility-group protein I(Y) and transcription of the cyclooxygenase-2 gene in human vascular endothelium.

Cyclooxygenases catalyze a rate-limiting step in the synthesis of vascular endothelial prostaglandins. Expression of the inducible cyclooxygenase-2 (COX-2) gene is increased by hypoxia in human vascular endothelial cells via the nuclear factor (NF)-kappaB p65 transcription factor, which is necessary but not sufficient to fully induce COX-2 transcription in response to hypoxia. After finding that cytoplasmic NF-kappaB p65 and IkappaBalpha (an inhibitory protein that binds NF-kappaB p65 precursors) levels are not changed by hypoxia, we hypothesized that other factors might play a role in regulating the COX-2 promoter, like the high-mobility-group (HMG) I(Y) family of proteins, which features multiple A.T hooks and is associated with NF-kappaB-mediated transactivation. Nuclear protein obtained from human umbilical vein endothelial cells (HUVECs) was supplemented with HMG I(Y) during electrophoretic mobility shift assays using an NF-kappaB-3' element probe. These data suggested that HMG I(Y) proteins interact with NF-kappaB p65 to induce COX-2 promoter activity. We also found that TATA-box DNA demonstrated increased electrophoretic shifting indicative of DNA binding after incubation with either hypoxic HUVEC nuclear protein or normoxic nuclear protein supplemented with HMG I(Y). Transfection of HUVECs with an expression vector containing the COX-2 promoter ligated to HMG I(Y) cDNA demonstrated positive feedback on COX-2 promoter activity in hypoxia. We confirmed that COX-2 is transcriptionally regulated by hypoxia using a nuclear runoff assay. Hypoxia increased steady-state cellular levels of HMG I(Y) mRNA as an early event, corresponding with increases in HMG I(Y) protein. Overexpression of HMG I(Y) was associated in a dose-response relationship with increasing prevalence of the COX-2 protein in hypoxic HUVECs. Furthermore, sense (and antisense) HMG I(Y) overexpression caused stimulation (or inhibition) of COX-2 promoter activity as measured by luciferase reporter gene expression. The physiological significance of these findings was demonstrated by cyclooxygenase-dependent release of prostaglandin E2 by HUVECs in hypoxia. We concluded that hypoxia increases expression of HMG I(Y) proteins while facilitating transactivation of the COX-2 promoter. The HMG I(Y) family of proteins may therefore function as part of a hypoxia-induced enhanceosome that helps to promote transcription of COX-2.

Cell Hypoxia↗

PubMed1997

Antithrombotic potency of hirudin is increased in nonhuman primates by fibrin targeting.

BACKGROUND: Inhibition of thrombin by either the indirect thrombin inhibitor heparin or by more potent direct thrombin inhibitors such as hirudin reduces thrombus formation after arterial injury. The present study was designed to determine if a fibrin-specific thrombin inhibitor could, by local thrombin inhibition, prevent thrombosis more effectively. METHODS AND RESULTS: We first studied antithrombotic potency in vitro, comparing fibrin-targeted hirudin (recombinant hirudin covalently linked to the Fab' fragment of the anti-fibrin monoclonal antibody 59D8) to recombinant hirudin in baboon plasma. Fibrin-targeted hirudin was nine times more effective than recombinant hirudin in inhibiting fibrin deposition on experimental clot surfaces in baboon plasma (P < .01). The potency of fibrin-targeted hirudin was then compared with that of recombinant hirudin in a baboon model of thrombus formation. 111In-labeled platelet deposition was measured in a synthetic graft segment of an extracorporeal arteriovenous shunt in control animals and in animals receiving either fibrin-targeted hirudin or hirudin. In these experiments, fibrin-targeted hirudin was 10-fold more potent than hirudin in inhibiting platelet deposition and thrombus formation (P < .05). CONCLUSIONS: These data indicate that targeting a thrombin inhibitors such as hirudin to an epitope present in thrombi results in increased antithrombotic potency.

Animals↗

PubMed1997

Hypoxia induces cyclooxygenase-2 via the NF-kappaB p65 transcription factor in human vascular endothelial cells.

The inducible cyclooxygenase, COX-2, has been associated with vascular inflammation and cellular proliferation. We have discovered that hypoxia increases expression of the COX-2 gene in human vascular endothelial cells in culture independent of other stimuli. Western analysis of human umbilical vein endothelial cells (HUVEC) revealed a greater than 4-fold induction of protein by hypoxia (1% O2). The steady-state level of COX-2 mRNA was correspondingly elevated by both Northern blot and reverse transcriptase-polymerase chain reaction analysis. Using electrophoretic mobility shift assays with antibody supershifting, we also found that hypoxia causes increased binding of NF-kappaB p65 (Rel A) to the one out of the two NF-kappaB consensus elements in the COX-2 promoter which is closest to the transcription start site of the COX-2 gene. Transfection of an immortalized human microvascular endothelial cell line (HMEC-1) with mutation reporter gene constructs and HUVEC with both mutation and deletion reporter gene constructs suggested that transcription of the COX-2 gene was enhanced by hypoxia. In transcription factor decoy experiments, hypoxic HUVEC were exposed in culture to 20 microM of the same NF-kappaB element found to bind NF-kappaB protein. The wild type transcription factor decoy prevented hypoxic induction of COX-2, presumably by binding with cytoplasmic p65; however, mutated or scrambled oligonucleotides did not prevent the increase in COX-2 protein expression by hypoxia. Thus, the intracellular signaling mechanism that leads to induction of COX-2 by hypoxia includes binding of p65 to the relatively 3' NF-kappaB consensus element in the COX-2 upstream promoter region in human vascular endothelial cells.

Base Sequence↗

PubMed1996

pH is critical to the regulation of expression of the beta 2-adrenergic receptor gene in hypoxia.

Expression and function of the beta 2-adrenergic receptor (beta 2-AR), a critical modulator of motor function, is altered in ischemic tissues. However, the mechanism by which ischemia influences gene expression remains controversial, in part because of the conflicting results reported by numerous investigators. To determine the relative importance of hypoxia and acidosis on beta 2-AR expression and function, steady-state mRNA levels and receptor function were measured in DDT1MF-2 hamster smooth muscle cells grown in 10% serum and 3 nM epinephrine in 5% CO2 (pH 7.50) and then exposed for 48 h to either combined hypoxia with acidosis (through incubation in 2% O2, 10% CO2, mean pH 7.14 at 48 h), hypoxia alone (2% O2, 2.5% CO2, pH 7.36), normoxia-acidosis (21% O2, 10% CO2, pH 7.12) or continued normoxia (21% O2, 2.5% CO2, pH 7.49). Combined hypoxia-acidosis downregulated the beta 2-AR membrane density by 50% compared to hypoxia alone and normoxia alone at 48 h. beta 2-AR coupling in these cells, as measured by cellular cAMP production in response to 10(-4) M isoproterenol, was decreased by hypoxia but increased by acidosis. The effect of hypoxia-acidosis on Bmax was abolished by inhibiting transcription with 1.0 microgram/ml actinomycin D. A quantitative reverse transcriptase polymerase chain reaction assay demonstrated a decrease in steady-state mRNA concentration with hypoxia-acidosis. Our experiments demonstrate an important distinction between the effects of modeled hypoxia and ischemia on beta 2-AR gene expression.

Animals↗

PubMed1996

Evidence of hypoxia-inducible factor-1 in vascular endothelial and smooth muscle cells.

The hypoxia-inducible element (HIE-1), a 50-bp region just 3' to the human erythropoietin gene, has been found to regulate transcription in cells that do not ordinarily synthesize erythropoietin. We hypothesized that the HIE-1 and associated protein factors may have a role in transcriptional regulation in hypoxic vascular tissues. Therefore, tissues of vascular origin were grown in culture and exposed to hypoxia (1% 02, 5% CO2, balance N2) or normoxia (21% O2, 5% CO2). Human microvascular endothelial cells (HMEC-1) studied with electrophoretic mobility shifting demonstrated that HIE-1 was bound to a protein induced by hypoxia in these cells. HMEC-1 and rat aortic smooth muscle cells (RASM) were transfected with the vector pGL2-HIE-1. HMEC-1 reporter gene expression was 3.7 +/- 0.5-fold increased at 12 hours and 3.7 +/- 1.3-fold increased at 24 hours by hypoxia. RASM reporter gene expression was 18.6 +/- 6.5-fold (SEM) increased at 12 hours and 2.0 +/- 0.7-fold increased at 24 hours by hypoxia. These findings provide indirect evidence of a hypoxia-inducible factor in vascular cellular transcriptional regulation.

Aerobiosis↗

PubMed1996

Cardiovascular deconditioning through head-down tilt bed rest increases blood pressure variability and plasma renin activity.

BACKGROUND: The renin-angiotensin-aldosterone system may be of primary importance in the mechanism of bed rest cardiovascular deconditioning. HYPOTHESIS: This study was designed to test the hypothesis that bed rest cardiovascular deconditioning does not result simply from plasma volume loss, but is also at least partially attributable to a persistent disequilibrium of the neuroendocrine mediators of plasma volume homeostasis. We examined whether changes in the renin-angiotensin-aldosterone system occur in association with the cardiovascular deconditioning and hemodynamic instability induced by antiorthostatic 6 degrees head-down tilt bed rest. METHODS: Normal male volunteers (n = 10) were tested before, during, and after 14 d of head-down tilt with a high (150 mEq.d-1) salt intake, using head-down tilt as a model of cardiovascular deconditioning and lower body negative pressure (LBNP) as a model of orthostatic stress. RESULTS: Resting plasma renin activity was 2.22 +/- 0.85 ng.ml-1.h-1 (+/- SD) at baseline and increased to 4.14 +/- 1.21 ng.ml-1.h-1 at the end of head-down tilt (p < 0.05), but urine aldosterone, plasma aldosterone, and urine sodium did not change with head-down tilt. Although the plasma norepinephrine response to LBNP was accentuated, resting adrenergic tone did not change during head-down tilt. Cardiovascular deconditioning was associated with an increase in blood pressure variability during LBNP as assessed by both beat-to-beat standard deviation and spectral analysis. CONCLUSIONS: These data support a proposed link between blood pressure variability and the renin-angiotensin system in cardiovascular deconditioning.

Adult↗

PubMed1996

Restenosis following percutaneous revascularization--the potential role of thrombin and the thrombin receptor.

Thrombin is present at sites of vascular injury and elicits many effects which may contribute to neointimal growth. Further studies are needed to order to determine steps involved in thrombin-induced effects and to identify potential sites to regulate these effects. The failure of the Helvetica trial to demonstrate an effect of treatment with hirudin on restenosis may relate more to our inability to safely inhibit thrombin than to a lack of a role for thrombin in restenosis. A therapy which enables safe and effective control of thrombin-induced responses following vascular injury may yet prove effective at reducing restenosis following percutaneous coronary revascularization.

Angioplasty, Balloon, Coronary↗

PubMed1995

Role of the thrombin receptor in restenosis and atherosclerosis.

Thrombus generation is central to thrombosis at vascular lesion sites, including post-PCTA acute reocclusion and chronic restenosis. Thrombin stimulates platelet activation, monocyte and neutrophil chemotaxis, and endothelial production of prothrombotic factors. The varied physiologic effects of thrombin are due to the widespread presence of thrombin receptors in many cell types. The receptor is uniquely activated: thrombin binds to the receptor at the thrombin anion-binding exosite, the receptor ligand ("tethered ligand") apparently being a sequence of 6 amino acids (SFLLRN). Thus, peptides corresponding to the sequence of the tethered ligand can stimulate almost all functions of native thrombin itself. Several intracellular signaling pathways have been identified as important in the restenosis process: the G protein-related pathway, cyclic adenosine monophosphate (cAMP) mediator pathway, and tyrosine kinase activation pathway. In situ hybridization has demonstrated an increase in thrombin receptor mRNA throughout the period of neointimal and vascular lesion development. The mechanism of this increase is unknown, but may be mediated by multiple inflammatory modulators. Several strategies have been tested in animal models for inhibiting thrombin: (1) Hirudin not only prevents thrombin from cleaving fibrinogen, but also prevents thrombin receptor activation. (2) Thrombin receptor antagonist peptides block platelet aggregation effects of thrombin. (3) Mono- and polyclonal antibodies inhibit thrombin receptor activation. (4) Antisense oligonucleotides block thrombin receptor expression.

Acute Disease↗

PubMed1995

Reciprocity of hemodynamic changes during lower body negative and positive pressure.

Lower body negative pressure (LBNP) and lower body positive pressure (LBPP) are two opposite environments for the lower abdomen and lower extremities. We examined RR interval (RRI), blood pressure, forearm blood flow, cardiac output, plasma atrial natriuretic factor, and plasma catecholamines during sequentially administered LBNP and LBPP in order to define the cardiovascular and neuroendocrine reciprocity of these models of orthostasis (LBNP) and anti-orthostasis (LBPP). Six healthy young men were studied with a ramped onset of LBNP followed by a similarly ramped LBPP, spaced between periods of data acquisition during rest. Forearm blood flow decreased during LBNP and increased during LBPP. However, LBNP and LBPP did not produce reciprocal changes in cardiac output. No reciprocal changes were found in plasma catecholamines and plasma atrial natriuretic factor. The variability of RRI and systolic/diastolic blood pressures were analyzed within two bandwidths of interest: the low bandwidth, corresponding to the frequency of baroreflex-mediated variability (0.04 to 0.12 Hz), and the high bandwidth, representative of parasympathetically mediated variability at the frequency of respiration (0.22 to 0.28 Hz). The ratio of high to low bandwidth RRI variability was reciprocally diminished during LBNP and increased during LBPP, reflecting a relative decrease in the level of parasympathetic tone during LBNP and an increase during LBPP. In addition, the variability of the DBP within the low baroreflex bandwidth increased during LBNP and decreased during LBPP. The phase lag of RRI variability in relation to BP variability did not change reciprocally.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1994

Left ventricular pseudoaneurysm.

Cardiorrhexis, or rupture of the myocardium, is an uncommon event after acute myocardial infarction. It has serious consequences such as acute hemopericardium, cardiac tamponade, and sudden death. If the rupture remains enclosed by surrounding adhesions or an intact visceral pericardium, a pseudoaneurysm may develop. We describe a rather typical presentation of a pseudoaneursym of the left ventricle with resultant congestive heart failure after a silent myocardial infarction, and we point out the utility of prompt ultrasound examination in this clinical setting.

Adult↗

PubMed1991

Preservation of oscillations in postocclusive reactive hyperemia.

Oscillations in skin blood flow (SkBF) during postocclusive reactive hyperemia are believed to be due to locally mediated events in the microcirculation. We characterized the activity of these oscillations in nine healthy young men who underwent 0, 10, and 40 Torr of lower body negative pressure (LBNP). Postocclusive SkBF was estimated in both forearms simultaneously in a stable thermal environment with laser-Doppler velocimetry. Periodic behavior of SkBF was characterized by frequency-domain power spectral analysis. LBNP at 40 Torr increased heart rate, decreased forearm blood flow, and decreased postocclusive SkBF amplitude but did not change the periodicity of SkBF in the frequency response range that is characteristic of postischemic SkBF oscillations (0.11 +/- 0.04 Hz). We observed that LBNP did not alter the frequency response of the postocclusive SkBF as quantified in the periodogram, even though the amplitude of the SkBF was markedly diminished as a part of the general decrease in arm blood flow. We found inferential evidence for a disseminated common pacemaker mechanism that performs similarly at distant sites. We conclude that the LBNP baroreflex-mediated modulation of SkBF reduces the amplitude but does not change the frequency behavior of postocclusive SkBF. We propose on the basis of our findings that the preservation of vasomotion suggests that this phenomenon is an adaptation to the ischemic changes induced by disruption of blood flow.

Adult↗

PubMed1990

Autonomic pathophysiology in heart failure patients. Sympathetic-cholinergic interrelations.

We conducted this study in an effort to characterize and understand vagal abnormalities in heart failure patients whose sympathetic activity is known. We measured sympathetic (peroneal nerve muscle sympathetic recordings and antecubital vein plasma norepinephrine levels) and vagal (R-R intervals and their standard deviations) activities in eight heart failure patients and eight age-matched healthy volunteers, before and after parasympathomimetic and parasympatholytic intravenous doses of atropine sulfate. At rest, sympathetic and parasympathetic outflows were related reciprocally: heart failure patients had high sympathetic and low parasympathetic outflows, and healthy subjects had low sympathetic and high parasympathetic outflows. Low dose atropine, which is known to increase the activity of central vagal-cardiac motoneurons, significantly increased R-R intervals in healthy subjects, but did not alter R-R intervals in heart failure patients. Thus, our data document reciprocal supranormal sympathetic and subnormal parasympathetic outflows in heart failure patients and suggest that these abnormalities result in part from abnormalities within the central nervous system.

Adult↗

PubMed1990

Correlation of plasma norepinephrine and plasma atrial natriuretic factor during lower body negative pressure.

Plasma atrial natriuretic factor (ANF) is released in proportion to intra-atrial pressures. Plasma norepinephrine (NE) levels are considered to be an indirect reflection of sympathetic tone. These two mediators were studied during human regulation of intravascular volume in the course of exposure to fluid shifts associated with a model of gravitational stress, lower body negative pressure (LBNP.) Blood was drawn from 10 normal subjects before and after exposure to 2 min of a graduated increase in LBNP to a level of 55 mmHg followed by 5 min at 55 mmHg. Plasma ANF was measured by RIA and catecholamines by HPLC-ECD. NE increased from 358 +/- 44 (S.E.M) pg/ml to 511 +/- 48 pg/ml (p = 0.03.) Although ANF only decreased from 27.3 +/- 2.4 pg/ml to 23.5 +/- 2.9 pg/ml (p = 0.33,) a statistically significant negative correlation was observed (r = -0.70, p = 0.02) between the changes in NE and ANF induced by LBNP. The modelling of physiologic responses to gravitational stress in this experiment revealed a negative correlation between changes in sympathetic tone (as reflected by plasma NE) and ANF levels.

Adult↗

PubMed1988

Effects of scopolamine and dextroamphetamine on human performance.

The effects of two drugs used to prevent symptoms of motion sickness in the operational environment were examined in this study of human performance as measured by computer-based tests of cognitive and psychomotor skills. Each subject was exposed repetitively to five tests: Symbol-Digit Substitution, Simple Reaction Time, Pattern Recognition, Digit Span Memory, and Pattern Memory. Although there have been previous reports of decreases in human performance in similar testing with higher dosages of scopolamine or dextroamphetamine, no significant decrements were observed with the operational-level combined dose used in this study (0.4 mg oral scopolamine and 5.0 mg oral dextroamphetamine.) The controversy over the use of combination drug therapy in this environment is discussed along with the indications for further research based on the findings.

Attention↗

PubMed1987

Localization of ectopic pheochromocytomas by magnetic resonance imaging.

Magnetic resonance imaging (MRI) was utilized to localize ectopic pheochromocytomas in two patients. In both cases, MRI proved superior to computed tomography. Differentiation from surrounding tissue was excellent. MRI permits spatial localization and by assessment of signal contrast intensity provides a suggestion as to the characteristics of the tumor without the use of invasive procedures or intravenous injections. MRI should be considered as an alternative to computed tomography when evaluating patients with a suspected pheochromocytoma.

Adrenal Gland Neoplasms↗

PubMed1981

Noradrenergic sympathetic innervation of lymphoid tissue in the rabbit appendix: further evidence for a link between the nervous and immune systems.

The rabbit appendix, a region of gut with well organized zones of lymphoid tissue, was examined with the glyoxylic acid histofluorescence technique for the localization of noradrenergic fibers, with high performance liquid chromatography with electrochemical detection (LCEC) for the quantitation of norepinephrine and serotonin, with Bielshowski and Giemsa stains for additional information about neural supply, and with acetylcholinesterase histochemistry for the localization of this hydrolytic enzyme. Fluorescent plexuses entered the serosal surface of the appendix associated with blood vessels, traveled longitudinally inside the muscularis interna, mainly in association with blood vessels but adjacent to enteric smooth muscle, and branched into long, linear, varicose plexuses that ran inward in a radial orientation towards the lumen in the internodular septa. As these fibers approached the interdomal regions near the epithelial surface, they passed through thymus-dependent cell zones, and arborized extensively throughout the interdomal region. A high density of varicosities was found in the subepithelial region where immunoglobulin-secreting plasma cells are found. These varicosities were sparse at 21 days of age, but were increased in number at 42 days of age. They were even further increased in number and density in adults. These fibers were further identified with a Bielshowski silver stain, and also demonstrated acetylcholinesterase activity. The noradrenergic varicosities in the interdomal regions of the adult rabbit appendix were closely associated with numerous yellow fluorescent cells of 25-40 microns diameter, which sometimes demonstrated fine varicose processes. The adult rabbit appendix contained a moderate concentration of norepinephrine (163.0 +/- 22.9 ng/g wet weight) and a very high concentration of serotonin (3981 +/- 283 ng/g wet weight). Levels in neonates were considerably lower, suggesting that the yellow fluorescent cells may contain serotonin. Acetylcholinesterase was associated with neural fibers and with non-neural regions of the lymph nodules and the domes, perhaps playing a protective role for these regions of the cellular immune system. The rabbit appendix is a well organized region of lymphoid tissue with specific zones of noradrenergic innervation and possible "paraneuronal" activity, with a readily accessible lumen for the isolation and collections of secretions. We propose this model as an excellent structure for further exploration of interactions between the nervous and immune systems.

Acetylcholinesterase↗

PubMed1981

Sympathetic innervation of murine thymus and spleen: evidence for a functional link between the nervous and immune systems.

Sympathetic innervation was demonstrated in both perivascular and parenchymal regions of murine thymus and spleen. Catecholamine varicosities were associated with mast cells in these areas. The antibody response to sheep red blood cells of 7 week old mice that had been sympathectomized with six-hydroxydopamine (6OHDA) at birth was significantly elevated compared with saline treated controls. Alpha-methyl tyrosine (alpha-MT) and 6OHDA treatment of mice, producing a more complete sympathectomy, showed a significantly enhanced anti-SRBC response with respect to mice treated with alpha-MT or 6OHDA alone. Catecholamine levels in thymus, spleen, and adrenals of both experimental and control mice were measured using liquid chromatography with electrochemical detection (LCEC). The present study suggests that the sympathetic nervous system has a functional role in modulating the humoral immune response in vivo.

Adrenergic Fibers↗