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Biomedical subjects

Werner Seeger

Publications and source records attributed to Werner Seeger.

At least 37 records · Page 2Linked to original sources
PubMed2006

Antioxidant vitamin C improves endothelial function in obstructive sleep apnea.

RATIONALE: Obstructive sleep apnea (OSA) is associated with oxidative stress, endothelial dysfunction, and increased cardiovascular morbidity and mortality. OBJECTIVE: We tested the hypothesis that endothelial dysfunction in patients with OSA is linked to oxidative stress. METHODS: In the present study, we measured flow-mediated dilation (FMD) of the brachial artery by ultrasound in 10 otherwise healthy, untreated patients with OSA and 10 age-and sex-matched control subjects without sleep-disordered breathing before and after intravenous injection of the antioxidant vitamin C. The investigator performing the FMD measurements was blinded to the status of the patients. RESULTS: When compared with control subjects, baseline FMD was significantly reduced in the patients with OSA. After intravenous injection of 0.5 g vitamin C, vasoreactivity remained unchanged in the control subjects. In the patients with OSA, ascorbate led to an increase in FMD to a level comparable to that observed in the control group. CONCLUSION: The reduced endothelial-dependent vasodilation in untreated patients with OSA acutely improves by the free radical scavenger vitamin C. These results are in favor of oxidative stress being responsible for the endothelial dysfunction in OSA. Antioxidant strategies should be explored for the treatment of OSA-related cardiovascular disease.

Administration, Sublingual↗
PubMed2006

Human RELMbeta is a mitogenic factor in lung cells and induced in hypoxia.

RELMbeta (resistin-like molecule) represents the most related human homologue of mouse RELMalpha, also known as hypoxic-induced mitogenic factor (HIMF). In this study, we isolated RELMbeta cDNA from human lung tissue and performed regulatory and functional expression studies. RELMbeta mRNA was upregulated in hypoxia in human lung A549 cell line as well as primary cultured adventitial fibroblasts and smooth muscle cells (SMC) of pulmonary arteries. Upon transfection of a RELMbeta encoding expression plasmid into these cells, we observed significant induction of proliferation particularly in SMC and A549 cells, which could be blocked by phosphatidyl-inositol 3-kinase (PI3K) inhibitors LY294002 and wortmannin. The results suggest that human RELMbeta may contribute to hypoxic-induced pulmonary vascular remodeling processes or hypoxia related fibrotic lung disease.

Androstadienes↗
PubMed2006

Genetic association of the serotonin transporter in pulmonary arterial hypertension.

RATIONALE: The bone morphogenetic receptor type II gene is the major genetic determinant for the inherited form of pulmonary arterial hypertension. However, deleterious mutations of this gene are not observed in the majority of subjects who develop the condition spontaneously and familial disease displays age- and sex-dependent penetrance, indicating the requirement for additional environmental and/or genetic modifiers for disease development. METHODS: We investigated polymorphic variation of the serotonin transporter gene, a biological candidate for predisposition to this vascular disorder. RESULTS: No significant evidence of association between alleles of the serotonin transporter gene and pulmonary hypertension was detected, nor did we observe a relationship with age of onset in familial and idiopathic disease. CONCLUSIONS: Variation of the serotonin transporter gene appears unlikely to confer significant susceptibility to pulmonary arterial hypertension. This study emphasizes the need for adequately powered cohorts for association analyses to identify not only genetic determinants of disease susceptibility but also inherited modifiers for disease development.

Adolescent↗
PubMed2006

Activation of soluble guanylate cyclase reverses experimental pulmonary hypertension and vascular remodeling.

BACKGROUND: Severe pulmonary hypertension is a disabling disease with high mortality, characterized by pulmonary vascular remodeling and right heart hypertrophy. Using wild-type and homozygous endothelial nitric oxide synthase (NOS3(-/-)) knockout mice with pulmonary hypertension induced by chronic hypoxia and rats with monocrotaline-induced pulmonary hypertension, we examined whether the soluble guanylate cyclase (sGC) stimulator Bay41-2272 or the sGC activator Bay58-2667 could reverse pulmonary vascular remodeling. METHODS AND RESULTS: Both Bay41-2272 and Bay58-2667 dose-dependently inhibited the pressor response of acute hypoxia in the isolated perfused lung system. When wild-type (NOS3(+/+)) or NOS3(-/-) mice were housed under 10% oxygen conditions for 21 or 35 days, both strains developed pulmonary hypertension, right heart hypertrophy, and pulmonary vascular remodeling, demonstrated by an increase in fully muscularized peripheral pulmonary arteries. Treatment of wild-type mice with the activator of sGC, Bay58-2667 (10 mg/kg per day), or the stimulator of sGC, Bay41-2272 (10 mg/kg per day), after full establishment of pulmonary hypertension from day 21 to day 35 significantly reduced pulmonary hypertension, right ventricular hypertrophy, and structural remodeling of the lung vasculature. In contrast, only minor efficacy of chronic sGC activator therapies was noted in NOS3(-/-) mice. In monocrotaline-injected rats with established severe pulmonary hypertension, both compounds significantly reversed hemodynamic and structural changes. CONCLUSIONS: Activation of sGC reverses hemodynamic and structural changes associated with monocrotaline- and chronic hypoxia-induced experimental pulmonary hypertension. This effect is partially dependent on endogenous nitric oxide generated by NOS3.

Animals↗
PubMed2006

Mutations of the TGF-beta type II receptor BMPR2 in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is clinically characterized by a sustained elevation in mean pulmonary artery pressure leading to significant morbidity and mortality. The disorder is typically sporadic, and in such cases the term idiopathic PAH (IPAH) is used. However, cases that occur within families (familial PAH (FPAH)) display similar clinical and histopathological features, suggesting a common etiology. Heterozygous mutations of a type II member of the TGF-beta cell signaling superfamily known as BMPR2 on chromosome 2q33 have been identified in many kindreds with FPAH, yet display both reduced penetrance and sex bias. This report presents the compilation of data for 144 distinct mutations that alter the coding sequence of the BMPR2 gene identified in 210 independent PAH subjects. This large data set characterizes the extent of sequence variation and reveals that the majority (71%) of mutations in FPAH and IPAH comprise nonsense, frameshift, and splice-site defects, and gene rearrangements. These predict premature termination of the transcript with likely loss through the process of nonsense-mediated decay (NMD). A total of 44 missense mutations were identified that substitute amino acid residues at highly conserved sites within recognized functional domains of the mature receptor. We assess this category of mutations in the context of their heterogeneous effects on cell signaling when assayed by in vitro cell-based systems. Disease-causing mutation hot-spots within BMPR2 are summarized. Taken together, these observations are likely to aid in the development of targeted mutation detection strategies relevant for patient management. Finally, we examine the age- and sex-dependent reduced penetrance of BMPR2 mutations by reviewing bmpr2 animal models and the requirement for additional genetic and/or environmental modifiers of disease. In conclusion, these data provide compelling genetic evidence that haploinsufficiency is the predominant molecular mechanism underlying disease predisposition, and support the concept of a critical threshold of signaling activity below which disease may be precipitated.

Bone Morphogenetic Protein Receptors, Type II↗
PubMed2006

Comparison of pharmacokinetics and vasodilatory effect of nebulized and infused iloprost in experimental pulmonary hypertension: rapid tolerance development.

Aerosolized iloprost has been suggested for selective pulmonary vasodilatation in severe pulmonary hypertension, but its pharmacokinetic profile is largely unknown. In perfused rabbit lungs, continuous infusion of the thromboxane mimetic U46619 was employed for establishing stable pulmonary hypertension. Delivery of a total amount of 75, 300, and 900 ng of iloprost to the bronchoalveolar space by a 10 min-aerosolization maneuver caused a dose-dependent pulmonary vasodilatation. Similarly, dose-dependent appearance of iloprost in the recirculating perfusate was noted, with maximum intravascular concentrations of iloprost ranging at 140, 510, and 1163 pg/mL at the same time period. Comparing pharmacokinetics and pharmacodynamics in a more detailed fashion, the following aspects were of interest. (i) The bioavailability (i.e., the percentage of aerosolized iloprost appearing intravascularly) decreased from 76% at the lowest to 33% at the highest iloprost dosage. (ii) The pulmonary vasodilatory response commenced already during the nebulization maneuver and preceded the perfusate entry of iloprost. (iii) After 3-3.5 h, the pulmonary vasodilatory response to aerosolized iloprost had virtually completely leveled off, whereas approximately two-thirds of the maximum iloprost perfusate levels were still detectable. A corresponding loss of vasodilatory response was also noted in experiments with continuous iloprost perfusion for clamping of the intravascular concentration of this prostanoid. We conclude that aerosolized iloprost causes dose-dependent vasodilatation and iloprost entry into the vascular space in a pulmonary hypertension model. Limited bioavailability in the higher dose range may suggest active prostanoid transport processes, and the early pulmonary vasodilatory response appears to be independent of prostanoid entry into the vessel lumen. Surprisingly, rapid tolerance development to the vasodilatory effect of iloprost is noted, occurring even with fully maintained perfusate levels of this agent.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed2006

Free arachidonic versus eicosapentaenoic acid differentially influences the potency of bacterial exotoxins to provoke myocardial depression in isolated rat hearts.

OBJECTIVE: Staphylococcal alpha-toxin and Escherichia coli hemolysin (ECH) evoke cardiac dysfunction in isolated rat hearts by provoking myocardial synthesis of arachidonic acid-derived thromboxane A2 or the cysteinyl-leukotrienes, LTC4, LTD4, and LTE4, respectively. We investigated whether low doses of either toxin, which fail to induce cardiac depression by themselves, induce cardiac dysfunction when combined with free arachidonic acid. DESIGN: Prospective, experimental study. SETTING: Research laboratory at a university hospital. SUBJECTS: Isolated hearts from male Wistar rats. INTERVENTIONS: Hearts were perfused with low doses of ECH or alpha-toxin in the absence or presence of arachidonic acid or the alternative eicosanoid precursor eicosapentaenoic acid (EPA). MEASUREMENTS AND MAIN RESULTS: Application of low-dose ECH with arachidonic acid increased coronary perfusion pressure, depressed left ventricular contractile function, provoked electrical instability, and induced a release of creatine kinase concomitant with the liberation of LTC4, LTD4, and LTE4 into the perfusate. All events were abolished when formation of cysteinyl-leukotrienes was blocked by the 5-lipoxygenase activity inhibitor MK-886, targeting 5-lipoxygenase activating protein. In the presence of arachidonic acid, low doses of alpha-toxin caused an increase in cerebral perfusion pressure and a decline of contractile performance, attributable to the release of thromboxane A2, as both events were mitigated by the cyclooxygenase-inhibitor indomethacin. High doses of ECH caused cardiac dysfunction even in the absence of arachidonic acid. However, in the presence of EPA, the cardiodepressant effect of ECH was blunted. Release of EPA-derived LTE5 at the expense of arachidonic acid-derived LTC4, LTD4, and LTE4 was noted in these hearts. CONCLUSIONS: The potency of the bacterial exotoxins ECH and alpha-toxin to cause coronary vasoconstriction and myocardial depression is dependent on the availability of free arachidonic acid and may be influenced by supplying omega-3 fatty acids as alternative lipid precursors.

Animals↗
PubMed2006

Lipoteichoic acid (LTA) from Staphylococcus aureus stimulates human neutrophil cytokine release by a CD14-dependent, Toll-like-receptor-independent mechanism: Autocrine role of tumor necrosis factor-[alpha] in mediating LTA-induced interleukin-8 generation.

OBJECTIVE: In sepsis, Gram-positive and Gram-negative bacteria provoke similar inflammatory processes. Whereas lipopolysaccharides (LPSs) are acknowledged as the principal immunostimulatory components of Gram-negative bacteria, the effect of the Gram-positive cell wall component lipoteichoic acid (LTA) is less well characterized. In the present study, we investigated the effect of highly purified LTA from Staphylococcus aureus on cytokine generation by isolated human neutrophils. SUBJECTS: Isolated human neutrophils from healthy volunteers. INTERVENTIONS: Incubation of neutrophils with purified LTA from S. aureus in the absence or presence of interleukin (IL)-10, anti-CD14, or anti-Toll-like-receptor antibodies. MEASUREMENTS: Measurement of tumor necrosis factor (TNF)-alpha, IL-1beta, and IL-8 by enzyme-linked immunosorbent assay. Analysis of IL-8 mRNA by reverse transcriptase polymerase chain reaction. CONCLUSIONS: The LTA challenge provoked a dramatic release of cytokines, with an early appearance of TNF-alpha and IL-1beta and a delayed liberation of IL-8. The first phase of IL-8 production was induced directly by LTA, whereas the second phase was endogenously mediated by TNF-alpha, as it was largely abrogated by neutralizing anti-TNF-alpha antibodies. In contrast, IL1-beta was not involved in LTA-induced IL-8 generation. Interestingly, the late phase of IL-8 generation could also be attenuated by exogenous IL-10, probably as a consequence of its downregulatory effects on TNF-alpha generation. When investigating the mechanism of LTA-induced cellular activation, activity-neutralizing antibodies demonstrated that CD14 was involved in LTA-mediated neutrophil cytokine generation. Using antibodies that neutralize the activity of Toll-like receptor 2 (TLR2) or 4 (TLR4), we also show that CD14-dependent, LTA-induced neutrophil activation did not proceed via TLR2- or TLR4-mediated pathways. In conclusion, LTA is a potent activator of human neutrophil cytokine generation, with the synthesis of the chemokine IL-8 being largely dependent on TNF-alpha generation in an autocrine fashion. This LTA-induced effect was inhibited by IL-10, dependent on CD14, and independent of TLR 2 or 4.

Analysis of Variance↗
PubMed2006

Pulmonary surfactant in patients with Pneumocystis pneumonia and acquired immunodeficiency syndrome.

OBJECTIVE: Pneumocystis pneumonia (PCP) is a severe infection of the immunocompromised host, resulting in diffuse alveolar damage and life-threatening respiratory failure. We analyzed pulmonary surfactant composition and function in bronchoalveolar lavage fluid (BALF) from ventilated and spontaneously breathing HIV-positive patients with PCP. DESIGN: Prospective clinical trial. SETTING: University hospital intensive care unit. PATIENTS: Thirty-four spontaneously breathing (SB-PCP) and 20 ventilated HIV-positive patients with PCP (V-PCP), ten patients with acute respiratory distress syndrome (ARDS), 11 spontaneously breathing patients with bacterial pneumonia (PNEU), and 22 healthy volunteers. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Total phospholipid in BALF did not differ between any category vs. controls, whereas total protein increased approximately 14-fold in V-PCP and five-fold in SB-PCP compared with controls (p < .001). The relative content of large surfactant aggregates (LA) was reduced in SB-PCP and V-PCP compared with controls (p < .05). The phospholipid and fatty acid profiles showed a significant reduction in the relative content of phosphatidylcholine (PC), phosphatidylglycerol, and palmitic acid in PC in all patient categories compared with controls, with more in V-PCP (p < .001) compared with SB-PCP (p < .05). The neutral lipid-to-phospholipid ratio in LA was three-fold elevated in V-PCP (p < .01 compared with control) but not in SB-PCP. Analysis of neutral lipid classes showed a significant increase in the relative content of triglycerides and a reduction in free fatty acids in V-PCP compared with controls. BALF surfactant protein (SP)-A and SP-D significantly increased in V-PCP and SB-PCP, but not in ARDS and PNEU, compared with controls (p < .05). SP-B and SP-C content in LA remained unchanged in PCP compared with controls but decreased significantly in ARDS and PNEU. The minimum surface tension of LA was impaired (p < .001) in V-PCP more than in SB-PCP and was strongly correlated with the reduction in palmitic acid levels in PC LA (r = -.81). Reductions in phosphatidylglycerol strongly correlated with decreased Pao2/Fio2 values (r = .72). CONCLUSIONS: We conclude that severe alterations in surfactant function and composition occur in patients with PCP and are even more pronounced in ventilated patients than in nonventilated patients. Surfactant lipid changes in PCP, but not surfactant protein profiles, closely resemble those found in ARDS.

AIDS-Related Opportunistic Infections↗
PubMed2006

Fish oil in the critically ill: from experimental to clinical data.

PURPOSE OF REVIEW: The aim of this paper is to describe recent relevant literature concerning the role of n-3 lipids derived from fish oil in clinical nutrition in an intensive care setting. RECENT FINDINGS: N-3 fatty acids compete with arachidonic acid for metabolism to lipid mediators and exert profound effects on second mediator generation and dependent cellular functions. In experimental models, dietary and parenteral use of fish oil was shown to protect the gut by increasing its perfusion. In contrast, use of immunonutrition including fish oil in critical ill patients or patients with severe sepsis may exert an excess mortality. Using parenteral fish oil in surgical patients promising data became available. In septic patients, immunologic effects of fish oil-based lipid emulsions have been found and intravenous supplementation with fish oil may have a beneficial impact on mortality and length of stay. For both patient groups, however, prospective data from randomized trials are lacking. SUMMARY: N-3 lipids exhibit strong immunologic properties. They offer the possibility to counterbalance the negative effects of conventional n-6 fatty acids. Recent studies exhibit positive effects of intravenous use of fish oil on immunologic functions and clinical parameters in surgical and septic patients.

Clinical Trials as Topic↗
PubMed2006

Circulating adrenomedullin in obstructive sleep apnoea.

Adrenomedullin (AM) is a potent endothelial-derived vasodilator secreted under the influence of various stimuli such as hypoxia, shear stress and cytokines. As all of these stimuli might be active under the conditions of obstructive sleep apnoea (OSA), we hypothesized that vascular AM production is increased in these patients. The study included 41 consecutive OSA patients and 28 control subjects without sleep-disordered breathing who were recruited from a pool of patients hospitalized for other reasons. Both groups were matched for anthropometric and comorbid factors. In all patients, i.e. OSA and controls, peripheral venous blood samples were taken at 07:00 hours after diagnostic polysomnography. In subsets of OSA patients, this was repeated after two nights of continuous positive airway pressure (CPAP) therapy (n = 28) and after several months of constant CPAP use (n = 11). The controls and the untreated OSA patients did not have serial blood sampling. In all blood samples, plasma AM levels were measured by an enzyme immunoassay kit. At baseline, the OSA patients had markedly elevated AM concentrations when compared to the controls. There were no differences between normo- and hypertensive OSA patients. After two nights of CPAP therapy, AM levels significantly decreased. Patients on long-term CPAP treatment showed complete normalization of plasma AM concentrations. In conclusion, this pilot study suggests that circulating AM is increased in untreated OSA irrespective of coexistent arterial hypertension and declines after CPAP therapy. AM upregulation might be considered as an adaptive mechanism to counteract the emergence of OSA-related cardiovascular disease.

Adrenomedullin↗
PubMed2006

Effect of nitric oxide synthase (NOS) inhibition on macro- and microcirculation in a model of rat endotoxic shock.

Treatment of hemodynamic instability in septic shock often demands the administration of vasopressor agents, although these may have deleterious effects on microcirculatory homeostasis. Inhibition of nitric oxide synthase (NOS) has been suggested as an alternative therapeutic approach, as NO formation may be excessively increased in sepsis. To compare the effects of epinephrine titration, non-selective NOS inhibition by L-NMMA and selective inhibition of inducible NOS (iNOS) by 1400W on hemodynamics and on the regulation of microcirculation in a rat model of endotoxic shock, we intravenously injected endotoxin (LPS) or saline to male Wistar rats and after 2 hours randomized LPS treated rats into four different groups that received either saline, norepinephrine, L-NMMA or 1400W (n = 6 per group). Three hours after LPS administration, rats presented with severe systemic arterial hypotension (64 +/- 3 vs. 115 +/- 4 mmHg, p < 0.001), unresponsiveness to volume treatment, lactate acidosis and a marked increase in plasmatic nitrite and nitrate levels (15 +/- 8 vs. 263 +/- 47 microM, p < 0.001). Measurement of the tissue oxygenation in the ileum mucosal layer by the Erlangen micro-lightguide spectrophotometer (EMPHO) technique demonstrated marked heterogeneity of hemoglobin saturation, with appearance of low oxygenated areas. Norepinephrine, usually stabilizing blood pressure (99 +/- 7 vs. 67 +/- 4 mmHg 60 min after infusion, p < 0.01), increased lactate formation (7.9 +/- 0.2 vs. 3.7 +/- 0.5 mM, p < 0.001) and drastically increased low oxygenated regions in the ileum mucosal layer. L-NMMA similarly increased blood pressure (92 +/- 6 vs. 67 +/- 4 mmHg 60 min after infusion, p < 0.05), but did not enhance lactate acidosis. However, some further deterioration of mucosa oxygenation was again noted. 1400W forwarded stabilization of blood pressure (88 +/- 5 vs. 67 +/- 4 mmHg 60 min after injection, p < 0.05), reduced plasmatic nitrite and nitrate levels similar to L-NMMA, without an aggravation of lactate acidosis. In addition, mucosal oxygenation did not deteriorate in response to this agent. Thereby, we conclude that in a rat model of endotoxic shock selective iNOS inhibitors are superior to non-specific NOS inhibitors and in particular to norepinephrine for the treatment of macro- and microcirculatory abnormalities in experimental septic shock.

Amidines↗
PubMed2006

Hypoxic pulmonary vasoconstriction--triggered by an increase in reactive oxygen species?

Hypoxic pulmonary vasoconstriction (HPV) is an essential mechanism of the lung that matches perfusion to ventilation in order to optimize pulmonary gas exchange. Despite intensive research, the underlying mechanism has not yet been fully elucidated. Reactive oxygen species (ROS) have been proposed as key mediators of HPV. However, there is ongoing discussion as to whether ROS really contribute to HPV regulation and if so, whether an increase or a decrease in ROS occurs during alveolar hypoxia. In this overview, we summarize our data that have led us to conclude that alveolar hypoxia induces an increase in superoxide and subsequently H2O2, and thus elicits HPV. This conclusion is drawn from investigations employing various inhibitors that interfere with ROS in isolated buffer-perfused rabbit lungs challenged with 10-minute periods of alveolar hypoxia. Targeting possible sources of a hypoxia-induced increase in ROS, our data are only partially in accordance with the hypothesis that mitochondria are the hypoxia-dependent ROS generators, and suggest NADPH oxidases as an alternative source. From measurements of intracellular and exhaled H2O2, we hypothesize that total lung ROS release is reduced in alveolar hypoxia, but that in specialized cells or sub-cellular structures an increased ROS release may occur, triggering HPV.

Animals↗
PubMed2005

Impact of mitochondria and NADPH oxidases on acute and sustained hypoxic pulmonary vasoconstriction.

Hypoxic pulmonary vasoconstriction (HPV) matches lung perfusion with ventilation to optimize pulmonary gas exchange. However, it remains unclear whether acute HPV (occurring within seconds) and the vasoconstrictor response to sustained alveolar hypoxia (developing over several hours) are triggered by identical mechanisms. We investigated the effect of mitochondrial and NADPH oxidase inhibitors on both phases of HPV in intact rabbit lungs. These studies revealed that the sustained HPV is largely dependent on mitochondrial complex I and totally dependent on complex IV, whereas NADPH oxidase dependence was only observed for acute HPV. These findings were reinforced by an alternative approach employing lungs from mice deficient in the NADPH oxidase subunit p 47(phox). In these mice (which lack a subunit suggested to be important for the function of most NADPH oxidase isoforms), but not in gp 91(phox)-deficient mice (which represent only one isoform of NADPH oxidases), acute HPV was significantly reduced, while non-hypoxia-induced vasoconstrictions elicited by the thromboxane mimetic U46619 were not affected. We concluded that the acute phase and the sustained phase of HPV are differentially regulated, with NADPH oxidase activity predominating in the acute phase, while a strong dependence on mitochondrial participation was observed for the second phase.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed2005

Tropolysin, a new oligopeptidase from African trypanosomes.

Oligopeptidases are emerging as important pathogenic factors and therapeutic targets in trypanosome infections. We describe here the purification, cloning, and biochemical analysis of a new oligopeptidase from two pathogenic African trypanosomes. This oligopeptidase, which we have called tropolysin (encoded by the trn gene), represents an evolutionarily distant member of the M3A subfamily of metallopeptidases, ancestral to thimet oligopeptidase, neurolysin, and saccharolysin. The trn gene was present as a single copy per haploid genome, was expressed in both the mammalian and insect stages of the parasite life cycle, and encoded an 84 kDa protein. Both purified and hyperexpressed tropolysin hydrolyzed bradykinin-derived fluorogenic peptide substrates at restricted sites, with an alkaline pH optimum, and were activated by dithiothreitol and reduced glutathione and by divalent metal cations, in the order Zn(2+) > Co(2+) > Mn(2+). Under oxidizing conditions, tropolysin reversibly formed inactive multimers. Tropolysin exhibited a preference for acidic amino acid side chains in P(4), hydrophobic side chains in P(3), and hydrophobic or large uncharged side chains in P(1), P(1)', and P(3)', while the S(2)' site was unselective. Highly charged residues were not tolerated in P(1)'. Tropolysin was responsible for the bulk of the kinin-degrading activity in trypanosome lysates, potently (k(cat) approximately 119 s(-)(1)) inactivated the vasoactive kinins bradykinin and kallidin, and generated angiotensin(1-7) from angiotensin I. This hydrolysis both abolished the capacity of bradykinin to stimulate the bradykinin B(2) receptor and abrogated bradykinin prohypotensive properties in vivo, raising the possibility that tropolysin may play a role in the dysregulated kinin metabolism observed in the plasma of trypanosome-infected hosts.

Africa↗
PubMed2005

Impact of HIF-1alpha and HIF-2alpha on proliferation and migration of human pulmonary artery fibroblasts in hypoxia.

Proliferation of adventitial fibroblasts of small intrapulmonary arteries (FBPA) has been disclosed as an early event in the development of pulmonary hypertension and cor pulmonale in response to hypoxia. We investigated the role of hypoxia-inducible transcription factors (HIF) in human FBPA exposed to hypoxia. Primary cultures of FBPA displayed a strong mitogenic response to 24 h hypoxia, whereas the rate of apoptosis was significantly suppressed. In addition, the migration of FBPA was strongly increased under hypoxic conditions but not the expression of alpha-smooth muscle actin. Hypoxia induced a marked up-regulation (protein level) of both HIF-1alpha and HIF-2alpha, alongside with nuclear translocation of these transcription factors. Specific inhibition of either HIF-1alpha or HIF-2alpha was achieved by RNA interference technology, as proven by HIF-1alpha and HIF-2alpha mRNA and protein analysis and expression analysis of HIF downstream target genes. With the use of this approach, the hypoxia-induced proliferative response of the FBPA was found to be solely HIF-2alpha dependent, whereas the migratory response was significantly reduced by both HIF-1alpha and HIF-2alpha interference. In conclusion, HIF up-regulation is essential for hypoxic cellular responses in human pulmonary artery adventitial fibroblasts such as proliferation and migration, mimicking the pulmonary hypertensive phenotype in vivo. Differential HIF subtype dependency was noted, with HIF-2alpha playing a predominant role, which may offer future intervention strategies.

Actins↗
PubMed2005

Inhaled tolafentrine reverses pulmonary vascular remodeling via inhibition of smooth muscle cell migration.

BACKGROUND: The aim of the study was to assess the chronic effects of combined phosphodiesterase 3/4 inhibitor tolafentrine, administered by inhalation, during monocrotaline-induced pulmonary arterial hypertension (PAH) in rats. METHODS: CD rats were given a single subcutaneous injection of monocrotaline to induce PAH. Four weeks after, rats were subjected to inhalation of tolafentrine or sham nebulization in an unrestrained, whole body aerosol exposure system. In these animals (i) the acute pulmonary vasodilatory efficacy of inhaled tolafentrine (ii) the anti-remodeling effect of long-term inhalation of tolafentrine (iii) the effects of tolafentrine on the expression profile of 96 genes encoding cell adhesion and extracellular matrix regulation were examined. In addition, the inhibitory effect of tolafentrine on ex vivo isolated pulmonary artery SMC cell migration was also investigated. RESULTS: Monocrotaline injection provoked severe PAH (right ventricular systolic pressure increased from 25.9 +/- 4.0 to 68.9 +/- 3.2 after 4 weeks and 74.9 +/- 5.1 mmHg after 6 weeks), cardiac output depression and right heart hypertrophy. The media thickness of the pulmonary arteries and the proportion of muscularization of small precapillary resistance vessels increased dramatically, and the migratory response of ex-vivo isolated pulmonary artery smooth muscle cells (PASMC) was increased. Micro-arrays and subsequent confirmation with real time PCR demonstrated upregulation of several extracellular matrix regulation and adhesion genes, such as matrixmetalloproteases (MMP) 2, 8, 9, 10, 11, 12, 20, Icam, Itgax, Plat and serpinb2. When chronically nebulized from day 28 to 42 (12 daily aerosol maneuvers), after full establishment of severe pulmonary hypertension, tolafentrine reversed about 60% of all hemodynamic abnormalities, right heart hypertrophy and monocrotaline-induced structural lung vascular changes, including the proportion of pulmonary artery muscularization. The upregulation of extracellular matrix regulation and adhesion genes was reduced by nearly 80% by inhalation of the tolafentrine. When assessed in vitro, tolafentrine blocked the enhanced PASMC migratory response. CONCLUSION: In conclusion, we demonstrate for the first time that inhalation of combined PDE3/4 inhibitor reverses pulmonary hypertension fully developed in response to monocrotaline in rats. This "reverse-remodeling" effect includes structural changes in the lung vascular wall and key molecular pathways of matrix regulation, concomitant with 60% normalization of hemodynamics.

Administration, Inhalation↗