Ladislau Kiss

Increased levels and reduced catabolism of asymmetric and symmetric dimethylarginines in pulmonary hypertension

Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase (NOS) and has been implicated in endothelial dysfunction. ADMA is metabolized by the enzyme dimethylarginine dimethylaminohydrolase (DDAH), with DDAH2 representing the predominant endothelial DDAH isoform. Symmetric dimethylarginine (SDMA), also originating from arginine methylation by protein arginine methyltransferases, is an inhibitor of intracellular arginine uptake. In both chronic pulmonary hypertensive rats and patients suffering from idiopathic pulmonary arterial hypertension (IPAH; NYHA class III and IV), a marked increase in plasma ADMA and SDMA levels, as well as tissue levels of asymmetric and symmetric dimethylated proteins, was observed. Moreover, when comparing lung tissue from pulmonary hypertensive rats and IPAH patients to corresponding normal lung tissue, expression of DDAH2 was found to be reduced at both the mRNA and the protein level with no significant changes in DDAH1 expression. These findings were further supported by demonstrating a decrease in DDAH2 function in the experimental pulmonary hypertension model. Immunohistochemistry in human and rat control tissue demonstrated both isoforms of DDAH in the endothelial layer and in the alveolar epithelium. In contrast, in pulmonary hypertensive tissue, the immunoreactivity of DDAH2 in pulmonary endothelium was significantly decreased compared with DDAH1. Therefore, altogether we can conclude that enhanced dimethylarginine levels may contribute to vascular abnormalities in pulmonary arterial hypertension. Suppression of endothelial DDAH2 expression and function represents an important underlying mechanism.

Nifedipine Increases Cytochrome P4502C Expression and Endothelium-Derived Hyperpolarizing Factor–Mediated Responses in Coronary Arteries

In addition to NO and prostacyclin, endothelial cells release a factor that elicits vasodilatation by hyperpolarizing the underlying vascular smooth muscle cells. In some vascular beds, this so-called endothelium-derived hyperpolarizing factor (EDHF) displays the characteristics of a cytochrome P450 (CYP)-derived arachidonic acid metabolite, such as an epoxyeicosatrienoic acid. Native porcine and cultured human coronary artery endothelial cells were screened for CYP epoxygenases, and CYP2B, CYP2C, and CYP2J were detected with reverse transcription-polymerase chain reaction. The CYP inducer beta-naphthoflavone and the Ca(2+) antagonist nifedipine significantly increased CYP2C mRNA but did not change the expression of CYP2J or CYP2B. To determine the relationship between CYP2C expression and EDHF production in native endothelial cells, we incubated porcine coronary arteries with nifedipine. Nifedipine enhanced endothelial CYP2C protein expression, as well as the generation of 11,12-epoxyeicosatrienoic acid. In organ bath experiments, pretreatment with nifedipine enhanced bradykinin-induced, EDHF-mediated relaxations as well as the concomitant hyperpolarization of smooth muscle cells. The specific CYP2C9 inhibitor sulfaphenazole, on the other hand, significantly attenuated EDHF-mediated hyperpolarization and relaxation. These results demonstrate that in porcine coronary arteries, the elevated expression of a CYP epoxygenase, homologous to CYP2C8/9, is associated with enhanced EDHF-mediated hyperpolarization in response to bradykinin. Therefore, we propose that an isozyme of CYP2C is the most likely candidate for the CYP-dependent EDHF synthase in porcine coronary arteries.

Cytochrome P450 Epoxygenase Gene Function in Hypoxic Pulmonary Vasoconstriction and Pulmonary Vascular Remodeling

We assessed pulmonary cytochrome P450 (CYP) epoxygenase expression and activity during hypoxia and explored the effects of modulating epoxygenase activity on pulmonary hypertension. The acute hypoxic vasoconstrictor response was studied in Swiss Webster mice, who express CYP2C29 in their lungs. Animals were pretreated with vehicle, the epoxygenase inhibitor (N-methylsulfonyl-6-[2-propargyloxyphenyl] hexanamide) or an inhibitor of the soluble epoxide hydrolase. Whereas the epoxygenase inhibitor attenuated hypoxic pulmonary constriction (by 52%), the soluble epoxide hydrolase inhibitor enhanced the response (by 39%), indicating that CYP epoxygenase–derived epoxyeicosatrienoic acids elicit pulmonary vasoconstriction. Aerosol gene transfer of recombinant adenovirus containing the human CYP2C9 significantly elevated mean pulmonary artery pressure and total pulmonary resistance indices, both of which were sensitive to the inhibitor sulfaphenazole. The prolonged exposure of mice to hypoxia increased CYP2C29 expression, and transcript levels increased 5-fold after exposure to normobaric hypoxia (FIO2 0.07) for 2 hours. This was followed by a 2-fold increase in protein expression and by a significant increase in epoxyeicosatrienoic acid production after 24 hours. Chronic hypoxia (7 days) elicited pulmonary hypertension and pulmonary vascular remodeling, effects that were significantly attenuated in animals continually treated with N-methylsulfonyl-6-[2-propargyloxyphenyl] hexanamide (−46% and −55%, respectively). Our results indicate that endogenously generated epoxygenase products are associated with hypoxic pulmonary hypertension in mice and that selective epoxygenase inhibition significantly reduces acute hypoxic pulmonary vasoconstriction and chronic hypoxia-induced pulmonary vascular remodeling. These observations indicate potential novel targets for the treatment of pulmonary hypertension and highlight a pivotal role for CYP epoxygenases in pulmonary responses to hypoxia.

Adult respiratory distress syndrome: model systems using isolated perfused rabbit lungs.

Publisher Summary The adult respiratory distress syndrome (ARDS) is a persisting problem in modern intensive care medicine, still resulting in a high fatality rate. This chapter presents several studies that have demonstrated that, among the different initial events involved, sepsis and polytrauma represent the major risk factors for the development of this condition. Lung injury is triggered via mechanisms still not fully elucidated. Severe disturbances in pulmonary physiology occur, including a rise of pulmonary vascular resistance and an increase in the permeability of the capillary-endothelial and the alveoloepithelial barrier. These two key alterations result in the formation of protein-rich interstitial and subsequently intraalveolar edema, followed by deterioration of alveolar surfactant function. Mismatch in the adaptation of perfusion and ventilation because of “anarchic” vasoconstriction and vascular occlusion increase in shunt flow because of loss of alveolar spaces (edema, atelectasis), and hindrance of diffusion of gases results in severe deterioration of gas exchange with arterial hypoxemia. This early “exudative” phase of ARDS may then be followed by a protracted “proliferative” phase, with remodeling of lung structure and development of widespread lung fibrosis within a few weeks. Isolated, perfused lungs have long been used by investigators interested in the physiological, biochemical, and metabolic aspects of this complex organ. This technique has also been adopted to study pathogenetic events underlying the exudative phase of ARDS.

Leukotriene-mediated coronary vasoconstriction and loss of myocardial contractility evoked by low doses of Escherichia coli hemolysin in perfused rat hearts

ObjectiveEscherichia coli hemolysin has been implicated as an important pathogenic factor in extraintestinal E. coli infections including sepsis. We investigated the effects of coronary administration of E. coli hemolysin on cardiac function in isolated rat hearts perfused at constant flow. DesignProspective, experimental study. SettingResearch laboratory at a university hospital. SubjectsIsolated hearts from male Wistar rats. InterventionsIsolated hearts were perfused with purified E. coli hemolysin for 60 min. Measurements and Main ResultsLow concentrations of the toxin in the perfusate (0.1–0.2 hemolytic units/mL) caused a dose-dependent coronary vasoconstriction with a marked increase in coronary perfusion pressure, which was paralleled by a decrease in left ventricular developed pressure (and the maximum rate of left ventricular pressure increase). Moreover, 0.2 hemolytic units/mL E. coli hemolysin evoked ventricular fibrillation within 10 mins of toxin application. These events were accompanied by the liberation of leukotrienes (LTC4, LTD4, LTE4, and LTB4), thromboxane A2, prostaglandin I2, and the cell necrosis markers lactate dehydrogenase and creatine kinase into the recirculating perfusate. The lipoxygenase inhibitor MK-886 fully blocked the toxin-induced coronary vasoconstrictor response and the loss of myocardial contractility and reduced the release of lactate dehydrogenase and creatine kinase. In contrast to this, the cyclooxygenase inhibitor indomethacin was entirely ineffective. In addition, E. coli hemolysin elicited an increase in heart weight and left ventricular end-diastolic pressure, the latter again being suppressed by MK-886. ConclusionsLow doses of E. coli hemolysin cause strong coronary vasoconstriction, linked with loss of myocardial performance, release of cell injury enzymes, and electrical instability, with all events being largely attributable to toxin-elicited leukotriene generation in the coronary vasculature. Bacterial exotoxins such as E. coli hemolysin thus may be implicated in the cardiac abnormalities encountered in septic shock.

Targeting Lipopolysaccharides by the Nontoxic Polymyxin B Nonapeptide Sensitizes Resistant Escherichia coli to the Bactericidal Effect of Human Neutrophils

The nonapeptide of polymyxin B (PMBN) has been reported to sensitize various pathogenic gram-negative bacteria to the direct bactericidal effect of human serum. To investigate the impact of PMBN on human neutrophil-effected killing of the serum- and phagocytosis-resistant Escherichia coli strains C14 and O111, serum was coapplied with PMBN or with neutrophils, but this did not result in decreased numbers of viable bacteria. In contrast, the most potent bacterial killing occurred in the presence of neutrophils plus serum components plus PMBN. The effect of this on E. coli C14 was the appearance of inositol phosphates, diacylglycerol, respiratory burst, elastase liberation, and generation of lipid mediators (leukotriene B(4), 5-HETE, and platelet-activating factor). Strong neutrophil activation required early, but not late, complement components and was blocked by inhibition of phagocytosis with cytochalasin D. PMBN seems to cause dramatic support of natural host defense by complement-dependent sensitization of E. coli to the bactericidal efficacy of human neutrophils.

Direct and simultaneous profiling of epoxyeicosatrienoic acid enantiomers by capillary tandem column chiral-phase liquid chromatography with dual online photodiode array and tandem mass spectrometric detection

AbstractDespite first evidence for the cytochrome P450-mediated enantioselective biosynthesis and activity of cis-epoxyeicosatrienoic acids (EETs), as yet little is known about the stereospecifity of EET generation and physiology, because the existing chiral methods are time consuming, labor intensive, and not sensitive enough. We present a method for highly sensitive, direct, and simultaneous chiral analysis of all eight EET enantiomers consisting of (i) solid-phase extraction, (ii) reversed-phase high-performance liquid chromatographic purification followed by (iii) consecutive regio- and enantiomeric separation of the four underivatized EET regioisomers within one chromatographic run employing capillary tandem column chiral-phase liquid chromatography with (iv) reliable dual online photodiode array and gentle electrospray ionization tandem mass spectrometric identification and quantitation of the eluting optical antipodes. This one-step, simple, expeditious, and highly sensitive measurement allows profiling of all eight EET enantiomers at once, thus avoiding substance loss and enabling high sample throughput. Limits of quantification in the low picogram range were achieved by the use of capillary columns with typical high quantitative sensitivity instead of conventional columns with low chromatographic signal intensity employed by previous methods. Application to tissue homogenates demonstrated the suitability of this approach for routine and reliable “enantioprofiling” of free endogenous EETs, i.e., EETs not esterified into cellular membrane phospholipids, typically occurring at very low concentrations. The technique can readily be employed for preparative purification of enantiomers in the microgram range using large-inner-diameter columns. FigureDirect and simultaneous enantioprofiling of the four free endogenous epoxyeicosatrienoic acids (EETs) from a complex biological matrix, like the cardiopulmonary system, within one chromatographic run by highly sensitive, one-step capillary tandem column chiral-phase liquid chromatography with dual online photodiode array and tandem mass spectrometric detection (CapTC-CP-LC-PDAD-ESI-MS2) enables accurate, systematic, and routine correlation between the absolute configuration of EETs and their physiological actions