Depletion of Bone Marrow-Derived Fibrocytes Attenuates TAA-Induced Liver Fibrosis in Mice.

Bone marrow-derived fibrocytes (FC) represent a unique cell type, sharing features of both mesenchymal and hematopoietic cells. FC were shown to specifically infiltrate the injured liver and participate in fibrogenesis. Moreover, FC exert a variety of paracrine functions, thus possibly influencing the disease progression. However, the overall contribution of FC to liver fibrosis remains unclear. We aimed to study the effect of a specific FC depletion, utilizing a herpes simplex virus thymidine kinase (HSV-TK)/Valganciclovir suicide gene strategy. Fibrosis was induced by oral thioacetamide (TAA) administration in C57BL/6J mice. Hepatic hydroxyproline content was assessed for the primary readout. The HSV-TK model enabled the specific depletion of fibrocytes. Hepatic hydroxyproline content was significantly reduced as a result of the fibrocyte ablation (-7.8%; 95% CI: 0.7-14.8%; p = 0.033), denoting a reduced deposition of fibrillar collagens. Lower serum alanine transaminase levels (-20.9%; 95% CI: 0.4-36.9%; p = 0.049) indicate a mitigation of liver-specific cellular damage. A detailed mode of action, however, remains yet to be identified. The present study demonstrates a relevant functional contribution of fibrocytes to chronic toxic liver fibrosis, contradicting recent reports. Our results emphasize the need to thoroughly study the biology of fibrocytes in order to understand their importance for hepatic fibrogenesis.

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.

Despite 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.

Direct eicosanoid profiling of the hypoxic lung by comprehensive analysis via capillary liquid chromatography with dual online photodiode-array and tandem mass-spectrometric detection.

Eicosanoids are arachidonic acid-derived mediators, with partly contradictory, incompletely elucidated actions. Thus, epoxyeicosatrienoic acids (EETs) are controversially discussed as putative vasodilatative endothelium-derived hyperpolarizing factors in the cardiovascular compartment but reported as vasoconstrictors in the lung. Inconsistent findings concerning eicosanoid physiology may be because previous methods were lacking sensitivity, identification reliability, and/or have focused on special eicosanoid groups only, ignoring the overall mediator context, and thus limiting the correlation accuracy between autacoid formation and bioactivity profile. Therefore, we developed an approach which enables the simultaneous assessment of 44 eicosanoids, including all representatives of the arachidonic acid cascade, i.e., cytochrome P450, lipoxygenase, cyclooxygenase products, and free isoprostanes as in vivo markers of oxidative stress, in one 50-minute chromatographic run. The approach combines (i) source-specific sample extraction, (ii) rugged isocratic and high-sensitivity capillary liquid-chromatographic separation, and (iii) reliable dual online photodiode-array and electrospray ionization tandem mass-spectrometric identification and quantitation. High sensitivity with limits of quantification in the femtogram range was achieved by use of capillary columns with typical high peak efficiency, due to small inner diameters, and virtually complete substance transfer to the mass spectrometer, due to flow rates in the low microliter range, instead of large inner diameter columns with low chromatographic signal and only partial analyte transfer employed by previous methods. This expeditious, global and sensitive technique provides the prerequisite for new, accurate insights regarding the physiology of specific mediators, for example EETs, in the context of all relevant vasoactive autacoids under varying conditions of oxidative stress by direct comparison of all eicosanoid generation profiles. Indeed, application of comprehensive "eicoprofiling" to hypoxically ventilated rabbit lungs revealed at a glance the enhanced biosynthesis of free EETs in the overall mediator generation context, thus suggesting their hypothetical contribution to hypoxic pulmonary vasoconstriction.

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.

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.

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.

The synthesis of 20-HETE in small porcine coronary arteries antagonizes EDHF-mediated relaxation.

OBJECTIVE: Exogenous application of 20-hydroxyeicosatetraenoic acid (20-HETE) to small (300-500 microm) porcine coronary arteries elicits contraction by activating the Rho kinase and increasing the sensitivity of contractile proteins to Ca2+. Here, we determined whether 20-HETE is involved in the regulation of coronary artery tone as well as its role in the modulation of endothelium-derived hyperpolarizing factor (EDHF)-mediated responses. METHODS AND RESULTS: Small porcine coronary arteries expressed cytochrome P450 (CYP) 4A, as demonstrated by Western blot analysis, and generated 20-HETE. Moreover, 20-HETE production was increased two- and threefold over basal levels in response to isometric stretch or the thromboxane analogue U46619, respectively, and was inhibited by the CYP 4A inhibitor N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS). In vascular reactivity studies, DDMS attenuated U46619-induced contractions and induced a concentration-dependent but endothelium-independent relaxation of precontracted arterial rings. Endogenously generated 20-HETE significantly inhibited the EDHF-mediated relaxation of coronary arteries, which was potentiated by the phospholipase A2 inhibitors AACOCF3 and ONO-RS-082, as well as by the omega-hydroxylase inhibitors 17-octadecynoic acid and DDMS. EDHF-mediated relaxation was not affected by either the nonselective epoxygenase inhibitors miconazole and clotrimazole or the CYP 2C inhibitor sulfaphenazole but was abolished by the Na-K-ATPase inhibitor, ouabain. Exogenous application of 20-HETE inhibited EDHF-mediated relaxations and caused a concomitant increase in the phosphorylation of protein kinase Calpha (PKCalpha). This effect was reversed by the PKC inhibitor Ro-318220 and mimicked by the PKC activator phorbol-12 myristate 13-acetate. CONCLUSIONS: These results indicate that vascular tone in small porcine coronary arteries is partly determined by the endogenous production of 20-HETE. In addition, 20-HETE functionally antagonizes EDHF-mediated relaxation via a PKCalpha-dependent mechanism, probably involving the inhibition of the Na-K-ATPase.

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

OBJECTIVE: hemolysin has been implicated as an important pathogenic factor in extraintestinal infections including sepsis. We investigated the effects of coronary administration of hemolysin on cardiac function in isolated rat hearts perfused at constant flow. DESIGN: Prospective, experimental study. SETTING: Research laboratory at a university hospital. SUBJECTS: Isolated hearts from male Wistar rats. INTERVENTIONS: Isolated hearts were perfused with purified hemolysin for 60 min. MEASUREMENTS AND MAIN RESULTS: Low 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 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, hemolysin elicited an increase in heart weight and left ventricular end-diastolic pressure, the latter again being suppressed by MK-886. CONCLUSIONS: Low doses of 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 hemolysin thus may be implicated in the cardiac abnormalities encountered in septic shock.

Staphylococcal alpha-toxin provokes neutrophil-dependent cardiac dysfunction: role of ICAM-1 and cys-leukotrienes.

The role of polymorphonuclear neutrophils (PMN) in septic myocardial dysfunction is presently unknown. Staphylococcus aureus infections are frequently associated with septic sequelae. Therefore, we perfused isolated rat hearts with low doses of alpha-toxin, the major staphylococcal exotoxin, followed by application of human PMN, N-formyl-methionyl-leucyl-phenylalanine, and arachidonic acid. In contrast to sham-perfused hearts (no alpha-toxin), a rise in coronary perfusion pressure (CPP) and a reduction of contractile function were noted, and cardiac expression of intercellular adhesion molecule (ICAM)-1 was detected by immunohistochemical methods and real-time PCR. Histological analysis and myeloperoxidase activity indicated cardiac PMN accumulation in alpha-toxin-challenged hearts. Major quantities of cysteinyl (cys)-leukotrienes (LT), LTB4, and 5-hydroxyeicosatetraenoic acid (5-HETE) were found in the perfusate of alpha-toxin-exposed hearts. With an anti-ICAM-1 antibody, neutrophil accumulation, leukotriene (LT) synthesis, coronary vasoconstriction, and the accompanying cardiodepression were suppressed. Similarly, the lipoxygenase inhibitor MK-886 blocked LT synthesis and maintained cardiac function. We conclude that low-dose alpha-toxin provokes coronary endothelial ICAM-1 expression and neutrophil accumulation, with subsequent synthesis of cys-LTs, LTB4, and 5-HETE under conditions of appropriate stimulation. This response is linked with coronary vasoconstriction and contractile dysfunction, with cys-LT synthesis and maldistribution of perfusion offered as likely underlying mechanisms.