Increased intrahepatic resistance in severe steatosis: endothelial dysfunction, vasoconstrictor overproduction and altered microvascular architecture.

Non-alcoholic fatty liver disease can progress to steatohepatitis and fibrosis, and is also associated with impaired liver regeneration. The pathophysiology remains elusive. We recently showed that severe steatosis is associated with an increase in portal pressure, suggesting liver flow impairment. The objective of this study is to directly assess total intrahepatic resistance and its potential functional and structural determinants in an in situ perfusion model. Male Wistar rats fed a control (n = 30) or a methionine-choline-deficient (MCD) diet (n = 30) for 4 weeks were compared. Liver tissue and serum analysis, in vivo haemodynamic measurements, in situ perfusion experiments and vascular corrosion casts were performed. The MCD group showed severe steatosis without inflammation or fibrosis on histology. Serum levels and liver tissue gene expression of interleukin (IL)-6, tumour necrosis factor-α, IL-1ß and interferon-γ, liver tissue myeloperoxidase activity and liver immunohistochemistry with anti-CD68 and anti-α smooth muscle actin were comparable between groups, excluding significant inflammation. Flow-pressure curves were significantly different between groups for all flows (slope values: 0.1636 ± 0.0605 mm Hg/ml/min in controls vs 0.7270 ± 0.0408 mm Hg/ml/min in MCD-fed rats, P < 0.001), indicating an increased intrahepatic resistance, which was haemodynamically significant (portocaval pressure gradient 2.2 ± 1.1 vs 8.2 ± 1.3 mm Hg in controls vs MCD, P<0.001). Dose-response curves to acetylcholine were significantly reduced in MCD-fed rats (P < 0.001) as was the responsiveness to methoxamine (P<0.001). Vascular corrosion casts showed a replacement of the regular sinusoidal anatomy by a disorganized pattern with multiple interconnections and vascular extensions. Liver phosphorylated endothelial NO synthase (eNOS)/eNOS and serum nitrite/nitrate were not increased in severe steatosis, whereas liver thromboxane synthase expression, liver endothelin-1 (ET-1) expression and serum andothelin-1 concentration were significantly increased. Severe steatosis induces a haemodynamically significant increase in intrahepatic resistance, which precedes inflammation and fibrogenesis. Both functional (endothelial dysfunction and increased thromboxane and ET-1 synthesis) and structural factors are involved. This phenomenon might significantly contribute to steatosis-related disease.

Non-monooxygenase cytochromes P450 as potential human autoantigens in anticonvulsant hypersensitivity reactions.

Antibodies recognizing rat cytochrome P450 (CYP) 3A1 but not the closely related human CYPs 3A4/5 have been identified in the sera of patients with hypersensitivity reactions to phenytoin and carbamazepine. Comparison of the mapped epitope to the comparable region in CYP3A4 revealed that Leu361 was essential for antibody recognition because of L361V mutation (mimicking human EYLDMVVNETLRL) abolished immunoreactivity. To identify alternative human autoantigens, a site-directed mutagenesis strategy was employed to identify amino acids critical for antibody recognition. A protein database search with the consensus sequence, DxVLxETLxx, from immunoblot analysis produced CYP8 (prostacyclin synthase), CYP5A1 (thromboxane synthase), CYP27 and CYP7A1 (cholesterol 7 alpha-hydroxylase) as possible candidates; considerable homology was also observed with the fungal CYP52A subfamily. Immunoblotting with patient sera and fragments of each candidate autoantigen expressed as Escherichia coli gene 10 fusion proteins confirmed CYP8 and CYP5A1 as possible antigens, and revealed the presence of IgG1 and IgG3 antibodies against a construct mimicking fungal CYP52A10. All patient sera contained IgG4 antibodies against CYP8, CYP5A1 and the fungal mimic suggestive of continual antigenic challenge. In genetically susceptible individuals, prior infectious challenge may be a determinant of risk for the development of anticonvulsant hypersensitivity reactions and has been incorporated into a model investigating the pathogenesis of these events.

Intravillous eicosanoid compartmentalization and regulation of placental blood flow.

OBJECTIVE: To determine the roles of the eicosanoids thromboxane and prostacyclin, and their compartmentalization, in the regulation of placental blood flow. METHODS: First, the sites of production of thromboxane and prostacyclin were determined within the placental villus using immunohistochemical staining for thromboxane and prostacyclin synthetase. Second, the production of both eicosanoids was studied in cultured trophoblasts and compared with that in the villous core by measuring the metabolites thromboxane B2 and 6-keto-prostaglandin F 1 alpha. Finally, eicosanoid production was assessed in intact villi after stimulation by an acute change in oxygen content, 5% to 95%. RESULTS: Immunohistochemical staining showed that thromboxane production was primarily within the trophoblasts, whereas prostacyclin production was localized to the endothelial cells within the villi. In culture, we found preferential production of prostacyclin by the villous core cells and increased production of thromboxane by trophoblasts. Perifusion of intact villi demonstrated increased production of thromboxane by trophoblasts in response to an increase in oxygen content. Prostacyclin levels were too low to be detected. CONCLUSIONS: Placental blood flow appears to be regulated by compartmentalized eicosanoids, with thromboxane affecting primarily the maternal side of the placental circulation and prostacyclin affecting primarily the fetal side.

Activated microglia are the principal glial source of thromboxane in the central nervous system.

Thromboxane A2(TxA2) is a potent vasoconstrictor associated with cerebrovascular disease and is thought to be synthesized within tissues of the brain. In order to determine the cellular sources of TxA2 in the central nervous system (CNS), we measured the release of the stable metabolite TxB2 in cultures of mixed or highly enriched populations of brain glia. Using techniques which isolated large numbers of highly enriched microglia and astroglia, we found that only microglia release TxB2. Moreover, microglia, not astroglia, contain the requisite synthetic enzyme thromboxane synthase. Phagocytic signals and lipopolysaccharide are potent stimulants of microglial release of thromboxane, with lesser effects shown by platelet activating factor and substance P. We conclude that microglia, when activated, are the principal source of brain-derived thromboxane and may help to control vascular flow at sites of acute CNS injury.

Immunohistochemical localization of thromboxane receptor and thromboxane synthase in rat testis.

The cellular localization of thromboxane A2 receptor (TXR) and thromboxane synthase (TXS) in rat testes was examined with an antibody against the carboxyl-terminal tail of rat TXR and anti-porcine lung TXS antibody. By light microscopy immunoreactivity for TXR was shown to be present in spermatids, whereas spermatogonia, spermatocytes, and spermatozoa lacked the immunoreactivity. Immunoelectron microscopic analysis revealed that immunostainable TXR was present in acrosomes of spermatids. In contrast, immunoreactivity for TXS was present in all stages of spermatogenic cells; spermatogonia, spermatocytes, spermatids, and spermatozoa. TX system may possibly contribute to the formation of acrosomes or have some unrecognized functions in an autocrine/paracrine fashion.

The localization of thromboxane synthase in normal and pathological human kidney tissue using a monoclonal antibody Tü 300.

Thromboxane, excreted in the urine in increased amounts in glomerular, vascular and tubulo-interstitial diseases, is considered to originate from the kidney. The localization of thromboxane synthase, a key enzyme of arachidonic acid metabolism, was studied in the human kidney by immunohistology using the monoclonal antibody Tü 300. In the interstitial tissue dendritic reticulum cells surrounding the tubules expressed high concentrations of the enzyme. In glomeruli the enzyme was weakly expressed in podocytes. This was confirmed by co-localization with an antiserum directed to podocalyxin, a marker of the visceral epithelial cells. In the study of various kidney diseases, massive accumulation of thromboxane synthase containing cells was observed in interstitial diseases, whereas in glomerular diseases there were no differences from normal kidney; in a case of thrombotic microangiopathy podocytes exhibited an increase in thromboxane-synthase. The thromboxane-synthase positive infiltrating interstitial cells were shown by conventional light microscopy to be mononuclear phagocytic cells. The physiological sources of renal thromboxane are dendritic reticular cells and podocytes. In interstitial renal disease infiltrating cells of the monocyte/macrophage system constitute the major site of thromboxane synthesis. In glomerular disease, a characteristic alteration of thromboxane-synthase was not found.

Production and characterization of polyclonal and monoclonal antibodies against human thromboxane synthase.

Polyclonal and monoclonal antibodies (MoAbs) were raised against human platelet thromboxane (Tx) synthase. Neither the antiserum nor the MoAbs inhibited the enzyme activity significantly. Three MoAbs, Tü 300, Kon 6, and Kon 7, were purified and further characterized. They are monospecific as shown by activity precipitation or Western blot analysis, and recognized different epitopes on Tx-synthase. Tü 300 could precipitate the enzyme and recognized conformational epitopes, whereas Kon 6 and Kon 7 only reacted in Western blots. Antibody Tü 300 can be used in immunohistology but shows no crossreactivity with Tx-synthase from other species. In human lung tissue staining with peroxidase, coupled Tü 300 was only found in alveolar macrophages.

Monoclonal antibodies to thromboxane synthase from porcine lung. Production and application to development of a tandem immunoradiometric assay.

Two hybridoma cell lines secreting antibodies against thromboxane synthase of porcine lung were produced. Clone TS1 secretes IgG2a antibody of lower affinity, while clone TS2 secretes IgG1 antibody of higher affinity. Both antibodies (when bound to rabbit anti-mouse IgG-Staphylococcus aureus complex) can immunoprecipitate thromboxane synthase from crude enzyme preparations in an active form suggesting that binding was not directed at the active site. Each antibody showed a distinctive pattern of cross-reactivity with thromboxane synthase from different porcine tissues. Neither of the antibodies cross-reacted with the enzyme from tissues of other species tested, indicating the heterogeneous nature of the enzyme among species. Competitive binding assay revealed that TS1 and TS2 recognized different determinants on the enzyme. The fact that two antibodies bind to separate epitopes on the same enzyme allows the development of a sensitive tandem immunoradiometric assay. The assay, based on binding of 125I-TS2 to thromboxane synthase immobilized on TS1-S. aureus complex, was linear with 7.5 approximately 75 ng of purified lung thromboxane synthase as standards and applicable to enzyme preparations regardless of their purity. The concentration of immunoreactive thromboxane synthase in porcine tissues as determined by this assay followed the order of platelet greater than colon greater than duodenum greater than lung greater than kidney greater than stomach. The fact that gastrointestinal tract is enriched with thromboxane synthase suggests that thromboxane may have significant physiological roles to be recognized in these organs.

Immunoaffinity purification and characterization of thromboxane synthase from porcine lung.

Thromboxane synthase has been purified 620-fold from porcine lung microsomes by a three-step purification procedure including Lubrol-PX solubilization, reactive blue-agarose chromatography, and immunoaffinity chromatography. The purified enzyme exhibited a single protein band (53,000 daltons) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Rabbit antiserum raised against the purified enzyme immunoprecipitated thromboxane synthase activity from crude enzyme preparations of porcine lung, cow lung, and human platelets, indicating the existence of structural homology of the enzyme in these species. Immunoblotting experiment identified the same polypeptide (53,000 daltons) in porcine lung and a polypeptide of 50,000 daltons in human platelets, confirming the identity of the enzyme and the specificity of the antiserum. Purified thromboxane synthase is a hemoprotein with a Soret-like absorption peak at 418 nm. The enzyme reaction has a Km for 15-hydroxy-9 alpha, 11 alpha-peroxidoprosta-5, 13-dienoic acid of 12 microM, an optimal pH of 7.5, and an optimal temperature of reaction at 30 degrees C. Purified thromboxane synthase catalyzed the formation of both thromboxane B2 and 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT). The ratios of HHT to thromboxane B2 varied from 1.6 to 2.1 dependent on the reaction conditions. Except that HHT was formed at a greater rate, the formation of HHT and that of thromboxane responded identically to pH, temperature, substrate concentration, kinetics of formation, metal ions, and inhibitors suggesting that the two products are probably formed at the same active site via a common intermediate. Thromboxane synthase was irreversibly inactivated by 15-hydroxy-9 alpha, 11 alpha-peroxidoprosta-5,13-dienoic acid during catalysis and by treatment of 15-hydroperoxyeicosatetraenoic acid. The irreversible inactivation, however, could be protected by reversible inhibitors such as sodium (E)-3-[4-(1-imidazolylmethyl)phenyl]-2-propenoate and 15-hydroxy-11 alpha,9 alpha-(epoxymethano)-prosta-5,13-dienoic acid, suggesting that the inactivation occurred at the active site of the enzyme. The catalytic inactivation of thromboxane synthase and the greater rate of formation of HHT in thromboxane-synthesizing system may probably play important regulatory roles in the control of thromboxane synthesis.