thromboxane-a2 and Fibrosis

Topics: Adult; Animals; Antifibrotic Agents; Antihypertensive Agents; Aspirin; Biomarkers; Blood Platelets; Blood Pressure; Cardiomyopathies; Case-Control Studies; Cells, Cultured; Disease Models, Animal; Essential Hypertension; Female; Fibrosis; Humans; Male; Mice, Inbred C57BL; Mice, Knockout; Middle Aged; Myocytes, Cardiac; Myofibroblasts; Platelet Aggregation Inhibitors; Receptors, Epoprostenol; Receptors, Thromboxane; Thromboxane A2

All-trans retinoic acid (ATRA) has been used for the treatment of acute promyelocytic leukemia. It remains unclear, however, whether ATRA affects cyclooxygenase-2 (COX-2; an enzyme involved in prostaglandin production), PGE2, and thromboxane A2 (TXA2) (metabolic products of COX-2) by a transforming growth factor-β/Smad-signaling pathway, which plays important roles in mesangial-cell proliferation and renal fibrosis. In this study, the mRNA and protein of Smad3, Smad7, and COX-2 were detected by reverse transcription-polymerase chain reaction and Western blot, respectively, in mesangial cells stimulated by transforming growth factor-β (TGF-β) and treated with ATRA at various concentrations and times. The protein level of PGE2 and TXA2 was also measured by enzyme-linked immunosorbent assay. The localization of Smad3 and Smand7 was observed by confocal microscope. Cell proliferation was detected by MTT assay, while apoptosis was determined using Hoechest staining. The expression of Smad3, Smad7, and COX-2 mRNA and protein was increased by exogenous TGF-β, but inhibited by pretreatment of ATRA, in dose and time-dependent manners. In addition, the expression of Smad3 and Smad7 was significantly reduced not only by staurosporine, an inhibitor of threonine/serine protein kinases as well as smad, but also by NS-398, an inhibitor of COX-2. PGE2 and TXA2 were raised by TGF-β, but also decreased by ATRA, staurosporine, and NS-398. Moreover, ATRA reversed the translocation of Smad3 and Smad7 induced by TGF-β. Compared with the control, TGF-β also significantly enhanced proliferation and inhibited apoptosis of mesangial cells. ATRA dose-dependently inhibited TGF-β-induced cell proliferation, but had no significant effect on apoptosis in rat mesangial cells. Therefore, ATRA repressed COX-2, PGE2, and TXA2 via the TGF-β/Smad-signaling pathway and inhibited mesangial-cell proliferation, which might subsequently prevent renal fibrosis.

Topics: Animals; Apoptosis; Cell Line; Cell Proliferation; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Dinoprostone; Fibrosis; Glomerular Mesangium; Mesangial Cells; Nitrobenzenes; Protein Transport; Rats; RNA, Messenger; Signal Transduction; Smad3 Protein; Smad7 Protein; Staurosporine; Sulfonamides; Thromboxane A2; Transforming Growth Factor beta; Tretinoin

Thromboxane A2 (TXA2) has been suggested to play a significant role in the development of portal hypertension in fibrosis, and Kupffer cell (KC) derived TXA2 has been shown to mediate the hyperresponsiveness of the portal circulation to the vasoconstrictive actions of endothelin-1 (ET-1) during endotoxemia. The aim of this study was to determine whether the double stresses of prefibrotic changes and endotoxemia additively activate KC to increase release of TXA2 in response to ET-1, resulting in elevated portal resistance.. One week Bile duct ligation (BDL) rats and sham-operated controls were subjected to isolated liver perfusions following LPS or saline for 6h. In a separate experiment, KC were isolated from BDL or sham rats and incubated with LPS or saline for 6h before the ET-1 treatment.. The double stresses of early fibrosis and LPS resulted in a greater sustained increase in portal pressure in response to ET-1 in BDL rats, and this increase correlated well with the much enhanced release of TXA2 in the perfusate. Media from the cultured KC showed significantly greater TXA2 release in response to ET-1 in BDL group than those in sham group, and LPS exacerbated this effect. Protein levels of cytosolic phospholipase A2 (cPLA2), cyclooxygenase-2, and thromboxane synthase were also significantly elevated in KC from BDL rats. ET-1 produced a marked increase in cPLA2 activation as measured by the phosphorylation of cPLA2 in KC of both BDL and sham groups. LPS greatly exacerbated the activation of cPLA2.. The data suggest that the double stresses additively activate KC with an upregulation of the key enzymes in the TXA2 biosynthesis and release increased amount of TXA2 via the augmented activation of cPLA2 in response to ET-1, which leads to the increased portal resistance and ultimately hepatic microcirculatory dysfunction.

Topics: Animals; Cyclooxygenase 2; Endothelin-1; Enzyme Activation; Fibrosis; Group IV Phospholipases A2; Hypertension, Portal; In Vitro Techniques; Kupffer Cells; Lipopolysaccharides; Liver; Liver Cirrhosis, Experimental; Male; Microcirculation; Phospholipases A; Phospholipases A2; Phosphorylation; Portal Pressure; Rats; Rats, Sprague-Dawley; Thromboxane A2

In a separate study, we identified PGE2 as a potent inhibitor of TGF-beta1induced epithelial-mesenchymal transition (EMT) in cultured Madin-Darby canine kidney (MDCK) cells (Zhang A, Wang M-H, Dong Z, and Yang T. Am J Physiol Renal Physiol 291: F1323-F1331, 2006). This finding prompted us to examine the roles of other prostanoids: PGD2, PGF(2alpha), PGI2, and thromboxane A2 (TXA2). Treatment with 10 ng/ml TGF-beta1 for 3 days induced EMT as reflected by conversion to the spindle-like morphology, loss of E-cadherin, and activation of alpha-smooth muscle actin (alpha-SMA). Treatment with PGD2 remarkably preserved the epithelial-like morphology, restored the expression of E-cadherin, and abolished the activation of alpha-SMA. In contrast, PGF(2alpha), carbocyclic thromboxane A2, PGI2 and its stable analog beraprost were without an effect. MDCK cells expressed DP1 and DP2 receptors; however, the effect of PGD2 was neither prevented by DP1 antagonist BW-A868C or DP2 antagonist BAY-u3405 nor was mimicked by DP1 agonist BW-245C. cAMP-elevating agents forskolin and 8-Br-cAMP blocked EMT. However, cAMP blockers H89 and Rp-cAMP failed to block the effect of PGD2. PGD2 did not seem to act via its metabolites as 15-deoxy-Delta(12,14)-prostaglandin J2 (15d-PGJ2) levels in the medium following incubation with 3 microM PGD2 were well below the values predicted from the cross activity of the assay. Exposure to TGF-beta1 induced a threefold increase in reactive oxygen species production that was completely abolished by PGD2. We conclude that 1) PGD2, but not PGI2, PGF(2alpha), and TXA2 inhibit EMT, 2) PGD2 inhibits EMT independently of DP1 and DP2 receptors, and 3) PGD2 exhibits antioxidant property which may, in part, account for the antifibrotic action of this PG.

Topics: Actins; Animals; Cadherins; Cell Line; Cyclic AMP; Dinoprost; Dogs; Drug Interactions; Epithelial Cells; Epoprostenol; Fibrosis; Kidney Tubules; Mesoderm; Prostaglandin D2; Reactive Oxygen Species; Receptors, Prostaglandin; Thromboxane A2; Transforming Growth Factor beta1