Bradykinin (BK) is an important mediator in several inflammatory and vascular diseases that acts in part via induction of cyclooxygenase-2 (COX-2). The mechanisms involved in BK-mediated COX-2 induction are unclear.Here we characterized the transcriptional mechanisms involved in human pulmonary artery smooth muscle cells. BK stimulated the activity of a transiently transfected 966-bp (؊917 to ؉ 49) COX-2 promoter luciferase reporter construct. There was no reduction in BK-induced luciferase activity in cells transfected with COX-2 promoter constructs of 674, 407, 239, and 135 bp or constructs with mutated CCAAT/enhancer-binding proteinor NF-B-binding sites. In contrast luciferase activity was reduced in cells transfected with a 407-bp COX-2 promoter fragment containing a mutated cAMP response element (CRE)-binding site, suggesting that the CRE binding site is critical. Electrophoretic mobility shift assays using oligonucleotides specific for the CREbinding region of the COX-2 promoter and consensus oligonucleotides showed strong specific binding. Furthermore BK increased consensus cAMP-responsive luciferase reporter (p6CRE/luc)-mediated luciferase expression. CRE activation occurred by BK inducing cytosolic phospholipase A 2 -mediated arachidonic acid release and rapid prostaglandin E 2 (PGE 2 ) production, thereby increasing cAMP. Indomethacin inhibited BKinduced PGE 2 production, cAMP accumulation, and CRE/luc reporter and COX-2 promoter luciferase activity. Exogenous PGE 2 and EP2 (ONO-AE1 259) and EP4 (ONO-AE1 329) PGE 2 receptor agonists mimicked the effect of BK. Collectively these studies indicate that COX-2 induction by BK in human pulmonary artery smooth muscle cells is mediated by the CRE through a novel autocrine loop involving endogenous PGE 2 .

Methicillin-resistant Staphylococcus aureus (MRSA) is an important human pathogen that causes various infections. The increasing resistance of MRSA to different antibiotics is widely spreading; therefore, plant extracts may be novel therapeutic alternatives. The phytochemical profiling of Cupressus macrocarpa Hartw. ex Gordon leaves in vitro, and in vivo, antimicrobial potential of its extracts against MRSA clinical isolates were explored. A phytochemical tentative identification of 49 compounds was performed in the leaves using LC-ESI-MS/MS; in addition, isolation, and structure elucidation of hesperidin and eriocitrin were achieved for the first time. The diethyl ether extract (DEEL) exhibited the best antibacterial effect with MIC values ranging from 2 to 8 µg/mL, which significantly reduced the growth and efflux activity in 48.78% and 29.26% of isolates, respectively. qRT-PCR showed a significant down expression of norA and norB genes, which significantly affected the bacterial cell morphology and had a non-significant effect on membrane depolarization (using flow cytometry). In a rat model, four groups were wounded and treated with normal saline or DEEL, or infected with MRSA, or infected and treated with DEEL. The regeneration of the epidermis, maturation of granulation tissue, and reduction of inflammatory cell infiltration were observed after treatment with DEEL. Thus, C. macrocarpa leaves may be a promising source for new antimicrobials against MRSA.

Abstract-Increased levels of inflammatory cytokines contribute to the pathophysiology of pulmonary hypertension.Prostacyclin (PGI 2 ) analogues, which relax pulmonary vessels mainly through cAMP elevation, have a major therapeutic role. In this study, we show that prolonged incubation with bradykinin (BK), interleukin-1␤ (IL-1␤), and transforming growth factor-␤ 1 (TGF-␤ 1 ) markedly impairs cAMP accumulation in human pulmonary artery smooth muscle cells in response to short-term incubation with prostaglandin E 2 (PGE 2 ) and the PGI 2 analogues iloprost and carbaprostacyclin. A similar reduction in cAMP accumulation in response to a direct adenylyl cyclase activator, forskolin, suggested that the effect was attributable to downregulation of adenylyl cyclase. Reverse transcriptase-polymerase chain reaction studies showed downregulation of adenylyl cyclase isoforms 1, 2, and 4. The effect of IL-1␤, BK, and TGF-␤ 1 on cAMP levels was abrogated by the selective COX-2 inhibitor NS398. Furthermore, it was mimicked by prolonged incubation with the COX-2 product PGE 2 and PGI 2 analogues or the COX substrate arachidonic acid, suggesting that it was mediated by endogenous prostanoids produced by COX-2. Consistent with this, IL-1␤, BK, and TGF-␤ 1 all induced COX-2 and PGE 2 release. These results show that BK, IL-1␤, and TGF-␤ 1 downregulate adenylyl cyclase in human pulmonary artery smooth muscle cells via COX-2 induction and prostanoid release. This suggests a novel mechanism whereby mediators and cytokines produced in pulmonary hypertension may impair the therapeutic effects of prostacyclin analogues such as iloprost and carbaprostacyclin. Key Words: interleukin-1␤ Ⅲ transforming growth factor-␤ 1 Ⅲ bradykinin Ⅲ cAMP Ⅲ adenylyl cyclase P rostacyclin (PGI 2 ) analogues are effective treatment for pulmonary hypertension. 1 Long-term treatment with intravenous PGI 2 improved survival rates and reduced vascular resistance in primary and secondary pulmonary hypertension. 2 PGI 2 analogues act mainly through cAMP, a relaxant second messenger, 3 by binding to adenylyl cyclase-coupled prostacyclin receptors. 4 -6 PGI 2 analogues also regulate pulmonary artery remodeling. 7 Administration of PGI 2 analogues may compensate for defective PGI 2 production in pulmonary hypertension. Pulmonary vascular tone and remodeling is controlled by the balance between vasoconstrictor and vasodilator mediators. 8 In pulmonary hypertension, there is an imbalance with excess thromboxane A 2 and reduced dilator PGI 2 production 9 and pulmonary artery PGI 2 synthase expression. 10 Endothelin-1 (ET-1) plays an important role in pulmonary hypertension. ET-1 levels are elevated in patients with pulmonary hypertension. ET-1 has a growth regulatory effect on pulmonary smooth muscle cells, partly via K ϩ channels. 11 Inflammatory cytokines and mediators contribute to the increased pulmonary resistance and remodeling in pulmonary hypertension, 12 including interleukin-1 (IL-1), IL-6, ET-1, and prostanoids. 13,14 IL-1␤ is interesting because its elevate...