Background-Preeclampsia is characterized clinically by hypertension and proteinuria. Soluble Flt-1 (sFlt-1; also known as soluble vascular endothelial growth factor receptor-1 [VEGFR-1]) and soluble endoglin (sEng) are elevated in preeclampsia, and their administration to pregnant rats elicits preeclampsia-like symptoms. Heme oxygenase-1 (HO-1) and its metabolite carbon monoxide (CO) exert protective effects against oxidative stimuli. Thus, we hypothesized that HO-1 upregulation may offer protection against preeclampsia by inhibiting sFlt-1 and sEng release. Methods and Results-Preeclamptic villous explants secreted high levels of sFlt-1 and sEng. Adenoviral overexpression of HO-1 in endothelial cells inhibited VEGF-mediated sFlt-1 release and interferon-␥-and tumor necrosis factor-␣-induced sEng release, whereas HO-1 inhibition potentiated sFlt-1 and sEng production from endothelial cells and placental villous explants. Consistent with these findings, mice lacking HO-1 produced higher levels of sFlt-1 and sEng compared with wild-type mice. Using selective ligands (VEGF-E and placental growth factor) and a receptor-specific inhibitor (SU-1498), we demonstrated that VEGF-induced sFlt-1 release was VEGFR-2 dependent. Furthermore, CO-releasing molecule-2 (CORM-2) or CO decreased sFlt-1 release and inhibited VEGFR-2 phosphorylation. Treatment of endothelial cells with statins upregulated HO-1 and inhibited the release of sFlt-1, whereas vitamins C and E had no effect. Conclusions-The present study demonstrates that the HO-1/CO pathway inhibits sFlt-1 and sEng release, providing compelling evidence for a protective role of HO-1 in pregnancy, and identifies HO-1 as a novel target for the treatment of preeclampsia. Key Words: endothelium Ⅲ endothelium-derived factors Ⅲ heme oxygenase-1 Ⅲ preeclampsia Ⅲ pregnancy Ⅲ statins Ⅲ angiogenesis C ardiovascular disease and preeclampsia share some common risk factors, such as insulin resistance, obesity, diabetes mellitus, and inflammation. 1,2 The disruption of endothelial homeostasis and inflammation are fundamental to the initiation and progression of atherosclerosis 3 and preeclampsia. 4 Preeclampsia is a maternal systemic endothelial disease defined clinically as hypertension and proteinuria after 20 weeks' gestation that affects 3% to 8% of all pregnancies and women.5 Women with a history of preeclampsia and their offspring are at greater risk of developing cardiovascular disease later in life. 6,7 Clinical Perspective p 1797Preeclampsia involves dysregulated placental angiogenesis, 8 resulting in the release of soluble antiangiogenic factors that induce systemic endothelial dysfunction. 9 Two key antiangiogenic circulating factors that give the highest strength of association with preeclamptic outcome are soluble Flt-1 (sFlt-1) and soluble endoglin (sEng). 10 -12 Maternal serum levels of sFlt-1 are elevated 5 weeks before the clinical onset of preeclampsia. 10,13-16 sEng, a placenta-derived 65-kDa cleaved form of endoglin (also known as CD105), a coreceptor for transform...
We have used three distinct bioassay systems (rat aorta (RA) relaxation; rat gastric longitudinal muscle (LM) contraction; human embryonic kidney 293 (HEK293) cell calcium signal) to evaluate the activity and receptor selectivity of analogues of the receptor-activating peptides derived either from the thrombin receptor (TRAPs, based on the human receptor sequence, SFLLRNPNDK...) or the proteinase-activated receptor 2 (PAR2APs, based on the rat receptor sequence SLIGRL...). Our main focus was on the activation of PAR2 by PAR2APs and the cross-activation of PAR2 by the TRAPs. In the RA and LM assay systems, PAR2APs that were either N-acetylated (N-acetyl-SLIGRL-NH2) or had a reverse N-terminal sequence (LSIGRL-NH2) were inactive, either as agonists or antagonists. An alanine substitution at position 3 of the PAR2AP (SLAGRL-NH2) led to a dramatic reduction of biological activity, as did substitution of threonine for serine at position 1 (TLIGRL-NH2). However, alanine substitution at PAR2AP position 4 caused only a modest reduction in activity, resulting in a peptide (SLIARL-NH2) with a potency equivalent to that of the human PAR2AP, SLIGKV-NH2. The order of potency of the PAR2APs in the RA, LM, and HEK assay systems was SLIGRL-NH2 > SLIARL-NH2 > SLIGKV-NH2 > TLIGRL-NH2 > SLAGRL-NH2. In HEK cells, none of the PAR2APs activated the thrombin receptor (PAR1). However, in the HEK cell assay, the TRAP, SFLLR-NH2, activated or desensitized both PAR1 and PAR2 receptors, whereas the xenopus TRAP, TFRIFD-NH2, activated or desensitized selectively PAR1 but not PAR2. By constructing human-xenopus hybrid peptides, we found that the TRAPs, TFLLR-NH2, and SFLLFD-NH2 selectively activated the thrombin receptor in HEK cells without activating or desensitizing PAR2. In contrast, the TRAPs SFLLRD-NH2 and AFLLR-NH2 activated or desensitized both PAR1 and PAR2. The order of potency for the TRAPs in all bioassay systems was SFLLR-NH2 approximately equal to SFLLRD-NH2 approximately equal to TFLLR-NH2 > SFLLFD-NH2 > TFRIFD-NH2. We conclude that the N-terminal domain of the PAR2AP as well as positon 3 plays important roles for PAR2 activation. In contrast, the first and fifth amino acids in the TRAP motif, SFLLR-NH2, do not play a unique role in activating the thrombin receptor, but if appropriately modified can abrogate the ability of this peptide to cross-desensitize or activate PAR2, so as to be selective for PAR1. The PAR1- and PAR2-selective peptides that we have synthesized will be of use for the evaluation of the roles of the PAR1 and PAR2 receptor systems in vivo.
1 The biological activities of the proteinase-activated receptor number 2 (PAR-2)-derived peptides, SLIGRL (PP6) SLIGRL-NH2 (PP6-NH2) and were measured in mouse and rat gastric longitudinal muscle (LM) tissue and in a rat aortic ring preparation and the actions of the PAR-2-derived peptides were compared with trypsin and with the actions of the thrombin receptor activating peptide, SFLLR-NH2 (TP5-NH2). 2 From a neonatal rat intestinal cDNA library, and from intestinal and kidney-derived cDNA, the coding region of the rat PAR-2 receptor was cloned and sequenced, thereby establishing its close sequence identity with the previously described mouse PAR-2 receptor; and this information, along with a reverse-transcriptase (RT) polymerase chain reaction (PCR) analysis of cDNA derived from gastric and aortic tissue was used to establish the concurrent presence of PAR-2 and thrombin receptor mRNA in both tissues. 3 In the mouse and rat gastric preparations, the PAR-2-derived polypeptides, PP6, PP6-HN2 and PP5-NH2 caused contractile responses that mimicked the contractile actions of low concentrations of trypsin (5 u/ml-'; 10 nM) and that were equivalent to contractions caused by TP5-NH2. 4 The cumulative exposure of the rat LM tissue to PP6-NH2 led to a desensitization of the contractile response to this polypeptide, but not to TP5-NH2 and vice versa, so as to indicate a lack of crossdesensitization between the receptors responsive to the PAR-2 and thrombin receptor-derived peptides. 5 In the rat gastric preparation, the potencies of the PAR-2-activating peptides were lower than the potency of TP5-NH2 (potency order: TP5-NH2 >>PP6-NH2k PP6 > PP5-NH2); PP6 was a partial agonist in this preparation. 6 The contractile actions of PP6 and PP6-NH2 in the rat gastric preparation required the presence of extracellular calcium, were inhibited by nifedipine and were blocked by the cyclo-oxygenase inhibitor, indomethacin and by the tyrosine kinase inhibitor, genistein, but not by the kinase C inhibitor, GF109203X. The contractile responses were not blocked by atropine, chlorpheniramine, phenoxybenzamine, propranolol, ritanserin or tetrodotoxin. 7 In a precontracted rat aortic ring preparation, with an intact endothelium, all of the PAR-2-derived peptides caused a prompt relaxation response that was blocked by the nitric oxide synthase inhibitor, NwO-nitro-L-arginine-methyl ester (L-NAME) but not by D-NAME; in an endothelium-free preparation, which possessed mRNA for both the PAR-2 and thrombin receptors, the PAR-2-activating peptides caused neither a relaxation nor a contraction, in contrast with the contractile action of TP5-NH2. The relaxation response to PP6-NH2 was not blocked by atropine, chlorpheniramine, genistein, indomethacin, propranolol or ritanserin. 8 In the rat aortic preparation, the potencies of PP6, PP6-NH2 and PP5-NH2 were greater than those of the thrombin receptor activating peptide, TP5-NH2 (potency order: PP6-NH2 > PP6 > PP5-NH2 > TP5-NH2).9 In the rat aortic preparation, the relaxant actions of the PAR-2-...
The effects of PAR 2 -activating PAR 2 -activating peptides, SLIGRL (SL)-NH 2 , and trans-cinnamoyl-LIGRLO (tc)-NH 2 were compared with the action of trypsin, thrombin, and the PAR 1 selective-activating peptide: Alaparaf luoroPhe-Arg-cyclohexylAla-Citrulline-Tyr (Cit)-NH 2 for stimulating intestinal ion transport. These agonists were added to the serosa of stripped rat jejunum segments mounted in Ussing chambers, and short circuit current (Isc) was used to monitor active ion transport. The relative potencies of these agonists also were evaluated in two bioassays specific for the activation of rat PAR 2 : a cloned rat PAR 2 cell calciumsignaling assay (PAR 2 -KNRK cells) and an aorta ring relaxation (AR) assay. In the Isc assay, all agonists, except thrombin, induced an Isc increase. The SL-NH 2 -induced Isc changes were blocked by indomethacin but not by tetrodotoxin. The relative potencies of the agonists in the Isc assay (trypsin> >SL-NH 2 >tc-NH 2 >Cit-NH 2 ) were strikingly different from their relative potencies in the cloned PAR 2 -KNRK cell calcium assay (trypsin> >>tc-NH 2 Х SL-NH 2 > >>Cit-NH 2 ) and in the AR assay (trypsin> >>tc-NH 2 Х SL-NH 2 ). Furthermore, all agonists were maximally active in the PAR 2 -KNRK cell and AR assays at concentrations that were one (PAR 2 -activating peptides) or two (trypsin) orders of magnitude lower than those required to activate intestinal transport. Based on the distinct potency profile for these agonists and the considerable differences in the concentration ranges required to induce an Isc effect in the intestinal assay compared with the PAR 2 -KNRK and AR assays, we conclude that a proteinase-activated receptor, pharmacologically distinct from PAR 2 and PAR 1 , is present in rat jejunum and regulates intestinal transport via a prostanoid-mediated mechanism.
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