PGI2 Receptor Research Articles

Prostacyclin synthase deficiency exacerbates systemic inflammatory responses in lipopolysaccharide-induced septic shock in mice.

Sepsis is a systemic inflammatory disorder characterized by life-threateningorgan dysfunction resulting from a dysregulated host response to infection. Prostacyclin (PGI2) is a bioactive lipid produced by PGI synthase (PGIS) and is known to play important roles in inflammatory reactions as well as cardiovascular regulation. However, little is known about the roles of PGIS and PGI2 in systemic inflammatory responses such as septic shock. Systemic inflammation was induced by intraperitoneal injection of 5mg/kg lipopolysaccharide (LPS) in wild type (WT) or PGIS knockout (KO) mice. Selexipag, a selective PGI2 receptor (IP) agonist, was administered 2h before LPS injection and again given every 12h for 3days. Intraperitoneal injection of LPS induced diarrhea, shivering and hypothermia. These symptoms were more severe in PGIS KO mice than in WT micqe. The expression of Tnf and Il6 genes was notably increased in PGIS KO mice. In contrast, over 95% of WT mice survived 72h after the administration of LPS, whereas all of the PGIS KO mice had succumbed by that time. The mortality rate of LPS-administrated PGIS KO mice was improved by selexipag administration. Our study suggests that PGIS-derived PGI2 negatively regulates LPS-induced symptoms via the IP receptor. PGIS-derived PGI2-IP signaling axis may be a new drug target for systemic inflammation in septic shock.

Diverse effects of prostacyclin on angiogenesis-related processes in the porcine endometrium

Angiogenesis is important for endometrial remodeling in mature females. The endometrium synthesizes high amounts of prostacyclin (PGI2) but the role of PGI2 in angiogenesis-related events in this tissue was not fully described. In the present study, porcine endometrial endothelial (pEETH) cells and/or a swine umbilical vein endothelial cell line (G1410 cells) were used to determine the regulation of PGI2 synthesis and PGI2 receptor (PTGIR) expression by cytokines and to evaluate the effect of PGI2 on pro-angiogenic gene expression, intracellular signaling activation, cell proliferation and migration, cell cycle distribution, and capillary-like structure formation. We found that IL1β, IFNγ, and/or TNFα increased PGI2 secretion and PTGIR expression in pEETH cells. Iloprost (a PGI2 analogue) acting through PTGIR enhanced the transcript abundance of KDR, FGFR2, and ANGPT2 and increased proliferation of pEETH cells. This latter was mediated by PI3K and mTOR activation. In support, transfection of G1410 cells with siRNA targeting PGI2 synthase decreased pro-angiogenic gene expression and cell proliferation. Furthermore, iloprost accelerated the gap closure and promoted cell cycle progression. Intriguingly, the formation of capillary-like structures was inhibited but not completely blocked by iloprost. These findings point to a complex pleiotropic role of PGI2 in angiogenesis-related events in the porcine uterus.

Impaired luteal progesterone synthesis observed in gilts with PMSG/hCG-induced estrus affects the expression of steroid, prostaglandin, and cytokine receptors in the endometrium and myometrium during the peri-implantation period

Abstract The present study aimed to examine the effect of impaired progesterone (P4) synthesis, observed in gilts with gonadotropin-induced estrus, on the uterine expression of receptors important for pregnancy establishment. Twenty prepubertal gilts received 750 IU PMSG and 500 IU hCG 72 h later, while 18 prepubertal gilts in the control group were observed daily for estrus behavior. Gilts were inseminated in their first estrus and slaughtered on days 10, 12, and 15 of pregnancy to collect endometrial and myometrial tissues for mRNA analysis using real-time PCR. As we previously described, gilts with PMSG/hCG-induced estrus showed decreased luteal P4 synthesis on days 10 and 12 of pregnancy. PMSG/hCG treatment did not affect P4 receptor mRNA expression in either uterine tissue. In the endometrium, a greater mRNA transcript abundance of estrogen receptors (ESR1 and ESR2), androgen receptor (AR), prostaglandin (PG) E2 receptors (PTGER2 and PTGER4), PGF2α receptor (PTGFR), interleukin 6 receptor (IL6R), and tumor necrosis factor α receptors (TNFRSF1A and TNFRSF1B) was detected in gilts with natural than with PMSG/hCG-induced estrus (P<0.05). In the myometrium, the mRNA expression of AR, PTGER2, and PTGFR was lower, while PGI2 receptor (PTGIR) transcript abundance was elevated in the gilts treated with PMSG/hCG as compared with the control animals (P<0.05). In summary, a decreased luteal P4 level during the peri-implantation period in gonadotropin-stimulated pigs affects endometrial and myometrial receptor expression, with the endometrium being more sensitive to impaired P4 synthesis. Whether the observed changes alter uterine receptivity to local and systemic factors remains to be elucidated.

Prostacyclin Synthesis and Prostacyclin Receptor Expression in the Porcine Myometrium: Prostacyclin Potential to Regulate Fatty Acid Transporters, Cytokines and Contractility-Related Factors.

Simple SummaryProstacyclin (prostaglandin I2; PGI2) is an important modulator of vascular functions and is involved in various reproductive processes. PGI2 was also described as a modulator of uterine contractility in several species, including the pig. However, its synthesis and role in the myometrium of the porcine uterus are still not fully described. The objective of this study was to evaluate profiles of PGI2 synthesis and PGI2 receptor expression in the myometrium of gilts throughout the estrous cycle and during early pregnancy and to investigate the in vitro effect of PGI2 on the mRNA expression of factors engaged in smooth muscle contraction, nutrient transport, prostaglandin synthesis and action, and inflammatory response. The obtained results showed that the synthesis of PGI2 changes in the myometrium of pigs during both the estrous cycle and early pregnancy, resulting in much greater concentrations of PGI2 in cyclic than in pregnant gilts. Moreover, PGI2 stimulated the expression of fatty acid transporters and contractility-related calponin 1 and caldesmon 1, whereas it decreased cytokine expression. This study indicates that PGI2 may participate in the regulation of myometrial functions modulating the availability of factors involved in smooth muscle activity and inflammatory reaction in the uterus of pigs.Although prostacyclin (PGI2) has been well described as a regulator of smooth muscle activity, limited data are available concerning its role in the myometrium of pigs. The present research aimed to examine profiles of PGI2 synthase (PTGIS) and PGI2 receptor (PTGIR) expression and 6-keto PGF1α (a PGI2 metabolite) concentrations in the myometrium of gilts throughout the estrous cycle and during early pregnancy using qPCR, Western blot, and/or ELISA methods. Furthermore, myometrial explants were exposed to iloprost (a stable PGI2 analog) to investigate the effect of PGI2 on the mRNA expression of factors engaged in smooth muscle contraction, nutrient transport, prostaglandin synthesis and action, and inflammatory response. PTGIS mRNA expression was greater in cyclic than in pregnant gilts on days 11–12 after estrus and was accompanied by greater concentrations of 6-keto PGF1α detected in cyclic than in pregnant animals on days 11–20. Iloprost stimulated fatty acid transporters and contractility-related calponin 1 and caldesmon 1 mRNA expression and decreased interleukin 1β and tumor necrosis factor transcript abundance. The obtained results indicate a physiologically relevant role of PGI2 during the estrous cycle in the porcine myometrium with its importance for regulating the expression of contractility-, nutrient transport- and inflammatory response-related factors.

Hypoxia-hindered methylation of PTGIS in endometrial stromal cells accelerates endometriosis progression by inducing CD16− NK-cell differentiation

Prostacyclin (PGI2) plays key roles in shaping the immune microenvironment and modulating vasodilation, whereas its contribution to endometriosis (EMs) remains largely unclear. Our study suggested that prostacyclin synthase (PTGIS)-dependent PGI2 signaling was significantly activated in EMs, which was involved in the hypoxic microenvironment of ectopic lesions and deficient methylation status of the PTGIS promoter. Notably, in vitro assays, hypoxia promoted PTGIS expression through DNA methyltransferase 1 (DNMT1)-mediated DNA methylation deficiency in endometrial stromal cells (ESCs); PTGIS overexpression enhanced the adhesive ability of ESCs and led to elevated PGI2 production, and PGI2 triggered CD16− (encoded by FCGR3, Fc fragment of IgG receptor IIIa) natural killer (NK)-cell differentiation through PGI2 receptor (IP, PTGIR) in an ESC/NK-cell coculture system. Our rodent model experiment suggested that treatment with the PGI2 analog iloprost and adoptive transfer of fcgr3 knockout (fcgr3−/−) NK cells aggravated EMs progression and that genetic ablation of ptgis (ptgis−/−) in ectopic lesions and treatment with the PTGIR antagonist RO1138452 partially rescued this outcome. Thus, our findings identified the contribution of PGI2 to EMs progression via enhancement of the adhesive ability of ESCs and inhibition of the activity of NK cells. We hypothesized that PGI2 is a target for EMs intervention and provide a rationale for studying pharmacological PTGIR inhibition and PTGIS genetic depletion therapies as therapeutic strategies for EMs.

Nanomaterial Inhalation During Pregnancy Alters Systemic Vascular Function in a Cyclooxygenase-Dependent Manner.

Pregnancy requires rapid adaptations in the uterine microcirculation to support fetal development. Nanomaterial inhalation is associated with cardiovascular dysfunction, which may impair gestation. We have shown that maternal nano-titanium dioxide (nano-TiO2) inhalation impairs microvascular endothelial function in response to arachidonic acid and thromboxane (TXA2) mimetics. However, the mechanisms underpinning this process are unknown. Therefore, we hypothesize that maternal nano-TiO2 inhalation during gestation results in uterine microvascular prostacyclin (PGI2) and TXA2 dysfunction. Pregnant Sprague-Dawley rats were exposed from gestational day 10-19 to nano-TiO2 aerosols (12.17 ± 1.67 mg/m3) or filtered air (sham-control). Dams were euthanized on gestational day 20, and serum, uterine radial arterioles, implantation sites, and lungs were collected. Serum was assessed for PGI2 and TXA2 metabolites. TXB2, the stable TXA2 metabolite, was significantly decreased in nano-TiO2 exposed dams (597.3 ± 84.4 vs 667.6 ± 45.6 pg/ml), whereas no difference was observed for 6-keto-PGF1α, the stable PGI2 metabolite. Radial arteriole pressure myography revealed that nano-TiO2 exposure caused increased vasoconstriction to the TXA2 mimetic, U46619, compared with sham-controls (-41.3% ± 4.3% vs -16.8% ± 3.4%). Nano-TiO2 exposure diminished endothelium-dependent vasodilation to carbaprostacyclin, a PGI2 receptor agonist, compared with sham-controls (30.0% ± 9.0% vs 53.7% ± 6.0%). Maternal nano-TiO2 inhalation during gestation decreased nano-TiO2 female pup weight when compared with sham-control males (3.633 ± 0.064 vs 3.995 ± 0.124 g). Augmented TXA2 vasoconstriction and decreased PGI2 vasodilation may lead to decreased placental blood flow and compromise maternofetal exchange of waste and nutrients, which could ultimately impact fetal health outcomes.

The Prostacyclin Analog Iloprost Promotes Cementum Formation and Collagen Reattachment of Replanted Molars and Up-regulates Mineralization by Human Periodontal Ligament Cells

IntroductionThis study evaluated the use of the prostacyclin analog iloprost as a root surface treatment agent in promoting acellular cementum (AC) formation and collagen reattachment after tooth replantation in vivo. In addition, its effect on human periodontal ligament cell (hPDLC) mineralization was assessed in vitro. MethodsFirst molars of 8-week-old Wistar rats were extracted. In 1 group, the root surfaces were treated with Hank's balanced salt solution (HBSS), and the other group's root surfaces were treated with 10−6 mol/L iloprost before replantation. At day 30, maxillae were prepared for micro–computed tomographic imaging and histomorphometric analysis. The effect of iloprost on mineralization by hPDLCs was analyzed by mineralized nodule formation and quantitative polymerase chain reaction at 7 and 14 days. ResultsMicro–computed tomographic imaging demonstrated a significant higher bone volume in the iloprost groups, whereas the HBSS groups had extensive bone and root resorption. Histologic analysis revealed deposition of a thick AC layer along the root in the iloprost group with well-organized periodontal ligament fibers inserted into the cementum. The HBSS group demonstrated more osteoclasts than the iloprost group. In vitro, iloprost-treated hPDLCs had a significantly increased RUNX2, OSX, BSP, and ALP gene expression that coincided with an increased deposition of mineralized nodules. These effects were abrogated by a PGI2 receptor inhibitor. ConclusionsOur results revealed that iloprost promoted PDL regeneration in replanted molars. Furthermore, resorption of the roots was decreased, whereas AC deposition was stimulated. Iloprost-treatment increased hPDLC mineralization and was mediated by PGI2 receptor activation. These observations indicate that iloprost may be a promising root surface treatment agent.

Absence of prostacyclin greatly relieves cyclophosphamide-induced cystitis and bladder pain in mice.

Cyclophosphamide (CP) has been widely used in the treatment of various malignancies and autoimmune diseases, but acrolein, a byproduct of CP, causes severe hemorrhagic cystitis as the major side effect of CP. On the other hand, a large amount of prostacyclin (PGI2 ) is produced in bladder tissues, and PGI2 has been shown to play a critical role in bladder homeostasis. PGI2 is biosynthesized from prostaglandin (PG) H2 , the common precursor of PGs, by PGI2 synthase (PTGIS) and is known to also be involved in inflammatory responses. However, little is known about the roles of PTGIS-derived PGI2 in bladder inflammation including CP-induced hemorrhagic cystitis. Using both genetic and pharmacological approaches, we here revealed that PTGIS-derived PGI2 -IP (PGI2 receptor) signaling exacerbated CP-induced bladder inflammatory reactions. Ptgis deficiency attenuated CP-induced vascular permeability and chemokine-mediated neutrophil migration into bladder tissues and then suppressed hemorrhagic cystitis. Treatment with RO1138452, an IP selective antagonist, also suppressed CP-induced cystitis. We further found that cystitis-related nociceptive behavior was also relieved in both Ptgis-/- mice and RO1138452-treated mice. Our findings may provide new drug targets for bladder inflammation and inflammatory pain in CP-induced hemorrhagic cystitis.

HYPOXIA-HINDERED METHYLATION OF PTGIS IN ENDOMETRIAL STROMA CELLS ACCELERATES ENDOMETRIOSIS PROGRESSION BY INDUCING CD16- NK CELLS DIFFERENTIATION

To investigate the role of prostacyclin (PGI2) in the pathogenesis of endometriosis (EMS). Expression of prostacyclin synthase (PTGIS)/PGI2 in normal (nEM) and ectopic endometrium (eEM), normal (NESCs) and ovarian endometrial stroma cells (OESCs) were analyzed in Gene Expression Omnibus (GEO) and further verified with quantitative Real-Time PCR (qRT-PCR), immunohistochemistry (IHC), western blotting (WB), enzyme-linked immuno sorbent assay (ELISA) and flow cytometer (FCM) assays. Methylation status of PTGIS promoter was detected in NESCs, OESCs and hypoxia-conditioned human endometrial stroma cells (HESCs). PTGIS over-expressed HESCs underwent RNA-sequencing process and co-cultured with the human peripheral natural killer (pNK) cells, and percentage of CD3-CD56+CD16- (CD16-) pNK cells were detected. Additionally, role of PTGIS/PGI2 in EMS was explored in vivo by instructing PTGIS knocked out (KO) (PTGIS-/-) mice, CD16 KO (CD16-/-) mice and mouse spleen NK cells adoptive transferring EMS model. Lastly, statistical analysis was analyzed by Student's t-test, one-way analysis of variance (ANOVA) and Mann-Whitney test. The expression of PTGIS/PGI2 was augmented in EMS specimens, which attributed to hypoxia milieu in endometriotic lesions and hypoxia-inducible factor 1 alpha (HIF-1α) accelerated PTGIS transcription through DNMT1-mediated DNA methylation deficiency in PTGIS promoter. Furthermore, over-expression of PTGIS enhanced adhesion ability of ESCs and ESCs derived PGI2 promoted CD16- NK cells differentiation through PGI2 receptor (PTGIR) in vitro. More importantly, PGI2 analogue iloprost and CD16- mouse spleen NK cells adoptive transfer aggravated EMS severity, with PTGIS-/- uterus fragments recipient and PTGIR antagonist RO1138452 treatment EMS model being endowed with attenuated ectopic lesions in vivo. Endometriotic hypoxia milieu activated PTGIS/PGI2 signaling pathway through DNMT1-mediated DNA methylation depression in PTGIS promoter, and this pathway accelerated EMS progression by enhancing adhesion ability of ESCs and inducing CD16- NK cells differentiation.

Involvement of prostacyclin synthase in high-fat-diet-induced obesity

Prostacyclin (PGI2) synthase (PGIS) functions downstream of inducible cyclooxygenase COX-2 in the PGI2 biosynthetic pathway. Although COX-2 and PGI2 receptor (IP) are known to be involved in adipogenesis and obesity, the involvement of PGIS has not been fully elucidated. In this study, we examined the role of PGIS in adiposity by using PGIS-deficient mice. Although PGIS deficiency did not affect in vitro adipocyte differentiation, when fed a high-fat diet (HFD), PGIS knockout (KO) mice showed reductions in both body weight gain and epididymal fat mass relative to wild-type (WT) mice. PGIS deficiency might reduce HFD-induced obesity by suppressing PGI2 production. We further found that additional gene deletion of microsomal prostaglandin (PG) E synthase-1 (mPGES-1), one of the other PG terminal synthases that also functions downstream of COX-2, emphasized the metabolic phenotypes of PGIS-deficient mice. More marked reduction in obesity and improved insulin resistance were observed in PGIS/mPGES-1 double KO (DKO) mice. Since an additive increase in PGF2α level in epididymal fat was observed in DKO mice, mPGES-1 deficiency might affect adiposity by enhancing the production of PGF2α. Our immunohistochemical analysis further revealed that in adipose tissues, PGIS was expressed in vascular and stromal cells but not in adipocytes. These results suggested that PGI2 produced from PGIS-expressed stromal tissues might enhance HFD-induced obesity by acting on IP expressed in adipocytes. The balance of expressions of PG terminal synthases and the subsequent production of prostanoids might be critical for adiposity.

Functional evidence for biased inhibition of G protein signaling by YM-254890 in human coronary artery endothelial cells

Small molecular chemicals targeting individual subtype of G proteins including Gs, Gi/o and Gq has been lacking, except for pertussis toxin being an established selective peptide inhibitor of the Gi/o protein. Recently, a cyclic depsipeptide compound YM-254890 isolated from culture broth of Chromobacterium sp. was reported as a selective inhibitor for the Gq protein by blocking GDP exchange of GTP on the α subunit of Gq complex. However, functional selectivity of YM-254890 towards various G proteins was not fully characterized, primarily due to its restricted availability before 2017. Here, using human coronary artery endothelial cells as a model, we performed a systemic pharmacological evaluation on the functional selectivity of YM-254890 on multiple G protein-mediated receptor signaling. First, we confirmed that YM-254890, at 30 nM, abolished UTP-activated P2Y2 receptor-mediated Ca2+ signaling and ERK1/2 phosphorylation, indicating its potent inhibition on the Gq protein. However, we unexpectedly found that YM-254890 also significantly suppressed cAMP elevation and ERK1/2 phosphorylation induced by multiple Gs-coupled receptors including β2-adrenegic, adenosine A2 and PGI2 receptors. Surprisingly, although YM-254890 had no impact on CXCR4/Gi/o protein-mediated suppression of cAMP production, it abolished ERK1/2 activation. Further, no cellular toxicity was observed for YM-254890, and it neither affected A23187- or thapsigargin-induced Ca2+ signaling, nor forskolin-induced cAMP elevation and growth factor-induced MAPK signaling. We conclude that YM-254890 is not a selective inhibitor for Gq protein; instead, it acts as a broad-spectrum inhibitor for Gq and Gs proteins and exhibits a biased inhibition on Gi/o signaling, without affecting non-GPCR-mediated cellular signaling.

The selective PGI2 receptor agonist selexipag ameliorates Sugen 5416/hypoxia-induced pulmonary arterial hypertension in rats.

Pulmonary arterial hypertension (PAH) is a lethal disease characterized by a progressive increase in pulmonary artery pressure due to an increase in vessel tone and occlusion of vessels. The endogenous vasodilator prostacyclin and its analogs are used as therapeutic agents for PAH. However, their pharmacological effects on occlusive vascular remodeling have not been elucidated yet. Selexipag is a recently approved, orally available and selective prostacyclin receptor agonist with a non-prostanoid structure. In this study, we investigated the pharmacological effects of selexipag on the pathology of chronic severe PAH in Sprague-Dawley and Fischer rat models in which PAH was induced by a combination of injection with the vascular endothelial growth factor receptor antagonist Sugen 5416 and exposure to hypoxia (SuHx). Oral administration of selexipag for three weeks significantly improved right ventricular systolic pressure and right ventricular (RV) hypertrophy in Sprague-Dawley SuHx rats. Selexipag attenuated the proportion of lung vessels with occlusive lesions and the medial wall thickness of lung arteries, corresponding to decreased numbers of Ki-67-positive cells and a reduced expression of collagen type 1 in remodeled vessels. Administration of selexipag to Fischer rats with SuHx-induced PAH reduced RV hypertrophy and mortality caused by RV failure. These effects were probably based on the potent prostacyclin receptor agonistic effect of selexipag on pulmonary vessels. Selexipag has been approved and is used in the clinical treatment of PAH worldwide. It is thought that these beneficial effects of prostacyclin receptor agonists on multiple aspects of PAH pathology contribute to the clinical outcomes in patients with PAH.

Prostaglandin I2 mediates weak vasodilatation in human placental microvessels.

Human placental vessels (HPVs) play important roles in the exchange of metabolites and oxygen in maternal-fetal circulation. Endothelial-derived prostacyclin (prostaglandin I2, PGI2) is a critical endothelial vasodilator in the body. However, the physiological and pharmacological functions of endothelial PGI2 in the human placenta are still unclear. Human, sheep, and rat blood vessels were used in this study. Unlike non-placental vessels (non-PVs), the PGI2 synthesis inhibitor tranylcypromine (TCP) did not modify 5-hydroxytryptamine (5-HT)-induced vascular contraction, indicating that endothelial-derived PGI2 was weak in PVs. Vascular responses to exogenous PGI2 showed slight relaxation followed by a significant contraction at a higher concentration in HPV, which was inhibited by the thromboxane-prostanoid (TP) receptors antagonist SQ-29,548. Testing PVs and non-PVs from sheep also showed similar functional results. More TP receptors than PGI2 (IP) receptors were observed in HPVs. The whole-cell K+ current density of HPVs was significantly weaker than that of non-PVs. This study demonstrated the specific characteristics of the placental endogenous endothelial PGI2 system and the patterns of placental vascular physiological/pharmacological response to exogenous PGI2, showing that placental endothelial PGI2 does not markedly contribute to vascular dilation in the human placenta, in notable contrast to non-PVs. The results provide crucial information for understanding the endothelial roles of HPVs, which may be helpful for further investigations of potential targets in the treatment of diseases such as preeclampsia.

In vivo response of the corpus luteum to progesterone treatment of gilts during early gestation

Supplementation of progesterone (P4) in pregnant gilts increases concentrations of circulating P4 and stimulates the secretory activity of the endometrium. In this study, there was examination of the consequences of exogenous P4 administration on luteal P4 content and the expression of genes related to the corpus luteum (CL) function. Gilts with gonadotropin-induced estrus were administered daily injections of corn oil (n = 8) or P4 (n = 8) on days 3 through 10 after insemination. The animals were slaughtered on day 12 of pregnancy to obtain corpora lutea for real-time polymerase chain reaction analyses of selected genes and for enzyme immunoassay of P4. Injections with P4 had no effect on the concentration of P4 and the relative abundance of mRNA transcripts of cholesterol transport-related proteins, steroidogenic enzymes, and receptors for luteotropic factors in the luteal tissue. The abundance of prostaglandin (PG) endoperoxide synthase 2, PGI2 synthase, PGI2 receptor, fibroblast growth factor 2, peroxisome proliferator-activated receptor γ, and tumor necrosis factor α receptor type I transcripts increased after P4 treatment. In contrast, the relative abundance of angiopoietin 2 mRNA decreased in response to P4 administration. In summary, P4 supplementation in pregnant gilts does not affect luteal steroidogenesis but modulates the abundance of factors related to vascular function. Given that the endometrium is the main target tissue for P4, an indirect uterine-mediated effect of exogenous P4 on CL function is likely.

INSPIRE: Final Results from a Phase 3, Open-Label, Pivotal Study to Evaluate the Safety and Tolerability of LIQ861 in Pulmonary Arterial Hypertension

Significant advantages are associated with inhaled therapies for pulmonary arterial hypertension (PAH), including enhanced delivery of drug to the lungs at a lower overall dose, improved ventilation-perfusion matching, and decreased risk of systemic vasodilation and other systemic adverse effects. LIQ861 is an investigational, novel, dry-powder formulation of treprostinil (TRE) designed using PRINT® technology to enhance deep-lung drug deposition and enable delivery of TRE doses in 1 to 2 breaths per capsule 4 times per day while using a convenient dry-powder inhaler. The primary objective of the INSPIRE study is to evaluate the safety and tolerability of LIQ861 in PAH patients transitioning to LIQ861 from a stable dose of Tyvaso® (Transition Group) and those adding LIQ861 to therapy with ≤2 non-prostacyclin (PGI) oral agents (Add-on Group). Final results from INSPIRE will be presented. INSPIRE is a phase 3, open-label, multicenter study that enrolled WHO Group I PAH patients classified as New York Heart Association (NYHA) functional class (FC) II-IV. Patients in the Transition Group received an initial dose of LIQ861 that was believed to be comparable to their Tyvaso® dose, while those in the Add-on Group initiated LIQ861 at a dose of 25 mcg capsule strength QID. LIQ861 dose increases in both groups were titrated in 25 mcg capsule strength QID increments to tolerance and symptom relief. The primary endpoint is the incidence of treatment-emergent adverse events and serious adverse events at Month 2. Exploratory endpoints are changes from baseline through Month 2 in physician assessment of stability; six-minute walk distance; NYHA FC, N-terminal B-type natriuretic peptide levels; and patient-reported quality of life. An additional endpoint for the Transition Group is the proportion of patients maintaining a sustained transition through Month 2, with sustained transition defined as continued LIQ861 therapy after discontinuation of Tyvaso®, no interruptions in LIQ861 therapy for ≥7 days; and no treatment with any other PGI analog or PGI receptor agonist.

Relaxin reduces endothelium-derived vasoconstriction in hypertension: Revealing new therapeutic insights.

Endothelium-derived vasoconstriction is a hallmark of vascular dysfunction in hypertension. In some cases, an overproduction of endothelium-derived prostacyclin (PGI2 ) can cause contraction rather than relaxation. Relaxin is well known for its vasoprotective actions, but the possibility that this peptide could also reverse endothelium-derived vasoconstriction has never been investigated. We tested the hypothesis that short-term relaxin treatment mitigates endothelium-derived vasoconstriction in spontaneously hypertensive rats (SHR). Male Wistar Kyoto rats (WKY) and SHR were subcutaneously infused with either vehicle (20mmol·L-1 sodium acetate) or relaxin (13.3μg·kg-1 ·hr-1 ) using osmotic minipumps for 3days. Vascular reactivity to the endothelium-dependent agonist ACh was assessed in vitro by wire myography. Quantitative PCR and LC-MS were used to identify changes in gene expression of prostanoid pathways and PG production, respectively. Relaxin treatment ameliorated hypertension-induced endothelial dysfunction by increasing NO-dependent relaxation and reducing endothelium-dependent contraction. Notably, short-term relaxin treatment up-regulated mesenteric PGI2 receptor (IP) expression, permitting PGI2 -IP-mediated vasorelaxation. In the aorta, reversal of contraction was accompanied by suppression of the hypertension-induced increase in prostanoid-producing enzymes and reduction in PGI2 -evoked contractions. Relaxin has region-dependent vasoprotective actions in hypertension. Specifically, relaxin has distinct effects on endothelium-derived contracting factors and their associated vasoconstrictor pathways in mesenteric arteries and the aorta. Taken together, these observations reveal the potential of relaxin as a new therapeutic agent for vascular disorders that are associated with endothelium-derived vasoconstriction including hypertension.

Repeated bronchoconstriction attenuates the cough response to bronchoconstriction in naïve guinea pigs

BackgroundCough variant asthma (CVA) is recognized as a precursor of bronchial asthma (BA). However, the cough response to bronchoconstriction differs between these similar diseases. Repeated bronchoconstriction and the resulting imbalance of endogenous lipid mediators may impact the cough response. MethodsWe investigated the influence of repeated bronchoconstriction on the cough response to bronchoconstriction using naïve guinea pigs. Bronchoconstriction was induced for 3 consecutive days and changes in the cough response and lipid mediators, such as PGE2, PGI2, and cysteinyl-LTs (Cys-LTs), in BAL fluid (BALF) were assessed. We investigated the effect of endogenous PGI2 on the cough response by employing a PGI2 receptor antagonist. In order to investigate the cough response over a longer period, we re-evaluated the cough response 2 weeks after repeated bronchoconstriction. ResultsThe number of coughs induced by bronchoconstriction were significantly decreased by repeated bronchoconstriction. The levels of PGE2, PGI2, and Cys-LTs, and the ratio of PGI2/PGE2 were significantly increased, following repeated bronchoconstriction. This decrease in the cough response was suppressed by pretreatment with a PGI2 receptor antagonist. Two weeks after repeated bronchoconstriction, the cough response returned to the same level as before repeated bronchoconstriction along with a concomitant return of lipid mediators, such as PGE2, PGI2, and Cys-LTs and the ratio of PGI2/PGE2. ConclusionsOur results suggest that repeated bronchoconstriction and the resulting imbalance of endogenous lipid mediators contribute to the difference in cough responses to bronchoconstriction in CVA and BA.

Migraine-provoking substances evoke periorbital allodynia in mice

BackgroundAdministration of endogenous mediators or exogenous chemicals in migraine patients provoke early headaches and delayed migraine-like attacks. Although migraine provoking substances are normally vasodilators, dilation of arterial vessels does not seem to be the sole contributing factor, and the underlying mechanisms of the delayed migraine pain are mostly unknown. Sustained mechanical allodynia is a common response associated with the local administration of various proalgesic substances in experimental animals and humans. Here, we investigated the ability of a series of endogenous mediators which provoke or do not provoke migraine in patients, to cause or not cause mechanical allodynia upon their injection in the mouse periorbital area.MethodsMechanical allodynia was assessed with the von Frey filament assay. Stimuli were given by subcutaneous injection in the periorbital area of C57BL/6J mice; antagonists were administered by local and systemic injections.ResultsCalcitonin gene related peptide (CGRP), but not adrenomedullin and amylin, pituitary adenylyl cyclase activating peptide (PACAP), but not vasoactive intestinal polypeptide (VIP), histamine, prostaglandin E2 (PGE2) and prostacyclin (PGI2), but not PGF2α, evoked a dose-dependent periorbital mechanical allodynia. The painful responses were attenuated by systemic or local (periorbital) administration of antagonists for CGRP (CLR/RAMP1), PACAP (PAC-1), histamine H1, PGE2 (EP4), and PGI2 (IP) receptors, respectively.ConclusionsThe correspondence between substances that provoke (CGRP; PACAP, histamine, PGE2, PGI2), or do not provoke (VIP and PGF2α), migraine-like attacks in patients and periorbital allodynia in mice suggests that the study of allodynia in mice may provide information on the proalgesic mechanisms of migraine-provoking agents in humans. Results underline the ability of migraine-provoking substances to initiate mechanical allodynia by acting on peripheral terminals of trigeminal afferents.

The PGI2 Analog Cicaprost Inhibits IL-33-Induced Th2 Responses, IL-2 Production, and CD25 Expression in Mouse CD4+ T Cells.

IL-33 has pleiotropic functions in immune responses and promotes the development of allergic diseases and asthma. IL-33 induces Th2 differentiation and enhances type 2 cytokine production by CD4+ T cells. However, the regulation of IL-33-driven type 2 cytokine responses is not fully defined. In this study, we investigated the effect of PGI2, a lipid mediator formed in the cyclooxygenase pathway of arachidonic acid metabolism, on naive CD4+ T cell activation, proliferation, and differentiation by IL-33. Using wild-type and PGI2 receptor (IP) knockout mice, we found that the PGI2 analog cicaprost dose-dependently inhibited IL-33-driven IL-4, IL-5, and IL-13 production by CD4+ T cells in an IP-specific manner. In addition, cicaprost inhibited IL-33-driven IL-2 production and CD25 expression by CD4+ T cells. Furthermore, IP knockout mice had increased IL-5 and IL-13 responses of CD4+ T cells to Alternaria sensitization and challenge in mouse lungs. Because IL-33 is critical for Alternaria-induced type 2 responses, these data suggest that PGI2 not only inhibits IL-33-stimulated CD4+ Th2 cell responses in vitro but also suppresses IL-33-induced Th2 responses caused by protease-containing allergens in vivo.

Iloprost, a prostacyclin analog, inhibits the invasion of ovarian cancer cells by downregulating matrix metallopeptidase-2 (MMP-2) through the IP-dependent pathway

Recent studies have shown that a bioactive lipid prostacyclin (PGI2) plays a role in various cancers, including lung cancer. However, the specific function of PGI2 in ovarian cancer progression has not been determined. This study investigated the effects of PGI2 on cell growth, migration, and invasion in ovarian cancer cells using iloprost, a stable PGI2 analog. Iloprost significantly inhibited migration and invasion, but not cell growth, in a dose-dependent manner in human ovarian cancer cells (A2780 and SKOV3). Interestingly, the cell surface Gs protein-coupled PGI2 receptor IP was enhanced in human ovarian cancer cells. The inhibitory effect of iloprost on migration and invasion was entirely reversed by an IP antagonist (CAY10449) and IP siRNA, whereas the knockdown of peroxisome proliferator-activated receptor δ (PPARδ), a nuclear receptor of PGI2, did not rescue the effect of iloprost. Additionally, iloprost markedly decreased the expression of matrix metallopeptidase-2 and -9 (MMP-2 and MMP-9), which may be induced in the process of ovarian cancer metastasis. IP siRNA inhibited iloprost-reduced MMP-2 expression but not MMP-9 expression. Moreover, inhibition of protein kinase A (PKA) and overexpression of Akt and p38 rescued the inhibition of invasion and the reduction of MMP-2 expression by iloprost. Furthermore, iloprost-induced activation of PKA was associated with PKA-mediated Akt and p38 inactivation in ovarian cancer cells. Taken together, these results demonstrate that iloprost inhibits ovarian cancer cell invasion by downregulating MMP-2 expression via the IP-mediated PKA pathway. This study is the first to reveal a novel role for iloprost and to clarify its underlying mechanism in human ovarian cancer cells.