IP Knockout Mice Research Articles

Superiority of CS585 as a selective prostacyclin receptor agonist in prevention of thrombosis

Abstract Background A major challenge with development of new targets for antithrombotic treatment is the sustained effect on the platelet and the target selectivity. While increasing cAMP in the platelet through activation of the prostacyclin (IP) receptor represents a viable antiplatelet target for prevention of thrombosis, current IP receptor agonists lack stability and selectivity. Development of a selective IP receptor agonist that is stable in the blood and selective for the IP receptor would represent a new approach for prevention of platelet activation and thrombosis in the blood and expand the utility of IP receptor agonists which are currently used for the treatment of pulmonary arterial hypertension (PAH). Purpose Develop an IV and oral IP receptor agonist, CS585, with sustained activity in vivo in the blood and eliminating the off-target effects observed with other IP agonists not having selective activation of the IP receptor. These characteristics would make CS585 an attractive new therapeutic approach for limiting platelet activation and thrombosis. Methods We assessed platelet activation and thrombosis in human blood ex vivo and mouse models in vivo. For mouse models, platelets and fibrin were labelled and accumulation at the site of injury was measured using intravital microscopy. Thrombosis in the vessel was assessed in the laser-induced cremaster thrombosis assay. Selectivity was assessed in human blood pharmacologically using prostaglandin receptor inhibitors and in mouse with an IP receptor knockout mouse. Flow cytometry, aggregometry, and western blot techniques were used to assess selectivity. Results IV and oral administration of CS585 resulted in sustained inhibited clot formation and fibrin formation at the site of injury in the laser-induced cremaster arteriole thrombosis assay in wild-type mice but was unable to alter platelet function in IP knockout mice. CS585 inhibition of platelet activation was fully prevented in platelets from IP knockout mice as assessed by flow cytometry (integrin activation and granule secretion) and by VASP phosphorylation. Inhibition of human platelet activation by CS585 was inhibited by pharmacological inhibition of the IP receptor, but not by inhibition of the DP1, EP2, or EP4 receptors as assessed by aggregation, flow cytometry, and VASP phosphorylation. Conclusions We have shown for the first time that CS585, a novel IP receptor agonist, not only inhibits platelet activation and clot formation, but gives sustained inhibition through multiple routes of administration in an in vivo mouse model of thrombosis. Importantly, the challenge in selectivity observed with other IP receptor agonists is not observed with CS585 supporting its development for a number of diseases in the cardiovascular system including thrombosis, VTE, secondary prevention after MI and stroke, and PAH.CS585 inhibition of thrombosis in vivoCS585 selectively signals through IP

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.

Prostaglandin I2 suppresses the development of diet-induced nonalcoholic steatohepatitis in mice.

Nonalcoholic steatohepatitis (NASH) is a hepatic manifestation of metabolic syndrome. Although the prostaglandin (PG)I2 receptor IP is expressed broadly in the liver, the role of PGI2-IP signaling in the development of NASH remains to be determined. Here, we investigated the role of the PGI2-IP system in the development of steatohepatitis using mice lacking the PGI2 receptor IP [IP-knockout (IP-KO) mice] and beraprost (BPS), a specific IP agonist. IP-KO and wild-type (WT) mice were fed a methionine- and choline-deficient diet (MCDD) for 2, 5, or 10 wk. BPS was administered orally to mice every day during the experimental periods. The effect of BPS on the expression of chemokine and inflammatory cytokines was examined also in cultured Kupffer cells. WT mice fed MCDD developed steatohepatitis at 10 wk. IP-KO mice developed steatohepatitis at 5 wk with augmented histologic derangements accompanied by increased hepatic monocyte chemoattractant protein-1 (MCP-1) and TNF-α concentrations. After 10 wk of MCDD, IP-KO mice had greater hepatic iron deposition with prominent oxidative stress, resulting in hepatocyte damage. In WT mice, BPS improved histologic and biochemical parameters of steatohepatitis, accompanied by reduced hepatic concentration of MCP-1 and TNF-α. Accordingly, BPS suppressed the LPS-stimulated Mcp-1 and Tnf-α mRNA expression in cultured Kupffer cells prepared from WT mice. PGI2-IP signaling plays a crucial role in the development and progression of steatohepatitis by modulating the inflammatory response, leading to augmented oxidative stress. We suggest that the PGI2-IP system is an attractive therapeutic target for treating patients with NASH.-Kumei, S., Yuhki, K.-I., Kojima, F., Kashiwagi, H., Imamichi, Y., Okumura, T., Narumiya, S., Ushikubi, F. Prostaglandin I2 suppresses the development of diet-induced nonalcoholic steatohepatitis in mice.

12-HETrE inhibits platelet reactivity and thrombosis in part through the prostacyclin receptor.

The dihomo-γ-linolenic acid (DGLA)-derived metabolite of 12-lipoxygenase, 12-hydroxy-eicosatrienoic acid (12-HETrE), was recently shown to potently inhibit thrombus formation without prolonging bleeding in murine models. Although 12-HETrE was found to inhibit platelet activation via the Gαs signaling pathway, the Gαs-coupled receptor by which 12-HETrE mediates its antiplatelet effects has yet to be identified. Defining the receptor by which 12-HETrE exerts its effects is key to determining its therapeutic potential as an antiplatelet drug. Therefore, the goal of this study was to determine the Gαs-coupled platelet receptor through which 12-HETrE exerts its antiplatelet effects. In this study, we showed that pharmacological inhibition of the prostacyclin (IP) receptor in human platelets or genetic ablation of IP in murine platelets prevented 12-HETrE from blocking aggregation in vitro. Furthermore, the antithrombotic effects of 12-HETrE were significantly diminished in IP knockout mice in vivo. Together these data demonstrate that the antiplatelet effects of 12-HETrE are at least partially dependent on IP signaling. Importantly, this work identified 12-HETrE as a novel regulator of IP signaling that may aid in the rationale for design of novel therapeutics to inhibit platelet function. Additionally, this study provides further insight into the mechanism by which DGLA supplementation inhibits platelets function.

Potent Anti-Platelet Metabolite, 12-HETrE, Inhibits Platelet Activation and Thrombosis In Vivo Via Activation of the IP Receptor

Platelet activation is a critical component of clot formation. Under pathophysiological conditions platelet-mediated clot formation can lead to an occlusive thrombotic event resulting in myocardial infarction and stroke. Regulation of platelet reactivity relies on an understanding of the complex intra-platelet signaling that takes place following activation of various receptors on the platelet surface. Our lab recently identified a novel oxidized metabolite known as 12-HETrE that is formed from 12-lipoxygenase (12-LOX) oxidation of the ω-6 fatty acid DGLA in the platelet. Since DGLA constitutes one of the predominant fatty acids contained in the phospholipids on the platelet membrane and is also a rich source of dietary fatty acid intake through a variety of plant oils, we had originally sought to determine if this metabolite contained inherent regulatory properties on the human platelet. We successfully showed not only that 12-HETrE potently regulates hemostasis and thrombosis in vivo, but additionally identified that the metabolite signals inhibition of platelet activity through a GαS-linked GPCR resulting in formation of cAMP and activation of PKA and VASP. In the current study, we identified for the first time the receptor activated by 12-HETrE in the human platelet. To identify which GPCR is activated by 12-HETrE, we utilized both pharmacologic as well as genetic approaches. Our ex vivo pharmacological data suggested 12-HETrE predominantly signals through the IP receptor in the human platelet. This finding was confirmed using an IP receptor knockout mouse as well as human cell lines expressing or absent IP receptor on their surface. Finally, these findings were confirmed in vivo with wild type or IP knockout mice and assessing the ability of 12-HETrE to inhibit laser-induced thrombosis. Hence, these findings show for the first time that 12-HETrE does in fact regulate inhibition of platelets through the IP receptor. Since 12-HETrE has already been shown to inhibit thrombosis without a significant increase in bleeding diathesis, the current study has elucidated a new target for IP receptor activation and an inhibition of platelet activity and occlusive thrombus formation and should be evaluated for its potential as a new class of anti-thrombotic therapeutics. DisclosuresNo relevant conflicts of interest to declare.

Cyclooxygenase inhibition abrogates aeroallergen-induced immune tolerance by suppressing prostaglandin I2 receptor signaling

The prevalence of allergic diseases has doubled in developed countries in the past several decades. Cyclooxygenase (COX)-inhibiting drugs augmented allergic diseases in mice by increasing allergic sensitization and memory immune responses. However, whether COX inhibition can promote allergic airway diseases by inhibiting immune tolerance is not known. To determine the role of the COX pathway and prostaglandin I2 (PGI2) signaling through the PGI2 receptor (IP) in aeroallergen-induced immune tolerance. Wild-type (WT) BALB/c mice and IP knockout mice were aerosolized with ovalbumin (OVA) to induce immune tolerance prior to immune sensitization with an intraperitoneal injection of OVA/alum. The COX inhibitor indomethacin or vehicle was administered in drinking water to inhibit enzyme activity during the sensitization phase. Two weeks after sensitization, the mice were challenged with OVA aerosols. Mouse bronchoalveolar lavage fluid was harvested for cell counts and TH2 cytokine measurements. WT mice treated with indomethacin had greater numbers of total cells, eosinophils, and lymphocytes, and increased IL-5 and IL-13 protein expression in BAL fluid compared to vehicle-treated mice. Similarly, IP knockout mice had augmented inflammation and TH2 cytokine responses compared to WT mice. In contrast, the PGI2 analog cicaprost attenuated the anti-tolerance effect of COX inhibition. COX inhibition abrogated immune tolerance by suppressing PGI2 IP signaling, suggesting that PGI2 signaling promotes immune tolerance and that clinical use of COX-inhibiting drugs may increase the risk of developing allergic diseases.

Prostacyclin mediates neuropathic pain through interleukin 1β-expressing resident macrophages

Prostacyclin is an important mediator of peripheral pain sensation. Here, we investigated its potential participation in mediating neuropathic pain and found that prostacyclin receptor (IP) knockout mice exhibited markedly decreased pain behavior. Application of an IP antagonist to the injury site or selective IP deficiency in myeloid cells mimicked the antinociceptive effect observed in IP knockout mice. At the site of nerve injury, IP was expressed in interleukin (IL) 1β-containing resident macrophages, which were less common in IP knockout mice. Local administration of the IP agonist cicaprost inhibited macrophage migration in vitro and promoted accumulation of IP- and IL1β-expressing cells as well as an increase of IL1β concentrations at the application site in vivo. Fittingly, the IL1-receptor antagonist anakinra (IL-1ra) decreased neuropathic pain behavior in wild-type mice but not in IP knockout mice. Finally, continuous, but not single administration, of the cyclooxygenase inhibitor meloxicam early after nerve injury decreased pain behavior and the number of resident macrophages. Thus, early synthesis of prostacyclin at the site of injury causes accumulation of IL1β-expressing macrophages as a key step in neuropathic pain after traumatic injury.

Prostaglandin I2 Signaling Drives Th17 Differentiation and Exacerbates Experimental Autoimmune Encephalomyelitis

BackgroundProstaglandin I2 (PGI2), a lipid mediator currently used in treatment of human disease, is a critical regulator of adaptive immune responses. Although PGI2 signaling suppressed Th1 and Th2 immune responses, the role of PGI2 in Th17 differentiation is not known.Methodology/Principal FindingsIn mouse CD4+CD62L+ naïve T cell culture, the PGI2 analogs iloprost and cicaprost increased IL-17A and IL-22 protein production and Th17 differentiation in vitro. This effect was augmented by IL-23 and was dependent on PGI2 receptor IP signaling. In mouse bone marrow-derived CD11c+ dendritic cells (BMDCs), PGI2 analogs increased the ratio of IL-23/IL-12, which is correlated with increased ability of BMDCs to stimulate naïve T cells for IL-17A production. Moreover, IP knockout mice had delayed onset of a Th17-associated neurological disease, experimental autoimmune encephalomyelitis (EAE), and reduced infiltration of IL-17A-expressing mononuclear cells in the spinal cords compared to wild type mice. These results suggest that PGI2 promotes in vivo Th17 responses.ConclusionThe preferential stimulation of Th17 differentiation by IP signaling may have important clinical implications as PGI2 and its analogs are commonly used to treat human pulmonary hypertension.

Abstract 1576: Prostaglandin I2 signaling regulates micro-metastasis in lung cancer

Abstract Background: Bone marrow-derived cells (BMDCs) play an important role in the initiation of neo-angiogenesis, and contribute to the diseases including ischemic diseases, retinopathy, and cancer. We have reported that BMDCs express prostaglandin I2 (PGI2) specific receptor, IP and the PGI2-IP signaling system maintains the function of BMDCs. Additionally, we have reported that the PGI2-IP signaling in BMDCs play an important role for tumor growth, by using mouse bone marrow transplanted models and tumor xenograft mouse models. Objectives: The purpose of this study was to test the hypothesis that PGI2-IP signaling system regulates micro-metastasis in lung cancer. Methods: In vitro study, 5-bromo-2’-deoxy-uridine (BrdU) cell proliferation assay and clonogenic cell assay were used to clarify the effect on lung cancer cell (A549, PC9) proliferation and cellular form in the existence of IP receptor agonist or antagonist. Human cytokine antibody array was performed to detect the expression of angiogenic cytokines and growth factors secreted by the treated cells. In vivo study, we employed both a mouse lung metastasis model and a mouse bone marrow transplantation model. We used GFP-labeled or DsRed-labeled lung cancer cells to distinguish cancer cells from BMDCs. Lung cancer cells were injected into the tail vein of mice (ICR nu/nu), which were transplanted with bone marrow cells from GFP-labeled wild-type or IP knockout mice. Real-time RT-PCR was used to quantify lung metastasis of human cancer cells in nude mice, and the expression levels of housekeeping genes (human and mouse GAPDH) in the lungs were measured. Immunofluorescence assay was performed to evaluate angiogenesis in pulmonary metastatic tumors. Results: In vitro study, PGI2-IP signaling didn't impair proliferation of lung cancer cells and production of angiogenic cytokines secreted by tumors. In the mice lung metastasis model, micro-metastasis in lung cancer was regulated by PGI2-IP signaling system. RT-PCR assay showed that PGI2-IP signaling changed the expression levels of human lung cancer cells in mice lungs. In the immunofluorescence assay, PGI2-IP signaling influenced the expression of pericyte markers around the tumors. Conclusions: The present study demonstrated that PGI2-IP signaling system regulates micro-metastasis in lung cancer. These results suggest that PGI2-IP system may become a novel therapeutic target in lung metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1576. doi:10.1158/1538-7445.AM2011-1576

Lack of prostanoid receptor involvement in ischemic acute renal failure in mice

Background. Thromboxane (TX) A2 inhibition or prostaglardin (PGI2) infusion prevents ischemic acute renal failure (ARF) in animal models. However, the pathophysiological roles of the prostanoid receptors in the development of ischemic ARF are not fully understood, partly because of the limited specificity of their inhibitors or antagonists. Methods. We investigated whether targeted disruption of the PGI2 receptor (IP) or TXA2 receptor (TP) genes conferred susceptibility to renal ischemic-reperfusion injury, using IP and TP knockout mice. Results. Serum creatinine concentration in TP knockout mice was not significantly different from that in wild-type controls. There were no significant histological differences between TP knockout and wild-type mice. Likewise, IP knockout mice showed no significant differences from the wild-type controls in regard to creatinine concentration or histological damage. Conclusions. Lack of TP or IP had no influence on postischemic ARF in mice, indicating that receptors for TXA2 or PGI2 may have minimal roles in the development of this mouse model of ischemic ARF.

O-109 - Reduced adaptive cytoprotection by hyperosmotic saline in IP knockout mice

O-109 - Reduced adaptive cytoprotection by hyperosmotic saline in IP knockout mice