Yujun Shen

Vitamin D inhibits COX-2 expression and inflammatory response by targeting thioesterase superfamily member 4

Background: Vitamin D insufficiency has been associated with chronic inflammatory diseases. However, the underlying mechanisms remain unclear. Results: Vitamin D inhibits COX-2-mediated inflammatory response by modulating the Akt/NF-κB signaling pathway via direct up-regulation of thioesterase superfamily member 4. Conclusion: Vitamin D plays novel roles in anti-inflammation. Significance: Supplemental vitamin D could protect against chronic inflammatory diseases by targeting THEM4/Akt/NF-κB signaling. Inadequate vitamin D status has been linked to increased risk of type 2 diabetes and cardiovascular disease. Inducible cyclooxygenase (COX) isoform COX-2 has been involved in the pathogenesis of such chronic inflammatory diseases. We found that the active form of vitamin D, 1,25(OH)2D produces dose-dependent inhibition of COX-2 expression in murine macrophages under both basal and LPS-stimulated conditions and suppresses proinflammatory mediators induced by LPS. Administration of 1,25(OH)2D significantly alleviated local inflammation in a carrageenan-induced paw edema mouse model. Strikingly, the phosphorylation of both Akt and its downstream target IκBα in macrophages were markedly suppressed by 1,25(OH)2D in the presence and absence of LPS stimulation through up-regulation of THEM4 (thioesterase superfamily member 4), an Akt modulator protein. Knockdown of both vitamin D receptor and THEM4 attenuated the inhibitory effect of 1,25(OH)2D on COX-2 expression in macrophages. A functional vitamin D-responsive element in the THEM4 promoter was identified by chromatin immunoprecipitation and luciferase reporter assay. Our results indicate that vitamin D restrains macrophage-mediated inflammatory processes by suppressing the Akt/NF-κB/COX-2 pathway, suggesting that vitamin D supplementation might be utilized for adjunctive therapy for inflammatory disease.

Cyclooxygenase-2–Derived Prostaglandin E2 Promotes Injury-Induced Vascular Neointimal Hyperplasia Through the E-prostanoid 3 Receptor

Rationale: Vascular smooth muscle cell (VSMC) migration and proliferation are the hallmarks of restenosis pathogenesis after angioplasty. Cyclooxygenase (COX)-derived prostaglandin (PG) E2 is implicated in the vascular remodeling response to injury. However, its precise molecular role remains unknown. Objective: This study investigates the impact of COX-2–derived PGE2 on neointima formation after injury. Methods and Results: Vascular remodeling was induced by wire injury in femoral arteries of mice. Both neointima formation and the restenosis ratio were diminished in COX-2 knockout mice as compared with controls, whereas these parameters were enhanced in COX-1>COX-2 mice, in which COX-1 is governed by COX-2 regulatory elements. PG profile analysis revealed that the reduced PGE2 by COX-2 deficiency, but not PGI2, could be rescued by COX-1 replacement, indicating COX-2–derived PGE2 enhanced neointima formation. Through multiple approaches, the EP3 receptor was identified to mediate the VSMC migration response to various stimuli. Disruption of EP3 impaired VSMC polarity for directional migration by decreasing small GTPase activity and restricted vascular neointimal hyperplasia, whereas overexpression of EP3&agr; and EP3&bgr; aggravated neointima formation. Inhibition or deletion of EP3&agr;/&bgr;, a G&agr;i protein–coupled receptor, activated the cAMP/protein kinase A pathway and decreased activation of RhoA in VSMCs. PGE2 could stimulate phosphatidylinositol 3-kinase/Akt/glycogen synthase kinase3&bgr; signaling in VSMCs through G&bgr;&ggr; subunits on EP3&agr;/&bgr; activation. Ablation of EP3 suppressed phosphatidylinositol 3-kinase signaling and reduced GTPase activity in VSMCs and altered cell polarity and directional migration. Conclusions: COX-2–derived PGE2 facilitated the neointimal hyperplasia response to injury through EP3&agr;/&bgr;-mediated cAMP/protein kinase A and phosphatidylinositol 3-kinase pathways, indicating EP3 inhibition may be a promising therapeutic strategy for percutaneous transluminal coronary angioplasty.

EP3 receptor deficiency attenuates pulmonary hypertension through suppression of Rho/TGF-β1 signaling

Pulmonary arterial hypertension (PAH) is commonly associated with chronic hypoxemia in disorders such as chronic obstructive pulmonary disease (COPD). Prostacyclin analogs are widely used in the management of PAH patients; however, clinical efficacy and long-term tolerability of some prostacyclin analogs may be compromised by concomitant activation of the E-prostanoid 3 (EP3) receptor. Here, we found that EP3 expression is upregulated in pulmonary arterial smooth muscle cells (PASMCs) and human distal pulmonary arteries (PAs) in response to hypoxia. Either pharmacological inhibition of EP3 or Ep3 deletion attenuated both hypoxia and monocrotaline-induced pulmonary hypertension and restrained extracellular matrix accumulation in PAs in rodent models. In a murine PAH model, Ep3 deletion in SMCs, but not endothelial cells, retarded PA medial thickness. Knockdown of EP3α and EP3β, but not EP3γ, isoforms diminished hypoxia-induced TGF-β1 activation. Expression of either EP3α or EP3β in EP3-deficient PASMCs restored TGF-β1 activation in response to hypoxia. EP3α/β activation in PASMCs increased RhoA-dependent membrane type 1 extracellular matrix metalloproteinase (MMP) translocation to the cell surface, subsequently activating pro-MMP-2 and promoting TGF-β1 signaling. Activation or disruption of EP3 did not influence PASMC proliferation. Together, our results indicate that EP3 activation facilitates hypoxia-induced vascular remodeling and pulmonary hypertension in mice and suggest EP3 inhibition as a potential therapeutic strategy for pulmonary hypertension.

COX-1-derived thromboxane A2 plays an essential role in early B-cell development via regulation of JAK/STAT5 signaling in mouse.

Cyclooxygenases (COXs) and their prostanoid products play important roles in a diverse range of physiological processes, including in the immune system. Here, we provide evidence that COX-1 is an essential regulator in early stages of B-cell development. COX-1-deficient mice displayed systematic reduction in total B cells, which was attributed to the arrest of early B-cell development from pro-B to pre-B stage. We further demonstrated that this defect was mediated through downregulation of the Janus kinase/signal transducer and activator of transcription 5 (JAK/STAT5) signaling and its target genes, including Pax5, in COX-1(-/-) mice. Mechanistic studies revealed that COX-1-derived thromboxane A2 (TxA2) could regulate JAK3/STAT5 signaling through the cyclic adenosine monophosphate-protein kinase A pathway, via binding with its receptor thromboxane A2 receptor (TP). Administration of the TP agonist could rescue the defective B-cell development and JAK/STAT5 signaling activity in COX-1-deficient mice. Moreover, administration of low-dose aspirin caused a significant reduction in total B cells in peripheral blood of healthy human volunteers, coincidentally with reduced TxA2 production and downregulation of JAK/STAT5 signaling. Taken together, our results demonstrate that COX-1-derived TxA2 plays a critical role in the stage transition of early B-cell development through regulation of JAK/STAT5 signaling and indicate a potential immune-suppressive effect of low-dose aspirin in humans.

Thromboxane Governs the Differentiation of Adipose-Derived Stromal Cells Toward Endothelial Cells In Vitro and In Vivo

RATIONALE Autologous adipose-derived stromal cells (ASCs) offer great promise as angiogenic cell therapy for ischemic diseases. Because of their limited self-renewal capacity and pluripotentiality, the therapeutic efficacy of ASCs is still relatively low. Thromboxane has been shown to play an important role in the maintenance of vascular homeostasis. However, little is known about the effects of thromboxane on ASC-mediated angiogenesis. OBJECTIVE To explore the role of the thromboxane-prostanoid receptor (TP) in mediating the angiogenic capacity of ASCs in vivo. METHODS AND RESULTS ASCs were prepared from mouse epididymal fat pads and induced to differentiate into endothelial cells (ECs) by vascular endothelial growth factor. Cyclooxygenase-2 expression, thromboxane production, and TP expression were upregulated in ASCs on vascular endothelial growth factor treatment. Genetic deletion or pharmacological inhibition of TP in mouse or human ASCs accelerated EC differentiation and increased tube formation in vitro, enhanced angiogenesis in in vivo Matrigel plugs and ischemic mouse hindlimbs. TP deficiency resulted in a significant cellular accumulation of β-catenin by suppression of calpain-mediated degradation in ASCs. Knockdown of β-catenin completely abrogated the enhanced EC differentiation of TP-deficient ASCs, whereas inhibition of calpain reversed the suppressed angiogenic capacity of TP re-expressed ASCs. Moreover, TP was coupled with Gαq to induce calpain-mediated suppression of β-catenin signaling through calcium influx in ASCs. CONCLUSION Thromboxane restrained EC differentiation of ASCs through TP-mediated repression of the calpain-dependent β-catenin signaling pathway. These results indicate that TP inhibition could be a promising strategy for therapy utilizing ASCs in the treatment of ischemic diseases.

E-Prostanoid 3 Receptor Mediates Sprouting Angiogenesis Through Suppression of the Protein Kinase A/β-Catenin/Notch Pathway

Objective— Angiogenesis is a hallmark of embryonic development and various ischemic and inflammatory diseases. Prostaglandin E2 receptor subtype 3 (EP3) plays an important role in pathophysiologic angiogenesis; however, the precise mechanisms remain unknown. Here, we investigated the role of EP3 in zebra fish embryo and mouse retina angiogenesis and evaluated the underlying mechanisms. Approach and Results— The EP3 receptor was highly expressed in the vasculature in both zebra fish embryos and murine fetal retinas. Pharmacological inhibition or genetic deletion of EP3 significantly reduced vasculature formation in zebra fish embryos and mouse retinas. Further characterization revealed reduced filopodia extension of tip cells in embryonic retinas in EP3-deficient mice. EP3 deletion activated Notch activity by upregulation of delta-like ligand 4 expression in endothelial cells (ECs). Inhibition of Notch signaling rescued the angiogenic defects in EP3-deficient mouse retinas. Moreover, EP3 deficiency led to a significant increase in &bgr;-catenin phosphorylation at Ser675 and nuclear accumulation of &bgr;-catenin in ECs. Knockdown or inhibition of &bgr;-catenin restored the impaired sprouting angiogenesis resulting from EP3 deficiency in ECs. The EP3 receptor depressed protein kinase A activity in ECs by coupling to G&agr;i. Inhibition of protein kinase A activity significantly reduced Ser675 phosphorylation and nuclear translocation of &bgr;-catenin, abolished the increased delta-like ligand 4 expression, and subsequently restored the impaired angiogenic capacity of EP3-deficient ECs both in vitro and in vivo. Conclusions— Activation of the EP3 receptor facilitates sprouting angiogenesis through protein kinase A–dependent Notch signaling, suggesting that EP3 and its downstream pathways maybe potential therapeutic targets in the treatment of ischemic diseases.

Rare SNP rs12731181 in the miR-590-3p Target Site of the Prostaglandin F2α Receptor Gene Confers Risk for Essential Hypertension in the Han Chinese Population

Objective— To investigate whether rs12731181 (A→G) interrupted miR-590-3p–mediated suppression of the prostaglandin F2&agr; receptor (FP) and whether it is associated with essential hypertension in the Chinese population. Approach and Results— We found that miR-590-3p regulates human FP gene expression by binding to its 3′-untranslated region. rs12731181 (A→G) altered the binding affinity between miR-590-3p and its FP 3′-untranslated region target, thus reducing the suppression of FP expression, which, in turn, enhanced FP receptor–mediated contractility of vascular smooth muscle cells. Overexpression of FP augmented vascular tone and elevated blood pressure in mice. An association study was performed to analyze the relationship between the FP gene and essential hypertension in the Han Chinese population. The results indicated that the rs12731181 G allele was associated with susceptibility to essential hypertension. Carriers of the AG genotype exhibited significantly higher blood pressure than those of the AA genotype. FP gene expression was significantly higher in human peripheral leukocytes from individuals with the AG genotype than that in leukocytes from individuals with the AA genotype. Conclusions— rs12731181 in the seed region of the miR-590-3p target site is associated with increased risk of essential hypertension and represents a new paradigm for FP involvement in blood pressure regulation.

COX-2-Derived PGE2 Promotes Injury-Induced Vascular Neointimal Hyperplasia Through the EP3 Receptor

Rationale: Vascular smooth muscle cell (VSMC) migration and proliferation are the hallmarks of restenosis pathogenesis after angioplasty. Cyclooxygenase (COX)-derived prostaglandin (PG) E2 is implicated in the vascular remodeling response to injury. However, its precise molecular role remains unknown. Objective: This study investigates the impact of COX-2–derived PGE2 on neointima formation after injury. Methods and Results: Vascular remodeling was induced by wire injury in femoral arteries of mice. Both neointima formation and the restenosis ratio were diminished in COX-2 knockout mice as compared with controls, whereas these parameters were enhanced in COX-1>COX-2 mice, in which COX-1 is governed by COX-2 regulatory elements. PG profile analysis revealed that the reduced PGE2 by COX-2 deficiency, but not PGI2, could be rescued by COX-1 replacement, indicating COX-2–derived PGE2 enhanced neointima formation. Through multiple approaches, the EP3 receptor was identified to mediate the VSMC migration response to various stimuli. Disruption of EP3 impaired VSMC polarity for directional migration by decreasing small GTPase activity and restricted vascular neointimal hyperplasia, whereas overexpression of EP3&agr; and EP3&bgr; aggravated neointima formation. Inhibition or deletion of EP3&agr;/&bgr;, a G&agr;i protein–coupled receptor, activated the cAMP/protein kinase A pathway and decreased activation of RhoA in VSMCs. PGE2 could stimulate phosphatidylinositol 3-kinase/Akt/glycogen synthase kinase3&bgr; signaling in VSMCs through G&bgr;&ggr; subunits on EP3&agr;/&bgr; activation. Ablation of EP3 suppressed phosphatidylinositol 3-kinase signaling and reduced GTPase activity in VSMCs and altered cell polarity and directional migration. Conclusions: COX-2–derived PGE2 facilitated the neointimal hyperplasia response to injury through EP3&agr;/&bgr;-mediated cAMP/protein kinase A and phosphatidylinositol 3-kinase pathways, indicating EP3 inhibition may be a promising therapeutic strategy for percutaneous transluminal coronary angioplasty.

Inhibition of CRTH2-mediated Th2 activation attenuates pulmonary hypertension in mice

Pulmonary arterial hypertension (PAH) is a life-threatening disease characterized by progressive pulmonary artery (PA) remodeling. T helper 2 cell (Th2) immune response is involved in PA remodeling during PAH progression. Here, we found that CRTH2 (chemoattractant receptor homologous molecule expressed on Th2 cell) expression was up-regulated in circulating CD3+CD4+ T cells in patients with idiopathic PAH and in rodent PAH models. CRTH2 disruption dramatically ameliorated PA remodeling and pulmonary hypertension in different PAH mouse models. CRTH2 deficiency suppressed Th2 activation, including IL-4 and IL-13 secretion. Both CRTH2+/+ bone marrow reconstitution and CRTH2+/+ CD4+ T cell adoptive transfer deteriorated hypoxia + ovalbumin–induced PAH in CRTH2−/− mice, which was reversed by dual neutralization of IL-4 and IL-13. CRTH2 inhibition alleviated established PAH in mice by repressing Th2 activity. In culture, CRTH2 activation in Th2 cells promoted pulmonary arterial smooth muscle cell proliferation through activation of STAT6. These results demonstrate the critical role of CRTH2-mediated Th2 response in PAH pathogenesis and highlight the CRTH2 receptor as a potential therapeutic target for PAH.

Cyclooxygenase-2–Derived Prostaglandin E2 Promotes Injury-Induced Vascular Neointimal Hyperplasia Through the E-prostanoid 3 ReceptorNovelty and Significance

Rationale: Vascular smooth muscle cell (VSMC) migration and proliferation are the hallmarks of restenosis pathogenesis after angioplasty. Cyclooxygenase (COX)-derived prostaglandin (PG) E2 is implicated in the vascular remodeling response to injury. However, its precise molecular role remains unknown. Objective: This study investigates the impact of COX-2–derived PGE2 on neointima formation after injury. Methods and Results: Vascular remodeling was induced by wire injury in femoral arteries of mice. Both neointima formation and the restenosis ratio were diminished in COX-2 knockout mice as compared with controls, whereas these parameters were enhanced in COX-1>COX-2 mice, in which COX-1 is governed by COX-2 regulatory elements. PG profile analysis revealed that the reduced PGE2 by COX-2 deficiency, but not PGI2, could be rescued by COX-1 replacement, indicating COX-2–derived PGE2 enhanced neointima formation. Through multiple approaches, the EP3 receptor was identified to mediate the VSMC migration response to various stimuli. Disruption of EP3 impaired VSMC polarity for directional migration by decreasing small GTPase activity and restricted vascular neointimal hyperplasia, whereas overexpression of EP3&agr; and EP3&bgr; aggravated neointima formation. Inhibition or deletion of EP3&agr;/&bgr;, a G&agr;i protein–coupled receptor, activated the cAMP/protein kinase A pathway and decreased activation of RhoA in VSMCs. PGE2 could stimulate phosphatidylinositol 3-kinase/Akt/glycogen synthase kinase3&bgr; signaling in VSMCs through G&bgr;&ggr; subunits on EP3&agr;/&bgr; activation. Ablation of EP3 suppressed phosphatidylinositol 3-kinase signaling and reduced GTPase activity in VSMCs and altered cell polarity and directional migration. Conclusions: COX-2–derived PGE2 facilitated the neointimal hyperplasia response to injury through EP3&agr;/&bgr;-mediated cAMP/protein kinase A and phosphatidylinositol 3-kinase pathways, indicating EP3 inhibition may be a promising therapeutic strategy for percutaneous transluminal coronary angioplasty.