Juan Tang

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.

Enhancing the precision of genetic lineage tracing using dual recombinases

The Cre–loxP recombination system is the most widely used technology for in vivo tracing of stem or progenitor cell lineages. The precision of this genetic system largely depends on the specificity of Cre recombinase expression in targeted stem or progenitor cells. However, Cre expression in nontargeted cell types can complicate the interpretation of lineage-tracing studies and has caused controversy in many previous studies. Here we describe a new genetic lineage tracing system that incorporates the Dre–rox recombination system to enhance the precision of conventional Cre–loxP-mediated lineage tracing. The Dre–rox system permits rigorous control of Cre–loxP recombination in lineage tracing, effectively circumventing potential uncertainty of the cell-type specificity of Cre expression. Using this new system we investigated two topics of recent debates—the contribution of c-Kit+ cardiac stem cells to cardiomyocytes in the heart and the contribution of Sox9+ hepatic progenitor cells to hepatocytes in the liver. By overcoming the technical hurdle of nonspecific Cre–loxP-mediated recombination, this new technology provides more precise analysis of cell lineage and fate decisions and facilitates the in vivo study of stem and progenitor cell plasticity in disease and regeneration.

I prostanoid receptor-mediated inflammatory pathway promotes hepatic gluconeogenesis through activation of PKA and inhibition of AKT

Nonsteroidal anti-inflammatory drugs (NSAIDs), including acetylsalicylic acid (ASA), improve glucose metabolism in diabetic subjects, although the underlying mechanisms remain unclear. In this study, we observed dysregulated expression of cyclooxygenase-2, prostacyclin biosynthesis, and the I prostanoid receptor (IP) in the liver’s response to diabetic stresses. High doses of ASA reduced hepatic prostaglandin generation and suppressed hepatic gluconeogenesis in mice during fasting, and the hypoglycemic effect of ASA could be restored by IP agonist treatment. IP deficiency inhibited starvation-induced hepatic gluconeogenesis, thus inhibiting the progression of diabetes, whereas hepatic overexpression of IP increased gluconeogenesis. IP deletion depressed cAMP-dependent CREB phosphorylation and elevated AKT phosphorylation by suppressing PI3K-γ/PKC-ζ–mediated TRB3 expression, which subsequently downregulated the gluconeogenic genes for glucose-6-phosphatase (G6Pase) and phosphoenol pyruvate carboxykinase 1 in hepatocytes. We therefore conclude that suppression of IP modulation of hepatic gluconeogenesis through the PKA/CREB and PI3K-γ/PKC-ζ/TRB3/AKT pathways contributes to the effects of NSAIDs in diabetes.

Fibroblasts in an endocardial fibroelastosis disease model mainly originate from mesenchymal derivatives of epicardium

Endocardial fibroelastosis (EFE) refers to the thickening of the ventricular endocardium as a result of de novo deposition of subendocardial fibrous tissue layers during neonatal heart development. The origin of EFE fibroblasts is proposed to be postnatal endocardial cells that undergo an aberrant endothelial-to-mesenchymal transition (EndMT). Genetic lineage tracing of endocardial cells with the inducible endocardial Cre line Npr3-CreER and the endothelial cell tracing line Cdh5-CreER on an EFE-like model did not reveal any contribution of neonatal endocardial cells to fibroblasts in the EFE-like tissues. Instead, lineage tracing of embryonic epicardium by Wt1-CreER suggested that epicardium-derived mesenchymal cells (MCs) served as the major source of EFE fibroblasts. By labeling MCs using Sox9-CreER, we confirmed that MCs of the embryonic heart expand and contribute to the majority of neonatal EFE fibroblasts. During this pathological process, TGFβ signaling, the key mediator of fibroblasts activation, was highly upregulated in the EFE-like tissues. Targeting TGFβ signaling by administration of its antagonist bone morphogenetic protein 7 effectively reduced fibroblast accumulation and tissue fibrosis in the EFE-like model. Our study provides genetic evidence that excessive fibroblasts in the EFE-like tissues mainly originate from the epicardium-derived MCs through epicardial to mesenchymal transition (EpiMT). These EpiMT-derived fibroblasts within the EFE-like tissues could serve as a potential therapeutic target.

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.

Prostaglandin E2 promotes hepatic bile acid synthesis by an E prostanoid receptor 3‐mediated hepatocyte nuclear receptor 4α/cholesterol 7α‐hydroxylase pathway in mice

Prostaglandin E2 (PGE2) is an important lipid mediator of inflammation. However, whether and how PGE2 regulates hepatic cholesterol metabolism remains unknown. We found that expression of the PGE2 receptor, E prostanoid receptor 3 (EP3) expression is remarkably increased in hepatocytes in response to hyperlipidemic stress. Hepatocyte‐specific deletion of EP3 receptor (EP3hep–/–) results in hypercholesterolemia and augments diet‐induced atherosclerosis in low‐density lipoprotein receptor knockout (Ldlr–/–) mice. Cholesterol 7α‐hydroxylase (CYP7A1) is down‐regulated in livers of EP3hep–/–Ldlr−/− mice, leading to suppressed hepatic bile acid (BA) biosynthesis. Mechanistically, hepatic‐EP3 deficiency suppresses CYP7A1 expression by elevating protein kinase A (PKA)‐dependent Ser143 phosphorylation of hepatocyte nuclear receptor 4α (HNF4α). Disruption of the PKA‐HNF4α interaction and BA sequestration rescue impaired BA excretion and ameliorated atherosclerosis in EP3hep–/–Ldlr−/− mice. Conclusion: Our results demonstrated an unexpected role of proinflammatory mediator PGE2 in improving hepatic cholesterol metabolism through activation of the EP3‐mediated PKA/HNF4α/CYP7A1 pathway, indicating that inhibition of this pathway may be a novel therapeutic strategy for dyslipidemia and atherosclerosis. (Hepatology 2017;65:999‐1014)

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.

Genetic Lineage Tracing of Non-Myocyte Population by Dual Recombinases

Background: Whether the adult mammalian heart harbors cardiac stem cells for regeneration of cardiomyocytes is an important yet contentious topic in the field of cardiovascular regeneration. The putative myocyte stem cell populations recognized without specific cell markers, such as the cardiosphere-derived cells, or with markers such as Sca1+, Bmi1+, Isl1+, or Abcg2+ cardiac stem cells have been reported. Moreover, it remains unclear whether putative cardiac stem cells with unknown or unidentified markers exist and give rise to de novo cardiomyocytes in the adult heart. Methods: To address this question without relying on a particular stem cell marker, we developed a new genetic lineage tracing system to label all nonmyocyte populations that contain putative cardiac stem cells. Using dual lineage tracing system, we assessed whether nonmyocytes generated any new myocytes during embryonic development, during adult homeostasis, and after myocardial infarction. Skeletal muscle was also examined after injury for internal control of new myocyte generation from nonmyocytes. Results: By this stem cell marker–free and dual recombinases–mediated cell tracking approach, our fate mapping data show that new myocytes arise from nonmyocytes in the embryonic heart, but not in the adult heart during homeostasis or after myocardial infarction. As positive control, our lineage tracing system detected new myocytes derived from nonmyocytes in the skeletal muscle after injury. Conclusions: This study provides in vivo genetic evidence for nonmyocyte to myocyte conversion in embryonic but not adult heart, arguing again the myogenic potential of putative stem cell populations for cardiac regeneration in the adult stage. This study also provides a new genetic strategy to identify endogenous stem cells, if any, in other organ systems for tissue repair and regeneration.