Perivascular adipose tissue-derived extracellular vesicle miR-221-3p mediates vascular remodeling.

Adipose tissue-secreted extracellular vesicles (EVs) containing microRNAs (miRNAs) convey intercellular message signaling. The biogenesis of EV-miRNAs from perivascular adipose tissue (PVAT) and their roles in intercellular communication in response to obesity-associated inflammation have not yet been fully explored. By feeding mice a high-fat diet for 16 wk, we established obesity-associated, chronic low-grade inflammation in PVAT, characterized as hypertrophy of perivascular adipocytes, decreased adipogenesis, and proinflammatory macrophage infiltration. We show that PVAT-derived EVs and their encapsulated miRNAs can be taken up into vascular smooth muscle cells (VSMCs) in vivo and in vitro. miR-221-3p is one of the highly enriched miRNAs in obese PVAT and PVAT-derived EVs. Transfer and direct overexpression of miR-221-3p dramatically enhances VSMC proliferation and migration. Peroxisome proliferator-activated receptor γ coactivator 1α is identified as a miR-221-3p target in VSMC phenotypic modulation. Obese mice secrete abundant miRNA-containing EVs, evoking inflammatory responses in PVAT and vascular phenotypic switching in abdominal aorta of lean mice. Local delivery of miR-221-3p mimic in femoral artery causes vascular dysfunction by suppressing the contractile genes in the arterial wall. Our findings provide an EV-miR-221-3p-mediated mechanism by which PVAT triggers an early-stage vascular remodeling in the context of obesity-associated inflammation.-Li, X., Ballantyne, L. L., Yu, Y., Funk, C. D. Perivascular adipose tissue-derived extracellular vesicle miR-221-3p mediates vascular remodeling.

Differential compensation of two cyclooxygenases in renal homeostasis is independent of prostaglandin-synthetic capacity under basal conditions.

The distinct functions of each cyclooxygenase (COX) isoform in renal homeostasis have been the subject of intense investigation for many years. We took the novel approach of using 3 characterized mouse lines, where the prostaglandin (PG)-endoperoxide synthase genes 1 and 2 ( Ptgs1 and Ptgs2) substitute for one another to delineate distinct roles and the potential for COX isoform substitution. Flipped Ptgs genes generate a reversed COX-expression pattern in the kidney, where the knockin COX-2 is highly expressed. Normal nephrogenesis was sustained in all 3 strains at the postnatal stage d 8 (P8). Knockin COX-1 can temporally restore renal function and delay but not prevent renal pathology consequent to COX-2 deletion. Loss of COX-2 in adult COX-1 > COX-2 mice results in severe nephropathy, which leads to impaired renal function. These defects are partially rescued by the knockin COX-2 in Reversa mice, whereas COX-2 can compensate for the loss of COX-1 in COX-2 > COX-1 mice. Intriguingly, the highly expressed knockin COX-2 enzyme barely makes any PGs or thromboxane in neonatal P8 or adult mice, demonstrating that prostanoid biosynthesis requires native COX-1 and cannot be rescued by the knockin COX-2. In summary, the 2 COX isoforms can preferentially compensate for some renal functions, which appears to be independent of the PG-synthetic capacity.-Li, X., Mazaleuskaya, L. L., Ballantyne, L. L., Meng, H., FitzGerald, G. A., Funk, C. D. Differential compensation of two cyclooxygenases in renal homeostasis is independent of prostaglandin-synthetic capacity under basal conditions.

Resolvin E1 attenuates injury-induced vascular neointimal formation by inhibition of inflammatory responses and vascular smooth muscle cell migration.

Mechanical insults, such as stent implantation, can induce endothelial injury, vascular inflammation, and ultimately lead to vascular neointimal hyperplasia. Resolvin E1 (RvE1), derived from the ω3 fatty acid eicosapentaenoic acid, can facilitate the resolution of inflammation in many settings. We therefore aimed to determine if there was a role for RvE1 in preventing neointimal formation after arterial injury and to understand the underlying mechanisms. Vascular inflammation and neointimal hyperplasia were induced by wire injury in the femoral arteries of mice. Administration of exogenous RvE1 and endogenously generated RvE1 via dietary supplementation with eicosapentaenoic acid and aspirin markedly reduced vascular neointima formation in this model. Mechanistically, RvE1 was found to inhibit vascular neutrophil infiltration, promote macrophage polarization toward an M2-like phenotype, suppress T-cell trafficking by reducing RANTES secretion from vascular smooth muscle cells, and inhibit vascular smooth muscle cell migration. In summary, RvE1 demonstrated a protective role against vascular inflammation and remodeling in response to mechanical injury, suggesting that it may serve as an adjuvant therapeutic agent for percutaneous coronary interventions, such as stent implantation.-Liu, G., Gong, Y., Zhang, R., Piao, L., Li, X., Liu, Q., Yan, S., Shen, Y., Guo, S., Zhu, M., Yin, H., Funk, C. D., Zhang, J., Yu, Y. Resolvin E1 attenuates injury-induced vascular neointimal formation by inhibition of inflammatory responses and vascular smooth muscle cell migration.

Genomic and lipidomic analyses differentiate the compensatory roles of two COX isoforms during systemic inflammation in mice.

Both cyclooxygenase (COX)-1 and COX-2, encoded by Ptgs1 and Ptgs2, function coordinately during inflammation. But the relative contributions and compensations of COX-1 and COX-2 to inflammatory responses remain unanswered. We used three engineered mouse lines where the Ptgs1 and Ptgs2 genes substitute for one another to discriminate the distinct roles and interchangeability of COX isoforms during systemic inflammation. In macrophages, kidneys, and lungs, "flipped" Ptgs genes generate a "reversed" COX expression pattern, where the knock-in COX-2 is expressed constitutively and the knock-in COX-1 is lipopolysaccharide inducible. A panel of eicosanoids detected in serum and kidney demonstrates that prostaglandin (PG) biosynthesis requires native COX-1 and cannot be rescued by the knock-in COX-2. Our data further reveal preferential compensation of COX isoforms for prostanoid production in macrophages and throughout the body, as reflected by urinary PG metabolites. NanoString analysis indicates that inflammatory networks can be maintained by isoform substitution in inflamed macrophages. However, COX-1>COX-2 macrophages show reduced activation of inflammatory signaling pathways, indicating that COX-1 may be replaced by COX-2 within this complex milieu, but not vice versa. Collectively, each COX isoform plays a distinct role subject to subcellular environment and tissue/cell-specific conditions, leading to subtle compensatory differences during systemic inflammation.

Flipping the cyclooxygenase (Ptgs) genes reveals isoform-specific compensatory functions.

Two prostaglandin (PG) H synthases encoded by Ptgs genes, colloquially known as cyclooxygenase (COX)-1 and COX-2, catalyze the formation of PG endoperoxide H2, the precursor of the major prostanoids. To address the functional interchangeability of these two isoforms and their distinct roles, we have generated COX-2>COX-1 mice whereby Ptgs2 is knocked in to the Ptgs1 locus. We then "flipped" Ptgs genes to successfully create the Reversa mouse strain, where knock-in COX-2 is expressed constitutively and knock-in COX-1 is lipopolysaccharide (LPS) inducible. In macrophages, flipping the two Ptgs genes has no obvious impact on COX protein subcellular localization. COX-1 was shown to compensate for PG synthesis at high concentrations of substrate, whereas elevated LPS-induced PG production was only observed for cells expressing endogenous COX-2. Differential tissue-specific patterns of expression of the knock-in proteins were evident. Thus, platelets from COX-2>COX-1 and Reversa mice failed to express knock-in COX-2 and, therefore, thromboxane (Tx) production in vitro and urinary Tx metabolite formation in COX-2>COX-1 and Reversa mice in vivo were substantially decreased relative to WT and COX-1>COX-2 mice. Manipulation of COXs revealed isoform-specific compensatory functions and variable degrees of interchangeability for PG biosynthesis in cells/tissues.

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 ß-catenin by suppression of calpain-mediated degradation in ASCs. Knockdown of ß-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 ß-catenin signaling through calcium influx in ASCs. CONCLUSION: Thromboxane restrained EC differentiation of ASCs through TP-mediated repression of the calpain-dependent ß-catenin signaling pathway. These results indicate that TP inhibition could be a promising strategy for therapy utilizing ASCs in the treatment of ischemic diseases.

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 EP3α and EP3ß aggravated neointima formation. Inhibition or deletion of EP3α/ß, a Gαi 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 kinase3ß signaling in VSMCs through Gßγ subunits on EP3α/ß 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 EP3α/ß-mediated cAMP/protein kinase A and phosphatidylinositol 3-kinase pathways, indicating EP3 inhibition may be a promising therapeutic strategy for percutaneous transluminal coronary angioplasty.

Multiple-site activation of the cysteinyl leukotriene receptor 2 is required for exacerbation of ischemia/reperfusion injury.

OBJECTIVE: Transgenic overexpression of the human cysteinyl leukotriene receptor 2 (CysLT2R) in murine endothelium exacerbates vascular permeability and ischemia/reperfusion injury. Here, we explore the underlying mechanisms of CysLT2R activation-mediated inflammation and delineate the relative contributions of endogenous murine CysLT2R and the transgene-derived receptor. APPROACH AND RESULTS: We created a novel mouse with only endothelial-expressed CysLT2R (endothelium-targeted overexpression mice [EC]/CysLT2R-knockout mice [KO]) by crossing EC with KO to dissect the role of endothelial CysLT2R in tissue injury. Surprisingly, we discovered that damage in EC/KO mice was not elevated (24% versus 47% EC) after ischemia/reperfusion. We examined vascular permeability and leukocyte recruitment/rolling responses in the cremaster vasculature after cysteinyl leukotriene (cysLT) stimulation. Mice possessing transgenic endothelial CysLT2R overexpression, whether EC or EC/KO, when stimulated with cysLTs, exhibited vascular hyperpermeability, declining leukocyte flux, and a transient increase in slow-rolling leukocyte fraction. Mice lacking endogenous CysLT2R (both KO [20 ± 3 cells/min] EC/KO [24 ± 3]) showed lower-rolling leukocyte flux versus wild-type (38 ± 6) and EC (35 ± 6) mice under unstimulated conditions. EC/KO mice differed from EC counterparts in that vascular hyperpermeability was not present in the absence of exogenous cysLTs. CONCLUSIONS: These results indicate that endothelial and nonendothelial CysLT2R niches have separate roles in mediating inflammatory responses. Endothelial receptor activation results in increased vascular permeability and leukocyte slow-rolling, facilitating leukocyte transmigration. Nonendothelial receptors, likely located on resident/circulating leukocytes, facilitate endothelial receptor activation and leukocyte transit. Activation of both receptor populations is required for injury exacerbation.

Myeloid-derived suppressor cell function is diminished in aspirin-triggered allergic airway hyperresponsiveness in mice.

BACKGROUND: Myeloid-derived suppressor cells (MDSCs) have recently been implicated in the pathogenesis of asthma, but their regulation in patients with aspirin-intolerant asthma (AIA) remains unclear. OBJECTIVE: We sought to characterize MDSC accumulation and pathogenic functions in allergic airway inflammation mediated by COX-1 deficiency or aspirin treatment in mice. METHODS: Allergic airway inflammation was induced in mice by means of ovalbumin challenge. The distribution and function of MDSCs in mice were analyzed by using flow cytometry and pharmacologic/gene manipulation approaches. RESULTS: CD11b(+)Gr1(high)Ly6G(+)Ly6C(int) MDSCs (polymorphonuclear MDSCs [PMN-MDSCs]) recruited to the lungs are negatively correlated with airway inflammation in allergen-challenged mice. Aspirin-treated and COX-1 knockout (KO) mice showed significantly lower accumulation of PMN-MDSCs in the inflamed lung and immune organs accompanied by increased TH2 airway responses. The TH2-suppressive function of PMN-MDSCs was notably impaired by COX-1 deletion or inhibition, predominantly through downregulation of arginase-1. COX-1-derived prostaglandin E2 promoted PMN-MDSC generation in bone marrow through E prostanoid 2 and 4 receptors (EP2 and EP4), whereas the impaired arginase-1 expression in PMN-MDSCs in COX-1 KO mice was mediated by dysregulation of the prostaglandin E2/EP4/cyclic AMP/protein kinase A pathway. EP4 agonist administration alleviated allergy-induced airway hyperresponsiveness in COX-1 KO mice. Moreover, the immunosuppressive function of PMN-MDSCs from patients with AIA was dramatically decreased compared with that from patients with aspirin-tolerant asthma. CONCLUSION: The immunosuppressive activity of PMN-MDSCs was diminished in both allergen-challenged COX-1 KO mice and patients with AIA, probably through an EP4-mediated signaling pathway, indicating that activation of PMN-MDSCs might be a promising therapeutic strategy for asthma, particularly AIA.

A mutation interfering with 5-lipoxygenase domain interaction leads to increased enzyme activity.

5-Lipoxygenase (5-LOX) catalyzes two steps in conversion of arachidonic acid to proinflammatory leukotrienes. Lipoxygenases, including human 5-LOX, consist of an N-terminal C2-like ß-sandwich and a catalytic domain. We expressed the 5-LOX domains separately, these were found to interact in the yeast two-hybrid system. The 5-LOX structure suggested association between Arg(101) in the ß-sandwich and Asp(166) in the catalytic domain, due to electrostatic interaction as well as hydrogen bonds. Indeed, mutagenic replacements of these residues led to loss of two-hybrid interaction. Interestingly, when Arg(101) was mutated to Asp in intact 5-LOX, enzyme activity was increased. Thus, higher initial velocity of the reaction (vinit) and increased final amount of products were monitored for 5-LOX-R101D, at several different assay conditions. In the 5-LOX crystal structure, helix α2 and adjacent loops (including Asp(166)) of the 5-LOX catalytic domain has been proposed to form a flexible lid controlling access to the active site, and lid movement would be determined by bonding of lid residues to the C2-like ß-sandwich. The more efficient catalysis following disruption of the R101-D166 ionic association supports the concept of such a flexible lid in human 5-LOX.

Cyclooxygenase-2 induction in macrophages is modulated by docosahexaenoic acid via interactions with free fatty acid receptor 4 (FFA4).

Cyclooxygenase-2 (COX-2)-derived prostaglandins are implicated in numerous inflammatory disorders. The purpose of these studies was to examine previously unexplored interactions between COX-2 induction and docosahexaenoic acid (DHA) via the free fatty acid receptor 4 (FFA4) signaling pathway in murine RAW 264.7 cells and peritoneal macrophages challenged with lipopolysaccharide (LPS). DHA dose (IC50=18 µM)- and time-dependently reduced COX-2 expression, without affecting COX-1. DHA (25 µM for 24 h) decreased LPS-induced prostaglandin E2 (PGE2) synthesis by 81%, primarily through reducing COX-2 (60%), as well as down-regulating microsomal prostaglandin E synthase-1 (46%), but independently of peroxisome proliferator-activated receptors. FFA4 knockdown abrogated DHA effects on COX-2 induction, PGE2 production, and interleukin 6 (IL-6) gene expression. In the presence of inhibitors of eicosanoid metabolism via COX-2, 12/15-lipoxygenase and CYP450s (rofecoxib (1 µM), PD146176 (2 µM), or MS-PPOH (20 µM)), DHA was still effective in attenuating COX-2 induction. Moreover, Toll-like receptor 4 signaling via Akt/JNK phosphorylation and p65 nuclear translocation was repressed by DHA-activated FFA4 coupling with ß-arrestin 2, which was reversed by FFA4 knockdown. These data support DHA modulation of COX-2 expression and activity, in part, via FFA4, which provides a new mechanistic explanation for some of the anti-inflammatory effects of DHA.

Prostaglandin receptor EP4 in abdominal aortic aneurysms.

Abdominal aortic aneurysm (AAA) pathogenesis is distinguished by vessel wall inflammation. Cyclooxygenase (COX)-2 and microsomal prostaglandin E synthase-1, key components of the most well-characterized inflammatory prostaglandin pathway, contribute to AAA development in the 28-day angiotensin II infusion model in mice. In this study, we used this model to examine the role of the prostaglandin E receptor subtype 4 (EP4) and genetic knockdown of COX-2 expression (70% to 90%) in AAA pathogenesis. The administration of the prostaglandin receptor EP4 antagonist AE3-208 (10 mg/kg per day) to apolipoprotein E (apoE)-deficient mice led to active drug plasma concentrations and reduced AAA incidence and severity compared with control apoE-deficient mice (P < 0.01), whereas COX-2 genetic knockdown/apoE-deficient mice displayed only a minor, nonsignificant decrease in incidence of AAA. EP4 receptor protein was present in human and mouse AAA, as observed by using Western blot analysis. Aortas from AE3-208-treated mice displayed evidence of a reduced inflammatory phenotype compared with controls. Atherosclerotic lesion size at the aortic root was similar between all groups. In conclusion, the prostaglandin E(2)-EP4 signaling pathway plays a role in the AAA inflammatory process. Blocking the EP4 receptor pharmacologically reduces both the incidence and severity of AAA in the angiotensin II mouse model, potentially via attenuation of cytokine/chemokine synthesis and the reduction of matrix metalloproteinase activities.

The cysteinyl leukotriene 2 receptor mediates retinal edema and pathological neovascularization in a murine model of oxygen-induced retinopathy.

Leukotrienes have been implicated in the pathogenesis of degenerative diabetic retinopathy, with research focusing primarily on leukotriene B(4), with little attention devoted to the cysteinyl leukotrienes (cysLTs), which act through cysLT receptors (CysLT(1)R and CysLT(2)R). We demonstrate here the presence of CysLT(2)R in pericytes and endothelial cells of superficial retinal vasculature using an indirect assay by assessment of ß-galactosidase expression in CysLT(2)R-knockout (KO) mice. Retinal damage was induced in KO and wild-type (WT) mice using an established oxygen-induced retinopathy (OIR) model. CysLT(2)R expression following OIR was intensely up-regulated compared to sham-treated controls. Staining with Griffonia simplicifolia lectin revealed enhanced tissue damage (as assessed by vasoobliteration/vasoproliferation) in KO mice compared to WT controls, yet the opposite was true with respect to retinal edema. However, vascular endothelial growth factor receptor 1 (VEGFR1) transcripts were increased by OIR similarly with respect to genotype. Intravitreal application of exogenous cysLTs elicited greater vasculature leakage (assessed ex vivo) in eyes from WT mice compared to KO mice. While mRNA encoding enzymes for various components of the leukotriene cascade were detected in sham- and OIR-treated retinas, only prostaglandins and hydroxyeicosatetraenoic acids, but not leukotrienes, were detected in A23187-treated retina preparations. Together, these results implicate the CysLT(2)R in the progression of ischemic retinopathy.

Genetic model of selective COX2 inhibition reveals novel heterodimer signaling.

Selective inhibitors of cyclooxygenase-2 (COX2) have attracted widespread media attention because of evidence of an elevated risk of cardiovascular complications in placebo-controlled trials, resulting in the market withdrawal of some members of this class. These drugs block the cyclooxygenase activity of prostaglandin H synthase-2 (PGHS2), but do not affect the associated peroxidase function. They were developed with the rationale of conserving the anti-inflammatory and analgesic actions of traditional nonsteroidal anti-inflammatory drugs (tNSAIDs) while sparing the ability of PGHS1-derived prostaglandins to afford gastric cytoprotection. PGHS1 and PGHS2 coexist in the vasculature and in macrophages, and are upregulated together in inflammatory tissues such as rheumatoid synovia and atherosclerotic plaque. They are each believed to function as homodimers. Here, we developed a new genetic mouse model of selective COX2 inhibition using a gene-targeted point mutation, resulting in a Y385F substitution. Structural modeling and biochemical assays showed the ability of PGHS1 and PGHS2 to heterodimerize and form prostaglandins. The heterodimerization of PGHS1-PGHS2 may explain how the ductus arteriosus closes normally at birth in mice expressing PGHS2 Y385F, but not in PGHS2-null mice.

Characterization of the cysteinyl leukotriene 2 receptor in novel expression sites of the gastrointestinal tract.

Cysteinyl leukotrienes (cysLTs: LTC4, LTD4, and LTE4) are pro-inflammatory lipid molecules synthesized from arachidonic acid. They exert their actions on at least two cysLT receptors (CysLT1R and CysLT2R). Endothelial expression and activation of these receptors is linked to vasoactive responses and to the promotion of vascular permeability. Here we track the expression pattern of CysLT2R in a loss-of-function murine model (CysLT2R-LacZ) to neurons of the myenteric and submucosal plexus in the small intestine, colonic myenteric plexus, dorsal root ganglia, and nodose ganglion. Cysteinyl leukotriene (LTC4/D4) stimulation of colonic submucosal venules elicited a greater permeability response in wild-type mice. In a dextran sulfate sodium-induced colon inflammation model, the disease activity index and colonic edema (measured by wet:dry weights and submucosal thickness) were significantly reduced in knockout (KO) mice compared to controls. Tumor necrosis factor-α levels in colon tissue were significantly lower in KO mice; however, myeloperoxidase activity was similar in both the KO and wild-type groups. Finally, patch-clamp recordings of basal neuronal activity of colonic-projecting nociceptive neurons from dorsal root ganglia (T9-13) revealed significantly higher excitability in KO neurons compared to wild type. These results suggest that a lack of neuronal expression of CysLT2R in the murine colonic myenteric plexus attenuates colitis disease progression via a reduction in inflammation-associated tissue edema and increases neuronal sensitivity to nociceptive stimuli.

Cyclooxygenase-2-dependent prostacyclin formation and blood pressure homeostasis: targeted exchange of cyclooxygenase isoforms in mice.

RATIONALE: Cyclooxygenase (COX)-derived prostanoids (PGs) are involved in blood pressure homeostasis. Both traditional nonsteroidal antiinflammatory drugs (NSAIDs) that inhibit COX-1 and COX-2 and NSAIDs designed to be selective for inhibition of COX-2 cause sodium retention and elevate blood pressure. OBJECTIVE: To elucidate the role of COX-2 in blood pressure homeostasis using COX-1>COX-2 mice, in which the COX-1 expression is controlled by COX-2 regulatory elements. METHODS AND RESULTS: COX-1>COX-2 mice developed systolic hypertension relative to wild types (WTs) on a high-salt diet (HSD); this was attenuated by a PGI(2) receptor agonist. HSD increased expression of COX-2 in WT mice and of COX-1 in COX-1>COX-2 mice in the inner renal medulla. The HSD augmented in all strains urinary prostanoid metabolite excretion, with the exception of the major PGI(2) metabolite that was suppressed on regular chow and unaltered by the HSD in both mutants. Furthermore, inner renal medullary expression of the receptor for PGI(2), but not for other prostanoids, was depressed by HSD in WT and even more so in both mutant strains. Increasing osmolarity augmented expression of COX-2 in WT renal medullary interstitial cells and again the increase in formation of PGI(2) observed in WTs was suppressed in cells derived from both mutants. Intramedullary infusion of the PGI(2) receptor agonist increased urine volume and sodium excretion in mice. CONCLUSIONS: These studies suggest that dysregulated expression of the COX-2 dependent, PGI(2) biosynthesis/response pathway in the renal inner renal medulla undermines the homeostatic response to a HSD. Inhibition of this pathway may contribute directly to the hypertensive response to NSAIDs.

Targeted exchange of an expression cassette encoding cyclooxygenase-2 at the Ptgs1 locus.

Defining the multi-faceted roles of prostaglandins has been facilitated by studying mice with manipulated expression of the two enzymes encoding cyclooxygenase (COX) via gene targeting, with either knocked down expression of COX-1 or COX-2, a knocked-in COX-2 active site mutation and exchange of COX isoforms by insertion of a cassette encoding COX-1 into the COX-2 (Ptgs2) gene to create COX-1>COX-2 mice. Here, we sought to extend these studies by creating a new induced mutant strain with manipulated COX expression. We carried out gene targeting at the Ptgs1 locus to knock-in an expression cassette encoding COX-2 under Ptgs1 regulatory elements in a manner analogous used in COX-1>COX-2 targeting. While successful gene targeting at the Ptgs1 locus was achieved, the strategy did not yield a "basal" increase of COX-2 under Ptgs1 gene regulatory control in various cells and tissues from COX-2>COX-1 mice but rather resulted in a Ptgs1 null allele. Possible explanations as to why this strategy was unsuccessful include non-functionality of the hybrid signal peptide and aberrant transcript processing. Since a similar strategy had previously worked (i.e. COX-1 cDNA knocked-in to the Ptgs2 locus; COX-1>COX-2 mice) interpretations of our findings on murine COX biology and gene targeting are discussed.

Differential signaling of cysteinyl leukotrienes and a novel cysteinyl leukotriene receptor 2 (CysLT2) agonist, N-methyl-leukotriene C4, in calcium reporter and ß arrestin assays.

The cysteinyl leukotrienes (cysLTs) LTC4, LTD4, and LTE4 are lipid mediators with physiological and pathophysiological functions. They exert their effects through G protein-coupled receptors (GPCRs), most notably via CysLT1 and CysLT2 receptor. The roles of the CysLT2 receptor are beginning to emerge. Both LTC4 and LTD4 are potent agonists for the CysLT2 receptor; however, LTC4 is rapidly converted to LTD4, which is also the main endogenous ligand for the CysLT1 receptor. A selective and potent agonist at the CysLT2 receptor would facilitate studies to discern between receptor subtypes. We show here that N-methyl LTC4 (NMLTC4), a metabolically stable LTC4 mimetic, is a potent and selective CysLT2 receptor agonist. Two expression systems were used to evaluate the functional activity of NMLTC4 at human and/or mouse CysLT1 and CysLT2 receptors. Through the aequorin cell-based assay for calcium-coupled GPCRs, NMLTC4 was almost equipotent to LTC4 at CysLT2 receptors but was the least efficacious at CysLT2 receptors. In a ß-galactosidase-ß-arrestin complementation assay, the human (h) CysLT2 receptor can couple with ß-arrestin-2, and NMLTC4 is slightly more potent for eliciting ß-arrestin-2 binding compared with cysLTs. Furthermore, LTE4 is nearly inactive in this assay compared with its weak partial agonist activity in the aequorin system. In a vascular leakage assay, NMLTC4 is potent and active in mice overexpressing hCysLT2 receptor in endothelium, whereas the response is abrogated in CysLT2 receptor knockout mice. Therefore, NMLTC4 is a potent subtype selective agonist for the CysLT2 receptor in vitro and in vivo, and it will be useful to elucidate its biological roles.