Differential Impact In Vivo of Pf4-ΔCre-Mediated and Gp1ba-ΔCre-Mediated Depletion of Cyclooxygenase-1 in Platelets in Mice.

BACKGROUND: Low-dose aspirin is widely used for the secondary prevention of cardiovascular disease. The beneficial effects of low-dose aspirin are attributable to its inhibition of platelet Cox (cyclooxygenase)-1-derived thromboxane A2. Until recently, the use of the Pf4 (platelet factor 4) Cre has been the only genetic approach to generating megakaryocyte/platelet ablation of Cox-1 in mice. However, Pf4-ΔCre displays ectopic expression outside the megakaryocyte/platelet lineage, especially during inflammation. The use of the Gp1ba (glycoprotein 1bα) Cre promises a more specific, targeted approach. METHODS: To evaluate the role of Cox-1 in platelets, we crossed Pf4-ΔCre or Gp1ba-ΔCre mice with Cox-1flox/flox mice to generate platelet Cox-1-/- mice on normolipidemic and hyperlipidemic (Ldlr-/-; low-density lipoprotein receptor) backgrounds. RESULTS: Ex vivo platelet aggregation induced by arachidonic acid or adenosine diphosphate in platelet-rich plasma was inhibited to a similar extent in Pf4-ΔCre Cox-1-/-/Ldlr-/- and Gp1ba-ΔCre Cox-1-/-/Ldlr-/- mice. In a mouse model of tail injury, Pf4-ΔCre-mediated and Gp1ba-ΔCre-mediated deletions of Cox-1 were similarly efficient in suppressing platelet prostanoid biosynthesis. Experimental thrombogenesis and attendant blood loss were similar in both models. However, the impact on atherogenesis was divergent, being accelerated in the Pf4-ΔCre mice while restrained in the Gp1ba-ΔCres. In the former, accelerated atherogenesis was associated with greater suppression of PGI2 biosynthesis, a reduction in the lipopolysaccharide-evoked capacity to produce PGE2 (prostaglandin E) and PGD2 (prostanglandin D), activation of the inflammasome, elevated plasma levels of IL-1ß (interleukin), reduced plasma levels of HDL-C (high-density lipoprotein receptor-cholesterol), and a reduction in the capacity for reverse cholesterol transport. By contrast, in the latter, plasma HDL-C and α-tocopherol were elevated, and MIP-1α (macrophage inflammatory protein-1α) and MCP-1 (monocyte chemoattractant protein 1) were reduced. CONCLUSIONS: Both approaches to Cox-1 deletion similarly restrain thrombogenesis, but a differential impact on Cox-1-dependent prostanoid formation by the vasculature may contribute to an inflammatory phenotype and accelerated atherogenesis in Pf4-ΔCre mice.

PGF2α signaling drives fibrotic remodeling and fibroblast population dynamics in mice.

Idiopathic pulmonary fibrosis (IPF) is a chronic parenchymal lung disease characterized by repetitive alveolar cell injury, myofibroblast proliferation, and excessive extracellular matrix deposition for which unmet need persists for effective therapeutics. The bioactive eicosanoid, prostaglandin F2α, and its cognate receptor FPr (Ptgfr) are implicated as a TGF-ß1-independent signaling hub for IPF. To assess this, we leveraged our published murine PF model (IER-SftpcI73T) expressing a disease-associated missense mutation in the surfactant protein C (Sftpc) gene. Tamoxifen-treated IER-SftpcI73T mice developed an early multiphasic alveolitis and transition to spontaneous fibrotic remodeling by 28 days. IER-SftpcI73T mice crossed to a Ptgfr-null (FPr-/-) line showed attenuated weight loss and gene dosage-dependent rescue of mortality compared with FPr+/+ cohorts. IER-SftpcI73T/FPr-/- mice also showed reductions in multiple fibrotic endpoints for which administration of nintedanib was not additive. Single-cell RNA-Seq, pseudotime analysis, and in vitro assays demonstrated Ptgfr expression predominantly within adventitial fibroblasts, which were reprogrammed to an "inflammatory/transitional" cell state in a PGF2α /FPr-dependent manner. Collectively, the findings provide evidence for a role for PGF2α signaling in IPF, mechanistically identify a susceptible fibroblast subpopulation, and establish a benchmark effect size for disruption of this pathway in mitigating fibrotic lung remodeling.

Disruption of Prostaglandin F2α Receptor Signaling Attenuates Fibrotic Remodeling and Alters Fibroblast Population Dynamics in A Preclinical Murine Model of Idiopathic Pulmonary Fibrosis.

Idiopathic Pulmonary Fibrosis (IPF) is a chronic parenchymal lung disease characterized by repetitive alveolar cell injury, myofibroblast proliferation, and excessive extracellular matrix deposition for which unmet need persists for effective therapeutics. The bioactive eicosanoid, prostaglandin F2α, and its cognate receptor FPr (Ptfgr) are implicated as a TGFß1 independent signaling hub for IPF. To assess this, we leveraged our published murine PF model (IER - SftpcI73T) expressing a disease-associated missense mutation in the surfactant protein C (Sftpc) gene. Tamoxifen treated IER-SftpcI73T mice develop an early multiphasic alveolitis and transition to spontaneous fibrotic remodeling by 28 days. IER-SftpcI73T mice crossed to a Ptgfr null (FPr-/-) line showed attenuated weight loss and gene dosage dependent rescue of mortality compared to FPr+/+ cohorts. IER-SftpcI73T/FPr-/- mice also showed reductions in multiple fibrotic endpoints for which administration of nintedanib was not additive. Single cell RNA sequencing, pseudotime analysis, and in vitro assays demonstrated Ptgfr expression predominantly within adventitial fibroblasts which were reprogrammed to an "inflammatory/transitional" cell state in a PGF2α/FPr dependent manner. Collectively, the findings provide evidence for a role for PGF2α signaling in IPF, mechanistically identify a susceptible fibroblast subpopulation, and establish a benchmark effect size for disruption of this pathway in mitigating fibrotic lung remodeling.

Sexual dimorphism in the response to chronic circadian misalignment on a high-fat diet.

Longitudinal studies associate shiftwork with cardiometabolic disorders but do not establish causation or elucidate mechanisms of disease. We developed a mouse model based on shiftwork schedules to study circadian misalignment in both sexes. Behavioral and transcriptional rhythmicity were preserved in female mice despite exposure to misalignment. Females were protected from the cardiometabolic impact of circadian misalignment on a high-fat diet seen in males. The liver transcriptome and proteome revealed discordant pathway perturbations between the sexes. Tissue-level changes were accompanied by gut microbiome dysbiosis only in male mice, biasing toward increased potential for diabetogenic branched chain amino acid production. Antibiotic ablation of the gut microbiota diminished the impact of misalignment. In the United Kingdom Biobank, females showed stronger circadian rhythmicity in activity and a lower incidence of metabolic syndrome than males among job-matched shiftworkers. Thus, we show that female mice are more resilient than males to chronic circadian misalignment and that these differences are conserved in humans.

Circadian disruption in lung fibroblasts enhances NF-κB activity to exacerbate neutrophil recruitment.

Fibroblasts are stromal cells abundant throughout tissues, including the lungs. Fibroblasts are integral coordinators of immune cell recruitment through chemokine secretion. Circadian rhythms direct the recruitment of immune cells to the lung, which in turn impacts response to infection and survival. Although fibroblasts display robust circadian rhythms, the contribution of the fibroblast molecular clock to lung-specific migration of immune cells and recruitment remains to be established. Mice challenged intranasally with lipopolysaccharide (LPS) at dusk showed increased expression of the pro-inflammatory cytokine IL-1ß and chemokine CXCL5 in the lung, which was accompanied by increased neutrophil recruitment. Primary lung fibroblasts with knockdown of the core clock gene Bmal1 and immortalized Bmal1-/- lung fibroblasts also displayed increased Cxcl5 expression under IL-1ß stimulation. Conditioned media obtained from IL-1ß-stimulated Bmal1-/- immortalized fibroblasts-induced greater neutrophil migration compared with Bmal1+/+ lung fibroblast controls. Phosphorylation of the NF-κB subunit, p65, was enhanced in IL-1ß-stimulated Bmal1-/- lung fibroblasts, and pharmacological inhibition of NF-κB attenuated the enhanced CXCL5 production and neutrophil recruitment observed in these cells. Collectively, these results demonstrate that Bmal1 represses NF-κB activity in lung fibroblasts to control chemokine expression and immune cell recruitment during an inflammatory response.

The roles of lipids in SARS-CoV-2 viral replication and the host immune response.

The significant morbidity and mortality associated with severe acute respiratory syndrome coronavirus 2 infection has underscored the need for novel antiviral strategies. Lipids play essential roles in the viral life cycle. The lipid composition of cell membranes can influence viral entry by mediating fusion or affecting receptor conformation. Upon infection, viruses can reprogram cellular metabolism to remodel lipid membranes and fuel the production of new virions. Furthermore, several classes of lipid mediators, including eicosanoids and sphingolipids, can regulate the host immune response to viral infection. Here, we summarize the existing literature on the mechanisms through which these lipid mediators may regulate viral burden in COVID-19. Furthermore, we define the gaps in knowledge and identify the core areas in which lipids offer therapeutic promise for severe acute respiratory syndrome coronavirus 2.

Considerations for the Safe Operation of Schools During the Coronavirus Pandemic.

During the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, providing safe in-person schooling has been a dynamic process balancing evolving community disease burden, scientific information, and local regulatory requirements with the mandate for education. Considerations include the health risks of SARS-CoV-2 infection and its post-acute sequelae, the impact of remote learning or periods of quarantine on education and well-being of children, and the contribution of schools to viral circulation in the community. The risk for infections that may occur within schools is related to the incidence of SARS-CoV-2 infections within the local community. Thus, persistent suppression of viral circulation in the community through effective public health measures including vaccination is critical to in-person schooling. Evidence suggests that the likelihood of transmission of SARS-CoV-2 within schools can be minimized if mitigation strategies are rationally combined. This article reviews evidence-based approaches and practices for the continual operation of in-person schooling.

Distinguishing Intrapulmonary Immune Cells from Intravascular Immune Cell Populations: the Intrajugular Approach.

Circadian rhythms refer to oscillations in various biological process that occur with a 24 h period. At the molecular level, such rhythms are comprised of a web of transcriptional-translational feedback loops (TTFL) of core clock genes. Individual tissues and organ systems, including the immune system, have their own clock. In the systemic circulation, various members of the CD45+ population oscillate across the day; however, many of these rhythms are not identical or even similar in the tissue resident CD45+ leukocyte population. When studying the role of circadian regulation of lung inflammation, CD45+ within the lung may need to be investigated. However, despite optimized perfusion methods, leukocytes trapped from the circulation persist in the lungs. The goal in designing this protocol was to distinguish between intravascular and intraparenchymal leukocytes. Towards this end, mice are injected with a fluorescent tagged CD45 antibody intrajugularly shortly before lung harvest. Thereafter, the lung is digested using a customized lung digestion technique to obtain a single cell suspension. The sample is stained for the regular panel of antibodies for intraparenchymal immune cells (including another CD45 antibody). Flowcytometric analyses shows a clear elucidation of the populations. Thus, the method of labeling and defining intrapulmonary CD45+ cells will be particularly important where the behavior of intrapulmonary and circulating immune cells are numerically and functionally distinct.

Bmal1 Deletion in Myeloid Cells Attenuates Atherosclerotic Lesion Development and Restrains Abdominal Aortic Aneurysm Formation in Hyperlipidemic Mice.

OBJECTIVE: Although the molecular components of circadian rhythms oscillate in discrete cellular components of the vasculature and many aspects of vascular function display diurnal variation, the cellular connections between the molecular clock and inflammatory cardiovascular diseases remain to be elucidated. Previously we have shown that pre- versus postnatal deletion of Bmal1 (brain and muscle aryl hydrocarbon receptor nuclear translocator-like 1), the nonredundant core clock gene has contrasting effects on atherogenesis. Here we investigated the effect of myeloid cell Bmal1 deletion on atherogenesis and abdominal aortic aneurysm formation in mice. Approach and Results: Mice lacking Bmal1 in myeloid cells were generated by crossing Bmal1 flox/flox mice with lysozyme 2 promoter-driven Cre recombinase mice on a hyperlipidemic low-density lipoprotein receptor-deficient background and were fed on a high-fat diet to induce atherosclerosis. Atherogenesis was restrained, concomitant with a reduction of aortic proinflammatory gene expression in myeloid cell Bmal1 knockout mice. Body weight, blood pressure, blood glucose, triglycerides, and cholesterol were unaltered. Similarly, myeloid cell depletion of Bmal1 also restrained Ang II (angiotensin II) induced formation of abdominal aortic aneurysm in hyperlipidemic mice. In vitro, RNA-Seq analysis demonstrated a proinflammatory response in cultured macrophages in which there was overexpression of Bmal1. CONCLUSIONS: Myeloid cell Bmal1 deletion retards atherogenesis and restrains the formation of abdominal aortic aneurysm and may represent a potential therapeutic target for inflammatory cardiovascular diseases.

LDL-mimetic lipid nanoparticles prepared by surface KAT ligation for in vivo MRI of atherosclerosis.

Low-density lipoprotein (LDL)-mimetic lipid nanoparticles (LNPs), decorated with MRI contrast agents and fluorescent dyes, were prepared by the covalent attachment of apolipoprotein-mimetic peptide (P), Gd(iii)-chelate (Gd), and sulforhodamine B (R) moieties on the LNP surface. The functionalized LNPs were prepared using the amide-forming potassium acyltrifluoroborate (KAT) ligation reaction. The KAT groups on the surface of LNPs were allowed to react with the corresponding hydroxylamine (HA) derivatives of P and Gd to provide bi-functionalized LNPs (PGd-LNP). The reaction proceeded with excellent yields, as observed by ICP-MS (for B and Gd amounts) and MALDI-TOF-MS data, and did not alter the morphology of the LNPs (mean diameter: ca. 50 nm), as shown by DLS and cryoTEM analyses. With the help of the efficient KAT ligation, a high payload of Gd(iii)-chelate on the PGd-LNP surface (ca. 2800 Gd atoms per LNP) was successfully achieved and provided a high r 1 relaxivity (r 1 = 22.0 s-1 mM-1 at 1.4 T/60 MHz and 25 °C; r 1 = 8.2 s-1 mM-1 at 9.4 T/400 MHz and 37 °C). This bi-functionalized PGd-LNP was administered to three atherosclerotic apoE -/- mice to reveal the clear enhancement of atherosclerotic plaques in the brachiocephalic artery (BA) by MRI, in good agreement with the high accumulation of Gd in the aortic arch as shown by ICP-MS. The parallel in vivo MRI and ex vivo studies of whole mouse cryo-imaging were performed using triply functionalized LNPs with P, Gd, and R (PGdR-LNP). The clear presence of atherosclerotic plaques in BA was observed by ex vivo bright field cryo-imaging, and they were also observed by high emission fluorescent imaging. These directly corresponded to the enhanced tissue in the in vivo MRI of the identical mouse.

Comparative evaluation of RNA-Seq library preparation methods for strand-specificity and low input.

Library preparation is a key step in sequencing. For RNA sequencing there are advantages to both strand specificity and working with minute starting material, yet until recently there was no kit available enabling both. The Illumina TruSeq stranded mRNA Sample Preparation kit (TruSeq) requires abundant starting material while the Takara Bio SMART-Seq v4 Ultra Low Input RNA kit (V4) sacrifices strand specificity. The SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (Pico) by Takara Bio claims to overcome these limitations. Comparative evaluation of these kits is important for selecting the appropriate protocol. We compared the three kits in a realistic differential expression analysis. We prepared and sequenced samples from two experimental conditions of biological interest with each of the three kits. We report differences between the kits at the level of differential gene expression; for example, the Pico kit results in 55% fewer differentially expressed genes than TruSeq. Nevertheless, the agreement of the observed enriched pathways suggests that comparable functional results can be obtained. In summary we conclude that the Pico kit sufficiently reproduces the results of the other kits at the level of pathway analysis while providing a combination of options that is not available in the other kits.

Myeloid Cell mPges-1 Deletion Attenuates Mortality Without Affecting Remodeling After Acute Myocardial Infarction in Mice.

Selective deletion of microsomal prostaglandin E2 synthase-1 (mPges-1) in myeloid cells retards atherogenesis and suppresses the vascular proliferative response to injury, while it does not predispose to thrombogenesis or hypertension. However, studies using bone marrow transplants from irradiated mice suggest that myeloid cell mPGES-1 facilitates cardiac remodeling and prolongs survival after experimental myocardial infarction (MI). Here, we addressed this question using mice lacking mPges-1 in myeloid cells, particularly macrophages [Mac-mPges-1-knockout (KO)], generated by crossing mPges-1 floxed mice with LysMCre mice and subjecting them to coronary artery ligation. Cardiac structure and function were assessed by morphometric analysis, echocardiography, and invasive hemodynamics 3, 7, and 28 days after MI. Despite a similar infarct size, in contrast to the prior report, the post-MI survival rate was markedly improved in the Mac-mPges-1-KO mice compared with wild-type controls. Left ventricular systolic (reflected by ejection fraction, fractional shortness end systolic volume, and +dP/dt) and diastolic function (reflected by end diastolic volume, -dP/dt, and Tau), cardiac hypertrophy (reflected by left ventricular dimensions), and staining for fibrosis did not differ between the groups. In conclusion, we found that Cre-loxP-mediated deletion of mPges-1 in myeloid cells has favorable effects on post-MI survival, with no detectable adverse influence on post-MI remodeling. These results add to evidence that targeting macrophage mPGES-1 may represent a safe and efficacious approach to the treatment and prevention of cardiovascular inflammatory disease.

Cold-Induced Browning of Inguinal White Adipose Tissue Is Independent of Adipose Tissue Cyclooxygenase-2.

Previous studies using genetic mouse models have implicated COX-2 in the browning of white adipose tissues (WATs) in mice during cold exposure. However, COX-2 is important during development, and conventional knockouts (KOs) exhibit many defects, conditioned by genetic background. Similarly, the physiological relevance of transgenic overexpression of COX-2 is questionable. In the present study, we utilized mice in which COX-2 was deleted postnatally, bypassing the consequences of enzyme deficiency during development. Despite activation of thermogenesis and browning of inguinal WAT, cold exposure failed to increase COX-2 expression in the adipose tissues of mice with different genetic backgrounds, and the body temperature response to cold was unaltered in postnatal global COX-2 KOs. Selective disruption of COX-2 in adipose tissues also failed detectably to impact systemic prostaglandin biosynthesis. Browning of inguinal WATs induced by exposure to cold is independent of adipose tissue COX-2.

Cyclooxygenase-2, Asymmetric Dimethylarginine, and the Cardiovascular Hazard From Nonsteroidal Anti-Inflammatory Drugs.

BACKGROUND: Large-scale, placebo-controlled trials established that nonsteroidal anti-inflammatory drugs confer a cardiovascular hazard: this has been attributed to depression of cardioprotective products of cyclooxygenase (COX)-2, especially prostacyclin. An alternative mechanism by which nonsteroidal anti-inflammatory drugs might constrain cardioprotection is by enhancing the formation of methylarginines in the kidney that would limit the action of nitric oxide throughout the vasculature. METHODS: Targeted and untargeted metabolomics were used to investigate the effect of COX-2 deletion or inhibition in mice and in osteoarthritis patients exposed to nonsteroidal anti-inflammatory drugs on the l-arginine/nitric oxide pathway. RESULTS: Analysis of the plasma and renal metabolome was performed in postnatal tamoxifen-inducible Cox-2 knockout mice, which exhibit normal renal function and blood pressure. This revealed no changes in arginine and methylarginines compared with their wild-type controls. Moreover, the expression of genes in the l-arginine/nitric oxide pathway was not altered in the renal medulla or cortex of tamoxifen inducible Cox-2 knockout mice. Therapeutic concentrations of the selective COX-2 inhibitors, rofecoxib, celecoxib, and parecoxib, none of which altered basal blood pressure or renal function as reflected by plasma creatinine, failed to elevate plasma arginine and methylarginines in mice. Finally, plasma arginine or methylarginines were not altered in osteoarthritis patients with confirmed exposure to nonsteroidal anti-inflammatory drugs that inhibit COX-1 and COX-2. By contrast, plasma asymmetrical dimethylarginine was increased in mice infused with angiotensin II sufficient to elevate blood pressure and impair renal function. Four weeks later, blood pressure, plasma creatinine, and asymmetrical dimethylarginine were restored to normal levels. The increase in asymmetrical dimethylarginine in response to infusion with angiotensin II in celecoxib-treated mice was also related to transient impairment of renal function. CONCLUSIONS: Plasma methylarginines are not altered by COX-2 deletion or inhibition but rather are elevated coincident with renal compromise.

Cardiovascular Consequences of Prostanoid I Receptor Deletion in Microsomal Prostaglandin E Synthase-1-Deficient Hyperlipidemic Mice.

BACKGROUND: Inhibitors of cyclooxygenase-2 alleviate pain and reduce fever and inflammation by suppressing the biosynthesis of prostacyclin (PGI2) and prostaglandin E2. However, suppression of these prostaglandins, particularly PGI2, by cyclooxygenase-2 inhibition or deletion of its I prostanoid receptor also predisposes to accelerated atherogenesis and thrombosis in mice. By contrast, deletion of microsomal prostaglandin E synthase 1 (mPGES-1) confers analgesia, attenuates atherogenesis, and fails to accelerate thrombogenesis, while suppressing prostaglandin E2, but increasing biosynthesis of PGI2. METHODS: To address the cardioprotective contribution of PGI2, we generated mice lacking the I prostanoid receptor together with mPges-1 on a hyperlipidemic background (low-density lipoprotein receptor knockouts). RESULTS: mPges-1 depletion modestly increased thrombogenesis, but this response was markedly further augmented by coincident deletion of the I prostanoid receptor (n=10-18). By contrast, deletion of the I prostanoid receptor had no effect on the attenuation of atherogenesis by mPGES-1 deletion in the low-density lipoprotein receptor knockout mice (n=17-21). CONCLUSIONS: Although suppression of prostaglandin E2 accounts for the protective effect of mPGES-1 deletion in atherosclerosis, augmentation of PGI2 is the dominant contributor to its favorable thrombogenic profile. The divergent effects on these prostaglandins suggest that inhibitors of mPGES-1 may be less likely to cause cardiovascular adverse effects than nonsteroidal anti-inflammatory drugs specific for inhibition of cyclooxygenase-2.

Myeloid cell microsomal prostaglandin E synthase-1 fosters atherogenesis in mice.

Microsomal prostaglandin E synthase-1 (mPGES-1) in myeloid and vascular cells differentially regulates the response to vascular injury, reflecting distinct effects of mPGES-1-derived PGE2 in these cell types on discrete cellular components of the vasculature. The cell selective roles of mPGES-1 in atherogenesis are unknown. Mice lacking mPGES-1 conditionally in myeloid cells (Mac-mPGES-1-KOs), vascular smooth muscle cells (VSMC-mPGES-1-KOs), or endothelial cells (EC-mPGES-1-KOs) were crossed into hyperlipidemic low-density lipoprotein receptor-deficient animals. En face aortic lesion analysis revealed markedly reduced atherogenesis in Mac-mPGES-1-KOs, which was concomitant with a reduction in oxidative stress, reflective of reduced macrophage infiltration, less lesional expression of inducible nitric oxide synthase (iNOS), and lower aortic expression of NADPH oxidases and proinflammatory cytokines. Reduced oxidative stress was reflected systemically by a decline in urinary 8,12-iso-iPF2α-VI. In contrast to exaggeration of the response to vascular injury, deletion of mPGES-1 in VSMCs, ECs, or both had no detectable phenotypic impact on atherogenesis. Macrophage foam cell formation and cholesterol efflux, together with plasma cholesterol and triglycerides, were unchanged as a function of genotype. In conclusion, myeloid cell mPGES-1 promotes atherogenesis in hyperlipidemic mice, coincident with iNOS-mediated oxidative stress. By contrast, mPGES-1 in vascular cells does not detectably influence atherogenesis in mice. This strengthens the therapeutic rationale for targeting macrophage mPGES-1 in inflammatory cardiovascular diseases.

Cyclooxygenase-2 in endothelial and vascular smooth muscle cells restrains atherogenesis in hyperlipidemic mice.

BACKGROUND: Placebo-controlled trials of nonsteroidal anti-inflammatory drugs selective for inhibition of cyclooxygenase-2 (COX-2) reveal an emergent cardiovascular hazard in patients selected for low risk of heart disease. Postnatal global deletion of COX-2 accelerates atherogenesis in hyperlipidemic mice, a process delayed by selective enzyme deletion in macrophages. METHODS AND RESULTS: In the present study, selective depletion of COX-2 in vascular smooth muscle cells and endothelial cells depressed biosynthesis of prostaglandin I2 and prostaglandin E2, elevated blood pressure, and accelerated atherogenesis in Ldlr knockout mice. Deletion of COX-2 in vascular smooth muscle cells and endothelial cells coincided with an increase in COX-2 expression in lesional macrophages and increased biosynthesis of thromboxane. Increased accumulation of less organized intimal collagen, laminin, α-smooth muscle actin, and matrix-rich fibrosis was also apparent in lesions of the mutants. CONCLUSIONS: Although atherogenesis is accelerated in global COX-2 knockouts, consistent with evidence of risk transformation during chronic nonsteroidal anti-inflammatory drug administration, this masks the contrasting effects of enzyme depletion in macrophages versus vascular smooth muscle cells and endothelial cells. Targeting delivery of COX-2 inhibitors to macrophages may conserve their efficacy while limiting cardiovascular risk.

Vascular COX-2 modulates blood pressure and thrombosis in mice.

Prostacyclin (PGI(2)) is a vasodilator and platelet inhibitor, properties consistent with cardioprotection. More than a decade ago, inhibition of cyclooxygenase-2 (COX-2) by the nonsteroidal anti-inflammatory drugs (NSAIDs) rofecoxib and celecoxib was found to reduce the amount of the major metabolite of PGI(2) (PGI-M) in the urine of healthy volunteers. This suggested that NSAIDs might cause adverse cardiovascular events by reducing production of cardioprotective PGI(2). This prediction was based on the assumption that the concentration of PGI-M in urine likely reflected vascular production of PGI(2) and that other cardioprotective mediators, especially nitric oxide (NO), were not able to compensate for the loss of PGI(2). Subsequently, eight placebo-controlled clinical trials showed that NSAIDs that block COX-2 increase adverse cardiovascular events. We connect tissue-specific effects of NSAID action and functional correlates in mice with clinical outcomes in humans by showing that deletion of COX-2 in the mouse vasculature reduces excretion of PGI-M in urine and predisposes the animals to both hypertension and thrombosis. Furthermore, vascular disruption of COX-2 depressed expression of endothelial NO synthase and the consequent release and function of NO. Thus, suppression of PGI(2) formation resulting from deletion of vascular COX-2 is sufficient to explain the cardiovascular hazard from NSAIDs, which is likely to be augmented by secondary mechanisms such as suppression of NO production.

Disruption of the 5-lipoxygenase pathway attenuates atherogenesis consequent to COX-2 deletion in mice.

Suppression of cyclooxygenase 2 (COX-2)-derived prostacyclin (PGI(2)) is sufficient to explain most elements of the cardiovascular hazard from nonsteroidal antinflammatory drugs (NSAIDs). However, randomized trials are consistent with the emergence of cardiovascular risk during chronic dosing with NSAIDs. Although deletion of the PGI(2) receptor fosters atherogenesis, the importance of COX-2 during development has constrained the use of conventional knockout (KO) mice to address this question. We developed mice in which COX-2 was deleted postnatally, bypassing cardiorenal defects exhibited by conventional KOs. When crossed into ApoE-deficient hyperlipidemic mice, COX-2 deletion accelerated atherogenesis in both genders, with lesions exhibiting leukocyte infiltration and phenotypic modulation of vascular smooth muscle cells, as reflected by loss of α-smooth muscle cell actin and up-regulation of vascular cell adhesion molecule-1. Stimulated peritoneal macrophages revealed suppression of COX-2-derived prostanoids and augmented 5-lipoxygenase product formation, consistent with COX-2 substrate rediversion. Although deletion of the 5-lipoxygenase activating protein (FLAP) did not influence atherogenesis, it attenuated the proatherogeneic impact of COX-2 deletion in hyperlipidemic mice. Chronic administration of NSAIDs may increasingly confer a cardiovascular hazard on patients at low initial risk. Promotion of atherogenesis by postnatal COX-2 deletion affords a mechanistic explanation for this observation. Coincident inhibition of FLAP may offer an approach to attenuating such a risk from NSAIDs.