Orina Belton

Cyclooxygenase-1 and -2-dependent prostacyclin formation in patients with atherosclerosis

BACKGROUND The formation of prostacyclin (PGI(2)), thromboxane (TX) A(2), and isoprostanes is markedly enhanced in atherosclerosis. We examined the relative contribution of cyclooxygenase (COX)-1 and -2 to the generation of these eicosanoids in patients with atherosclerosis. METHODS AND RESULTS The study population consisted of 42 patients with atherosclerosis who were undergoing surgical revascularization. COX-2 mRNA was detected in areas of atherosclerosis but not in normal blood vessel walls, and there was evidence of COX-1 induction. The use of immunohistochemical studies localized the COX-2 to proliferating vascular smooth muscle cells and macrophages. Twenty-four patients who did not previously receive aspirin were randomized to receive either no treatment or nimesulide at 24 hours before surgery and then for 3 days. Eighteen patients who were receiving aspirin were continued on a protocol of either aspirin alone or a combination of aspirin and nimesulide. Urinary levels of 11-dehydro-TXB(2) and 2,3-dinor-6-keto-PGF(1alpha), metabolites of TXA(2) and PGI(2), respectively, were elevated in patients with atherosclerosis compared with normal subjects (3211+/-533 versus 679+/-63 pg/mg creatinine, P<0.001; 594+/-156 versus 130+/-22 pg/mg creatinine, P<0.05, respectively), as was the level of the isoprostane 8-iso-PGF(2alpha). Nimesulide reduced 2, 3-dinor-6-keto-PGF(1alpha) excretion by 46+/-5% (378.3+/-103 to 167+/-37 pg/mg creatinine, P<0.01) preoperatively and blunted the increase after surgery. Nimesulide had no significant effect on 11-dehydro-TXB(2) before (2678+/-694 to 2110+/-282 pg/mg creatinine) or after surgery. The levels of both products were lower in patients who were taking aspirin, and no further reduction was seen with the addition of nimesulide. None of the treatments influenced urinary 8-iso-PGF(2alpha) excretion. CONCLUSIONS Both COX-1 and -2 are expressed and contribute to the increase in PGI(2) in patients with atherosclerosis, whereas TXA(2) is generated by COX-1.

Mesenchymal stem cell inhibition of T-helper 17 cell- differentiation is triggered by cell–cell contact and mediated by prostaglandin E2 via the EP4 receptor

Mesenchymal stem cells (MSCs) inhibit T‐cell activation and proliferation but their effects on individual T‐cell‐effector pathways and on memory versus naïve T cells remain unclear. MSC influence on the differentiation of naïve and memory CD4+ T cells toward the Th17 phenotype was examined. CD4+ T cells exposed to Th17‐skewing conditions exhibited reduced CD25 and IL‐17A expression following MSC co‐culture. Inhibition of IL‐17A production persisted upon re‐stimulation in the absence of MSCs. These effects were attenuated when cell–cell contact was prevented. Th17 cultures from highly purified naïve‐ and memory‐phenotype responders were similarly inhibited. Th17 inhibition by MSCs was reversed by indomethacin and a selective COX‐2 inhibitor. Media from MSC/Th17 co‐cultures contained increased prostaglandin E2 (PGE2) levels and potently suppressed Th17 differentiation in fresh cultures. MSC‐mediated Th17 inhibition was reversed by a selective EP4 antagonist and was mimicked by synthetic PGE2 and a selective EP4 agonist. Activation‐induced IL‐17A secretion by naturally occurring, effector‐memory Th17 cells from a urinary obstruction model was also inhibited by MSC co‐culture in a COX‐dependent manner. Overall, MSCs potently inhibit Th17 differentiation from naïve and memory T‐cell precursors and inhibit naturally‐occurring Th17 cells derived from a site of inflammation. Suppression entails cell‐contact‐dependent COX‐2 induction resulting in direct Th17 inhibition by PGE2 via EP4.

Cyclooxygenase Isoforms and Platelet Vessel Wall Interactions in the Apolipoprotein E Knockout Mouse Model of Atherosclerosis

Background—Cyclooxygenase (COX) activity is induced in human atherosclerosis, and the products formed may modify the disease directly or through an effect on platelets. We examined the role of COX-1 and -2 on platelet vessel wall interactions and development of atherosclerosis in a murine model. Methods and Results—Apolipoprotein E–deficient (apoE−/−) mice fed a 1% cholesterol diet were treated with a selective COX-1 inhibitor (SC-560), a selective COX-2 inhibitor (SC-236), or vehicle. Urinary prostacyclin and thromboxane metabolites (2,3-dinor-6-keto-PGF1&agr; and 2,3-dinor-TXB2) were increased in the apoE−/− knockout mouse. There was also induction of both COX isoforms in the vascular lesions formed, which stained for CD41, a platelet-specific marker, and for CD40L. Selective inhibition of COX-2 had no effect on lesion formation and, despite selective reduction in prostacyclin generation, had no effect on platelet activity, as measured by thromboxane formation or platelet deposition. Selective inhibition of COX-1 reduced 2,3-dinor-TXB2 generation and lesion formation. However, platelet deposition on the vessel wall persisted, with well-defined monolayers seen. There was also persistent expression of the macrophage marker CD68 and increased expression of the cell death protein Bax. In contrast to lesion development, the selective COX-1 inhibitor had no effect on the regression of evolving lesions. Conclusions—COX-1 plays an important role in the early stages of lesion development in the apoE−/− knockout model of atherosclerosis, preventing gross lesion formation in the face of continued vascular injury and inflammation. Despite the inhibition of prostacyclin, COX-2 inhibition had no effect on lesion development or platelet–vessel wall interactions.

Intravascular thrombosis after hypoxia-induced pulmonary hypertension: regulation by cyclooxygenase-2.

Arachidonic acid metabolism leads to the generation of biologically active metabolites that regulate cell growth and proliferation, as well as survival and apoptosis. We have demonstrated previously that platelet-type 12-lipoxygenase (LOX) regulates the growth and survival of a number of cancer cells. In this study, we show that overexpression of platelet-type 12-LOX in prostate cancer PC3 cells or epithelial cancer A431 cells significantly extended their survival and delayed apoptosis when cultured under serum-free conditions. These effects were shown to be a result of enhanced surface integrin expression, resulting in a more spread morphology of the cells in culture. PC3 cells transfected with 12-LOX displayed increased alpha(v)beta(3) and alpha(v)beta(5) integrin expression, whereas other integrins were unaltered. Transfected A431 cells did not express alpha(v)beta(3); however, alpha(v)beta(5) integrin expression was increased. Treatment of both transfected cell lines with monoclonal antibody to alpha(v)beta(5) (and in the case of PC3 cells, anti-alpha(v)beta(3)) resulted in significant apoptosis. In addition, treatment with 100 nM 12(S)-hydroxy-eicosatetraenoic acid, the end product of platelet-type 12-LOX, but not other hydroxy-eicosatetraenoic acids, enhanced the survival of wild-type PC3 and A431 cells and resulted in increased expression of alpha(v)beta(5). Furthermore, Baicalein or N-benzyl-N-hydroxy-5-phenylpentamide, specific 12-LOX inhibitors, significantly decreased alpha(v)beta(5)-mediated adhesion and survival in 12-LOX-overexpressing cells. The results show that 12-LOX regulates cell survival and apoptosis by affecting the expression and localization of the vitronectin receptors, alpha(v)beta(3) and alpha(v)beta(5), in two cancer cell lines.Background—Pulmonary hypertension induced by chronic hypoxia is characterized by thickening of pulmonary artery walls, elevated pulmonary vascular resistance, and right-heart failure. Prostacyclin analogues reduce pulmonary pressures in this condition; raising the possibility that cycloxygenase-2 (COX-2) modulates the response of the pulmonary vasculature to hypoxia. Methods and Results—Sprague-Dawley rats in which pulmonary hypertension was induced by hypobaric hypoxia for 14 days were treated concurrently with the selective COX-2 inhibitor SC236 or vehicle. Mean pulmonary arterial pressure (mPAP) was elevated after hypoxia (28.1±3.2 versus 17.2±3.1 mm Hg; n=8, P<0.01), with thickening of small pulmonary arteries and increased COX-2 expression and prostacyclin formation. Selective inhibition of COX-2 aggravated the increase in mPAP (42.8±5.9 mm Hg; n=8, P<0.05), an effect that was attenuated by the thromboxane (TX) A2/prostaglandin endoperoxide receptor antagonist ifetroban. Urinary TXB2 increased during hypoxia (5.9±0.9 versus 1.2±0.2 ng/mg creatinine; n=6, P<0.01) and was further increased by COX-2 inhibition (8.5±0.7 ng/mg creatinine; n=6, P<0.05). In contrast, urinary excretion of the prostacyclin metabolite 6-ketoprostaglandin F1&agr; decreased with COX-2 inhibition (8.6±3.0 versus 27.0±4.8 ng/mg creatinine; n=6, P<0.05). Platelet activation was enhanced after chronic hypoxia. COX-2 inhibition further reduced the PFA-100 closure time and enhanced platelet deposition in the smaller pulmonary arteries, effects that were attenuated by ifetroban. Mice with targeted disruption of the COX-2 gene exposed to chronic hypoxia had exacerbated right ventricular end-systolic pressure, whereas targeted disruption of COX-1 had no effect. Conclusions—COX-2 expression is increased and regulates platelet activity and intravascular thrombosis in hypoxia-induced pulmonary hypertension.

Contribution of cyclooxygenase-1 to thromboxane formation, platelet–vessel wall interactions and atherosclerosis in the ApoE null mouse

OBJECTIVE Prostaglandin and thromboxane (TXA(2)) generation is increased in atherosclerosis. Studies with selective inhibitors attribute the enhanced prostacyclin (PGI(2)) generation to both cyclooxygenase-1 (COX-1) and COX-2 whereas the increased TXA(2) generation reflects platelet COX-1 expression. However, TXA(2) formation remains elevated in patients with cardiovascular disease on doses of aspirin that fully suppress platelet COX-1, suggesting other tissue sources for TXA(2) formation. Disruption of the thromboxane receptor gene suppresses the development of atherosclerosis. Notwithstanding this, the role of COX-1 in atherosclerosis is unclear, as it is widely distributed and contributes to a number of products, including those that potentially contribute to the resolution of inflammation. METHODS AND RESULTS We examined the role of COX-1 on prostaglandin generation, development of atherosclerosis and platelet-vessel wall interactions in the apoE(-/-) murine model by disrupting the COX-1 gene. ApoE(-/-)/COX-1(+/+), ApoE(-/-)/COX-1(+/-) and ApoE(-/-)/COX-1(-/-), were administered a 1% cholesterol diet for 8 weeks. Stable urinary metabolites of PGI(2) and TXA(2), which were markedly increased in the ApoE(-/-)/COX-1(+/+) were reduced by disruption of COX-1. Deletion of one or both copies of the COX-1 gene suppressed lesion formation. Assessment of platelet-vessel wall interactions by intravital microscopy showed a significant decrease in firm adhesion of platelets in the apoE/COX-1 double knockout (DKO). CONCLUSION COX-1 contributes to the enhanced formation of both PGI(2) and TXA(2) in atherosclerosis, and to the development of the disease. Non-platelet sources of COX-1 and TXA(2) that are inaccessible to standard doses of aspirin may contribute to the development of atherosclerosis.

Induction of cyclooxygenase-1 and -2 modulates angiogenic responses to engagement of αvβ3

Summary. Prostaglandins and cyclooxygenase (COX) have been implicated in the angiogenesis that occurs around tumours, but how they are induced is unclear. Prostaglandin formation is regulated by the availability of arachidonic acid and/or COX activity that in turn are controlled by activation of G‐protein‐coupled receptors or kinase receptors. Adhesion receptors provide another potential level of control as they transduce a variety of ‘outside‐in’ signals implicated in inflammation. We examined whether engagement of the vitronectin receptor (αvβ3) modulated prostacyclin (PGI2) formation in human umbilical vein endothelial cells (EC). Engagement of EC αvβ3 by vitronectin (versus fibronectin or gelatin) or by monoclonal antibodies (mAbs) LM609 and LIBS6, enhanced PGI2 generation and also induced expression of both COX‐1 and ‐2 isoforms. αvβ3 engagement also led to vascular endothelial cell growth factor (VEGF) generation and EC proliferation that was attenuated by inhibition of both COX‐1 and COX‐2. COX‐1 inhibition also prevented new vessel formation in an in vitro model of angiogenesis that is αvβ3 dependent. Inhibition of angiogenesis by the COX‐1 inhibitor was partially reversed by removal of the inhibitor or by addition of the stable analogue of PGI2, iloprost. These findings strongly indicate that αvβ3‐mediated angiogenesis is partly due to induction of both isoforms of COX.

IL-10 mediates the immunoregulatory response in conjugated linoleic acid-induced regression of atherosclerosis

Conjugated linoleic acid (CLA) induces regression of preestablished atherosclerosis in the ApoE–/– mouse. Understanding the mechanisms involved may help in identifying novel pathways associated with the regression of human disease. Animals were administered a 1% cholesterol diet for 12 wk, with 1% CLA supplementation from wk 8 to 12. ApoE–/– mice fed only the 1% cholesterol diet for 12 wk were employed as controls. Transcriptomic analysis of mouse aorta showed that many of the components of the IL‐10 signaling pathway were modified during CLA‐induced regression. Real‐time PCR and Western blot analysis showed increased IL‐10 receptor expression, phosphorylation of STAT3, and downstream target gene expression in the aorta, alongside an increase in serum IL‐10 (79.8±22.4 vs. 41.9±5.5 pg/ml, n=10; P<0.01). CLA ‐supplementation also increased IL‐10 production in bone marrow‐derived macrophages (143.6±28.6 vs. 94±5.6 pg/ml, n=5; P<0.05). To explore the mechanisms for altered IL‐10 production, we examined the profile of monocyte/macrophage phenotype in the vessel wall, bone marrow, and spleen. CLA increased macrophage polarization toward an anti‐inflammatory M2 phenotype in vivo, increasing the population of Ly6Clo monocytes (29 vs. 77±14, n=5, P < 0.05) in the aorta. CLA had similar effects on monocytes/macrophages differentiated from marrow‐derived progenitor cells and on splenocytes. The induction of IL‐10 on CLA supplementation in this model may reflect a systemic alteration toward an anti‐inflammatory phenotype, which, in turn promotes increased vascular infiltration by Ly6Clo monocytes. These cells may contribute to CLA‐induced disease regression.—McCarthy, C., Duffy, M. M., Mooney, D., James, W. G., Griffin, M. D., Fitzgerald, D. J., Belton, O. IL‐10 mediates the immunoregulatory response in conjugated linoleic acid‐induced regression of atherosclerosis. FASEB J. 27, 499–510 (2013). www.fasebj.org

Milk-derived bioactive peptides inhibit human endothelial-monocyte interactions via PPAR-γ dependent regulation of NF-κB

BackgroundMilk-derived bioactive peptides retain many biological properties and have therapeutic effects in cardiovascular disorders such as atherosclerosis. Under inflammatory conditions the expression of endothelial cells adhesion molecules is induced, increasing monocyte adhesion to human vessel wall, a critical step in the pathogenesis of atherosclerosis. In the present work we explored the effects of milk-derived bioactive peptides on the expression of the inflammatory phenotype of human endothelial cells and their effects on monocyte adherence to endothelial cells.ResultsTreatment of endothelial cells with milk-derived hydrolysate inhibited their production of inflammatory proteins MCP-1 and IL-8 and expression of VCAM-1, ICAM-1 and E-selectin. Milk derived hydrolysate also attenuated the adhesion of human monocytes to activated endothelial cells. The effect was similar to that obtained in endothelial cells treated with troglitazone, a ligand of peroxisome proliferators-activator receptor-gamma (PPAR-γ). PPAR-γ is a transcription factor which when activated antagonises the pro-inflammatory capability of nuclear factor κB (NF-κB). We further examined whether the effects of milk-derived hydrolysates on endothelial cells may be mediated through NF-κB activation via a PPAR-γ dependent mechanism. The specific PPAR-γ inhibitor, GW9662 blocked the effects of the hydrolysate on the NF-κB-mediated chemokines and adhesion molecules expression in endothelial cells.ConclusionsThese results suggest that milk-derived bioactive peptides work as anti-atherogenic agents through the inhibition of endothelial-dependent adhesive interactions with monocytes by inhibiting the NF-κB pathway through a PPAR-γ dependent mechanism.

M1- and M2-Type Macrophage Responses Are Predictive of Adverse Outcomes in Human Atherosclerosis.

Atherosclerosis is an inflammatory disease caused by endothelial injury, lipid deposition, and oxidative stress. This progressive disease can be converted into an acute clinical event by plaque rupture and thrombosis. In the context of atherosclerosis, the underlying cause of myocardial infarction and stroke, macrophages uniquely possess a dual functionality, regulating lipid accumulation and metabolism and sustaining the chronic inflammatory response, two of the most well-documented pathways associated with the pathogenesis of the disease. Macrophages are heterogeneous cell populations and it is hypothesized that, during the pathogenesis of atherosclerosis, macrophages in the developing plaque can switch from a pro-inflammatory (MΦ1) to an anti-inflammatory (MΦ2) phenotype and vice versa, depending on the microenvironment. The aim of this study was to identify changes in macrophage subpopulations in the progression of human atherosclerotic disease. Established atherosclerotic plaques from symptomatic and asymptomatic patients with existing coronary artery disease undergoing carotid endarterectomy were recruited to the study. Comprehensive histological and immunohistochemical analyses were performed to quantify the cellular content and macrophage subsets of atherosclerotic lesion. In parallel, expression of MΦ1 and MΦ2 macrophage markers were analyzed by real-time PCR and Western blot analysis. Gross analysis and histological staining demonstrated that symptomatic plaques presented greater hemorrhagic activity and the internal carotid was the most diseased segment, based on the predominant prevalence of fibrotic and necrotic tissue, calcifications, and hemorrhagic events. Immunohistochemical analysis showed that both MΦ1 and MΦ2 macrophages are present in human plaques. However, MΦ2 macrophages are localized to more stable locations within the lesion. Importantly, gene and protein expression analysis of MΦ1/MΦ2 markers evidenced that MΦ1 markers and Th1-associated cytokines are highly expressed in symptomatic plaques, whereas expression of the MΦ2 markers, mannose receptor (MR), and CD163 and Th2 cytokines are inversely related with disease progression. These data increase the understanding of atherosclerosis development, identifying the cellular content of lesions during disease progression, and characterizing macrophage subpopulation within human atherosclerotic plaques.

Macrophage PPAR gamma Co-activator-1 alpha participates in repressing foam cell formation and atherosclerosis in response to conjugated linoleic acid

Conjugated linoleic acid (CLA) has the unique property of inducing regression of pre‐established murine atherosclerosis. Understanding the mechanism(s) involved may help identify endogenous pathways that reverse human atherosclerosis. Here, we provide evidence that CLA inhibits foam cell formation via regulation of the nuclear receptor coactivator, peroxisome proliferator‐activated receptor (PPAR)‐γ coactivator (PGC)‐1α, and that macrophage PGC‐1α plays a role in atheroprotection in vivo. PGC‐1α was identified as a hub gene within a cluster in the aorta of the apoE−/− mouse in the CLA‐induced regression model. PGC‐1α was localized to macrophage/foam cells in the murine aorta where its expression was increased during CLA‐induced regression. PGC‐1α expression was also detected in macrophages in human atherosclerosis and was inversely linked to disease progression in patients with the disease. Deletion of PGC‐1α in bone marrow derived macrophages promoted, whilst over expression of the gene inhibited foam cell formation. Importantly, macrophage specific deletion of PGC‐1α accelerated atherosclerosis in the LDLR−/− mouse in vivo. These novel data support a functional role for PGC‐1α in atheroprotection.