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International audience; Medullary and extra-medullary hematopoiesis has been shown to govern inflammatory cell infiltration and subsequently cardiac remodeling and function after acute myocardial infarction (MI). Emerging evidence positions adipose tissue (AT) as an alternative source of immune cell production. We, therefore, hypothesized that AT could act as a reservoir of inflammatory cells that participate in cardiac homeostasis after MI. To reveal the distinct role of inflammatory cells derived from AT or bone marrow (BM), chimeric mice were generated using standard repopulation assays. We showed that AMI increased the number of AT-derived macrophages in the cardiac tissue. These macrophages exhibit pro-inflammatory characteristics and their specific depletion improved cardiac function as well as decreased infarct size and interstitial fibrosis. We then reasoned that the alteration of AT-immune compartment in type 2 diabetes could, thus, contribute to defects in cardiac remodeling. However, in these conditions, myeloid cells recruited in the infarcted heart mainly originate from the BM, and AT was no longer used as a myeloid cell reservoir. Altogether, we showed here that a subpopulation of cardiac inflammatory macrophages emerges from myeloid cells of AT origin and plays a detrimental role in cardiac remodeling and function after MI. Diabetes abrogates the ability of AT-derived myeloid cells to populate the infarcted heart.

AimsThe cardioprotective effects of human induced pluripotent stem cell-derived cardiovascular progenitor cells (CPC) are largely mediated by the paracrine release of extracellular vesicles (EV). We aimed to assess the immunological behaviour of EV-CPC, which is a prerequisite for their clinical translation.Methods and resultsFlow cytometry demonstrated that EV-CPC expressed very low levels of immune relevant molecules including HLA Class I, CD80, CD274 (PD-L1), and CD275 (ICOS-L); and moderate levels of ligands of the natural killer (NK) cell activating receptor, NKG2D. In mixed lymphocyte reactions, EV-CPC neither induced nor modulated adaptive allogeneic T cell immune responses. They also failed to induce NK cell degranulation, even at high concentrations. These in vitro effects were confirmed in vivo as repeated injections of EV-CPC did not stimulate production of immunoglobulins or affect the interferon (IFN)-γ responses from primed splenocytes. In a mouse model of chronic heart failure, intra-myocardial injections of EV-CPC, 3 weeks after myocardial infarction, decreased both the number of cardiac pro-inflammatory Ly6Chigh monocytes and circulating levels of pro-inflammatory cytokines (IL-1α, TNF-α, and IFN-γ). In a model of acute infarction, direct cardiac injection of EV-CPC 2 days after infarction reduced pro-inflammatory macrophages, Ly6Chigh monocytes, and neutrophils in heart tissue as compared to controls. EV-CPC also reduced levels of pro-inflammatory cytokines IL-1α, IL-2, and IL-6, and increased levels of the anti-inflammatory cytokine IL-10. These effects on human macrophages and monocytes were reproduced in vitro; EV-CPC reduced the number of pro-inflammatory monocytes and M1 macrophages, while increasing the number of anti-inflammatory M2 macrophages.ConclusionsEV-CPC do not trigger an immune response either in in vitro human allogeneic models or in immunocompetent animal models. The capacity for orienting the response of monocyte/macrophages towards resolution of inflammation strengthens the clinical attractiveness of EV-CPC as an acellular therapy for cardiac repair.

Background: Ischemic cardiovascular diseases, particularly acute myocardial infarction (MI), is one of the leading causes of mortality worldwide. Indoleamine 2, 3-dioxygenase 1 (IDO) catalyzes 1 rate-limiting step of L-tryptophan metabolism, and emerges as an important regulator of many pathological conditions. We hypothesized that IDO could play a key role to locally regulate cardiac homeostasis after MI. Methods: Cardiac repair was analyzed in mice harboring specific endothelial or smooth muscle cells or cardiomyocyte or myeloid cell deficiency of IDO and challenged with acute myocardial infarction. Results: We show that kynurenine generation through IDO is markedly induced after MI in mice. Total genetic deletion or pharmacological inhibition of IDO limits cardiac injury and cardiac dysfunction after MI. Distinct loss of function of IDO in smooth muscle cells, inflammatory cells, or cardiomyocytes does not affect cardiac function and remodeling in infarcted mice. In sharp contrast, mice harboring endothelial cell–specific deletion of IDO show an improvement of cardiac function as well as cardiomyocyte contractility and reduction in adverse ventricular remodeling. In vivo kynurenine supplementation in IDO-deficient mice abrogates the protective effects of IDO deletion. Kynurenine precipitates cardiomyocyte apoptosis through reactive oxygen species production in an aryl hydrocarbon receptor–dependent mechanism. Conclusions: These data suggest that IDO could constitute a new therapeutic target during acute MI.

Background and Purpose The side effects of cyclooxygenase-2 (COX-2) inhibitors on the cardiovascular system could be associated with reduced prostaglandin (PG)I2 synthesis. Microsomal PGE synthase-1 (mPGES-1) catalyses the formation of PGE2 from COX-derived PGH2. This enzyme is induced under inflammatory conditions and constitutes an attractive target for novel anti-inflammatory drugs. However, it is not known whether mPGES-1 inhibitors could be devoid of cardiovascular side effects. The aim of this study was to compare, in vitro, the effects of mPGES-1 and COX-2 inhibitors on vascular tone in human blood vessels. Experimental Approach The vascular tone and prostanoid release from internal mammary artery (IMA) and saphenous vein (SV) incubated for 30 min with inhibitors of mPGES-1 or COX-2 were investigated under normal and inflammatory conditions. Key Results In inflammatory conditions, mPGES-1 and COX-2 proteins were more expressed, and increased levels of PGE2 and PGI2 were released. COX-2 and NOS inhibitors increased noradrenaline induced vascular contractions in IMA under inflammatory conditions while no effect was observed in SV. Interestingly, the mPGES-1 inhibitor significantly reduced (30–40%) noradrenaline-induced contractions in both vessels. This effect was reversed by an IP (PGI2 receptor) antagonist but not modified by NOS inhibition. Moreover, PGI2 release was increased with the mPGES-1 inhibitor and decreased with the COX-2 inhibitor, while both inhibitors reduced PGE2 release. Conclusions and Implications In contrast to COX-2 inhibition, inhibition of mPGES-1 reduced vasoconstriction by increasing PGI2 synthesis. Targeting mPGES-1 could provide a lower risk of cardiovascular side effects, compared with those of the COX-2 inhibitors. Linked Articles This article is part of a themed section on Targeting Inflammation to Reduce Cardiovascular Disease Risk. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.22/issuetoc and http://onlinelibrary.wiley.com/doi/10.1111/bcp.v82.4/issuetoc

International audience; Ischemic cardiovascular diseases, particularly acute myocardial infarction (MI) is one of the leading cause of mortality worldwide. Indoleamine 2, 3-dioxygenase 1 (IDO) catalyzes one rate-limiting step of L-Tryptophan (Trp) metabolism, and emerges as an important regulator of many pathological conditions. We hypothesized that IDO could play a key role to locally regulate cardiac homeostasis after MI. METHODS: Cardiac repair was analyzed in mice harboring specific endothelial or smooth muscle cells or cardiomyocyte or myeloid cell deficiency of IDO and challenged with acute myocardial infarction. RESULTS: We show that Kynurenine (Kyn) generation through IDO is markedly induced after MI in mice. Total genetic deletion or pharmacological inhibition of IDO limits cardiac injury and cardiac dysfunction after MI. Distinct loss of function of IDO in smooth muscle cells, inflammatory cells, or cardiomyocytes does not impact cardiac function and remodeling in infarcted mice. In sharp contrast, mice harboring endothelial cell-specific deletion of IDO show an improvement of cardiac function, as well as cardiomyocyte contractility and reduction in adverse ventricular remodeling. In vivo Kyn supplementation in IDO-deficient mice abrogates the protective effects of IDO deletion. Notably, Kyn precipitates cardiomyocyte apoptosis through reactive oxygen species production in an aryl hydrocarbon receptor-dependent mechanism. CONCLUSIONS: These data suggest that IDO could constitute a new therapeutic target during acute MI.

Introduction Myocardial infarction (MI) is a severe ischemic disease precipitating long-term adverse remodeling and heart failure. The chemokine CXCL12/SDF-1 is essential for cardiovascular system development and plays a prominent role in physio-pathological processes such as inflammation, angiogenesis and tissue fibrosis. In addition to the binding to its cognate receptors CXCR4 and CXCR7, CXCL12 interacts with heparan-sulfates (HS) which coordinate its biological activity. We have previously highlighted the essential role of CXCL12/HS interactions in vascular growth and remodeling in the setting of critical limb ischemia. In addition, studies in experimental model of MI revealed a protective role for the CXCL12α isoform, through the regulation of cardiomyocyte survival and recruitment of inflammatory cells. However, in mice, three CXCL12 isoforms (α, β and γ) have been identified and, among them, the CXCL12γ isoform shows an unchallenged ability to cooperate with HS, suggesting a putative pivotal role in tissue repair. Objectives The aim of the study is to analyze the role of CXCL12γ isoform and the importance of CXCL12/HS interactions in post-ischemic cardiac remodeling in an acute model of MI. Methods MI was induced by permanent ligation of the left ascending coronary artery in mice carrying a Cxcl12 gene mutation that precludes interactions with HS (Cxcl12Gagtm) and in Cxcl12γ knock-in animals (Cxcl12γ-KI) harboring CXCL12γ deficiency. Alternatively, the impact of CXCL12γ overexpression and the importance of its interactions with HS was also evaluated in wild-type (WT) mice receiving transcutaneous echo-guided injections of adenovirus encoding WT Cxcl12γ or HS-binding-disabled Cxcl12γ in cardiac tissue. Cardiac function and remodeling have been assessed through echocardiography analysis, evaluation of infarct size, interstitial fibrosis, vascular growth (capillary and arteriole densities) and inflammatory cell infiltration into the cardiac tissue. Results After MI, Cxcl12Gagtm and Cxcl12γ-KI animals exhibit reduction in cardiac function and adverse left ventricular remodeling when compared to their respective WT littermates. Interestingly, overexpression of CXCL12γ in WT mice cardiac restored cardiac function by reducing the size of the infarcted area, interstitial fibrosis and promoting vascular growth. In sharp contrast, HS–binding disabled CXCL12gamma mutants failed to improve cardiac function and to abrogate adverse left ventricular remodeling. Conclusion We show that CXCL12γ isoform plays an important role in the regulation of post-ischemic cardiac function and remodeling and that its interactions with HS are essential for adequate cardiac repair in the setting of acute MI.

Background Myocardial infarction (MI) is a severe ischemic disease responsible for heart failure and sudden death. Mature B lymphocytes have been shown to exacerbate tissue injury and deterioration of cardiac function after MI. However, the cellular and molecular mechanisms governing B cell deleterious effects in the ischemic milieu remain to be defined. Purpose In this study, we speculate that endogenous activation of the miR21/HIFα-related pathways mediates the effect of B lymphocytes on post-ischemic cardiac remodeling. Methods Acute MI was induced by permanent ligation of the left anterior descending artery in mice. Cardiac function and remodeling was determined by echocardiography and immunohistochemistry. Inflammatory cell number and phenotype were defined by FACS analysis. To evaluate the role of HIFα isoforms in B cells, we generated mice with B cell lineage specific (Cd79aCre/+) conditional deletion of HIF1α (HIF1αflox/flox), HIF2α (HIF2αflox/flox), or both isoforms (HIF1α-HIF2αflox/flox). Results Acute MI increased miR21 levels in B cells. miR21 deficient mice showed reduced B cell numbers in the spleen, blood and subsequently in the injured cardiac tissue. Transplantation of bone marrow derived cells isolated from miR21-deficient mice (miR21−/−) improved cardiac function and remodeling when compared to administration of wild-type (WT) bone marrow cells. Similarly, in Rag1−/− immunodeficient mice with acute MI, re-supplementation with miR21−/− B lymphocytes restored cardiac repair and function when compared to injection of WT B cells. miR21 abrogated PTEN contents and subsequently enhanced HIF1α levels in cultured B cells. B cell deletion of HIF1α, but not that of HIF2α, reduced B cell accumulation and improved cardiac function after MI. Mice, which were equally deficient in HIF1α and HIF2α, also exhibited abrogation of adverse ventricular remodeling and showed recovery of cardiac function after MI. Toll like receptor agonist, CpG, fostered the release of the monocyte chemo-attractant protein, Ccl7, in cultured WT B cells but not in miR21- or HIF1α- deficient B cells. Ccl7 circulating levels were also reduced in miR21−/− and Cd79aCre/+/HIF1α flox/flox animals after acute MI. Ccl7 down-regulation hampered Ly6Chigh monocyte infiltration in the ischemic myocardium, leading to decreased infarct size and interstitial fibrosis, supporting cardiac repair. Conclusion This work reveals a novel function for miR21/HIF1α related pathways in B lymphocyte dependent effect on cardiac function and remodeling in the setting of acute MI.

Neutrophils have been implicated in the pathogenesis of atherosclerosis, a lipid-driven disease of arteries, but they are seldom found in atherosclerotic plaques. To resolve this longstanding paradox, we investigated whether neutrophil-derived microvesicles may influence arterial pathophysiology. Clinical and pre-clinical studies revealed that levels of circulating neutrophil microvesicles were enhanced by exposure to a high fat diet, a known risk factor for atherosclerosis. Neutrophil microvesicles accumulated at disease-prone regions of arteries that are exposed to complex flow patterns, and they promoted vascular inflammation and atherosclerosis in a murine model. Using cultured endothelial cells exposed to disturbed flow, it was demonstrated that neutrophil microvesicles promoted inflammatory gene expression by delivering a microRNA (miR-155) that enhanced NF-κB activation. Similary, neutrophil microvesicles increased miR-155 and enhanced NF-κB at disease-prone sites of disturbed flow in arteries of mice. We conclude that delivery of microvesicles carrying miR-155 to disease-prone regions of arteries provides a novel mechanism by which neutrophils contribute to vascular inflammation and atherogenesis.

Perivascular adipose tissue (PVAT) surrounds most vessels and has now been recognized as a regulator of vascular functions. This effect of PVAT has been mostly demonstrated in vessels obtained from rats and mice. Thus, the aim of this study was to investigate anti-contractile effect of PVAT surrounding human coronary bypass grafts such as saphenous vein (SV) and internal mammary artery (IMA). Moreover, we aimed to determine the involvement of prostanoids in the anticontractile effect of PVAT. Human SV and IMA preparations were set up in an organ bath. The presence of PVAT in SV and IMA preparations significantly attenuated the contractile response to noradrenaline (NA). Preincubation with indomethacin, a cyclooxygenase inhibitor, increased NA contraction in SV preparations with PVAT. This effect was not observed in IMA preparation with PVAT incubated with indomethacin. The lower measurements of prostaglandin E2 (PGE2) released from PVAT surrounding IMA versus SV supported these effects. In conclusion, our results show that PVAT of SV could attenuate NA-induced contraction by releasing both PGE2 and prostacyclin (PGI2). In contrast to SV, PVAT of IMA exerts its anti-contractile effect independently from prostanoids. These observations suggest that retaining PVAT in human SV and IMA preparations may have potential clinical implications to improve coronary bypass graft patency.

Prostacyclin (PGI2) and its mimetics (iloprost, treprostinil, beraprost and MRE-269) are potent vasodilators (via IP-receptor activation) and a major therapeutic intervention for pulmonary hypertension (PH). These PGI2 mimetics have anti-proliferative and potent vasodilator effects on pulmonary vessels. We compared the relaxant effects induced by these recognized IP-agonists in isolated human pulmonary arteries (HPA) and veins (HPV). In addition, using selective antagonists, the possible activation of other prostanoid relaxant receptors (DP, EP4) was investigated. Iloprost and treprostinil were the more potent relaxant agonists when both vessels were analyzed. HPA were significantly more sensitive to iloprost than to treprostinil, pEC50 values: 7.94 ± 0.06 (n = 23) and 6.73 ± 0.08 (n = 33), respectively. In contrast, in HPV these agonists were equipotent. The relaxations induced by treprostinil were completely or partially inhibited by IP-antagonists in HPA or HPV, respectively. The effects of the IP-agonists were not significantly modified by the EP4 antagonist. Finally, DP-antagonists inhibited the relaxations induced by treprostinil in HPV, suggesting that the DP-receptor plays a role in treprostinil-induced relaxation in the HPV. These data suggest that iloprost and treprostinil should be the most effective clinically available agonists to decrease pulmonary vascular resistance and to prevent oedema formation (by similar decrease in HPA and HPV resistance) in PH patients.

Prostaglandins (PG) are the product of a cascade of enzymes such as cyclooxygenases and PG synthases. Among PG, PGE 2 is produced by 3 isoforms of PGE synthase (PGES) and through activation of its cognate receptors (EP1–4), this PG is involved in the pathophysiology of vascular diseases. Some anti-inflammatory drugs (e.g. glucocorticoids, nonsteroidal anti-inflammatory drugs) interfere with its metabolism or effects. Vascular cells can initiate many of the responses associated with inflammation. In human vascular tissue, PGE 2 is involved in many physiological processes, such as increasing vascular permeability, cell proliferation, cell migration and control of vascular smooth muscle tone. PGE 2 has been shown to contribute to the pathogenesis of atherosclerosis, abdominal aortic aneurysm but also in physiologic/adaptive processes such as angiogenesis. Understanding the roles of PGE 2 and its cognate receptors in vascular diseases could help to identify diagnostic and prognostic biomarkers. In addition, from these recent studies new promising therapeutic approaches like mPGES-1 inhibition and/or EP4-antagonism should be investigated.

The side effects of cyclooxygenase-2 (COX-2) inhibitors on the cardiovascular system could be associated with reduced prostaglandin (PG)IThe vascular tone and prostanoid release from internal mammary artery (IMA) and saphenous vein (SV) incubated for 30 min with inhibitors of mPGES-1 or COX-2 were investigated under normal and inflammatory conditions.In inflammatory conditions, mPGES-1 and COX-2 proteins were more expressed, and increased levels of PGEIn contrast to COX-2 inhibition, inhibition of mPGES-1 reduced vasoconstriction by increasing PGIThis article is part of a themed section on Targeting Inflammation to Reduce Cardiovascular Disease Risk. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.22/issuetoc and http://onlinelibrary.wiley.com/doi/10.1111/bcp.v82.4/issuetoc.

Arterial vascularization of the spinal cord may be mechanically or functionally altered during thoraco-abdominal surgery/intravascular procedures. Increased arterial pressure has been shown to restore spinal perfusion and function probably by increasing the blood flow through the intercostal arteries. The regulation of human intercostal artery (HICA) vascular tone is not well documented. Prostaglandin (PG)E 2 concentration is increased during inflammatory conditions and has been shown to regulate vascular tone in many preparations. In this context, the pharmacological response of HICA to PGE 2 and the characterization of the PGE 2 receptor subtypes (EP 1 , EP 2 , EP 3 or EP 4 ) involved are of importance and that is the aim of this study. Rings of HICA were prepared from 29 patients and suspended in organ baths for isometric recording of tension. Cumulative concentration–response curves were performed in these preparations with various EP receptor agonists in the absence or presence of different receptor antagonists or inhibitors. PGE 2 induced the contraction of HICA (E max = 7.28 ± 0.16 g; pEC 50 value = 0.79 ± 0.18; n = 17); contractions were also observed with the EP 3 receptor agonists, sulprostone, 17-phenyl-PGE 2 , misoprostol or ONO-AE-248. In conclusion, PGE 2 induced vasoconstriction of HICA via EP 3 receptor subtypes and this result was confirmed by the use of selective EP receptor antagonists (L-826266, ONO-8713, SC-51322) and by a strong detection of EP 3 mRNA. These observations suggest that in the context of perioperative inflammation, increased PGE 2 concentrations could trigger vasoconstriction of HICA and possibly alter spinal vascularization.

BACKGROUND AND PURPOSE Human internal mammary arteries (IMA) and saphenous veins (SV) are frequently used for coronary artery bypass graft surgery. Intra- and postoperatively, the bypass grafts are exposed to inflammatory conditions, under which there is a striking increase in the synthesis of prostaglandin E2 (PGE2). In this context, the physiological response of these vascular grafts to PGE2 is highly relevant. The aim of this study was thus to characterize the PGE2 receptor subtypes (EP1, EP2, EP3 or EP4) involved in modulation of the vascular tone in these two vessels. EXPERIMENTAL APPROACH Rings of IMA and SV were prepared from 48 patients. The rings were mounted in organ baths for isometric recording of tension, and a pharmacological study was performed, together with associated reverse transcriptase PCR and immunohistochemistry experiments. KEY RESULTS PGE2 induced contractions of IMA (Emax= 1.43 ± 0.20 g; pEC50= 7.50 ± 0.10); contractions were also observed with the EP3 receptor agonists, sulprostone, 17-phenyl-PGE2, misoprostol or ONO-AE-248. In contrast, PGE2 induced relaxation of the precontracted SV (Emax=–0.22 ± 0.02 g; pEC50= 7.14 ± 0.09), as did the EP4 receptor agonist, ONO-AE1-329. These results were confirmed by the use of selective EP receptor antagonists (GW627368X, L-826266, ONO-8713, SC-51322) and by molecular biology and immunostaining. CONCLUSIONS AND IMPLICATIONS PGE2 induced potent and opposite effects on the human vascular segments used for grafting, namely vasoconstriction of the IMA and vasodilatation of the SV via EP3 and EP4 receptors respectively. These observations suggest that EP3 and EP4 receptors could constitute therapeutic targets to increase vascular graft patency.

Hydrogen sulfide (H2S) is a mediator with demonstrated protective effects for the cardiovascular system. On the other hand, prostaglandin (PG)E2 is involved in vascular wall remodeling by regulating matrix metalloproteinase (MMP) activities. We tested the hypothesis that endogenous H2S may modulate PGE2, MMP-1 activity and endogenous tissue inhibitors of MMPs (TIMP-1/-2). This regulatory pathway could be involved in thinning of abdominal aortic aneurysm (AAA) and thickening of saphenous vein (SV) varicosities. The expression of the enzyme responsible for H2S synthesis, cystathionine-γ-lyase (CSE) and its activity, were significantly higher in varicose vein as compared to SV. On the contrary, the endogenous H2S level and CSE expression were lower in AAA as compared to healthy aorta (HA). Endogenous H2S was responsible for inhibition of PGE2 synthesis mostly in varicose veins and HA. A similar effect was observed with exogenous H2S and consequently decreasing active MMP-1/TIMP ratios in SV and varicose veins. In contrast, in AAA, higher levels of PGE2 and active MMP-1/TIMP ratios were found versus HA. These findings suggest that differences in H2S content in AAA and varicose veins modulate endogenous PGE2 production and consequently the MMP/TIMP ratio. This mechanism may be crucial in vascular wall remodeling observed in different vascular pathologies (aneurysm, varicosities, atherosclerosis and pulmonary hypertension).

Background: Atherosclerosis is the most common underlying cause of cardiovascular disease. Many cells types are involved in the initiation and progression of atherosclerotic plaques but, until recently, neutrophils were not considered to be among them. Despite their rare detection in plaques, neutrophil depletion has been shown to reduce plaque formation and monocyte recruitment to the vessel wall. Hypothesis: Neutrophils may influence plaque initiation and progression through the formation of microvesicles. Aim: To determine the role of neutrophil-derived-microvesicles in the early stage of atherosclerosis. Methods and Results: Mouse peripheral blood neutrophils were isolated by negative magnetic separation. Following stimulation for 1h with fMLP (3x105M), supernatants were subjected to differential centrifugation (twice at 300g for 6 min followed by 20,000g for 20 mins) to isolate the microvesicle population. Microvesicles were quantified using flow cytometry calibrated with MegaMix and Spherocount beads. Apo E mice were fed a Western diet for 6 weeks and injected twice a week with PBS or neutrophil microvesicles (4.52x106 ± 3.05x105) isolated from control mice. After 6 weeks, blood was collected for neutrophil and microvesicle quantification. The aorta was dissected and stained by conventional procedures using Oil Red. Areas of plaque formation was determined using computer based imaging and semi-quantitative scoring. Injection with microvesicles increased circulating neutrophil number (PBS treated = 67.3 ± 3.9 neutrophils per μl; microvesicle treated = 88.25 ± 4.0 neutrophils per μl, P = 0.031; n = 3) and neutrophil reactivity with respect to microvesicle formation (PBS treated = 4.5 ± 0.8 microvesicles per neutrophil; microvesicle treated = 8.3±0.7 microvesicles per neutrophil, P = 0.0249; n = 3). After 6 weeks, the plaque formation was more advanced in the MVs group (% lesion area in the arch in mice injected with PBS = 0.4563 ± 0.09452, with MVs = 1.643 ± 0.3891, P = 0.0118, n=7). Conclusion: Neutrophil microvesicles induce increased plaque formation and increase circulating neutrophil levels in a mouse model of atherosclerosis. This suggests neutrophils may be involved in atherogenesis through the release of microvesicles.

Background Many cell types are involved in the initiation and progression of atherosclerosis but neutrophils are rarely considered to be among them. However, despite their infrequent detection in plaques, neutrophils have been shown to facilitate the movement of monocytes into the vessel wall and increase plaque growth. One possible mechanism for this is through release of microvesicles that contain cargo such as microRNAs (miRNAs). Hypothesis Neutrophils influence plaque initiation and progression by releasing miRNA-containg microvesicles. Aim To determine whether neutrophil microvesicles i) influence atherosclerotic plaque development and ii) contain miRNAs relevant to atherosclerosis. Methods and results Mouse blood neutrophils were stimulated for 1h with fMLP (3 × 10 5 M) and supernatants subjected to differential centrifugation (twice at 300 g for 6 min followed by 20,000 g for 20 mins). Microvesicles were quantified using calibrated flow cytometry. ApoE –/– mice were fed Western diet for 6 weeks and injected twice a week with sterile PBS or microvesicles (4 × 10 6 ) isolated from wild type mice. After 6 weeks aortae were dissected and stained with Oil Red-O to identify areas of plaque formation. To detect miRNA, human neutrophils were isolated and stimulated with fMLP (10 –5 M), AcLDL (20 µg/ml) or PBS for 2 h and microvesicles pelleted by differential centrifugation. MiRNA extraction and quantitative RT-PCR were performed in microvesicles, HUVEC and HUVEC incubated with microvesicles for 2 h. The number of neutrophils isolated from mice injected with microvesicles was increased (PBS = 67.3 ± 3.9/μl; microvesicles = 88.25 ± 4.0/μl, P = 0.031; n = 3 in each group). In addition neutrophil reactivity with respect to microvesicle formation was also increased (PBS = 4.5 ± 0.8 microvesicles per neutrophil; microvesicle = 8.3 ± 0.7 microvesicles per neutrophil, P = 0.0249; n = 3). Consequently plaque formation was more advanced in mice injected with microvesicles (% lesion area in aortic arch: PBS = 0.4563 ± 0.09452; microvesicles = 1.643 ± 0.3891, P = 0.0118; n = 7 in each group). MiR-223 and miR-150, found to be constitutively expressed in microvesicles and not in HUVEC, are involved in inflammation and atherosclerosis. These miRNAs were increased in after fMLP and AcLDL stimulation compared to PBS and were found to be significantly increased in HUVEC after 2h incubation with microvesicles (miR-223: 1.74 ± 0.44, P = 0.0305; 2.68 ± 0.50, P = 0.0034 and 6.79 ± 0.99, P = 0.0012 fold increase with microvesicles from PBS, fMLP and AcLDL stimulated neutrophils respectively and for miR-150: 3.79 ± 0.21, P = 0.0131 and 3.89 ± 0.78, P = 0.0109 fold increase with microvesicles from fMLP and AcLDL stimulated neutrophils respectively; n = 5 for each group). Conclusion Neutrophil microvesicles induce increased plaque formation and contain miRNA that may be transferred to endothelial cells suggesting neutrophils may be involved in atherogenesis through the release of microvesicles.

Varicose veins affect one-third of the adult population in western countries, but their pathogenesis is incompletely characterized. One of the most controversial issues is the role of inflammation. It is well known that inflammation involves an increased expression/activity of inflammatory mediators.The aim of this study was to investigate the presence or absence of mediators of inflammation in varicose as compared to healthy veins.Using immunohistofluorescence on varicose and healthy veins, we investigated the presence of inflammatory cells. They were not detectable. Venous wall C-reactive protein (CRP), fibrinogen (EIA) and pentraxin-3 (Western blot) content were measured. CRP was significantly lower in varicose veins, but no difference was found for fibrinogen or pentraxin-3 between varicose and healthy veins. No difference was observed for enzymes involved in inflammation and responsible for arachidonic acid metabolism such as the acute phase reactant secreted phospholipase A₂-IIA and cyclooxygenase-2, as determined in varicose and healthy veins by Western blot and real-time qRT-PCR.Our experiments demonstrate no increase in the presence of mediators of inflammation in varicose as compared to healthy veins, suggesting that inflammation may not be an important contributor to the pathogenesis of varicose veins.

Prostaglandin E(2) is produced in inflammatory responses via the cyclooxygenase pathway and regulates a variety of physiological and pathological reactions through four different receptor subtypes; EP(1), EP(2), EP(3) and EP(4). The role of the classical prostanoid receptors stimulated by prostaglandin I(2) and thromboxane A(2) in the blood circulation has been largely studied, whereas the other receptors such as EP activated by prostaglandin E(2), have been recently shown to be also implicated. There is now increasing evidence suggesting an important role of EP(3) and EP(4) receptor subtypes in the control of the human vascular tone and remodeling of the vascular wall as well in platelet aggregation and thrombosis. These receptors are implicated in vascular homeostasis and in the development of some pathological situations, such as atherosclerosis, aneurysms and hypertension. The use of specific EP agonists/antagonists would provide a novel cardiovascular therapeutic approach. In this review, we discuss the role of prostaglandin E(2) receptors in the control of human blood and vascular cells.

Arterial vascularization of the spinal cord may be mechanically or functionally altered during thoraco-abdominal surgery/intravascular procedures. Increased arterial pressure has been shown to restore spinal perfusion and function probably by increasing the blood flow through the intercostal arteries. The regulation of human intercostal artery (HICA) vascular tone is not well documented. Prostaglandin (PG)E(2) concentration is increased during inflammatory conditions and has been shown to regulate vascular tone in many preparations. In this context, the pharmacological response of HICA to PGE(2) and the characterization of the PGE(2) receptor subtypes (EP(1), EP(2), EP(3) or EP(4)) involved are of importance and that is the aim of this study. Rings of HICA were prepared from 29 patients and suspended in organ baths for isometric recording of tension. Cumulative concentration-response curves were performed in these preparations with various EP receptor agonists in the absence or presence of different receptor antagonists or inhibitors. PGE(2) induced the contraction of HICA (E(max)=7.28 ± 0.16 g; pEC(50) value=0.79 ± 0.18; n=17); contractions were also observed with the EP(3) receptor agonists, sulprostone, 17-phenyl-PGE(2), misoprostol or ONO-AE-248. In conclusion, PGE(2) induced vasoconstriction of HICA via EP(3) receptor subtypes and this result was confirmed by the use of selective EP receptor antagonists (L-826266, ONO-8713, SC-51322) and by a strong detection of EP(3) mRNA. These observations suggest that in the context of perioperative inflammation, increased PGE(2) concentrations could trigger vasoconstriction of HICA and possibly alter spinal vascularization.

Background Asthma and chronic obstructive pulmonary disease are characterized by inappropriate constriction of the airway smooth muscle. In this context, the physiological response of the human airways to selective relaxant agonists like PGE 2 is highly relevant. The aim of this study was thus to characterize the PGE 2 receptor subtypes (EP 2 or EP 4 ) involved in the relaxation of human bronchial preparations. Methods Human bronchial preparations cut as rings were mounted in organ baths for isometric recording of tension and a pharmacological study was performed using selective EP 2 or EP 4 ligands. Results In the presence of a thromboxane TP receptor antagonist and indomethacin, PGE 2 induced the relaxation of human bronchi (E max = 86 ± 04% of papaverine response; pEC 50 value = 7.06 ± 0.13; n = 6). This bronchodilation was significantly blocked by a selective EP 4 receptor antagonist (GW627368X, 1 and 10 μmol/L) with a pK B value of 6.38 ± 0.19 ( n = 5). In addition, the selective EP 4 receptor agonists (ONO-AE1-329; L-902688), but not the selective EP 2 receptor agonist (ONO-AE1-259), induced potent relaxation of bronchial preparations pre-contracted with histamine or anti-IgE. Conclusion PGE 2 and EP 4 agonists induced potent relaxations of human bronchial preparations via EP 4 receptor. These observations suggest that EP 4 receptor agonists could constitute therapeutic agents to treat the increased airway resistance in asthma.