Search

Background: Rapid and accurate new biomarkers to predict risk of cardiovascular disease (CVD) are essential. The utility of extracellular vesicles in predicting the CVD risk is postulated, yet it remains unknown whether their expression is altered in response to statin therapy. Methods: We performed in-vitro studies with human umbilical vein endothelial cells (HUVEC) and vascular smooth muscle cells (hVSMC), and conducted a nested case-control study (nCCS) in hypertensive patients (n = 40) randomized to either atorvastatin or placebo in the ASCOT-LLA. Cases had a major adverse cardiovascular event or death (MACE) during 3.5 years of follow-up (median) from the time of extracellular vesicle characterization while controls, matched for age and duration of treatment, remained event-free. Conditional logistic regression models determined the risk of MACE. Additionally, the relationship of extracellular vesicle levels with statin therapy was assessed. Results: Added to HUVEC, extracellular vesicles increased neutrophil recruitment, and to hVSMC, aggravated calcification and proliferation. In the nCCS, compared with controls, cases (i.e. with MACE) had preceding higher levels of CD14+ and CD14+/CD41+ extracellular vesicles (P = 0.009 and P = 0.012, respectively) and a significant reduction in the median size of the vesicles (P = 0.037). On matched analysis, higher CD14+ extracellular vesicles were associated with a 3.7-fold increased risk of MACE (P = 0.032). Patients treated with atorvastatin (vs. placebo) had both reduced size of extracellular vesicles and the proportion of CD146+ extracellular vesicles (P = 0.034 and P = 0.020, respectively). Conclusion and relevance: These pilot analyses suggest a mechanistic role for extracellular vesicles in the development of CVD, with significant and differential changes in extracellular vesicles amongst those at risk of MACE, and those on atorvastatin therapy.

Supplemental Digital Content is available in the text., Background: Ischemia reperfusion injury (I/RI) is a common complication of cardiovascular diseases. Resolution of detrimental I/RI-generated prothrombotic and proinflammatory responses is essential to restore homeostasis. Platelets play a crucial part in the integration of thrombosis and inflammation. Their role as participants in the resolution of thromboinflammation is underappreciated; therefore we used pharmacological and genetic approaches, coupled with murine and clinical samples, to uncover key concepts underlying this role. Methods: Middle cerebral artery occlusion with reperfusion was performed in wild-type or annexin A1 (AnxA1) knockout (AnxA1−/−) mice. Fluorescence intravital microscopy was used to visualize cellular trafficking and to monitor light/dye–induced thrombosis. The mice were treated with vehicle, AnxA1 (3.3 mg/kg), WRW4 (1.8 mg/kg), or all 3, and the effect of AnxA1 was determined in vivo and in vitro. Results: Intravital microscopy revealed heightened platelet adherence and aggregate formation post I/RI, which were further exacerbated in AnxA1−/− mice. AnxA1 administration regulated platelet function directly (eg, via reducing thromboxane B2 and modulating phosphatidylserine expression) to promote cerebral protection post-I/RI and act as an effective preventative strategy for stroke by reducing platelet activation, aggregate formation, and cerebral thrombosis, a prerequisite for ischemic stroke. To translate these findings into a clinical setting, we show that AnxA1 plasma levels are reduced in human and murine stroke and that AnxA1 is able to act on human platelets, suppressing classic thrombin-induced inside-out signaling events (eg, Akt activation, intracellular calcium release, and Ras-associated protein 1 [Rap1] expression) to decrease αIIbβ3 activation without altering its surface expression. AnxA1 also selectively modifies cell surface determinants (eg, phosphatidylserine) to promote platelet phagocytosis by neutrophils, thereby driving active resolution. (n=5–13 mice/group or 7–10 humans/group.) Conclusions: AnxA1 affords protection by altering the platelet phenotype in cerebral I/RI from propathogenic to regulatory and reducing the propensity for platelets to aggregate and cause thrombosis by affecting integrin (αIIbβ3) activation, a previously unknown phenomenon. Thus, our data reveal a novel multifaceted role for AnxA1 to act both as a therapeutic and a prophylactic drug via its ability to promote endogenous proresolving, antithromboinflammatory circuits in cerebral I/RI. Collectively, these results further advance our knowledge and understanding in the field of platelet and resolution biology.

Rationale Microvesicles (MV) act as a nonsoluble means of intercellular communication, with effector roles in disease pathogenesis and potentially as biomarkers. Previously, we reported that neutrophil MV expressing alpha-2-macroglobulin (A2MG) are protective in experimental sepsis and associate with survival in a small cohort of patients with sepsis due to community acquired pneumonia (CAP). Objectives To characterize MV profiles in sepsis due to CAP or fecal peritonitis (FP) and determine their relation to outcome. To investigate the effects of novel sepsis treatments (granulocyte-macrophage colony stimulating factor (GM-CSF) and interferon-υ (IFN-γ)) on MV production and functions in vitro. Methods Flow cytometry analysis of MV identified the cell of origin and the proportion of A2MG expression in the plasma of patients with sepsis secondary to CAP (n = 60) or FP (n = 40) and compared with healthy volunteers (HV, n = 10). The association between MV subsets and outcome was examined. The ability of GM-CSF and IFN-γ on A2MG MV production from whole blood was examined together with the assessment of their effect on neutrophil and endothelial functions. Results Circulating cell-derived and A2MG MV were higher in CAP compared with FP and HV. A2MG MV were higher in survivors of CAP, but not in FP. GM-CSF and IFN-γ enhanced A2MG MV production, with these MV eliciting pathogen clearance in vitro. Conclusions Plasma MV profiles vary according to the source of infection. A2MG MV are associated with survival in CAP but not FP. We propose specific MV subsets as novel biomarkers in sepsis and potential effector for some of the actions of experimental therapeutic interventions.

Objective— Neutrophil recruitment is a key process in the pathogenesis of stroke, and may provide a valuable therapeutic target. Targeting the melanocortin (MC) receptors has previously shown to inhibit leukocyte recruitment in peripheral inflammation, however, it is not known whether treatments are effective in the unique cerebral microvascular environment. Here, we provide novel research highlighting the effects of the MC peptides on cerebral neutrophil recruitment, demonstrating important yet discrete roles for both MC 1 and MC 3 . Approach and Results— Using intravital microscopy, in 2 distinct murine models of cerebral ischemia-reperfusion (I/R) injury, we have investigated MC control for neutrophil recruitment. After global I/R, pharmacological treatments suppressed pathological neutrophil recruitment. MC 1 selective treatment rapidly inhibited neutrophil recruitment while a nonselective MC agonist provided protection even when coadministered with an MC 3/4 antagonist, suggesting the importance of early MC 1 signaling. However, by 2-hour reperfusion, MC 1 -mediated effects were reduced, and MC 3 anti-inflammatory circuits predominated. Mice bearing a nonfunctional MC 1 displayed a transient exacerbation of neutrophil recruitment after global I/R, which diminished by 2 hours. However importantly, enhanced inflammatory responses in both MC 1 mutant and MC 3 −/− mice resulted in increased infarct size and poor functional outcome after focal I/R. Furthermore, we used an in vitro model of leukocyte recruitment to demonstrate these anti-inflammatory actions are also effective in human cells. Conclusions— These studies reveal for the first time MC control for neutrophil recruitment in the unique pathophysiological context of cerebral I/R, while also demonstrating the potential therapeutic value of targeting multiple MCs in developing effective therapeutics.

Rationale: Giant-cell arteritis (GCA) is a large-vessel vasculitis characterized by immune cell infiltration, yet the potential involvement of neutrophils has rarely been studied. Objective: We investigated whether alterations in neutrophil reactivity occurred in the pathogenesis of GCA or during its clinical management with a canonical glucocorticoid dose regimen during a 6-month period. Methods and Results: Blood samples were taken within 48 hours of therapy commencement and at weeks 1, 4, and 24 after glucocorticoid dose. Flow cytometric analysis revealed 3 distinct neutrophil populations and phenotypes. Within 48 hours of steroid treatment, neutrophils displayed an AnxA1 hi CD62L lo CD11b hi phenotype, whereas week 1 neutrophils were AnxA1 hi CD62L lo CD11b lo and displayed minimal adhesion to endothelial monolayers under flow, and week 24 (ie, lowest glucocorticoid dose) neutrophils were AnxA1 hi CD62L hi CD11b hi with increased endothelial adhesion under flow. Week 24 plasma analyses showed high levels of C-X-C motif chemokine ligand 5, interleukin (IL) 8, IL-17, and IL-6. Importantly, comparison of week 1 and week 24 samples revealed a suppressive neutrophil effect on T-cell proliferation at the former time point only. Finally, in vitro incubation of naive neutrophils with concentrations of IL-6 and IL-17 quantified in GCA plasma at weeks 1 and 24 replicated this differential modulation of lymphocyte proliferation. Conclusions: This translational study highlights a novel clinical manifestation of GCA, with evidence for a neutrophil component and an escaped proinflammatory phenotype when glucocorticoid therapy is tapered. These results indicate potential involvement of neutrophils in GCA pathogenesis.

Objective— Resolvin D1 (RvD1) limits neutrophil recruitment during acute inflammation and is derived from omega-3 docosahexaenoic acid to promote catabasis. The contribution of its specific receptors, the lipoxin A 4 /Annexin-A1 receptor formyl-peptide receptor 2 (FPR2/ALX) and the orphan receptor G-protein–coupled receptor 32 (GPR32) are of considerable interest. Methods and Results— RvD1 reduced human polymorphonuclear leukocytes recruitment to endothelial cells under shear conditions as quantified using a flow chamber system. Receptor-specific antibodies blocked these anti-inflammatory actions of RvD1, with low (1 nmol/L) concentrations sensitive to GPR32 blockade, while the higher (10 nmol/L) concentration appeared FPR2/ALX-specific. Interestingly, polymorphonuclear leukocytes surface expression of FPR2/ALX but not GPR32 increased following activation with pro-inflammatory stimuli, corresponding with secretory vesicle mobilization. Lipid mediator metabololipidomics carried out with 24-hour exudates revealed that RvD1 in vivo gave a significant reduction in the levels of a number of pro-inflammatory mediators including prostaglandins and leukotriene B 4 . These actions of RvD1 were abolished in fpr2 null mice. Conclusion— Pro-resolving lipid mediators and their receptors, such as RvD1 and the 2 G-protein–coupled receptors, studied here regulate resolution and may provide new therapeutic strategies for diseases with a vascular inflammatory component.

Objective— The anti-inflammatory properties of the female sex hormone estrogen have been linked to a reduced incidence of cardiovascular disease. In the present study, we addressed whether estrogen could activate vasculoprotective mechanisms via annexin A1 (AnxA1) mobilization in human polymorphonuclear cells (PMNs). Methods and Results— Using whole-blood flow cytometry, we demonstrated that premenopausal women expressed higher levels of surface AnxA1 on circulating PMNs compared with males. This correlated with high plasma estrogen during the menstrual cycle. The addition of estrogen in vitro to male PMNs induced rapid mobilization of AnxA1, optimal at 5 ng/mL and a 30-minute incubation period; this effect was abolished in the presence of the estrogen receptor antagonist ICI182780. Estrogen addition to human PMNs induced a distinct AnxA1 hi CD62L lo CD11b lo phenotype, and this was associated with lower cell activation as measured by microparticle formation. Treatment of human PMNs with E 2 inhibited cell adhesion to an endothelial cell monolayer under shear, which was absent when endogenous AnxA1 was neutralized. Of interest, addition of estrogen to PMNs flowed over the endothelial monolayer amplified its upregulation of AnxA1 localization on the cell surface. Finally, in a model of intravital microscopy, estrogen inhibition of white blood cell adhesion to the postcapillary venule was absent in mice nullified for AnxA1. Conclusion— We unveil a novel AnxA1-dependent mechanism behind the inhibitory properties of estrogen on PMN activation, describing a novel phenotype with a conceivable impact on the vasculoprotective effects of this hormone.

Objective— To determine whether the inhibitory action of the antiallergic cromone “mast cell stabilizing” drugs on polymorphonuclear leukocyte (PMN) trafficking is mediated through an annexin-A1 (Anx-A1) dependent mechanism. Methods and Results— Intravital microscopy was used to monitor the actions of cromones in the inflamed microcirculation. Reperfusion injury provoked a dramatic increase in adherent and emigrated leukocytes in the mesenteric vascular bed, associated with augmented tissue levels of myeloperoxidase. Nedocromil, 2 to 20 mg/kg, significantly ( P −/− mice. Short pretreatment of human PMNs with nedocromil, 10 nmol/L, inhibited cell adhesion ( P Conclusion— We propose a novel mechanism to explain the antiinflammatory actions of cromones on PMN trafficking, an effect that has long puzzled investigators.

Objective— The proinflammatory phenotype induced by low laminar shear stress (LSS) is implicated in atherogenesis. The kinin B1 receptor (B1R), known to be induced by inflammatory stimuli, exerts many proinflammatory effects including vasodilatation and leukocyte recruitment. We investigated whether low LSS is a stimulus for endothelial B1R expression and function. Methods and Results— Human and mouse atherosclerotic plaques expressed high level of B1R mRNA and protein. In addition, B1R expression was upregulated in the aortic arch (low LSS region) of ApoE −/− mice fed a high-fat diet compared to vascular regions of high LSS and animals fed normal chow. Of interest, a greater expression of B1R was noticed in endothelial cells from regions of low LSS in aortic arch of ApoE −/− mice. B1R was also upregulated in human umbilical vein endothelial cells (HUVECs) exposed to low LSS (0 to 2 dyn/cm 2 ) compared to physiological LSS (6 to 10 dyn/cm 2 ): an effect similarly evident in murine vascular tissue perfused ex vivo. Functionally, B1R activation increased prostaglandin and CXCL5 expression in cells exposed to low, but not physiological, LSS. IL-1β and ox-LDL induced B1R expression and function in HUVECs, a response substantially enhanced under low LSS conditions and inhibited by blockade of NFκB activation. Conclusion— Herein, we show that LSS is a major determinant of functional B1R expression in endothelium. Furthermore, whereas physiological high LSS is a powerful repressor of this inflammatory receptor, low LSS at sites of atheroma is associated with substantial upregulation, identifying this receptor as a potential therapeutic target.