Increased levels of microparticles originating from endothelial cells, platelets and erythrocytes in subjects with metabolic syndrome: relationship with oxidative stress

Background and aims The Metabolic Syndrome (MetS) is associated with increased cardiovascular risk. Circulating microparticles (MP) are involved in the pathogenesis of atherothrombotic disorders and are raised in individual with CVD. We measured their level and cellular origin in subjects with MetS and analyzed their associations with 1/anthropometric and biological parameters of MetS, 2/inflammation and oxidative stress markers. Methods and results Eighty-eight subjects with the MetS according to the NCEP-ATPIII definition were enrolled in a bicentric study and compared to 27 healthy controls. AnnexinV-positive MP (TMP), MP derived from platelets (PMP), erythrocytes (ErMP), endothelial cells (EMP), leukocytes (LMP) and granulocytes (PNMP) were determined by flow cytometry. MetS subjects had significantly higher counts/μl of TMP (730.6 ± 49.7 vs 352.8 ± 35.6), PMP (416.0 ± 43.8 vs 250.5 ± 23.5), ErMP (243.8 ± 22.1 vs 73.6 ± 19.6) and EMP (7.8 ± 0.8 vs 4.0 ± 1.0) compared with controls. LMP and PNMP were not statistically different between groups. Multivariate analysis demonstrated that each criterion for the MetS influenced the number of TMP. Waist girth was a significant determinant of PMP and EMP level and blood pressure was correlated with EMP level. Glycemia positively correlated with PMP level whereas dyslipidemia influenced EMP and ErMP levels. Interestingly, the oxidative stress markers, plasma glutathione peroxydase and urinary 8-iso-prostaglandin F2 α, independently influenced TMP and PMP levels whereas inflammatory markers did not, irrespective of MP type. Conclusion Increased levels of TMP, PMP, ErMP and EMP are associated with individual metabolic abnormalities of MetS and oxidative stress. Whether MP assessment may represent a marker for risk stratification or a target for pharmacological intervention deserves further investigation.

Effect of olive oil on immune function in middle-aged men

Consumption of diets rich in monounsaturated fatty acids (MUFAs) has been linked with a low prevalence of atherosclerosis and there has been great interest in the effects of MUFAs on lipoprotein metabolism. Less attention has been paid to the effects of MUFAs on the immune system, yet cells of the immune system are an inherent part of the inflammatory events involved in atherosclerosis and several animal studies showed that olive oil has some potent immunomodulatory actions. We therefore considered it important to investigate the effects of chronic consumption of MUFAs on several immune cell functions in healthy humans. Healthy middle-aged males entered a doubleblind, randomized, controlled trial in which they consumed either a MUFA diet or a control diet for 2 mo. There was a significant decrease in the expression of intercellular adhesion molecule 1 by peripheral blood mononuclear cells from subjects consuming the MUFA diet. Consumption of the MUFA diet did not affect natural killer cell activity or proliferation of mitogen-stimulated leukocytes. The effects of a MUFA-rich diet on adhesion molecule expression may have implications for the influence of dietary fat on inflammatory diseases, including atherosclerosis.

Collagen, convulxin, and thrombin stimulate aggregation-independent tyrosine phosphorylation of CD31 in platelets. Evidence for the involvement of Src family kinases

Platelet endothelial cell adhesion molecule-1 (CD31) is a 130-kDa glycoprotein receptor present on the surface of platelets, neutrophils, monocytes, certain T-lymphocytes, and vascular endothelial cells. CD31 is involved in adhesion and signal transduction and is implicated in the regulation of a number of cellular processes. These include transendothelial migration of leukocytes, integrin regulation, and T-cell function, although its function in platelets remains unclear. In this study, we demonstrate the ability of the platelet agonists collagen, convulxin, and thrombin to induce tyrosine phosphorylation of CD31. Furthermore, we show that this event is independent of platelet aggregation and secretion and is accompanied by an increase in surface expression of CD31. A kinase capable of phosphorylating CD31 was detected in CD31 immunoprecipitates, and its activity was increased following activation of platelets. CD31 tyrosine phosphorylation was reduced or abolished by the Src family kinase inhibitor PP2, suggesting a role for these enzymes. In accordance with this, each of the Src family members expressed in platelets, namely Fyn, Lyn, Src, Yes, and Hck, was shown to co-immunoprecipitate with CD31. The involvement of Src family kinases in this process was confirmed through the study of mouse platelets deficient in Fyn.

Expression of cyclooxygenase-2 in intestine of pigs of different ages and hygiene status

Prostaglandins (PG) are bioactive lipids derived from the metabolism of membrane polyunsaturated fatty acids (PUFA), and play important roles in a number of biological processes including cell division, immune responses and wound healing. Cyclooxygenase (COX) is the key enzyme in PG synthesis from arachidonic acid. The hypothesis of the present study was that expression of COX-2 in porcine intestine was dependent on the microbial load and the age of piglets. Piglets were obtained from sows raised either on outdoor free-range farms or on indoor commercial farms, and littermates were divided into three treatments: One group of piglets suckled the sow, a second group was put into an isolator and fed a milk formula, and a third group was put into the isolator fed milk formula and injected with broad spectrum antibiotics. Samples were collected from the 75% level of the small intestine at day 5, 28 and 56 of age. Tissue section from four piglets from each of these six treatment groups was analysed by immunofluorescence for COX-2 and type-IV collagen (basement membrane, defining lamina propria (LP)). Image analysis was used to determine the number of positive pixels expressing LP and epithelial COX-2. COX-2 expressing cells were observed in LP and epithelium in all porcine intestinal samples. When analysing images obtained on day 28, injection of antibiotics seemed to reduce the COX-2 expression in intestinal samples of piglets when compared to other treatments (P=0.053). No significant effect of farm, treatments or age of piglets was observed on COX-2 expressing data when analysing all data of images obtained at day 28 and 56. By double-labelling experiments, COX-2 was found not to be expressed on cell co-expressing CD45, CD16, CD163 or CD2, thus indicating that mucosal leukocytes, including dendritic cells, macrophages and NK cells did not express COX-2. Future research should investigate the role of COX-2 expression in the digestive tract in relation to pig health.

Flavonoid inhibitory pharmacodynamics on platelet function in physiological environments

The complex relationship between flavonoid-based nutrition and cardiovascular disease may be dissected by understanding the activities of these compounds in biological systems. The aim of the present study was to explore a hierarchy for the importance of dietary flavonoids on cardiovascular health by examining the structural basis for inhibitory effects of common, dietary flavonoids (quercetin, apigenin, and naringenin) and the plasma metabolite, tamarixetin. Understanding flavonoid effects on platelets in vivo can be informed by investigations of the ability of these compounds to attenuate the function of these cells. Inhibition of platelet function in whole blood and plasma was structure-dependent. The order of potency was apigenin > tamarixetin > quercetin = naringenin indicating that in vivo, important functional groups are potentially a methylated B ring, and a non-hydroxylated, planar C ring. Apigenin and the methylated metabolite of quercetin, tamarixetin significantly reduced thrombus volume at concentrations (5 μM) that suggested their reported physiological levels (0.1-1 μM) may exert low levels of inhibition. Flavonoid interactions with erythrocytes, leukocytes and human serum albumin in whole blood reduce their inhibitory activities against platelet function. The diminished inhibitory activity of flavonoids that we observed in whole blood and plasma indicated that these interactions do not overcome the attenuating effects of these compounds. Furthermore, inhibition of platelet aggregation by flavonoids was enhanced with increases in exposure time, indicating the potential for measurable inhibitory effects during resident plasma times. We conclude that flavonoid structures may be a major influence of their activities in vivo with methylated metabolites and those of flavones being more potent than those of flavonols and flavanones.

Targeting stem cell niches and trafficking for cardiovascular therapy.

Regenerative cardiovascular medicine is the frontline of 21st-century health care. Cell therapy trials using bone marrow progenitor cells documented that the approach is feasible, safe and potentially beneficial in patients with ischemic disease. However, cardiovascular prevention and rehabilitation strategies should aim to conserve the pristine healing capacity of a healthy organism as well as reactivate it under disease conditions. This requires an increased understanding of stem cell microenvironment and trafficking mechanisms. Engagement and disengagement of stem cells of the osteoblastic niche is a dynamic process, finely tuned to allow low amounts of cells move out of the bone marrow and into the circulation on a regular basis. The balance is altered under stress situations, like tissue injury or ischemia, leading to remarkably increased cell egression. Individual populations of circulating progenitor cells could give rise to mature tissue cells (e.g. endothelial cells or cardiomyocytes), while the majority may differentiate to leukocytes, affecting the environment of homing sites in a paracrine way, e.g. promoting endothelial survival, proliferation and function, as well as attenuating or enhancing inflammation. This review focuses on the dynamics of the stem cell niche in healthy and disease conditions and on therapeutic means to direct stem cell/progenitor cell mobilization and recruitment into improved tissue repair.

Protease-activated receptor 2 mediates eosinophil infiltration and hyperreactivity in allergic inflammation of the airway

Trypsin and mast cell tryptase can signal to epithelial cells, myocytes, and nerve fibers of the respiratory tract by cleaving proteinase-activated receptor 2 (PAR2). Since tryptase inhibitors are under development to treat asthma, a precise understanding of the contribution of PAR2 to airway inflammation is required. We examined the role of PAR2 in allergic inflammation of the airway by comparing OVA-sensitized and -challenged mice lacking or overexpressing PAR2. In wild-type mice, immunoreactive PAR2 was detected in airway epithelial cells and myocytes, and intranasal administration of a PAR2 agonist stimulated macrophage infiltration into bronchoalveolar lavage fluid. OVA challenge of immunized wild-type mice stimulated infiltration of leukocytes into bronchoalveolar lavage and induced airway hyperreactivity to inhaled methacholine. Compared with wild-type animals, eosinophil infiltration was inhibited by 73% in mice lacking PAR2 and increased by 88% in mice overexpressing PAR2. Similarly, compared with wild-type animals, airway hyperreactivity to inhaled methacholine (40 micro g/ml) was diminished 38% in mice lacking PAR2 and increased by 52% in mice overexpressing PAR2. PAR2 deletion also reduced IgE levels to OVA sensitization by 4-fold compared with those of wild-type animals. Thus, PAR2 contributes to the development of immunity and to allergic inflammation of the airway. Our results support the proposal that tryptase inhibitors and PAR2 antagonists may be useful therapies for inflammatory airway disease.

Targeted activation of toll-like receptors: conjugation of a toll-like receptor 7 agonist to a monoclonal antibody maintains antigen binding and specificity

Therapeutic activation of Toll-like receptors (TLR) has potential for cancer immunotherapy, for augmenting the activity of anti-tumor monoclonal antibodies (mAbs), and for improved vaccine adjuvants. A previous attempt to specifically target TLR agonists to dendritic cells (DC) using mAbs failed because conjugation led to non-specific binding and mAbs lost specificity. We demonstrate here for the first time the successful conjugation of a small molecule TLR7 agonist to an anti-tumour mAb (the anti-hCD 20 rituximab) without compromising antigen specificity. The TLR7 agonist UC-1V150 was conjugated to rituximab using two conjugation methods and yield, molecular substitution ratio, retention of TLR7 activity and specificity of antigen binding were compared. Both conjugation methods produced rituximab-UC-1V150 conjugates with UC-1V150 : rituximab ratio ranging from 1:1 to 3:1 with drug loading quantified by UV spectroscopy and drug substitution ratio verified by MALDI TOF mass spectroscopy. The yield of purified conjugates varied with conjugation method, and dropped as low as 31% using a method previously described for conjugating UC-1V150 to proteins, where a bifunctional crosslinker was firstly reacted with rituximab, and secondly to the TLR7 agonist. We therefore developed a direct conjugation method by producing an amine-reactive UV active version of UC-1V150, termed NHS:UC-1V150. Direct conjugation with NHS:UC-1V150 was quick and simple and gave improved conjugate yields of 65-78%. Rituximab-UC-1V150 conjugates had the expected pro-inflammatory activity in vitro (EC50 28-53 nM) with a significantly increased activity over unconjugated UC-1V150 (EC50 547 nM). Antigen binding and specificity of the rituxuimab-UC-1V150 conjugates was retained, and after incubation with human peripheral blood leukocytes, all conjugates bound strongly only to CD20-expressing B cells whilst no non-specific binding to CD20-negative cells was observed. Selective targeting of Toll-like receptor activation directly within tumors or to DC is now feasible.