Objective-Endothelial progenitor cells (EPCs) participate in vascular homeostasis and angiogenesis. The aim of the present study was to explore EPC number and function in relation to cardiovascular risk, gender, and reproductive state. Methods and Results-As measured by flow-cytometry in 210 healthy subjects, CD34ϩ KDR ϩ EPCs were higher in fertile women than in men, but were not different between postmenopausal women and age-matched men. These gender gradients mirrored differences in cardiovascular profile, carotid intima-media thickness, and brachial artery flowmediated dilation. Moreover, EPCs and soluble c-kit ligand varied in phase with menstrual cycle in ovulatory women, suggesting cyclic bone marrow mobilization. Experimentally, hysterectomy in rats was followed by an increase in circulating EPCs. EPCs cultured from female healthy donors were more clonogenic and adherent than male EPCs. Treatment with 17␤-estradiol stimulated EPC proliferation and adhesion, via estrogen receptors. Finally, we show that the proangiogenic potential of female EPCs was higher than that of male EPCs in vivo. Conclusions-EPCs

Human peripheral-blood monocytes are used as an established in vitro system for generating macrophages. For several reasons, monocytic cell lines such as THP-1 have been considered as a possible alternative. In view of their distinct developmental origins and phenotypic attributes, we set out to assess the extent to which human monocyte-derived macrophages (MDMs) and phorbol-12-myristate-13-acetate (PMA)-differentiated THP-1 cells were overlapping across a variety of responses to activating stimuli. Resting (M0) macrophages were polarized toward M1 or M2 phenotypes by 48-h incubation with LPS (1 μg/ml) and IFN-γ (10 ng/ml) or with IL-4 (20 ng/ml) and IL-13 (5 ng/ml), respectively. At the end of stimulation, MDMs displayed more pronounced changes in marker gene expression than THP-1. Upon assaying an array of 41 cytokines, chemokines and growth factors in conditioned media (CM) using the Luminex technology, secretion of 29 out of the 41 proteins was affected by polarized activation. While in 12 of them THP-1 and MDM showed comparable trends, for the remaining 17 proteins their responses to activating stimuli did markedly differ. Quantitative comparison for selected analytes confirmed this pattern. In terms of phenotypic activation markers, measured by flow cytometry, M1 response was similar but the established MDM M2 marker CD163 was undetectable in THP-1 cells. In a beads-based assay, MDM activation did not induce significant changes, whereas M2 activation of THP-1 decreased phagocytic activity compared to M0 and M1. In further biological activity tests, both MDM and THP-1 CM failed to affect proliferation of mouse myogenic progenitors, whereas they both reduced adipogenic differentiation of mouse fibro-adipogenic progenitor cells (M2 to a lesser extent than M1 and M0). Finally, migration of human umbilical vein endothelial cells was enhanced by CM irrespective of cell type and activation state except for M0 CM from MDMs. In summary, PMA-differentiated THP-1 macrophages did not entirely reproduce the response spectrum of primary MDMs to activating stimuli. We suggest that THP-1 be regarded as a simplified model of human macrophages when investigating relatively straightforward biological processes, such as polarization and its functional implications, but not as an alternative source in more comprehensive immunopharmacology and drug screening programs.

AIM/HYPOTHESIS: Monocytes/macrophages play important roles in adipose and vascular tissues and can be polarised as inflammatory M1 or anti-inflammatory M2. We sought to analyse monocyte polarisation status in type 2 diabetes, which is characterised by chronic inflammation. METH-ODS: We enrolled 60 individuals without diabetes and 53 patients with type 2 diabetes. We quantified standard monocyte subsets defined by cluster of differentiation (CD)14 and CD16. In addition, based on the phenotype of polarised macrophages in vitro, we characterised and quantified more definite M1 (CD68(+)CCR2(+)) and M2 (CX3CR1(+)CD206(+)/CD163(+)) monocytes. We also analysed bone marrow (BM) samples and the effects of granulocyte-colony stimulating factor (G-CSF) stimulation in diabetic and control individuals. RESULTS: We found no alterations in standard monocyte subsets (classical, intermediate and non-classical) when comparing groups. For validation of M1 and M2 phenotypes, we observed that M2 were enriched in non-classical monocytes and had lower TNF-content, higher LDL scavenging and lower transendothelial migratory capacity than M1. Diabetic patients displayed an imbalanced M1/M2 ratio compared with the control group, attributable to a reduction in M2. The M1/M2 ratio was directly correlated with waist circumference and HbA1c and, among diabetic patients, M2 reduction and M1/M2 increase were associated with microangiopathy. A decrease in M2 was also found in the BM from diabetic patients, with a relative M2 excess compared with the bloodstream. BM stimulation with G-CSF mobilised M2 macrophages in diabetic but not in healthy individuals. CON-CLUSIONS/INTERPRETATION: We show that type 2 diabetes markedly reduces anti-inflammatory M2 monocytes through a dysregulation in bone-marrow function. This defect may have a negative impact on microangiopathy. DOI: https://doi.org/10.1007/s00125-013-2918-9Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-83908 Accepted Version Originally published at: Fadini, G P; de Kreutzenberg, S Vigili; Boscaro, E; Albiero, M; Cappellari, R; Kränkel, N; Landmesser, U; Toniolo, A; Bolego, C; Cignarella, A; Seeger, F; Dimmeler, S; Zeiher, A; Agostini, C; Avogaro, A (2013). An unbalanced monocyte polarisation in peripheral blood and bone marrow of patients with type 2 diabetes has an impact on microangiopathy. Diabetologia, 56 (8) (CX3CR1+CD206+/CD163+) monocytes. We also analysed bone marrow (BM) samples and the effects of G-CSF stimulation in type 2 diabetic and control subjects.Results. We found no alterations in standard monocyte subsets (classical, intermediate, and nonclassical) between groups. For validation of M1 and M2 phenotypes, we show that M2 were enriched in nonclassical monocytes, had lower TNF-α content, higher LDL scavenging and lower transendothelial migratory capacity than M1. Diabetic patients displayed an imbalanced M1/M2 ratio compared to controls, which was attributable to a reduction in M2. The M1/M2 ratio was d...

A brief challenge of rat astrocytes with either a,b-methyleneATP (a,b-meATP) or basic ®broblast growth factor (bFGF) resulted, three days later, in morphological di erentiation of cells, as shown by marked elongation of astrocytic processes. The P2 receptor antagonist suramin prevented a,b-meATPbut not bFGF-induced astrocytic elongation. Similar e ects on astrocytic elongation were also observed with ATP and other P2 receptor agonists (b,gmeATP, ADPbS, 2meSATP and, to a lesser extent, UTP). 2 Pertussis toxin completely abolished a,b-meATP-but not bFGF-induced e ects. No e ects were exerted by a,b-meATP on cyclic AMP production; similarly, neomycin had no e ects on elongation of processes induced by the purine analogue, suggesting that adenylyl cyclase and phospholipase C are probably not involved in a,b-meATP-induced e ects (see also the accompanying paper by Centemeri et al., 1997). The tyrosine-kinase inhibitor genistein greatly reduced bFGF-but not a,b-meATP-induced astrocytic elongation. 3 Challenge of cultures with a,b-meATP rapidly and concentration-dependently increased [ 3 H]-arachidonic acid (AA) release from cells, suggesting that activation of phospholipase A 2 (PLA 2 ) may be involved in the long-term functional e ects evoked by purine analogues. Consistently, exogenously added AA markedly elongated astrocytic processes. Moreover, various PLA 2 inhibitors (e.g. mepacrine and dexamethasone) prevented both the early a,b-meATP-induced [ 3 H]-AA release and/or the associated long-term morphological changes, without a ecting the astrocytic elongation induced by bFGF. Finally, the protein kinase C (PKC) inhibitor H7 fully abolished a,b-meATP-but not bFGF-induced e ects. 4 Both a,b-meATP and bFGF rapidly and transiently induced the nuclear accumulation of Fos and Jun. Both c-fos and c-jun induction by the purine analogue could be fully prevented by pretreatment with suramin. In contrast, the e ects of bFGF were una ected by this P2 receptor antagonist. 5 It was concluded that a,b-meATP-and bFGF-morphological di erentiation of astrocytes occurs via independent transductional pathways. For the purine analogue, signalling involves a G i /G o proteincoupled P2Y-receptor which may be linked to activation of PLA 2 (involvement of an arachidonatesensitive PKC is speculated); for bFGF, a tyrosine kinase receptor is involved. Both pathways merge on some common intracellular target, as suggested by induction of primary response genes, which in turn may regulate late response genes mediating long-term phenotypic changes of astroglial cells. 6 These ®ndings implicate P2 receptors as novel targets for the pharmacological regulation of reactive astrogliosis, which has intriguing implications in nervous system diseases characterized by degenerative events.

Estrogen plays an important role in the regulation of cardiovascular physiology and the immune system by inducing direct effects on multiple cell types including immune and vascular cells. Sex steroid hormones are implicated in cardiovascular protection, including endothelial healing in case of arterial injury and collateral vessel formation in ischemic tissue. Estrogen can exert potent modulation effects at all levels of the innate and adaptive immune systems. Their action is mediated by interaction with classical estrogen receptors (ERs), ERα and ERβ, as well as the more recently identified G-protein coupled receptor 30/G-protein estrogen receptor 1 (GPER1), via both genomic and non-genomic mechanisms. Emerging data from the literature suggest that estrogen deficiency in menopause is associated with an increased potential for an unresolved inflammatory status. In this review, we provide an overview through the puzzle pieces of how 17β-estradiol can influence the cardiovascular and immune systems.

Smoking is a major cause of coronary heart disease for both men and women and a positive correlation between tobacco use and cerebrovascular disease has been also described. In addition, cigarette smoking is the most powerful risk factor predisposing to atherosclerotic peripheral artery disease. More recently, passive smoking has been also shown to represent an important risk factor for coronary artery disease. Moreover, the incidence of coronary artery and cerebrovascular diseases in ex-smokers consistently decreases after cessation, further underlying the relevance of smoking as a risk factor for these pathological conditions. The effects of cigarette smoking on atherosclerosis initiation and progression as well on its complications are mostly responsible for the enhanced cardio- and cerebrovascular risk observed in smoking compared to non-smoking subjects. Since hormonal status may also play a role in the development and stability of the atherosclerotic plaque, smoking habits could influence the clinical complications of atheroclerosis in a gender dependent manner. Up to now, however, few studies have investigated the relative importance of smoking as a risk factor for fatal and non-fatal diseases in the two sexes within the same study population. On the basis of available clinical data, this review will discuss the risk of fatal and non-fatal diseases among smoking men and women with special emphasis on cardiovascular and cerebrovascular disease which also represents the most common cause of death among smokers. A description of the mechanisms involved in the tobacco-induced atherosclerotic damage will be also given in order to underline possible gender-related differences.

Abstract:The inflammatory component of atherogenesis has been increasingly recognized over the last decade. Inflammation participates in all stages of atherosclerosis, not only during initiation and during evolution of lesions, but also with precipitation of acute thrombotic complications. The metabolic syndrome is associated with increased risk for development of both cardiovascular disease and type-2 diabetes in humans. Central obesity and insulin resistance are thought to represent common underlying factors of the syndrome, which features a chronic low-grade inflammatory state. Diagnosis of the metabolic syndrome occurs using defined threshold values for waist circumference, blood pressure, fasting glucose and dyslipidemia. The metabolic syndrome appears to affect a significant proportion of the population. Therapeutic approaches that reduce the levels of proinflammatory biomarkers and address traditional risk factors are particularly important in preventing cardiovascular disease and, potentially, diabetes. The primary management of metabolic syndrome involves healthy lifestyle promotion through moderate calorie restriction, moderate increase in physical activity and change in dietary composition. Treatment of individual components aims to control atherogenic dyslipidemia using fibrates and statins, elevated blood pressure, and hyperglycemia. While no single treatment for the metabolic syndrome as a whole yet exists, emerging therapies offer potential as future therapeutic approaches. Keywords: metabolic syndrome, systemic inflammation, coronary artery disease Metabolic syndromeIt has been known for more than 40 years that the risk factors for coronary heart disease (CHD) include hypertension, elevated levels of low-density lipoprotein cholesterol (LDL-C), smoking, and type-2 diabetes. Yet, extensive research has established that several of these cardiovascular risk factors cluster to a greater degree than can be explained by chance. In the last few years, this clustering of symptoms has been ascribed to a specific condition: the metabolic syndrome (MS). The MS is a cluster of the most dangerous heart attack risk factors: diabetes or prediabetes, abdominal obesity, changes in cholesterol and high blood pressure (Eckel et al 2005). The basis for this aggregation of metabolic disorders has been investigated in an epidemiological analysis carried out in the adult population of a small Pacific island, leading to the identification of four independent factors that explained 73% of the total variance (Shmulewitz et al 2001). This refinement in the clustering, however, is lost when factors are all packaged together. In a re-analysis of the West of Scotland Coronary Prevention Study (WOSCOPS), risk prediction increased with the number of metabolic abnormalities (Sattar et al 2003). Evidence suggests that it is the exact nature of the cluster which appears to bring additional risk over and above that which would be expected from each of the components (Reilly and Rader 2003). It has been argued, though, that the comb...

Estrogen treatment prevented LPS/IFN-γ action on human M2 macrophage markers and cytokine production, whereas menopausal estrogen loss was associated with an impaired response to alternative activation, suggesting that these mechanisms affect the cardiovascular risk profile in relation to menopausal status.