Systemic and vascular inflammation in experimental allergic asthma.

Allergic asthma and atherosclerosis are inflammatory diseases characterized by similar sets of circulating inflammatory cells, in addition to mast cells in the airway and vessel wall. Animal models and human studies provide evidence of a potential interaction between the two apparently unrelated diseases. The main objective of this study was to determine whether experimental allergic asthma is accompanied by inflammatory responses, measured as the activation of the vasculature and the presence of immune cells in the perivascular adipose tissue. For this purpose, male Dunkin Hartley guinea pigs weighing 250 - 300 g were sensitized twice with 10 µg ovalbumin dissolved in aluminium hydroxide (Al(OH)3). Allergen inhalation was performed 10 days after the second immunization and continued 5 days a week for 2 months. After that period, T cell and macrophage content was measured by flow cytometry. The aortic expression of inflammatory markers was studied by real-time PCR. The number of T cells in the peripheral blood was significantly greater in the allergic group in comparison to the sham group. We did not find any significant differences in the leukocyte content of the perivascular adipose tissue between the groups. Nor did we identify significant changes in the expression of inflammatory markers (tumor necrosis factor, monocyte chemoattractant protein-1) and adhesion molecules (intercellular adhesion molecules and vascular cell adhesion molecules) in the aorta. Interestingly, we observed a significantly decreased expression of the endothelial nitric oxide synthase (eNOS) mRNA in the aortic vessel of the allergic group compared to the sham group.

Nox1/4 inhibition exacerbates age dependent perivascular inflammation and fibrosis in a model of spontaneous hypertension.

Hypertension is associated with oxidative stress and perivascular inflammation, critical contributors to perivascular fibrosis and accelerated vascular ageing. Oxidative stress can promote vascular inflammation, creating options for potential use of NADPH oxidase inhibitors in pharmacological targeting of perivascular inflammation and its consequences. Accordingly, we characterized age-related changes in oxidative stress and immune cell infiltration in normotensive (WKY) and spontaneously hypertensive rats (SHRs). Subsequently, we used pharmacological inhibitors of Nox1 (ML171) and Nox1/Nox4 (GKT137831; 60 mg/kg), to modulate NADPH oxidase activity at the early stage of spontaneous hypertension and investigated their effects on perivascular inflammation and fibrosis. RESULTS: Ageing was associated with a progressive increase of blood pressure as well as an elevation of the total number of leukocytes, macrophages and NK cells infiltrating perivascular adipose tissue (PVAT) in SHRs but not in WKY. At 1 month of age, when blood pressure was not yet different, only perivascular NK cells were significantly higher in SHR. Spontaneous hypertension was also accompanied by the higher perivascular T cell accumulation, although this increase was age independent. Aortic Nox1 and Nox2 mRNA expression increased with age only in SHR but not in WKY, while age-related increase of Nox4 mRNA in the vessels has been observed in both groups, it was more pronounced in SHRs. At early stage of hypertension (3-months) the most pronounced differences were observed in Nox1 and Nox4. Surprisingly, GKT137831, dual inhibitor of Nox1/4, therapy increased both blood pressure and perivascular macrophage infiltration. Mechanistically, this was linked to increased expression of proinflammatory chemokines expression (CCL2 and CCL5) in PVAT. This inflammatory response translated to increased perivascular fibrosis. This effect was likely Nox4 dependent as the Nox1 inhibitor ML171 did not affect the development of spontaneous hypertension, perivascular macrophage accumulation, chemokine expression nor adventitial collagen deposition. In summary, spontaneous hypertension promotes ageing-associated perivascular inflammation which is exacerbated by Nox4 but not Nox1 pharmacological inhibition.

Significance of sphingosine-1-phosphate in cardiovascular physiology and pathology.

Sphingosine-1-phosphate (S1P) is a signaling lipid, synthetized by sphingosine kinases (SPHK1 and SPHK2), that affects cardiovascular function in various ways. S1P signaling is complex, particularly since its molecular action is reliant on the differential expression of its receptors (S1PR1, S1PR2, S1PR3, S1PR4, S1PR5) within various tissues. Significance of this sphingolipid is manifested early in vertebrate development as certain defects in S1P signaling result in embryonic lethality due to defective vasculo- or cardiogenesis. Similar in the mature organism, S1P orchestrates both physiological and pathological processes occurring in the heart and vasculature of higher eukaryotes. S1P regulates cell fate, vascular tone, endothelial function and integrity as well as lymphocyte trafficking, thus disbalance in its production and signaling has been linked with development of such pathologies as arterial hypertension, atherosclerosis, endothelial dysfunction and aberrant angiogenesis. Number of signaling mechanisms are critical - from endothelial nitric oxide synthase through STAT3, MAPK and Akt pathways to HDL particles involved in redox and inflammatory balance. Moreover, S1P controls both acute cardiac responses (cardiac inotropy and chronotropy), as well as chronic processes (such as apoptosis and hypertrophy), hence numerous studies demonstrate significance of S1P in the pathogenesis of hypertrophic/fibrotic heart disease, myocardial infarction and heart failure. This review presents current knowledge concerning the role of S1P in the cardiovascular system, as well as potential therapeutic approaches to target S1P signaling in cardiovascular diseases.

The role of chemokine CCL5/RANTES and metalloproteinase-9 as inflammatory modulators in symptomatic internal carotid artery stenosis.

Up to 80% of all ischemic strokes (IS) attributed to internal carotid athero-occlusive artery stenosis (ICAS) are related to a thromboembolic mechanism. One athero-occlusive ischemic event increases the risk for ischemia in another vascular territory, resulting from inflammation within the atherosclerotic plaque induced by cytokines. Thus, ultrasonographic characteristics of vulnerable plaques in ICAS, including plaque echolucency and ulceration might correspond to cytokine activity. The present study aimed to investigate the associations between serum cytokines and atherosclerotic plaque characteristics and the 3-year risk of a major adverse coronary and carotid ischemic event (MACCE) in symptomatic patients treated for ICAS. Plaque characteristics on ultrasonography, serum levels of C-C motif chemokine ligand 5 (CCL5)/regulated on activation, normal T-cell expressed and secreted (RANTES), metalloproteinase-9 (MMP-9), interleukin-6 (IL-6), transforming growth factor beta (TGF-ß), C-X-C motif chemokine ligand 16 (CXCL16), FAS ligand (FASL) and high sensivity C-reactive protein (hs-CRP) were analyzed in 103 symptomatic patients with ICAS prior to carotid revascularization. The incidence of MACCE: cardiovascular death (CVD), myocardial infarction (MI) and recurrent ischemic stroke (IS) were recorded prospectively for up to 5 years (median 37; IQR 21 - 40 months). Echolucent plaques, in comparison to echogenic plaques, displayed lower median levels of RANTES (P = 0.042) but higher median levels of IL-6 (P = 0.039). There was no relationship between plaque characteristics and median levels of MMP-9, TGF ß, CXCL16, FASL, or hs-CRP (P = NS). During follow-up, MACCE occurred in 15 (14.6%) patients. Univariate Cox proportional hazard analysis indicated median RANTES levels < 45.5ng/mL (hazard ratio (HR) = 3.95; 95%CI = 1.10 - 14.2; P = 0.035), MMP-9 > 0.6 µg/mL (HR 4.5; 95%CI = 1.4 - 13.9; P = 0.009), renal impairment (HR 3.48; 95%CI = 1.29 - 9.34; P = 0.013) as potential MACCE risk factors. On multivariate Cox proportional hazard analysis, MMP-9 > 0.6 µg/mL and RANTES < 45.5 ng/ml were associated with a 4.72-fold (95%CI = 1.3 - 17.0; P = 0.017) and a 3.8-fold risk increase (95%CI = 1.07 - 13.89; P = 0.038) of MACCE. Kaplan-Meier analysis showed significant differences in MACCE-free survival rates depending on RANTES and MMP-9 median levels. We conclude that serum RANTES, IL-6, and MMP-9 were associated with plaque vulnerability and predicted adverse MACCE in patients treated for ICAS.

Serum rantes, transforming growth factor-ß1 and interleukin-6 levels correlate with cardiac muscle fibrosis in patients with aortic valve stenosis.

Regulated on Activation Normal T Expressed and Secreted (RANTES) chemokine is involved in the initiation of inflammation and immune-cell recruitment. Interleukin -6 (IL-6) is used as a general index of severity of the chronic inflammatory process. Finally, transforming growth factor-ß (TGF-ß) is an immune biomarker potentially involved in the regulation of valve fibrosis and calcification. The aim of this study was to analyze selected biomarkers associated with the different stages of immune-pathogenesis in aortic stenosis. Forty consecutive patients with moderate to severe aortic stenosis (AS) and without previous myocardial infarction history were included in the study and divided into two groups. Two imaging techniques, echocardiography and magnetic resonance, were used to estimate the degree of AS and left ventricular muscle function. Inflammatory biomarker serum levels including CCL5/RANTES, IL-6, and TGF-ß1 were determined based on ELISA measurements. Mean levels of RANTES, IL-6, and TGF-ß1 did not significantly differ between both groups. A negative correlation was found between RANTES serum level and left ventricle (LV) mass as assessed by MRI (r = -0.3358, P = 0.0341). A positive correlation (r = 0.3283, P = 0.0387) was found between IL-6 serum levels and LV mass as measured by MRI. In addition, a positive correlation (r = 0.6803, P = 0.01) was seen between IL-6 serum levels and LV muscle mass with positive late gadolinium enhancement (LGE). There was a positive correlation between TGF-ß1 serum level and ejection fraction as measured by echocardiography (r = 0.3217, P = 0.043). The relationship between selected inflammatory biomarkers, LV ejection fraction, LV mass, and LV muscle mass with LGE appeared to be independent of valvular pathobiologic process severity, as we did not observe differences in IL-6, RANTES, or TGF-ß1 between groups differing in severity. On the contrary, these markers appear to be linked to myocardial function and remodeling, which may provide valuable insights into the pathobiology of AS and provide a basis for future detection strategies of AS.

Effects of controlled physical activity on immune cell phenotype in peripheral blood in prehypertension - studies in preclinical model and randomised crossover study.

Hypertension (HT) is a global public health issue. There are many behavioural risk factors including unhealthy diet, tobacco use and alcohol consumption as well physical inactivity that contribute to the development of high blood pressure (BP) and its complications. Favourable effect of regular physical activity on treatment or prevention of hypertension by improvement of endothelial function is widely accepted however little is known about its relationship with immune system. Thus, the aim of this study was to assess the role of moderate regular physical activity on immune cell phenotype. T cell and monocyte subsets were characterised in 31 subjects with prehypertension (130 - 139 mmHg systolic and 85 - 89 mmHg diastolic blood pressure) who participated in moderate training (3 times/week) on cyclometers for 3 months in crossover study design. Complementary study was performed in murine model of Ang II-induced hypertension and ten-week-old animals were trained on a treadmill (5 times/week, 1 hour) for 2 weeks before and 1.5 weeks after minipumps implantation. In the context of elevated blood pressure regular physical activity had modest influence on immune cell phenotype. Both in human study and murine model we did not observe effects of applied exercise that can explain the mechanism of BP reduction after short-term regular training. Twelve-weeks regular training did not affect the activation status of T lymphocytes measured as expression of CD69, CD25 and CCR5 in human study. Physical activity resulted in higher expression of adhesion molecule CD11c on CD16+ monocytes (especially CD14 high) without any changes in leukocytes subpopulation counts. Similar results were observed in murine model of hypertension after the training. However the training caused significant decrease of CCR5 and CD25 expressions (measured as a mean fluorescence intensity) on CD8+ T cells infiltrating perivascular adipose tissue. Our studies show modest regulatory influence of moderate training on inflammatory markers in prehypertensive subjects and murine model of Ang II induced hypertension.

Anti-atherosclerotic effect of the angiotensin 1-7 mimetic AVE0991 is mediated by inhibition of perivascular and plaque inflammation in early atherosclerosis.

BACKGROUND AND PURPOSE: Inflammation plays a key role in atherosclerosis. The protective role of angiotensin 1-7 (Ang-(1-7)) in vascular pathologies suggested the therapeutic use of low MW, non-peptide Ang-(1-7) mimetics, such as AVE0991. The mechanisms underlying the vaso-protective effects of AVE0991, a Mas receptor agonist, remain to be explored. EXPERIMENTAL APPROACH: We investigated the effects of AVE0991 on the spontaneous atherosclerosis in apolipoprotein E (ApoE)-/- mice, in the context of vascular inflammation and plaque stability. KEY RESULTS: AVE0991 has significant anti-atherosclerotic properties in ApoE-/- mice and increases plaque stability, by reducing plaque macrophage content, without effects on collagen. Using the descending aorta of chow-fed ApoE-/- mice, before significant atherosclerotic plaque develops, we gained insight to early events in atherosclerosis. Interestingly, perivascular adipose tissue (PVAT) and adventitial infiltration with macrophages and T-cells precedes atherosclerotic plaque or the impairment of endothelium-dependent NO bioavailability (a measure of endothelial function). AVE0991 inhibited perivascular inflammation, by reducing chemokine expression in PVAT and through direct actions on monocytes/macrophages inhibiting their activation, characterized by production of IL-1ß, TNF-α, CCL2 and CXCL10, and differentiation to M1 phenotype. Pretreatment with AVE0991 inhibited migration of THP-1 monocytes towards supernatants of activated adipocytes (SW872). Mas receptors were expressed in PVAT and in THP-1 cells in vitro, and the anti-inflammatory effects of AVE0991 were partly Mas dependent. CONCLUSIONS AND IMPLICATIONS: The selective Mas receptor agonist AVE0991 exhibited anti-atherosclerotic and anti-inflammatory actions, affecting monocyte/macrophage differentiation and recruitment to the perivascular space during early stages of atherosclerosis in ApoE-/- mice. 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.

Combining genomewide association study and lung eQTL analysis provides evidence for novel genes associated with asthma.

BACKGROUND: Genomewide association studies (GWASs) of asthma have identified single-nucleotide polymorphisms (SNPs) that modestly increase the risk for asthma. This could be due to phenotypic heterogeneity of asthma. Bronchial hyperresponsiveness (BHR) is a phenotypic hallmark of asthma. We aim to identify susceptibility genes for asthma combined with BHR and analyse the presence of cis-eQTLs among replicated SNPs. Secondly, we compare the genetic association of SNPs previously associated with (doctor's diagnosed) asthma to our GWAS of asthma with BHR. METHODS: A GWAS was performed in 920 asthmatics with BHR and 980 controls. Top SNPs of our GWAS were analysed in four replication cohorts, and lung cis-eQTL analysis was performed on replicated SNPs. We investigated association of SNPs previously associated with asthma in our data. RESULTS: A total of 368 SNPs were followed up for replication. Six SNPs in genes encoding ABI3BP, NAF1, MICA and the 17q21 locus replicated in one or more cohorts, with one locus (17q21) achieving genomewide significance after meta-analysis. Five of 6 replicated SNPs regulated 35 gene transcripts in whole lung. Eight of 20 asthma-associated SNPs from previous GWAS were significantly associated with asthma and BHR. Three SNPs, in IL-33 and GSDMB, showed larger effect sizes in our data compared to published literature. CONCLUSIONS: Combining GWAS with subsequent lung eQTL analysis revealed disease-associated SNPs regulating lung mRNA expression levels of potential new asthma genes. Adding BHR to the asthma definition does not lead to an overall larger genetic effect size than analysing (doctor's diagnosed) asthma.

Dietary factors and lung function in the general population: wine and resveratrol intake.

Wine intake is associated with a better lung function in the general population, yet the source of this effect is unknown. Resveratrol, a polyphenol in wine, has anti-inflammatory properties in the lung, its effects being partially mediated via induction of Sirtuin (SIRT)1 activity. We assessed the impact of wine and resveratrol intake, and SIRT1 single-nucleotide polymorphisms (SNPs) on lung function in the general population. Effects of red and white wine and resveratrol intake on forced expiratory volume in 1 s (FEV(1)), forced vital capacity (FVC) and FEV(1)/FVC were analysed in the population-based Doetinchem cohort (n=3,224). Associations of four tagging SIRT1 SNPs with lung function were analysed in the Doetinchem (n=1,152) and Vlagtwedde-Vlaardingen (n=1,390) cohorts. Resveratrol intake was associated with higher FVC levels, and white wine intake with higher FEV(1) levels and lower risk of airway obstruction. SIRT1 SNPs were not significantly associated with level or course of lung function, either directly or indirectly via wine or resveratrol intake. This study shows a positive association of resveratrol intake with lung function in the general population, confirms the previously reported positive association of white wine intake with higher levels of FEV(1), and additionally shows an association with a higher FEV(1)/FVC ratio. These effects probably do not run via SNPs in SIRT1.

Genetic variation in TIMP1 but not MMPs predict excess FEV1 decline in two general population-based cohorts.

BACKGROUND: An imbalance in matrix metalloproteases (MMPs) and tissue inhibitors of MMPs (TIMPs) contributes to chronic obstructive pulmonary disease (COPD) development. Longitudinal studies investigating Single Nucleotide Polymorphisms (SNPs) in MMPs and TIMPs with respect to COPD development and lung function decline in the general population are lacking. METHODS: We genotyped SNPs in MMP1 (G-1607GG), MMP2 (-1306 C/T), MMP9 (3 tagging SNPs), MMP12 (A-82G and Asn357Ser) and TIMP1 (Phe124Phe and Ile158Ile) in 1390 Caucasians with multiple FEV1 measurements from a prospective cohort study in the general population. FEV1 decline was analyzed using linear mixed effect models adjusted for confounders. Analyses of the X-chromosomal TIMP1 gene were stratified according to sex. All significant associations were repeated in an independent general population cohort (n=1152). RESULTS: MMP2 -1306 TT genotype carriers had excess FEV1 decline (-4.0 ml/yr, p=0.03) compared to wild type carriers. TIMP1 Ile158Ile predicted significant excess FEV1 decline in both males and females. TIMP1 Phe124Phe predicted significant excess FEV1 decline in males only, which was replicated (p=0.10) in the second cohort. The MMP2 and TIMP1 Ile158Ile associations were not replicated. Although power was limited, we did not find associations with COPD development. CONCLUSIONS: We for the first time show that TIMP1 Phe124Phe contributes to excess FEV1 decline in two independent prospective cohorts, albeit not quite reaching conventional statistical significance in the replication cohort. SNPs in MMPs evidently do not contribute to FEV1 decline in the general population.

Superoxide dismutases, lung function and bronchial responsiveness in a general population.

Oxidative stress is an important causative factor in the onset and progression of smoking-related lung diseases, such as chronic obstructive pulmonary disease (COPD). Superoxide dismutases (SODs) can prevent an increase in oxidative burden. A total of 1,390 subjects from the prospective Vlagtwedde-Vlaardingen cohort were genotyped for two single nucleotide polymorphisms (SNPs) in SOD2 and four SNPs in SOD3, which were further analysed for associations with the presence of bronchial hyperresponsiveness (BHR; provocative concentration causing a 10% fall in the forced expiratory volume in one second (FEV(1); PC(10)