Systemic and local vascular inflammation and arterial reactive oxygen species generation in patients with advanced cardiovascular diseases.

Background: Systemic inflammation may cause endothelial activation, mediate local inflammation, and accelerate progression of atherosclerosis. We examined whether the levels of circulating inflammatory cytokines reflect local vascular inflammation and oxidative stress in two types of human arteries. Methods: Human internal mammary artery (IMA) was obtained in 69 patients undergoing coronary artery bypass graft (CABG) surgery and left anterior descending (LAD) artery was obtained in 17 patients undergoing heart transplantation (HTx). Plasma levels of tumor necrosis factor α (TNF-α), interleukin-6 (IL-6) and interleukin-1ß (IL-1ß) were measured using ELISA, high-sensitivity C-reactive protein (hs-CRP) was measured using Luminex, and mRNA expression of proinflammatory cytokines in the vascular tissues was assessed. Furthermore, formation of superoxide anion was measured in segments of IMA using 5 uM lucigenin-dependent chemiluminescence. Vascular reactivity was measured using tissue organ bath system. Results: TNF-α, IL-6 and IL-1ß mRNAs were expressed in all studied IMA and LAD segments. Plasma levels of inflammatory cytokines did not correlate with vascular cytokine mRNA expression neither in IMA nor in LAD. Plasma TNF-α and IL-6 correlated with hs-CRP level in CABG group. Hs-CRP also correlated with TNF-α in HTx group. Neither vascular TNF-α, IL-6 and IL-1ß mRNA expression, nor systemic levels of either TNF-α, IL-6 and IL-1ß were correlated with superoxide generation in IMAs. Interestingly, circulating IL-1ß negatively correlated with maximal relaxation of the internal mammary artery (r = -0.37, p = 0.004). At the same time the mRNA expression of studied inflammatory cytokines were positively associated with each other in both IMA and LAD. The positive correlations were observed between circulating levels of IL-6 and TNF-α in CABG cohort and IL-6 and IL-1ß in HTx cohort. Conclusions: This study shows that peripheral inflammatory cytokine measurements may not reflect local vascular inflammation or oxidative stress in patients with advanced cardiovascular disease (CVD). Circulating pro-inflammatory cytokines generally correlated positively with each other, similarly their mRNA correlated in the arterial wall, however, these levels were not correlated between the studied compartments.

The Role of Bradykinin Receptors in Hereditary Angioedema Due to C1-Inhibitor Deficiency.

BACKGROUND: Hereditary angioedema (HAE) is a rare, genetic disease caused by the decreased level or function of the C1 inhibitor. The primary mediator of symptoms in HAE is bradykinin acting through its two receptors, namely receptors 1 (BR1) and 2 (BR2). Although BR2 is well characterized, the role of BR1 remains unclear. OBJECTIVE: To study the role of bradykinin receptors 1 (BR1) in the etiopathogenesis of HAE. METHODS: A total of 70 individuals, 40 patients with HAE, and 30 healthy subjects were recruited to the study. HAE was diagnosed in accordance with the international guideline. The level of bradykinin receptors was determined in populations of CD3+, CD4+, CD8+, and CD14++CD16-, CD14++CD16+ monocytes. In addition, the level of disease activity-specific markers was measured. RESULTS: There were statistically significant differences in the subpopulation of lymphocytes and monocytes between patients with HAE compared to healthy subjects. The level of BR1 and BR2 on PBMCs was comparable in healthy subjects and HAE patients during remission with significant overexpression of both receptors, triggered by HAE attack. Moreover, a significant increase in TNF-alpha and IL-1 plasma levels was observed among HAE patients. CONCLUSIONS: BR1 expression may play an important role in the pathomechanism of HAE.

Breast cancer chemotherapy induces vascular dysfunction and hypertension through a NOX4-dependent mechanism.

Cardiovascular disease is the major cause of morbidity and mortality in breast cancer survivors. Chemotherapy contributes to this risk. We aimed to define the mechanisms of long-term vascular dysfunction caused by neoadjuvant chemotherapy (NACT) and identify novel therapeutic targets. We studied arteries from postmenopausal women who had undergone breast cancer treatment using docetaxel, doxorubicin, and cyclophosphamide (NACT) and from women with no history of such treatment matched for key clinical parameters. We explored mechanisms in WT and Nox4-/- mice and in human microvascular endothelial cells. Endothelium-dependent, NO-mediated vasodilatation was severely impaired in patients after NACT, while endothelium-independent responses remained normal. This was mimicked by a 24-hour exposure of arteries to NACT agents ex vivo. When applied individually, only docetaxel impaired endothelial function in human vessels. Mechanistic studies showed that NACT increased inhibitory eNOS phosphorylation of threonine 495 in a Rho-associated protein kinase-dependent (ROCK-dependent) manner and augmented vascular superoxide and hydrogen peroxide production and NADPH oxidase activity. Docetaxel increased expression of the NADPH oxidase NOX4 in endothelial and smooth muscle cells and NOX2 in the endothelium. A NOX4 increase in human arteries may be mediated epigenetically by diminished DNA methylation of the NOX4 promoter. Docetaxel induced endothelial dysfunction and hypertension in mice, and these were prevented in Nox4-/- mice and by pharmacological inhibition of Nox4 or Rock. Commonly used chemotherapeutic agents and, in particular, docetaxel alter vascular function by promoting the inhibitory phosphorylation of eNOS and enhancing ROS production by NADPH oxidases.

Low-grade chronic inflammation and immune alterations in childhood and adolescent cancer survivors: A contribution to accelerated aging?

BACKGROUND: The long-term consequences of chemotherapy and radiotherapy result in a high prevalence and early onset of age-related chronic diseases in survivors. We aimed to examine whether childhood and adolescent cancer survivors (CS) demonstrate biomarkers of accelerated aging. METHODS: We evaluated 50 young adult CS at 11 [8-15] years after cancer diagnosis, and 30 healthy, age and sex-matched controls, who were unexposed to cancer therapy. Using a machine-learning approach, we assessed factors discriminating CS from controls and compared selected biomarkers and lymphocyte subpopulations with data from the Framingham Heart Study (FHS) cohort and the Genotype Tissue Expression (GTEx) project. RESULTS: Survivors compared with controls had higher levels of C-reactive protein and fibrinogen. The surface expression of CD38 on T cells was increased, and there was an increase in the percentage of memory T cells in survivors, compared with the unexposed group. The relationships between above cell subpopulations and age were consistent in CS, FHS, and GTEx cohorts, but not in controls. CONCLUSIONS: Young pediatric cancer survivors differ from age-related controls in terms of activation of the adaptive immune system and chronic, low-grade inflammation. These changes resemble aging phenotype observed in older population. Further research in biomarkers of aging in young, adult childhood cancer survivors is warranted, as it may facilitate screening and prevention of comorbidities in this population.

Electrochemical Degradation of Industrial Dyes in Wastewater through the Dissolution of Aluminum Sacrificial Anode of Cu/Al Macro-Corrosion Galvanic Cell.

This paper reports on the process of industrial-type wastewater purification carried-out through continuous anodic dissolution of aluminum alloy sacrificial anode for artificially aerated Cu-Al alloy galvanic (macro-corrosion) cells and synthetically prepared wastewater solutions. Electrochemical experiments were performed by means of a laboratory size electrolyzer unit, where the electrocoagulation process along with surface-induced electrooxidation phenomena were examined for wastewater containing Acid Mixture and Disperse Red 167 dyes. Final reduction of the dyes concentrations came to 32 and 99% for Acid Mixture and Disperse Red 167, correspondingly. The above was visualized through the employment of electrochemical (cyclic voltammetry and a.c. impedance spectroscopy techniques) and instrumental spectroscopy analyses.

Oleacein and Foam Cell Formation in Human Monocyte-Derived Macrophages: A Potential Strategy Against Early and Advanced Atherosclerotic Lesions.

BACKGROUND: Oleacein is a secoiridoid group polyphenol found mostly in Olea europea L. and Ligustrum vulgare L. (Oleaceae). The aim of the present study was to investigate a potential role of oleacein in prevention of the foam cell formation. MATERIALS AND METHODS: Oleacein was isolated from Ligustrum vulgare leaves. Human monocyte-derived macrophages were obtained from monocytes cultured with Granulocyte-macrophage colony-stimulating factor (GM-CSF)Then, cells were incubated with 20 M or 50 M of oleacein and with oxidized low-density lipoprotein (oxLDL) (50 g/mL). Visualization of lipid deposition within macrophages was carried out using Oil-Red-O. Expression of CD36, Scavenger receptor A1 (SRA1) and Lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1) was determined by Reverse transcription polymerase chain reaction (RT-PCR) and by flow cytometry. Apoptosis was determined by flow cytometry using Annexin V assay. STAT3 and Acyl-coenzyme A:cholesterol acyltransferase type 1 (ACAT1)levels were determined by ELISA. P-STAT3, P-JAK1, P-JAK2 expressions were determined by Western blot (WB). RESULTS: Oleacein in dose-dependent manner significantly reduced lipid deposits in macrophages as well as their expression of selected scavenger receptors. The highest decrease of expression was found for CD36 and SRA1 receptors, from above 20% to more than 75% compared to oxLDL and the lowest for LOX-1 receptor, from approx. 8% to approx. 25% compared to oxLDL-stimulated macrophages. Oleacein significantly reduced (2.5-fold) early apoptosis of oxLDL-stimulated macrophages. Moreover, oleacein significantly increased the protein expression of JAK/STAT3 pathway and had no effect on ACAT1 level. CONCLUSIONS: Our study demonstrates, for the first time, that oleacein inhibits foam cell formation in human monocyte-derived macrophages and thus can be a valuable tool in the prevention of early and advanced atherosclerotic lesions.

T Cells Are Dominant Population in Human Abdominal Aortic Aneurysms and Their Infiltration in the Perivascular Tissue Correlates With Disease Severity.

Abdominal Aortic Aneurysm (AAA) is a major cause of cardiovascular mortality. Adverse changes in vascular phenotype act in concert with chronic inflammation to promote AAA progression. Perivascular adipose tissue (PVAT) helps maintain vascular homeostasis but when inflamed and dysfunctional, can also promote vascular pathology. Previous studies suggested that PVAT may be an important site of vascular inflammation in AAA; however, a detailed assessment of leukocyte populations in human AAA, their anatomic location in the vessel wall and correlation to AAA size remain undefined. Accordingly, we performed in depth immunophenotyping of cells infiltrating the pathologically altered perivascular tissue (PVT) and vessel wall in AAA samples at the site of maximal dilatation (n = 51 patients). Flow cytometry revealed that T cells, rather than macrophages, are the major leukocyte subset in AAA and that their greatest accumulations occur in PVT. Both CD4+ and CD8+ T cell populations are highly activated in both compartments, with CD4+ T cells displaying the highest activation status within the AAA wall. Finally, we observed a positive relationship between T cell infiltration in PVT and AAA wall. Interestingly, only PVT T cell infiltration was strongly related to tertiles of AAA size. In summary, this study highlights an important role for PVT as a reservoir of T lymphocytes and potentially as a key site in modulating the underlying inflammation in AAA.

1,2,3,4,6-Penta-O-galloyl-ß-d-glucose modulates perivascular inflammation and prevents vascular dysfunction in angiotensin II-induced hypertension.

BACKGROUND AND PURPOSE: Hypertension is a multifactorial disease, manifested by vascular dysfunction, increased superoxide production, and perivascular inflammation. In this study, we have hypothesized that 1,2,3,4,6-penta-O-galloyl-ß-d-glucose (PGG) would inhibit vascular inflammation and protect from vascular dysfunction in an experimental model of hypertension. EXPERIMENTAL APPROACH: PGG was administered to mice every 2 days at a dose of 10 mg·kg-1 i.p during 14 days of Ang II infusion. It was used at a final concentration of 20 µM for in vitro studies in cultured cells. KEY RESULTS: Ang II administration increased leukocyte and T-cell content in perivascular adipose tissue (pVAT), and administration of PGG significantly decreased total leukocyte and T-cell infiltration in pVAT. This effect was observed in relation to all T-cell subsets. PGG also decreased the content of T-cells bearing CD25, CCR5, and CD44 receptors and the expression of both monocyte chemoattractant protein 1 (CCL2) in aorta and RANTES (CCL5) in pVAT. PGG administration decreased the content of TNF+ and IFN-γ+ CD8 T-cells and IL-17A+ CD4+ and CD3+ CD4- CD8- cells. Importantly, these effects of PGG were associated with improved vascular function and decreased ROS production in the aortas of Ang II-infused animals independently of the BP increase. Mechanistically, PGG (20 µM) directly inhibited CD25 and CCR5 expression in cultured T-cells. It also decreased the content of IFN-γ+ CD8+ and CD3+ CD4- CD8- cells and IL-17A+ CD3+ CD4- CD8- cells. CONCLUSION AND IMPLICATION: PGG may constitute an interesting immunomodulating strategy in the regulation of vascular dysfunction and hypertension. LINKED ARTICLES: This article is part of a themed section on Immune Targets in Hypertension. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.12/issuetoc.

TNF-α Inhibitors Decrease Classical CD14hiCD16- Monocyte Subsets in Highly Active, Conventional Treatment Refractory Rheumatoid Arthritis and Ankylosing Spondylitis.

Monocytes are pivotal cells in inflammatory joint diseases. We aimed to determine the effect of TNF-α inhibitors (TNFi) on peripheral blood monocyte subpopulations and their activation in ankylosing spondylitis (AS) and rheumatoid arthritis (RA) patients with high disease activity. To address this, we studied 50 (32 AS, 18 RA) patients with highly active disease with no prior history of TNFi use who were recruited and assigned to TNFi or placebo treatment for 12 weeks. Cytometric and clinical assessment was determined at baseline, four, and 12 weeks after initiation of TNFi treatment. We observed that treatment with TNFi led to a significant decrease in CD14hiCD16- monocytes in comparison to placebo, while circulating CD14dimCD16+ monocytes significantly increased. The TNFi-induced monocyte subset shifts were similar in RA and AS patients. While the percentage of CD14dimCD16+ monocytes increased, expression of CD11b and CD11c integrins on their surface was significantly reduced by TNFi. Additionally, CD45RA+ cells were more frequent. The shift towards nonclassical CD14dimCD16+ monocytes in peripheral blood due to TNFi treatment was seen in both AS and RA. This may reflect reduced recruitment of these cells to sites of inflammation due to lower inflammatory burden, which is associated with decreased disease activity.

Microvascular dysfunction in ankylosing spondylitis is associated with disease activity and is improved by anti-TNF treatment.

Ankylosing spondylitis (AS) is associated with high cardiovascular morbidity and mortality. Recent studies indicate that microvascular dysfunction may underlie cardiovascular risk in AS. We hypothesized, that microvascular morphology and dysfunction is linked to AS activity and is modifiable by TNF-α inhibitor (TNFi) treatment. Functional Laser Doppler Flowmetry with post-occlusive reactive hyperemia, and structural nailfold capillaroscopy were performed in 54 patients with AS and 28 matched controls. Active AS was diagnosed based on BASDAI ≥ 4 (n = 37). Effects of 3-month TNFi on microcirculation in active AS were studied. AS was associated with prolonged time to peak hyperemia compared to healthy controls. High disease activity was associated with increased time to peak hyperemia and decreased peak hyperemia when compared to patients with inactive AS. In capillaroscopy, AS was associated with morphological abnormalities indicating increased neoangiogenesis and pericapillary edema compared to controls. Microvascular function improved following 3 months of TNFi in reference to basal flow as well as post-occlusive parameters. TNFi reduced pericapillary edema, while other parameters of capillary morphology remained unchanged. Microvascular dysfunction and capillary neovascular formation are associated with disease activity of AS. Anti-TNF-α treatment may restore microcirculation function and capillary edema but does not modify microvascular structural parameters.

Hypertension and increased endothelial mechanical stretch promote monocyte differentiation and activation: roles of STAT3, interleukin 6 and hydrogen peroxide.

Aims: Monocytes play an important role in hypertension. Circulating monocytes in humans exist as classical, intermediate, and non-classical forms. Monocyte differentiation can be influenced by the endothelium, which in turn is activated in hypertension by mechanical stretch. We sought to examine the role of increased endothelial stretch and hypertension on monocyte phenotype and function. Methods and results: Human monocytes were cultured with confluent human aortic endothelial cells undergoing either 5% or 10% cyclical stretch. We also characterized circulating monocytes in normotensive and hypertensive humans. In addition, we quantified accumulation of activated monocytes and monocyte-derived cells in aortas and kidneys of mice with Angiotensin II-induced hypertension. Increased endothelial stretch enhanced monocyte conversion to CD14++CD16+ intermediate monocytes and monocytes bearing the CD209 marker and markedly stimulated monocyte mRNA expression of interleukin (IL)-6, IL-1ß, IL-23, chemokine (C-C motif) ligand 4, and tumour necrosis factor α. STAT3 in monocytes was activated by increased endothelial stretch. Inhibition of STAT3, neutralization of IL-6 and scavenging of hydrogen peroxide prevented formation of intermediate monocytes in response to increased endothelial stretch. We also found evidence that nitric oxide (NO) inhibits formation of intermediate monocytes and STAT3 activation. In vivo studies demonstrated that humans with hypertension have increased intermediate and non-classical monocytes and that intermediate monocytes demonstrate evidence of STAT3 activation. Mice with experimental hypertension exhibit increased aortic and renal infiltration of monocytes, dendritic cells, and macrophages with activated STAT3. Conclusions: These findings provide insight into how monocytes are activated by the vascular endothelium during hypertension. This is likely in part due to a loss of NO signalling and increased release of IL-6 and hydrogen peroxide by the dysfunctional endothelium and a parallel increase in STAT activation in adjacent monocytes. Interventions to enhance bioavailable NO, reduce IL-6 or hydrogen peroxide production or to inhibit STAT3 may have anti-inflammatory roles in hypertension and related conditions.

CD14+CD16++ "nonclassical" monocytes are associated with endothelial dysfunction in patients with coronary artery disease.

Endothelial dysfunction and inflammation are key mechanisms of vascular disease. We hypothesised that heterogeneity of monocyte subpopulations may be related to the development of vascular dysfunction in coronary artery disease (CAD). Therefore, we examined the relationships between monocyte subsets (CD14++CD16- "classical - Mon1", CD14++CD16+ "intermediate - Mon2" and CD14+CD16++ "nonclassical - Mon3"), endothelial function and risk factor profiles in 130 patients with CAD undergoing coronary artery bypass grafting. This allowed for direct nitric oxide (NO) bioavailability assessment using isometric tension studies ex vivo (acetylcholine; ACh- and sodium-nitropruside; SNP-dependent) in segments of internal mammary arteries. The expression of CD14 and CD16 antigens and activation markers were determined in peripheral blood mononuclear cells using flow cytometry. Patients with high CD14+CD16++ "nonclassical" and low CD14++CD16- "classical" monocytes presented impaired endothelial function. High frequency of CD14+CD16++ "nonclassical" monocytes was associated with increased vascular superoxide production. Furthermore, endothelial dysfunction was associated with higher expression of activation marker CD11c selectively on CD14+CD16++ monocytes. Nonclassical and classical monocyte frequencies remained independent predictors of endothelial dysfunction when major risk factors for atherosclerosis were taken into account (ß=0.18 p=0.04 and ß=-0.19 p=0.03, respectively). In summary, our data indicate that CD14+CD16++ "nonclassical" monocytes are associated with more advanced vascular dysfunction measured as NO- bioavailability and vascular reactive oxygen species production.

Malignant hypertension: new aspects of an old clinical entity.

Malignant hypertension (MHT), also known as accelerated-malignant hypertension or malignant-phase hypertension, is the most severe form of arterial hypertension. It is defined clinically as high blood pressure (BP) levels associated with lesions of the retinal fundus (flame-shaped hemorrhages, exudates, or cotton wool spots, with or without papilledema). Despite the availability of a vast range of antihypertensive agents, MHT continues to be a significant clinical challenge. Although its prevalence is very low, the absolute number of new cases has not changed over the past decades. While the role of the activation of the renin-angiotensin-aldosterone system and endothelial dysfunction in the pathogenesis of MHT has been well described, recent studies have indicated that the immune system may also play an important role in the development of this condition. Patients with MHT are characterized by pronounced target organ damage, including structural and functional cardiac abnormalities. MHT is frequently complicated by renal insufficiency and end-stage renal disease. The survival rates for patients with MHT have improved considerably with increased availability of antihypertensive treatment. However, renal insufficiency and end-stage renal disease still remain a significant cause of morbidity and mortality in this patient group. In conclusion, MHT is not a "vanishing disease" because there is a relatively stable number of new cases per year. Nonetheless, prognosis and survival rates in these patients have improved significantly owing to earlier detection, stricter BP control, lower BP targets, better choice of antihypertensive drugs, and availability of hemodialysis and renal transplantation.

Denture-related stomatitis is associated with endothelial dysfunction.

UNLABELLED: Oral inflammation, such as periodontitis, can lead to endothelial dysfunction, accelerated atherosclerosis, and vascular dysfunction. The relationship between vascular dysfunction and other common forms of oral infections such as denture-related stomatitis (DRS) is unknown. Similar risk factors predispose to both conditions including smoking, diabetes, age, and obesity. Accordingly, we aimed to investigate endothelial function and major vascular disease risk factors in 44 consecutive patients with dentures with clinical and microbiological features of DRS (n = 20) and without DRS (n = 24). While there was a tendency for higher occurrence of diabetes and smoking, groups did not differ significantly in respect to major vascular disease risk factors. Groups did not differ in main ambulatory blood pressure, total cholesterol, or even CRP. Importantly, flow mediated dilatation (FMD) was significantly lower in DRS than in non-DRS subjects, while nitroglycerin induced vasorelaxation (NMD) or intima-media thickness (IMT) was similar. Interestingly, while triglyceride levels were normal in both groups, they were higher in DRS subjects, although they did not correlate with either FMD or NMD. CONCLUSIONS: Denture related stomatitis is associated with endothelial dysfunction in elderly patients with dentures. This is in part related to the fact that diabetes and smoking increase risk of both DRS and cardiovascular disease.

Blood monocyte heterogeneity and markers of endothelial activation in ankylosing spondylitis.

OBJECTIVE: Ankylosing spondylitis (AS) is associated with excessive cardiovascular (CV) morbidity. Interactions between activated endothelium and monocytes precede atherosclerotic plaques. Our aim was to quantify blood monocyte subsets in relation to endothelial activation and inflammatory activity in subjects with AS who were free of clinical atherosclerotic CV disease. METHODS: Markers of inflammation and endothelial activation were measured in 47 patients with AS receiving no disease-modifying antirheumatic drugs, and 22 healthy controls. Exclusion criteria included atherosclerotic CV disease and traditional risk factors. Flow cytometry was used to identify monocyte subsets: classical CD14(++)CD16(-), intermediate CD14(++)CD16(+), and nonclassical CD14(+)CD16(++) monocytes and to evaluate their expression of CD11b and CD11c. RESULTS: Traditional risk factors were comparable among the groups, except for lower high-density lipoprotein cholesterol in AS (p = 0.007). Relative to controls, in subjects with AS counts of classical monocytes were higher (84.3 ± 5.4 vs 78.9 ± 5.3% of blood monocytes, p < 0.001) and nonclassical monocytes lower (2.9 ± 2.2 vs 5.5 ± 2.3%, p < 0.001). In AS we observed increased soluble intercellular adhesion molecule-1 [251 (224-293) vs 202 (187-230) ng/ml, p = 0.002], an endothelial ligand for monocytic ß2-integrin CD11b/CD18. CD11b expression on all 3 monocyte subsets was elevated in 21 AS subjects with a Bath Ankylosing Spondylitis Disease Activity Index score ≥ 4 versus the remaining patients (p = 0.005-0.03). C-reactive protein, interleukin 6 (IL-6), and pentraxin-3 were increased in AS, in contrast to tumor necrosis factor-α and IL-18. IL-6 correlated with classical monocytes numbers in AS (r = 0.56, p < 0.0001) but not in the controls (r = 0.10, p = 0.65). CONCLUSION: Our findings suggest a contribution of immune dysregulation to enhanced monocyte-endothelial interactions in AS, especially in patients with active disease, which possibly can accelerate atherogenesis on a longterm basis.

Markers of thrombogenesis and fibrinolysis and their relation to inflammation and endothelial activation in patients with idiopathic pulmonary arterial hypertension.

BACKGROUND: Chronic anticoagulation is a standard of care in idiopathic pulmonary arterial hypertension (IPAH). However, hemostatic abnormalities in this disease remain poorly understood. Therefore, we aimed to study markers of thrombogenesis and fibrinolysis in patients with IPAH. METHODS: We studied 27 consecutive patients (67% female) with IPAH aged 50.0 years (IQR: 41.0-65.0) and 16 controls without pulmonary hypertension. Prothrombin fragment 1+2 (F1+2) and thrombin-antithrombin (TAT) complexes were measured to assess thrombogenesis; tissue-type plasminogen activator (tPA) antigen and plasmin-anti-plasmin complex to characterize activation of fibrinolysis; plasminogen activator inhibitor 1 (PAI-1) to measure inhibition of fibrinolysis; and endothelin-1 (ET-1) and interleukin-6 (IL-6) to assess endothelial activation and systemic inflammation, respectively. In addition, in treatment-naive IPAH patients these markers were assessed after 3 months of PAH-specific therapies. RESULTS: TPA (10.1[6.8-15.8] vs 5.2[3.3-7.3] ng/ml, p<0.001), plasmin-anti-plasmin (91.5[60.3-94.2] vs 55.8[51.1-64.9] ng/ml, p<0.001), IL-6 (4.9[2.5-7.9] vs 2.1[1.3-3.8] pg/ml, p=0.001) and ET-1 (3.7 [3.3-4.5] vs 3.4[3.1-3.5], p= 0.03) were higher in patients with IPAH than in controls. In IPAH patients plasmin-anti-plasmin and tPA correlated positively with IL-6 (r=0.39, p=0.04 and r=0.63, p<0.001, respectively) and ET-1 (r=0.55, p=0.003 and r=0.59, p=0.001, respectively). No correlation was found between tPA or plasmin-anti-plasmin and markers of thrombogenesis. Plasmin-anti-plasmin decreased after 3 months of PAH specific therapy while the other markers remained unchanged. CONCLUSIONS: In the present study we showed that markers of fibrynolysis were elevated in patients with IPAH however we did not find a clear evidence for increased thrombogenesis in this group of patients. Fibrinolysis, inflammation, and endothelial activation were closely interrelated in IPAH.

Elevated markers of inflammation and endothelial activation and increased counts of intermediate monocytes in adult survivors of childhood acute lymphoblastic leukemia.

BACKGROUND: Adult survivors of childhood malignancy are prone to accelerated atherogenesis and late cardiovascular complications. Plaque formation is initiated by recruitment of monocytes and T-cells into the intima, mediated by adhesion molecules and chemokines expressed by activated endothelial cells. AIM: To assess markers of inflammatory activity, endothelial activation as well as monocyte heterogeneity in adult survivors of childhood acute lymphoblastic leukemia (ALL) who had been treated with chemotherapy without cranial irradiation. METHODS AND RESULTS: We studied 27 (age: 18-28 years) healthy survivors of childhood ALL and 20 controls (age: 20-31 years). Flow cytometry was used to identify monocyte subsets: classical CD14(++)CD16(-), intermediate CD14(++)CD16(+) and nonclassical CD14(+)CD16(++) monocytes which were further characterized by their expression of HLA-DR and ß2-integrins CD11b/CD18 and CD11c/CD18. In ALL survivors we found increased levels of pentraxin-3 (median [interquartile range]: 0.63 [0.36-0.94] vs. 0.40 [0.32-0.57] ng/ml; p = 0.03), soluble vascular cell adhesion molecule-1 (687 [597-761] vs. 558 [534-702]ng/ml; p = 0.02), osteoprotegerin (mean ± SD: 5.24 ± 1.00 vs. 4.42 ± 1.34 pmol/l; p = 0.02) and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (107.0 ± 23.6 vs. 89.5 ± 18.9 pg/ml; p = 0.01), whereas C-reactive protein, interleukin 6 and 18, TNF-α, monocyte chemotactic protein-1 and soluble intercellular adhesion molecule-1 were unchanged. Former ALL patients exhibited elevated counts of intermediate monocytes (6.3 ± 4.0 vs. 4.3 ± 1.5% of blood monocytes; p = 0.03). CD11b/CD18 and CD11c/CD18 expression on intermediate monocytes tended to be higher in ALL survivors (1917 ± 993 vs. 1396 ± 673 MFI [median fluorescence intensity]; p = 0.06 and 3883 ± 1445 vs. 3185 ± 645 MFI; p = 0.05, respectively). CONCLUSION: Our findings suggest chronic inflammatory activation and immune dysregulation in adult survivors of childhood ALL, which can translate into late cardiovascular morbidity.

Local inflammation is associated with aortic thrombus formation in abdominal aortic aneurysms. Relationship to clinical risk factors.

Intraluminal thrombus formation in aortic abdominal aneurysms (AAA) is associated with adverse clinical prognosis. Interplay between coagulation and inflammation, characterised by leukocyte infiltration and cytokine production, has been implicated in AAA thrombus formation. We studied leukocyte (CD45+) content by flow cytometry in AAA thrombi from 27 patients undergoing surgical repair. Luminal parts of thrombi were leukocyte-rich, while abluminal segments showed low leukocyte content. CD66b+ granulocytes were the most prevalent, but their content was similar to blood. Monocytes (CD14+) and T cells (CD3+) were also abundant, while content of B lymphocytes (CD19+) and NK cells (CD56+CD16+) were low. Thrombi showed comparable content of CD14highCD16- monocytes and lower CD14highCD16+ and CD14dimCD16+, than blood. Monocytes were activated with high CD11b, CD11c and HLA-DR expression. Total T cell content was decreased in AAA thrombus compared to peripheral blood but CD8 and CD3+CD4-CD8- (double negative T cell) contents were increased in thrombi. CD4+ cells were lower but highly activated (high CD69, CD25 and HLA-DR). No differences in T regulatory (CD4+CD25+FoxP3+) cell or pro-atherogenic CD4+CD28null lymphocyte content were observed between thrombi and blood. Thrombus T cells expressed high levels of CCR5 receptor for chemokine RANTES, commonly released from activated platelets. Leukocyte or T cell content in thrombi was not correlated with aneurysm size. However, CD3+ content was significantly associated with smoking in multivariate analysis taking into account major risk factors for atherosclerosis. In conclusion, intraluminal AAA thrombi are highly inflamed, predominantly with granulocytes, CD14highCD16- monocytes and activated T lymphocytes. Smoking is associated with T cell infiltration in AAA intraluminal thrombi.

Interaction of human peripheral blood monocytes with apoptotic polymorphonuclear cells.

Macrophages have the potential to recognize apoptotic neutrophils and phagocytose them while the same function for monocytes is uncertain. In fact, early findings indicated that monocytes started to phagocytose neutrophils on the third day of differentiation to macrophages. Here we show, using flow cytometry and confocal microscopy, that peripheral blood monocytes phagocytose apoptotic but not freshly isolated granulocytes. Recognition of apoptotic cells is predominantly connected with CD16(+) monocytes (CD14(high) CD16(+) and CD14(dim) CD16(+)) and requires CD36. Clearance of apoptotic polymorphonuclear leucocytes appears to be independent of the CD14 mechanism. Uptake of apoptotic Jurkat T cells by monocytes is CD14 and CD36 dependent. Liposomes containing phosphatidyl-l-serine reduce binding of apoptotic polymorphonuclear leucocytes. Lipopolysaccharide-activated subpopulations of monocytes while in contact with apoptotic cells produce more anti-inflammatory cytokine interleukin-10 whereas the production of pro-inflammatory cytokines, tumour necrosis factor-alpha and interleukin-1beta is reduced.

Phagocytosis of Staphylococcus aureus by macrophages exerts cytoprotective effects manifested by the upregulation of antiapoptotic factors.

It is becoming increasingly apparent that Staphylococcus aureus are able to survive engulfment by macrophages, and that the intracellular environment of these host cells, which is essential to innate host defenses against invading microorganisms, may in fact provide a refuge for staphylococcal survival and dissemination. Based on this, we postulated that S. aureus might induce cytoprotective mechanisms by changing gene expression profiles inside macrophages similar to obligate intracellular pathogens, such as Mycobacterium tuberculosis. To validate our hypothesis we first ascertained whether S. aureus infection could affect programmed cell death in human (hMDMs) and mouse (RAW 264.7) macrophages and, specifically, protect these cells against apoptosis. Our findings indicate that S. aureus-infected macrophages are more resistant to staurosporine-induced cell death than control cells, an effect partly mediated via the inhibition of cytochrome c release from mitochondria. Furthermore, transcriptome analysis of human monocyte-derived macrophages during S. aureus infection revealed a significant increase in the expression of antiapoptotic genes. This was confirmed by quantitative RT-PCR analysis of selected genes involved in mitochondria-dependent cell death, clearly showing overexpression of BCL2 and MCL1. Cumulatively, the results of our experiments argue that S. aureus is able to induce a cytoprotective effect in macrophages derived from different mammal species, which can prevent host cell elimination, and thus allow intracellular bacterial survival. Ultimately, it is our contention that this process may contribute to the systemic dissemination of S. aureus infection.