Neutrophil Infiltration Characterized by Upregulation of S100A8, S100A9, S100A12 and CXCR2 Is Associated With the Co-Occurrence of Crohn's Disease and Peripheral Artery Disease.

Background: Crohn's disease (CD) and peripheral arterial disease (PAD) are closely related. The pathophysiological mechanisms underlying the coexistence of CD and PAD are unknown. The aim of this study was to investigate the key molecules and pathways mediating the co-occurrence of CD and PAD through quantitative bioinformatic analysis of a public RNA sequencing database. Methods: Datasets of CD (GSE111889) and PAD (GSE120642) were downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were analyzed using the 'edgeR' and 'limma' packages of R. Gene Ontology and Kyoto Encyclopedia analyses of common DEGs were performed to explore the functions of DEGs. Protein-protein interaction (PPI) networks were established by the Search Tool for the Retrieval of Interacting Genes (STRING) database and visualized by Cytoscape. Hub genes were selected using the plugin cytoHubba. Hub gene validation was performed in GSE95095 for CD and GSE134431 for PAD. Receiver operating characteristic curves were used to evaluate the predictive values of the hub genes. Gene set enrichment analysis and immune infiltration of the hub genes were performed. Results: A total of 54 common DEGs (2 downregulated and 52 upregulated) were identified. Pathways of neutrophil chemotaxis, neutrophil migration and cytokine and cytokine receptors were enriched in CD and PAD. S100A8, S100A9, S100A12 and CXCR2 were identified as hub genes after validation, with all area under the curve > 0.7 for both CD and PAD. Neutrophil infiltration was associated with upregulation of the hub genes. Pathways of immune processes, including neutrophil activation, neutrophil chemotaxis, neutrophil migration were significantly correlated with high expression of S100A8, S100A9, S100A12 and CXCR2 in both CD and PAD. Conclusions: This bioinformatic study elucidates S100A8, S100A9, S100A12 and CXCR2 as hub genes for the co-occurrence of Crohn's disease and peripheral artery disease. Inflammation and immune regulation modulated by neutrophil infiltration play a central role in the development of CD and PAD and may be potential targets for diagnosis and treatment.

Cohort Study Examining the Association of Immunosuppressant Drug Prescription With Major Adverse Cardiovascular and Limb Events in Patients With Peripheral Artery Disease.

AIM: Immune activation is strongly implicated in atherosclerotic plaque instability, however, the effect of immunosuppressant drugs on cardiovascular events in patients with peripheral artery disease (PAD) is not known. The aim of this study was to assess whether prescription of one or more immune suppressant drugs was associated with a lower risk of major adverse cardiovascular (MACE; i.e. myocardial infarction, stroke or cardiovascular events) or limb events (MALE; i.e. major amputation or requirement for peripheral revascularization) in patients with PAD. METHODS: A total of 1506 participants with intermittent claudication (n = 872) or chronic limb threatening ischemia (CLTI; n = 634) of whom 53 (3.5%) were prescribed one or more immunosuppressant drugs (prednisolone 41; methotrexate 17; leflunomide 5; hydroxychloroquine 3; azathioprine 2; tocilizumab 2; mycophenolate 1; sulfasalazine 1; adalimumab 1) were recruited from 3 Australian hospitals. Participants were followed for a median of 3.9 (inter-quartile range 1.2, 7.3) years. The association of immunosuppressant drug prescription with MACE or MALE was examined using Cox proportional hazard analyses. RESULTS: After adjusting for other risk factors, prescription of an immunosuppressant drug was associated with a significantly greater risk of MACE (Hazard ratio, HR, 1.83, 95% confidence intervals, CI, 1.11, 3.01; P = 0.017) but not MALE (HR 1.32, 95% CI 0.90, 1.92; P = 0.153). In a sub-analysis restricted to participants with CLTI findings were similar: MACE (HR 2.44, 95% CI 1.32, 4.51; P = 0.005); MALE (HR 1.38, 95% CI 0.87, 2.19; P = 0.175); major amputation (HR 1.37, 95% CI 0.49, 3.86; P = 0.547). CONCLUSIONS: This cohort study suggested that immunosuppressant drug therapy is associated with a greater risk of MACE amongst patients with PAD.

Strategies to Overcome the Barrier of Ischemic Microenvironment in Cell Therapy of Cardiovascular Disease.

The transplantation of various immune cell types are promising approaches for the treatment of ischemic cardiovascular disease including myocardial infarction (MI) and peripheral arterial disease (PAD). Major limitation of these so-called Advanced Therapy Medicinal Products (ATMPs) is the ischemic microenvironment affecting cell homeostasis and limiting the demanded effect of the transplanted cell products. Accordingly, different clinical and experimental strategies have been evolved to overcome these obstacles. Here, we give a short review of the different experimental and clinical strategies to solve these issues due to ischemic cardiovascular disease.

Location of recurrent cardiovascular events and anticardiolipin antibodies.

BACKGROUND: The relationship between anticardiolipin (aCL) antibodies and cardiovascular events is uncertain and may vary according to arterial location. MATERIALS AND METHODS: FRENA is an ongoing registry of stable outpatients with symptomatic coronary artery disease (CAD), cerebrovascular disease (CVD) or peripheral artery disease (PAD). The rate of subsequent ischaemic events was cross-referenced with the presence of aCL antibodies (any isotype, IgG or IgM). RESULTS: As of June 2017, 1387 stable outpatients were recruited. Of these, 120 (8.7%) showed positive levels of aCL antibodies. Over an average follow-up of 18 months, 250 patients developed subsequent events: 101 myocardial infarction, 57 ischaemic stroke and 92 critical leg events. Patients with positive aCL antibodies had a higher risk of distal artery events (a composite of ischaemic stroke or critical leg events) than patients with undetectable or low levels (rate ratio: 1.66; 95% CI: 1.07-2.60). However, an association with central coronary events was not found. The multivariate Cox analysis after adjustment for relevant clinical covariates showed that positivity of aCL antibodies is an independent risk factor for distal events (hazard ratio: 1.60; 95% CI: 1.01-2.55; P < .05). CONCLUSIONS: Positivity of aCL antibodies is associated with an increased risk of subsequent distal artery ischaemic events (cerebral or leg arteries) but not coronary artery events. Anticardiolipin antibodies appear to have a different relationship on the localisation of ischaemic events in patients with symptomatic artery disease.

The novel role of IL-37 to enhance the anti-inflammatory response of regulatory T cells in patients with peripheral atherosclerosis.

OBJECTIVES: Regulatory T cells (Tregs) mediate immunomodulation and protect against atherosclerosis. It is considered that reducing the amount of pro-inflammatory mediators could be achieved by enhancing the anti-inflammatory response, and this may be considered one of the main targets for therapy development. The inhibitory cytokines secreted by Tregs mainly include interleukin-10 (IL-10) and transforming growth factor-beta (TGF-ß). Based on its known immunosuppressive involvement with other inflammatory disorders, we hypothesized that the newly characterized cytokine interleukin-37 (IL-37) might be associated with the inhibitory functions of Treg in atherosclerosis. Immune regulatory functions of IL-37 have not been completely clarified. Accordingly, we speculated that IL-37 might play a regulatory role in the immunosuppression of Tregs in atherosclerotic disease. METHODS: Real-time polymerase chain reaction and enzyme linked immunosorbent assay were used to test gene expression and protein levels of IL-37 in peripheral blood and localized freshly resected arterial tissues from 84 patients with peripheral arterial occlusive disease and 50 non-atherosclerotic subjects. Results were correlated to disease hallmarks. We also evaluated the ability of recombinant IL-37 to modulate Treg cytokine secretion and T cell inhibition in relation to atherosclerotic disorder in vitro.Results: Our results revealed that IL-37 was increased in patients with chronic lower limb atherosclerotic ischemia, compared to non-atherosclerotic controls. In addition, the expression levels of circulating IL-37 correlated with disease severity of chronic lower limb ischemia. Supplementation with rIL-37 augmented levels of released IL-10 and TGF-ß in supernatants of T cells co-cultured with Tregs in the enrolled patients.Conclusions: Results suggest a role for IL-37 in mediating anti-inflammatory functions in the atherosclerotic process, potentially involving enhancement of Treg inhibitory function and anti-inflammatory cytokine secretion with a particularly marked direct response in severe disease.

Diet is associated with ankle-brachial index, inflammation, and ambulation in patients with intermittent claudication.

OBJECTIVE: The aims of this investigation were to determine whether the daily dietary intake of nutrients by patients with peripheral artery disease (PAD) and intermittent claudication (IC) met recommended levels for adults older than 50 years and to determine whether meeting recommended levels of nutrients was associated with ankle-brachial index (ABI), inflammation, and ambulation of patients with PAD and IC. METHODS: A total of 48 patients were assessed on their dietary intake of 20 nutrients during a 3-day period. Patients were further characterized on demographic variables, comorbid conditions, cardiovascular risk factors, ABI, 6-minute walk distance (6MWD), and high-sensitivity C-reactive protein (hsCRP) concentration. RESULTS: Few patients met the daily recommended intakes for calcium (4%), fiber (6%), vitamin E (6%), trans fatty acids (13%), vitamin A (15%), total sugars (19%), potassium (23%), sodium (29%), saturated fat (29%), and vitamin C (31%), and none of the patients met the daily recommended intake of vitamin D (0%). Overall, patients met few of the 20 dietary recommendations as the median score was seven recommendations. Only 17 of 48 patients met more than seven of the recommendations. For the ABI regression model adjusted for age, sex, race, smoking, hypertension, dyslipidemia, body mass index, and percentage body fat, the only significant predictor was total sugars (P < .001); patients who did not meet the recommendation had lower ABI values. For the hsCRP-adjusted regression model, the strongest significant predictor was omega-3 polyunsaturated fatty acids (P = .001), indicating that those who did not meet the recommendation had higher hsCRP values. Finally, for the 6MWD-adjusted regression model, folate (P = .011) and dietary score index (P = .014) were significant predictors; those who did not meet the recommendation for folate and those who met 5 or fewer of the 20 recommendations had shorter 6MWD. CONCLUSIONS: Patients with PAD and IC consume a low-nutrient-dense diet that is deficient in many vitamins, calcium, fruits, and vegetables and contains too much added sugar, saturated and trans fats, and processed foods. In addition, more severe PAD, greater inflammation, and ambulatory dysfunction are independently associated with aspects of a low-nutrient-dense diet, such as too much intake of added sugars, low intake of omega-3 polyunsaturated fatty acids and folate, and meeting the recommended intakes of only five or fewer nutrients.

Changes and significance of T helper-9 cells and interleukin-9 in patients with atherosclerotic chronic lower limb ischemia: Effect on IL-17 release.

OBJECTIVES: Atherosclerosis is considered as a chronic inflammatory disorder where the central role of T cells in its pathogenesis is well known. T helper-9 cells have a distinctive effect upon the inflammatory processes. They stimulate macrophages via secretion of their cytokine interleukin-9. Based on its known involvement with other inflammatory disorders, we hypothesized that interleukin-9 might be associated with the inflammatory limb of peripheral atherosclerotic disease. METHODS: We tested this hypothesis on peripheral blood mononuclear cells (PBMCs) and freshly resected arterial tissues from 84 patients with peripheral arterial occlusive disease (PAOD) and 50 non-atherosclerotic subjects. A number of experimental methods were used including flow cytometry analysis of T helper-9 cells using anti-CD3, anti-CD4, and anti-interleukin-9monoclonal antibodies as well as real-time polymerase chain reaction for the assessment of gene expression of interleukin-9. In addition, circulating serum levels of interleukin-9 were measured using enzyme linked immunosorbent assay. We also evaluated the ability of recombinant interleukin-9 to modulate IL-17 release in cultured isolated CD3+ T cells with relation to atherosclerotic disorder in vitro. RESULTS AND CONCLUSIONS: Here we report increased percentages of T helper-9 cells and interleukin-9 levels in patients with chronic lower limb atherosclerotic ischemia, compared to healthy controls. Through investigation of different atherosclerotic patient populations with different disease stages, we found elevated interleukin-9 level both systemically and within the lesion and increased expression of cells in severe disease stages. The current study also revealed enhanced expression of mRNA levels of interleukin-9 within the atherosclerotic lesion when compared with non-atherosclerotic vessels. Levels of released IL-17 in CD3+ T cell culture supernatants supplemented with interleukin-9 were significantly positively correlated in the enrolled patients. The results suggest a role for T helper-9 cells and IL-9 in atherosclerotic process, potentially involving IL-17-mediated mechanisms. Indeed, we found that interleukin-9 promoted IL-17 release in PBMCs, with a particularly marked response in severe disease.

Impact of Femoral Ossification on Local and Systemic Cardiovascular Patients' Condition.

BACKGROUND: Vascular calcifications are associated with a high cardiovascular morbi-mortality in the coronary territory. In parallel, femoral arteries are more calcified and develop osteoid metaplasia (OM). This study was conducted to assess the predictive value of OM and local inflammation on the occurrence of mid- and long-term adverse cardiovascular events. METHOD: Between 2008 and 2015, 86 atheromatous samples were harvested during femoral endarterectomy on 81 patients and processed for histomorphological analyses of calcifications and inflammation (monocytes and B cells). Histological findings were compared with the long-term follow-up of patients, including major adverse cardiac event (MACE), major adverse limb event (MALE), and mortality. Frequencies were presented as percentage, and continuous data, as mean and standard deviation. A P-value < 0.05 was considered statistically significant. RESULTS: Median follow-up was 42.4 months (26.9-58.8). Twenty-eight percent of patients underwent a MACE; a MALE occurred in 18 (21%) limbs. Survival rate was 87.2% at 36 months. OM was found in 41 samples (51%), without any significant impact on the occurrence of MACE, MALE, or mortality. Preoperative white blood cell formulae revealed a higher rate of neutrophils associated with MACE (P = 0.04) and MALE (P = 0.0008), correlated with higher B cells counts in plaque samples. CONCLUSIONS: OM is part of femoral calcifications in almost 50% of the cases but does not seem to be an independent predictive variable for MACE or MALE. However, a higher rate of B cell infiltration of the plaque and preoperative neutrophil blood count may be predictive of adverse events during follow-up.

Autologous CD34+ Cell Therapy for Ischemic Tissue Repair.

In 1997, the seminal manuscript by Asahara, Murohara, Isner et al outlined the evidence for the existence of circulating, bone marrow-derived cells capable of stimulating and contributing to the formation of new blood vessels. Consistent with the paradigm shift that this work represented, it triggered much scientific debate and controversy, some of which persists 2 decades later. In contrast, the clinical application of autologous CD34 cell therapy has been marked by a track record of consistent safety and clinical benefit in multiple ischemic conditions. In this review, we summarize the preclinical and clinical evidence from over 700 patients in clinical trials of CD34 cell therapy.

The Role of the Microbiota in the Diabetic Peripheral Artery Disease.

Vascular complications of diabetes mellitus represent a major public health problem. Although many steps forward have been made to define the causes and to find the best possible therapies, the problem remains crucial. In recent years, more and more evidences have defined a link between microbiota and the initiation, promotion, and evolution of atherosclerotic disease, even in the diabetic scenario. There is an urgency to develop the knowledge of modern medicine about the link between gut microbiota and its host's metabolic pathways, and it would be useful to understand and justify the interindividual diversity of clinical disease presentation of diabetic vascular complication even if an optimization of pharmacological treatment has been made or in the case of young patients where hypertension, dyslipidemia, and diabetes are not able to justify a very quick progress of atherosclerotic process. The aim of the present review is to gather all the best available evidence in this regard and to define a new role of the microbiota in this field, from biomarker to possible therapeutic target.

Fish Oil Increases Specialized Pro-resolving Lipid Mediators in PAD (The OMEGA-PAD II Trial).

BACKGROUND: N-3 polyunsaturated fatty acid (PUFA) supplementation has been associated with reduced mortality and inflammation in patients with cardiovascular disease. There are limited data on the effects of n-3 PUFA supplementation in patients with peripheral artery disease (PAD). MATERIALS AND METHODS: The OMEGA-PAD II trial was a double-blinded, randomized, placebo-controlled trial to assess the effect of 3 mo of high-dose oral n-3 PUFA supplementation on inflammation, endothelial function, and walking ability in patients with PAD. RESULTS: Twenty-four patients with claudication received 4.4 g/d of fish oil or placebo for 3 mo. Outcomes measured included high-sensitivity C-reactive protein levels, the omega-3 index, endothelial function as measured via flow-mediated vasodilation, walking impairment questionnaire, and a 6-min walk test. Plasma levels of specialized pro-resolving lipid mediators (SPMs) were measured by liquid-chromatography-tandem mass spectrometry. In patients treated with fish oil, the absolute mean omega-3 index significantly increased from baseline (fish oil: 7.2 ± 1.2%, P < 0.001; placebo: -0.4 ± 0.9%, P = 0.31; between-group P < 0.001). Furthermore, there were significant increases in several pathway markers of SPM biosynthesis, including several mono-hydroxyeicosapentaenoic acids and mono-hydroxydocosahexaenoic acids. We also observed significant increases in the SPM lipoxin A5 (fish oil: 0.57 ± 0.70 pg/mL, P = 0.05; placebo: 0.01 ± 0.38 pg/mL, P = 0.93; between-group P = 0.04) and resolvin E3 (fish oil: 154 ± 171 pg/mL, P = 0.04; placebo: 32 ± 54 pg/mL, P = 0.08; between-group P = 0.04). There were no significant changes in high-sensitivity C-reactive protein, flow-mediated vasodilation, walking impairment questionnaire, or 6-min walk test in the fish oil group. CONCLUSIONS: Fish oil increases SPMs in plasma of patients with PAD. Further studies are required to determine whether these early changes translate to clinical improvements in patients with PAD.

Anti-phosphorylcholine IgM, an Anti-inflammatory Mediator, Predicts Peripheral Vein Graft Failure: A Prospective Observational Study.

OBJECTIVES: One third of infrainguinal vein bypasses may fail within the first 1.5 years. Pro- and anti-inflammatory mechanisms are thought to be involved in these graft stenoses and occlusions. In previous studies, low levels of anti-phosphorylcholine IgM (anti-PC IgM, an innate anti-inflammatory IgM) have been associated with increased cardiovascular events. In this study, the peri-operative dynamics of anti-PC IgM levels were established during leg bypass surgery, and associations assessed between anti-PC IgM levels and primary graft patency. DESIGN AND METHODS: This was a prospective, observational cohort study of infrainguinal autogenous vein bypass for peripheral arterial occlusive disease involving four university affiliated hospitals. Plasma cytokine and anti-PC IgM levels were measured pre- and post-operatively. The outcome of interest was loss of primary graft patency because of occlusion or intervention for graft stenosis. RESULTS: One hundred and forty-two consecutive patients were enrolled: mean age 66 (46-91); 91% white race and male; 72.5% critical limb ischaemia (Fontaine III or IV). Median pre-operative anti-PC IgM levels were 49 units/mL (IQR 32.3-107.7, mean 89.8 + 101 sd). During follow up of an average of 1.8 years (1 month-7.4 years), 50 (35.2%) grafts lost primary patency. Pre-operative levels of interleukin 6 or C-reactive protein did not predict graft failure. Patients with pre-operative anti-PC IgM values in the lowest quartile had a twofold increased risk of graft failure (multivariable Cox proportional hazard, p = .03, HR 2.11, 95% CI 1.09-4.07), even after accounting for the other significant factors of conduit diameter, distal anastomosis, smoking, and the severity of leg ischaemia. CONCLUSIONS: Low levels of anti-PC IgM are associated with vein bypass graft failure. This biological mediator may be a useful marker to identify patients at higher risk, and offers the potential for novel, directed therapies for vascular inflammation and its consequences.

Autoantibodies to ox-LDL in Sjögren's syndrome: are they atheroprotective?

OBJECTIVES: The higher incidence of atherosclerosis and cardiovascular disease (CVD) in patients with systemic autoimmune diseases cannot be attributed exclusively to traditional risk factors for CVD. Antibodies to oxidised Low Density Lipoprotein (ox-LDL) seem to have a crucial role in atherogenesis. METHODS: Sera from 63 consecutive patients with primary Sjögren's syndrome (pSS), 121 with systemic lupus erythematosus (SLE), 79 with rheumatoid arthritis (RA) and 26 apparently healthy individuals were evaluated for the presence of antibodies to ox-LDL by ELISA. The femoral and/or carotid intima media thickness (IMT) and plaque formation as well as traditional CVD risk factors and disease-related features were recorded for all study participants. RESUKTS: Anti-ox-LDL antibody levels were significantly reduced in SS and RA patients, but not in SLE patients, compared to their healthy counterparts. Subsequently, SS patients were divided into two groups according to antibody levels to ox-LDL, using as cut off the median of each group studied. SS patients with high titres of antibodies to ox-LDL displayed higher rates of autoantibodies to Ro/SSA and La/SSB antigens, purpura, low complement levels and increased SS activity index. On the other hand, the high anti-ox-LDL group was characterised by reduced rates of carotid and/or femoral plaque after adjusting for potential confounders (OR [95%CI]: 0.14 [0.03-0.72]). Such associations were not shown in all other groups included in the study. CONCLUSIONS: These findings suggest that antibodies to ox-LDL, possibly resulting from B cell hyperactivity, might exert a protective role in the development of atherosclerosis among primary SS patients.

Involvement of CD8+ T cell subsets in early response to vascular injury in patients with peripheral artery disease in vivo.

AIMS: Adaptive immunity is critical in vascular remodelling following arterial injury. We hypothesized that acute changes in T cells at a percutaneous transluminal angioplasty (PTA) site could serve as an index of their potential interaction with the injured vascular wall. METHODS AND RESULTS: T cell subsets were characterised in 45 patients with Rutherford 3-4 peripheral artery disease (PAD) undergoing PTA. Direct angioplasty catheter blood sampling was performed before and immediately after the procedure. PTA was associated with an acute reduction of α/ß-TcR CD8+ T cells. Further characterisation revealed significant reduction in pro-atherosclerotic CD28nullCD57+ T cells, effector (CD45RA+CCR7-) and effector memory (CD45RA-CCR7-) cells, in addition to cells bearing activation (CD69, CD38) and tissue homing/adhesion markers (CD38, CCR5). CONCLUSIONS: The acute reduction observed here is likely due to the adhesion of cells to the injured vascular wall, suggesting that immunosenescent, activated effector CD8+ cells have a role in the early vascular injury immune response following PTA in PAD patients.

Cardiovascular disease in systemic lupus erythematosus is associated with increased levels of biomarkers reflecting receptor-activated apoptosis.

BACKGROUND AND AIMS: There is convincing evidence that adaptive immune responses affect the development of atherosclerosis and thrombosis and several autoimmune diseases are associated with increased cardiovascular risk. However, our understanding of the underlying mechanisms remains limited. We investigated how biomarkers reflecting four aspects of autoimmunity: apoptosis, inflammation, tissue degradation and repair, associate with cardiovascular disease (CVD) in subjects with systemic lupus erythematosus (SLE). METHODS: We investigated 484 well-characterized SLE patients, 69 of whom had CVD (coronary artery disease, cerebrovascular disease or peripheral artery disease), and 253 controls. Occurrence of carotid plaques was investigated with ultrasound. Plasma levels of biomarkers reflecting apoptosis (Fas, TNF receptor 1, TRAIL receptor 2), inflammation (IL-6, IL-8, monocyte chemotactic protein-1), tissue degradation (matrix metalloproteinase (MMP)-1, MMP-3, MMP-7), and tissue repair (platelet-derived growth factor, epidermal growth factor and stem cell factor) were analyzed by Proximity Extension Assay. RESULTS: Subjects with SLE had markedly elevated plasma levels of biomarkers reflecting apoptosis, inflammation and tissue degradation as compared to controls. SLE patients with CVD had higher levels of Fas, TNF receptor 1, TRAIL receptor 2, MMP-1 and -7 than those without CVD. The same associations were found for the presence of a carotid plaque. When controlling for the factors included in the Framingham risk score, all biomarkers except MMP-1 remained associated with the presence of a carotid plaque, while only TRAIL receptor 2 levels remained significantly associated with CVD. CONCLUSIONS: Our findings argue that the cardiovascular risk in SLE is associated with increased cell death by apoptosis and tissue degradation.

Prognostic significance of circulating leukocyte subtype counts in patients with coronary artery disease.

BACKGROUND AND AIMS: This study investigated the association of leukocyte subtype counts with vascular endothelial dysfunction and future cardiovascular events in patients with coronary artery disease (CAD). METHODS: The study included 389 consecutive CAD patients (259 male, 130 female; mean age, 70.1 ± 9.9 years). The patients underwent coronary angiography, and measurement of blood parameters, including leukocyte subtype counts. RESULTS: There were 84 cardiovascular events during a mean follow-up of 586 ± 378 days. Kaplan-Meier analysis showed a higher probability of cardiovascular events in the high-monocyte group (≥360/mm3) compared with the low-monocyte group (<360/mm3) (log-rank test, p = 0.047). Multivariate Cox hazard analysis identified a high monocyte count as an independent predictor of cardiovascular events (hazard ratio: 1.63, 95% confidence interval:1.05-2.51, p = 0.028). Peripheral endothelial function in 355 of the CAD patients was assessed by reactive hyperemia peripheral arterial tonometry index (RHI) to examine the association of ln-RHI with leukocyte subtype counts. Total leukocyte, monocyte and neutrophil counts were significantly higher in CAD patients with low ln-RHI (<0.57: the mean ln-RHI value) compared with those with high ln-RHI (≥0.57). Univariate analyses revealed that ln-RHI in CAD patients was positively correlated with ln-total leukocyte (r = -0.187, p < 0.001), ln-monocyte (r = 0.316, p < 0.001), and neutrophil (r = -0.175, p = 0.001) counts. Multiple regression analysis showed that the monocyte count was a significant and independent factor associated with ln-RHI (adjusted R2 = 0.126, p < 0.001). CONCLUSIONS: A high monocyte count was an independent and incremental predictor of cardiovascular events in CAD patients. The monocyte count was also significantly correlated with peripheral endothelial dysfunction in CAD patients.

Antiphospholipid antibodies and lower extremity peripheral arterial disease - a systematic review and meta-analysis.

BACKGROUND: Despite being an important risk factor for venous thromboembolism and ischaemic stroke, the role of antiphospholipid antibodies in patients with peripheral arterial disease remains a matter of debate. The aim of this study was to evaluate the association of persistently elevated antiphospholipid antibodies and lower extremity peripheral arterial disease. METHODS: We conducted a systematic review of electronic databases including MEDLINE, EUROPUBMED and EMBASE to assess the prevalence of antiphospholipid antibodies in patients with lower extremity peripheral arterial disease. Case-control studies were included if they reported the prevalence of antiphospholipid antibodies in patients with lower extremity peripheral arterial disease. Two reviewers (FV and EG) independently assessed the eligibility of all articles. The primary outcome measure was the odds ratio (OR) for the prevalence of antiphospholipid antibodies patients with lower extremity peripheral arterial disease, along with the corresponding 95 % confidence intervals (CIs). RESULTS: Our initial electronic search identified 128 relevant abstracts, of which two studies were included. Antiphospholipid antibodies were found in 50/571 patients with lower extremity peripheral arterial disease and 13/490 of the controls, OR 3.32 (95 % CI = 1.49 to 7.4). In those with critical limb ischaemia, the prevalence of antiphospholipid antibodies was elevated compared to controls, pooled OR 4.78 (95 % CI = 2.37 to 9.65). CONCLUSIONS: Our systematic review and meta-analysis suggests that the prevalence of persistently elevated levels of antiphospholipid antibodies is increased in patients with lower extremity peripheral diseases when compared to healthy controls, especially in those with critical limb ischaemia.

Effect of Simvastatin Treatment on "In Vitro" NLRP1 Inflammasome Expression in Peripheral Arterial Disease.

BACKGROUND: Inflammatory stress stimuli in the plasma of patients with peripheral artery disease (PAD) are able to trigger the expression of NLRP1 inflammasome in human aortic endothelial cells (HAECs). Our objective was to elucidate the effect of simvastatin treatment on NLRP1 inflammasome expression in endothelial cells exposed to the plasma of PAD patients. METHODS: The study included 81 patients with PAD, 24 of them treated with simvastatin (20 mg/day) and 57 without statin therapy. HAECs between passages 3 and 6 were stimulated for 2 hr using the plasma samples of the study participants. NLRP1 gene transcription of HAECs exposed to the plasma of PAD patients was quantificated. RESULTS: HAECs exposed to the plasma of PAD patients with simvastatin therapy showed significantly higher expression of the NLRP1 gene compared with those exposed to the plasma of PAD patients without this treatment (relative quantitation [RQ] 1.12 ± 0.06 vs. 1.06 ± 0.07, P = 0.03). Furthermore, HAECs exposed to the plasma of patients with critical limb ischemia and treated with simvastatin responded with a higher NLRP1 expression than those exposed to the plasma of simvastatin-treated patients with claudication (RQ 1.1 ± 0.3 vs. 0.99 ± 0.14, P < 0.001). CONCLUSION: Simvastatin intake in PAD patients increases in vitro reactivity of NLRP1 inflammasome gene expression in HAECs, especially in critical limb ischemia patients.

The Toll of Vascular Insufficiency: Implications for the Management of Peripheral Arterial Disease.

Peripheral artery disease (PAD) can result in limb loss within six months of diagnosis in a subset of patients who cannot undergo endovascular or surgical revascularization yet continues to maintain a marginal position in cardiovascular research. While a body of literature continues to grow describing the role of danger signaling and innate immunity in cardiac biology, the role of these pathways in the ischemic myopathy associated with PAD has not been extensively studied. The following report will review the current literature on the role of Toll-like receptor (TLR) signaling in cardiovascular biology as well as in nonischemic myopathy. While attenuation of TLR signaling has not been shown to be clinically useful in the treatment of infectious inflammation, it may show promise in the management of severe arterial insufficiency.

Increased Number of Mast Cells in Atherosclerotic Lesions Correlates with the Presence of Myeloid but not Plasmacytoid Dendritic Cells as well as Pro-inflammatory T Cells.

BACKGROUND: Atherosclerosis is an inflammatory disease of the vessel wall promoted by different immune cells and inflammatory mediators. METHODS: In this study, 26 human plaques and 12 control vessels without atherosclerosis were immunohistochemically stained to analyze the emergence of mast cells dependent on plaque morphology and to correlate mast cell occurrence with the emergence of myeloid as well as plasmacytoid dendritic cells. Also, mast cell emergence was correlated with the number of pro-inflammatory T cells. For this, plaques were classified as stable or unstable according to established histological criteria. RESULTS: As expected, atherosclerotic lesions showed significantly higher numbers of tryptase+, chymase+, and cathepsin G+ mast cells compared to control vessels, particularly in lesions with unstable morphology. As a novel finding, we detected significant correlations between mast cells and myeloid dendritic cells (fascin, CD83, r > 0.3, p < 0.01), but not plasmacytoid dendritic cells (CD123, CD304). Also, we observed significant correlations of mast cells and different subgroups of pro-inflammatory T cells (CD3, CD8, CD161, CD25; r > 0.35, p < 0.05). CONCLUSIONS: Overall, the higher number of mast cells in plaques, particularly with unstable morphology, suggests that mast cells might be involved in the progression of atherosclerosis. The correlation of mast cells with other immune cells that are pivotal in atherogenesis, e.g., myeloid dendritic cells and pro-inflammatory T cells, also suggests an interplay leading to plaque destabilization. Therefore, modulating local mast cell function and invasion into the plaque might be a therapeutic tool for plaque stabilization.