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Supplemental Digital Content is available in the text., Rationale: Despite increasing understanding of the prognostic importance of vascular stiffening linked to perivascular fibrosis in hypertension, the molecular and cellular regulation of this process is poorly understood. Objectives: To study the functional role of microRNA-214 (miR-214) in the induction of perivascular fibrosis and endothelial dysfunction driving vascular stiffening. Methods and Results: Out of 381 miRs screened in the perivascular tissues in response to Ang II (angiotensin II)-mediated hypertension, miR-214 showed the highest induction (8-fold, P=0.0001). MiR-214 induction was pronounced in perivascular and circulating T cells, but not in perivascular adipose tissue adipocytes. Global deletion of miR-214−/− prevented Ang II-induced periaortic fibrosis, Col1a1, Col3a1, Col5a1, and Tgfb1 expression, hydroxyproline accumulation, and vascular stiffening, without difference in blood pressure. Mechanistic studies revealed that miR-214−/− mice were protected against endothelial dysfunction, oxidative stress, and increased Nox2, all of which were induced by Ang II in WT mice. Ang II-induced recruitment of T cells into perivascular adipose tissue was abolished in miR-214−/− mice. Adoptive transfer of miR-214−/− T cells into RAG1−/− mice resulted in reduced perivascular fibrosis compared with the effect of WT T cells. Ang II induced hypertension caused significant change in the expression of 1380 T cell genes in WT, but only 51 in miR-214−/−. T cell activation, proliferation and chemotaxis pathways were differentially affected. MiR-214−/− prevented Ang II-induction of profibrotic T cell cytokines (IL-17, TNF-α, IL-9, and IFN-γ) and chemokine receptors (CCR1, CCR2, CCR4, CCR5, CCR6, and CXCR3). This manifested in reduced in vitro and in vivo T cell chemotaxis resulting in attenuation of profibrotic perivascular inflammation. Translationally, we show that miR-214 is increased in plasma of patients with hypertension and is directly correlated to pulse wave velocity as a measure of vascular stiffness. Conclusions: T-cell–derived miR-214 controls pathological perivascular fibrosis in hypertension mediated by T cell recruitment and local profibrotic cytokine release.

Aims Inflammation is an important driver of hypertension. Periodontitis is a chronic inflammatory disease, which could provide a mechanism for pro-hypertensive immune activation, but evidence of a causal relationship in humans is scarce. We aimed to investigate the nature of the association between periodontitis and hypertension. Methods and results We performed a two-sample Mendelian randomization analysis in the ∼750 000 UK-Biobank/International Consortium of Blood Pressure-Genome-Wide Association Studies participants using single nucleotide polymorphisms (SNPs) in SIGLEC5, DEFA1A3, MTND1P5, and LOC107984137 loci GWAS-linked to periodontitis, to ascertain their effect on blood pressure (BP) estimates. This demonstrated a significant relationship between periodontitis-linked SNPs and BP phenotypes. We then performed a randomized intervention trial on the effects of treatment of periodontitis on BP. One hundred and one hypertensive patients with moderate/severe periodontitis were randomized to intensive periodontal treatment (IPT; sub- and supragingival scaling/chlorhexidine; n = 50) or control periodontal treatment (CPT; supragingival scaling; n = 51) with mean ambulatory 24-h (ABPM) systolic BP (SBP) as primary outcome. Intensive periodontal treatment improved periodontal status at 2 months, compared to CPT. This was accompanied by a substantial reduction in mean SBP in IPT compared to the CPT (mean difference of −11.1 mmHg; 95% CI 6.5–15.8; P Conclusion A causal relationship between periodontitis and BP was observed providing proof of concept for development of clinical trial in a large cohort of hypertensive patients. ClinicalTrials.gov: NCT02131922.

Recent studies have emphasized the role of perivascular inflammation in cardiovascular disease. We studied mechanisms of perivascular leukocyte infiltration in angiotensin II (Ang II)-induced hypertension and their links to vascular dysfunction. Chronic Ang II infusion in mice increased immune cell content of T cells (255 ± 130 to 1664 ± 349 cells/mg; P < 0.01), M1 and M2 macrophages, and dendritic cells in perivascular adipose tissue. In particular, the content of T lymphocytes bearing CC chemokine receptor (CCR) 1, CCR3, and CCR5 receptors for RANTES chemokine was increased by Ang II (CCR1, 15.6 ± 1.5% vs. 31 ± 5%; P < 0.01). Hypertension was associated with an increase in perivascular adipose tissue expression of the chemokine RANTES (relative quantification, 1.2 ± 0.2 vs. 3.5 ± 1.1; P < 0.05), which induced T-cell chemotaxis and vascular accumulation of T cells expressing the chemokine receptors CCR1, CCR3, and CCR5. Mechanistically, RANTES−/− knockout protected against vascular leukocyte, and in particular T lymphocyte infiltration (26 ± 5% in wild type Ang II vs. 15 ± 4% in RANTES−/−), which was associated with protection from endothelial dysfunction induced by Ang II. This effect was linked with diminished infiltration of IFN-γ-producing CD8+ and double-negative CD3+CD4−CD8− T cells in perivascular space and reduced vascular oxidative stress while FoxP3+ T-regulatory cells were unaltered. IFN-γ ex vivo caused significant endothelial dysfunction, which was reduced by superoxide anion scavenging. In a human cohort, a significant inverse correlation was observed between circulating RANTES levels as a biomarker and vascular function measured as flow-mediated dilatation (R = −0.3, P < 0.01) or endothelial injury marker von Willebrand factor (R = +0.3; P < 0.01). Thus, chemokine RANTES is important in the regulation of vascular dysfunction through modulation of perivascular inflammation.—Mikolajczyk, T. P., Nosalski, R., Szczepaniak, P., Budzyn, K., Osmenda, G., Skiba, D., Sagan, A., Wu, J., Vinh, A., Marvar, P. J., Guzik, B., Podolec, J., Drummond, G., Lob, H. E., Harrison, D. G., Guzik, T. J. Role of chemokine RANTES in the regulation of perivascular inflammation, T-cell accumulation, and vascular dysfunction in hypertension.

Systemic immune activation has been recently linked to chronic inflammatory disorders of the oral cavity, particularly to periodontitis. The purpose of this study was to determine whether treatment of a fungus-induced oral inflammation, namely denture-related stomatitis (DRS), can affect the activation of the systemic immune response.Peripheral blood from patients with denture-related stomatitis caused by Candida albicans infection (n = 15) was collected at three time points: before treatment with nystatin, at the end of therapy and 2 months after finishing therapy. Activation of T cells and monocytes was assessed by flow cytometry.The percentages of peripheral lymphocytes, T cells and their subpopulations, as well as monocytes were similar before, immediately following and two months after nystatin treatment. Cells expressing early activation marker CD69 and RANTES C-C chemokine receptor type 5 significantly increased immediately after treatment and returned to baseline levels after two months. Th17 cells, which have been implicated in the pathogenesis of DRS, remained unchanged. Central memory CD4+ subset and intermediate subset of monocytes were lower after therapy and this effect was sustained for two months.Treatment of denture-related stomatitis does not seem to affect the general state of the cellular components of the immune system. The results suggest a potential proinflammatory effect of the antifungal agent, nystatin. Although transient and not intense, this effect might be of particular clinical importance, because of relationships between inflammation and certain diseases. Further studies are required to clarify this aspect.

Vascular dysfunction is an important phenomenon in hypertension. We hypothesized that angiotensin II (AngII) affects transcriptome in the vasculature in a region-specific manner, which may help to identify genes related to vascular dysfunction in AngII-induced hypertension. Mesenteric artery and aortic transcriptome was profiled using Illumina WG-6v2.0 chip in control and AngII infused (490 ng/kg/min) hypertensive mice. Gene set enrichment and leading edge analyses identified Sphingosine kinase 1 (Sphk1) in the highest number of pathways affected by AngII. Sphk1 mRNA, protein and activity were up-regulated in the hypertensive vasculature. Chronic sphingosine-1-phosphate (S1P) infusion resulted in a development of significantly increased vasoconstriction and endothelial dysfunction. AngII-induced hypertension was blunted in Sphk1−/− mice (systolic BP 167 ± 4.2 vs. 180 ± 3.3 mmHg, p Sphk1−/− mice. Pharmacological inhibition of S1P synthesis reduced vasoconstriction of mesenteric arteries. While Sphk1 is important in mediating vasoconstriction in hypertension, Sphk1−/− mice were characterized by enhanced endothelial dysfunction, suggesting a local protective role of Sphk1 in the endothelium. S1P serum level in humans was correlated with endothelial function (arterial tonometry). Thus, vascular transcriptome analysis shows that S1P pathway is critical in the regulation of vascular function in AngII-induced hypertension, although Sphk1 may have opposing roles in the regulation of vasoconstriction and endothelium-dependent vasorelaxation.

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.

Chronic inflammatory disorders of the oral cavity, such as periodontitis, were recently linked to systemic immune activation. Since fungal oral infections have not yet been studied in this respect, the aim of our study is to determine whether the local inflammation caused by oral fungal infection of the palatal tissue (denture stomatitis-DS) is associated with the systemic inflammatory response. This question is becoming essential as the population ages.Peripheral blood of DS patients (n = 20) and control patients (n = 24) was assessed with flow cytometry to determine lymphocyte and monocyte profiles. Intracellular cytometric analysis was carried out to establish cytokine production by T cells. DS was diagnosed based on clinical symptoms of DS such as swelling and redness of oral mucosa, confirmed by microbiological swabs for fungal colonization with Candida species. The control group was recruited from denture users without clinical and microbiological signs of oral infections.Percentages of peripheral lymphocytes, T cells, monocytes, and their subpopulations were similar in both studied groups. The exception was median percentages of CD25+ T cell subsets, which were significantly lower in DS patients than in control subjects. This reduction was observed in both CD4 T cell subset (16.7% and 28.1%; p = 0.0006) and CD8 T cell subset (4.6% and 7.0%; p = 0.007) CONCLUSIONS: While DS and associated local fungal infection do not overtly affect activation of monocytes or lymphocytes, the number of CD 25+ T cells is significantly lower in the DS patients, possibly indicating limited potential for the infection clearance in denture-using aging patients.

Background Systemic lupus erythematosus (SLE) is characterized by increased cardiovascular morbidity and mortality. SLE patients have increased prevalence of subclinical atherosclerosis, although the mechanisms of this observation remain unclear. Considering the emerging role of monocytes in atherosclerosis, we aimed to investigate the relationship between subclinical atherosclerosis, endothelial dysfunction and the phenotype of peripheral blood monocytes in SLE patients. Methods We characterized the phenotype of monocyte subsets defined by the expression of CD14 and CD16 in 42 patients with SLE and 42 non-SLE controls. Using ultrasonography, intima-media thickness (IMT) of carotid arteries and brachial artery flow-mediated dilation (FMD) as well as nitroglycerin-induced dilation (NMD) were assessed. Results Patients with SLE had significantly, but only modestly, increased IMT when compared with non-SLE controls (median (25th/75th percentile) 0.65 (0.60/0.71) mm vs 0.60 (0.56/0.68) mm; p Conclusions Our observations suggest that CD14dimCD16+monocytes are associated with subclinical atherosclerosis in SLE, although the mechanism appears to be independent of endothelial dysfunction.

ObjectiveMutations in the glucokinase (GCK) gene, along with hepatocyte nuclear factor 1A (HNF1A) gene mutations, are the most frequent cause of maturity-onset diabetes of the young (MODY). GCK-MODY patients are typically characterized by a moderate fasting hyperglycemia; however, little is known about atherosclerosis and intermediate-related phenotypes in these subjects.DesignTo examine carotid artery intima-media thickness (IMT) and endothelial function assessed by brachial artery flow-mediated dilatation (FMD) in GCK gene mutations carriers and HNF1A-MODY.MethodsA total of 64 subjects with GCK gene mutations, and 52 HNF1A gene mutation carriers as well as 53 nondiabetic controls were examined. IMT and FMD were assessed by ultrasonography. Appropriate statistical tests were performed to assess differences between the groups, and multivariate linear regression was done for the association with IMT and FMD.ResultsThe clinical characteristics of all groups were similar with the mean age at examination of 35.1, 41.1, and 39.5 years for GCK, HNF1A and the control group respectively. The highest mean IMT value was in the HNF1A-MODY group: 7.0±1.4 mm, whereas it reached 6.3±1.4 mm in GCK mutation carriers and 6.3±1.3 mm in controls (P=0.008). After adjustment for possible clinical and biochemical cofounders, IMT remained higher in HNF1A-MODY patients as compared with GCK-MODY patients (P=0.02) and controls (P=0.0003). FMD was significantly lower in HNF1A (9.9±4.6%) and GCK-MODY (11.1±4.6%) patients in comparison with controls (13.9±4.7%; P=0.0001). After adjustment, FMD remained lower in HNF1A-MODY (P=0.0005) and GCK-MODY patients (P=0.01) as compared with controls.ConclusionsBoth examined MODY groups demonstrated evidence of endothelial dysfunction. In addition, HNF1-MODY patients seem to be more prone to an early atherosclerotic phenotype.

Objectives. To evaluate blood monocyte subsets and functional monocyte properties in patients with rheumatoid arthritis (RA) of short duration in the context of cardiovascular (CV) risk and disease activity.Methods. We studied conventional markers of CV risk, intima media thickness (IMT), and blood monocyte subsets in 27 patients aged 41 ± 10 years with RA of short duration (median 12 months) and 22 healthy controls. The RA subjects were divided into low (DAS28: 2.6–5.1) and high (DAS28 > 5.1) disease activity.Results. RA patients exhibited increased levels of intermediate (CD14++CD16+) monocytes with decreased CD45RA expression compared to controls, increased counts of classical (CD14++CD16−) monocytes, and decreased percentages of nonclassical (CD14+CD16++) monocytes. Patients with high disease activity had lower HLA DR expression on classical monocytes compared to low disease activity patients. There were no differences in monocyte subsets between subjects with DAS > 5.1 and DAS ≤ 5.1. There were no significant intergroup differences in IMT and the majority of classical CV risk factors.Conclusions. Patients with RA of short duration show alteration in peripheral blood monocyte subsets despite the fact that there is no evidence of subclinical atherosclerosis. Disease activity assessed with DAS28 was associated with impaired functional properties but not with a shift in monocyte subpopulations.

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.

Efficient removal of apoptotic polymorphonuclear leukocytes (PMNs) is an important step in the resolution of inflammation, which protects tissues from the noxious contents of dying cells. While the impairment of apoptotic PMNs removal has been demonstrated for macrophages in systemic lupus erythematosus (SLE), recent studies show that monocytes are also capable of such phagocytosis, although their involvement in SLE is not clear. Therefore, we characterized phagocytosis of apoptotic PMNs by monocytes in 22 patients with SLE and 22 healthy controls. Using flow cytometry we demonstrate that in SLE peripheral blood monocytes show impaired phagocytosis of autologous apoptotic PMNs, while they efficiently engulf apoptotic PMNs isolated from healthy subjects. Monocytes CD14highCD16+ and CD14dimCD16+ more efficiently interacted with apoptotic neutrophils than CD16– cells both in SLE and healthy subjects. Monocytes in SLE showed modestly decreased expression of CD35 and CD91 and increased expression of T Cell Ig- and mucin-domain-containing molecule-3 (TIM-3); however, these differences were evident mainly in selected subsets of monocytes (CD16+) while defects in phagocytosis were observed in all monocyte subsets. Apoptotic cell-dependent induction of lipopolysaccharide (LPS) stimulated production of anti-inflammatory cytokine IL-10 by peripheral blood mononuclear cells (PBMC) was blunted in SLE while the production of pro-inflammatory cytokine TNF-α was unchanged.

Background:\ud \ud The genetic background of atherosclerosis in type 2 diabetes mellitus (T2DM) is complex and poorly understood. Studying genetic components of intermediate phenotypes, such as endothelial dysfunction and oxidative stress, may aid in identifying novel genetic components for atherosclerosis in diabetic patients.\ud \ud Methods:\ud \ud Five polymorphisms forming two haplotype blocks within the GTP cyclohydrolase 1 gene, encoding a rate limiting enzyme in tetrahydrobiopterin synthesis, were studied in the context of flow and nitroglycerin mediated dilation (FMD and NMD), intima-media thickness (IMT), and plasma concentrations of von Willebrand factor (vWF) and malondialdehyde (MDA).\ud \ud Results:\ud \ud Rs841 was associated with FMD (p = 0.01), while polymorphisms Rs10483639, Rs841, Rs3783641 (which form a single haplotype) were associated with both MDA (p = 0.012, p = 0.0015 and p = 0.003, respectively) and vWF concentrations (p = 0.016, p = 0.03 and p = 0.045, respectively). In addition, polymorphism Rs8007267 was also associated with MDA (p = 0.006). Haplotype analysis confirmed the association of both haplotypes with studied variables.\ud \ud Conclusions:\ud \ud Genetic variation of the GCH1 gene is associated with endothelial dysfunction and oxidative stress in T2DM patients.

INTRODUCTION Endothelial dysfunction, characterized by the loss of nitric oxide bioavailability, is a key element in the pathogenesis of atherosclerosis and an important prognostic factor in cardiovascular diseases. Therefore, the development of reliable, safe, and noninvasive methods of endothelial function assessment is important for their use in cardiovascular risk stratification. Brachial artery flow‑mediated dilation (FMD) is widely used in research but technical difficulties and problems with calibration between laboratories limit its clinical use. Reactive hyperemia–peripheral artery tonometry (RH‑PAT, EndoPAT) has been developed as a simpler, cheaper, and potentially more reproducible method.\ud OBJECTIVES We aimed to investigate associations between RH‑PAT and FMD in relation to atherosclerotic risk factor profile.\ud PATIENTS AND METHODS The study involved 80 subjects (52 men, 28 women) aged 43.6 ±14.8 years, with moderate‑to‑low cardiovascular risk (mean SCORE, 2.2% ±2%), in whom FMD, RH‑PAT, and intima–media thickness (IMT) were determined.\ud RESULTS The reactive hyperemia index (RHI) measured by RH‑PAT correlated with FMD (r = 0.35, P

Background Microangioathy and macroangiopathy in type 2 diabetes mellitus (T2DM) frequently coexist. Both types of vascular complications share traditional risk factors. It is not clear whether the presence of microangiopathy, such as diabetic retinopathy (DR), constitutes a predictor of atherosclerosis in T2DM. Here we described the search for the association between DR and intima-media thickness (IMT) in T2DM. We also compared endothelial function in subjects with and without DR. Material and methods We examined 182 consecutive patients with T2DM for at least 5 years (mean age at examination 56·3 ± 6·52 years). We assessed (i) IMT of carotid artery by ultrasound and (ii) endothelial function by flow-mediated dilatation (FMD) method as well as by measurement of concentrations of von Willebrand factor (vWF) and s-ICAM-1. All patients underwent ophthalmological examination. Statistical analysis included Student's, Mann–Whitney, chi-square, Fisher tests and multiple regression. Results DR was found in 71 (39·0%) subjects. IMT was higher in patients with DR than those without DR (0·87 mm vs. 0·79 mm, respectively, P = 0·0001). FMD was lower in the complication group than in subjects without DR (8·38% vs. 10·45%, respectively, P = 0·0023). Concentrations of s-ICAM-1 and vWF were not different between the groups. In multiple regression analysis, DR was among the predictors of increased IMT (P = 0·016) and decreased FMD (P = 0·002). We did not find a significant association of DR with vWF and s-ICAM-1 (P = 0·09 and P = 0·11, respectively). Conclusions DR is associated with increased IMT and endothelial dysfunction in T2DM. Impaired endothelial function may be a common denominator of pathogenesis of microvascular complications and atherosclerosis in T2DM.