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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.

Animal models, human observational and genetic studies have shown that immune and inflammatory mechanisms play a key role in hypertension and its complications. We review the effects of immunomodulatory interventions on blood pressure, target organ damage and cardiovascular risk in humans. In experimental and small clinical studies both non-specific immunomodulatory approaches, such as mycophenolate mofetil and methotrexate, and medications targeting T and B lymphocytes, such as tacrolimus, cyclosporine, everolimus, rituximab, lower blood pressure and reduce organ damage. Mechanistically targeted immune interventions include isolevuglandin (isoLG) scavengers to prevent neo-antigen formation, co-stimulation blockade (abatacept, belatacept), and anti-cytokine therapies (secukinumab, tocilizumab, canakinumab, TNF-α inhibitors). In many studies, trial designs have been complicated by a lack of blood pressure related endpoints, inclusion of largely normotensive study populations, polypharmacy, and established comorbidities. Among a wide range of interventions reviewed, TNF-α inhibitors have provided the most robust evidence of blood pressure lowering. Treatment of periodontitis also appears to deliver non-pharmacological antihypertensive effects. Evidence of immunomodulatory drugs influencing hypertension-mediated organ damage are discussed. Animal model, observational studies, and trial data in humans support the therapeutic potential of immune targeted therapies in blood pressure lowering and in hypertension-mediated organ damage. Targeted studies are now needed to address their effects on blood pressure in hypertensive individuals.

The novel coronavirus disease (COVID-19) outbreak, caused by SARS-CoV-2, represents the greatest medical challenge in decades. We provide a comprehensive review of the clinical course of COVID-19, its comorbidities, and mechanistic considerations for future therapies. While COVID-19 primarily affects the lungs, causing interstitial pneumonitis and severe acute respiratory distress syndrome (ARDS), it also affects multiple organs, particularly the cardiovascular system. Risk of severe infection and mortality increase with advancing age and male sex. Mortality is increased by comorbidities: cardiovascular disease, hypertension, diabetes, chronic pulmonary disease, and cancer. The most common complications include arrhythmia (atrial fibrillation, ventricular tachyarrhythmia, and ventricular fibrillation), cardiac injury [elevated highly sensitive troponin I (hs-cTnI) and creatine kinase (CK) levels], fulminant myocarditis, heart failure, pulmonary embolism, and disseminated intravascular coagulation (DIC). Mechanistically, SARS-CoV-2, following proteolytic cleavage of its S protein by a serine protease, binds to the transmembrane angiotensin-converting enzyme 2 (ACE2) —a homologue of ACE—to enter type 2 pneumocytes, macrophages, perivascular pericytes, and cardiomyocytes. This may lead to myocardial dysfunction and damage, endothelial dysfunction, microvascular dysfunction, plaque instability, and myocardial infarction (MI). While ACE2 is essential for viral invasion, there is no evidence that ACE inhibitors or angiotensin receptor blockers (ARBs) worsen prognosis. Hence, patients should not discontinue their use. Moreover, renin–angiotensin–aldosterone system (RAAS) inhibitors might be beneficial in COVID-19. Initial immune and inflammatory responses induce a severe cytokine storm [interleukin (IL)-6, IL-7, IL-22, IL-17, etc.] during the rapid progression phase of COVID-19. Early evaluation and continued monitoring of cardiac damage (cTnI and NT-proBNP) and coagulation (D-dimer) after hospitalization may identify patients with cardiac injury and predict COVID-19 complications. Preventive measures (social distancing and social isolation) also increase cardiovascular risk. Cardiovascular considerations of therapies currently used, including remdesivir, chloroquine, hydroxychloroquine, tocilizumab, ribavirin, interferons, and lopinavir/ritonavir, as well as experimental therapies, such as human recombinant ACE2 (rhACE2), are discussed.

Supplemental Digital Content is available in the text., High blood pressure is a risk factor for cardiovascular diseases. Ang II (angiotensin II), a key pro-hypertensive hormone, mediates target organ consequences such as endothelial dysfunction and cardiac hypertrophy. S1P (sphingosine-1-phosphate), produced by Sphk1 (sphingosine kinase 1), plays a pivotal role in the pathogenesis of hypertension and downstream organ damage, as it controls vascular tone and regulates cardiac remodeling. Accordingly, we aimed to examine if pharmacological inhibition of Sphk1 using selective inhibitor PF543 can represent a useful vasoprotective and cardioprotective anti-hypertensive strategy in vivo. PF543 was administered intraperitoneally throughout a 14-day Ang II-infusion in C57BL6/J male mice. Pharmacological inhibition of Sphk1 improved endothelial function of arteries of hypertensive mice that could be mediated via decrease in eNOS (endothelial nitric oxide synthase) phosphorylation at T495. This effect was independent of blood pressure. Importantly, PF543 also reduced cardiac hypertrophy (heart to body weight ratio, 5.6±0.2 versus 6.4±0.1 versus 5.9±0.2 mg/g; P

Perivascular adipose tissue (PVAT) plays a critical role in the pathogenesis of cardiovascular disease. In vascular pathologies perivascular adipose tissue increases in volume and becomes dysfunctional, with altered cellular composition and molecular characteristics. PVAT dysfunction is characterized by its inflammatory character, oxidative stress, diminished production of vaso-protective adipocyte derived relaxing factors (ADRFs) and increased production of paracrine factors such as resistin, leptin, cytokines (IL-6, TNF-α) and chemokines (RANTES, MCP-1). These adipocyte-derived factors initiate and orchestrate inflammatory cell infiltration including primarily T cells, macrophages, dendritic cells (DCs), B cells as well as NK cells. Protective factors such as adiponectin can reduce NADPH oxidase superoxide production and increase nitric oxide bioavailability in the vessel wall, while inflammation (e.g. IFN-γ or IL-17) induces vascular oxidases and eNOS dysfunction in the endothelium, vascular smooth muscle cells and adventitial fibroblasts. All of these events link dysfunctional perivascular fat to vascular dysfunction. These mechanisms are important in the context of a number of cardiovascular disorders including atherosclerosis, hypertension, diabetes and obesity. Inflammatory changes in PVAT molecular and cellular responses are uniquely different from classical visceral or subcutaneous adipose tissue or from adventitia, emphasizing unique structural and functional features of this adipose tissue compartment. Therefore, it is essential to develop techniques for monitoring PVAT characteristics and assessing its inflammation. This will lead to better understanding of the early stages of vascular pathologies and development of new therapeutic strategies focusing on perivascular adipose tissue.

Background and Purpose: Inflammation plays a key role in atherosclerosis. The protective role of angiotensin 1–7 (Ang-(1–7)) in vascular pathologies suggested the therapeutic use of low MW, non-peptide Ang-(1–7) mimetics, such as AVE0991. The mechanisms underlying the vaso-protective effects of AVE0991, a Mas receptor agonist, remain to be explored. Experimental Approach: We investigated the effects of AVE0991 on the spontaneous atherosclerosis in apolipoprotein E (ApoE)−/− mice, in the context of vascular inflammation and plaque stability. Key Results: AVE0991 has significant anti-atherosclerotic properties in ApoE−/− mice and increases plaque stability, by reducing plaque macrophage content, without effects on collagen. Using the descending aorta of chow-fed ApoE−/− mice, before significant atherosclerotic plaque develops, we gained insight to early events in atherosclerosis. Interestingly, perivascular adipose tissue (PVAT) and adventitial infiltration with macrophages and T-cells precedes atherosclerotic plaque or the impairment of endothelium-dependent NO bioavailability (a measure of endothelial function). AVE0991 inhibited perivascular inflammation, by reducing chemokine expression in PVAT and through direct actions on monocytes/macrophages inhibiting their activation, characterized by production of IL-1β, TNF-α, CCL2 and CXCL10, and differentiation to M1 phenotype. Pretreatment with AVE0991 inhibited migration of THP-1 monocytes towards supernatants of activated adipocytes (SW872). Mas receptors were expressed in PVAT and in THP-1 cells in vitro, and the anti-inflammatory effects of AVE0991 were partly Mas dependent. Conclusions and Implications: The selective Mas receptor agonist AVE0991 exhibited anti-atherosclerotic and anti-inflammatory actions, affecting monocyte/macrophage differentiation and recruitment to the perivascular space during early stages of atherosclerosis in ApoE−/− mice. Linked Articles: This article is part of a themed section on Targeting Inflammation to Reduce Cardiovascular Disease Risk. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.22/issuetoc and http://onlinelibrary.wiley.com/doi/10.1111/bcp.v82.4/issuetoc

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-β-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.

Purpose 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. Materials and Methods 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. Results 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.

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.

Introduction: Hypertension is associated with enhanced oxidative stress and perivascular inflammation. Although, that aging and oxidative stress are major factors in the development of hypertension their effect on perivascular inflammation remains unclear. Methods: Using flow cytometry we studied leukocytes infiltrating perivascular adipose tissue (PVAT) in 1-, 3-, 6- and 12-month-old SHR (Spontaneously Hypertensive Rats) and normotensive WKY (Wistar-Kyoto) rats. Additionally, 1-month-old rats were treated with GKT137831 (60mg/kg) or ML171 (NOX1/4 and NOX1 inhibitor, respectively) for 4 weeks. Blood pressure (PB) was measured by the tail cuff. Results: Aging in SHRs was associated with elevation of BP (139±4 vs 180±4 vs 202±2 vs 208±2 mmHg; 1 vs 3 vs 6 vs 12-month-old, respectively) when this effect was not seen in WKY rats. While the total number of leukocytes infiltrating PVAT were comparable between 1-month-old WKY and SHR (p=0.8) aging escalated their numbers only in SHRs (2096±164 vs 1994±296 vs 2311±470 vs 3255±408 cell/mg; p int int int int Conclusions: Aging in spontaneous hypertension is associated with elevation of BP and aggravation of perivascular inflammation which are hastened after NOX1/4 inhibitor treatment.

Transient Receptor Potential Melastatin 7 (TRPM7) cation channel is a chanzyme (channel + kinase) that influences cellular Mg2+ homeostasis and vascular signalling. However, the pathophysiological significance of TRPM7 in the cardiovascular system is unclear. The aim of this study was to investigate the role of this chanzyme in the cardiovascular system focusing on inflammation and fibrosis.TRPM7-deficient mice with deletion of the kinase domain (TRPM7+/Δkinase) were studied and molecular mechanisms investigated in TRPM7+/Δkinase bone marrow-derived macrophages (BMDM) and co-culture systems with cardiac fibroblasts. TRPM7-deficient mice had significant cardiac hypertrophy, fibrosis, and inflammation. Cardiac collagen and fibronectin content, expression of pro-inflammatory mediators (SMAD3, TGFβ) and cytokines [interleukin (IL)-6, IL-10, IL-12, tumour necrosis factor-α] and phosphorylation of the pro-inflammatory signalling molecule Stat1, were increased in TRPM7+/Δkinase mice. These processes were associated with infiltration of inflammatory cells (F4/80+CD206+ cardiac macrophages) and increased galectin-3 expression. Cardiac [Mg2+]i, but not [Ca2+]i, was reduced in TRPM7+/Δkinase mice. Calpain, a downstream TRPM7 target, was upregulated (increased expression and activation) in TRPM7+/Δkinase hearts. Vascular functional and inflammatory responses, assessed in vivo by intra-vital microscopy, demonstrated impaired neutrophil rolling, increased neutrophil: endothelial attachment and transmigration of leucocytes in TRPM7+/Δkinase mice. TRPM7+/Δkinase BMDMs had increased levels of galectin-3, IL-10, and IL-6. In co-culture systems, TRPM7+/Δkinase macrophages increased expression of fibronectin, proliferating cell nuclear antigen, and TGFβ in cardiac fibroblasts from wild-type mice, effects ameliorated by MgCl2 treatment.We identify a novel anti-inflammatory and anti-fibrotic role for TRPM7 and suggest that its protective effects are mediated, in part, through Mg2+-sensitive processes.

RATIONALE: Giant cell arteritis (GCA), a primary vasculitis of medium and large arteries, is associated with vessel wall damage, elastic membrane fragmentation and vascular remodeling. Proteinases are believed to contribute to pathogenesis by degrading extracellular matrix and causing tissue injury. OBJECTIVE: The matrix metalloproteinase 9 (MMP-9), a type IV collagenase, is produced in the vasculitic lesions of GCA. It is unknown which pathogenic processes are MMP-9 dependent. METHODS AND RESULTS: The tissue transcriptome of GCA-affected temporal arteries contained high amounts of MMP-9 transcripts and immunostaining for pro-MMP-9 localized the enzyme to wall-infiltrating macrophages. MMP-2 and MMP-9 transcripts were also abundant in monocytes and monocyte-derived macrophages from GCA patients. Patient-derived monocytes outperformed healthy monocytes in passing through engineered basement membranes. GCA CD4(+) T cells required MMP-9-producing monocytes to penetrate through matrix built from type IV collagen. In vivo functions of MMP-9 were tested in a human artery-SCID chimera model by blocking enzyme activity with a highly specific monoclonal antibody or by injecting recombinant MMP-9. Inhibiting MMP-9 activity profoundly suppressed vascular injury, decreased the density of inflammatory infiltrates (p

Objective: Hypertension (HT) is associated with perivascular inflammation and increased vascular fibrosis. MicroRNAs (miR) are a novel gene expression regulation mechanism and play a pivotal role in a range of pathological processes. The role and mechanism of miR214 in vascular fibrosis is unknown. Methods: 3-month-old C57BL/6, miR214KO and wild-type littermates were treated with angiotensin II (AngII, 490ng/kg/min; n=6-10) or control buffer for 14 days. PVATs from C57BL/6 animals were analysed using TaqMan_Rodent_microRNA_Arrays. Histological studies, wire myography, lucigenin-enhanced luminometry and cytometrical analysis was conducted, followed by statistical analysis with ANOVA or t-test. Data are expressed as a mean±SEM. Results: Out of 381 miRs, 16 were significantly overexpressed in C57BL/6 AngII animals, with only miR214 showing 8-fold induction (p Conclusions: MiR-214 plays a major role in modulation of aortic fibrosis, vascular function, oxidative stress and perivascular inflammation.

Purpose of Review: \ud \ud Hypertension is a common disorder with substantial impact on public health due to highly elevated cardiovascular risk. The mechanisms still remain unclear and treatments are not sufficient to reduce risk in majority of patients. Inflammatory mechanisms may provide an important mechanism linking hypertension and cardiovascular risk. We aim to review newly identified immune and inflammatory mechanisms of hypertension with focus on their potential therapeutic impact.\ud \ud Recent Findings: \ud \ud In addition to the established role of the vasculature, kidneys and central nervous system in pathogenesis of hypertension, low-grade inflammation contributes to this disorder as indicated by experimental models and GWAS studies pointing to SH2B3 immune gene as top key driver of hypertension. Immune responses in hypertension are greatly driven by neoantigens generated by oxidative stress and modulated by chemokines such as RANTES, IP-10 and microRNAs including miR-21 and miR-155 with other molecules under investigation. Cells of both innate and adoptive immune system infiltrate vasculature and kidneys, affecting their function by releasing pro-inflammatory mediators and reactive oxygen species.\ud \ud Summary: \ud \ud Immune and inflammatory mechanisms of hypertension provide a link between high blood pressure and increased cardiovascular risk, and reduction of blood pressure without attention to these underlying mechanisms is not sufficient to reduce risk.

Perivascular adipose tissue (PVAT) plays a critical role in the pathogenesis of cardiovascular disease. In vascular pathologies, perivascular adipose tissue increases in volume and becomes dysfunctional, with altered cellular composition and molecular characteristics. PVAT dysfunction is characterized by its inflammatory character, oxidative stress, diminished production of vaso-protective adipocyte-derived relaxing factors and increased production of paracrine factors such as resistin, leptin, cytokines (IL-6 and TNF-α) and chemokines [RANTES (CCL5) and MCP-1 (CCL2)]. These adipocyte-derived factors initiate and orchestrate inflammatory cell infiltration including primarily T cells, macrophages, dendritic cells, B cells and NK cells. Protective factors such as adiponectin can reduce NADPH oxidase superoxide production and increase NO bioavailability in the vessel wall, while inflammation (e.g. IFN-γ or IL-17) induces vascular oxidases and eNOS dysfunction in the endothelium, vascular smooth muscle cells and adventitial fibroblasts. All of these events link the dysfunctional perivascular fat to vascular dysfunction. These mechanisms are important in the context of a number of cardiovascular disorders including atherosclerosis, hypertension, diabetes and obesity. Inflammatory changes in PVAT's molecular and cellular responses are uniquely different from classical visceral or subcutaneous adipose tissue or from adventitia, emphasizing the unique structural and functional features of this adipose tissue compartment. Therefore, it is essential to develop techniques for monitoring the characteristics of PVAT and assessing its inflammation. This will lead to a better understanding of the early stages of vascular pathologies and the development of new therapeutic strategies focusing on perivascular adipose tissue.This article is part of a themed section on Molecular Mechanisms Regulating Perivascular Adipose Tissue - Potential Pharmacological Targets? To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.20/issuetoc.

TRPM7 is a cationic ion channel and with a serine/threonine kinase important for cellular Mg 2+ homeostasis. We recently showed that TRPM7-kinase plays a role in aldosterone-mediated vascular effects and inflammation. Here we explored the role of TRPM7-kinase in cardiac fibrosis and vascular function in aldosterone-induced hypertension in mice. Wild-type (WT) or heterozygote TRPM7-kinase domain (TRPM7+/-) were treated with infused aldosterone (600 μg/Kg/day) and NaCl 1% in drinking water (aldo/salt) for 4 weeks. Blood pressure (BP) was evaluated by tail-cuff. Vessel function was investigated in mesenteric arteries by wire and pressure myography. Protein expression was assessed in cardiac tissue by western-blot and histology. Aldo/salt increased BP in TRPM7+/- and WT to similar levels (137mmHg vs control 118mmHg). Mesenteric arteries from untreated TRPM7+/- mice were more sensitive to relaxation induced by acetylcholine (LogEC50: 7.6±0.1 vs 7.1±0.2, TRPM7+/- and WT, respectively), effects that were reduced by Aldo/salt treatment (LogEC50: 7.2±0.1). Phenylephrine-contraction and sodium nitroprusside-relaxation curves were similar among groups. Pressure myography showed that in WT, aldo/salt increase the diameter (26%) and cross-sectional area (40%), resulting in hypertrophic outward remodelling, whereas in TRPM7+/-, the treatment decreased the diameter (16%) and increase the wall/lumen ration (82%), resulting in eutrophic inward remodelling. Hearts from TRPM7+/- presented decreased expression of annexin-1, which is a target protein of TRPM7-kinase, that was further decreased by aldo-salt. Hearts from untreated TRPM7+/- mice had increased fibrotic markers: plasma galectin-3 (2.5ng/mL) vs WT (1.4ng/mL) and protein expression for fibronectin (2.4-fold) and TGFβ (2-fold), and the aging marker p-P66Sch (47%) which were similar to WT-aldo/salt. Aldo/salt induced higher collagen expression in TRPM7+/- than in WT animals (15%), as observed by picrosirius red staining. Our findings provide some insights into aldosterone signalling through TRPM7-kinase and suggest that this chanzyme may have protective actions, which when downregulated, promotes vascular remodelling and cardiac fibrosis in aldosterone-induced hypertension.

TRPM7 is a Mg2 + channel linked to a kinase domain important in cell proliferation and survival. We demonstrated that cells deficient in TRPM7-kinase domain are prone to aldosterone (aldo)-induced oxidative stress and inflammation. Here, we investigated whether TRPM7-kinase plays a role in inflammatory responses in aldo-induced hypertension. Wild-type (WT) or heterozygote TRPM7-kinase domain (TRPM7+/-) mice were infused with aldo (600μg/Kg/day - osmotic minipumps) and 1% NaCl in the drinking water (aldo-salt) for 4 weeks. Inflammatory responses were evaluated by examining T cells (CD4+ and CD8+) and macrophages (M1- and M2-phenotype) infiltration in kidneys and spleens, using flow cytometry. Gene and protein expression was assessed by real-time PCR and immunoblot respectively. ROS was evaluated by lucigenin chemiluminescence. Aldo-salt increased kidney mass and urinary levels of albumin, Ca 2+ , Mg 2+ and K + , similarly in WT and TRPM7+/- (p6 vs WT 0.22x10 6 cells/g), TCD4+ cells (27% vs WT 19%), and macrophages (47% vs WT 31%) (p8 vs WT 2.0x10 8 cells/mg). Splenic macrophages were higher in untreated TRPM7+/- (8.8% vs WT 5.9%) and presented an increase in CD206 M2-marker (16% vs WT 7%), higher TCD4+ (68% vs WT 50%) and TCD8+ (26% vs WT 17%), values that were similar to WT aldo-salt. Our data provide insights into the importance of the TRPM7-kinase domain in the immune system activation, which when down regulated provokes an increase in inflammatory cell infiltration in kidneys and spleens in aldo-salt-induced hypertension.

Objective: We have previously characterised the stroke prone spontaneously hypertensive rat (SHRSP) as a model of deficient uterine artery remodelling and identified an increase in pro-inflammatory TNFα relative to the normotensive WKY strain during pregnancy. Method: SHRSP were treated with etanercept (0.8 mg/kg) or vehicle at gestational day (GD) 0, 6, 12 and 18. Animals were sacrificed at GD18. Results: Etanercept reduced systolic blood pressure in the SHRSP after GD12 (ΔSBP GD 10-21 SHRSP 12.0 ± 4.17 vs. ETN 25.8 ± 4.27 mmHg; pLow 14.32 ± 0.77 % granzyme B+; p Conclusions: Excess TNFα plays a causative role in adverse pregnancy outcome in the SHRSP. One source of this TNFα is an increase in NK cells during gestation in the SHRSP. Etanercept targets NK cells in the SHRSP placenta and down-regulates cytotoxic granzyme B production.

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.

SummaryIntraluminal 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.

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.

TRPM7 is cation channel with intrinsic kinase activity important for cellular Mg2+ homeostasis. We recently showed that TRPM7-kinase plays a role in aldosterone-mediated vascular effects and inflammation. Here we explored the putative role of TRPM7-kinase in cardiac fibrosis and vascular function in aldosterone-induced hypertension in mice. Wild-type (WT) or heterozygote TRPM7-kinase domain (TRPM7+/-) mice were treated with infused aldosterone (600 μg/Kg/day) and NaCl 1% in drinking water (aldo/salt) for 4 weeks. Blood pressure (BP) was evaluated by tail-cuff. Vessel function was investigated in mesenteric resistance arteries by wire and pressure myography. Protein expression of pro-fibrotic molecules was assessed in cardiac tissue by western-blot and histology. Aldo/salt increased BP in TRPM7+/- and WT animals to similar levels (137mmHg vs control group 118mmHg). Mesenteric arteries from untreated TRPM7+/- mice are more sensitive to relaxation induced by acetylcholine (LogEC50: 7.6 ± 0.1 vs 7.1 ± 0.2, TRPM7+/- and WT, respectively), effects that were reduced by Aldo/salt treatment (LogEC50: 7.2 ± 0.1). Phenylephrine-contraction and sodium nitroprusside-relaxation curves were similar among groups. Pressure myography showed that in WT, aldo/salt increase the diameter (26%) and cross-sectional area (40%), resulting in hypertrophic outward remodeling, whereas in TRPM7+/-, the treatment decreased the diameter (16%) and increase the wall/lumen ration (82%), resulting in eutrophic inward remodeling. Regarding to fibrotic markers, untreated TRPM7+/- mice had increased plasma galectin-3 (2.5ng/mL) vs WT (1.4ng/mL) and protein expression for fibronectin (2.4-fold) and TGFβ (2-fold) in hearts, which were similar to WT-aldo/salt. Aldo/salt treatment induced higher collagen expression in TRPM7+/- than in WT animals (15%). Our results suggest anti-fibrotic effects of the TRPM7-kinase domain, since TRPM7+/- mice presented increase in heart fibrotic markers and vascular remodeling compared to WT. Our findings provide some insights into aldosterone signaling through TRPM7-kinase and suggest that this chanzyme may have protective actions, which when downregulated, promotes cardiac fibrosis in aldosterone-induced hypertension.

Background Cardiac dysfunction is one of the hallmarks of hypertension. It is characterised by cardiac fibrosis, diastolic dysfunction and development of heart failure. The mechanisms of hypertensive heart disease are not fully understood. Recent studies suggest a role of miR-214 in regulation of fibrosis. Thus, the aim of this work is to investigate the role miR-214 has in the cardiac sequelae of hypertension. Methods Hypertension was induced in three month old miR-214 knock out (KO) and wild type (WT) littermates using 490 ng/kg/min angiotensin II (Ang II) for 14 or 28 days (n=4–6). Blood pressure was monitored by tail cuff plethysmography. Cardiac fibrosis was assessed by picrosirius red and Massons trichrome staining. Mir214 expression was assessed by Taqman and in situ hybridisation (ISH). Cardiac function was assessed by echocardiography. Effects of Ang II on fibrosis genes was studied in primary cardiac fibroblasts. Results 14 day Ang II infusion caused 4-fold induction of miR-214 in the left ventricle of C57bl/6 mice (p COL1A1 mRNA compared with their WT littermates (p in vivo observations, in vitro fibroblast studies show that KO fibroblasts showed significantly reduced induction of COL1A1 (p COL3A1 (p Conclusions Cardiac fibrosis, hypertrophy and diastolic dysfunction are enhanced in miR-214 KO animals. The mechanisms of this observation are not caused by mir214 effects in cardiac fibroblasts, but through other mechanisms that need to be further addressed.

Angiotensin 1-7 (Ang 1-7) has anti-atherosclerotic effects, possibly through its anti-inflammatory properties. As perivascular inflammation is an important component of early atherosclerosis, we aimed to determine whether angiotensin-(1-7) non peptide mimetic (AVE0991) can modulate these inflammatory mechanisms in the early development of atherosclerosis. Twelve-week old ApoE-/- mice and controls (C57BL/6J) on chow diet were treated with placebo or AVE0991 (0.58μM/g/day, per os). Mice were analysed at 16, 20 and 24 weeks of age. The level of perivascular leukocyte and T cell infiltration was measured by flow cytometry. Atherosclerotic plaque area was assessed by Oil Red O staining of en-face aortic preparations. Endothelial dysfunction was measured by wire myography and ROS production in the aorta by lucigenin-enhanced chemiluminescence (5μM). RESULTS: Levels of endogenous Ang 1-7, measured by mass spectrometry in the perivascular adipose tissue were low in both WT and ApoE-/- (32.9±1.2 vs 31.1±1.1 pg/mg; p=0.1). AVE0991 significantly reduced plaque area in ApoE-/- mice at all time points (14.2±1.9% vs 7.25±1.3% at 24 weeks; p

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.

Endothelial dysfunction plays an important role in the pathogenesis of many common diseases, like atherosclerosis and hypertension. The key role of the interaction between oxidative stress and inflammation in causal mechanisms of these diseases is widely accepted. Until recently, perivascular adipose tissue was not taken into account while looking at mechanisms of these disorders. However, it has recently been demonstrated that most processes involved in endothelial dysfunction development are taking place in this tissue. Adipocytes are an important source of free radicals and pro-inflammatory cytokines. These molecules lead to further enhancement of oxidative stress, through uncoupling of endothelial nitric oxide synthase (eNOS) and production of peroxynitrite radical instead of nitric oxide which further disrupts eNOS function. In addition, macrophages and T lymphocytes infiltrate adipose tissue as a result of chemotactic proteins release, upon oxidative stress activation, which further enhances inflammation. Thus, the chronic inflammation, which develops in this compartment of adipose tissue in patients with obesity, is the first step in the development of atherosclerotic plaque or hypertension. That is why comprehensive understanding of ongoing processes within perivascular adipocytes is so important.

Hypertension is associated with perivascular inflammation and increased collagen deposition in adventitia. MicroRNAs (miR) are a novel mechanism for gene expression regulation and play a pivotal role in a wide range of pathological processes such as vascular fibrosis.The role and mechanism of miRs in this irreversible process are still unknown. 3-months-old C57BL/6, miR-214KO and wild type littermates were treated with angiotensin II (AngII, 490ng/kg/min; n = 6-10) or control buffer for 14 days. Perivascular adipose tissues (PVAT) from C57BL/6 animals were analysed using TaqMan_Rodent_microRNA_Arrays. Histological studies, wire myography, lucigenin enhanced luminometry and cytometrical analysis was conducted. Statistical analysis was performed using ANOVA or t-test. Data are expressed as a meanSEM. Out of 381 miRs, 16 were significantly overexpressed in C57BL/6 AngII animals after global normalisation. Mir-214 was the only significantly overexpressed miR with 8-fold induction (p

Objective Pregnancy induces extensive yet relatively rapid remodelling of the cardiovascular (CV) system, however, little is known about this adaptation in women with pre-existing CV disease. We have previously characterised the stroke prone spontaneously hypertensive rat (SHRSP) as a model of deficient uterine artery remodelling and identified an increase in pro-inflammatory TNFI ± relative to the normotensive WKY strain during pregnancy. Design and method SHRSP were treated with etanercept (0.8 mg/kg) or vehicle at gestational day (GD) 0, 6, 12 and 18. Animals were sacrificed at GD18. SHRSP, SHRSP treated with etanercept (ETN) and WKY (n = 6) were used for vascular studies. An independent set of animals (n = 6) were used for flow cytometry analysis. Results Etanercept significantly reduced systolic blood pressure in the SHRSP after GD 12 (I”SBP GD 10–21 SHRSP 12.0 ± 4.17 vs. ETN 25.8 ± 4.27 mmHg; p Conclusions Etanercept improves uterine artery function and pregnancy outcome in the SHRSP. We propose that this is through the limitation of both damaging TNFI ± release and cytotoxicity from NK cells.