Ryszard Nosalski

Role of chemokine RANTES in the regulation of perivascular inflammation, T-cell accumulation, and vascular dysfunction in hypertension

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. FASEB J. 30, 1987–1999 (2016). www.fasebj.org

Perivascular adipose tissue inflammation in vascular disease.

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 PVATs 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.

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

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

Novel Immune Mechanisms in Hypertension and Cardiovascular Risk

Purpose of ReviewHypertension 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.Recent FindingsIn 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.SummaryImmune 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.

Antiatherosclerotic effect of Ang- (1-7) non-peptide mimetic (AVE 0991) is mediated by inhibition of perivascular and plaque inflammation in early atherosclerosis.

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.

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

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.

Vascular transcriptome profiling identifies Sphingosine kinase 1 as a modulator of angiotensin II-induced vascular dysfunction

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 < 0.05), which was associated with decreased aortic and mesenteric vasoconstriction in hypertensive 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.

Role of Tumor Necrosis Factor-α and Natural Killer Cells in Uterine Artery Function and Pregnancy Outcome in the Stroke-Prone Spontaneously Hypertensive Rat

Women with chronic hypertension are at increased risk of maternal and fetal morbidity and mortality. We have previously characterized the stroke-prone spontaneously hypertensive rat (SHRSP) as a model of deficient uterine artery function and adverse pregnancy outcome compared with the control Wistar–Kyoto. The activation of the immune system plays a role in hypertension and adverse pregnancy outcome. Therefore, we investigated the role of tumor necrosis factor-&agr; in the SHRSP phenotype in an intervention study using etanercept (0.8 mg/kg SC) at gestational days 0, 6, 12, and 18 in pregnant SHRSP compared with vehicle-treated controls (n=6). Etanercept treatment significantly lowered systolic blood pressure after gestational day 12 and increased litter size in SHRSP. At gestational day 18, etanercept improved the function of uterine arteries from pregnant SHRSP normalizing the contractile response and increasing endothelium-dependent relaxation, resulting in increased pregnancy-dependent diastolic blood flow in the uterine arteries. We identified that the source of excess tumor necrosis factor-&agr; in the SHRSP was a pregnancy-dependent increase in peripheral and placental CD3– CD161+ natural killer cells. Etanercept treatment also had effects on placental CD161+ cells by reducing the expression of CD161 receptor, which was associated with a decrease in cytotoxic granzyme B expression. Etanercept treatment improves maternal blood pressure, pregnancy outcome, and uterine artery function in SHRSP by antagonizing signaling from excess tumor necrosis factor-&agr; production and the reduction of granzyme B expression in CD161+ natural killer cells in SHRSP.

Denture-Related Stomatitis Is Associated with Endothelial Dysfunction

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.

Systemic T Cells and Monocyte Characteristics in Patients with Denture Stomatitis.

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.