Agnieszka Sagan

Mechanisms of oxidative stress in human aortic aneurysms — Association with clinical risk factors for atherosclerosis and disease severity

Aortic abdominal aneurysms (AAA) are important causes of cardiovascular morbidity and mortality. Oxidative stress may link multiple mechanisms of AAA including vascular inflammation and increased metalloproteinase activity. However, the mechanisms of vascular free radical production remain unknown. Accordingly, we aimed to determine sources and molecular regulation of vascular superoxide (O2•−) production in human AAA. Methods and results AAA segments and matched non-dilated aortic samples were obtained from 40 subjects undergoing AAA repair. MDA levels (determined by HPLC/MS) were greater in plasma of AAA subjects (n = 16) than in risk factor matched controls (n = 16). Similarly, superoxide production, measured by lucigenin chemiluminescence and dihydroethidium fluorescence, was increased in aneurysmatic segments compared to non-dilated aortic specimens. NADPH oxidases and iNOS are the primary sources of O2•− in AAA. Xanthine oxidase, mitochondrial oxidases and cyclooxygenase inhibition had minor or no effect. Protein kinase C inhibition had no effect on superoxide production in AAA. NADPH oxidase subunit mRNA levels for p22phox, nox2 and nox5 were significantly increased in AAAs while nox4 mRNA expression was lower. Superoxide production was higher in subjects with increased AAA repair risk Vanzetto score and was significantly associated with smoking, hypercholesterolemia and presence of CAD in AAA cohort. Basal superoxide production and NADPH oxidase activity were correlated to aneurysm size. Conclusions Increased expression and activity of NADPH oxidases are important mechanisms underlying oxidative stress in human aortic abdominal aneurysm. Uncoupled iNOS may link oxidative stress to inflammation in AAA. Oxidative stress is related to aneurysm size and major clinical risk factors in AAA patients.

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

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.

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.

[OP.2D.04] PERIVASCULAR T REGULATORY CELLS AND ENDOTHELIAL DYSFUNCTION IN HUMAN ATHEROSCLEROSIS.

Objective: Atherosclerosis is an inflammatory disease associated with an imbalance between pro- and anti-inflammatory mechanisms. While ample evidence is available in animal models, less is known about links between local vascular inflammation in human disease. T regulatory cells (Treg) have been implicated in atherosclerosis in mice but links with human vascular inflammation are poorly understood. Accordingly, we aimed to investigate the relationship between T regs in peripheral blood and locally in perivascular adipose tissue (pVAT) with endothelial function as a key mechanism in pathogenesis of vascular disease. Design and method: Treg (CD3+/CD4+/CD25+/FoxP3+) infiltration was studied in pVAT of atherosclerotic coronary artery (CORO), non-atherosclerotic internal mammary artery (IMA), as well as subcutaneous AT and peripheral blood were quantified using flow cytometry from 50 CABG patients (38 M;12F;age 65y+/-1) with typical atherosclerosis risk factor profile. Vascular function was assessed in IMA segments ex vivo by isometric tension studies of vasorelaxations to acetylcholine and ROS production was measured in vascular segments with 5uM lucigenin enhanced chemiluminescence. Results: Treg infiltration was observed in both IMA and CORO, but was significantly higher in IMA pVAT than in pVAT surrounding CORO (13,58 ± 15,6 vs. 4,74 ± 6,2 cells/mg; p < 0,01). Moreover, there was a significant correlation between these two AT depots suggesting systemic pVAT regulation (R = +0,53; p = 0,001). Indeed we observed a significant correlation between number of risk factors for atherosclerosis and Treg content (Rs = +0,33 p = 0,02). Importantly, there was an inverse correlation between Treg content in peripheral blood and Ach-induced vasorelaxations (R = -0,31 p < 0,05). Local T reg infiltration in pVAT was significantly correlated with indices of NO production, as measured by chemiluminescence (R = +0,58 p = 0,007). However, NO produced was scavenged by ROS (measured by ratio of L-NAME enhanced/basal superoxide levels) and Treg infiltration in pVAT did not correlate with IMA endothelial function (R = -0.02 p = 0,92) or total vascular ROS production (R = -0,12 p = 0,53). Conclusions: Atherosclerosis is accompanied by local decrease in T regulatory cell content in atherosclerotic vs. non-atherosclerotic arteries, although their amount is linked with classical risk factors for atherosclerosis. Higher peripheral blood T regs are associated with better endothelial function, although it is not achieved through locally infiltrating Tregs.

Inflammatory aortic abdominal aneurysm – immunophenotypic characterization of inflammatory infiltrate

Abdominal aortic aneurysm (AAAs) constitute an important clinical problem as they occur in up to 8% of male patients over 60 years of age, and their complications such as aneurysm rupture are associated with 80% mortality [1]. While the pathogenesis and epidemiology of classical AAAs are relatively well studied, inflammatory aneurysms (IAAA) create a particularly interesting subset which represents 5% to 10% of AAAs and are characterized by a firm, thick aortic wall with a shiny white appearance and peri-aneurysmal infiltration or retroperitoneal fibrosis which can be detected by computed tomography (CT) scan or ultrasonography (USG), as a cuff of soft tissue inflammation surrounding the aneurysm [2, 3]. Their development involves a predominant immune response within the vessel wall [2], although its nature remains poorly defined. Inflammatory markers (C-reactive protein (CRP) or leukocytes (WBC)) are elevated [2]. The inflammatory process leads to more complicated IAAA repair and subsequent post-operative period. There are a number of differences in the epidemiology and clinical presentation of IAAA. Inflammatory abdominal aortic aneurysm AAAs are generally larger than AAAs [4]. The risk factors, such as male gender and smoking, have even stronger effects in relation to IAAA than for typical atherosclerotic AAA [4, 5]. The male : female ratio is almost 5-fold higher than for atherosclerotic aneurysm (30 : 1 vs. 6 : 1) [4, 5]. Although almost 90% of IAAA patients have symptoms such as abdominal or back pain, weight loss or elevated inflammatory markers, such as erythrocyte sedimentation rate, C-reactive protein or white blood cell level [6], these are nonspecific, which makes the diagnosis of IAAA difficult. Computed tomography (CT) scan and ultrasonography (USG) are useful and reliable imaging methods used for diagnosis of both inflammatory and atherosclerosis aneurysm. In this article we present an immunophenotyping method which might be useful for IAAA diagnosis and confirmation. We studied IAAA wall inflammatory infiltrate obtained from a 60-year-old man, with significant obesity (body mass index (BMI) 30 kg/m 2), in whom AAA was diagnosed 24 months prior to surgical repair. The IAAA diameter increased in this time from 4 to 6 cm. Smoking history includes 42 pack-years but was stopped 3 months prior to surgery. Diffuse coronary artery disease was present with myocardial infarction (1995) and percutaneous coronary intervention (PCI) (2013) for which they received aspirin, clopidogrel, atorvastatin, bisoprolol and ramipril. Ankle brachial index was 0.9. Computed tomography demonstrated a clearly thickened wall of the abdominal aorta suggestive of IAAA (Figure 1B), but the increase of inflammatory parameters was very modest (CRP 5.2 mg/l and WBC 6800/µl). During surgical repair the aneurysm wall was hard, “ivory” colored and thickened (100 mm), and was embedded in the surrounding infiltrate. After clamping the aorta, the aneurysm was repaired using a simple aneurysm graft. Upon releasing the clamp, no pulse was detected in the left groin, and an additional prosthesis between the aneurysm graft and common femoral artery was implanted. The postoperative course was complicated by a febrile (39°C) state, which subsided after 4 days, and the patient was discharged in good condition. Figure 1 A – IAAA wall in the context of perivascular tissues and atherosclerotic plaque. The size bar indicates 15 mm. B – Angio-CT image showing an abdominal aorta aneurysm. Arrows mark lines showing the thickened and inflamed wall of the abdominal ... A fragment of the wall of the aneurysm was obtained at the site of maximal dilatation. After harvesting, the sample was placed in ice cold (4°C) phosphate buffered saline (PBS, Gibco, Invitrogen, Carlsbad, CA, USA). Additionally, before the surgery, a blood sample was collected in EDTA containing tubes (Becton Dickinson) from peripheral access for the determination of reference immunophenotype. Samples were transported to the laboratory immediately. The Local Research Ethics Committee approved sample collection, written informed consent was obtained. Peripheral blood was lysed using RBC Lysis Buffer (eBioscience, San Diego, CA, USA) (8 min, room temperature) and was washed three times with ice-cold PBS. Cells were incubated with fluorescently labeled antibodies anti-CD45 PeCy7, anti-CD3 PerCP, anti-CD4 APC, anti-CD8 APC-H7, as well as anti-CD25 PE, anti-CD69 FITC, anti-CD28 APC and anti-CCR5 PE (BD Biosciences) for 20 min at 4°C. After washing, cells were re-suspended in PBS with 1% fetal bovine serum (FBS) (Gibco), studied on a FACSVerse (BD Biosciences) and analyzed using FlowJo software. Atherosclerotic plaque and perivascular tissue were separated from the wall (Figure 1A). The wall was delicately mechanically disrupted and digested with a mix of digestion enzymes, collagenases and hyaluronidase in PBS with calcium-magnesium containing 20 mM HEPES, at 37°C, for 20 min, with gentle agitation to isolate residual cells infiltrating the wall. The resulting cell suspension was passed through a 70-µm strainer (BD Pharmingen, San Diego, CA, USA). Cells were incubated with the same fluorescently labeled antibodies using the same labeling procedures as were applied for blood. The initial selection of blood cells was performed on FSC/SSC scatter, allowing CD45 antigen to be applied for leukocyte confirmation. Subsequently T cells (CD3+ positive) and their subpopulations, CD4 and CD8 positive, were identified (Figure 2A). Additionally we studied the presence of antigens on T cells, such as CD25, CD69, CD28 and CCR5 (Figure 2C). To study activation markers on T cells we used the isotype control (KI) or fluorescent minus one (FMO) method. Figure 2 A – Examples of flow cytometric determination of leukocytes (CD45), T cells (CD3), CD4+ and CD8+ cells in wall and peripheral blood. B – Leukocyte subpopulations content per mg of wall tissue. C – Flow cytometric determination ... As for blood, tissue cells were first gated on FSC/SSC scatter and then CD45-positive cells were identified. T cells, their subpopulations and characteristics were identified using the same markers as for blood (Figures 2A, ​,CC). The aneurysmal wall was significantly infiltrated by leukocytes which importantly differed from peripheral blood. In particular, T cell infiltration was strongly increased in IAAA, constituting 68% of infiltrating leukocytes. These were predominantly CD4+ lymphocytes (Figures 2A–B). Both CD4+ and CD8+ T cells were activated in the IAAA wall and expressed the early activation marker (CD69) (30% in IAAA vs. 1% in blood). CD25 (typically late activation) was increased only on CD8+ cells. In line with this, > 50% of CD8+ T cells in blood of the patient showed the senescent phenotype (lack of CD28 marker – CD8 + CD28null). This population rarely exceeds 30–40% in healthy individuals. In contrast, CD4 + CD28null T cells, characteristic for atherosclerotic plaques, were not found in the IAAA wall. T cells showed in turn high expression of the CCR5 receptor for the chemokine RANTES, which could in part explain high T cell recruitment (Figure 2C). In the IAAA patient described here, the clinical presentation included only abdominal pain from the usual (80%) triad including abdominal/lumbar pain, severe weight loss and elevated CRP [2]. Despite this, during surgery we observed a characteristic macroscopic view of IAAA with strong peri-aortic infiltration, consistent with the classic Walker description [3, 7, 8]. Importantly, angio-CT could indicate the inflammatory nature of the AAA process. Therefore it should be kept in mind that diagnostic imaging is a very important tool, which can suggest pathology within the vascular wall and may guide optimal patient management and help predict possible complications [9]. Interestingly, the imaging technique was a more reliable indication of inflammatory nature than routine blood tests (CRP, WBC). This may be due to the local nature of inflammation or treatment with concomitant medications of an anti-inflammatory nature: statins/ACE-inhibitor/NSAID [8]. Interestingly, the patient developed peri-operative fever, which was not associated with infection and could be related to the inflammatory nature of the aneurysm. Recent studies highlight the possible role of infections in initiating IAAA [2, 10]. Presence of senescent CD8+ T cells could point towards a role of viral infection or an occult endogenous auto-immune process. While in the blood of IAAA patients we observed increased granulocytes and decreased total mononuclear cells when compared to values reported for atherosclerotic AAA blood, the IAAA wall was heavily infiltrated by leukocytes (ca. 80% of stromal fraction, vs. 40% in atherosclerotic AAAs; unpublished) which were predominantly activated T lymphocytes. Moreover, the presented case brings attention to the senescent CD8 + TCD28null cells as players in IAAA. These cells have recently been described in human hypertension. Our study may show that the chemokine RANTES, overproduced in IAAA, may play an important role in inflammation and thus may constitute a future important target. In conclusion, in spite of the lack of a typical high rise of inflammatory markers (CRP, WBC), IAAA wall inflammation is characterized by activation of T cells, with a possible role of cytotoxic CD8+ cells, which have a senescent phenotype. This infiltrate is better reflected by the immunophenotypic method than typical inflammatory markers such as CRP or WBC.