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Background Coronary atherosclerosis is a frequent complication of type 2 diabetes mellitus (DM2). Considering the contiguity with the vascular wall, perivascular adipose tissue (PVAT) could play a crucial role in the pathogenic microenvironment of atherosclerosis. The PVAT attenuation index (p-FAI) is a non-invasive marker that reveals the change in peri-coronary adipose tissue (PCAT). High values of p-FAI are associated with increased cardiovascular mortality and poor prognosis. Emerging as an indication, contributor to, and therapeutic target for atherosclerosis, PCAT warrants further investigation in DM2. Purpose We aimed to characterize the association of PCAT by p-FAI and DM2, and to compare coronary inflammation in DM2 versus non DM2 patients with coronary artery disease (CAD), and versus healthy controls. Methods 15 consecutive DM2 patients (9 male, age 63±10 years) without symptoms/signs of cardiovascular disease were included in the study and compared to 8 non DM2 patients with CAD and 13 healthy volunteers without cardiovascular diseases, matched for age and sex. All patients and controls underwent coronary computed tomography angiography (CCTA) for the evaluation of coronary arteries and p-FAI. All scans were performed using a 320-slice multidetector computed tomography (Toshiba Aquilion) and a prospective ECG-triggered sequential acquisition. p-FAI analysis was performed using a dedicated workstation (Aquarius iNtuition Edition version 4.4.13. P3; TeraRecon Inc., Foster City, CA, USA). The proximal 40-mm segment of the right coronary artery (RCA) was identified and the inner and the outer wall were automatically traced, excluding the 10 mm from the ostium. The adipose tissue localized within a radial distance from the outer wall equal to a medium diameter of the RCA was evaluated. Voxel histograms of CT attenuation were traced and included between −190 to −30 HU within the PCAT volume. p-FAI was calculated as the median CT attenuation value of PCAT of the proximal 40-mm segment of the RCA (Figure 1). Results CAD was present in 10 DM2 patients (5 males, aged 63.1±10.5 years); in 5 DM2 patients (4 males, aged 63±11 years) epicardial coronary arteries were normal. p-FAI was higher in DM2 patients than in healthy controls (p=0.004). The presence of CAD did not impact on p-FAI in DM2 patients, presenting a comparable value (p=0.37). p-FAI was higher in DM2 patients with CAD than in non DM2 patients with CAD (p=0.04). Moreover, p-FAI was higher in DM2 patients without CAD than in non DM2 patients with CAD (p=0.002, Figure 2). Finally, p-FAI was not different in non DM2 patients with CAD compared to healthy controls (p=0.65), suggesting the limited role of CAD in the progression of peri-coronary inflammation when compared to DM2. Conclusions Coronary inflammation evaluated by p-FAI measurement was higher in DM2 patients, also without CAD. Therefore, our results suggest that DM2 is a determinant of coronary inflammation stronger than CAD. Funding Acknowledgement Type of funding sources: None. Figure 1Figure 2

Introduction Hypertension is associated with enhanced NADPH oxidase activation and increased accumulation of immune cells leading to perivascular inflammation. However, while aging and oxidative stress are a major factor for the development of hypertension, their effect on perivascular inflammation remains unclear. Purpose We aimed to establish the interaction between aging, oxidative stress and perivascular inflammation. We hypothesize that the modulation of oxidative stress, by NADPH oxidase inhibition, could affect perivascular inflammation during the aging. Methods Using flow cytometry, we studied leukocyte infiltration in the perivascular adipose tissue (PVAT) in 1-, 3-, 6- and 12-month-old SHR (Spontaneously Hypertensive Rats) and normotensive WKY (Wistar-Kyoto) rats (n=5–12). Additionally, 1-month-old rats were treated with GKT137831 or ML171 (60mg/kg; NOX1/4 and NOX1 inhibitor, respectively) for 4 weeks. Blood pressure (PB) was measured by tail cuff. Statistical analysis was performed using two-way ANOVA or t-test. Data are presented as means±SEM. Results Aging in SHRs was associated with an increase of BP (139±4 vs. 180±4 vs. 203±3 vs. 209±3, mmHg) and an elevation of PVAT leukocytes (2090±164 vs. 2255±359 vs. 2502±496 vs. 3255±408, cell/mg) in 1-, 3-, 6- and 12-month-old rats, respectively. These effects were not seen in WKY. These were associate with similar changes of NK cells (p int While an age-related increase of Nox4 mRNA in the vessels was observed in both groups, this increase was more dynamic in SHRs, (p int Interestingly, GKT137831 increased BP in both WKY and SHR rats (p Conclusion Aging and spontaneous hypertension is associated with a trajectory of BP elevation and perivascular inflammation. This state is hastened after NOX1/4 inhibition, while ML171 treatment protects against perivascular inflammation. Acknowledgement/Funding National Science Centre 2013/11/N/NZ4/00310

Background/Introduction Perivascular inflammation plays an important role in the pathogenesis of pulmonary arterial hypertension (PAH). Recent studies have demonstrated that damaged mitochondrial DNA induces sterile inflammation by activating toll-like receptor (TLR)9 in spontaneous hypertensive rats. However, it remains unclear whether TLR9 is involved in perivascular inflammation and subsequent development of PAH. Purpose The purpose of the present study is to investigate whether chronic inhibition of TLR9 can ameliorate monocrotaline (MCT)-induced PAH in rats. Methods Male Sprague-Dawley rats were injected with MCT (60 mg/kg). First, we conducted immunohistochemistory to examine which cell types express TLR9 in lungs of normal rats and MCT-exposed rats. Second, we extracted cell-free DNA from plasma of rats and amplified genes of COX2 by real-time PCR to detect circulating cell-free mitochondrial DNA, a ligand of TLR9. Third, the administration of a selective TLR9 inhibitor (E6446, 10mg/kg/day, drinking water) or non-selective TLR9 inhibitor (chloroquine: 50mg/kg/day, ip) started three days before MCT injection and sacrificed on day 21. We assessed hemodynamic data and histopathological analysis (EVG stain for medial wall thickness (MWT) in pulmonary arteries (outer diameter: 50 ∼ 100 μm) and CD68 for macrophage accumulation around pulmonary arteries (outer diameter: Results TLR9 was expressed dominantly in pulmonary endothelial cells and macrophages in the lungs of both normal rats and MCT-exposed rats. Compared with normal rats, MCT-exposed rats showed increased gene expression of COX2 (0.048±0.001 vs. 0.052±0.001 expressed by 1/Ct) in plasma on day 14. MCT-exposed rats also had increased right ventricular systolic pressure (RVSP: 21±1 vs. 60±2 mmHg), total pulmonary vascular resistance index (TPRI: 0.07±0.01 vs. 0.43±0.02 mmHg/min/mL/kg), MWT (0.07±0.01 vs. 0.26±0,02) and accumulation of macrophages (1.6±0.3 vs. 20.0±1.7 cells/HPF) on day 21. In the prevention protocol, either E6446 or chloroquine significantly prevented the elevations of RVSP (49±4 or 48±3 mmHg) and TPRI (0.29±0.04 or 0.27±0.03 mmHg/min/mL/kg) with reducing MWT (0.18±0.01 or 0.18±0.01) and macrophage accumulation (9.7±1.3 or 9.8±2.5 cell/HPF) on day 21. In addition, these drugs significantly reduced the levels of IL-6 mRNA compared with MCT group (4.4±1.0 or 4.8±1.4 vs. 11.9±1.0). In the reversal protocol, the treatment of E6446 had significantly increased the survival rate (50 vs. 10%). Conclusions TLR9 largely contributes to the development of PAH by reducing perivascular inflammation. Inhibition of TLR9 could be a novel therapeutic target for PAH.