Supplementary Material for: In situ Evidence of Collagen V and Interleukin-6/Interleukin-17 Activation in Vascular Remodeling of Experimental Pulmonary Hypertension

Several studies have reported the pathophysiologic and molecular mechanisms responsible for pulmonary arterial hypertension (PAH). However, the in situ evidence of collagen V (Col V) and interleukin-17 (IL-17)/interleukin-6 (IL-6) activation in PAH has not been fully elucidated. We analyzed the effects of collagen I (Col I), Col V, IL-6, and IL-17 on vascular remodeling and hemodynamics and its possible mechanisms of action in monocrotaline (MCT)-induced PAH. Twenty male Wistar rats were randomly divided into two groups. In the PAH group, animals received MCT 60 mg/kg intraperitoneally, whereas the control group (CTRL) received saline. On day 21, the pulmonary blood pressure (PAP) and right ventricular systolic pressure (RVSP) were determined. Lung histology (smooth muscle cell proliferation [α-smooth muscle actin; α-SMA] and periadventitial fibrosis), immunofluorescence (Col I, Col V, and α-SMA), immunohistochemistry (IL-6, IL-17, and transforming growth factor-beta [TGF-β]), and transmission electron microscopy to detect fibronexus were evaluated. The RVSP (40 ± 2 vs. 24 ± 1 mm Hg, respectively; p < 0.0001), right ventricle hypertrophy index (65 ± 9 and 25 ± 5%, respectively; p < 0.0001), vascular periadventitial Col I and Col V, smooth muscle cell α-SMA+, fibronexus, IL-6, IL-17, and TGF-β were higher in the MCT group than in the CTRL group. In conclusion, our findings indicate in situ evidence of Col V and IL-6/IL-17 activation in vascular remodeling and suggest that increase of Col V may yield potential therapeutic targets for treating patients with PAH.

已有多项研究报道了肺动脉高压（pulmonary arterial hypertension, PAH）的病理生理及分子机制。然而，PAH中胶原V（collagen V, Col V）与白细胞介素-17（interleukin-17, IL-17）/白细胞介素-6（interleukin-6, IL-6）活化的原位证据尚未完全阐明。本研究针对野百合碱（monocrotaline, MCT）诱导的PAH模型，分析了胶原I（collagen I, Col I）、Col V、IL-6及IL-17对血管重构与血流动力学的影响及其潜在作用机制。将20只雄性Wistar大鼠随机分为两组：PAH组大鼠腹腔注射60 mg/kg野百合碱，对照组（CTRL）给予等量生理盐水。造模第21天，检测各组大鼠的肺动脉血压（pulmonary blood pressure, PAP）与右心室收缩压（right ventricular systolic pressure, RVSP）。随后通过肺组织病理学检测（以α-平滑肌肌动蛋白[α-smooth muscle actin, α-SMA]作为平滑肌细胞增殖标志物，同时检测血管外膜周纤维化情况）、免疫荧光染色（检测Col I、Col V与α-SMA的表达）、免疫组化染色（检测IL-6、IL-17与转化生长因子-β[transforming growth factor-beta, TGF-β]的表达）以及透射电镜观察纤维连接体（fibronexus）的形态与表达，对各组样本进行评估。结果显示，与对照组相比，野百合碱组大鼠的RVSP（40±2 vs. 24±1 mmHg，P<0.0001）、右心室肥厚指数（65±9% vs. 25±5%，P<0.0001）、血管外膜周Col I与Col V沉积、平滑肌细胞α-SMA阳性表达、纤维连接体数量以及IL-6、IL-17和TGF-β水平均显著升高。综上，本研究为PAH中Col V与IL-6/IL-17活化参与血管重构提供了原位实验证据，并提示Col V表达上调或可成为PAH患者潜在的治疗靶点。