HLA-B35 upregulates ET-1 and downregulates eNOS via ER stresss response in endothelial cells. Homo sapiens

The presence of the HLA-B35 allele has emerged as an important risk factor for the development of isolated pulmonary hypertension (iPHT) in patients with Scleroderma (SSc), however the mechanisms underlying this association have not been fully elucidated. The goal of our study was to determine the molecular mechanisms that mediate the biological effects of HLA-B35 in endothelial cells (ECs). Our data demonstrate that HLA-B35 expression at the physiological levels using via adenoviral vector resulted in a significantly increased endothelin-1 (ET-1) and a significantly decreased endothelial nitric oxide synthase (eNOS) mRNA and protein levels. Furthermore, HLA-B35 greatly upregulated expression of cytoplasmic chaperones, including heat shock proteins (HSPs) HSP70 (HSPA1A and HSPA1B) and HSP40 (DNAJB1 and DNAJB9), suggesting that HLA-B35 induced the ER stress and unfolded protein response (UPR) in ECs. Examination of selected mediators of the UPR response, including BiP (GRP78), CHOP (GADD153), ERO1 (endoplasmic reticulum oxidase) and PDI (protein disulfide isomerase) has revealed a consistent increase of BiP expression levels. Accordingly, Thapsigargin (TG), a known ER stress inducer, stimulated ET-1 mRNA and protein levels in ECs. This study suggests that HLA-B35 could contribute to endothelial cell dysfunction via ER stress mediated induction of ET-1 in patients with PHT. Overall design: Three samples: endothelial cells transduced with Adenovirus GFP; cells without gene insert, cell with HLA-B35, and cells with HLA-B8.

人类白细胞抗原B35（HLA-B35）等位基因的存在，已被证实为硬皮病（Scleroderma, SSc）患者罹患孤立性肺动脉高压（isolated pulmonary hypertension, iPHT）的重要危险因素，然而二者关联背后的分子机制尚未完全阐明。本研究旨在阐明介导HLA-B35在内皮细胞（endothelial cells, ECs）中发挥生物学效应的分子机制。

我们的实验数据显示，通过腺病毒载体在生理水平表达HLA-B35，可使内皮素-1（endothelin-1, ET-1）的表达显著升高，同时使内皮型一氧化氮合酶（endothelial nitric oxide synthase, eNOS）的mRNA及蛋白水平显著降低。进一步研究发现，HLA-B35可显著上调胞质分子伴侣的表达，包括热休克蛋白（heat shock proteins, HSPs）HSP70（HSPA1A与HSPA1B）及HSP40（DNAJB1与DNAJB9），这表明HLA-B35可在内皮细胞中诱导内质网应激（ER stress）与未折叠蛋白反应（unfolded protein response, UPR）。

对UPR通路的部分关键介质——包括BiP（GRP78）、CHOP（GADD153）、内质网氧化酶（endoplasmic reticulum oxidase, ERO1）以及蛋白质二硫键异构酶（protein disulfide isomerase, PDI）——的检测结果显示，BiP的表达水平呈一致性升高。相应地，经典内质网应激诱导剂毒胡萝卜素（Thapsigargin, TG）可刺激内皮细胞中ET-1的mRNA及蛋白水平上调。

本研究表明，在肺动脉高压患者中，HLA-B35可能通过内质网应激介导的ET-1诱导通路，参与内皮细胞功能异常的发生发展。

整体实验设计：共设置四组样本，分别为经腺病毒绿色荧光蛋白（Adenovirus GFP）转导的内皮细胞、未携带基因插入片段的空白对照组细胞、转导HLA-B35的细胞以及转导HLA-B8的细胞。