Dysfunction of the pulmonary endothelium is associated with most lung diseases. Extracellular nucleotides modulate a plethora of endothelial functions in the lung such as vessel integrity, vasodilatation, inflammatory, and thrombotic responses as well as survival and DNA repair, mostly via Ca²⁺ signaling pathways. However, a comprehensive analysis of the molecular components of the underlying P2 receptor-mediated Ca²⁺ signaling pathways in the lung has not been conducted so far. Therefore, our aim was to identify the principal P2 receptor Ca²⁺ signalosome in the human pulmonary endothelium and investigate potential dysregulation in pulmonary vascular disease. Comparative transcriptomics and quantitative immunohistochemistry were performed on publicly available RNA sequencing and protein datasets to identify the specific expression profile of the P2-receptor Ca²⁺ signalosome in the healthy human pulmonary endothelium and endothelial cells (EC) dysfunctional due to loss of or defective bone morphogenetic protein receptor (BMPR2). Functional expression of signalosome components was tested by single cell Ca²⁺ imaging. Comparative transcriptome analysis of 11 endothelial cell subtypes revealed a specific P2 receptor Ca²⁺ signalosome signature for the pulmonary endothelium. Pulmonary endothelial expression of the most abundantly expressed Ca²⁺ toolkit genes CALM1, CALM2, VDAC1, and GNAS was confirmed by immunohistochemistry (IHC). P2RX1, P2RX4, P2RY6, and P2YR11 showed strong lung endothelial staining by IHC, P2X5, and P2Y1 were found to a much lesser extent. Very weak or no signals were detected for all other P2 receptors. Stimulation of human pulmonary artery (HPA) EC by purine nucleotides ATP, ADP, and AMP led to robust intracellular Ca²⁺ signals mediated through both P2X and P2Y receptors. Pyrimidine UTP and UDP-mediated Ca²⁺ signals were generated almost exclusively by activation of P2Y receptors. HPAEC made dysfunctional by siRNA-mediated BMPR2 depletion showed downregulation of 18 and upregulation of 19 P2 receptor Ca²⁺ signalosome genes including PLCD4, which was found to be upregulated in iPSC-EC from BMPR2-mutant patients with pulmonary arterial hypertension. In conclusion, the human pulmonary endothelium expresses a distinct functional subset of the P2 receptor Ca²⁺ signalosome. Composition of the P2 receptor Ca²⁺ toolkit in the pulmonary endothelium is susceptible to genetic disturbances likely contributing to an unfavorable pulmonary disease phenotype found in pulmonary arterial hypertension.

中文翻译：

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P2受体介导的人肺内皮Ca2 +信号小体-对肺动脉高压的影响。

肺内皮功能障碍与大多数肺部疾病有关。细胞外核苷酸主要通过Ca ²⁺信号通路调节肺中的大量内皮功能，例如血管完整性，血管扩张，炎症和血栓形成反应以及存活和DNA修复。但是，到目前为止，尚未对肺中潜在的P2受体介导的Ca ²⁺信号通路的分子成分进行全面分析。因此，我们的目的是确定主要的P2受体Ca ²⁺人肺内皮细胞中的信号小体，并研究肺血管疾病的潜在失调。在可公开获得的RNA测序和蛋白质数据集上进行了比较转录组学和定量免疫组化分析，以鉴定P2-受体Ca ²⁺信号体在健康人肺内皮和由于骨丢失或缺损而功能失调的内皮细胞（EC）中的特异性表达谱。形态发生蛋白受体（BMPR2）。通过单细胞Ca ²⁺成像测试信号体组分的功能表达。对11种内皮细胞亚型进行的转录组比较分析显示特定的P2受体Ca ²⁺肺血管内皮的信号体特征。免疫组织化学（IHC）证实了表达最丰富的Ca ²⁺工具箱基因CALM1，CALM2，VDAC1和GNAS的肺血管内皮表达。P2RX1，P2RX4，P2RY6和P2YR11在IHC，P2X5和P2Y1上显示出较强的肺内皮染色，但程度较轻。对于所有其他P2受体，检测到的信号非常弱或没有信号。嘌呤核苷酸ATP，ADP和AMP对人肺动脉（HPA）EC的刺激导致强大的细胞内Ca ²⁺信号通过P2X和P2Y受体介导。嘧啶UTP和UDP介导的Ca ²⁺信号几乎完全是通过激活P2Y受体产生的。siRNA介导的BMPR2耗竭使HPAEC功能失调，显示18的下调和19的P2受体Ca ²⁺信号体基因（包括PLCD4）的上调，这在BPSR2突变的肺动脉高压患者的iPSC-EC中被上调。总之，人肺内皮表达P2受体Ca ²⁺信号小体的独特功能子集。肺内皮中P2受体Ca ²⁺工具箱的组成易受遗传干扰的影响，可能导致在肺动脉高压中发现不利的肺部疾病表型。