Cellular sex has rarely been considered as a biological variable in preclinical research, even when the pathogenesis of diseases with predictable sex differences is studied. In this perspective, proteomics, and “omics” approaches in general, can provide powerful tools to obtain comprehensive cellular maps, thus favoring the discovery of still unknown sex-biased physio-pathological mechanisms. We performed proteomic and Gene Ontology (GO) analyses of the secretome from human serum-deprived male and female endothelial cells (ECs) followed by ELISA validation. Apoptosis was detected by FACS and Western blot techniques and efferocytosis through the ability of the macrophage cell line RAW 264.7 to engulf apoptotic ECs. PTX3 mRNA levels were measured by RT-qPCR. Proteomic and GO analyses of the secretome from starved human male and female ECs demonstrated a significant enrichment in proteins related to cellular responses to stress and to the regulation of apoptosis in the secretome of male ECs. Accordingly, a higher percentage of male ECs underwent apoptosis in response to serum deprivation in comparison with female ECs. Among the secreted proteins, we reliably found higher levels of PTX3 in the male EC secretome. The silencing of PTX3 suggested that male ECs were dependent on its expression to properly carry out the efferocytotic process. At variance, female EC efferocytosis seemed to be independent on PTX3 expression. Our results demonstrated that serum-starved male and female ECs possess different secretory phenotypes that might take part in the sex-biased response to cellular stress. We identified PTX3 as a crucial player in the male-specific endothelial response to an apoptotic trigger. This novel and sex-related role for secreted proteins, and mainly for PTX3, may open the way to the discovery of still unknown sex-specific mechanisms and pharmacological targets for the prevention and treatment of endothelial dysfunction at the onset of atherosclerosis and cardiovascular disease.

中文翻译：

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人内皮细胞分泌组的性别依赖性差异

在临床前研究中，细胞性别很少被视为生物学变量，即使在研究具有可预测性别差异的疾病的发病机制时也是如此。从这个角度来看，蛋白质组学和一般的“组学”方法可以提供强大的工具来获得全面的细胞图谱，从而有利于发现仍然未知的性别偏见生理病理机制。我们对来自人血清剥夺的男性和女性内皮细胞 (EC) 的分泌组进行了蛋白质组学和基因本体论 (GO) 分析，然后进行了 ELISA 验证。通过 FACS 和蛋白质印迹技术检测细胞凋亡，并通过巨噬细胞系 RAW 264.7 吞噬凋亡 EC 的能力进行胞吞作用。PTX3 mRNA 水平通过 RT-qPCR 测量。来自饥饿的人类男性和女性 ECs 的分泌组的蛋白质组学和 GO 分析表明，与细胞对压力的反应和男性 ECs 分泌组的细胞凋亡调节相关的蛋白质显着富集。因此，与女性 ECs 相比，更高百分比的男性 ECs 因血清剥夺而发生细胞凋亡。在分泌的蛋白质中，我们可靠地发现雄性 EC 分泌组中 PTX3 水平较高。PTX3 的沉默表明雄性 ECs 依赖于它的表达来正确地进行胞吞过程。在不同情况下，雌性 EC 胞吞作用似乎与 PTX3 表达无关。我们的结果表明，血清饥饿的雄性和雌性 ECs 具有不同的分泌表型，可能参与对细胞压力的性别偏见反应。我们将 PTX3 确定为男性特异性内皮细胞对凋亡触发的反应中的关键参与者。分泌蛋白的这种新的和性别相关的作用，主要是 PTX3，可能为发现仍然未知的性别特异性机制和药理学靶点开辟道路，用于预防和治疗动脉粥样硬化和心血管疾病发作时的内皮功能障碍。