Female fibroblast activation is estrogen-mediated in sex-specific 3D-bioprinted pulmonary artery adventitia models

Pulmonary arterial hypertension (PAH) impacts male and female patients in different ways. Female patients exhibit a greater susceptibility to disease (4:1 female-to-male ratio) but live longer after diagnosis than male patients. This complex sexual dimorphism is known as the estrogen paradox. Prior studies suggest that estrogen signaling may be pathologic in the pulmonary vasculature and protective in the heart, yet the mechanisms underlying these sex-differences in PAH remain unclear. PAH is a form of a pulmonary vascular disease that results in scarring of the small blood vessels, leading to impaired blood flow and increased blood pressure. Over time, this increase in blood pressure causes damage to the heart. Many previous studies of PAH relied on male cells or cells of undisclosed origin for in vitro modeling. Here we present a dynamic, 3D-bioprinted model that incorporates cells and circulating sex hormones from female patients to specifically study how female patients respond to changes in microenvironmental stiffness and sex hormone signaling. Poly(ethylene glycol)-alpha methacrylate (PEGαMA)-based hydrogels containing female human pulmonary artery adventitia fibroblasts (hPAAFs) from idiopathic PAH (IPAH) or control donors were 3D bioprinted to mimic pulmonary artery adventitia. These biomaterials were initially soft, like healthy blood vessels, and then stiffened using light to mimic vessel scarring in PAH. These 3D-bioprinted models showed that stiffening the microenvironment around female IPAH hPAAFs led to hPAAF activation. On both the protein and gene-expression levels, cellular activation markers significantly increased in stiffened samples and were highest in IPAH patient-derived cells. Treatment with a selective estrogen receptor modulator reduced expression hPAAF activation markers, demonstrating that hPAAF activation is a one pathologic response mediated by estrogen signaling in the vasculature, validating that drugs currently in clinical trials could be evaluated in sex-specific 3D-bioprinted pulmonary artery adventitia models.

肺动脉高压（Pulmonary arterial hypertension, PAH）在男性与女性患者中呈现出截然不同的致病特征。女性患者的患病易感性更高（女性与男性的患病比例约为4:1），但确诊后的生存期却长于男性患者。这种复杂的性别二态性被称为雌激素悖论（estrogen paradox）。既往研究表明，雌激素信号通路在肺血管中可能具有致病性，而在心脏中则发挥保护作用，但肺动脉高压中这类性别差异的具体分子机制仍未明确。肺动脉高压是一类肺血管疾病，会引发肺部小血管瘢痕形成，进而导致血流灌注受损、血压升高。随着病情进展，血压持续升高会进一步造成心脏损伤。既往多数肺动脉高压相关的体外建模研究，仅采用男性细胞或来源未明确标注的细胞。本研究构建了一款动态3D生物打印模型，纳入女性患者来源的细胞与循环性激素，旨在专门探究女性患者对微环境刚度变化及性激素信号通路的响应机制。我们采用聚乙二醇-α-甲基丙烯酸酯（Poly(ethylene glycol)-alpha methacrylate, PEGαMA）制备水凝胶载体，搭载特发性肺动脉高压（idiopathic PAH, IPAH）患者或健康对照者来源的女性人肺动脉外膜成纤维细胞（human pulmonary artery adventitia fibroblasts, hPAAFs），通过3D生物打印技术模拟肺动脉外膜结构。该生物材料初始状态柔软，与健康血管的力学特性相符，随后可通过光照调控实现刚度提升，以模拟肺动脉高压患者的血管瘢痕形成过程。实验结果表明，升高女性特发性肺动脉高压来源的hPAAFs所处微环境的刚度，可诱导hPAAF激活。在蛋白质与基因表达层面，刚度升高的样本中细胞激活标志物水平均显著上调，且在特发性肺动脉高压患者来源的细胞中上调幅度最为显著。经选择性雌激素受体调节剂处理后，hPAAF激活标志物的表达水平显著降低，这证明hPAAF激活是血管内皮雌激素信号通路介导的病理性应答过程，同时验证了当前处于临床试验阶段的药物，可通过该性别特异性3D生物打印肺动脉外膜模型开展疗效评估。