Female Fibroblast Activation Is Estrogen-Mediated in Sex-Specific 3D-Bioprinted Pulmonary Artery Adventitia Models

Pulmonary arterial hypertension (PAH) is a form of pulmonary vascular disease characterized by scarring of the small blood vessels that results in reduced blood flow and increased blood pressure in the lungs. Over time, this increase in blood pressure causes damage to the heart. Idiopathic (IPAH) impacts male and female patients differently, with female patients showing a higher disease susceptibility (4:1 female-to-male ratio) but experiencing longer survival rates postdiagnosis compared to male patients. This complex sex 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 IPAH remain unclear. Many previous studies of PAH relied on male cells or cells of undisclosed origin for in vitro modeling. Here, we present a dynamic, three-dimensional (3D)-bioprinted model incorporating cells and circulating sex hormones from female patients to specifically study how female patients respond to changes in microenvironmental stiffness and sex hormone signaling on the cellular level. Poly(ethylene glycol)-α methacrylate (PEGαMA)-based hydrogels containing female human pulmonary artery adventitia fibroblasts (hPAAFs) from 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, which is currently in clinical trials for IPAH treatment, reduced the expression of hPAAF activation markers, demonstrating that hPAAF activation is one pathologic response mediated by estrogen signaling in the vasculature. These results showed the utility of sex-specific, 3D-bioprinted pulmonary artery adventitia models for preclinical drug discovery and validation.