Dynamically stiffening biomaterials reveal serum age and sex influence pulmonary arterial adventitial fibroblast activation in vitro

Respiratory diseases like pulmonary arterial hypertension (PAH) frequently exhibit sexual dimorphism. Female patients are more susceptible to, but have increased survival for, PAH, and the underlying mechanisms are not fully understood. This phenomenon is known as the estrogen paradox. Historic underrepresentation of female subjects in clinical trials, failure of many studies to identify the sex of animals in in vivo studies, and similar lack of information about tissue and cell sex in benchtop research have perpetuated a long-standing disparity in healthcare outcomes for female patients. During PAH progression in vivo, human pulmonary arterial adventitial fibroblasts (hPAAF) differentiate into an activated phenotype. These cells produce excess, aberrant extracellular matrix proteins that stiffen the surrounding pulmonary arterial tissues. Here, we employed dynamic poly(ethylene glycol)-alpha methacrylate (PEGαMA)-based biomaterials to study how the age and sex of human serum influenced hPAAF activation in response to microenvironmental stiffening in vitro. Results showed female and male cells responded differently to increases in microenvironmental stiffness and serum composition. Male hPAAFs were less activated than female cells on soft hydrogels and more responsive to increases in microenvironmental stiffness regardless of serum composition. Female hPAAF activation followed this same pattern only when cultured in younger (age < 50) female serum or when older (age ≥ 50) female serum was supplemented with estradiol. Otherwise, female hPAAF activation was relatively high on both soft and stiffened hydrogels, with little difference in activation between the two conditions. Collectively, these results suggest that it may be possible to model the estrogen paradox observed in PAH in vitro and that it is critical for researchers to report cell sex and serum source when conducting in vitro experimentation.