Engineering dynamic microenvironments to model sex-specific fibroblast activation in pulmonary arterial hypertension

Introduction:: Respiratory diseases like pulmonary arterial hypertension (PAH) frequently show sexual dimorphisms [1]. 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 [2]. Indeed, the National Institutes of Health have recently tried to address these inequities in healthcare research by calling for researchers to include sex as a variable in funded studies [3]. The research outlined here investigates sex-specific cell behavior in the context of PAH using hydrogel biomaterials.

Hydrogel biomaterials are a powerful tool for creating synthetic tissue culture platforms. Poly(ethylene glycol)-alpha methacrylate (PEGαMA) is the backbone of a dynamic hydrogel system that undergoes two polymerization reactions – the first generates an initially soft hydrogel that recapitulates healthy pulmonary artery adventitia and the second stiffens the material to elastic modulus (E) values measured in fibrotic pulmonary artery adventitia [4]. This phototunable biomaterial system is ideal for recapitulating the physiological changes in the microenvironment of fibrotic tissues and study sexual dimorphisms in diseases like PAH.

Here, we measure the influence of hydrogel stiffness, hormonal milieu, and dimensionality on human pulmonary artery adventitia fibroblast (hPAAF) activation, a hallmark of PAH.


Materials and Methods:: PEGαMA Hydrogel: Eight-arm 10kg mol^-1 PEGαMA was synthesized as previously described [5]. DTT and KCGPQGIWGQCK (MMP-2 degradable peptide) were used as crosslinkers, and a fibronectin mimetic (CGRGDS) was added to facilitate cell adhesion. For 2D hydrogels, the precursor solution was placed in 90 µL drops between a silanated 18-mm glass coverslip and a glass slide treated with SigmaCote and allowed to polymerize for 30 min. 3D Bioprinting: Vessel constructs were 3D bioprinted using the Freeform Reversible Embedding of Suspended Hydrogels method [6]. HPAAFs from control donors and donors with idiopathic pulmonary arterial hypertension (IPAH) were suspended in hydrogel precursor solution, then printed in a support bath and allowed to polymerize. Support material was melted and replaced with growth media containing 1% sex-matched human serum. Cellular Activation: Female and male hPAAFs were seeded at 20,000 cells cm^-2 onto 2D hydrogels or within 3D hydrogels during bioprinting. Basal medium with 1% FBS or pooled human serum supplemented the cells. HPAAFs were grown on or in soft materials for 7 days. Photoinitiator was added to the media on day 6 and UV light was applied on day 7 to stiffen half of the hydrogels. HPAAFs were grown on or in stiffened hydrogels for 2 days. Proliferation was quantified by EdU (Click-iT EdU Assay) incorporation and nuclear counterstaining (Hoescht). Samples were immunostained for α-smooth muscle actin (αSMA; mouse anti-human αSMA monoclonal antibody, goat anti-mouse Alexa Fluor-555), f-actin (ActinGreen 488 Ready Probes), and nuclei (DAPI), imaged using fluorescent microscopy, and analyzed with ImageJ.


Results, Conclusions, and Discussions:: Female hPAAFs in 2D exhibited increased levels of activation on soft and stiffened conditions, unless cultured in medium containing younger (age < 50) female serum. Results suggested that higher sex-hormone concentrations increased female hPAAF sensitivity to increased microenvironmental stiffness. Male hPAAFs were more activated on stiffened substrates than on soft regardless of the concentration of the sex hormones. Results found that male hPAAFs were more sensitive to increases to microenvironmental stiffness, and that sensitivity increased with higher sex hormone concentrations, especially in response to progesterone, which is commonly found fetal bovine serum, a widely used cell culture media additive.

Fibroblast proliferation, as measured by EdU incorporation, and activation were greater in stiffened bioprinted constructs compared to soft in 3D. This was true in male and female cells and in cells from control and IPAH donors. Significant increases in the percentage of αSMA-positive cells cultured within stiffened hydrogels were consistent with prior research conducted on 2D hydrogels. The inclusion of an MMP-degradable peptide crosslinker allowed fibroblast spreading prior to stiffening and facilitated activation [7]. IPAH female HPAAFs exhibited a greater percentage of activation compared to cells cultured in other conditions, which was consistent with epidemiological research showing increased prevalence of PAH in female patients. Prior research has also shown increased αSMA positivity in fibroblasts treated with female serum compared to male serum [8]. 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.


Acknowledgements (Optional): :

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