Abstract Body: Introduction: Pulmonary arterial hypertension (PAH) is characterized by pathologic vascular remodeling that increases stiffness in proximal and distal pulmonary arteries. Although stiffness is a known predictor of right heart failure, the impact of incorporating a stiffened microenvironment into in vitro models of PAH remains poorly defined. Our lab has biofabricated a 3D vessel model incorporating human smooth muscle cells (SMCs) within a phototunable, perfusable hydrogel. We hypothesize that increasing construct stiffness will activate pro-inflammatory and pro-fibrotic remodeling pathways.

Methods: We analyzed publicly available human single-cell RNA-sequencing (scRNA-seq) datasets from healthy controls and PAH patients to identify clinically relevant stiffness-associated pathways associated with PAH. Constructs were hardened by varying crosslinking method (UV vs blue light) and time exposure. Stiffness of constructs was characterized using microindentation with a Mach-1 system. A custom macro was developed to process Mach-1 output files and spatially map stiffness measurements across the 3D constructs on Paraview.

Results: Analysis of scRNA-seq datasets revealed that fibrotic markers, such as JUN, FOS, and NFKB1, were more strongly associated with PAH patients compared to healthy controls. Following one minute of photo-crosslinking the top and bottom surfaces of the constructs, we characterized stiffness at the outer surfaces and inner lumen. Constructs stiffened using broad UV exposure exhibited significantly greater stiffness than those exposed to blue light (12.8 kPa, n = 2 vs. 10.1 kPa, n = 3; p < 0.001). No significant differences in surface stiffness were observed between light-exposed and unexposed regions within either UV- or blue-light–treated constructs. In contrast, luminal stiffness was significantly higher than surface stiffness for both blue-light constructs (11.7 kPa vs. 9.2 kPa, p < 0.05, n = 3) and UV-light constructs (15.7 kPa vs. 11.8 kPa, p < 0.01, n = 2). Future studies will perfuse vessel constructs with variable stiffness levels to evaluate how stiffness influences vascular remodeling pathway activation.

Conclusions: We established a method for tunable and spatially defined stiffness in a 3D biofabricated pulmonary artery model. This system provides a physiologically relevant in vitro platform to investigate how increased vessel stiffness contributes to vascular remodeling in pulmonary arterial hypertension.