Abstract Body (Do not enter title and authors here): Background:
Pulmonary arterial hypertension (PAH) is a progressive vasculopathy driven by endothelial cell (EC) dysfunction, smooth muscle cell (SMC) proliferation, and medial thickening. While endoplasmic reticulum (ER) stress is a key contributor to PAH vascular remodeling, mechanistic studies and therapeutic development are limited by a lack of physiologically relevant patient models. Engineered 3D tissue platforms offer an opportunity to recapitulate disease-relevant architecture and biomechanics.
Hypothesis:
We hypothesize that elevated pressure and hypoxia-induced ER stress activate remodeling pathways in SMCs, and that a bioengineered human bilayered EC-SMC 3D vessel can serve as a model for early-stage PAH remodeling.
Methods:
We designed a CAD-guided, casted bilayer vessel composed of human pulmonary artery ECs and SMCs embedded in a gelatin methacrylate (gelMA)-based hydrogel supplemented with bioactive additives (1.0% LAP, 1.0 mM tartrazine, elastin). SMCs were suspended in gelMA bioink and cast into a custom bioreactor with rods to define lumens, which were then seeded with ECs. Viability was assessed via Live/Dead staining; structural fidelity by lumen diameter measurements across 1 mm, 4 mm, and 4-->1 mm gradient constructs; and stiffness via Mach-1 compression testing. Inlet pressures were measured during perfusion to evaluate physiologic pressure capacity. To inform 3D experiments, ECs and SMCs were exposed to hypoxia in 2D culture and assessed for ER stress responses.
Results:
Casted vessels showed high structural fidelity of the lumen diameters (4 mm: 91.3%, 1 mm: 112.3%) and robust cell viability. Inlet pressures in perfused constructs modeled both physiologic and pathologic pulmonary arterial conditions, exceeding 20 mm Hg with added downstream resistance. Construct stiffness (9-12 kPa) approximated ex vivo PA tissue (11-14 kPa). SMCs remained viable and well-integrated throughout the medial layer, supporting stable incorporation. Hypoxia upregulated ER stress marker ATF4 and remodeling genes in 2D ECs and SMCs, establishing conditions for future 3D modeling.
Conclusions:
We present a bioengineered, human-relevant 3D vessel platform that supports coculture, mimics physiologic mechanical properties, and accommodates pathologic pulmonary pressures. This system offers a scalable and adaptable foundation for studying SMC-driven remodeling in PAH and supports future applications in therapeutic screening and personalized disease modeling.