Variations in mechanical stiffness alter microvascular sprouting and stability in a PEG hydrogel model of idiopathic pulmonary fibrosis

Abstract

ObjectiveMicrovascular remodeling is governed by biomechanical and biochemical cues which are dysregulated in idiopathic pulmonary fibrosis. Understanding how these cues impact endothelial cell‐pericyte interactions necessitates a model system in which both variables can be independently and reproducibly modulated. In this study we develop a tunable hydrogel‐based angiogenesis assay to study how varying angiogenic growth factors and environmental stiffness affect sprouting and vessel organization.MethodsLungs harvested from mice were cut into 1 mm long segments then cultured on hydrogels having one of seven possible stiffness and growth factor combinations. Time course, brightfield, and immunofluorescence imaging were used to observe and quantify sprout formation.ResultsOur assay was able to support angiogenesis in a comparable manner to Matrigel in soft 2 kPa gels while enabling tunability to study the effects of stiffness on sprout formation. Matrigel and 2 kPa groups contained significantly more samples with sprouts when compared to the stiffer 10 and 20 kPa gels. Growth factor treatment did not have as obvious an effect, although the 20 kPa PDGF + FGF‐treated group had significantly longer vessels than the vascular endothelial growth factor‐treated group.ConclusionsWe have developed a novel, tunable hydrogel assay for the creation of lung explant vessel organoids which can be modulated to study the impact of specific environmental cues on vessel formation and maturation.

Multiscale computational model predicts how environmental changes and drug treatments affect microvascular remodeling in fibrotic disease

“…019079; The Jackson Laboratory, Bar Harbor, ME) and ROSA floxed STOP tdTomato mice (007914; The Jackson Laboratory), on C57BL/6J background. Expression of tdTomato in cells of the myosin heavy chain 11 (Myh11) lineage (including pericytes and vascular smooth muscle cells) was induced by feeding 6 to 8 week old male mice a tamoxifen-containing diet (Envigo) for 14 days 50, 68, 118, 119 . Only male mice were used in this study, as the Cre gene cassette was inserted on the Y chromosome.…”

“…We performed a lung explant assay as previously described 68 . After the tamoxifen clearance period, when mice were approximately 12-16 weeks of age, three mice were humanely euthanized via carbon dioxide asphyxiation.…”

“…On Day 7, five plates were imaged using brightfield imaging on the Leica THUNDER Imager using the 10X objective (Leica). Images were then sorted into four groups, as previously described 68 , based on the identification of organized vessel-like structures versus disorganized cell infiltration. The number of images in each category were averaged in each well and then across wells of the same condition in a plate.…”

“…For the nintedanib treated simulations the nintedanib treated were compared to their respective control group using an unpaired one-tailed student’s t-test. For the experimental results statistics was performed as previously described 68 . In short, each 24-well plate was considered an independent sample, N, and all images acquired for that plate were averaged for each experimental sub-group.…”

Engineering pre-vascularized 3D tissue and rapid vascular integration with host blood vessels via co-cultured spheroids-laden hydrogel

“…When considering in vitro vascularization using co-culture method requires the consideration of several parameters, including selection of appropriate types of ECs and pericytes, coculture ratio, duration of in vitro pre-culture, and mechanical modulation of the matrix [8][9][10]. For example, changing the pericyte source from dental pulp stem cells to bone marrow-derived stromal cells (BMSCs) co-cultured with human umbilical vein cells (HUVECs), the co-culture ratio for HUVECs and fibroblasts from 1:1 to 1:2, the pre-culture period of co-cultured stem cells and HUVECs from 7 to 21 d, and the modulus of gelatin methacrylate (GelMA) hydrogel-encapsulating smooth muscle cells and HUVECs from 20 to 2 kPa consistently resulted in faster sprouting, greater elongation of ECs, and more complex vascular network formation after the alternations [11][12][13]. However, in vitro vasculatures are often characterized by a bare lumen structure, disconnected branches, limited EC sprouting, and poor angiogenesis outside of the construct because of insufficient cell-cell interactions, paracrine signaling, extracellular matrix (ECM) expression, and ECMremodeling abilities [14].…”