Effect of Stiffness and Growth Factor Composition on Microvascular Sprouting in PEG Hydrogel Model of Idiopathic Pulmonary Fibrosis

Introduction:: In diseases such as idiopathic pulmonary fibrosis (IPF), a progressive fibrotic lung disease, biomechanical and biochemical cues are dysregulated and disrupt microvascular stability. The mechanical stiffness of the lung in IPF is heterogenous and ranges from stiffnesses close to healthy lung, 2 kPa, up to that of mature fibrosis, 20 kPa. Angiogenic growth factors, such as vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF), and fibroblast growth factor (FGF), are also dysregulated perturbing vascular stability. Understanding how these cues impact the cells that make up the microvasculature, endothelial cells and pericytes, necessitates a model system in which both variables can be independently and reproducibly modulated. In this study we develop a tunable PEG hydrogel-based lung explant angiogenesis assay to study how varying angiogenic growth factors and environmental stiffness affect sprouting and vessel organization.


Materials and Methods:: Lungs harvested from 12-16 weeks old TdTomato Myh11-Cre mice were sectioned, allowed to adhere to the surfaces of protease-sensitive PEGDA hydrogels of varying stiffness (2-20 kPa) that either included matrix-bound VEGF or a combination of PDGF+FGF and cultured for 7 days in Endothelial Growth Medium-2 (Lonza). Brightfield and immunofluorescent images of capillary sprouts and branching were quantified using Leica Las X software, AngioTool, and ImageJ. The gels were sensitive to MMP-9 degradation therefore we performed an ELISA on spent media from Days 2, 4, and 6 of culture to quantify MMP-9 driven matrix remodeling.


Results, Conclusions, and Discussions:: Using immunofluorescent imaging we confirmed that our hydrogel system was able to support sprouting of endothelial cells, labelled with CD31, and pericyte migration onto sprouts by expression of TdTomato (Figure 1A&B). Our softest gels (2kPa) treated with VEGF or PDGF+FGF both performed similarly to the Matrigel control with over 75% of samples in all three groups containing sprouts (Figure 1C) with an average of over 7 sprouts per sample in all groups (Figure 1D). These similarities to Matrigel, a commonly used hydrogel in angiogenesis assays, indicated that the 2kPa PEGDA gels supported angiogenesis in a comparable manner but is also tailorable to the user’s specific design needs. When comparing across stiffnesses for healthy lung (2kPa), fibrotic foci (10kPa), and mature fibrosis (20kPa) we observed significantly more sprout formation at one week in the 2kPa group compared to both the 10 and 20 kPa groups in both growth factor treatments. Interestingly, in the PDGF+FGF treated group both 10kPa and 20kPa had similar average number of samples containing sprouts and average number of sprouts per sample. To investigate this group further we performed MMP-9 ELISA analysis on Days 2, 4, and 6 and the presence of MMP-9 was confirmed in all gels. The only significant change in MMP-9 levels was observed in the 2kPa group where the amount of MMP-9 on Day 2 was significantly higher than Days 4 or 6, suggesting that MMP-9 may play an important role in early phase of sprouting.



Overall, our lung explant angiogenesis assay was able to recapitulate the pro-angiogenic, soft environment of Matrigel, was MMP-degradable, and allowed for tunability to interrogate the effects of different growth factors and stiffnesses on sprout formation to better model the IPF microenvironment. Future work will include the use of lungs from mice treated with bleomycin, the standard murine model of IPF, to investigate how fibrosis in the explants affects vessel sprouting. Additionally, to further quantify how the sprouts are modulating the microenvironment of the gel we will perform atomic force microscopy (AFM) to analyze changes in gel stiffness in areas of high sprout concentration.


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