(413e) A Poly(ethylene glycol) Diacrylate Hydrogel Angiogenesis Platform for Idiopathic Pulmonary Fibrosis

Microvascular remodeling is governed by the tightly regulated coordination of biochemical and mechanical signaling which becomes dysregulated in pathological environments such as idiopathic pulmonary fibrosis (IPF). IPF is characterized by progressive stiffening of the alveolar interstitium due to excess deposition of type I collagen and is often associated with a poor prognosis (mean survival of 2-5 years after diagnosis). Excessive stiffening of the extracellular interstitium due to uncontrolled extracellular matrix (ECM) deposition contributes to vascular crowding and architectural collapse at the tissue scale and aberrant mechanotransduction at the cellular scale, compromising both the function of the alveolar epithelium in respiration and the innate ability of the surrounding tissue to respond to injury.

Ex vivo recapitulation of the fibrotic extracellular environment utilizing tailorable, synthetic biomaterials provides an opportunity to study the mechanisms by which angiogenic remodeling is affected by pathological microenvironments, the unique characteristics of which are otherwise inseparable in vivo (e.g., matrix stiffness or the presence and concentration of exogeneous signals). The engineering of three-dimensional tissue mimetic models from synthetic polymers such as poly(ethylene glycol) (PEG), as opposed to natural materials such as collagen or Matrigel, allows for precise control over material properties such as degradation kinetics or architecture and deliberate cell-material interactions such as adhesivity or presentation of bioactive molecules. Additionally, synthetic materials offer improved reproducibility, versatility, and scalability compared to biologically sourced materials, which may vary in composition or mechanical properties between batches or manufacturers.

Here, we report a novel angiogenesis assay designed to simulate the mechanical and biochemical environment of IPF to evaluate the effects of microenvironmental stiffness on recovery of microvascular sprouting and angiogenic signaling in the fibrotic murine lung. The assay consists of a biochemically modular poly(ethylene glycol) diacrylate (PEGDA) hydrogel system with tunable mechanics capable of recovering microvascular sprouting from fibrotic murine lung tissue.

Briefly, 3.4 kDa monoacrylate poly(ethylene glycol) succinimidyl valerate (PEG-SVA) was modified via ester amidation with the peptide adhesion motif RGDS and GGGPQGIWGQGK (PQ) as a proteolytically degradable linker to form PEG-RGDS and PEG-PQ. Pro-angiogenic signaling proteins platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF), similarly coupled to PEG-SVA to form PEG-PDGF and PEG-FGF, were covalently integrated into the matrix via acrylate crosslinking to promote matrix-mediated pericyte recruitment and endothelial cell migration and assembly. Gel elastic moduli were tuned by incorporation of alloc-protected lysine monomers to replicate stiffnesses of healthy (2kPa), intermediate (10kPa), and fibrotic (20kPa) lung tissue.

Precursor solutions were prepared in 1 mL sterile HEPES-buffered saline (HBS, pH 7.4) from 50 mg/mL PEG-PQ, 3.5 mM PEG-RGDS and the alloc-protected lysine in HBS, then sterile-filtered using a 0.2 µm syringe filter. To each precursor solution, 0.7 nmol/mL PEG-PDGF and 0.27 PEG-FGF were added as matrix-bound angiogenic factors. 35 μl of PEG precursor solution containing PEG-RGDS, PEG-PDGF, PEG-FGF, and 10 μL/mL of the photoinitiator 2-dimethoxy-2-phenylacetophenone, prepared by dissolving 300 mg acetophenone in 1 ml of N-vinylpyrrolidone (NVP), was pipetted into the center of a 6 mm ID PDMS mold, and the plate exposed to UV light (365 nm, 10 mW/cm²) for 30 seconds. Lung segments from mice treated with bleomycin to stimulate fibrosis or littermate controls were cultured on biofunctionalized hydrogels for one week followed by immunofluorescence imaging and cytokine and MMP analysis.

MMP-2 levels increased for all conditions, indicating elevated matrix remodeling. Fibrotic tissue segments exhibited a two-fold increase in MMP-2 secretion over controls for all time points, corroborating that stiffness upregulates MMP-2 production and matrix degradation. MMP-9 levels fell for all conditions, possibly due to stiffness-modulated TIMP-1 expression. Controls contained more vessel sprouts than bleomycin samples in all but the 20kPa condition. Expression of hematopoietic growth factor and monocyte chemoattractant protein-1 was significantly upregulated in diseased tissue, indicating dysregulation of angiogenic signaling irrespective of environmental stiffness. Overall, stiffer matrices diminished sprouting from healthy lungs, soft matrices were able to recover sprouting from diseased lungs, and diseased lungs were primed to withstand the stiffest hydrogels to allow for vascular spouting.

This study highlights the critical role of microenvironmental stiffness in microvascular remodeling and angiogenesis in the context of fibrotic lung disease and provides insight into the use of synthetic biomaterials as a platform for disease modeling in 3D microenvironments. Our observations demonstrate that excessive matrix stiffness, characteristic of fibrotic environments, leads to dysregulated angiogenic signaling and compromised microvascular recovery, and that soft microenvironments are capable of rescuing angiogenic sprouting in the fibrotic lung. Importantly, our novel angiogenesis assay provides a tailorable platform for investigating the mechanisms underlying angiogenic dysfunction in pathological or tissue-mimetic systems through biomaterial design and fabrication. This study contributes to the greater understanding of the pathophysiology of IPF and emphasizes the need for rational design of synthetic vascular ECM for tissue engineering of pathological models.