High-throughput platform to investigate effects of dynamic presentation of mechanical and biochemical matrix cues on VIC phenotype

Abstract

Event Abstract Back to Event High-throughput platform to investigate effects of dynamic presentation of mechanical and biochemical matrix cues on VIC phenotype Megan E. Schroeder1, Kelly M. Mabry2, Samuel Z. Payne2 and Kristi S. Anseth1, 2 1 University of Colorado Boulder, Materials Science and Engineering, United States 2 University of Colorado Boulder, Chemical and Biological Engineering, United States Introduction: Aortic stenosis occurs in heart valves when fibroblasts (valvular interstitial cells, VICs) transition to an activated myofibroblast state and engage in pathological matrix remodeling. Mechanotransduction is essential in maintaining valve homeostasis and contributes to the diseased state when misregulated[1]. To better understand how the extracellular matrix and biochemical cues influence VIC activation, we have implemented a high-throughput (HT) platform capable of producing and imaging cell-laden PEG hydrogels functionalized with adhesion peptides. The approach allows one to dynamically alter the culture environment (e.g., add cytokines, alter matrix adhesivity, stiffen the matrix) while simultaneously imaging cellular responses. This HT platform is used to investigate potential synergies between mechanical and biochemical cues on VIC phenotype. Materials and Methods: VICs were isolated from porcine hearts. Hydrogels were synthesized with a thiol-ene photo-click reaction of a norbornene end-functionalized PEG with a thiol containing MMP-degradable crosslinker (KCGPQG*IWGQCK). Concentrations of 0.75mM PEG and 1.875mM MMP led to pendant ene functionalities available for on-demand reactions with thiolated biomacromolecules. Dosing of gels with solutions containing CRGDS (1.25mM or 5mM) resulted in photoinitiated tethering of either 0.4mM or 1.5mM CRGD as determined by fluorescent labeling. A robotic liquid handling system (EpMotion M5073) dispensed 10μL gel droplets on 96-well plates that were then photopolymerized (2.5mW/cm2 @ 365 nm light). VIC morphology, proliferation and expression of α-smooth muscle actin (α-SMA), a marker of the myofibroblast phenotype, were observed with time. A high-content confocal imager (Operetta, Perkin Elmer) was used for all live cell and immunofluorescent imaging. Results and Discussion: VICs were encapsulated in gels of varying adhesivity (with 1mM or no CRGD (Day 0)). On Day 3, either 0.4mM or 1.5mM CRGD was tethered into a subset of the gels. Controls show VICs in gels without CRGD remained rounded, while those in gels with 1mM CRGD elongated up to Day 5. Upon dynamic addition of CRGD, VICs rapidly elongated in 24 hours, which may indicate local matrix degradation occurred prior to its addition. VIC activation to the myofibroblast required the presence of CRGD in the matrix to form contractile α-SMA stress fibers. Tethering CRGD at Day 3 resulted in slightly elevated levels of α-SMA compared to gels originally formed with 1mM CRGD, which relates to increases of fibronectin in diseased valves[2]. Deeper investigations to characterize the persistent vs. transient myofibroblast phenotype using cytokine cocktails and altering matrix modulus are in progress. We seek to understand factors that lead to an irreversible myofibroblast phenotype to identify potential pathways involved in aortic stenosis. Conclusion: This HT cell culture platform enables studies of the dynamic presentation of mechanical and biochemical matrix cues in directing VIC phenotype and assessing pathways involved in the myofibroblast transition. NSF Graduate Research Fellowship; Howard Hughes Medical Institute (HHMI)