Conservation of mechanosignaling: responses of human adult mesenchymal stem cells and differentiated vascular cells to applied physical forces
Doyle, Adele Marion
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Mesenchymal stem cells (MSCs) may benefit vascular cell-based therapies as smooth muscle or endothelial cell substitutes or through paracrine actions to repair, replace, or regenerate vascular tissue. Previous studies have demonstrated that MSCs can adopt traits of smooth muscle cells (SMCs) or endothelial cells (ECs), as well as secrete specific factors that tune signaling and material properties in the local environment. Few studies have investigated the cell signaling response of MSCs to mechanical forces present in the vasculature: specifically, shear stress due to blood flow and cyclic strain due to pulsatile blood flow. Thus, the central objective of this dissertation was to determine the signaling responses of MSCs to vascular-relevant applied physical forces, in comparison with that of differentiated vascular cells. Vascular-relevant mechanosignaling of MSCs was assessed through two comparisons: (1) MSC and SMC responses to applied cyclic strain and (2) MSC and EC responses to applied fluid shear stress. MSCs and SMCs were seeded on fibronectin-coated silicone and subjected in vitro to cyclic strain (10%, 1 Hz) or parallel static culture using a custom-built equibiaxial cyclic strain device. Gene expression analysis of 84 signal transduction molecules demonstrated both cell types respond with significant (p<0.05, n=3) fold-changes (|FC|≥ 1.5) within 24 hours of applied equibiaxial strain. Most strain-responsive genes identified were significantly strain-responsive in only one cell type. A signaling trio of Interleukin 8, Vascular cell adhesion molecule 1, and Heme oxygenase 1 was significantly altered in both MSCs and SMCs, suggesting cyclic strain regulates immune and inflammatory functions in both cell types. The response to shear stress of MSCs and ECs was compared using cells seeded on type I collagen or fibronectin and exposed to steady laminar shear stress (5 or 15 dyn/sq-cm) using a parallel plate shear chamber system. Gene expression was compared in MSCs and ECs for a panel of immune and inflammation-related markers. Expression of Cox-2 and Hmox-1 increased significantly (p<0.05, n≥3; |FC|≥1:5) in both cell types. Reduced shear stress-responses of Mcp-1, Pecam-1, and VE-Cad in MSCs relative to ECs suggests that MSCs promote less inflammation and immune activation in response to shear stress than ECs. Mechanosensitivity profiles for MSCs and differentiated vascular cells were broadened using whole genome microarrays. These high-throughput studies confirmed that (1) signaling profiles between sample groups vary significantly more (p<0.05, n=3) with cell type than applied force condition and (2) a subset of conserved mechanosensitive genes alter expression levels significantly and in the same direction fold-change in multiple cell types. Bioinformatics analysis of these conserved mechanoresponsive genes highlighted oxidative stress, cell cycle, and DNA replication as functions regulated by vascular-relevant mechanical cues. These studies demonstrate that MSCs partially reproduce differentiated vascular cell mechanosignaling, while simultaneously altering expression of genes not typically force-responsive in vascular cells. This work defines a role for conserved mechanosignals, based on genes whose expression in response to applied force alters significantly (p<0.05, n≥3) and by at least 1.5-fold change in multiple cell types and/or force types. Comparisons completed for this dissertation motivate future studies to track the functional impact of specific similar or unique MSC mechanoresponses. This work contributes to design of MSC-based vascular therapies and an understanding of stem and differentiated cell mechanobiology.