Hydrogels preserve native phenotypes of valvular fibroblasts through an elasticity-regulated PI3K/AKT pathway

Abstract

Matrix elasticity regulates proliferation, apoptosis, and differentiation of many cell types across various tissues. In particular, stiffened matrix in fibrotic lesions has been shown to promote pathogenic myofibroblast activation. To better understand the underlying pathways by which fibroblasts mechano-sense matrix elasticity, we cultured primary valvular interstitial cells (VICs) isolated from porcine aortic valves on poly(ethylene glycol)-based hydrogels with physiologically relevant and tunable elasticities. We show that soft hydrogels preserve the quiescent fibroblast phenotype of VICs much better than stiff plastic plates. We demonstrate that the PI3K/AKT pathway is significantly upregulated when VICs are cultured on stiff gels or tissue culture polystyrene compared with freshly isolated VICs. In contrast, myofibroblasts de-activate and pAKT/AKT decreases as early as 2 h after reducing the substrate modulus. When PI3K or AKT is inhibited on stiff substrates, myofibroblast activation is blocked. When constitutively active PI3K is overexpressed, the myofibroblast phenotype is promoted even on soft substrates. These data suggest that valvular fibroblasts are sensing the changes in matrix elasticity through the PI3K/AKT pathway. This mechanism may be used by other mechano-sensitive cells in response to substrate modulus, and this pathway may be a worthwhile target for treating matrix stiffness-associated diseases. Furthermore, hydrogels can be designed to recapitulate important mechanical cues in native tissues to preserve aspects of the native phenotype of primary cells for understanding basic cellular responses to biophysical and biochemical signals, and for tissue-engineering applications.mechanosensing | tissue stiffening | phosphatidylinositide 3-kinase signaling E very cell has its distinct microenvironment. Fibroblasts reside in the interstitial mesenchyme, maintaining the balance and structure of the matrix; muscle satellite cells reside between the basal lamina and muscle fibers, waiting to be activated upon injury. In a basic sense, the cellular microenvironment is defined as the extracellular space surrounding and supporting the cell. This microenvironment is often comprised of extracellular matrix (ECM), soluble chemical factors, and neighboring cells. The ECM in particular does not just act as a passive scaffold, but instructs cell fate through the coordinated and dynamic presentation of biochemical and biophysical cues. For example, in fibrotic tissues stiffened by excessive collagen deposition, resident fibroblasts acquire an activated myofibroblast phenotype with α-smooth muscle actin (αSMA) stress fibers (1). The integrity and signaling of the ECM are essential for regulating normal cellular function and maintaining organ homeostasis.The aortic valve, which controls the unidirectional flow of blood during heart contractions, is composed of elastin-, proteoglycanand collagen-rich layers of ECM (2) and supports survival and metabolism of valvular interstitial cells (VICs). VICs are the main fib...

Dynamic stiffening of poly(ethylene glycol)-based hydrogels to direct valvular interstitial cell phenotype in a three-dimensional environment

“…Early efforts to study VIC-matrix interactions and mechanotransduction have observed that seeding VICs on softer (~7 kPa) hydrogel substrates better preserves their native phenotype compared to culture on tissue culture polystyrene (TCPS) (33). This study identified thousands of genes that were differentially regulated on TCPS compared to freshly isolated cells and found that three of the genes upregulated on TCPS (αSMA, CTGF, and Col1A1) were expressed at levels closer to freshly isolated cells when the VICs were instead cultured on the soft hydrogel.…”

Palladin Mediates Stiffness-Induced Fibroblast Activation in the Tumor Microenvironment

“…The stromal tissue surrounding the tumor, known as the tumor microenvironment (TME), is also typically stiffer than normal tissue, although usually only three to five times stiffer than normal stroma (1,2). This increase in stromal stiffness has been implicated in changes in both tumor cells and in tumor associated stromal cells such as fibroblasts, which are sensitive to mechanical properties of the extracellular matrix (ECM) (1)(2)(3)(4)(5)(6)(7)(8).…”

Synthetic Mimics of the Extracellular Matrix: How Simple is Complex Enough?

“…identified the PI3K/AKT pathway as one of the key signaling pathways that regulate mechanosensing and activation of primary valvular interstitial cells (VICs). 55 This study showed that through a reduction in the material stiffness the myofibroblast to fibroblast activation of VICs could be reversed. As another example, Gilbert et al also illustrated the efficacy of recapitulating appropriate ECM substrate stiffness on muscle stem cell (MuSC) function.…”

“…As discussed previously, these user-tunable hydrogels have advanced our understanding of cellular mechanotransduction and mechanical ‘memory’. 55,59 Photodegradable hydrogels have also been used to study how cells receive mechanical information from the environment by spatially controlling cell adhesion on a sub-cellular length scale. Tibbitt et al used photodegradable PEG hydrogels to study cytoskeletal pretension through sub-cellular focal adhesion detachment of encapsulated and spread cells within 3D hydrogels.…”