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

Significance

Valvular interstitial cells (VICs) are the principal cellular component of cardiac valves and maintain normal valve homeostasis. During valvular fibrosis, VICs differentiate into myofibroblasts and stiffen the valve matrix. The results in this report demonstrate that standard techniques of culturing VICs on supraphysiologically stiff, tissue-culture polystyrene cause a dramatic induction of myofibroblast differentiation. In contrast, culturing VICs on soft, poly(ethylene glycol)-based hydrogels preserves the native, quiescent phenotype. A detailed study of VIC mechano-sensing reveals that matrix elasticity elicits pathologic changes in VICs through PI3K/AKT signaling. A more complete understanding of the molecular mechanisms of VIC mechano-biology may facilitate development of novel therapeutics targeting downstream signaling in matrix-stiffness–associated diseases, and may be applicable to fibrotic diseases in different tissues.

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 up-regulated 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.

Every 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-, proteoglycan- and collagen-rich layers of ECM (2) and supports survival and metabolism of valvular interstitial cells (VICs). VICs are the main fibroblast population in cardiac valves that secrete ECM-related proteins for tissue development and homeostasis (3). Normal valves maintain a bulk elastic modulus (E, a measure of the deformation of a material to an external force) around 0.8–8 kPa (4). As valve stenosis develops, characterized by fibrotic stiffening and nodule-like calcification, the tissue can become as stiff as collagenous bone (E, ∼27 ± 10 kPa) (5, 6). VICs have been shown to differentiate from quiescent fibroblasts in normal valves into pathogenic myofibroblasts and osteoblast-like cells in diseased valves (7). Consistently, VICs are activated to myofibroblasts in vitro in response to a high substrate modulus (E > 15 kPa), but this phenotype can be reverted when the substrate modulus is reduced (8, 9). To better mimic the ECM environment of aortic valves, we examined poly(ethylene glycol) (PEG)-based hydrogels as culture substrates, which have physiologically relevant moduli (E, ∼7 kPa to ∼32 kPa), spanning the range of normal and diseased valves. Furthermore, the moduli of these PEG hydrogels can be reduced in situ with light from 32 kPa to 7 kPa in ∼5 min (10), enabling close monitoring of changes in intracellular signaling in response to real-time modulus reduction.

Despite the recognized importance of elasticity in regulating the function and differentiation of many cell types, including fibroblasts (1), mesenchymal stem cells (11), and myoblasts (12), the effect of matrix stiffness is still debated (13, 14) and many questions remain about how the mechanical cue of elasticity is translated into intracellular signaling. Cells often bind to the matrix via integrins, mechano-sensitive transmembrance proteins that stimulate intracellular biochemical signaling, including FAK, ERK, and JNK (15). Dupont et al. showed that YAP and TAZ, two Hippo-related transcription factors, can serve as sensors of ECM stiffness in mesenchymal stem cells (11). Huang et al. showed that lung fibroblasts initiate nuclear translocation of megakaryoblastic leukemia factor-1 and RhoA/Rock activation on stiff substrata (16). All of these results suggest that matrix elasticity can elicit complex cellular signaling and transcriptional responses, but further characterization is needed. In this study, we set out to develop a tissue-mimicking culture platform that better preserves aspects of the native VIC phenotype to study how VICs respond to matrix elasticity changes via the PI3K/AKT pathway.

Results

Tissue-Culture Polystyrene Culture Alters the Transcriptional Profile of VICs.

Based on porcine genome microarrays, tissue-culture polystyrene (TCPS) culture alters mRNA expression genome-wide compared with freshly isolated (passage 0, P0) VICs (Fig. 1A): 2,173 gene probes were up-regulated and 1,926 gene probes were down-regulated in VICs cultured on TCPS compared with P0 VICs (fold-change ≥ 2 and P value ≤ 0.01) (Fig. 1B). Gene expression was validated using quantitative real time-PCR (qRT-PCR) and the fold-change detected by qRT-PCR followed the magnitude of change observed in the microarrays (Fig. S1). The most significantly up-regulated gene functions for TCPS VICs relative to P0 VICs were cell cycle, cytoskeleton, and mitochondrion. In contrast, the most significantly down-regulated gene functions included extracellular region, regulation of transcription from RNA polymerase II promoter, polysaccharide binding, and inflammatory response (Fig. 1C and Table S1). These results suggest that conventional TCPS cultures, which involve passaging and culturing cells in media, do not preserve the quiescent fibroblast phenotype of VICs in the native valve matrix. Although substrate properties, media components, cell density, and so forth could all affect gene expression, we focus here on investigating how substrates affect VIC phenotypes in culture.

Fig. 1.

Culture on TCPS changes the whole-genome transcriptional profile of VICs isolated from porcine aortic valves. (A) Hierarchical clustering based on microarray gene-expression data for freshly isolated VICs (P0 VICs) and VICs cultured on TCPS (TCPS VICs). (B) For this experiment, 2,173 gene probes were significantly up-regulated (≥twofold) and 1,926 gene probes were significantly down-regulated (≥twofold) in TCPS VICs compared with P0 VICs, with P < 0.01. (C) DAVID functional annotation showed that genes related to mitochondrion, cell cycle, and cytoskeleton were significantly up-regulated, whereas genes related to extracellular region, regulation of transcription from RNA polymerase II promoter, inflammatory response, and polysaccharide binding were down-regulated in TCPS VICs compared with P0 VICs. The P value of each gene ontology (GO) functional group follows the names of the GO terms. The percentage in the pie charts refers to the fraction of genes that fall into each functional category.

Hydrogels Preserve Aspects of the Native VIC Phenotype.

When P0 VICs were cultured on soft PEG hydrogels or TCPS, they maintained similar fibroblast morphology; however, at the molecular level, they showed very distinct gene expression. We examined αSMA, connective tissue growth factor (CTGF), and collagen type 1 α1 (Col1A1), as these are important myofibroblast markers (17–20) and both αSMA and CTGF were significantly up-regulated by TCPS culture in the microarray data (Table S2). VICs cultured on TCPS had significantly higher expression of these profibrogenic genes, including ∼86-fold increase in αSMA, ∼7-fold increase in CTGF, and ∼2-fold increase in Col1A1, compared with P0 VICs (Fig. 2A). However, VICs cultured on soft gels or native valve matrix maintained low expression levels of these genes, comparable to P0 VICs (Fig. 2A). From the microarrays (Fig. 1), tissue inhibitor of metalloproteinase-3 (TIMP3) was significantly down-regulated by culture on TCPS, whereas TIMP2 was not changed significantly and can thus serve as a control, unregulated gene (Table S2). As shown in Fig. 2A, TIMP3 was highly expressed in P0 VICs and valve cultures but was significantly down-regulated with culture on soft gels and TCPS, whereas TIMP2 was maintained at a similar level in all isolated VIC cultures (except for the intact valve culture condition). At the protein level, P0 VICs and VICs cultured on soft gels expressed a much lower level of αSMA than those cultured on TCPS (Fig. 2B). Consistently, a low percentage of VICs in native valves or cultured on soft gels stained positive for αSMA. However, a majority of VICs cultured on TCPS formed αSMA⁺ stress fibers, indicative of the myofibroblast phenotype (Fig. 2C). Because myofibroblast activation results in contractility, VICs cultured on TCPS were treated with blebbistatin, an actomyosin inhibitor. This process resulted in a potent inhibition of myofibroblast activation (Fig. S2). These data support that soft hydrogels mimic the native matrix more closely than TCPS in preserving aspects of the unactivated fibroblast phenotype of VICs.

Fig. 2.

Soft hydrogels preserve aspects of the native phenotype of VICs better than TCPS. (A) αSMA, CTGF, and Col1A1 expression levels, revealed by qRT-PCR, were significantly up-regulated when VICs were cultured on TCPS, but were preserved at levels similar to native cells when cultured on soft hydrogels and in valve culture. TIMP3 was expressed at high levels in both P0 VICs and valve culture, but was inhibited for VICs cultured on both soft gels and TCPS. *Significantly different from P0 with P < 0.05. ^†Significantly different from the soft gel condition with P < 0.05. (B) Based on Western blot analysis, VICs cultured on soft gels, like P0 VICs, expressed significantly less αSMA protein than those cultured on TCPS. (C) Representative staining of αSMA for VICs residing in the native valve (P0), cultured on soft hydrogels or on TCPS. Green: αSMA; blue: nuclei. (Scale bars, 100 μm.)

The PI3K Pathway Is Activated During TCPS Culture.

Because culturing VICs on TCPS activates the myofibroblast phenotype, we hypothesized that certain pathogenic pathways are activated by unnaturally stiff TCPS. We used Ingenuity Pathway Analysis (IPA), which builds pathways based on differentially regulated genes from microarrays and databases of known molecular connections, to detect the gene networks that were activated by TCPS culture relative to P0 VICs. We found that cell cycle, DNA replication, lipid metabolism, cell morphology, cellular assembly, and organization were among the top pathways to be activated (Fig. 3A and Fig. S3). Interestingly, the PI3K complex was up-regulated as part of the gene network for cell morphology, cellular assembly, and organization (Fig. 3A) (21). Based on Western blots, we confirmed that pAKT/AKT, which is downstream of PI3K, was significantly up-regulated by TCPS culture, compared with P0 VICs, but not FAK or p38 MAPK (Fig. 3B). Although these pathways could be regulated by multiple variables associated with the TCPS cultures (i.e., substrate stiffness, media components, cell density), we focused on the effects of substrate stiffness on the PI3K pathway.

Fig. 3.

The PI3K pathway is activated during TCPS culture. Ingenuity pathway analysis was performed to construct gene networks based on the genes that were significantly up-regulated by TCPS culture by ≥twofold with P < 0.01. In A, a gene network associated with cell morphology, cellular assembly, and organization was detected with significant changes and revealed up-regulation of the PI3K complex (blue highlights). The darker the red, the higher the fold change in gene expression. Values under each gene are the P value and the fold changes. (B) AKT, which is a main downstream target of PI3K, was activated by the TCPS culture, compared with P0 VICs, but not FAK or p38 MAPK.

PI3K Inhibition Blocks Myofibroblast Activation.

We set out to study whether PI3K activation is necessary for myofibroblast activation on stiff substrates. P0 VICs were cultured on TCPS or stiff gels for 3 d and then treated with a small molecule inhibitor of PI3K, LY294002, for 2 d. The initial culture of P0 VICs on the substrates for 3 d was to allow the primary cells to recover and seed evenly before starting inhibition. As expected, LY294002 reduced the ratio of pAKT/AKT (Fig. 4A). With PI3K inhibition, VICs demonstrated decreased myofibroblast activation on stiff substrates. First, αSMA protein levels were significantly reduced in response to increasing concentrations of LY294002 for VICs cultured on TCPS (Fig. 4A). Second, at the mRNA level, a number of myofibroblast activation genes, including αSMA, CTGF, and Col1A1, were significantly down-regulated with LY294002 treatment on TCPS (Fig. 4B). Third, the percentage of myofibroblasts quantified by αSMA⁺ stress fiber staining was significantly reduced with PI3K inhibition on both TCPS and stiff gels (Fig. 4C). Specifically, the percentage of myofibroblasts was reduced from 46.2 ± 0.9% to 16.9 ± 8.0% on TCPS and from 35.6 ± 1.4% to 11.4 ± 6.2% on stiff gels with 50 μM LY294002. In addition, LY294002 completely blocked TGF-β1–induced nodule formation on both TCPS and stiff gels (Fig. 4D). Furthermore, wortmannin, a second PI3K inhibitor, inhibited myofibroblast differentiation on TCPS (Fig. S4), and MK2206, an AKT inhibitor, suppressed the myofibroblast phenotypes on stiff substrates (Fig. S5).

Fig. 4.

PI3K inhibition by LY294002 blocks myofibroblast activation on stiff substrates. (A) On TCPS, LY294002 treatment reduced the pAKT/AKT level as well as αSMA protein expression in a dose-dependent manner. (B) Expression of profibrogenic genes, αSMA, Col1A1, and CTGF, was also inhibited with 50 μM LY294002 in VICs cultured on TCPS. An asterisk (*) indicates significantly different from DMSO with P < 0.05. (C) Consistently, immunostaining for αSMA showed that LY294002 treatment inhibited αSMA⁺ stress fiber formation in VICs cultured on stiff substrates, both TCPS and stiff gels. Green: αSMA; blue: nuclei. (Scale bars, 100 μm.) An asterisk (*) indicates significantly different from DMSO with P < 0.05. (D) Contraction-mediated nodule formation induced by TGF-β1 was significantly reduced by 50 μM LY294002. An asterisk (*) indicates significantly different from DMSO with P < 0.05. (Scale bars, 100 μm.)

Endogeneous PI3K/AKT Activity Is Correlated with Changes in Substrate Modulus.

As PI3K activation is required for myofibroblast activation on stiff substrates, we then tested whether endogeneous PI3K/AKT activity is regulated by substrate modulus. As shown in Fig. 5A, VICs cultured on stiff gels displayed the characteristic myofibroblast phenotype with αSMA⁺ stress fibers (green). However, those cultured on soft or stiff-to-soft gels (softened on day 3) had a significantly lower percentage of myofibroblasts (Fig. 5A). Interestingly, pAKT/AKT was significantly reduced on stiff-to-soft gels compared with stiff gels, but not on soft gels (Fig. 5B). To better characterize the kinetic changes of myofibroblast deactivation and pAKT/AKT, stiff gels were softened on day 5 and collected over 2, 6, or 48 h after softening. The percentage of αSMA⁺ myofibroblasts was reduced, but only significantly at 48 h after softening (Fig. 5C). In contrast, pAKT/AKT levels were decreased on stiff-to-soft gels as early as 2 h after softening and maintained at a low level at 48 h, compared with that on stiff gels (Fig. 5D). Neither the pFAK/FAK nor the pp38 MAPK/p38 MAPK ratio changed significantly on materials with different elasticities (Fig. S6). However, when VICs were cultured on stiff substrates for 7 d, they did not de-activate by 48 h after softening the gels (Fig. 5E), indicating that the lineage plasticity of the myofibroblast-to-fibroblast transition may have a time or dose limit based on the culture conditions.

Fig. 5.

Endogeneous PI3K/AKT activity is correlated with changes in substrate modulus. (A) VICs were cultured on stiff gels for 3 d before softening for 2 d. VICs cultured on stiff gels adopted the activated myofibroblast phenotype with striated αSMA⁺ stress fibers (green), but VICs cultured on soft gels or stiff-to-soft gels displayed reduced myofibroblast activation. Green: αSMA; blue: nuclei. (Scale bars, 100 μm.) Percent of myofibroblasts was quantified in the bar graph below. *P < 0.05. (B) Meanwhile, pAKT/AKT was significantly lower in the stiff-to-soft condition than the stiff condition. *P < 0.05. (C) To study the detailed kinetics of pAKT/AKT changes from 2 to 48 h after softening, we softened gels on day 5. Percent of myofibroblasts was not significantly reduced at either 2 or 6 h, but was significantly reduced at 48 h after softening. In contrast, pAKT/AKT was reduced as early as 2 h after gel softening and maintained at low levels until 48 h (D). *P < 0.05. (E) However, myofibroblasts were not effectively de-activated after VICs have been cultured on stiff gels for 7 d and then softened. *P < 0.05.

Constitutively Active PI3K Promotes Nodule Formation.

We then asked whether constitutively active PI3K (caPI3K) is sufficient to drive myofibroblast activation on soft substrates. When VICs were infected with caPI3K adenoviruses, these cells had higher pAKT/AKT levels and higher expression of αSMA than the GFP-infected cells (Fig. 6A). Furthermore, VICs infected with caPI3K formed significantly more nodules on soft gels (Fig. 6B) and TCPS (Fig. S7) than those infected with GFP. These nodules stained positive for αSMA, but quantifying stress fibers was difficult when cells formed nodules. On both soft gels and TCPS, we tracked cellular movement after viral infection in real time and found that nodules were induced by caPI3K through contraction of VICs (Movies S1–S4) and may eventually lead to matrix calcification (Fig. S7). In Fig. 6C, a contraction event that led to nodule formation (white arrow) was recorded for VICs infected with caPI3K on soft gels.

Fig. 6.

Constitutively active PI3K promotes contraction-mediated nodule formation by VICs on soft substrates. (A) caPI3K increased phosphorylation of AKT, one of the main downstream effectors of PI3K. Similarly, the αSMA protein level was increased by caPI3K overexpression. (B) Four days after viral infection, increased nodule formation was observed for VICs treated with caPI3K but cultured on soft gels. Cell nodules induced by caPI3K stained positive for αSMA. *P < 0.05. (Scale bars, 100 µm.) (C) Real-time tracking of nodule formation in caPI3K-infected VICs revealed that nodule formation was mediated by cell contraction. White arrows highlight a contraction event (Movie S2).

Discussion

Studies of the cellular microenvironment have broadened our understanding of how various extracellular cues regulate cell function at the molecular level. In this study, we have begun to elucidate how the physical cue of matrix elasticity regulates the fibroblast-myofibroblast transition. We find that stiff TCPS (E, ∼3 GPa) spontaneously activates the pathogenic myofibroblast phenotypes, whereas soft hydrogels (E, ∼7 kPa) inhibit these traits. To understand how soft hydrogels preserve the unactivated fibroblast phenotype of VICs, we analyzed microarray data and found that the PI3K/AKT pathway is activated in VICs cultured on TCPS and is necessary for myofibroblast activation on stiff substrates. Inhibition of PI3K with LY294002 prevents myofibroblast differentiation and contraction-mediated nodule formation on both stiff gels and TCPS. Inhibition of AKT also inhibits some myofibroblast phenotypes. We also observed that, as myofibroblasts de-activate in response to reducing substrate modulus in situ, endogenous pAKT is decreased as early as 2 h after gel softening and continues to decrease over a time course of 48 h. Overexpression of caPI3K leads to increased αSMA protein expression and can drive myofibroblast-mediated nodule formation on soft hydrogels and TCPS. These data suggest a unique link between substrate modulus and intracellular biochemical signaling through the PI3K pathway and indicate that PI3K inhibition may be used to prevent valve fibrosis, wherein the stiffened tissue reinforces myofibroblast activation.

TCPS has been the traditional substrate for mammalian cell culture because of its commercial availability, ease of use, and support for basic cell survival and function. However, TCPS culture significantly alters the whole genome transcriptional profile of primary porcine aortic VICs and regulates genes of different functional categories (Fig. 1). The functional annotation analysis in Fig. 1C helps us narrow down the major cellular functions for further study, and we discuss the implications in the SI Materials and Methods. In contrast, soft PEG hydrogels (E, ∼7 kPa) preserve aspects of the native phenotype of VICs much better than TCPS (Fig. 2). The data suggest that the substrate environment may have an overriding effect beyond that of the media in controlling fibroblast gene activation and demonstrate that these soft gels do not fully mimic the native scaffold, because TIMP3 expression was not preserved. However, these experiments highlight the importance of the cellular context to VIC phenotype and motivate the fabrication of material microenvironments that better recapitulate the quiescent valve niche. For example, the design of PEG hydrogels might be improved by incorporating binding epitopes as presented in the native niche, including protein ligands, integrin binding sequences, and proteoglycans, or by encapsulating VICs in 3D culture. Nevertheless, these data support that soft hydrogels can serve as a basic substrate for propagating unactivated VICs for in vitro studies and may be applicable to quiescent fibroblasts from other tissues.

Although the mechanoresponsive nature of fibroblasts from the valve, liver, and lung has been elucidated previously (1, 4, 8, 22, 23), the manner by which these mechanical signals are integrated in cell signaling to direct cellular phenotype is not completely understood. Based on bioinformatic analysis and biochemical experiments, we discovered that the PI3K/AKT pathway is associated with VIC activation on stiff substrates. Our drug-treatment study indicates that AKT could be a main effector protein downstream of PI3K in mediating myofibroblast activation on stiff substrates (Fig. 4 and Figs. S4 and S5). Consistently, Conte et al. have shown that LY294002 can also inhibit myofibroblast differentiation of human lung fibroblasts on TCPS (24). The PI3K/AKT pathway is not only necessary for myofibroblast activation on stiff substrates; endogenous AKT activation is correlated with substrate modulus reduction and cell phenotypes. The pAKT/AKT level is increased when P0 VICs are cultured on TCPS (Fig. 3B) and decreased as early as 2 h after gel softening, which happens before decreases in αSMA⁺ stress fibers (Fig. 5 C and D). The time-course experiments suggest a potential direct link between de-activation of AKT and reduction in substrate modulus. However, we did not observe a significant difference in pAKT/AKT between stiff gels and static soft gels (Fig. 5B). We hypothesize that pAKT/AKT may be responding to dynamic elasticity changes in a time-dependent manner. It is possible that, as the cells have been cultured on static soft gels for 5 d, they adapt to their matrix environment and show a sustained level of pAKT/AKT. Because our photodegradable-PEG hydrogels have been extensively characterized to ensure that light-mediated degradation does not lead to significant changes in ligand density or substrate topography (9, 25–27), VICs should be primarily sensing the reduction in substrate modulus.

PI3K is a phospholipid kinase that specifically phosphorylates the 3-OH group of the inositol ring and generates secondary messengers, such as phosphatidylinositol (3-5)-trisphosphate (PIP3) that is bound to the inner plasma membrane (28). Downstream effector proteins then bind to PIP3 through the pleckstrin-homology (PH) domain and activate signaling events regulating actin polymerization, cell growth, cell cycle, and apoptosis, among other functions (21). Interestingly, the PI3K pathway has been shown to be mechano-sensitive in muscle tissues. Elevated AKT phosphorylation has been observed in rat skeletal muscles in response to exercise or passive stretch and in the mouse diaphragm in response to tissue stretch and aging (29). In addition, cardiac-specific overexpression of active AKT has been shown to enhance myocardial contractility (30). Our study shows that PI3K in fibroblasts is mechano-responsive to elasticity changes of culture substrates. When softening gels from ∼32 kPa to ∼7 kPa, there was reduced PI3K activity measured by pAKT/AKT, indicating that PI3K can respond bidirectionally to mechanical forces (i.e., in response to substrate modulus reduction or increased mechanical load during muscle stretch and contraction).

Interestingly, we also observed that overexpressing caPI3K can overcome some effects of soft substrates and promote myofibroblast phenotypes on soft gels (Fig. 6 A and B). It has been shown that cell nodules form via contraction in an αSMA-dependent manner when VICs are cultured on TCPS (31), and blebbistatin, which inhibits myosin II-mediated actomyosin contraction, significantly blocks myofibroblast differerntiation (Fig. S2). We observed similar patterns of nodule formation for VICs infected with caPI3K adenoviruses on soft gels and TCPS (Movies S2 and S4). This finding indicates that caPI3K may have increased αSMA-mediated contractility of VICs. Reif et al. have shown that activation of PI3K promotes Rac- and Rho-dependent stress fiber formation, focal-adhesion development and membrane ruffling in Swiss 3T3 cells (32). It is possible that PI3K may activate small GTPases or other myosin-related proteins in these fibroblasts to increase their contractility and tension with their substrates. These data point to a potential role of the PI3K/AKT pathway in transmitting external biomechanical cues of elasticity to intracellular cytoskeleton remodeling, gene regulation, and cell fate determination.

One intriguing question is how VICs mechano-sense the matrix elasticity changes and transmit that into PI3K/AKT signaling. Substrate modulus could be acting through multiple mechano-sensitive cell surface proteins and converge on the downstream PI3K signaling (Fig. S8). For example, focal adhesions are the main connection points between cells and their matrix, and serve as sensors to various mechanical cues (33, 34). Various proteins in the focal-adhesion machinery could affect PI3K activation, such as integrins (35) and focal-adhesion kinase (36). Furthermore, a large screening study has shown that siRNA-mediated knockdown of 85 human protein tyrosine kinases induced distinct alterations in the focal-adhesion morphology on different stiffnesses (37), and tyrosine phosphorylation levels are linked to mechanically induced changes controlling different cellular functions (38). As protein tyrosine kinases are common upstream receptors which activate PI3K (39), it is possible that their regulation, which is linked with focal-adhesion formation, could affect PI3K/AKT phosphorylation. Another important group of mechano-senstive proteins are mechano-sensitive ion channels. Our preliminary data support that they may be involved in the elasticity-regulated myofibroblast activation, as treatment with their inhibitor reduced VIC myofibroblast differentiation on TCPS (Fig. S8). For example, the TRPC family cation channels can be induced by plasma membrane deformation (40) and lead to Ca²⁺ influx into the cells, which can then affect a number of signaling events intracellularly, including PI3K (41). This result supports some basic hypotheses about potential upstream mechano-sensing events and provides a future candidate to explore as the field seeks to better understand the continuum of mechanical signaling from the ECM to the nucleus, especially related to the upstream mechano-sensing machinery in VICs.

During normal wound healing of mesenchymal tissues, the fibroblast-to-myofibroblast transition is an essential process, because myofibroblasts contract and secrete ECM proteins to repair the tissue. Toward the end of healing, apoptosis of myofibroblasts has been observed to put a brake on active tissue remodeling (42, 43). When activated cells fail to undergo apoptosis, chronic fibrosis can occur. Therefore, temporal regulation of myofibroblast apoptosis and survival is crucial in determining healthy and diseased tissue regeneration. PI3K/AKT signaling antagonizes apoptosis and promotes cell survival and proliferation. This process is largely mediated by the effector protein AKT, which inhibits apoptosis by directly phosphorylating BAD and Forkhead transcription factors (44). Two independent reports have shown that AKT activation confers an apoptosis-resistant phenotype to myofibroblasts (45, 46). In fibrotic tissues, elevated AKT activity in myofibroblasts, induced either by stiffened matrix or proinflammatory cytokines, could be an important cause of persistent activation. Therefore, targeting the PI3K/AKT pathway may accelerate the turnover of myofibroblasts through apoptosis.

Unlike a decade ago, tissue engineering and regenerative medicine have not been restricted to the laboratory, but have been implemented in the clinic (47). It is realized that the scaffold does not need to completely recapitulate the native tissue structures and functionalities before implantation, but can be instructive because cells and scaffolds are plastic and mutually responsive. However, important properties of these cell-culture scaffolds can be informed by basic cell biology research. For example, the elasticity of the materials can control key cell signaling events and it may be important for the elasticity to match the changing environment in vivo (48, 49). Over the years, PEG hydrogels have been studied to incorporate different functional and dynamic modules to serve as a better surrogate scaffold for cell culture and tissue regeneration (50). Even though one can change intracellular signaling by chemical cues and transcription factors easily, it is still necessary to learn how matrix mechanical cues can be used to regulate cellular functions to understand basic cell biology and for clinical applications. This outside-in programming may reveal unique manners to intervene during disease progression.

Materials and Methods

Primary VICs were harvested from porcine aortic valves based on a sequential collagenase digestion. These cells were cultured either on TCPS or PEG hydrogels, which were manufactured as previously published (8). Hydrogels were made with two different moduli, ∼7 kPa (soft) and ∼32 kPa (stiff), or degraded in situ from ∼32 kPa to ∼7 kPa (stiff-to-soft) over time. Cells cultured on different substrates were then fixed for immunocytochemistry analysis or lysed to collect RNA or protein for gene expression quantification based on real-time PCR and microarrays or protein analysis based on Western blot. Porcine genome microarrays (Affymetrix) were performed based on standard hybridization methods and analyzed using multiple bioinformatics tools. Experimental data were based on at least three biological repeats and analyzed using a one-way ANOVA statistical test. Please refer to SI Materials and Methods for detailed methods.