Hang on tight: reprogramming the cell with microstructural cues.

Cells interact intimately with complex microdomains in their extracellular matrix (ECM) and maintain a delicate balance of mechanical forces through mechanosensitive cellular components. Tissue injury results in acute degradation of the ECM and disruption of cell-ECM contacts, manifesting in loss of cytoskeletal tension, leading to pathological cell transformation and the onset of disease. Recently, microscale hydrogel constructs have been developed to provide cells with microdomains to form focal adhesion binding sites, which enable restoration of cytoskeletal tension. These synthetic anchors can recapitulate the complex 3D architecture of the native ECM to provide microtopographical cues. The mechanical deformation of proteins at the cell surface can activate signaling cascades to modulate downstream gene-level transcription, making this a unique materials-based approach for reprogramming cell behavior. An overview of the mechanisms underlying these mechanosensitive interactions in fibroblasts, stem and other cell types is provided to review their effects on cellular reprogramming. Recent investigations on the fabrication, functionalization and implementation of these materials and microtopographical features for drug testing and therapeutic applications are discussed.

Lipid signaling affects primary fibroblast collective migration and anchorage in response to stiffness and microtopography.

Cell migration is regulated by several mechanotransduction pathways, which consist of sensing and converting mechanical microenvironmental cues to internal biochemical cellular signals, such as protein phosphorylation and lipid signaling. While there has been significant progress in understanding protein changes in the context of mechanotransduction, lipid signaling is more difficult to investigate. In this study, physical cues of stiffness (10, 100, 400 kPa, and glass), and microrod or micropost topography were manipulated in order to reprogram primary fibroblasts and assess the effects of lipid signaling on the actin cytoskeleton. In an in vitro wound closure assay, primary cardiac fibroblast migration velocity was significantly higher on soft polymeric substrata. Modulation of PIP2 availability through neomycin treatment nearly doubled migration velocity on 10 kPa substrata, with significant increases on all stiffnesses. The distance between focal adhesions and the lamellar membrane (using wortmannin treatment to increase PIP2 via PI3K inhibition) was significantly shortest compared to untreated fibroblasts grown on the same surface. PIP2 localized to the leading edge of migrating fibroblasts more prominently in neomycin-treated cells. The membrane-bound protein, lamellipodin, did not vary under any condition. Additionally, fifteen micron-high micropost topography, which blocks migration, concentrates PIP2 near to the post. Actin dynamics within stress fibers, measured by fluorescence recovery after photobleaching, was not significantly different with stiffness, microtopography, nor with drug treatment. PIP2-modulating drugs delivered from microrod structures also affected migration velocity. Thus, manipulation of the microenvironment and lipid signaling regulatory drugs might be beneficial in improving therapeutics geared toward wound healing.

Variation in stiffness regulates cardiac myocyte hypertrophy via signaling pathways.

Much diseased human myocardial tissue is fibrotic and stiff, which increases the work that the ventricular myocytes must perform to maintain cardiac output. The hypothesis tested is that the increased load due to greater stiffness of the substrata drives sarcomere assembly of cells, thus strengthening them. Neonatal rat ventricular myocytes (NRVM) were cultured on polyacrylamide or polydimethylsiloxane substrates with stiffness of 10 kPa, 100 kPa, or 400 kPa, or glass with stiffness of 61.9 GPa. Cell size increased with stiffness. Two signaling pathways were explored, phosphorylation of focal adhesion kinase (p-FAK) and lipids by phosphatidylinositol 4,5-bisphosphate (PIP2). Subcellular distributions of both were determined in the sarcomeric fraction by antibody localization, and total amounts were measured by Western or dot blotting, respectively. More p-FAK and PIP2 distributed to the sarcomeres of NRVM grown on stiffer substrates. Actin assembly involves the actin capping protein Z (CapZ). Both actin and CapZ dynamic exchange were significantly increased on stiffer substrates when assessed by fluorescence recovery after photobleaching (FRAP) of green fluorescent protein tags. Blunting of actin FRAP by FAK inhibition implicates linkage from mechano-signalling pathways to cell growth. Thus, increased stiffness of cardiac disease can be modeled with polymeric materials to understand how the microenvironment regulates cardiac hypertrophy.

Cyclic mechanical strain of myocytes modifies CapZß1 post translationally via PKCε.

The heart is exquisitely sensitive to mechanical stimuli and adapts to increased demands for work by enlarging the cardiomyocytes. In order to determine links between mechano-transduction mechanisms and hypertrophy, neonatal rat ventricular myocytes (NRVM) were subjected to physiologic strain for analysis of the dynamics of the actin capping protein, CapZ, and its post-translational modifications (PTM). CapZ binding rates were assessed after strain by fluorescence recovery after photobleaching (FRAP) of green fluorescent protein (GFP) expressed by a GFP-CapZß1 adenovirus. To assess the role of the protein kinase C epsilon isoform (PKCε), rest or cyclic strain were combined with specific PKCε activation by constitutively active PKCε, or by inhibition with dominant negative PKCε (dnPKCε) expression. Significant increases of CapZ FRAP kinetics with strain were blunted by dnPKCε, suggesting that PKCε is involved in mechano-transduction signaling. Similar combinations of strain and PKC regulation in NRVMs were studied by PTM profiles of CapZß1 using quantitative two-dimensional gel electrophoresis. The significantly increased charge on CapZ seen with mechanical strain was reversed by the addition of dnPKCε. Potential clinical relevance was confirmed in vivo by PTMs of CapZ in the failing heart of one-year old transgenic mice over-expressing PKCε. Furthermore, with strain there was significant PKCε translocation to the Z-disc and co-localization with CapZß1 or α-actinin, which was quantified on confocal images. A hypothetical model is presented proposing that one destination of the mechanotransduction signaling pathways might be for PTMs of CapZ thereby regulating actin capping and filament assembly.

PKC epsilon signaling effect on actin assembly is diminished in cardiomyocytes when challenged to additional work in a stiff microenvironment.

The stiffness of the microenvironment surrounding a cell can result in cytoskeletal remodeling, leading to altered cell function and tissue macrostructure. In this study, we tuned the stiffness of the underlying substratum on which neonatal rat cardiomyocytes were grown in culture to mimic normal (10 kPa), pathological stiffness of fibrotic myocardium (100 kPa), and a nonphysiological extreme (glass). Cardiomyocytes were then challenged by beta adrenergic stimulation through isoproterenol treatment to investigate the response to acute work demand for cells grown on surfaces of varying stiffness. In particular, the PKCɛ signaling pathway and its role in actin assembly dynamics were examined. Significant changes in contractile metrics were seen on cardiomyocytes grown on different surfaces, but all cells responded to isoproterenol treatment, eventually reaching similar time to peak tension. In contrast, the assembly rate of actin was significantly higher on stiff surfaces, so that only cells grown on soft surfaces were able to respond to acute isoproterenol treatment. Förster Resonance Energy Transfer of immunofluorescence on the cytoskeletal fraction of cardiomyocytes confirmed that the molecular interaction of PKCɛ with the actin capping protein, CapZ, was very low on soft substrata but significantly increased with isoproterenol treatment, or on stiff substrata. Therefore, the stiffness of the culture surface chosen for in vitro experiments might mask the normal signaling and affect the ability to translate basic science more effectively into human therapy.

Injectable hyaluronic acid based microrods provide local micromechanical and biochemical cues to attenuate cardiac fibrosis after myocardial infarction.

Repairing cardiac tissue after myocardial infarction (MI) is one of the most challenging goals in tissue engineering. Following ischemic injury, significant matrix remodeling and the formation of avascular scar tissue significantly impairs cell engraftment and survival in the damaged myocardium. This limits the efficacy of cell replacement therapies, demanding strategies that reduce pathological scarring to create a suitable microenvironment for healthy tissue regeneration. Here, we demonstrate the successful fabrication of discrete hyaluronic acid (HA)-based microrods to provide local biochemical and biomechanical signals to reprogram cells and attenuate cardiac fibrosis. HA microrods were produced in a range of physiological stiffness and shown to degrade in the presence of hyaluronidase. Additionally, we show that fibroblasts interact with these microrods in vitro, leading to significant changes in proliferation, collagen expression and other markers of a myofibroblast phenotype. When injected into the myocardium of an adult rat MI model, HA microrods prevented left ventricular wall thinning and improved cardiac function at 6 weeks post infarct.