Igor Titushkin

Regulation of Cell Cytoskeleton and Membrane Mechanics by Electric Field: Role of Linker Proteins

Cellular mechanics is known to play an important role in the cell homeostasis including proliferation, motility, and differentiation. Significant variation in the mechanical properties between different cell types suggests that control of the cell metabolism is feasible through manipulation of the cell mechanical parameters using external physical stimuli. We investigated the electrocoupling mechanisms of cellular biomechanics modulation by an electrical stimulation in two mechanically distinct cell types--human mesenchymal stem cells and osteoblasts. Application of a 2 V/cm direct current electric field resulted in approximately a twofold decrease in the cell elasticity and depleted intracellular ATP. Reduction in the ATP level led to inhibition of the linker proteins that are known to physically couple the cell membrane and cytoskeleton. The membrane separation from the cytoskeleton was confirmed by up to a twofold increase in the membrane tether length that was extracted from the cell membrane after an electrical stimulation. In comparison to human mesenchymal stem cells, the membrane-cytoskeleton attachment in osteoblasts was much stronger but, in response to the same electrical stimulation, the membrane detachment from the cytoskeleton was found to be more pronounced. The observed effects mediated by an electric field are cell type- and serum-dependent and can potentially be used for electrically assisted cell manipulation. An in-depth understanding and control of the mechanisms to regulate cell mechanics by external physical stimulus (e.g., electric field) may have great implications for stem cell-based tissue engineering and regenerative medicine.

oxLDL-induced decrease in lipid order of membrane domains is inversely correlated with endothelial stiffness and network formation

Oxidized low-density lipoprotein (oxLDL) is a major factor in development of atherosclerosis. Our earlier studies have shown that exposure of endothelial cells (EC) to oxLDL increases EC stiffness, facilitates the ability of the cells to generate force, and facilitates EC network formation in three-dimensional collagen gels. In this study, we show that oxLDL induces a decrease in lipid order of membrane domains and that this effect is inversely correlated with endothelial stiffness, contractility, and network formation. Local lipid packing of cell membrane domains was assessed by Laurdan two-photon imaging, endothelial stiffness was assessed by measuring cellular elastic modulus using atomic force microscopy, cell contractility was estimated by measuring the ability of the cells to contract collagen gels, and EC angiogenic potential was estimated by visualizing endothelial networks within the same gels. The impact of oxLDL on endothelial biomechanics and network formation is fully reversed by supplying the cells with a surplus of cholesterol. Furthermore, exposing the cells to 7-keto-cholesterol, a major oxysterol component of oxLDL, or to another cholesterol analog, androstenol, also results in disruption of lipid order of membrane domains and an increase in cell stiffness. On the basis of these observations, we suggest that disruption of lipid packing of cholesterol-rich membrane domains plays a key role in oxLDL-induced changes in endothelial biomechanics.

Physicochemical control of adult stem cell differentiation: shedding light on potential molecular mechanisms.

Realization of the exciting potential for stem-cell-based biomedical and therapeutic applications, including tissue engineering, requires an understanding of the cell-cell and cell-environment interactions. To this end, recent efforts have been focused on the manipulation of adult stem cell differentiation using inductive soluble factors, designing suitable mechanical environments, and applying noninvasive physical forces. Although each of these different approaches has been successfully applied to regulate stem cell differentiation, it would be of great interest and importance to integrate and optimally combine a few or all of the physicochemical differentiation cues to induce synergistic stem cell differentiation. Furthermore, elucidation of molecular mechanisms that mediate the effects of multiple differentiation cues will enable the researcher to better manipulate stem cell behavior and response.

Mode- and cell-type dependent calcium responses induced by electrical stimulus

Intracellular calcium ion concentration ([Ca/sup 2+/]/sub i/) is known to affect numerous molecular signaling events. Regulation of the [Ca/sup 2+/]/sub i/ could therefore be used to control important cellular and molecular responses, including cell functionality, survival, differentiation, and proliferation. Because changes in the membrane electrical potential (MEP) activate plasma membrane Ca/sup 2+/ influx pathways and increase the [Ca/sup 2+/]/sub i/ level, electrical stimulus (ES) has been used to induce such MEP changes. It now appears that while large ES can directly activate voltage-gated Ca/sup 2+/ channels (VGCCs) by depolarizing the cell membrane, smaller and noninvasive ES could also be applied to activate the Ca/sup 2+/ entry pathways. Elucidation of the electrocoupling mechanisms that control [Ca/sup 2+/]/sub i/ increase is complicated by the observations that not only the mode (e.g., dc or oscillatory) of ES application but also the cell type (e.g., excitable or nonexcitable) regulates calcium responses. The aim of this review article is therefore to provide a comparative description for ES mode- and cell type dependent changes in [Ca/sup 2+/]/sub i/.

Controlling cellular biomechanics of human mesenchymal stem cells

The therapeutic efficacy of human mesenchymal stem cells (hMSCs) depends on proper characterization and control of their unique biological, mechanical and physicochemical properties. For example, cellular biomechanics and environmental mechanical cues have been shown to critically influence cell commitment to a particular lineage. We characterized biomechanical properties of hMSCs including cytoskeleton elasticity and plasma membrane/cytoskeleton coupling. As expected, during osteogenic differentiation of hMSCs, the cellular biomechanics is remodeled, and such remodeling precedes up-regulation of the osteogenic markers. Further, application of an electrical stimulation modulates the cellular biomechanics and therefore may be used to facilitate stem cell differentiation for stem cell-based tissue engineering.

Control of adipogenesis by ezrin, radixin and moesin-dependent biomechanics remodeling

We have recently shown that altered stem cell biomechanics can regulate the lineage commitment through a family of the membrane-cytoskeleton linker proteins (ERM; ezrin, radixin, moesin). The ERM proteins not only modulate the cell stiffness and actin cytoskeleton organization, but also rearrange focal adhesions and therefore influence the biochemically-directed stem cell differentiation. Combining silencing RNA, atomic force microscopy, and fluorescence microscopy, the role of the ERM proteins involved in the regulation of stem cell biomechanics and adipogenic differentiation was quantitatively determined. Transient ERM knockdown by RNAi caused disassembly of actin stress fibers and focal adhesions and a decrease in the cell stiffness. The silencing RNA treatment not only induced mechanical changes in stem cells but impaired adipogenesis in a time-dependent manner. While siRNA ERM treatment at day 0 substantially interfered with adipogenesis, the same treatment at day 3 of adipogenic differentiation significantly facilitated adipogenesis, as assessed by the expression of adipocyte-specific markers. The intact biomechanics homeostasis appears to be critical for the adipogenic induction. These findings may lead to potential biomechanical intervention techniques and methodologies to control the fate and extent of adipogenesis that would likely be involved in stem cell-based therapeutics for soft tissue repair and regeneration.

Endothelial invasive response in a co-culture model with physically-induced osteodifferentiation

Manipulation of stem cells using physicochemical stimuli has emerged as an important tool in regenerative medicine. While 2D substrates with tunable elasticity have been studied for control of stem cell differentiation, we recently developed a stratified co‐culture model of angiogenesis of human mesenchymal stem cells (hMSCs) that differentiate on a tunable polydimethylsiloxane (PDMS) substrate, thereby creating a physiologic context for elasticity‐induced differentiation. Endothelial cells (EC) were cultured on top of the hMSC construct on a collagen gel to monitor network formation. Media composition influenced EC invasion due to the conditioning media, the reduction of serum and supplemental growth factors, and the addition of recombinant growth factors. Conditioned media, recombinant growth factors and direct co‐culture were compared for endothelial cell invasive response using quantitative image analysis. As anticipated, use of recombinant vascular endothelial growth factor (VEGF) induced the deepest EC invasions while direct co‐culture caused shallow invasions compared to other conditions. However, endothelial cells displayed lumen‐like morphology, suggesting that cell‐cell interaction in the co‐culture model could mimic sprouting behaviour. In summary, an engineered suitable biochemical and physical environment facilitated endothelial cells to form 3D vessel structures onto hMSCs. These structures were plated on a stiff surface known to induce osteodifferentiation of stem cells. This low cost co‐culture system, with its minimal chemical supplementation and physically controllable matrix, could potentially model in vivo potential in engineered and pre‐vascularized bone grafts. Copyright

Structure and Biology of the Cellular Environment: The Extracellular Matrix

The extracellular matrix (ECM) represents a complex organization of macromolecules that surrounds the cell and comprises the substratum onto which the cell may be attached. The properties and functions of the ECM depend ultimately on its structure, molecular components, architecture, and dynamic modulation. Because the critical role of ECM involved in cell biology and physiology has long been recognized, the structure and biology of the ECM have been extensively studied (Yurchenco and Birk 1994; Ayad et al. 1998; Robert 2001). The diversity found in the structure and organization of the ECM appears to be tissue specific and regulates the properties and function of each tissue.

Oxldl-Induced Decrease in Lipid Order of Membrane Domains is Inversely Correlated with Endothelial Stiffness and Network Formation

Oxidized Low Density Lipoprotein (OxLDL) is a major factor in development of atherosclerosis. Our earlier studies have shown that exposure of endothelial cells (EC) to oxLDL increases EC stiffness, facilitates the ability of the cells to generate force and facilitates EC networks formation in 3D collagen gels. In this study, we show that oxLDL induces a decrease in lipid order of membrane domains and that this effect is inversely correlated with endothelial stiffness, contractility and network formation. Local lipid packing of cell membrane domains is assessed by Laurdan two-photon imaging, endothelial stiffness was assessed by measuring cellular elastic modulus using Atomic Force Microscopy (AFM), cell contractility was estimated by measuring the ability of the cells to contract collagen gels and EC angiogenic potential was estimated by visualizing endothelial networks within the same gels. Furthermore, we also show that the impact of oxLDL on endothelial biomechanics and network formation is fully reversed by supplying the cells with a surplus of cholesterol suggesting that changes in membrane cholesterol underlie oxLDL-induced effects on endothelial biomechanics. In contrast, exposure to sphingomyelinase C (SMase C) has no effect on endothelial stiffness and network formation, indicating that hydrolysis of sphingomyelin cannot be responsible for these effects. Based on these observations, we suggest that disruption of lipid packing of cholesterol-rich membrane domains plays a key role in oxLDL-induced changes in endothelial biomechanics.