Interactive role of tyrosine kinase,protein kinase C,and Rho/Rho kinase systems in the mechanotransduction of vascular smooth muscles

Blood vessels are always subjected to hemodynamic stresses including blood pressure and blood flow. The cerebral artery is particularly sensitive to hemodynamic stresses such as pressure and stretch, and shows contractions that are myogenic in nature; i.e., the mechanical response is generated by the vascular smooth muscle itself. The artery constricts in response to an increase in intraluminal pressure, and dilates in response to a decrease in the intraluminal pressure. We provide herein some insights into the mechanotransduction of vascular tissue; i.e., we discuss how the tissue is receptive to mechanical force and how the latter induces the specific signals leading to myogenic contraction in terms of mechanosensor action and subsequent intracellular signaling. The interactive role of tyrosine kinase, protein kinase C, and Rho/Rho-kinase systems in the mechanotransduction process is discussed, which systems also seem to play an important role in the development of experimental cerebral vasospasm. The study of the mechanotransduction in vascular tissue may aid in clarifying the mechanisms underlying vasospastic episodes and pathologic remodeling in cardiovascular diseases, and may potentially have therapeutic consequences.     

Protein kinases as mediators of fluid shear stress stimulated signal transduction in endothelial cells: A hypothesis for calcium-dependent and calcium-independent events activated by flow

Fluid shear stress regulates endothelial cell function, but the signal transduction mechanisms involved in mechanotransduction remain unclear. Recent findings demonstrate that several intracellular kinases are activated by mechanical fórces. In particular, members of the mitogen-activated protein (MAP) kinase family are stimulated by hyperosmolárity, stretch, and stress such as heat shock. We propose a model for mechanotransduction in endothelial cells involving calcium-dependent and calcium-independent protein kinase pathways. The calcium-dependent pathway involves activation of phospholipase C, hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP₂), increases in intracellular calcium and stimulation of kinases such as calcium-calmodulin and C kinases (PKC). The calcium-independent pathway involves activation of a small GTP-binding protein and stimulation of calcium-independent PKC and MAP kinases. The calcium-dependent pathway mediates the rapid, transient response to fluid shear stress including activation of nitric oxide synthase (NOS) and ion transport. In contrast, the calcium-independent pathway mediates a slower response including the sustained activation of NOS and changes in cell morphology and gene expression. We propose that focal adhesion complexes link the calcium-dependent and calcium-independent pathways by regulating activity of phosphatidylinositol 4-phosphate (PIP) 5-kinase (which regulates PIP₂ levels0 and p125 focal adhesion kinase (FAK, which phosphorylates paxillin and interacts with cytoskeletal proteins). This model predicts that dynamic interactions between integrin molecules present in focal adhesion complexes and membrane events involved in mechanotransduction will be integrated by calcium-dependent and calcium-independent kinases to generate intracellular signals involved in the endothelial cell response to flow.     

Interstitial fluid flow in the osteon with spatial gradients of mechanical properties: a finite element study

Bone remodelling is the process that maintains bone structure and strength through adaptation of bone tissue mechanical properties to applied loads. Bone can be modelled as a porous deformable material whose pores are filled with cells, organic material and interstitial fluid. Fluid flow is believed to play a role in the mechanotransduction of signals for bone remodelling. In this work, an osteon, the elementary unit of cortical bone, is idealized as a hollow cylinder made of a deformable porous matrix saturated with an interstitial fluid. We use Biot’s poroelasticity theory to model the mechanical behaviour of bone tissue taking into account transverse isotropic mechanical properties. A finite element poroelastic model is developed in the COMSOL Multiphysics software. Elasticity equations and Darcy’s law are implemented in this software; they are coupled through the introduction of an interaction term to obtain poroelasticity equations. Using numerical simulations, the investigation of the effect of spatial gradients of permeability or Poisson’s ratio is performed. Results are discussed for their implication on fluid flow in osteons: (i) a permeability gradient affects more the fluid pressure than the velocity profile; (ii) focusing on the fluid flow, the key element of loading is the strain rate; (iii) a Poisson’s ratio gradient affects both fluid pressure and fluid velocity. The influence of textural and mechanical properties of bone on mechanotransduction signals for bone remodelling is also discussed.     

Altered PKC expression and phosphorylation in response to the nature, direction, and magnitude of mechanical stretch

Protein kinase C (PKC) isozymes have been shown to play a role in mechanotransduction in a variety of cell types. We sought to identify the PKC isozymes involved in transducing mechanical (cyclic vs. static), direction and intensity of stretch by examining changes in protein expression and phosphorylation. We used a 3-dimensional culture system with aligned neonatal rat cardiac myocytes on silastic membranes. Myocytes were subjected to either cyclic stretch at 5 cycles/min or static stretch for a period of 24 h at intensities of 0%, 2.5%, 5%, or 10% of full membrane length. Stretch was applied in perpendicular or parallel directions to myocyte alignment. PKC delta was most sensitive to stretch applied perpendicular to myocyte alignment regardless of the nature of stretch, while phospho PKC delta T505 increased in response to static-perpendicular stretch. PKC epsilon expression was altered by cyclic stretch but not static stretch, while phospho PKC epsilon S719 remained unchanged. PKC alpha expression was not altered by stretch; however, phospho PKC alpha S657 increased in a dose-dependent manner following cyclic-perpendicular stretch. Our results indicate that changes in PKC expression and phosphorylation state may be a mechanism for cardiac myocytes to discriminate between the nature, direction, and intensity of mechanical stretch.     

Contribution of the cytoskeleton and the phospholipase C signaling pathway to fluid stream-induced membrane currents

A fluid stream induced by a concentration clamp system evokes in Xenopus oocytes a deformation of the membrane which results in transient chloride currents of high amplitude (stream-evoked inward current, I(i,st)) during calcium-activated chloride current oscillations. The involvement of cytoskeleton elements and of components of the phospholipase C-dependent signaling pathway on the generation of the I(i,st) were investigated. Incubation of the oocytes with cytoskeleton-disrupting agents exerted no effects on generation of the I(i,st), suggesting that the mechanotransduction is not mediated by these structures. The fluid stream induced an elevation of the submembraneous calcium concentration, as measured by an increase of Fluo-4-mediated fluorescence after the stimulus. Lowering the intracellular calcium concentration by injection of calcium chelators or depleting inositol 1,4,5-triphosphate (InsP(3))-sensitive calcium stores by blockers of the calcium pumps suppressed the generation of the I(i,st) in most cases. Furthermore, the phospholipase C inhibitor U73122 reversibly blocked the I(i,st). The results suggest that the fluid stream leads to a membrane stretch which modulates directly or indirectly the activity of a membrane-bound phospholipase C. The phospholipase C transiently elevates the InsP(3) concentration, in turn releasing calcium from InsP(3)-sensitive internal calcium stores, thus evoking an enhanced calcium-sensitive chloride current.     

Cyclic stretch induces reorientation of cells in a Src family kinase- and p130Cas-dependent manner

Recognition of external mechanical signals by cells is an essential process for life. One important mechanical signal experienced by various cell types, e.g. around blood vessels, within the lung epithelia or around the intestine, is cyclic stretch. As a response, many cell types reorient their actin cytoskeleton and main cell axis almost perpendicular to the direction of stretch. Despite the vital necessity of cellular adaptation to cyclic stretch, the underlying mechanosensory signal cascades are far from being understood. Here we show an important function of Src-family kinase activity in cellular reorientation upon cyclic stretch. Deletion of all three family members, namely c-Src, Yes and Fyn (SYF), results in a strongly impaired cell reorientation of mouse embryonic fibroblasts with an only incomplete reorientation upon expression of c-Src. We further demonstrate that this reorientation phenotype of SYF-depleted cells is not caused by affected protein exchange dynamics within focal adhesions or altered cell force generation. Instead, Src-family kinases regulate the reorientation in a mechanotransduction-dependent manner, since knock-down and knock-out of p130Cas, a putative stretch sensor known to be phosphorylated by Src-family kinases, also reduce cellular reorientation upon cyclic stretch. This impaired reorientation is identical in intensity upon mutating stretch-sensitive tyrosines of p130Cas only. These statistically highly significant data pinpoint early events in a Src family kinase- and p130Cas-dependent mechanosensory/mechanotransduction pathway.     

Design of a Novel Equi-Biaxial Stretcher for Live Cellular and Subcellular Imaging

Cells in the body experience various mechanical stimuli that are often essential to proper cell function. In order to study the effects of mechanical stretch on cell function, several devices have been built to deliver cyclic stretch to cells; however, they are generally not practical for live cell imaging. We introduce a novel device that allows for live cell imaging, using either an upright or inverted microscope, during the delivery of cyclic stretch, which can vary in amplitude and frequency. The device delivers equi-biaxial strain to cells seeded on an elastic membrane via indentation of the membrane. Membrane area strain was calibrated to indenter depth and the device showed repeatable and accurate delivery of strain at the scale of individual cells. At the whole cell level, changes in intracellular calcium were measured at different membrane area strains, and showed an amplitude-dependent response. At the subcellular level, the mitochondrial network was imaged at increasing membrane area strains to demonstrate that stretch can lead to mitochondrial fission in lung fibroblasts. The device is a useful tool for studying transient as well as long-term mechanotransduction as it allows for simultaneous stretching and imaging of live cells in the presence of various chemical stimuli.     

Functional coupling of ion channels in cellular mechanotransduction

The major players in the processes of cellular mechanotransduction are considered to be mechanosensitive (MS) or mechano-gated ion channels. Non-selective Ca²⁺-permeable channels, whose activity is directly controlled by membrane stretch (stretch-activated channels, SACs) are ubiquitously present in mammalian cells of different origin. Ca²⁺ entry mediated by SACs presumably has a significant impact on various Ca²⁺-dependent intracellular and membrane processes. It was proposed that SACs could play a crucial role in the different cellular reactions and pathologies, including oncotransformation, increased metastatic activity and invasion of malignant cells. In the present work, coupling of ion channels in transformed fibroblasts in course of stretch activation was explored with the use of patch-clamp technique. The combination of cell-attached and inside-out single-current experiments showed that Ca²⁺ influx via SACs triggered the activity of Ca²⁺-sensitive K⁺ channels indicating functional compartmentalization of different channel types in plasma membrane. Importantly, the analysis of single channel behavior demonstrated that K⁺ currents could be activated by the rise of intracellular calcium but displayed no direct mechanosensitivity. Taken together, our data imply that local changes in Ca²⁺ concentration due to SAC activity may provide a functional link between various Ca²⁺-dependent molecules in the processes of cellular mechanotransduction.     

Pharmacological investigation of the bioluminescence signaling pathway of the dinoflagellate Lingulodinium polyedrum: evidence for the role of stretch‐activated ion channels

Dinoflagellate bioluminescence serves as a whole‐cell reporter of mechanical stress, which activates a signaling pathway that appears to involve the opening of voltage‐sensitive ion channels and release of calcium from intracellular stores. However, little else is known about the initial signaling events that facilitate the transduction of mechanical stimuli. In the present study using the red tide dinoflagellate Lingulodinium polyedrum (Stein) Dodge, two forms of dinoflagellate bioluminescence, mechanically stimulated and spontaneous flashes, were used as reporter systems to pharmacological treatments that targeted various predicted signaling events at the plasma membrane level of the signaling pathway. Pretreatment with 200 μM Gadolinium III (Gd³⁺), a nonspecific blocker of stretch‐activated and some voltage‐gated ion channels, resulted in strong inhibition of both forms of bioluminescence. Pretreatment with 50 μM nifedipine, an inhibitor of L‐type voltage‐gated Ca²⁺ channels that inhibits mechanically stimulated bioluminescence, did not inhibit spontaneous bioluminescence. Treatment with 1 mM benzyl alcohol, a membrane fluidizer, was very effective in stimulating bioluminescence. Benzyl alcohol‐stimulated bioluminescence was inhibited by Gd³⁺ but not by nifedipine, suggesting that its role is through stretch activation via a change in plasma membrane fluidity. These results are consistent with the presence of stretch‐activated and voltage‐gated ion channels in the bioluminescence mechanotransduction signaling pathway, with spontaneous flashing associated with a stretch‐activated component at the plasma membrane.     

Changes in membrane properties during in-vitro meiotic maturation of the limpet Patella vulgata

Changes in the membrane properties of the oocyte of the mollusk, Patella vulgata, were analyzed following the induction of meiosis reinitiation by paleopedial ganglia extract or by the weak base ammonia. During maturation it was possible to distinguish between an early phase characterized by an initial hyperpolarization and a late phase consisting of a depolarization which triggers an action potential with a long-term overshoot (20 minutes) of the membrane potential. Major changes in individual ionic permeabilities were studied using both current and voltage clamp conditions. The depolarizing phase appears to depend on decreases in K+ membrane permeability. Finally we observed that the overshoot did not appear to be directly related to germinal vesicle breakdown (GVBD) since it was absent in Na-deprived artificial sea water and could be elicited in the presence of TEA bromide, which did not induce maturation. This last observation suggests that it may result from a change in specific K+ ion permeability due to the possible activation of stretch channels.     

Diacylglycerols as activators of protein kinase C

Diacylglycerols are generated in the membrane as the result of extracellular signals and are able to stimulate the activity of protein kinase C, acting as membrane second messengers. Diacylglycerols are recognized by protein kinases C through the C1 domain and established models propose that they will stabilize the translocation of the protein to the membrane. However, diacylglycerols also act by modulating the physical properties of the membrane, thus favouring the translocation of the enzyme. This is done through alteration of the membrane surface curvature, dehydration of the surface and the separation of phospholipid surface groups. Good correlations have been observed between the physical state of the membrane and protein kinase C activity.     

Cyclic stretch downregulates arterial vascular connexin43 protein expression: an ex vivo study

Vascular cells may communicate through gap junctions that are formed by connexin (Cx) proteins. We investigated differential regulation of arterial gap junctions by steady and cyclic stretch and the underlying mechanotransduction pathways. Ex vivo culture of rabbit thoracic aortas was used to investigate regulation of Cx43 by cyclic stretch. After culturing for 6 or 24 h, Cx43 protein levels were quantified using Western blot. Cultures under a pulsatile pressure (mean 80 mmHg, pulse 30 mmHg) decreased Cx43 protein at both 6 and 24 h as compared with cultures under a steady pressure (80 mmHg). The regulation of Cx43 protein was mediated by pulsatile pressure-induced cyclic stretch, not by cyclic stress. Protein levels of active and total Src were also decreased by cyclic stretch at 24 h. The Src- specific inhibitor PP1 in steady culture only or in both steady and pulsatile culture conditions eliminated the difference in Cx43 protein levels between the two culture conditions. Addition of reactive oxygen species inhibitor apocynin to the pulsatile culture abolished the differences in Src and Cx43 protein levels between the two cultures. Thus, Src and reactive oxygen species appear to play a role in cyclic stretch-mediated regulation of Cx43 protein. These results are likely to have important implications in cardiovascular physiology and pathophysiology under conditions wherein significant alterations in the level of cyclic stretch are present.     

Muscle spindle autogenetic inhibition in the extraocular muscles of lamb

The role of extraocular muscle (EOM) proprioceptors on eye motility has been investigated in lambs on "encéphale isolé", by evaluating the tension of EOMs at various lengths and velocities of stretch before and after proprioceptive blocks. The EOM tension, in the absence of proprioceptive input, was higher than in normal conditions. Such an effect occurred at lengthening values greater than 3 mm of stretch from resting muscle length, corresponding to 18 degrees of eye deviation and was dependent on the velocity of the stretch, being more effective at high velocity. The muscle receptors responsible for this effect was determined by comparing the sensitivity to vibratory stimulation of spindles and tendon organs to the amount of inhibition provoked by the same stimulation on an EOM electromyographic activity. The tension inhibition appeared to be correlated to muscle spindle activation. Thus, the presence of muscle spindles can determine a reduction of the tension within the stretched muscles. This result suggests that the EOM length and velocity signals operate moment to moment reduction on the stiffness of the muscle which antagonizes eye displacement, thus facilitating the ocular movements.     

Diacylglycerols as activators of protein kinase C (Review)

Diacylglycerols are generated in the membrane as the result of extracellular signals and are able to stimulate the activity of protein kinase C, acting as membrane second messengers. Diacylglycerols are recognized by protein kinases C through the C1 domain and established models propose that they will stabilize the translocation of the protein to the membrane. However, diacylglycerols also act by modulating the physical properties of the membrane, thus favouring the translocation of the enzyme. This is done through alteration of the membrane surface curvature, dehydration of the surface and the separation of phospholipid surface groups. Good correlations have been observed between the physical state of the membrane and protein kinase C activity.     

Biophysical regulation of histone acetylation in mesenchymal stem cells

Histone deacetylation and acetylation are catalyzed by histone deacetylase (HDAC) and histone acetyltransferase, respectively, which play important roles in the regulation of chromatin remodeling, gene expression, and cell functions. However, whether and how biophysical cues modulate HDAC activity and histone acetylation is not well understood. Here, we tested the hypothesis that microtopographic patterning and mechanical strain on the substrate regulate nuclear shape, HDAC activity, and histone acetylation. Bone marrow mesenchymal stem cells (MSCs) were cultured on elastic membranes patterned with parallel microgrooves 10 μm wide that kept MSCs aligned along the axis of the grooves. Compared with MSCs on an unpatterned substrate, MSCs on microgrooves had elongated nuclear shape, a decrease in HDAC activity, and an increase of histone acetylation. To investigate anisotropic mechanical sensing by MSCs, cells on the elastic micropatterned membranes were subjected to static uniaxial mechanical compression or stretch in the direction parallel or perpendicular to the microgrooves. Among the four types of loads, compression or stretch perpendicular to the microgrooves caused a decrease in HDAC activity, accompanied by the increase in histone acetylation and slight changes of nuclear shape. Knocking down nuclear matrix protein lamin A/C abolished mechanical strain-induced changes in HDAC activity. These results demonstrate that micropattern and mechanical strain on the substrate can modulate nuclear shape, HDAC activity, and histone acetylation in an anisotropic manner and that nuclear matrix mediates mechanotransduction. These findings reveal a new mechanism, to our knowledge, by which extracellular biophysical signals are translated into biochemical signaling events in the nucleus, and they will have significant impact in the area of mechanobiology and mechanotransduction.     

Stretch increases alveolar epithelial permeability to uncharged micromolecules

We measured stretch-induced changes in transepithelial permeability in vitro to uncharged tracers 1.5–5.5 Å in radius to identify a critical stretch threshold associated with failure of the alveolar epithelial transport barrier. Cultured alveolar epithelial cells were subjected to a uniform cyclic (0.25 Hz) biaxial 12, 25, or 37% change in surface area (SA) for 1 h. Additional cells served as unstretched controls. Only 37% SA (100% total lung capacity) produced a significant increase in transepithelial tracer permeability, with the largest increases for bigger tracers. Using the permeability data, we modeled the epithelial permeability in each group as a population of small pores punctuated by occasional large pores. After 37% SA, increases in paracellular transport were correlated with increases in the radii of both pore populations. Inhibition of protein kinase C and tyrosine kinase activity during stretch did not affect the permeability of stretched cells. In contrast, chelating intracellular calcium and/or stabilizing F-actin during 37% SA stretch reduced but did not eliminate the stretch-induced increase in paracellular permeability. These results provide the first in vitro evidence that large magnitudes of stretch increase paracellular transport of micromolecules across the alveolar epithelium, partially mediated by intracellular signaling pathways. Our monolayer data are supported by whole lung permeability results, which also show an increase in alveolar permeability at high inflation volumes (20 ml/kg) at the same rate for both healthy and septic lungs. ventilator-induced lung injury; acute lung injury; barrier properties