Pattern formation of vascular smooth muscle cells subject to nonuniform fluid shear stress: mediation by gradient of cell density

Smooth muscle cells (SMCs) are organized in various patterns in blood vessels. Whereas straight blood vessels mainly contain circumferentially aligned SMCs, curved blood vessels are composed of axially aligned SMCs in regions with vortex blood flow. The vortex flow-dependent feature of SMC alignment suggests a role for nonuniform fluid shear stress in regulating the pattern formation of SMCs. Here, we demonstrate that, in experimental models with vascular polymer implants designed for the observation of neointima formation and SMC migration under defined fluid shear stress, nonuniform shear stress possibly plays a role in regulating the direction of SMC migration and alignment in the neointima of the vascular implant. It was found that fluid shear stress inhibited cell growth, and the presence of nonuniform shear stress influenced the distribution of total cell density and induced the formation of cell density gradients, which in turn directed SMC migration and alignment. In contrast, uniform fluid shear stress in a control model influenced neither the distribution of total cell density nor the direction of SMC migration and alignment. In both the uniform and nonuniform shear models, the gradient of total cell density was consistent with the alignment of SMCs. These observations suggest that nonuniform shear stress may regulate the pattern formation of SMCs, possibly via mediating the gradient of cell density in the neointima of vascular polymer implants.     

Pattern formation of vascular smooth muscle cells subject to nonuniform fluid shear stress: role of PDGF-beta receptor and Src

Blood vessels are subject to fluid shear stress, a hemodynamic factor that inhibits the mitogenic activities of vascular cells. The presence of nonuniform shear stress has been shown to exert graded suppression of cell proliferation and induces the formation of cell density gradients, which in turn regulate the direction of smooth muscle cell (SMC) migration and alignment. Here, we investigated the role of platelet-derived growth factor (PDGF)-beta receptor and Src in the regulation of such processes. In experimental models with vascular polymer implants, SMCs migrated from the vessel media into the neointima of the implant under defined fluid shear stress. In a nonuniform shear model, blood shear stress suppressed the expression of PDGF-beta receptor and the phosphorylation of Src in a shear level-dependent manner, resulting in the formation of mitogen gradients, which were consistent with the gradient of cell density as well as the alignment of SMCs. In contrast, uniform shear stress in a control model elicited an even influence on the activity of mitogenic molecules without modulating the uniformity of cell density and did not significantly influence the direction of SMC alignment. The suppression of the PDGF-beta receptor tyrosine kinase and Src with pharmacological substances diminished the gradients of mitogens and cell density and reduced the influence of nonuniform shear stress on SMC alignment. These observations suggest that PDGF-beta receptor and Src possibly serve as mediating factors in nonuniform shear-induced formation of cell density gradients and alignment of SMCs in the neointima of vascular polymer implants.     

Activation of Rac1 by shear stress in endothelial cells mediates both cytoskeletal reorganization and effects on gene expression

Hemodynamic shear stress is a fundamental determinant of vascular remodeling and atherogenesis. Changes in focal adhesions, cytoskeletal organization and gene expression are major responses of endothelial cells to shear stress. Here, we show that activation of the small GTPase Rac is essential for gene expression and for providing spatial information for shear stress-induced cell alignment. Fluorescence resonance energy transfer (FRET) localizes activated Rac1 in the direction of flow. This directional Rac1 activation is downstream of shear-induced new integrin binding to extracellular matrix. Additionally, Rac1 mediates flow-induced stimulation of nuclear factor kappaB (NF-kappaB) and the subsequent expression of intercellular cell adhesion molecule 1 (ICAM-1), an adhesion receptor involved in the recruitment of leukocytes to atherosclerotic plaque. These studies provide a unifying model linking three of the main responses to shear stress that mediate both normal adaptation to hemodynamic forces and inflammatory dysfunction of endothelial cells in atherosclerosis.     

Disruption of TGF-β signaling in smooth muscle cell prevents flow-induced vascular remodeling

Transforming growth factor-β (TGF-β) signaling has been prominently implicated in the pathogenesis of vascular remodeling, especially the initiation and progression of flow-induced vascular remodeling. Smooth muscle cells (SMCs) are the principal resident cells in arterial wall and are critical for arterial remodeling. However, the role of TGF-β signaling in SMC for flow-induced vascular remodeling remains unknown. Therefore, the goal of our study was to determine the effect of TGF-β pathway in SMC for vascular remodeling, by using a genetical smooth muscle-specific (SM-specific) TGF-β type II receptor (Tgfbr2) deletion mice model. Mice deficient in the expression of Tgfbr2 (MyhCre.Tgfbr2^f/f) and their corresponding wild-type background mice (MyhCre.Tgfbr2^WT/WT) underwent partial ligation of left common carotid artery for 1, 2, or 4 weeks. Then the carotid arteries were harvested and indicated that the disruption of Tgfbr2 in SMC provided prominent inhibition of vascular remodeling. And the thickening of carotid media, proliferation of SMC, infiltration of macrophage, and expression of matrix metalloproteinase (MMP) were all significantly attenuated in Tgfbr2 disruption mice. Our study demonstrated, for the first time, that the TGF-β signaling in SMC plays an essential role in flow-induced vascular remodeling and disruption can prevent this process.     

Flow effects on cultured vascular endothelial and smooth muscle cell functions.

Cultured vascular endothelial cells were exposed to fluid shear stress by means of a rotary-disc shear-loading device, and the physiological effects of the conditioned medium (CM) and the homogenate (HM) of the cells on migration, adhesion and growth of endothelial cells (EC) or smooth muscle cells (SMC) were studied. Effects of shear stress on the production and secretion of collagen, one of the extracellular matrices of EC, were also studied. CM stimulated the adhesion and growth of SMC, but not of EC themselves. The ability to stimulate SMC adhesion and growth was similar in CM obtained from the static and shear-loaded cells. HM of the shear-loaded EC stimulated SMC migration. Further, HM of the shear-loaded EC contained increased amounts of collagen compared with the static EC. These results suggest that: 1) EC produce and secrete accelerators for the adhesion and growth of SMC, 2) EC react to the physical stimulus of fluid shear stress to produce stimulators of SMC migration, and 3) EC produce collagen, the production of which is enhanced by fluid shear stress.     

Vascular smooth muscle cell glycocalyx modulates shear-induced proliferation, migration, and NO production responses

The endothelial cell glycocalyx, a structure coating the luminal surface of the vascular endothelium, and its related mechanotransduction have been studied by many over the last decade. However, the role of vascular smooth muscle cells (SMCs) glycocalyx in cell mechanotransduction has triggered little attention. This study addressed the role of heparan sulfate proteoglycans (HSPGs), a major component of the glycocalyx, in the shear-induced proliferation, migration, and nitric oxide (NO) production of the rat aortic smooth muscle cells (RASMCs). A parallel plate flow chamber and a peristaltic pump were employed to expose RASMC monolayers to a physiological level of shear stress (12 dyn/cm(2)). Heparinase III (Hep.III) was applied to selectively degrade heparan sulfate on the SMC surface. Cell proliferation, migration, and NO production rates were determined and compared among the following four groups of cells: 1) untreated with no flow, 2) Hep.III treatment with no flow, 3) untreated with flow of 12 dyn/cm(2) exposure, and 4) Hep.III treatment with flow of 12 dyn/cm(2) exposure. It was observed that flow-induced shear stress significantly suppressed SMC proliferation and migration, whereas cells preferred to aligning along the direction of flow and NO production were enhanced substantially. However, those responses were not found in the cells with Hep.III treatment. Under flow condition, the heparinase III-treated cells remained randomly oriented and proliferated as if there were no flow presence. Disruption of HSPG also enhanced wound closure and inhibited shear-induced NO production significantly. This study suggests that HSPG may play a pivotal role in mechanotransduction of SMCs.     

Tissue stretch decreases soluble TGF-beta1 and type-1 procollagen in mouse subcutaneous connective tissue: evidence from ex vivo and in vivo models

Vascular smooth muscle cells (SMC) may be directly exposed to blood flow after an endothelial-denuding injury. It is not known whether direct exposure of SMC to shear stress reduces SMC turnover and contributes to the low rate of restenosis after most vascular interventions. This study examines if laminar shear stress inhibits SMC proliferation or stimulates apoptosis. Bovine aortic SMC were exposed to arterial magnitudes of laminar shear stress (11 dynes/cm(2)) for up to 24 h and compared to control SMC (0 dynes/cm(2)). SMC density was assessed by cell counting, DNA synthesis by (3)[H]-thymidine incorporation, and apoptosis by TUNEL staining. Akt, caspase, bax, and bcl-2 phosphorylation were assessed by Western blotting; caspase activity was also measured with an in vitro assay. Analysis of variance was used to compare groups. SMC exposed to laminar shear stress had a 38% decrease in cell number (n = 4, P = 0.03), 54% reduction in (3)[H]-thymidine incorporation (n = 3, P = 0.003), and 15-fold increase in TUNEL staining (n = 4, P < 0.0001). Akt phosphorylation was reduced by 67% (n = 3, P < 0.0001), whereas bax/bcl-2 phosphorylation was increased by 1.8-fold (n = 3, P = 0.01). Caspase-3 activity was increased threefold (n = 5, P = 0.03). Pretreatment of cells with ZVAD-fmk or wortmannin resulted in 42% increased cell retention (n = 3, P < 0.01) and a fourfold increase in apoptosis (n = 3, P < 0.04), respectively. Cells transduced with constitutively-active Akt had twofold decreased apoptosis (n = 3, P < 0.002). SMC exposed to laminar shear stress have decreased proliferation and increased apoptosis, mediated by the Akt pathway. These results suggest that augmentation of SMC apoptosis may be an alternative strategy to inhibit restenosis after vascular injury.     

Shear stress induces endothelial transdifferentiation from mouse smooth muscle cells

Smooth muscle cells (SMCs) under shear stress may alter their gene expression patterns to adapt to a new hemodynamic environment. Their plasticity may play an important role in vascular development, healing, and remodeling as well as vascular lesion formation under abnormal environmental conditions. A mouse vascular SMC line (P53LMACO1) cultured under shear stress significantly increased the mRNA levels of endothelial cell markers including Platelet-endothelial cell adhesion molecule-1 (PECAM-1), von Willebrand factor (vWF), and VE-cadherin, while significantly decreasing the mRNA levels of SMC markers including alpha-smooth muscle actin (alpha-SMA), calponin-1, smooth muscle myosin heavy chain (SMMHC), and transgelin as compared to static control cells. Protein levels of PECAM-1 and vWF were significantly increased, while protein levels of alpha-SMA were substantially decreased in the shear stress-cultured cells. In addition, shear stress-cultured cells showed an enhanced capability to form capillary-like structures on Matrigel. Thus, shear stress may promote endothelial cell transdifferentiation from SMCs.     

Placenta growth factor expression is regulated by hydrogen peroxide in vascular smooth muscle cells

When supply arteries become occluded, blood is diverted through preexisting collateral vessels. Shear stress arising from this increase in blood flow provides the initial physiological stimulus for expansion of the collateral circulation, a process termed arteriogenesis. Endothelial cells (EC) respond to increased shear stress by releasing a variety of mediators that can act on underlying smooth muscle cells (SMC). Placenta growth factor (PLGF) is known to mediate certain aspects of arteriogenesis, such as recruitment of monocytes to the vessel wall. Therefore, we tested whether SMC PLGF expression is influenced by mediators released by EC. We used A10 SMC cultured with medium that had been conditioned by EOMA EC for 4 days as a model. We found that EC-conditioned medium is able to upregulate PLGF gene expression in A10 SMC. Further experiments identified hydrogen peroxide (H(2)O(2)) as a key mediator of this response. We confirmed the physiological relevance of this mechanism in primary human coronary artery SMCs by demonstrating that exogenous H(2)O(2) specifically upregulates PLGF gene and protein expression. We also demonstrated that the physiological stimulus of shear stress raises endogenous H(2)O(2) levels in media into the range found to increase PLGF expression. In this study, we demonstrate that EC-released H(2)O(2) acts as a positive regulator of PLGF gene and protein expression in vascular SMC. To our knowledge, this is the first study to describe H(2)O(2) as a regulator of PLGF expression and therefore an upstream mediator of PLGF-driven arteriogenesis.     

Hypo- and Hyperglycemia Impair Endothelial Cell Actin Alignment and Nitric Oxide Synthase Activation in Response to Shear Stress

Uncontrolled blood glucose in people with diabetes correlates with endothelial cell dysfunction, which contributes to accelerated atherosclerosis and subsequent myocardial infarction, stroke, and peripheral vascular disease. In vitro, both low and high glucose induce endothelial cell dysfunction; however the effect of altered glucose on endothelial cell fluid flow response has not been studied. This is critical to understanding diabetic cardiovascular disease, since endothelial cell cytoskeletal alignment and nitric oxide release in response to shear stress from flowing blood are atheroprotective. In this study, porcine aortic endothelial cells were cultured in 1, 5.55, and 33 mM D-glucose medium (low, normal, and high glucose) and exposed to 20 dynes/cm² shear stress for up to 24 hours in a parallel plate flow chamber. Actin alignment and endothelial nitric oxide synthase phosphorylation increased with shear stress for cells in normal glucose, but not cells in low and high glucose. Both low and high glucose elevated protein kinase C (PKC) levels; however PKC blockade only restored actin alignment in high glucose cells. Cells in low glucose instead released vascular endothelial growth factor (VEGF), which translocated β-catenin away from the cell membrane and disabled the mechanosensory complex. Blocking VEGF in low glucose restored cell actin alignment in response to shear stress. These data suggest that low and high glucose alter endothelial cell alignment and nitric oxide production in response to shear stress through different mechanisms.     

Enhanced Cell Adhesion and Alignment on Micro-Wavy Patterned Surfaces

Various micropatterns have been fabricated and used to regulate cell adhesion, morphology and function. Micropatterns created by standard photolithography process are usually rectangular channels with sharp corners (microgrooves) which provide limited control over cells and are not favorable for cell-cell interaction and communication. This paper proposes a new micropattern with smooth wavy surfaces (micro-waves) to control the position and orientation of cells. To characterize cell growth and responses on the micro-patterned substrates, bovine aortic endothelial cells were seeded onto surfaces with micro-grooves and micro-waves for 24 h. As a result, the cells on the micro-wavy pattern appeared to have a lower death rate and better alignment compared to those on the micro-grooved pattern. In addition, flow-induced shear stress was applied to examine the adhesion strength of cells on the micro-wavy pattern. Results showed that cells adhered to the wavy surface displayed both improved alignment and adhesion strength compared to those on the flat surface. The combination of increased alignment, lower death rate and enhanced adhesion strength of cells on the micro-wavy patterns will offer advantages in potential applications for cell phenotype, proliferation and tissue engineering.     

Biomechanical regulation of hedgehog signaling in vascular smooth muscle cells in vitro and in vivo

Hedgehog (Hh) signaling has recently been shown to be both responsive to mechanical loading in vitro and to control vascular development in vivo. We investigated the role of cyclic strain and pulsatile flow in modulating Hh signaling and growth of adult rat vascular smooth muscle cells (SMC) in culture. Exposure of SMC to defined equibiaxial cyclic strain (0% and 10% stretch, 60 cycles/min, for 24 h) significantly decreased sonic hedgehog (Shh) and patched 1 (Ptc1) expression while concurrently inhibiting Gli₂-dependent promoter activity and mRNA expression, respectively. Cyclic strain significantly decreased SMC proliferation (cell counts and proliferating cell nuclear antigen expression) concomitant with a marked increase in SMC apoptosis (fluorescence-activated cell sorter analysis, acridine orange staining of apoptotic nuclei and Bax/Bcl-x_L ratio). These strain-induced changes in proliferation and apoptosis were significantly attenuated following addition of either recombinant Shh (3.5 µg/ml) or overexpression of the Notch 3 intracellular domain (Notch IC). Further studies using a perfused transcapillary culture system demonstrated a significant decrease in Hh signaling in SMC following exposure of cells to increased pulsatile flow concomitant with a decrease in proliferation and an increase in apoptosis. Finally, the pulsatile flow-induced decreases in Hh signaling were validated in vivo following flow-induced rat carotid arterial remodeling after 28 days. These data suggest that Hh expression is diminished by biomechanical stimulation in vitro and in vivo and thus may play a fundamental role in arterial remodeling and atherogenesis in vivo. cyclic strain; apoptosis; proliferation     

A model for studying the effect of shear stress on interactions between vascular endothelial cells and smooth muscle cells

Vascular endothelial cells (ECs) are constantly subjected to blood flow-induced shear stress and the influences of neighboring smooth muscle cells (SMCs). In the present study, a coculture flow system was developed to study the effect of shear stress on EC-SMC interactions. ECs and SMCs were separated by a porous membrane with only the EC side subjected to the flow condition. When ECs were exposed to a shear stress of 12 dynes/cm2 for 24 h, the cocultured SMCs tended to orient perpendicularly to the flow direction. This perpendicular orientation of the cocultured SMCs to flow direction was not observed when ECs were exposed to a shear stress of 2 dynes/cm2. Under the static condition, long and parallel actin bundles were observed in the central regions of the cocultured SMCs, whereas the actin filaments localized mainly at the periphery of the cocultured ECs. After 24 h of flow application, the cocultured ECs displayed very long, well-organized, parallel actin stress fibers aligned with the flow direction in the central regions of the cells. Immunostaining of platelet endothelial cell adhesion molecule-1 confirmed the elongation and alignment of the cocultured ECs with the flow direction. Coculture with SMCs under static condition induced EC gene expressions of growth-related oncogene-alpha and monocyte chemotactic protein-1, and shear stress was found to abolish these SMC-induced gene expressions. Our results suggest that shear stress may serve as a down-regulator for the pathophysiologically relevant gene expression in ECs cocultured with SMCs.     

The role of deformability of endothelial cells in the arterial response to shear tension

Treatment of the femoral artery luminal surface with glutaraldehyde dimere or dithiosuccinimidyl propionate reduced or eliminated flow-induced dilation, the responses to acetylcholine and the ATP being preserved. The findings suggest that the endothelial cells perceive changes in shear stress and that the cell stiffness is a factor subject to the influence of the magnitude of flow-induced arterial dilation.     

Novel mechanical bioreactor for concomitant fluid shear stress and substrate strain

The two main types of mechanical stimuli used in cellular-level bone mechanotransduction studies are substrate strain and flow-induced shear stress. A subset of studies has investigated which of these stimuli induces the primary mechanotransduction effect on bone cells. The shortcomings of these experiments are twofold. First, in some experiments the magnitude of one loading type is able to be quantitatively measured while the other loading mode is only estimated. Second, the two loading modes are compared using different bioreactors, representing different cellular environments and substrates to which the cells are attached. In addition, none of these studies utilized bioreactors which apply controlled magnitudes of substrate strain and flow-induced shear stress differentially and simultaneously. This study presents the design of a multimodal loading device which can apply substrate stretch and fluid flow simultaneously while allowing for real-time cell imaging. The mechanical performance of the bioreactor is validated in this study by correlating the output levels of flow-induced shear stress and substrate strain with the input levels of displacement and displacement rate. The magnitudes of cross-talk loading (i.e. flow-induced strain, and strain-induced fluid flow) are also characterized and shown to be magnitudes lower than physiological levels of loading estimated to occur in bone in vivo.     

Perfused transcapillary smooth muscle and endothelial cell co-culture—a novelin vitro model

Summary As mostin vitro endothelial cell (EC)-vascular smooth muscle cell (SMC) co-culture studies have been performed utilizing static culture conditions, none have successfully mimicked the physical environment of these cellsin vivo. EC covering the inner surface of blood vessels are continuously exposed to a hemodynamically imposed mechanical stress resulting from the flow of blood, while SMC are affected by pressure, a flow-related force acting perpendicular to the surface. We have developed a perfused transcapillary co-culture system that permits the chronic exposure of EC and SMC to physiological shear stresses and pressures. SMC and EC co-cultures were successfully established and maintained in long-term culture (7 wk) on an enclosed perfused bundle of semipermeable polypropylene capillaries. By altering flow rate and/or viscosity, shear stresses of 0.07–20 dyn/cm² can be readily achieved in this system. Electron microscopic analysis revealed that SMC formed multilayers around the outside of the capillaries, whereas EC, subjected to 3 dyn/cm² shear stress, formed an intact closely adherent monolayer lining the capillary lumen. EC and SMC exhibited characteristic ultrastructural and gross morphology. EC were separated from SMC by the capillary wall (pore size 0.5 μm, width 150 μM) and while no direct cell-cell contact was evident some cells were seen to migrate into the capillary wall. Both EC and SMC are exposed to the same culture medium, allowing the interaction of substances released in both directions. Yet separate populations of cells are maintained and can be individually harvested for further analysis. This co-culture system that mimics the architecture and physical environment of the vessel wall should have many potential applications in vascular biology.     

Role of cyclic strain frequency in regulating the alignment of vascular smooth muscle cells in vitro

The arterial system is subjected to cyclic strain because of periodic alterations in blood pressure, but the effects of frequency of cyclic strain on arterial smooth muscle cells (SMCs) remain unclear. Here, we investigated the potential role of the cyclic strain frequency in regulating SMC alignment using an in vitro model. Aortic SMCs were subject to cyclic strain at one elongation but at various frequencies using a Flexercell Tension Plus system. It was found that the angle information entropy, the activation of integrin-β1, p38 MAPK, and F/G actin ratio of filaments were all changed in a frequency-dependent manner, which was consistent with SMC alignment under cyclic strain with various frequencies. A treatment with anti-integrin-β1 antibody, SB202190, or cytochalasin D inhibited the cyclic strain frequency-dependent SMC alignment. These observations suggested that the frequency of cyclic strain plays a role in regulating the alignment of vascular SMCs in an intact actin filament-dependent manner, and cyclic strain at 1.25 Hz was the most effective frequency influencing SMC alignment. Furthermore, integrin-β1 and p38 MAPK possibly mediated cyclic strain frequency-dependent SMC alignment.     

Flow-induced control of arterial lumen

Blood flow velocity is a factor that affects the diameter of arteries. In order to investigate the flow-induced arterial dilatation, the outer diameter of the femoral, common carotid or renal arteries of anaesthetized cats was measured during perfusion of these arteries with blood or plasma-substituting solutions under conditions of stabilized perfusion pressure. It has been shown that, whatever the perfusate, blood or a substituent, an increase in flow makes the artery to dilate. Consequently, the flow-induced dilatation is not due to any blood-borne humoral factor. As an increase in the solution's viscosity causes dilatation even at constant flow-rate and pressure in the artery, the effect is to be ascribed to the ability of the vascular wall to perceive shear stress. As far as removal of endothelium eliminates the dilatation evoked by increasing flow or fluid viscosity, it may be concluded that the flow-induced dilatation is due to the sensitivity to shear stress of the endothelium.