Changes In Fluid Shear Stress Research Articles

Shear-induced phenotypic transformation of microglia in vitro

The brain cells are affected by continuous fluid shear stress that is driven by varying hydrostatic and osmotic pressure conditions, depending on the brain’s pathophysiological conditions. Although all brain cells are sensitive to the subtle changes in various physicochemical factors in the microenvironment, microglia, the resident brain immune cells, exhibit the most significant morphodynamic transformation. However, little is known about the phenotypic alterations in microglia in response to changes in fluid shear stress. In this study, we established a flow-controlled microenvironment to investigate the effects of shear flow on microglial phenotypes, including morphology, motility, and activation states. We observed two distinct morphologies of microglia in a static condition: bipolar cells that oscillate along their long axis and unipolar cells that migrate persistently. When exposed to flow, a significant fraction of bipolar cells showed unstable oscillation with an increased amplitude of oscillation and a decreased frequency, which consequently led to the phenotypic transformation of oscillating cells into migrating cells. Furthermore, we observed that the level of proinflammatory genes increased in response to shear stress, although there were no significant changes in the level of antiinflammatory genes. Our findings suggest that an interstitial fluid-level stimulus can cause a dramatic phenotypic shift in microglia toward proinflammatory states, shedding light on the pathological outbreaks of severe brain diseases. Given that the fluidic environment in the brain can be locally disrupted in pathological circumstances, the mechanical stimulus by fluid flow should also be considered a crucial element in regulating the immune activities of the microglia in brain diseases.

Proximal tubule apical endocytosis is modulated by fluid shear stress via an mTOR-dependent pathway.

Cells lining the proximal tubule (PT) have unique membrane specializations that are required to maintain the high-capacity ion transport and endocytic functions of this nephron segment. PT cells in vivo acutely regulate ion transport in response to changes in glomerular filtration rate (GFR) to maintain glomerulotubular balance. PT cells in culture up-regulate endocytic capacity in response to acute changes in fluid shear stress (FSS); however, it is not known whether GFR modulates PT endocytosis to enable maximally efficient uptake of filtered proteins in vivo. Here, we show that cells cultured under continuous FSS develop an expanded apical endocytic pathway and increased endocytic capacity and lysosomal biogenesis. Furthermore, endocytic capacity in fully differentiated cells is rapidly modulated by changes in FSS. PT cells exposed to continuous FSS also acquired an extensive brush border and basolateral membrane invaginations resembling those observed in vivo. Culture under suboptimal levels of FSS led to intermediate phenotypes, suggesting a threshold effect. Cells exposed to FSS expressed higher levels of key proteins necessary for PT function, including ion transporters, receptors, and membrane-trafficking machinery, and increased adenine nucleotide levels. Inhibition of the mechanistic target of rapamycin (mTOR) using rapamycin prevented the increase in cellular energy levels, lysosomal biogenesis, and endocytic uptake, suggesting that these represent a coordinated differentiation program. In contrast, rapamycin did not prevent the FSS-induced increase in Na+/K+-ATPase levels. Our data suggest that rapid tuning of the endocytic response by changes in FSS may contribute to glomerulotubular balance in vivo. Moreover, FSS provides an essential stimulus in the differentiation of PT cells via separate pathways that up-regulate endocytosis and ion transport capacity. Variations in FSS may also contribute to the maturation of PT cells during kidney development and during repair after kidney injury.

Substrate stiffness and VE-cadherin mechano-transduction coordinate to regulate endothelial monolayer integrity.

The vascular endothelium is subject to diverse mechanical cues that regulate vascular endothelial barrier function. In addition to rigidity sensing through integrin adhesions, mechanical perturbations such as changes in fluid shear stress can also activate force transduction signals at intercellular junctions. This study investigated how extracellular matrix rigidity and intercellular force transduction, activated by vascular endothelial cadherin, coordinate to regulate the integrity of endothelial monolayers. Studies used complementary mechanical measurements of endothelial monolayers grown on patterned substrates of variable stiffness. Specifically perturbing VE-cadherin receptors activated intercellular force transduction signals that increased integrin-dependent cell contractility and disrupted cell-cell and cell-matrix adhesions. Further investigations of the impact of substrate rigidity on force transduction signaling demonstrated how cells integrate extracellular mechanics cues and intercellular force transduction signals, to regulate endothelial integrity and global tissue mechanics. VE-cadherin specific signaling increased focal adhesion remodeling and cell contractility, while sustaining the overall mechanical equilibrium at the mesoscale. Conversely, increased substrate rigidity exacerbates the disruptive effects of intercellular force transduction signals, by increasing heterogeneity in monolayer stress distributions. The results provide new insights into how substrate stiffness and intercellular force transduction coordinate to regulate endothelial monolayer integrity.

Flow stimulated endocytosis in the proximal tubule

The proximal tubule plays a critical role in the reabsorption of ions, solutes and low molecular weight proteins from the glomerular filtrate. Although the proximal tubule has long been known to acutely modulate ion reabsorption in response to changes in flow rates of the glomerular filtrate, it has only recently been discovered that proximal tubule cells can similarly adjust endocytic capacity in response to flow. This review synthesizes our current understanding of mechanosensitive regulation of endocytic capacity in proximal tubule epithelia and highlights areas of opportunity for future investigations. Recent studies have reported that the endocytic capacity of proximal tubule cells is dramatically increased upon exposure to flow and the accompanying fluid shear stress. Modulation of this pathway is dependent on increases in intracellular calcium initiated by bending of the primary cilium, and also requires purinergic receptor activation that is mediated by release of extracellular ATP. This article summarizes what is currently known about the signaling cascade that transduces changes in flow into alterations in endocytosis. We discuss the implications of this newly described regulatory pathway with respect to our understanding of protein retrieval by the kidney under normal conditions, and in diseases that present with low molecular weight proteinuria. Primary cilia act as mechanotransducers that modulate apical endocytic capacity in proximal tubule cells in response to changes in fluid shear stress.

Shear Stress Reverses Dome Formation in Confluent Renal Tubular Cells

Background/Aims. It has been shown that MDCK cells, a cell line derived from canine renal tubules, develop cell domes due to fluid pumped under cell monolayer and focal detachment from the adhesion surface. In vitro studies have shown that primary cilia of kidney tubular epithelial cells act as mechanosensors, increasing intracellular calcium within seconds upon changes in fluid shear stress (SS) on cell membrane. We then studied the effect of prolonged SS exposure on cell dome formation in confluent MDCK cell monolayers. Methods. A parallel plate flow chamber was used to apply laminar SS at 2 dynes/cm² to confluent cell monolayers for 6 hours. Control MDCK cell monolayers were maintained in static condition. The effects of Ca²⁺ blockade and cell deciliation on SS exposure were also investigated. Results. Seven days after reaching confluence, static cultures developed liquid filled domes, elevating from culture plate. Exposure to SS induced almost complete disappearance of cell domes (0.4±0.8 vs. 11.4±2.8 domes/mm², p < 0.01, n=14). SS induced dome disappearance took place within minutes to hours, as shown by time-lapse videomicroscopy. Exposure to SS importantly affected cell cytoskeleton altering actin stress fibers expression and organization, and the distribution of tight junction protein ZO-1. Dome disappearance induced by flow was completely prevented in the presence of EGTA or after cell deciliation. Conclusions. These data indicate that kidney tubular cells are sensitive to apical flow and that these effects are mediated by primary cilia by regulation of Ca²⁺ entry in to the cell. SS induced Ca²⁺ entry provokes contraction of cortical actin ring that tenses cell-cell borders and decreases basal stress fibers. These processes may increase paracellular permeability and decrease basal adhesion making dome disappear. Elucidation of the effects of apical fluid flow on tubular cell function may open new insights on the pathophysiology of kidney diseases associated with cilia dysfunction.

Rapid changes in shear stress induce dissociation of a Gαq/11–platelet endothelial cell adhesion molecule‐1 complex

It has been recently shown that endothelial platelet endothelial cell adhesion molecule-1 (PECAM-1) expression is pro-atherogenic. PECAM-1 is involved in sensing rapid changes in fluid shear stress but the mechanisms for activating signalling complexes at the endothelial cell junction have yet to be elucidated. Additional studies suggest the activation of membrane-bound G proteins G alpha(q/11) also mediate flow-induced responses. Here, we investigated whether PECAM-1 and G alpha(q/11) could act in unison to rapidly respond to fluid shear stress. With immunohistochemistry, we observed a co-localization of G alpha(q/11) and PECAM-1 at the cell-cell junction in the atheroprotected section of mouse aortae. In contrast, G alpha(q/11) was absent from junctions in atheroprone areas as well as in all arterial sections of PECAM-1 knockout mice. In primary human endothelial cells, temporal gradients in shear stress led to a rapid dissociation of the G alpha(q/11)-PECAM-1 complex within 30 s and a partial relocalization of the G alpha(q/11) staining to perinuclear areas within 150 min, whereas transitioning fluid flow devoid of temporal gradients did not disrupt the complex. Inhibition of G protein activation eliminated temporal gradient flow-induced G alpha(q/11)-PECAM-1 dissociation. These results allow us to conclude that G alpha(q/11)-PECAM-1 forms a mechanosensitive complex and its localization suggests the G alpha(q/11)-PECAM-1 complex is a critical mediator of vascular diseases.

The response of human embryonic stem cell‐derived endothelial cells to shear stress

An important physiological function of vascular endothelial cells is to detect and respond to physical stimuli. While many efforts have been made to derive endothelial cells from human embryonic stem cells (hESCs), the ability of these derivatives to respond to mechanical forces has yet to be ascertained. hESC-derived endothelial cells (hEECs) were obtained by coculturing hESCs with OP9 stromal cells. Here we applied physiologic levels of shear stress to hEECs in a parallel plate flow chamber and observed changes in cell morphology and gene expression, comparing the response to that of human umbilical vein endothelial cells (HUVECs) and human microvascular endothelial cells (HMVECs). Shear induced hEECs to elongate and align in the direction of flow, and their overall transcriptional response to shear was similar to the primary cells tested. In response to shear in hEECs, COX2 and MMP1 were upregulated four- and threefold, MCP1 and VCAM1 expression decreased over fivefold, and ICAM1 and TPA were downregulated almost threefold. TGFbeta1 and SOD2 transcription exhibited no change under the conditions tested. Additionally, preshearing of hEECs mitigated TNFalpha-induced VCAM1 surface expression. These findings suggest that hEECs are capable of functionally responding to changes in fluid shear stress by modulating gene expression and cell morphology.

Microfluidic System for Studying the Interaction of Nanoparticles and Microparticles with Cells

Nanoparticles and microparticles have many potential biomedical applications ranging from imaging to drug delivery. Therefore, in vitro systems that can analyze and optimize the interaction of such particles with cells may be beneficial. Here, we report a microfluidic system that can be used to study these interactions. As a model system, we evaluated the interaction of polymeric nanoparticles and microparticles and similar particles conjugated to aptamers that recognize the transmembrane prostate specific membrane antigen (PSMA), with cells seeded in microchannels. The binding of particles to cells that expressed or did not express the PSMA (LNCaP or PC3, respectively) were evaluated with respect to changes in fluid shear stress, PSMA expression on target cells, and particle size. Nanoparticle-aptamer bioconjugates selectively adhered to LNCaP but not PC3 cells at static and low shear (<1 dyn/cm2) but not higher shear (approximately 4.5 dyn/cm2) conditions. Control nanoparticles and microparticles lacking aptamers and microparticle-aptamer bioconjugates did not adhere to LNCaP cells, even under very low shear conditions (approximately 0.28 dyn/cm2). These results demonstrate that the interaction of particles with cells can be studied under controlled conditions, which may aid in the engineering of desired particle characteristics. The scalability, low cost, reproducibility, and high-throughput capability of this technology is potentially beneficial to examining and optimizing a wide array of cell-particle systems prior to in vivo experiments.

Cells in fluidic environments are sensitive to flow frequency

Virtually all cells accommodate to their mechanical environment. In particular, cells subject to flow respond to rapid changes in fluid shear stress (SS), cyclic stretch (CS), and pressure. Recent studies have focused on the effect of pulsatility on cellular behavior. Since cells of many different tissue beds are constantly exposed to fluid flows over a narrow range of frequencies, we hypothesized that an intrinsic flow frequency that is optimal for determining cell phenotype exists. We report here that cells from various tissue beds (bovine aortic endothelial cells (BAEC), rat small intestine epithelial cells (RSIEC), and rat lung epithelial cells (RLEC)) proliferate maximally when cultured in a perfusion bioreactor under pulsatile conditions at a specific frequency, independent of the applied SS. Vascular endothelial and pulmonary epithelial cell proliferation peaked under 1 Hz pulsatile flow. In contrast, proliferation of gastrointestinal cells, which in their physiological context are subject to no flow or higher wavelength signal, was maximum at 0.125 Hz or under no flow. Moreover, exposure of BAEC to pulsatile flow of varying frequency influenced their nitric oxide synthase activity and prostacyclin production, which reached maximum values at 1 Hz. Notably, the "optimal" frequencies for the cell types examined correspond to the physiologic operating range of the organs from where they were initially derived. These findings suggest that frequency, independent of shear, is an essential determinant of cell response in pulsatile environments.

Intact capillaries sensitive to rate, magnitude, and pattern of shear stress stimuli as assessed by hydraulic conductivity (l p)

Magnitude of abrupt changes in fluid shear stress (Δτ) correlates with capillary hydraulic conductivity (L p). Cultured endothelial cells differentiate between rate of change in and pattern of fluid stimulation; however, neither rate nor pattern sensitivity has been evaluated in individual capillaries. We hypothesized that L p would be greater following abrupt compared to slow Δτ and that magnitude of Δτ would correlate with L p regardless of rate. Mesenteric venular capillaries (VC, n = 41) located in pithed North American leopard frogs ( Rana pipiens) were cannulated either above or at in situ pressure to induce abrupt (<0.1 s) or slow (1–5 min) Δτ. L p was assessed always at the same pressure (30 cm H 2O) using the modified Landis technique. Mean ± SE L p was sixfold higher ( P = 0.005) following abrupt (19.0 ± 3.9 × 10 −7) compared to slow (2.7 ± 0.9 × 10 −7 cm/s/cm H 2O) Δτ after accounting for stimulus pattern variability. Linear relationships between L p and Δτ existed for both abrupt [L p = 0.026(Δτ)−1.6, R 2 = 0.90, P = 0.0001] and slow [L p = 0.005(Δτ)−0.3, R 2 = 0.82, P = 0.03] stimuli. These results suggest that frog mesenteric VC sense unique characteristics of Δτ and respond by altering L p.

Network Assessment of Capillary Hydraulic Conductivity after Abrupt Changes in Fluid Shear Stress

Intact capillaries are composed of endothelial cells that experience continuous fluid shear stress as blood flows. Endothelial cells grown in culture demonstrate changes in barrier function in response to changes in shear stress. Therefore, it was hypothesized that intact capillaries would alter filtration in response to changes in fluid shear stress. Capillaries (n= 25) located in frog mesentery were classified according to flow direction as arteriolar, true, or venular and cannulated at 30 cm H2O to induce an abrupt change in shear stress. Frog and human red blood cells acted as velocity markers before and after cannulation, respectively, for calculating fluid shear stress. Hydraulic conductivity (Lp) was measured at 30 cm H2O following a change in shear stress and ranged from 0.7 to 96.8 × 10−7cm s−1cm H2O−1. For true and venular capillaries,Lpwas related to the magnitude of the shear stress stimulus and accounted for the wide range in absolute values ofLp. Arteriolar capillaries did not respond to the mechanical stimulus. These data indicate that blood flow may modulate filtration in homogeneous subpopulations of capillaries in the capillary bed.

Submarine pollination in the marine angiosperm Zostera marina (Zosteraceae). I. The influence of floral morphology on fluid flow

An understanding of the process of submarine pollination should provide insight into the evolutionary and reproductive ecology of the marine angiosperms (seagrasses). The flow around the reproductive organs of the seagrass Zostera marina L. (Potamogetonales) was, therefore, examined in a flow chamber. The phenological emergence of flowers during (1) pollen capture and (2) pollen release, and by fruit during (3) seed release, led to a reduction in flow rate toward the inflorescence. This change in flow due to floral emergence was associated with a 50% increase in the fluid shear stress [tau = (2.2 _ 0.3) x 10-3 Pa for an immature flower vs. tau = (3.1 _ 0.5) x 10-3 Pa for a receptive flower]. The Reynolds number (Re) and fluid shear stress around inflorescences and infructescences were comparable, indicating a dynamic similarity in the processes of pollen capture and fruit dehiscence [Re = 47 _ 5, tau = (1.6 _ 0.3) x 10-3 Pa for inflorescences; Re = 38 _ 5, tau = (1.3 _ 0.1) x 10-3 Pa for infructescences]. These results indicate that the emergence of reproductive organs leads to changes in fluid shear stress, which will affect the release, transport, and capture of particles including pollen. Theoretical considerations of these observations using aerosol-filtration theory suggest that pollen capture in Z. marina occurs through direct interception of pollen by stigmas.

Shear stress alters pleural mesothelial cell permeability in culture.

The sliding motion of the lung against the chest wall creates a shear stress in the pleural space, which can be as high as 60 dyn/cm2, depending on the respiration rate. Such shear stresses may affect the mesothelial cells that line the pleural space on the lung (visceral pleura) and chest wall (parietal pleura). When exposed to shear stress (17 dyn/cm2) in a parallel-plate flow chamber for 22 h, rat visceral pleura mesothelial cells were not altered morphologically and did not align in the direction of flow, in contrast to the shape changes observed for bovine aortic endothelial cells. By using mesothelial cells cultured on porous microcarrier beads, we measured the permeability of the cells at different flows in a cell-column chromatography assay. The permeabilities to sodium fluorescein and cyanocobalamin increased from 8.2 +/- 1.0 and 7.8 +/- 0.7 x 10(-5) cm/s to 22.5 +/- 1.2 and 21.8 +/- 3.0 x 10(-5) cm/s, respectively, when the flow was increased from 0.9 to 3.5 ml/min (corresponding to average shear stresses of 4.7-18.4 dyn/cm2). The permeabilities returned to baseline values when the flow was reduced. Cytochalasin D stimulated an increase in permeability that was not augmented by a subsequent increase in shear stress. These results suggest that the barrier function of mesothelial cells is responsive to changes in fluid shear stress.

Effects of fluid flow on living vascular cells.

Shear stress produced by a flowing fluid has been found to cause changes in the structure and function of vascular endothelial cells. They tend to become elongated and aligned with the direction of flow. They respond to changes in fluid shear stress (either from low shear to high shear or vice versa) by transiently increasing fluid-phase endocytosis. And they are capable of producing intracellular actin and myosin filaments that are oriented in the flow direction.