Oscillatory Fluid Shear Stress Research Articles

PGC1α Regulates the Endothelial Response to Fluid Shear Stress via Telomerase Reverse Transcriptase Control of Heme Oxygenase-1.

Fluid shear stress (FSS) is known to mediate multiple phenotypic changes in the endothelium. Laminar FSS (undisturbed flow) is known to promote endothelial alignment to flow, which is key to stabilizing the endothelium and rendering it resistant to atherosclerosis and thrombosis. The molecular pathways responsible for endothelial responses to FSS are only partially understood. In this study, we determine the role of PGC1α (peroxisome proliferator gamma coactivator-1α)-TERT (telomerase reverse transcriptase)-HMOX1 (heme oxygenase-1) during shear stress in vitro and in vivo. Approach and Results: Here, we have identified PGC1α as a flow-responsive gene required for endothelial flow alignment in vitro and in vivo. Compared with oscillatory FSS (disturbed flow) or static conditions, laminar FSS (undisturbed flow) showed increased PGC1α expression and its transcriptional coactivation. PGC1α was required for laminar FSS-induced expression of TERT in vitro and in vivo via its association with ERRα(estrogen-related receptor alpha) and KLF (Kruppel-like factor)-4 on the TERT promoter. We found that TERT inhibition attenuated endothelial flow alignment, elongation, and nuclear polarization in response to laminar FSS in vitro and in vivo. Among the flow-responsive genes sensitive to TERT status, HMOX1 was required for endothelial alignment to laminar FSS. These data suggest an important role for a PGC1α-TERT-HMOX1 axis in the endothelial stabilization response to laminar FSS.

Abstract P154: Pgc1a Regulates The Endothelial Response To Fluid Shear Stress Via Telomerase Reverse Transcriptase Control Of Heme Oxygenase-1

Fluid shear stress (FSS) is known to mediate multiple phenotypic changes in the endothelium. Laminar FSS (undisturbed flow) is known to promote endothelial alignment to flow that is key to stabilizing the endothelium and rendering it resistant to atherosclerosis and thrombosis. The molecular pathways responsible for endothelial responses to FSS are only partially understood. Here we have identified peroxisome proliferator gamma coactivator-1α (PGC-1α) as a flow-responsive gene required for endothelial flow alignment in vitro and in vivo . Compared to oscillatory FSS (disturbed flow) or static conditions, laminar FSS (undisturbed flow) increased PGC-1α expression and its transcriptional co-activation. PGC-1α was required for laminar FSS-induced expression of telomerase reverse transcriptase (TERT) in vitro and in vivo via its association with ERRα and KLF4 on the TERT promoter. We found that TERT inhibition attenuated endothelial flow alignment, elongation, and nuclear polarization in response to laminar FSS in vitro and in vivo . Among the flow-responsive genes sensitive to TERT status was heme oxygenase-1 (HMOX1), a gene required for endothelial alignment to laminar FSS. Thus, these data suggest an important role for a PGC-1α-TERT-HMOX1 axis in the endothelial stabilization response to laminar FSS.

Single cell morphological metrics and cytoskeletal alignment regulate VCAM-1 protein expression

In the initial stages of atherosclerosis, vascular adhesion molecule-1 (VCAM-1) is a surface protein that mediates leukocyte adhesion to the endothelium’s luminal surface. VCAM-1 expression is upregulated on endothelial cells (ECs) under pro-inflammatory conditions and is known to be modulated by fluid shear stress (FSS). High, pulsatile FSS induces endothelial elongation and cytoskeletal alignment and downregulates pro-inflammatory induced VCAM-1 expression, which is associated with an athero-protective EC phenotype. In contrast, athero-prone ECs under low, oscillatory FSS fail to elongate and maintain a cobblestone morphology with random cytoskeletal alignment, while VCAM-1 expression is upregulated. Whether EC shape and cytoskeletal alignment play a role in the regulation of VCAM-1 protein expression independent of FSS has not been previously determined. The goal of this study was to determine the effect of EC morphology, specifically cell elongation and alignment, and cytoskeletal alignment on VCAM-1 protein expression using topographical micropatterning of an endothelial monolayer and single cell image analysis techniques. Elongated ECs with an aligned cytoskeleton significantly downregulated VCAM-1 protein expression in the absence of FSS compared to planar controls. In addition, linear correlations between morphological metrics and protein expression showed that actin alignment had a significantly stronger effect on VCAM-1 expression than cell elongation. Functionally, monocytic U937 cells statically adhered less on micropatterns compared to planar substrates, in a VCAM-1 dependent manner. Therefore, endothelial cellular elongation and alignment as well as cytoskeletal alignment regulate VCAM-1 protein expression and immunogenic functions to produce a less inflammatory phenotype in the absence of hemodynamic effects.

P2X7R-Panx1 Complex Impairs Bone Mechanosignaling under High Glucose Levels Associated with Type-1 Diabetes.

Type 1 diabetes (T1D) causes a range of skeletal problems, including reduced bone density and increased risk for bone fractures. However, mechanisms underlying skeletal complications in diabetes are still not well understood. We hypothesize that high glucose levels in T1D alters expression and function of purinergic receptors (P2Rs) and pannexin 1 (Panx1) channels, and thereby impairs ATP signaling that is essential for proper bone response to mechanical loading and maintenance of skeletal integrity. We first established a key role for P2X7 receptor-Panx1 in osteocyte mechanosignaling by showing that these proteins are co-expressed to provide a major pathway for flow-induced ATP release. To simulate in vitro the glucose levels to which bone cells are exposed in healthy vs. diabetic bones, we cultured osteoblast and osteocyte cell lines for 10 days in medium containing 5.5 or 25 mM glucose. High glucose effects on expression and function of P2Rs and Panx1 channels were determined by Western Blot analysis, quantification of Ca2+ responses to P2R agonists and oscillatory fluid shear stress (± 10 dyne/cm2), and measurement of flow-induced ATP release. Diabetic C57BL/6J-Ins2Akita mice were used to evaluate in vivo effects of high glucose on P2R and Panx1. Western blotting indicated altered P2X7R, P2Y2R and P2Y4R expression in high glucose exposed bone cells, and in diabetic bone tissue. Moreover, high glucose blunted normal P2R- and flow-induced Ca2+ signaling and ATP release from osteocytes. These findings indicate that T1D impairs load-induced ATP signaling in osteocytes and affects osteoblast function, which are essential for maintaining bone health.

Computational simulations predict a key role for oscillatory fluid shear stress in de novo valvular tissue formation

Previous efforts in heart valve tissue engineering demonstrated that the combined effect of cyclic flexure and steady flow on bone marrow derived stem cell-seeded scaffolds resulted in significant increases in engineered collagen formation [Engelmayr et al. Cyclic flexure and laminar flow synergistically accelerate mesenchymal stem cell-mediated engineered tissue formation: Implications for engineered heart valve tissues. Biomaterials 2006; 27(36): 6083–95]. Here, we provide a new interpretation for the underlying reason for this observed effect. In addition, another related investigation demonstrated the impact of fluid flow on DNA content and quantified the fluid-induced shear stresses on the engineered heart valve tissue specimens [Engelmayr et al. A Novel Flex-Stretch-Flow Bioreactor for the Study of Engineered Heart Valve Tissue Mechanobiology]. Annals of Biomedical Engineering 2008, 36, 1–13]. In this study, we performed more advanced CFD analysis with an emphasis on oscillatory wall shear stresses imparted on specimens when mechanically conditioned by a combination of cyclic flexure and steady flow. Specifically, we hypothesized that the dominant stimulatory regulator of the bone marrow stem cells is fluid-induced and depends on both the magnitude and temporal directionality of surface stresses, i.e., oscillatory shear stresses (OSS) acting on the developing tissues. Therefore, we computationally quantified the (i) magnitude of fluid-induced shear stresses as well as (ii) the extent of temporal fluid oscillations in the flow field using the oscillatory shear index (OSI) parameter. Noting that sample cyclic flexure induces a high degree of OSS, we incorporated moving boundary computational fluid dynamic simulations of samples housed within a bioreactor to consider the effects of: (1) No Flow, No Flexure (control group), (2) Steady Flow-alone, (3) Cyclic Flexure-alone and (4) Combined Steady flow and Cyclic Flexure environments. Indeed we found that the coexistence of both OSS and appreciable shear stress magnitudes explained the high levels of engineered collagen previously observed from combining cyclic flexure and steady flow states. On the other hand, each of these metrics on its own showed no association. This finding suggests that cyclic flexure and steady flow synergistically promote engineered heart valve tissue production via OSS, so long as the oscillations are accompanied by a critical magnitude of shear stress.

Mechanical loading in osteocytes induces formation of a Src/Pyk2/MBD2 complex that suppresses anabolic gene expression.

Mechanical stimulation of the skeleton promotes bone gain and suppresses bone loss, ultimately resulting in improved bone strength and fracture resistance. The molecular mechanisms directing anabolic and/or anti-catabolic actions on the skeleton during loading are not fully understood. Identifying molecular mechanisms of mechanotransduction (MTD) signaling cascades could identify new therapeutic targets. Most research into MTD mechanisms is typically focused on understanding the signaling pathways that stimulate new bone formation in response to load. However, we investigated the structural, signaling and transcriptional molecules that suppress the stimulatory effects of loading. The high bone mass phenotype of mice with global deletion of either Pyk2 or Src suggests a role for these tyrosine kinases in repression of bone formation. We used fluid shear stress as a MTD stimulus to identify a novel Pyk2/Src-mediated MTD pathway that represses mechanically-induced bone formation. Our results suggest Pyk2 and Src function as molecular switches that inhibit MTD in our mechanically stimulated osteocyte culture experiments. Once activated by oscillatory fluid shear stress (OFSS), Pyk2 and Src translocate to and accumulate in the nucleus, where they associate with a protein involved in DNA methylation and the interpretation of DNA methylation patterns –methyl-CpG-binding domain protein 2 (MBD2). OFSS-induced Cox-2 and osteopontin expression was enhanced in Pyk2 KO osteoblasts, while inhibition of Src enhanced osteocalcin expression in response to OFSS. We found that Src kinase activity increased in the nucleus of osteocytes in response to OFSS and an interaction activated between Src (Y418) and Pyk2 (Y402) increased in response to OFSS. Thus, as a mechanism to prevent an over-reaction to physical stimulation, mechanical loading may induce the formation of a Src/Pyk2/MBD2 complex in the nucleus that functions to suppress anabolic gene expression.

Elevation of glucose to levels associated with type I diabetes impairs bone cell mechanosignaling (1180.10)

It has been shown that patients with insulin dependent diabetes mellitus (IDDM; Type 1 diabetes) manifest a range of bone problems, including increased risk for bone fractures and defective bone healing. However, the underlying mechanisms are still not well understood. Given that the maintenance of skeletal integrity in response to daily physical activity relies homeostasis between bone forming cells, osteoblasts, and the primary load sensing cells, osteocytes, we hypothesized that bone loss in Type 1 diabetes is primarily triggered by exposure to high glucose levels which in turn compromises ability of bone cells to perceive and respond to mechanical loading as a result of altered expression of mechanosignaling mediators: specific purinergic receptor (P2Rs) and pannexin 1 (Panx1) channels. Our in situ (Akita, Type 1 diabetic and age‐matched wild type mouse model) morphometric analysis and in vitro (MLO‐Y4 osteocytes and MOB‐C osteoblasts) findings indicate that exposure to elevated glucose (10 days in α‐MEM containing 450 mg/dL (25 mM) glucose) alters P2R (specifically P2Y2R, P2Y4R and P2X7R) and Panx1 expression in both osteoblastic and osteocytic cells and blunts proper Ca2+ signaling in response to oscillatory fluid shear stress mechanical loading. Our findings indicate that Type 1 diabetes impairs the proper response of bone cells to mechanical loads, which is required to maintain bone health.Grant Funding Source: Supported by DK091466; DK081435; AR057139

A novel in vitro loading system to produce supraphysiologic oscillatory fluid shear stress

A multi-well fluid loading (MFL) system was developed to deliver oscillatory subphysiologic to supraphysiologic fluid shear stresses to cell monolayers in vitro using standard multi-well culture plates. Computational fluid dynamics modeling with fluid-structure interactions was used to quantify the squeeze film fluid flow between an axially displaced piston and the well plate surface. Adjusting the cone angle of the piston base modulated the fluid pressure, velocity, and shear stress magnitudes. Modeling results showed that there was near uniform fluid shear stress across the well with a linear drop in pressure across the radius of the well. Using the MFL system, RAW 264.7 osteoclastic cells were exposed to oscillatory fluid shear stresses of 0, 0.5, 1.5, 4, 6, and 17Pa. Cells were loaded 1h per day at 1Hz for two days. Compared to sub-physiologic and physiologic levels, supraphysiologic oscillatory fluid shear induced upregulation of osteoclastic activity as measured by tartrate-resistant acid phosphatase activity and formation of mineral resorption pits. Cell number remained constant across all treatment groups.

A new experimental system for simultaneous application of cyclic tensile strain and fluid shear stress to tenocytes in vitro

Tenocyte mechanotransduction has been of great interest to researchers in tendon mechanobiology and biomechanics. In vivo, tenocytes are subjected to tensile strain and fluid shear stress, but most studies of tenocyte mechanobiology have been to understand how tenocytes regulate their functions in response to tensile strain. Thus, there is still much to know about tenocyte responses to fluid shear stress, partly due to the difficulty of devising a suitable experimental set-up and understanding the exact magnitude of imposed fluid shear stress. Therefore, this study was performed to test a new experimental system, which is suitable for the application of tensile strain and fluid shear stress to tenocytes in vitro. It was experimentally and numerically confirmed that tenocytes could maintain their in situ morphology within microfabricated microgrooves; also, physiological tensile strain and a wide range of fluid shear stress magnitudes can be applied to these cells. Indeed, it was demonstrated that the combined stimulation of cyclic tensile strain and oscillatory fluid shear stress induced a greater synergetic effect on tenocyte calcium response and significantly increased the percentage of tenocyte exhibiting increases in intracellular Ca(2+) concentration compared to the solo applications of these two modes of mechanical stimulation. The experimental system presented here is suitable for research of tenocyte mechanobiology, particularly mechanotransduction events, which were difficult to study using previous experimental models like explants and cell monolayers.

Blockade of TNFR1 signaling: A role of oscillatory fluid shear stress in osteoblasts

Fluid shear stress protects cells from TNF-α-induced apoptosis. Oscillatory fluid shear stress (OFSS) is generally perceived as physiologically relevant biophysical signal for bone cells. Here we identify several cellular mechanisms responsible for mediating the protective effects of OFSS against TNF-α-induced apoptosis in vitro. We found that exposure of MC3T3-E1 osteoblast-like cells to as little as 5 min of OFSS suppressed TNF-α-induced activation of caspase-3, cleavage of PARP and phosphorylation of histone. In contrast, H(2)O(2)-induced apoptosis was not inhibited by OFSS suggesting that OFSS might not be protecting cells from TNF-α-induced apoptosis via stimulation of global pro-survival signaling pathways. In support of this speculation, OFSS inhibition of TNF-α-induced apoptosis was unaffected by inhibitors of several pro-survival signaling pathways including pI3-kinase (LY294002), MAPK/ERK kinase (PD98059 or U0126), intracellular Ca2+ release (U73122), NO production (L-NAME), or protein synthesis (cycloheximide) that were applied to cells during exposure to OFSS and during TNF-α treatment. However, TNF-α-induced phosphorylation and degradation of IκBα was blocked by pre-exposure of cells to OFSS suggesting a more specific effect of OFSS on TNF-α signaling. We therefore focused on the mechanism of OFSS regulation of TNF-receptor 1 (TNFR1) signaling and found that OFSS (1) reduced the amount of receptor on the cell surface, (2) prevented the association of ubiquitinated RIP in TNFR1 complexes with TRADD and TRAF2, and (3) reduced TNF-α-induced IL-8 promoter activity in the nucleus. We conclude that the anti-apoptotic effect of OFSS is not mediated by activation of universal pro-survival signaling pathways. Rather, OFSS inhibits TNF-α-induced pro-apoptotic signaling which can be explained by the down-regulation of TNFR1 on the cell surface and blockade of TNFR1 downstream signaling by OFSS.

Oscillatory fluid shear stress‐induced JNK activation via NADPH oxidase implicates mitochondrial superoxide production in endothelial cells

Oscillatory fluid shear stress (OSS) increases vascular oxidative stress and induces endothelial cell dysfunction. In our previous study, we demonstrated that OSS induced superoxide generation via activation of NADPH oxidase enzyme system. In this study, we examined the effect of OSS on mitochondrial dysfunction. Using chemical inhibitors against NADPH oxidase (Apocynin) and JNK (SP600125), we demonstrated that OSS induced JNK activation via NADPH oxidase in bovine aortic endothelial cells (BAEC). JNK activation peaked at 1h following treatment of OSS (±3dyn/cm2) by western blot analysis. Apocynin inhibited this induction and also inhibited OSS‐induced superoxide generation measured by dihydroethidium. We then examined the role of JNK activation in OSS‐induced mtO2·− production by using MitoSox Red dye specific for mtO2·−. We found that SP600125 inhibited ·− production as quantified by flow cytometry. OSS‐stimulated mtO2 Our data suggests that OSS‐activated NADPH oxidase induces intra‐cellular superoxide production and activates JNK, which in turn induces mtO2·− production..This study is supported by NIH HL083015 and HL091302.

Activation of NF-κB by fluid shear stress, but not TNF-α, requires focal adhesion kinase in osteoblasts

When bone is mechanically loaded fluid shear stress (FSS) is generated as a result of the movement of interstitial fluid across the membranes of osteoblasts and osteocytes. This external mechanical loading stimulates changes in the activity of cytoplasmic signaling molecules and alters gene expression in bone cells. This process, referred to as mechanotransduction, is vital for maintaining bone health in vivo by regulating the balance between bone formation and bone resorption. This current study focuses on the role of focal adhesions, sites of integrin-mediated cellular attachment to the extracellular matrix, and their proposed function as mechanosensors in bone cells. We examined the role of a key component of focal adhesions and of mechanotransduction, focal adhesion kinase (FAK) in regulation of FSS- and tumor necrosis factor-α (TNF-α)-induced activation of nuclear factor-kappa B (NF-κB) signaling in osteoblasts. Immortalized FAK +/+ and FAK − / − osteoblasts were exposed to periods of oscillatory fluid shear stress (OFF) and NF-κB activation was analyzed. We determined that FAK is required for OFF-induced nuclear translocation and activation of NF-κB in osteoblasts. In addition we found that OFF-induced phosphorylation of the IκB kinases (IKKα/β) in both FAK +/+ and FAK −/− osteoblasts, but only FAK +/+ osteoblasts demonstrated the resulting degradation of NF-κB inhibitors IκBα and IκBβ. OFF did not induce the degradation of IκBε or the processing of p105 in either FAK +/+ and FAK −/− osteoblasts. To compare the role of FAK in mediating OFF-induced mechanotransduction to the well characterized activation of NF-κB by inflammatory cytokines, we exposed FAK +/+ and FAK −/− osteoblasts to TNF-α. Interestingly, FAK was not required for TNF-α induced NF-κB activation in osteoblasts. In addition we determined that TNF-α treatment did not induce the degradation of IκBβ as did OFF. These data indicate a novel relationship between FAK and NF-κB activation in osteoblast mechanotransduction and demonstrates that the mechanism of FSS-induced NF-κB activation in osteoblasts differs from the well characterized TNF-α-induced activation.

Novel early response genes in osteoblasts exposed to dynamic fluid flow

Cyclic mechanical loads applied to the skeleton from habitual physical activity result in increased bone formation. These loads lead to dynamic pressure gradients and oscillatory flow of bone interstitial fluid, which, in turn, exposes cells resident in the bony matrix to oscillatory fluid shear stress. Dynamic fluid flow has previously been shown to be a potent anabolic stimulus for cultured osteoblasts. In this study, we used cDNA microarrays to examine early phase, broad-spectrum gene expression in MC3T3-E1 osteoblasts in response to physical stimulation. RNA was harvested at 30 min and 1 h post-stimulation. RNA was used for microarray hybridization as well as subsequent reverse transcription polymerase chain reaction (RT-PCR) validation of expression levels for selected genes. Microarray results were analysed by both functional and expression profile clustering. We identified a small number of genes at both the 30 min and 1 h timepoints that were either upregulated or downregulated with flow compared to no-flow control by twofold or more. From the group of genes upregulated at 30 min, we selected nine for RT-PCR confirmation. All were found to be upregulated by at least twofold. We identify a novel set of early response genes potentially involved in mediating the anabolic response of MC3T3 osteoblasts to flow, and provide functional groupings of these genes that may shed light on the relevant mechanosensory pathways involved.

Nmp4/CIZ inhibits mechanically induced β‐catenin signaling activity in osteoblasts

Cellular mechanotransduction, the process of converting mechanical signals into biochemical responses within cells, is a critical aspect of bone health. While the effects of mechanical loading on bone are well recognized, elucidating the specific molecular pathways involved in the processing of mechanical signals by bone cells represents a challenge and an opportunity to identify therapeutic strategies to combat bone loss. In this study we have for the first time examined the relationship between the nucleocytoplasmic shuttling transcription factor nuclear matrix protein-4/cas interacting zinc finger protein (Nmp4/CIZ) and beta-catenin signaling in response to a physiologic mechanical stimulation (oscillatory fluid shear stress, OFSS) in osteoblasts. Using calvaria-derived osteoblasts from Nmp4-deficient and wild-type mice, we found that the normal translocation of beta-catenin to the nucleus in osteoblasts that is induced by OFSS is enhanced when Nmp4/CIZ is absent. Furthermore, we found that other aspects of OFSS-induced mechanotransduction generally associated with the beta-catenin signaling pathway, including ERK, Akt, and GSK3beta activity, as well as expression of the beta-catenin-responsive protein cyclin D1 are also enhanced in cells lacking Nmp4/CIZ. Finally, we found that in the absence of Nmp4/CIZ, OFSS-induced cytoskeletal reorganization and the formation of focal adhesions between osteoblasts and the extracellular substrate is qualitatively enhanced, suggesting that Nmp4/CIZ may reduce the sensitivity of bone cells to mechanical stimuli. Together these results provide experimental support for the concept that Nmp4/CIZ plays an inhibitory role in the response of bone cells to mechanical stimulation induced by OFSS.

Cytoskeletal structure regulates endothelial cell immunogenicity independent of fluid shear stress

The cardiovascular disease atherosclerosis is directly linked to the functions of endothelial cells (ECs), which are affected by fluid shear stress (FSS). High, unidirectional FSS causes EC elongation with aligned cytoskeletal components and nonimmunogenic EC functions that protect against atherosclerosis. In contrast, low, oscillatory FSS is associated with cobblestone-shaped ECs with randomly oriented cytoskeletons and proinflammatory EC functions that promote atherosclerosis. Whether EC shape plays a role in EC immunogenic functions, independent of FSS, has not been previously determined. The goal of this study was to determine the effect of EC elongation and cytoskeletal alignment on the expression of inflammatory genes and functions. With the use of micropatterned lanes, EC elongation and cytoskeletal alignment were achieved in the absence of FSS. EC gene expression of key inflammation markers determined that the elongation and cytoskeletal alignment of micropattern-elongated ECs (MPECs) alone significantly downregulated VCAM-1 while having no effect on E-selectin and ICAM-1. The positive control of FSS-elongated ECs promoted E-selectin and VCAM-1 downregulation and upregulation of ICAM-1. Functionally, monocytic U937 cells formed weaker interactions on the surface of MPECs compared with cobblestone ECs. Interestingly, MPEC expression of the known FSS-dependent transcription factor krüppel-like factor 2 (KLF2), which promotes a nonimmunogenic EC phenotype, was significantly upregulated in MPECs compared with cobblestone ECs. Cytoskeletal regulation of KLF2 expression was shown to be dependent on microtubules. Therefore, the cellular elongation and cytoskeletal alignment of MPECs regulated immunogenic gene expression and functions and may act synergistically with FSS to create an EC surface with reduced inflammatory capability.

Endothelial Cell Elongation Impacts Thrombogenicity Independent of Fluid Shear Stress.

Atherosclerotic vascular disease and dysfunction of endothelial cells (ECs), which form the continuous lining of blood vessels, preferentially develop in regions where blood vessels are bifurcated and curved. In these regions, ECs are exposed to low, oscillatory fluid shear stress (FSS), are cobblestone in morphology, and have an athero-prone phenotype. In contrast, in regions where FSS is high and unidirectional, ECs are elongated parallel to the direction of flow and have an athero-protective phenotype. Although previous research has correlated FSS with EC morphology and phenotype, the effects of dramatic changes in cell morphology alone, i.e., in the absence of FSS differences, on EC functions remain largely unknown. To determine the role of EC shape on cell function, we investigated the regulation of EC hemostatic functions, an important measure of EC dysfunction and atherosclerosis, by elongated and cobblestone ECs (with shape independent of FSS). To separate EC shape from FSS-induced effects, surface engineering was used to create elongated ECs on micropatterned collagen I lanes (25 μm wide with 100 μm spacing). By 24 hrs, ECs elongated on these micropatterned lanes had a comparable shape index and cytoskeletal alignment as ECs elongated by exposure to 24 hrs of 12.5 dyn/cm2 FSS. qtPCR was used to determine the gene expression of the following markers of coagulant/hemostatic functions: tissue factor (TF), tissue factor pathway inhibitor (TFPI), endothelial nitric oxide synthase (eNOS), thrombomodulin (TM), and von Willebrand Factor (vWF). PCR results indicated that EC elongation alone upregulated expression of TF (1.41 ± 0.28) and decreased expression of eNOS (0.78 ± 0.07). vWF was downregulated (0.59 ± 0.11). Micropattern elongated ECs expressed TFPI and TM at levels comparable to cobblestoneappearing ECs (1.04 ± 0.08 and 1.12 ± 0.04, respectively). To determine whether these changes in gene expression had functional consequences, the generation of thrombin (factor X activation, FXa) and platelet adhesion were studied. Micropattern elongated ECs were able to convert more FX to FXa per cell compared to cobblestone ECs (0.88 ± 0.20 and 0.065 ± 0.01 pg/cell, respectively), indicating an increase in TF activity. This data is consistent with the increased TF gene expression seen in micropattern elongated ECs. Platelet adhesion studies also suggested a thrombogenic phenotype for micropattern elongated ECs, with more platelets adhering and spreading per cell on elongated (8.82 ± 1.47) versus cobblestone (4.64 ± 1.49) ECs. Overall, these findings suggest that EC shape is an independent variable that can regulate cell hemostatic functions, such as thrombotic potential. Surprisingly, elongated ECs exhibited a more thrombogenic phenotype, findings that contrast results obtained with FSS-elongated ECs, both in vitro and in vivo. Thus, both cell shape and FSS may play important and in some instances opposing roles in regulating EC hemostatic functions in the maintenance of vascular integrity.

Transmission of steady and oscillatory fluid shear stress across epithelial and endothelial surface structures

The glycocalyx on the apical surface of vascular endothelial cells and the microvilli and cilia on kidney epithelial cells have been modeled as surface layers with a hexagonal arrangement of structural elements. These elements have been proposed to serve a mechanosensory function in the initiation of intracellular signaling by fluid shear stress. In this paper we examine the response of these surface layers when steady or oscillating shear is applied at their outer edge. In the case of steady shear, our results show that the deflection of the structural elements is proportional to the product of the applied shear stress and their length L and inversely proportional to the natural damped vibration frequency of the structural element ωc. A fluid velocity boundary layer develops at the outer edge of the surface layers when the dimensionless Brinkman parameter α=L∕KP, where KP is the Darcy permeability, is asymptotically large. In the case of oscillating shear, we find that the motions of both the fluid and structural elements are in a quasisteady state at physiological conditions. No attenuation or phase shift of the torque is induced by the hydrodynamic drag when the applied frequency ω<ωc or ωr(=ω∕ωc)<1. However, the velocity at the tips of the structural element is π∕2 out of phase with the applied shear in this frequency range, due to the elastic recoil of the element. Furthermore, the fluid velocity at the tips can also be out of phase with the applied shear at large α if the closely spaced structural elements of the glycocalyx on endothelial cells or microvilli on proximal tubule cells transport substantial fluid with them.

Effect of Combined Cyclic Stretch and Fluid Shear Stress on Endothelial Cell Morphological Responses

Endothelial cells in vivo are normally subjected to multiple mechanical stimuli such as stretch and fluid shear stress (FSS) but because each stimulus induces magnitude-dependent morphologic responses, the relative importance of each stimulus in producing the normal in vivo state is not clear Using cultured human aortic endothelial cells, this study first determined equipotent levels of cyclic stretch, steady FSS, and oscillatory FSS with respect to the time course of cell orientation. We then tested whether these levels of stimuli were equipotent in combination with each other by imposing simultaneous cyclic stretch and steady FSS or cyclic stretch and oscillatory FSS so as to reinforce or counteract the cells' orientation responses. Equipotent levels of the three stimuli were 2% cyclic stretch at 2%/s, 80 dynes/cm2 steady FSS and 20 +/- 10 dynes/cm2 oscillatory FSS at 20 dyne/cm2-s. When applied in reinforcing fashion, cyclic stretch and oscillatory, but not steady, FSS were additive. Both pairs of stimuli canceled when applied in counteracting fashion. These results indicate that this level of cyclic stretch and oscillatory FSS sum algebraically so that they are indeed equipotent. In addition, oscillatory FSS is a stronger stimulus than steady FSS for inducing cell orientation. Moreover, arterial endothelial cells in vivo are likely receiving a stronger stretch than FSS stimulus.

Shear Stress-Induced Cytoskeleton Rearrangement Mediates NF-κB-Dependent Endothelial Expression of ICAM-1

When bone is mechanically loaded fluid shear stress (FSS) is generated as a result of the movement of interstitial fluid across the membranes of osteoblasts and osteocytes. This external mechanical loading stimulates changes in the activity of cytoplasmic signaling molecules and alters gene expression in bone cells. This process, referred to as mechanotransduction, is vital for maintaining bone health in vivo by regulating the balance between bone formation and bone resorption. This current study focuses on the role of focal adhesions, sites of integrin-mediated cellular attachment to the extracellular matrix, and their proposed function as mechanosensors in bone cells. We examined the role of a key component of focal adhesions and of mechanotransduction, focal adhesion kinase (FAK) in regulation of FSS- and tumor necrosis factor-α (TNF-α)-induced activation of nuclear factor-kappa B (NF-κB) signaling in osteoblasts. Immortalized FAK+/+ and FAK−/− osteoblasts were exposed to periods of oscillatory fluid shear stress (OFF) and NF-κB activation was analyzed. We determined that FAK is required for OFF-induced nuclear translocation and activation of NF-κB in osteoblasts. In addition we found that OFF-induced phosphorylation of the IκB kinases (IKKα/β) in both FAK+/+ and FAK−/− osteoblasts, but only FAK+/+ osteoblasts demonstrated the resulting degradation of NF-κB inhibitors IκBα and IκBβ. OFF did not induce the degradation of IκBε or the processing of p105 in either FAK+/+ and FAK−/− osteoblasts. To compare the role of FAK in mediating OFF-induced mechanotransduction to the well characterized activation of NF-κB by inflammatory cytokines, we exposed FAK+/+ and FAK−/− osteoblasts to TNF-α. Interestingly, FAK was not required for TNF-α induced NF-κB activation in osteoblasts. In addition we determined that TNF-α treatment did not induce the degradation of IκBβ as did OFF. These data indicate a novel relationship between FAK and NF-κB activation in osteoblast mechanotransduction and demonstrates that the mechanism of FSS-induced NF-κB activation in osteoblasts differs from the well characterized TNF-α-induced activation.

Influence of sickle hemoglobin polymerization and membrane properties on deformability of sickle erythrocytes in the microcirculation.

The rheological properties of normal erythrocytes appear to be largely determined by those of the red cell membrane. In sickle cell disease, the intracellular polymerization of sickle hemoglobin upon deoxygenation leads to a marked increase in intracellular viscosity and elastic stiffness as well as having indirect effects on the cell membrane. To estimate the components of abnormal cell rheology due to the polymerization process and that due to the membrane abnormalities, we have developed a simple mathematical model of whole cell deformability in narrow vessels. This model uses hydrodynamic lubrication theory to describe the pulsatile flow in the gap between a cell and the vessel wall. The interior of the cell is modeled as a Voigt viscoelastic solid with parameters for the viscous and elastic moduli, while the membrane is assigned an elastic shear modulus. In response to an oscillatory fluid shear stress, the cell--modeled as a cylinder of constant volume and surface area--undergoes a conical deformation which may be calculated. We use published values of normal and sickle cell membrane elastic modulus and of sickle hemoglobin viscous and elastic moduli as a function of oxygen saturation, to estimate normalized tip displacement, d/ho, and relative hydrodynamic resistance, Rr, as a function of polymer fraction of hemoglobin for sickle erythrocytes. These results show the transition from membrane to internal polymer dominance of deformability as oxygen saturation is lowered. More detailed experimental data, including those at other oscillatory frequencies and for cells with higher concentrations of hemoglobin S, are needed to apply fully this approach to understanding the deformability of sickle erythrocytes in the microcirculation. The model should be useful for reconciling the vast and disparate sets of data available on the abnormal properties of sickle cell hemoglobin and sickle erythrocyte membranes, the two main factors that lead to pathology in patients with this disease.