Oscillatory shear stress and hydrostatic pressure modulate cell-matrix attachment proteins in cultured endothelial cells

Summary Endothelial cells (ECs) may behave as hemodynamic sensors, translating mechanical information from the blood flow into biochemical signals, which may then be transmitted to underlying smooth muscle cells. The extracellular matrix (ECM), which provides adherence and integrity for the endothelium, may serve an important signaling function in vascular diseases such as atherogenesis, which has been shown to be promoted by low and oscillating shear stresses. In this study, confluent bovine aortic ECs (BAECs) were exposed to an oscillatory shear stress or to a hydrostatic pressure of 40 mmHg for time periods of 12 to 48 h. Parallel control cultures were maintained in static condition. Although ECs exposed to hydrostatic pressure or to oscillatory flow had a polygonal morphology similar to that of control cultures, these cells possessed more numerous central stress fibers and exhibited a partial loss of peripheral bands of actin, in comparison to static cells. In EC cultures exposed to oscillatory flow or hydrostatic pressure, extracellular fibronectin (Fn) fibrils were more numerous than in static cultures. Concomitantly, a dramatic clustering ofα ₅β₁ Fn receptors and of the focal contact-associated proteins vinculin and talin occurred. Laminin (Ln) and collagen type IV formed a network of thin fibrils in static cultures, which condensed into thicker fibers when BAECs were exposed to oscillatory shear stress or hydrostatic pressure. The ECM-associated levels of Fn and Ln were found to be from 1.5-to 5-fold greater in cultures exposed to oscillatory shear stress or pressure for 12 and 48 h, than in static cultures. The changes in the organization and composition of ECM and focal contacts reported here suggest that ECs exposed to oscillatory shear stress or hydrostatic pressure may have different functional characteristics from cells in static culture, even though ECs in either environment exhibit a similar morphology.     

Fluid shear stress stimulates prostaglandin and nitric oxide release in bone marrow-derived preosteoclast-like cells

Bone is a porous tissue that is continuously perfused by interstitial fluid. Fluid flow, driven by both vascular pressure and mechanical loading, may generate significant shear stresses through the canaliculi as well as along the bone lining at the endosteal surface. Both osteoblasts and osteocytes produce signaling factors such as prostaglandins and nitric in response to fluid shear stress (FSS); however, these humoral agents appear to have more profound affects on osteoclast activity at the endosteal surface. We hypothesized that osteoclasts and preosteoclasts may also be mechanosensitive and that osteoclast-mediated autocrine signaling may be important in bone remodeling. In this study, we investigated the effect of FSS on nitric oxide (NO), prostaglandin E(2) (PGE(2)), and prostacyclin (PGI(2)) release by neonatal rat bone marrow-derived preosteoclast-like cells. These cells were tartrate-resistant acid phosphatase (TRAP) positive, weakly nonspecific esterase (NSE) positive, and capable of fusing into calcitonin-responsive, bone-resorbing, multinucleated cells. Bone marrow-derived preosteoclast-like cells exposed for 6 h to a well-defined FSS of 16 dynes/cm(2) produced NO at a rate of 7.5 nmol/mg protein/h, which was 10-fold that of static controls. This response was completely abolished by 100 microM N(G)-amino-L-arginine (L-NAA). Flow also stimulated PGE(2) production (3.9 microg/mg protein/h) and PGI(2) production (220 pg/mg protein/h). L-NAA attenuated flow-induced PGE(2) production by 30%, suggesting that NO may partially modulate PGE(2) production. This is the first report demonstrating that marrow derived cells are sensitive to FSS and that autocrine signaling in these cells may play an important role in load-induced remodeling and signal transduction in bone.     

Fluid shear stress enhanced DNA synthesis in cultured endothelial cells during repair of mechanical denudation

We have previously observed a stimulatory effect of fluid shear stress on the regeneration of cultured endothelial cell layers after mechanical denudation. In this study we examined how fluid shear stress affects endothelial cell DNA synthesis during regeneration. Following mechanical denudation of narrow linear areas, monolayers of bovine aortic endothelial cells cultured on plastic dishes were subjected to shear stress of 1.3-4.1 dynes/cm2 for 24-48 hours in a specially designed apparatus. After the application of shear stress, cells were stained with propidium iodide, and its fluorescence intensity, reflecting cellular DNA content, was measured using photometric fluorescence microscopy. The DNA content of cells exposed to shear stress increased significantly more than that of paired, static control cells (p less than 0.005 to p less than 0.001). The DNA histogram showed that cells exposed to shear stress contained a relatively high proportion of cells located in the S, G2, and M phases of the cell cycle as compared with the static control. These data suggest that fluid shear stress enhances endothelial cell DNA synthesis during the repair of mechanical denudation.     

Fluid shear stress-induced alignment of cultured vascular smooth muscle cells

The study objectives were to quantify the time- and magnitude-dependence of flow-induced alignment in vascular smooth muscle cells (SMC) and to identify pathways related to the orientation process. Using an intensity gradient method, we demonstrated that SMC aligned in the direction perpendicular to applied shear stress, which contrasts with parallel alignment of endothelial cells under flow SMC alignment varied with the magnitude of and exposure time to shear stress and is a continuous process that is dependent on calcium and cycloskeleton based mechanisms. A clear understanding and control of flow-induced SMC alignment will have implications for vascular tissue engineering.     

Fluid shear stress in trabecular bone marrow due to low-magnitude high-frequency vibration

Low-magnitude high-frequency (LMHF) loading has recently received attention for its anabolic effect on bone. The mechanism of transmission of the anabolic signal is not fully understood, but evidence indicates that it is not dependent on bone matrix strain. One possible source of signaling is mechanostimulation of the cells in the bone marrow. We hypothesized that the magnitude of the fluid shear stress in the marrow during LMHF loading is in the mechanostimulatory range. As such, the goal of this study was to determine the range of shear stress in the marrow during LMHF vibration. The shear stress was estimated from computational models, and its dependence on bone density, architecture, permeability, marrow viscosity, vibration amplitude and vibration frequency were examined. Three-dimensional finite element models of five trabecular bone samples from different anatomic sites were constructed, and a sinusoidal velocity profile was applied to the models. In human bone models during axial vibration at an amplitude of 1 g, more than 75% of the marrow experienced shear stress greater than 0.5Pa. In comparison, in vitro studies indicate that fluid induced shear stress in the range of 0.5 to 2.0Pa is anabolic to a variety of cells in the marrow. Shear stress at the bone-marrow interface was as high as 5.0Pa. Thus, osteoblasts and bone lining cells that are thought to reside on the endosteal surfaces may experience very high shear stress during LMHF loading. However, a more complete understanding of the location of the various cell populations in the marrow is needed to quantify the effects on specific cell types. This study suggests the shear stress within bone marrow in real trabecular architecture during LMHF vibration could provide the mechanical signal to marrow cells that leads to bone anabolism.     

Hemodynamic shear stress stimulates endothelin production by cultured endothelial cells

We have examined the effect of shear stress on the production of endothelin by cultured porcine endothelial cells. Low shear stress stimulated the expression of endothelin mRNA in polygonal endothelial cells with a peak time of 2 to 4 hours and also increased the release of immunoreactive endothelin into the culture medium. The expression of endothelin mRNA in the ellipsoidal endothelial cells under higher shear stress was not different from that of the control level. Our results suggest a possible role for hemodynamic shear stress in the regulation of endothelin production in vascular endothelial cells.     

The elongation and orientation of cultured endothelial cells in response to shear stress

Vascular endothelial cells appear to be aligned with the flow in the immediate vicinity of the arterial wall and have a shape which is more ellipsoidal in regions of high shear and more polygonal in regions of low shear stress. In order to study quantitatively the nature of this response, bovine aortic endothelial cells grown on Thermanox plastic coverslips were exposed to shear stress levels of 10, 30, and 85 dynes/cm2 for periods up to 24 hr using a parallel plate flow chamber. A computer-based analysis system was used to quantify the degree of cell elongation with respect to the change in cell angle of orientation and with time. The results show that (i) endothelial cells orient with the flow direction under the influence of shear stress, (ii) the time required for cell alignment with flow direction is somewhat longer than that required for cell elongation, (iii) there is a strong correlation between the degree of alignment and endothelial cell shape, and (iv) endothelial cells become more elongated when exposed to higher shear stresses.     

Fluid shear stress induces differentiation of circulating phenotype endothelial progenitor cells

Endothelial progenitor cells (EPCs) are mobilized from bone marrow to peripheral blood, and contribute to angiogenesis in tissue. In the process, EPCs are exposed to shear stress generated by blood flow and tissue fluid flow. Our previous study showed that shear stress induces differentiation of mature EPCs in adhesive phenotype into mature endothelial cells and, moreover, arterial endothelial cells. In this study we investigated whether immature EPCs in a circulating phenotype differentiate into mature EPCs in response to shear stress. When floating-circulating phenotype EPCs derived from ex vivo expanded human cord blood were exposed to controlled levels of shear stress in a flow-loading device, the bioactivities of adhesion, migration, proliferation, antiapoptosis, tube formation, and differentiated type of EPC colony formation increased. The surface protein expression rate of the endothelial markers VEGF receptor 1 (VEGF-R1) and -2 (VEGF-R2), VE-cadherin, Tie2, VCAM1, integrin α(v)/β(3), and E-selectin increased in shear-stressed EPCs. The VEGF-R1, VEGF-R2, VE-cadherin, and Tie2 protein increases were dependent on the magnitude of shear stress. The mRNA levels of VEGF-R1, VEGF-R2, VE-cadherin, Tie2, endothelial nitric oxide synthase, matrix metalloproteinase 9, and VEGF increased in shear-stressed EPCs. Inhibitor analysis showed that the phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signal transduction pathway is a potent activator of adhesion, proliferation, tube formation, and differentiation in response to shear stress. Western blot analysis revealed that shear stress activated the VEGF-R2 phosphorylation in a ligand-independent manner. These results indicate that shear stress increases differentiation, adhesion, migration, proliferation, antiapoptosis, and vasculogenesis of circulating phenotype EPCs by activation of VEGF-R2 and the PI3K/Akt/mTOR signal transduction pathway.     

Cytoplasmic calcium response to fluid shear stress in cultured vascular endothelial cells

Summary Vascular endothelial cells modulate their structure and functions in response to changes in hemodynamic forces such as fluid shear stress. We have studied how endothelial cells perceive the shearing force generated by blood flow and the substance(s) that may mediate such a response. We identify cytoplasmic-free calcium ion (Ca⁺⁺), a major component of an internal signaling system, as a mediator of the cellular response to fluid shear stress. Cultured monolayers of bovine aortic endothelial cells loaded with the highly fluorescent Ca⁺⁺-sensitive dye Fura 2 were exposed to different levels of fluid shear stress in a specially designed flow chamber, and simultaneous changes in fluorescence intensity, reflecting the intracellular-free calcium concentration ([Ca⁺⁺] _i ), were monitored by photometric fluorescence microscopy. Application of shear stress to cells by fluid perfusion led to an immediate severalfold increase in fluorescence within 1 min, followed by a rapid decline for about 5 min, and finally a plateau somewhat higher than control levels during the entire period of the stress application. Repeated application of the stress induced similar peak and plateau levels of [Ca⁺⁺] _i but at reduced magnitudes of response. These responses were observed even in Ca⁺⁺-free medium. Thus, a shear stress transducer might exist in endothelial cells, which perceives the shearing force on the membrane as a stimulus and mediates the signal to increase cytosolic free Ca⁺⁺. This work was partly supported by a grant-in-aid, for Special Project Research no. 61132008, from the Japanese Ministry of Education, Science and Culture and a research fund from the Atherosclerosis Study Association.     

Fluid shear stress regulates the invasive potential of glioma cells via modulation of migratory activity and matrix metalloproteinase expression

Background

Glioma cells are exposed to elevated interstitial fluid flow during the onset of angiogenesis, at the tumor periphery while invading normal parenchyma, within white matter tracts, and during vascular normalization therapy. Glioma cell lines that have been exposed to fluid flow forces in vivo have much lower invasive potentials than in vitro cell motility assays without flow would indicate.

Methodology/Principal Findings

A 3D Modified Boyden chamber (Darcy flow through collagen/cell suspension) model was designed to mimic the fluid dynamic microenvironment to study the effects of fluid shear stress on the migratory activity of glioma cells. Novel methods for gel compaction and isolation of chemotactic migration from flow stimulation were utilized for three glioma cell lines: U87, CNS-1, and U251. All physiologic levels of fluid shear stress suppressed the migratory activity of U87 and CNS-1 cell lines. U251 motility remained unaltered within the 3D interstitial flow model. Matrix Metalloproteinase (MMP) inhibition experiments and assays demonstrated that the glioma cells depended on MMP activity to invade, and suppression in motility correlated with downregulation of MMP-1 and MMP-2 levels. This was confirmed by RT-PCR and with the aid of MMP-1 and MMP-2 shRNA constructs.

Conclusions/Significance

Fluid shear stress in the tumor microenvironment may explain reduced glioma invasion through modulation of cell motility and MMP levels. The flow-induced migration trends were consistent with reported invasive potentials of implanted gliomas. The models developed for this study imply that flow-modulated motility involves mechanotransduction of fluid shear stress affecting MMP activation and expression. These models should be useful for the continued study of interstitial flow effects on processes that affect tumor progression.     

Fluid shear stress suppresses interleukin 8 production by vascular endothelial cells.

The effects of shear stress on interleukin 8 (IL-8) production by human umbilical vein endothelial cells (HUVEC) were studied by subjecting the HUVEC to a steady flow laminar shear stress of up to 0.7 N/m(2) in a parallel plate flow chamber. Shear stress decreased IL-8 mRNA expression in a dose and time-dependent fashion. High glucose concentrations increased IL-8 mRNA levels in a MAPK-p38-dependent manner, which was suppressed by shear stress. Measurement of IL-8 protein in HUVEC culture media by ELISA demonstrated that IL-8 secretion was also increased by high glucose and suppressed by shear stress. These results suggest that the anti-atherogenic effect of shear stress arises partly from the suppression of the production of IL-8 which has been shown to trigger the adhesion of monocytes to a vascular endothelium and also acts as a mitogen and chemoattractant for vascular smooth muscle cells.     

Mood stabilizing drug lithium increases expression of endoplasmic reticulum stress proteins in primary cultured rat cerebral cortical cells

The mood stabilizing drug lithium is a highly effective treatment for bipolar disorder. Previous studies in our laboratory found that chronic treatment with the mood stabilizing drug valproate in rat brain increased the expression of endoplasmic reticulum (ER) stress proteins GRP78, GRP94 and calreticulin. We report here that in primary cultured rat cerebral cortical cells, expression of GRP78, GRP94 and calreticulin are increased not only by valproate, but also by lithium after chronic treatment for 1 week at therapeutically relevant concentrations. However, two other mood stabilizing drugs carbamazepine and lamotrigine had no effect on expression of GRP78, GRP94 or calreticulin. Chronic treatment with lithium for 1 week increased both mRNA and protein levels of ER stress proteins. In contrast to a classic GRP78 inducer thapsigargin, an inhibitor of the ER Ca2+ -ATPase, chronic treatment with lithium or valproate for 1 week modestly increased GRP78 expression in neuronal cells, had no effect on basal intracellular free Ca2+ concentration and does not induce cell death. These results indicate that lithium and valproate may increase expression of GRP78, GRP94 and calreticulin in primary cultured rat cerebral cortical cells without causing cell damage. These results also suggest that the mechanism of GRP78 increase induced by lithium and valproate may be different from that of thapsigargin.     

Fluid shear stress induces cell migration and invasion via activating autophagy in HepG2 cells

Fluid shear stress (FSS) regulates the metastasis of hepatocellular carcinoma (HCC). In the present study, we aimed to study the role of autophagy in HCC cells under FSS. The results showed that FSS upregulated the protein markers of autophagy, induced LC3B aggregation and formation of autophagosomes. Inhibition of integrin by Cliengitide (Cli) or inhibition of the microfilaments formation both inhibited the activation of autophagy in HepG2 under FSS. In addition, Cli inhibited the microfilaments formation and expressions of Rac1 and RhoA in HepG2 cells under FSS. Finally, inhibition of autophagy suppressed the cell migration and invasion in HepG2 under FSS. In conclusion, FSS induced autophagy to promote migration and invasion of HepG2 cells via integrin/cytoskeleton pathways.     

The expression of specific proteins in cultured renal collecting duct cells

It is still uncertain whether cell cultures attain the functional maturity of corresponding in vivo cells. The degree of differentiation of cultured collecting-duct (CD) epithelium cells was therefore examined using immunohistochemical procedures. Three monoclonal antibodies (mabs CD1, CD2, and CD3) were raised against proteins (PCD) isolated from the renal papilla. At Western-blot analysis, each of these antibodies reacted with a specific protein that was distinguishable according to its molecular weight [PCD1, 190 kilodaltons (kDa); PCD2, 210 kDa; PCD3, 50 kDa]. Using immunofluorescence, these proteins were found to be localized exclusively in the renal CD system. Other renal structures, such as the proximal or distal tubular portions, the glomeruli and the interstitial network, were not reactive. The mabs, CD2 and CD3, labeled both the cortical and medullary CD in a uniform way, whereas mab CD1 produced heterogeneous immunolabeling along the length of the cortical, medullary, and papillary CD. As revealed by immunohistochemistry, the mabs revealed differences with respect to the expression of the specific renal proteins in cultured CD cells. In polar-differentiated epithelium cultured for 5 days on a specific renal support, mab CD1 was unreactive, whereas mabs CD2 and CD3 were positive. This demonstrated the biochemical immaturity of this cultured epithelium with respect to CD1 reactivity. In morphologically dedifferentiated CD monolayer cells grown on the bottom of a culture dish, only a weak reaction for mab CD3 was observed. The loss of epithelial polarization in CD monolayer cells obviously coincides with the absence of the renal proteins PCD1 and PCD2.(ABSTRACT TRUNCATED AT 250 WORDS)     

The expression of specific proteins in cultured renal collecting duct cells

Summary It is still uncertain whether cell cultures attain the functional maturity of corresponding in vivo cells. The degree of differentiation of cultured collecting-duct (CD) epithelium cells was therefore examined using immunohistochemical procedures. Three monoclonal antibodies (mabs CD 1, CD 2, and CD 3) were raised against proteins (P_CD) isolated from the renal papilla. At Western-blot analysis, each of these antibodies reacted with a specific protein that was distinguishable according to its molecular weight [P_CD1, 190 kilodaltons (kDa); P_CD2, 210 kDa; P_CD3, 50 kDa]. Using immunofluorescence, these proteins were found to be localized exclusively in the renal CD system. Other renal structures, such as the proximal or distal tubular portions, the glomeruli and the interstitial network, were not reactive. The mabs, CD 2 and CD 3, labeled both the cortical and medullary CD in a uniform way, whereas mab CD 1 produced heterogeneous immunolabeling along the length of the cortical, medullary, and papillary CD. As revealed by immunohistochemistry, the mabs revealed differences with respect to the expression of the specific renal proteins in cultured CD cells. In polar-differentiated epithelium cultured for 5 days on a specific renal support, mab CD 1 was unreactive, whereas mabs CD 2 and CD 3 were positive. This demonstrated the biochemical immaturity of this cultured epithelium with respect to CD 1 reactivity. In morphologically dedifferentiated CD monolayer cells grown on the bottom of a culture dish, only a weak reaction for mab CD 3 was observed. The loss of epithelial polarization in CD monolayer cells obviously coincides with the absence of the renal proteins P_CD1 and P_CD2. Thus, our mabs proved to be valuable tools for the investigation of the differentiation and dedifferentiation of renal CD cells in culture.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday