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1Center for Cardiovascular Science, Albany Medical College, Albany 12208; 2Department of Biomedical Engineering, Rensselaer Polytechnic Institute, Troy, New York 12180; 3Vascular Biology and Angiogenesis Program, Sidney Kimmel Cancer Center, San Diego, California 92121; and 4Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104
Submitted 18 April 2002 ; accepted in final form 14 June 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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shear stress; endothelial cells
The most current understanding of hemodynamically induced responses in endothelium comes from experiments performed on cultured cells with the use of devices engineered to mimic the fluid forces generated in vivo. Although significant insights into endothelial mechanosignaling pathways have been obtained by exposure to flow in vitro, the removal of endothelial cells from their natural flow environment induces significant structural changes. There is a drift toward a dedifferentiated phenotype, including the loss of caveolae. Interestingly, cultured endothelial cells gradually change when returned to a physiologically relevant flow environment. Hemodynamic shear stress induces endothelial cell elongation and coaxial alignment of cytoskeletal elements with the direction of flow (10), focal adhesion rearrangement (6), spatial normalization of several junctional proteins (7, 19), and expression of a wide variety of genes (11, 18). As shown here, hemodynamic shear stress restores an important characteristic of endothelial cells in vivo, namely, a greater abundance of caveolae. Thus an appropriate model for investigating the effects of fluid forces on endothelial cell structure and function is to condition the cells to a particular hemodynamic environment before any subsequent flow challenge.
We have investigated flow preconditioning of cultured endothelial cells in relation to the expression and spatial location of caveolae and caveolar-residing proteins, including caveolin-1. We show that physiologically relevant levels of shear stress enhance the density of caveolae to a level that more closely resembles their distribution in vivo, and recruits them into mechanotransduction pathways in response to flow challenge. Thus the chronic hemodynamic environment may influence the abundance of caveolae signaling domains present at the cell surface, an effect that could have important regional functional consequences in the arterial tree.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Parallel plate apparatus. To assess both hemodynamic-dependent regulation of endothelial structure and function and the mechanisms governing endothelial responses to changing fluid forces, an in vitro system designed to mimic the forces imposed on the endothelium in vivo was used (7, 8). The flow chamber consisted of a Teflon upper plate and a stainless steel bottom plate held together by 12 screws. A medical-grade silicon gasket was used to seal the chamber and avoid fluid leakage. A precisely machined recess on the top plate defined the flow path in the chamber. The top plate also housed inlet and outlet ports and three quartz windows for light transmission and sample visualization. The bottom plate was machined flat and polished to a mirror finish with an opening for the placement of three endothelial slides. Because the channel height in the flow chamber is much less than its width, the flow is considered two-dimensional with a uniform wall shear stress given by the equation 
= 6 µU/H, where U is the mean velocity of the flow through the channel, H is the channel height, and µ is the dynamic viscosity of the fluid. The parallel plate chamber was connected to a recirculating flow circuit composed of a variable speed peristaltic pump, a fluid capacitor to dampen pulsation, and a reservoir containing culture medium. Temperature was maintained at 37°C, and pH and oxygen levels were controlled by a 95% O2-5% CO2 humidified gas mixture that was blown over the medium in the reservoir.
Shear stress protocols. For preconditioning, a uniform laminar pattern of flow to generate shear stress (10 dyn/cm2) was applied to endothelial cell monolayers for 6 h. At this magnitude and duration of shear stress, endothelial cells develop parallel arrays of stress fibers and begin to reacquire many in vivo characteristics indicating a moderate level of preconditioning. For the step-flow experiments, shear stress was increased by an additional 5 dyn/cm2 for 2 min at the end of the acclimation period. At the conclusion of the experiments, cells were processed for analysis as described below. A minimum of three independent experiments was performed at each flow condition.
Purification of endothelial luminal plasma membranes. To determine the subcellular distribution of caveolin and caveolar-residing proteins and to localize mechanotransduction events occurring in response to shear stress, luminal endothelial cell plasma membranes were isolated as previously described (27, 28). At the completion of the stress experiments, cells were rapidly cooled to 4°C by being rinsed with ice-cold 2-(N-morpholino)ethanesulfonic acid-buffered saline (MBS; pH 6.0) to stabilize enzymatic activity and the diffusion of molecules within the lipid bilayer of the plasma membrane. Cells were then incubated with a positively charged colloidal silica solution for 10 min at 4°C. Subsequent cross linking of the silica particles by incubation with polyacrylic acid (0.1%) served to create a stable adherent silica pellicle that permitted purification by centrifugation to separate the silica-coated endothelial cell plasma membranes from the whole cell homogenate. Silica-coated endothelial cell monolayers were scraped and pooled in 1 ml of HEPES-buffered sucrose containing a cocktail of protease inhibitors. After sample homogenization, whole cell homogenates were mixed with 102% (wt/vol) Nycodenz (Life Sciences) with 20 mM KCl to make a 50% final solution. The Nycodenz/homogenate solution was layered over a continuous 55–70% Nycodenz gradient containing 20 mM KCl and HEPES-buffered sucrose and centrifuged in a Beckman SW 55 rotor at 15,000 revolutions/min for 30 min at 4°C. The resulting pellet was resuspended in 0.5 ml of MBS.
Immunoaffinity isolation of caveolae. To determine whether mechanotransduction events occurred generally on the luminal plasma membrane or within plasma membrane micrdomains, caveolae were isloated as previously described (20). Briefly, goat anti-mouse IgG-coated magnetic beads (Dynal; Oslo, Norway) were preabsorbed with a specific monoclonal antibody that recognizes the oligomeric form of caveolin-1 (25 µg; Clone 2234 from Transduction Laboratories, Lexington, KY). The antibody-bead conjugates were washed and incubated for 1 h at 4°C with cell lysates prepared by sonication. Beads with any attached membranes were separated magnetically from unbound material, washed, and then processed for SDS-PAGE and immunoblotting.
Western blot analysis. Protein content of the whole cell homogenates or purified luminal membranes was determined by bicinchoninic acid analysis (Pierce). Equivalent amounts of protein from each sample were prepared and separated by SDS-PAGE (5–15% gradient gels), followed by electrotransfer to nitrocellulose filters. Immunoblotting with antibodies against phosphotyrosine (pY20; Transduction Laboratories or 4G10; Upstate) detected rapid mechanotransduction responses. Tyrosine phosphorylated caveolin-1 was detected with either mouse monoclonal (pY14, Transduction Laboratories) or goat polyclonal (Santa Cruz) primary antibodies. Activation of eNOS (Ser1179; Cell Signaling) and ERK1/2 mitogen-activated protein (MAP) kinases was assessed with primary antibody raised to detect only their phosphorylated form (Promega). Other primary antibodies were used to characterize expression and distribution of well-known caveolar proteins (caveolin-1 and eNOS; Transduction Laboratories, and Gq, a kind gift from Dr. David Manning, University of Pennsylvania) and the noncaveolar proteins 
-actin and 5'-nucleotidase. Proteins of interest were detected with the use of enhanced chemiluminescence substrate (Amersham). Autoradiograms were scanned and digitized (Molecular Dynamics; Sunnyvale, CA). Densitometric quantification of immunoblots with the use of Image-Quant software (Molecular Dynamics) enabled direct comparisons between no-flow control and shear stress stimulated cells. Results were normalized by arbitrarily setting the densitometry value of control cells to 1.0. Digital images were transferred to a personal computer and printed using Abode PhotoShop.
Fluorescence microscopy. Fluorescence microscopy served as an independent technique to qualitatively examine caveolae/caveolin expression and distribution within static and shear exposed endothelial cells. Cells were fixed with 3% parafomaldehyde and permeabilized with saponin, as previously described (17). After incubation in blocking solution (2% goat serum), cells were incubated for 1 h with 5 µg/ml caveolin-1 polyclonal antibody (Transduction Laboratories). A reporter secondary antibody conjugated to Texas red dye was used to indirectly label caveolin-1. Caveolin protein within control and sheared endothelial monolayers was visualized with the use of an inverted fluorescent microscope (model MD IRB; Leica) equipped with a x63 PlanApo objective.
To assess shear-induced morphological changes, the actin cytoskeleton was visualized via fluorophore-conjugated phalloidin binding to filamentous actin. Disappearance of cortical actin banding pattern and emergence of a network of fine actin fibers within the cell served to qualitatively define shear-induced changes in actin architecture. Fluorescent images were captured with the use of an Optronics digital camera operated by Magnafire software.
Transmission electron microscopy. To directly examine induction of caveolae expression and distribution, cells were processed for transmission electron microscopy (TEM). Briefly, after fixation for 1 h with cold 3% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3), and following several washes with 0.2 M cacodylate, the cells were gently scraped from the glass slides and pelleted by centrifugation. Cell pellets were then postfixed with 2% osmium tetroxide in 0.1 m cacodylate for 1 h at 4°C, stained en bloc with 2% uranyl acetate for an additional 1 h, rinsed with double-distilled water, dehydrated through a graded series of ethanol, and embedded in epoxy resin. Ultrathin sections (60–90 nm) were collected on copper grids and examined with the use of an electron microscope (model 100-CX; JEOL) operating at 80 kV. Random fields taken from individual endothelial cell samples (n = 25 for each group) were photographed at x35,000. Caveolae associated with the plasma membrane were quantified by counting the number of distinct flask-shaped, noncoated vesicles (50–90 nm in diameter) found on or within 100 nm of the plasma membrane. The morphometric data are expressed as number of caveolae per unit plasma membrane length measured in each micrograph. Differences in caveolae expression and distribution between groups were compared by a two-tailed Student's t-test.
Statistical analysis. Data from a minimum of three independent experiments were pooled according to group. Mean and standard deviation were calculated, and differences between groups were analyzed with an unpaired two-tailed Student's t-test using StatGraphics software (version 4.0, Statistical Graphics). Differences between control and experimental groups were considered to be significant at P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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-subunit Gq, which is enriched in caveolar microdomains (21), was unaltered by shear stress acclimatization. Proteins considered to reside outside of caveolae, such as the cytoskeletal protein -actin and the glycosylphosphatidylinositol (GPI)-anchored protein 5'-nucleotidase, also showed no change in their total cellular expression in response to laminar shear stress.
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In contrast, densitometric analyses of purified endothelial plasma membranes revealed a fivefold increase in caveolin expression in cells exposed to prolonged shear stress compared with static controls (Fig. 1B). In addition, the distribution of eNOS (3.8-fold), and Gq (3.2-fold) paralleled caveolin expression at the luminal cell surface (Fig. 1B). Expression of -actin was also enhanced (twofold) at the luminal cell surface, which serves to further demonstrate that cytoskeletal remodeling is an important adaptive response to shear stress. The GPI-anchored protein 5'-nucleotidase showed no difference in expression in plasma membranes purified from sheared and nonsheared monolayers. These data show that exposure to prolonged laminar shear stress enhances endothelial luminal cell surface caveolin and the caveolae resident molecules eNOS and Gq but not the noncaveolar protein 5'-nucleotidase.
Prolonged laminar shear stress alters caveolin-1 distribution within endothelial cells. Cultured endothelial cells subjected to physiological levels of laminar shear stress (10 dyn/cm2) for 6 h in a parallel plate device developed prominent stress fibers and demonstrated a differential pattern of caveolin-1 expression and distribution compared with controls when examined by fluorescence microscopy (Fig. 2). In no-flow control cells, the fluorescence signal for caveolin-1 was evident at cell borders (arrows), in an intracellular juxtanuclear compartment (arrowheads), possibly Golgi and/or endosomes, and as a low level of punctate labeling over the cell surface. This pattern is consistent with previously observed localization of caveolin in a variety of cultured cell types (4, 17, 25). However, shear-acclimated endothelial cells displayed a higher level of punctate staining for caveolin over the cell surface as well as enhanced caveolin labeling at the cell periphery. Concomitant with the apparent shift in caveolin distribution to the surface membrane was a loss of fluorescence signal from intracellular compartments.
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Caveolae surface densities increase as part of endothelial adaptive response to flow. Although caveolin-1 is generally accepted to be a molecular marker for caveolae based on observations that caveolin is necessary for the formation of invaginated caveolae (9, 26), we confirmed that shear-induced increases in plasma membrane caveolin resulted in morphologically distinct caveolae. Static and shear-conditioned BAEC were examined by TEM and the number of invaginated caveolae associated with each plasma membrane was quantified. Caveolae frequency was measured and found to be consistent with previous reports for BAEC (19) (0.2 ± 0.03 caveolae/µm of plasma membrane) (Fig. 3). Although present mostly as single, noncoated membrane invaginations, caveolae were also assembled as 3–5 vesicle clusters attached to the cell surface. After 6 h of laminar shear stress, the caveolae density at the plasma membrane increased sixfold (Fig. 3B).
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Mechanotransduction sensitivity of luminal plasma membrane is enhanced in shear-acclimated endothelial cells. Because the subcellular localization of caveolae to the surface of cultured endothelial cells appears to be influenced by prevailing hemodynamic forces, we asked whether the ability of the endothelium to transduce fluid-mechanical signals was subsequently enhanced. The luminal plasma membranes of endothelial cells were examined for changes of mechanosignaling sensitivity when subjected to shear stress. Induction of protein tyrosine phosphorylation is an indicator of shear stress and caveolar responses (28). Luminal endothelial cell surface plasma membranes purified from static cultured cells displayed a relatively weak compliment of phosphorylated tyrosine residues (Fig. 4A). Shear-acclimation further reduced the level of protein tyrosine phosphorylation at the cell surface. In contrast, an acute (2 min) increase in shear stress to 15 dyn/cm2 induced protein tyrosine phosphorylation at the cell surface in both static and shear-conditioned endothelial cells (Fig. 4A). From static culture conditions, exposure to 15 dyn/cm2 shear stress increased the phosphotyrosine signal on the luminal plasmalemma by 1.7-fold compared with no-flow controls. More striking, however, was a near sevenfold induction of phosphotyrosine signal in the plasma membranes of shear-conditioned cells after challenge by an additional 5 dyn/cm2 shear stress (Fig. 4A).
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Luminal plasma membrane fractions were also analyzed for shear-induced activation of specific caveolae associated molecules, caveolin-1, and eNOS. Similar to the general pattern observed for protein tyrosine phosphorylation, caveolin-1 phosphorylation was slight in cells cultured under static conditions (Fig. 4B). Shear stress applied at 5 dyn/cm2 for 2 min enhanced the tyrosine phosphorylation of luminal membrane caveolin by 1.3-fold (Fig. 4B). Caveolin-1 phosphorylation reached a twofold enhancement at 5 min and returned near baseline after 10 min of shear stress (data not shown). Control levels of caveolin-1 tyrosine phosphorylation were sustained through to 6 h of continuous exposure to laminar shear stress (Fig. 4B). After an acute, 2 min, step change of 5 dyn/cm2 shear stress to preconditioned endothelial cell cultures, caveolin-1 tyrosine phosphorylation increased by 5.8-fold over shear-preconditioned cells alone.
Shear stress rapidly activates eNOS activity through phosphorylation at Ser1179 (2). Consistent with these observations, we observed a 2.6-fold increase in eNOS Ser1179 phosphorylation within 2 min of shear stress applied to static cultures (Fig. 4B). Unlike caveolin-1 phosphorylation, eNOS phosphorylation remained elevated after 6 h of laminar shear stress (1.8-fold over static controls). Preconditioned BAEC subjected to a shear step of 5 dyn/cm2 for 2 min showed a 1.5-fold increase in Ser1179 phosphorylation compared with preconditioned cell cultures and a total of 3.3-fold over levels detected in static control cells (Fig. 4B).
Mechanotransduction occurs in caveolae. We previously reported (27, 28) that altering flow through the rat lung vasculature stimulates key signaling molecules in endothelial cell caveolae, resulting in localized signal transduction with activation eNOS and the Ras/Raf/MAP kinase pathway. To test whether the shear-initiated signaling responses observed at the luminal plasma membrane (Fig. 4) are localized to caveolar microdomains, caveolar vesicles were immunoaffinity purified from shear-acclimated and -challenged endothelial cells and analyzed for mechanotransduction events. Figure 5 illustrates the efficiency of caveolae isolation using the immunoaffinity methodology with nearly 95% of starting caveolin-1 signal (starting material; SM) recovered in the bound fraction (B). Consistent with the distribution of eNOS within rat lung endothelia (27), approximately 90% of detected eNOS colocalized to caveolar vesicles in shear-preconditioned cells. Interestingly, we found eNOS to be evenly distribution between bound and unbound fractions in static control cells (data not shown). These observations further support the concept that shear preconditioning directs endothelial cells toward their in vivo phenotype. Acute shear challenge to flow-acclimated BAEC again induced the phosphorylation of both caveolin-1 and eNOS (SM). Tyrosine phosphorylated caveolin-1 colocalized almost exclusively to the caveolae fraction (B), whereas 72% of phospho-Ser1179 eNOS was found in caveolae. That nearly 30% of phosphorylated eNOS was detected in the unbound fraction suggests that phosphorylation alters the enzymes ability to remain in caveolae.
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Shear preconditioning desensitizes ERK1/2 to further changes in laminar shear stress. Activation of ERK1/2 MAP kinases is considered a hallmark mechanotransduction response to flow. Shear-induced ERK1/2 activation occurs within 5–10 min of initiating shear stress in cultured endothelial cells (13, 23, 33) and within 1–3 min in endothelium subjected to flow in situ (28). The mechanotransduction mechanism leading to MAP kinase activation appears to involve cholesterol-sensitive microdomains of the plasma membrane including caveolae (23, 28). Because flow-preconditioned endothelial cells show enhanced caveolae expression at the plasma membrane, an observation that correlates with increased mechanosignaling sensitivity at the luminal cell surface, we sought to determine whether downstream ERK1/2 activation was also altered in shear-acclimated cells. As reported previously, ERK1/2 was weakly activated (1.4-fold) after 2 min of shear stress applied at 5 dyn/cm2 to static endothelial cell cultures (Fig. 6) (13, 23, 33). Shear stress of 15 dyn/cm2 applied for the same 2-min period showed a fourfold increase in ERK1/2 activity (data not shown), demonstrating a shear stress dose response for MAP kinase. In shear-conditioned monolayers, however, a 2-min exposure to a step change of 5 dyn/cm2 shear stress did not enhance ERK1/2 activity (Fig. 6). Thus shear-acclimated BAEC appears to have lost sensitivity to activate ERK1/2 MAP kinases.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Two recent reports using microarray technology reveal expression changes in a wide variety of human umbilical vein endothelial cell genes when the cell were subjected to laminar (18) and/or turbulent (11) shear stresses for intervals as long as 24 h. Some of the changes represent adaptation to a continuous period of flow, suggesting that some recapitulation of in vivo phenotype might be achieved by preconditioning endothelial monolayers to a relevant hemodynamic environment before initiating subsequent changes in flow. In steady flow, the endothelium eventually reacquires a morphology that more closely resembles that of cells present in nonbranching regions of an intact blood vessel (1). In addition, during flow-induced changes of cell shape, endothelial cells initially disassemble adherence junctions and then reassemble them as cell shape change reaches completion. The stable adherens plaques that form are structurally distinct from the junctions of endothelial cells observed under no-flow conditions (19). Another recent example of redifferentiation on acclimation to flow is the reassembly of connexin43 gap junctional structures in BAEC (7). Laminar flow induced an increase in connexin43 mRNA and protein expression that returned to baseline within 16 h. A consequence was restoration of cell-cell communication in the monolayer subjected to unidirectional laminar shear stress as measured by dye transfer between cells. Taken together, these data suggest that the endothelium possess plasticity in modulating its constitutive phenotype in response to the hemodynamic microenvironment.
The cell surface density of endothelial caveolae is greatly decreased in tissue culture when compared with cells in vivo (30) and is accompanied by the redistribution of caveolin, and possibly caveolar molecules such as eNOS, to other compartments of the cell such as the Golgi (V. Rizzo, unpublished observations). Like adherens and gap junctional proteins, recent studies (including data presented here) suggest that prolonged exposure to physiological shear stress can alter the expression and/or distribution of caveolae and caveolin within the endothelium (14, 32).
With the use of three-dimensional fluorescence microscopy, Sun et al. (32) showed that shear stress induced a time-dependent variation in caveolin-1 distribution. Endothelial cell cultures exposed to 4 h of laminar shear stress enhanced caveolin-1 intracellular distribution and by 12 h accumulated at the upstream side of the cell. This local concentration effect became significantly enhanced after 24 h of exposure to shear. The authors suggest a correlation between caveolin-1 translocation and hemodynamic force distribution because mechanical forces are asymmetrically imposed over the cell surface with the upstream side of the cell experiencing maximum hydrostatic pressure and high spatial gradients of shear stress. In a separate study, shear stress also induced the gradual relocation of caveolae and caveolin-1 to the endothelial cells up-stream edge (14). Unlike the former study, however, shear stress did not enhance the expression of caveolin-1 mRNA or protein levels in the cell, suggesting redistribution from an existing caveolin intracellular compartment (such as the Golgi) to the plasma membrane. Interestingly, a similar pattern of caveolin-1 redistribution was observed after wound induction to endothelial cell monolayers. Because both conditions can stimulate cell migration, it was hypothesized that polarization of caveolae on the cell surface is attributed to the migratory process.
Here, we provide further verification that prolonged exposure to physiological shear stress alters the distribution of caveolae and caveolin-1 within the endothelium. Purified luminal cell membranes markedly increased their content of caveolin-1 by preconditioning with shear stress for 6 h without a concomitant increase in total cellular caveolin-1 protein levels. In addition, the distribution of eNOS and Gq, which have been shown to be highly enriched in caveolae and to interact directly with caveolin (21, 23–27), parallels caveolin expression at the luminal cell surface. The spatial localization of caveolin was confirmed by fluorescence microscopy demonstrating enhanced surface labeling in shear-exposed cells. In contrast to the prior studies, local accumulation of caveolin-1 at the up-stream surface of the cell was not obvious after 6 h of shear stress. In agreement with Western blot analysis and the immunoflorescence observations of caveolin, TEM morphometric analysis of caveolae associated with the plasma membrane demonstrated a significantly increased frequency in cells preconditioned to flow. These results are in general agreement with a previous TEM study showing that exposure of BAEC to shear for 1 h increased caveolae density threefold (23). Taken together, these data suggest that cultured endothelial cells recruit caveolin-1 and caveolae to the cell surface during adaptation to flow, and, with sufficient time, relocates to the upstream cell surface. Because upstream clustering of caveolae is not a commonly described feature of endothelial cells in vivo, the significance of local accumulation of caveolae during flow adaptation bears further investigation.
Because luminally organized caveolae appear to function as mechano-signaling centers (23, 24, 27, 28), it is implicit in our findings that flow-conditioned endothelial cells may be more mechanosensitive. Here, we showed that the overall extent of protein tyrosine phosphorylation within the luminal membrane and the phosphorylation state of caveolar enriched molecules, caveolin-1 and eNOS was significantly more sensitive to an acute change of shear stress in preconditioned endothelial cells (2 min; 5 dyn/cm2). Although phosphorylation of caveolin-1 on tyrosine 14 has been described in cells exposed to osmotic and oxidative stress (35), our study is the first to demonstrate phosphorylation of caveolin-1 by acute changes in shear stress. This response was accelerated and more robust in endothelial cell cultures conditioned to6hof sustained laminar shear stress (Fig. 4B). To date, tyrosine-phosphorylated caveolin-1 has been shown to localize primarily to focal contact regions on the basal cell surface (35). Here, we observed that caveolin-1 in apical cell membrane fractions is also tyrosine phosphorylated after acute changes in shear stress (Fig. 4B). Furthermore, phosphocaveolin-1 could be detected within caveolar vesicles (Fig. 5). Although the functional significance of caveolin-1 tyrosine phosphorylation is currently unknown, it has been suggested that pY14 may serve as an SH2 domain for docking of signaling molecules (35). Therefore, elucidation of the spatial location and functional consequence of caveolin-1 phosphorylation by shear stress may bring important new insights into the mechanism of mechanotransduction.
In addition to caveolin-1 phosphorylation, an acute change in shear stress rapidly activated eNOS localized at the endothelial cell luminal surface (Fig. 4B). We observed that phosphorylation of eNOS Ser1179 remained above static control cell levels even after 6 h of flow preconditioning. These data indicate that a pool of total eNOS is phosphorylated, hence activated, by prevailing hemodynamic environment. This observation lends further support to the concept that chronic laminar shear stress induces sustained release of nitric oxide affording a mechanism for the atheroprotective effect attributed to laminar shear stress. As we observed in the rat lung profusion model, shear challenge to flow-preconditioned endothelial cell cultures enhanced and accelerated eNOS activation (Fig. 4B). Thus the mechanotransduction responses observed in shear-acclimated cells in vitro reflects, in some ways, the caveolae-mediated mechanosignaling responses to acute changes in shear stress that were observed in the in vivo system (27).
ERK1/2 signaling has been shown to be activated in both cultured BAEC and in human umbilical vein endothelial cells exposed to a step increase of shear stress (3–35 dyn/cm2), peaking within 5–15 min after the initiation of flow (13, 33). Similarly, an increase of flow through the lung vasculature activated ERK albeit in a more rapid manner (1–3 min) (28). Abrogation of caveolar architecture with cholesterol-sequestering compounds prevented the flow-induced signaling cascade that leads to MAP kinase activation (28) implicating a role for cholesterol-rich regions of the plasma membrane (these include caveoli) in the mechanotransduction mechanism. Corroborating studies (23) also demonstrated that shear activation of ERK was sensitive to membrane cholesterol content in cultured endothelial cells. Further work by Park et al. (24) showed that shear-induced MAP kinase activation could be blocked by introduction of polyclonal caveolin-1 antibody inside the cell. Because polyclonal caveolin-1 antibody targeted the scaffolding and oligomerization domains of caveolin-1, inhibition of shear induced MAP kinase activation was likely achieved through disruption of caveolin clusters or its interaction with signaling molecules involved in shear-sensitive ERK pathway. From these studies, we concluded that mechanical forces stimulate key signaling molecules in caveoli resulting in localized signal transduction through the activation of the MAP kinase pathway.
Laminar shear stress has been demonstrated to decrease mitotic index and cell-cycle progression of endothelial cells in vivo and in vitro (5). Because activation of ERK is usually associated with a proliferative response, it seems counterintuitive to expect laminar shear stress imposed on static culture cells to initiate an ERK response. In contrast, endothelial cells found at vascular branch points, which are regions prone to endothelial cell dysfunction and atherosclerotic plaque formation, show a higher proliferative phenotype (5). These observation support the concept that static endothelial cell more closely resemble endothelium found in disturbed flow regions in the vasculature and hence, do not represent "normal" endothelia.
Our present report demonstrates that shear preconditioned BAEC cultures did not respond to an additional challenge of shear stress with activation of the ERK1/2 MAP kinase pathway despite the enhanced placement of mechanosensitive caveolae at the luminal plasma membrane. That enhanced density of caveolae at the luminal cell surface did not translate to a concomitant change in ERK activation in shear preconditioned cells may reflect that other mechanosensing elements that influence ERK activation may also be altered during flow acclimation. Whether additional mediators of shear induced ERK activity, such as integrins (3, 34), vascular endothelial growth factor receptors (3), and platelet endothelial cell adhesion molecule (22), are affected during shear acclimation remains to be tested. Although the rat lung model used in our previous studies allows for an investigation of the endothelium in their native state, the ability to define the precise hemodynamic force parameters applied to the endothelial cell layer was lacking. Therefore, activation of the MAP kinase system in that model may be due to a combination of shear/pressure and/or stretch. Thus the need to define more appropriate in vivo models that will allow for precise control over hemodynamics imposed through the system is needed to make better comparisons between in vitro and in vivo findings.
In summary, endothelial caveolae are potentially an efficient means to regulate cell surface signaling. By concentrating a variety of signaling molecules and their immediate substrates within a small invaginated microdomain, the necessary proximity to propagate signals to downstream pathways is achieved. Therefore, the regulation of caveolar expression, and its subcellular distribution, is likely to be an important mechanism utilized by endothelial cells to adjust their sensitivity to mechanical stimuli. In the present studies, we have demonstrated that prolonged exposure to physiological shear stress alters the distribution but not the expression of the caveolar marker protein caveolin-1 within the endothelium. The subcellular localization of these molecules is indicative of their distribution in vivo and suggests that prolonged shear stress may induce endothelial cell maturation toward an in vivo (28) phenotype. Preconditioning the endothelium to flow greatly enhanced its ability to detect and respond to changes in shear stress conditions suggesting that shear stress may dynamically regulate both caveolae structure and signaling. Regional differences of blood flow would be expected to have important consequences in determining the endothelial cell signaling capacity through the modulation of the cell surface caveolae and this is of potential relevance to the regional localization of pathologies such as atherogenesis.
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DISCLOSURES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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