Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

Pulmonary endothelial cells (EC) form a semiselective barrier to macromolecule transport into the lung that is routinely exposed to biophysical forces in the form of shear stress (SS) and mechanical strain imposed by circulating blood and respiratory cycles. Pulmonary EC barrier function in vivo and in vitro is compromised by vasoactive substances such as thrombin, histamine, and tumor necrosis factor (1), events which result in life-threatening conditions such as pulmonary edema. However, in contrast to the effects of bioactive peptides, the role of mechanical factors in pulmonary EC signaling and barrier function is not well understood. Morphologic studies in situ reveal distinct orientation of systemic ECs along the direction of flow and alignment of stress fibers parallel to SS vector (5) that contrasts with the random cytoskeletal arrangement of EC culture grown under static conditions. In vitro endothelial cell perfusion at physiologic shear rates (10-20 dynes/cm²) reproduces a pattern of cell alignment observed in intact vessels (6). For example, actin stress fiber reorientation was noted after 6 h of SS (7), and cytoskeletal rearrangements and flow-directed reorientation of EC monolayer are complete after 12-24 h (7).

Numerous reports demonstrate intracellular signaling systems, such as ion channels, and G-protein-coupled signaling (10, 11), with rapid p38 and p42/44 MAP kinase (12), tyrosine kinase (15), and focal adhesion kinase (16) activation, events which potentially trigger the early cytoskeletal changes noted (16). Consistent with these in vitro findings, Kano and coworkers (20) recently demonstrated the surgical coarctation-induced rapid stress fiber formation and localized activation of protein tyrosine phosphorylation within endothelial monolayers in the areas of cell-cell interface. Decreased flow or flow disturbances correlate with compromised cell-cell communication and increased endothelial permeability both in animal models (21) and in vitro (22). Exposure of static EC culture to laminar flow in vitro may increase transendothelial hydraulic conductance and albumin clearance at the end of 3 h of perfusion (23). However, recent reports demonstrate a reduction of cell motility (24) and enhancement of barrier properties revealed by increased transendothelial electrical resistance (25) at early times of EC flow exposure, with preservation of endothelial monolayer integrity at later times of stimulation. Together, these findings suggest a potential importance of flow-dependent rearrangement of intercellular adhesions and cortical cytoskeleton in control of EC integrity and regulation of vascular permeability.

Non muscle actin rearrangement is a complex, spatially-defined process involving multiple members of a large actin-binding protein family whose activities are regulated by several diverse mechanisms, including serine/threonine- and tyrosine kinase-mediated phosphorylation. Recent reports demonstrate the role for the actin-binding protein cortactin in the regulation of cortical actin cytoskeletal rearrangement via interaction with the Arp-2,3 protein complex, resulting in the potentiation of actin polymerization (26, 27). Cortactin is highly expressed in a number of tumors and accounts for increased tumor invasiveness (28, 29). Mechanisms of cortactin accumulation in the lamellopodia of migrating cells (30, 31), or in response to stimulation with growth factors or reactive oxygen species (32), involve p60^Src-mediated tyrosine phosphorylation of three key tyrosine residues: Y⁴²¹, Y⁴⁶⁶, and Y⁴⁸² (35). However, the involvement of cortactin in SS-induced EC cytoskeletal response remains unknown.

In the present study we have characterized rapid cytoskeletal responses of pulmonary EC to acute SS, such as time-dependent F-actin rearrangement, accumulation of diphosphorylated regulatory myosin light chains (MLC) in the cortical actin cytoskeleton, and increased junctional protein tyrosine phosphorylation, and we have described a Rac-dependent mechanism of SS-induced cortactin translocation to the cortical cytoskeleton. These data define the role of Rac guanidine triphosphatase in SS-induced cytoskeletal rearrangement and provide mechanistic information regarding the adaptive stress fiber realignment along the direction of flow.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Materials and Reagents

Plasmids encoding the dominant negative mutants of Rac (N17Rac) bearing HA-tag, and its downstream effector p21-activated kinase (PAK)-1 (DN PAK1) bearing Myc-tag subcloned into pcDNA-3 vector, were generous gifts from Dr Margarita Nicolic (Harvard Medical School, Boston, MA). A plasmid encoding the tyrosine-deficient murine cortactin with mutations in the three major p60^Src phosphorylation sites Y/F⁴²¹, Y/F⁴⁶⁶, and Y/F⁴⁸² bearing Myc-tag has been previously described (35). Anti-diphospho-MLC specific antibodies, which recognize phosphorylated Ser¹⁹ and Thr¹⁸ of myosin regulatory light chains, were used for immunofluorescent staining and have been described previously (36). Anti-phospho-specific p38, anti-phospho-specific Erk-1,2, and horseradish peroxidase-conjugated secondary antibody were obtained from New England BioLabs (Beverly, MA). Anti-p38 antiserum was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-cortactin, anti-phospho-tyrosine (PY20), anti-Erk-1,2, anti-Myc, and anti- HA-tag antibodies were obtained from Upstate Biotechnology (Lake Placid, NY). The following reagents were also commercially obtained: the MEK inhibitor UO126 (Promega Corp., Madison, WI), myosin light chain kinase (MLCK) inhibitor ML-7, Rho-associated kinase (RhoK) inhibitor Y27632 (Tocris Cookson, Ballwin, MO), tyrosine kinase inhibitor genistein, p60^Src kinase family inhibitor PP-2 (Calbiochem, La Jolla, CA), Texas Red phalloidin, Alexa 488- or Alexa 546-conjugated secondary antibodies (Molecular Probes, Eugene, OR), Opti-MEM (GIBCO-BRL, Gaithersburg, MD), and fetal calf serum (Hyclone Laboratories, Logan, UT).

Cell Culture

Bovine pulmonary artery endothelial cells (CCL 209) were obtained from the American Tissue Culture Collection (Manassas, VA) and used at passages 14-18 as we have previously decribed (1). Cells were maintained in complete culture medium consisting of Dulbecco's modified Eagle's medium containing 10% bovine serum, endothelial cell growth supplement (17 µg/ml, H-neurext; Upstate Biotechnology), and 100 U/ml penicillin/streptomycin (GIBCO-BRL), and incubated at 37°C in humidified 5% CO₂ incubator. Human pulmonary artery endothelial cells were obtained from Clonetics, BioWhittaker Inc. (Frederick, MD), propagated in culture medium EGM-2 provided by Clonetics, BioWhittaker Inc., and used at passages 6-10.

Flow Chamber and SS Experiments

Endothelial cell monolayers grown on the gelatin-covered glass slides were placed in a parallel plate perfusion chamber for SS experiments (22). Cells on glass slides placed in D100 dishes and kept in the tissue culture incubator served as static controls. The flow chamber contains quartz windows for light transmission and sample visualization. Flow was imposed on the endothelial surface by connecting the parallel plate chamber to a flow circuit which consists of a variable speed peristaltic pump, a fluid capacitor that damps flow pulsation, and a fluid reservoir for recirculation. The flow chamber and flow loop were kept inside a tissue culture incubator (37°C, 5% CO₂ atmosphere) for the duration of experiments. When necessary, glass slides with the cells were placed into sterile D100 dishes with working dilutions of inhibitors in culture medium for 1 h before SS experiments. At the end of SS experiments, the flow chamber was disassembled, glass slides with cells removed, washed in ice-cold phosphate-buffered saline (PBS), and used for biochemical analysis or immunofluorescent staining.

Transient Transfection Protocol

Bovine EC grown on gelatin-covered glass slides at 70% confluence were transfected with the plasmid of interest. Each slide was incubated with 2 ml of OPTI-MEM medium containing 2 µg DNA and 20 µl of Fugene 6 (Roche Molecular Biochemicals, Indianapolis, IN) for 6 h in CO₂ incubator at 37°C followed by 18 h incubation with complete culture medium. Cells were used for shear stress experiments 24 h after transfection. Control transfections were performed with an empty vector plasmid.

Immunoblot Detection of Erk-1,2, p38 MAP Kinases, and Tyrosine Kinase Activation

Activation of Erk-1,2, p38 and protein tyrosine phosphorylation was assessed by determining the phosphorylation status of the proteins of interest by immunoblotting with a phospho-specific antibody. Briefly, after exposure to shear stress, cells were washed with ice-cold PBS twice and scraped in sodium dodecyl sulfate (SDS)- polyacrylamide gel electrophoresis (PAGE) sample buffer containing 500 mM Tris pH 6.8, 5 mM ethylenediaminetetraacetic acid, 50 mM sodium fluoride, 1 mM sodium orthovanadate, 2% SDS, 20% 2-mercaptoethanol, and 10% glycerol, boiled for 5 min, and samples were resolved on 8% SDS-PAGE and transferred to Immobilon membrane (Millipore, Bedford, MA). The membrane was probed with appropriate primary antibody and horseradish peroxidase-conjugated secondary antibody followed by immunodetection using enhanced chemiluminescence reagents (New England BioLabs) and autoradiography. To ensure equal loading, the membranes were stripped and reprobed with anti- Erk-1,2 or anti-p38 MAP kinase antibody.

MLC Phosphorylation in Intact Endothelium

Phosphorylation profiles of regulatory MLC were analyzed by glycerol/urea gel electrophoresis followed by electrotransfer and Western blotting of resolved unphospho-, mono-, and diphosphorylated MLC bands using anti-MLC antibody as decribed previously (1).

Immunofluorescent Staining

Following SS, cells were fixed in 3.7% formaldehyde solution in PBS for 10 min at 4°C, washed three times with PBS, permeabilized with 0.2% Triton X-100 in PBS for 5 min at 4°C, and blocked with 2% bovine serum albumin in PBS for 20 min. Incubation with antibodies was performed in blocking solution for 1 h at room temperature. Alexa 488- and Alexa 546-conjugated secondary antibodies (Molecular Probes) were used for immunodetection, and actin filaments were stained with Texas Red-conjugated phalloidin (Molecular Probes). After immunostaining procedure, the glass slides were analyzed using a Nikon video-imaging system consisting of a phase contrast inverted microscope connected to a digital camera and image processor. The images were recorded and processed using Adobe Photoshop 4.0 software.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

SS Induces Actin Cytoskeleton Rearrangement and MLC Phosphorylation

Pulmonary endothelial cells cultured under static conditions revealed the typical faint cortical F-actin arrangement and few stress fibers in the central area of the cell (Figure 1). We next applied a level of SS (10 dynes/cm²) believed to be relevant for the pulmonary artery circulation (37), and observed rapid cytoskeletal reorganization (15 min) with increased stress fiber formation in random orientation. More prolonged SS (24 h) resulted in cell reorientation in the direction of flow and re-establishment of the prominent cortical actin ring (Figure 1). Further analysis of the early SS-induced cytoskeletal events demonstrated a rapid (15 min) increase in the pool of mono- and diphosphorylated MLC that remained significantly elevated after 2 h of SS (Figure 2A) and returned to basal level by 24 h. SS-induced MLC phosphorylation was reduced ~ 30% after pretreatment of EC monolayer before flow exposure with MLCK inhibitor ML-7 (10 µM), and by 80% after pretreatment with Y27632 compound (5 µM), an inhibitor of RhoK (Figure 2A). These results are consistent with the ability of RhoK to either directly phosphorylate MLC, or inactivate myosin-associated phosphatase via phosphorylation of its myosin-binding subunit (38). Immunofluorescent studies utilizing an antibody specific for MLC Ser¹⁹ and Thr¹⁸, sites of MLCK-mediated MLC phosphorylation (39, 40), revealed robust SS-induced MLC diphosphorylation in bovine pulmonary artery endothelial cells which was preferentially colocalized with cortical actin filaments after 15 min of SS exposure (Figure 2B, middle panel ). In contrast, thrombin-stimulated EC revealed intense cytoplasmic stress fiber formation, and colocalization of diphospho-MLC to stress fibers (Figure 2B, lower panel ). Similar results were observed in human pulmonary artery endothelial cells (not shown).

View larger version (96K): [in this window] [in a new window]   Figure 1.   Actin cytoskeleton remodeling in bovine pulmonary artery endothelial cell cultures in response to SS. Bovine pulmonary artery EC were exposed to static conditions (upper panel), or to 10 dynes/cm2 SS for 15 min (middle panel) or for 24 h (lower panel). F-actin was visualized using Texas Red-conjugated phalloidin, as described in MATERIALS AND METHODS. Arrows show direction of flow. Bar = 10 µm. The results are representative of nine independent experiments.

View larger version (69K): [in this window] [in a new window]   Figure 2.   SS-induced MLC phosphorylation. (A) Cell lysates were prepared from bovine pulmonary artery EC subjected to SS, 10 dynes/cm2 in the flow chamber for the indicated periods of time, or after preincubation with myosin light chain kinase (MLCK) inhibitor ML-7 (10 µM) or Rho-associated kinase (RhoK) inhibitor Y27632 (5 µM) followed by 15 min exposure to SS. Unphosphorylated, mono-, and diphosphorylated MLC were detected as described in MATERIALS AND METHODS. (B) Intracellular localization of diphospho-MLC in SS-activated bovine pulmonary artery endothelial cells (BPAEC). After formaldehyde fixation of EC culture exposed to 15 min SS, diphospho-MLC were detected using specific anti-phospho-Ser19/phospho-Thr18 MLC antibody (36). Alternatively, BPAEC under static conditions were stimulated with 100 nM thrombin for 5 min. Note the preferential cortical localization of diphospho-MLC in response to SS shown by arrows and colocalization of diphospho-MLC to stress fibers in cells stimulated with thrombin. The results are representative of three independent experiments. Bar = 10 µm.

SS-Mediated Cortactin Translocation

These results indicate flow-induced activation of the pulmonary EC cortical cytoskeleton. A key element involved in the regulation of the cortical actin cytoskeleton is cortactin, an actin-binding protein, whose translocation to lamellipodia in response to growth factor stimulation correlates with activation of cell motility (30, 31) and invasion (41). Analysis of intracellular cortactin localization in unstimulated EC monolayers revealed diffuse cortactin distribution (Figure 3). The application of SS-induced the rapid but transient translocation of cortactin to the cortical actin layer with maximal translocation at 15 min, diminishing thereafter to negligible cortical staining after 24 h of SS (Figure 3). Thus, cortactin appears to spatially localize to sites of active cortical actin rearrangement after acute exposure to SS.

View larger version (157K): [in this window] [in a new window]   Figure 3.   SS-induced cortactin translocation in bovine pulmonary artery endothelial cell cultures. Cortactin was detected in static EC culture and in EC subjected to SS during 30 min, 2 h, or 24 h using immunofluorescent staining as described in MATERIALS AND METHODS. Note the transient cortactin translocation after 15 min of SS to the cell margins as an indication of early cytoskeletal rearrangements in response to SS. The results are representative of eight independent experiments.

Potential Mechanisms of SS-Mediated Cortactin Translocation and Actin Rearrangement

Mechanisms triggering cortactin translocation under various conditions include p60^Src-mediated cortactin phosphorylation (30, 42), Erk-1,2 MAP kinase-directed phosphorylation (43), or Rac GTPase-mediated pathways (31). SS-mediated intracellular signaling additionally engages MAP kinase family members (13, 14), and protein tyrosine kinases (16, 44), pathways known to be involved in cytoskeletal regulation (8, 14, 17). Consistent with studies of endothelial cells from the systemic circulation (aorta, large caliber arteries), we observed in pulmonary EC rapid SS-induced intracellular activation of Erk-1,2 MAP kinase within 2 min of SS with return to basal level by 20 min, which was completely abolished by inhibition of the Erk-1,2 MAP kinase upstream activator, MEK (Figure 4A). In contrast, SS-induced p38 MAP-kinase activation was detectable at 30 min, and remained elevated at 2 h of SS (Figure 4B). The time course of SS-induced Erk-1,2 and p38 MAP kinase activation was similar for human and bovine pulmonary endothelial cells (not shown). Western blot analysis revealed only moderate increases in total protein tyrosine phosphorylation in response to SS (Figure 4C), however, immunofluorescent staining revealed significant accumulation of tyrosine phosphorylated proteins within the cell cortical layer in close proximity to cell-cell junctions (Figure 5). Interestingly, flow-induced activation of protein tyrosine phosphorylation in the cell cortical layer returned to basal levels after 2 h of SS (data not shown) and remained reduced until completion of cell reorientation by 24 h of stimulation (Figure 5, lower panel ). Thus, these results suggest that SS-induced cortactin translocation may be temporally linked to activation of tyrosine kinase and Erk-1,2 MAP kinase signaling pathways. To identify the mechanism of SS-mediated cortactin translocation, we pretreated EC monolayers with pharmacologic inhibitors, including inhibitors of broad range tyrosine kinases (genistein), p60^Src family kinases (PP-2), MEK (UO126), MLCK (ML-7), and RhoK (Y27632). Preincubation with each of these pharmacologic inhibitors failed to significantly alter SS-induced cortactin translocation (Figure 6), despite the documented inhibition of their target kinases (Figures 2A and 4B), whereas SS-induced stress fiber formation was significantly attenuated by ML-7 and Y37632 (and marginally by PP-2) (Figure 7). These results suggest that neither Erk-1,2, MLCK, RhoK, nor tyrosine kinases including p60^Src are directly involved in SS-induced cortactin translocation in pulmonary EC monolayers. Finally, overexpression of a cortactin mutant with site-directed mutation of Tyr Phe of the three major p60^Src phosphorylation sites (Y⁴²¹, Y⁴⁶⁶, and Y⁴⁸²) did not prevent translocation of cortactin to the cortical cytoskeletal rim detected by anti-Myc-tag immunofluorescent staining (Figure 8, upper right panel ). Furthermore, the overexpression of tyrosine-deficient cortactin mutant did not alter human pulmonary artery endothelial cells reorientation after 24 h SS (Figure 9). These results confirm that tyrosine phosphorylation of cortactin may be necessary, but not sufficient, for cortactin translocation in response to SS. Together with inability of tyrosine kinase inhibitors (genistein and PP-2) to abolish the rapid SS-induced cortactin translocation (Figure 6, middle panels), these results indicate that additional molecular mechanisms are likely involved in both transient cortactin translocation and long-term cytoskeletal remodeling induced by SS.

View larger version (59K): [in this window] [in a new window]   Figure 4.   SS-induced activation of Erk-1,2, p38 MAP kinases, and protein tyrosine phosphorylation. Activation of Erk-1,2 (A) and p38 MAP kinase (C ) was assessed by immunoreactivity with anti-phospho-Erk-1,2 and anti-phospho-p38 antibody, respectively. Equal loadings were verified by detection of total Erk-1,2 and p38 protein levels in cell samples. An increase in phosphorylation state indicates time-dependent activation of Erk-1,2 with peak at 2-15 min and p38 MAP-kinase with peak at 2 h. SS-induced activation of Erk-1,2 was completely attenuated by 1-h EC preincubation with MEK inhibitor UO126 (5 µM) before SS exposure (B). (D) Western blot detection of protein tyrosine phosphorylation in response to SS using anti-phosphotyrosine antibody. Transient activation of protein tyrosine phosphorylation was observed during the first 30 min of SS. The results are representative of six independent experiments.

View larger version (171K): [in this window] [in a new window]   Figure 5.   SS-induced protein tyrosine phosphorylation is localized within the cell periphery. Intracellular localization of tyrosine phosphorylated proteins was examined by immunofluorescent staining using anti-phosphotyrosine primary antibody (left panels). Right panels depict F-Actin counterstaining using Texas Red-conjugated phalloidin, as outlined in MATERIALS AND METHODS. Note the colocalization of phosphoproteins with the cortical actin cytoskeleton at early times (15 min) of SS. The results are representative of three independent experiments.

View larger version (138K): [in this window] [in a new window]   Figure 6.   Effect of protein kinase inhibitors on SS-induced cortactin translocation. Human pulmonary artery EC were pretreated with p60Src inhibitor PP-2 (5 µM), tyrosine kinase inhibitor genistein (100 µM), MLCK inhibitor ML-7 (10 µM), MEK inhibitor UO126 (5 µM), or RhoK inhibitor Y27632 (5 µM) for 1 h before SS exposure. After 15 min of 10 dynes/cm2 SS, cells were fixed with 3.7% formaldehyde and anti-cortactin antibodies were used for immunofluorescent detection of cortactin, as described in MATERIALS AND METHODS. Nonimmune serum was used as negative control for immunofluorescent staining (lower right panel). Each of the inhibitors tested failed to significantly alter flow-induced cortactin translocation to the cell periphery. The results are representative of three independent experiments. Bar = 10 µm.

View larger version (194K): [in this window] [in a new window]   Figure 7.   Effect of serine/threonine and tyrosine protein kinase inhibitors on SS-induced F-actin rearrangement. Human pulmonary artery EC were preincubated with myosin light chain kinase inhibitor ML-7 (10 µM), RhoK inhibitor Y27632 (5 µM), p60Src kinase family inhibitor PP-2 (5 µM), or MEK inhibitor UO126 (5 µM) for 1 h before experiment. After 15 min of 10 dynes/cm2 SS, cells were fixed with 3.7% formaldehyde, and F-Actin was detected using Texas Red-conjugated phalloidin. ML-7 and Y27632 significantly attenuated SS-induced stress fiber formation. The results are representative of three independent experiments. Bar = 10 µm.

View larger version (147K): [in this window] [in a new window]   Figure 8.   Effect of 15 min SS on endothelial cells overexpressing wild-type or tyrosine-deficient cortactin. Human pulmonary artery EC were transfected with either plasmid encoding myc-tagged cortactin mutated on tyrosine residues (Y/F421, Y/F466, and Y/F482), critical sites for p60Src-mediated phosphorylation (upper panels), or wild-type myc-tagged cortactin (lower left panel ), or control vector encoding myc tag only (lower right panel ) 24 h before SS exposure. After 15 min SS at 10 dynes/cm2, cells were stained with anti-myc tag antibody to determine intracellular cortactin distribution. EC overexpressing the tyrosine-deficient cortactin mutant (n = 16 cells) exhibited SS-induced translocation of recombinant cortactin to cell periphery, as detected by immunofluorescent staining with anti-tag antibody. Upper left panel depicts diffuse intracellular localization of mutant cortactin under static conditions. The results are representative of three independent experiments. Bar = 10 µm.

View larger version (118K): [in this window] [in a new window]   Figure 9.   Overexpression of a tyrosine-deficient cortactin mutant does not affect SS-induced F-actin reorientation after 24 h SS. Human pulmonary artery EC were transfected with plasmid encoding cortactin mutated on tyrosine residues (Y/F421, Y/F466, and Y/F482), critical sites for p60Src-mediated phosphorylation (upper panels), or control vector (lower panels) 24 h before SS exposure. After 24 h SS at 10 dynes/cm2, cells were stained for F-actin (left column), and transfected cells were detected by immunofluorescent counterstaining using anti-tag antibody (right column). The results are representative of three independent experiments.

Role of Rac GTPase in SS-Mediated Cytoskeletal Rearrangement

The Rho family of small GTP-binding proteins that include Rho, Rac, and cdc42 GTPases, play an important role in cytoskeletal remodeling associated with cell shape changes and motility (45). Previous reports have identified the involvement of Rac in flow-dependent redox changes and activation of protein tyrosine phosphorylation (46). To evaluate the potential role of Rac in SS-mediated cortactin translocation, human pulmonary EC were transiently transfected with a plasmid encoding an epitope-tagged dominant negative Rac 1 GTPase (N17-Rac) before SS exposure. N17-Rac-expressing cells under static conditions revealed the same morphology and diffuse cortactin localization as control or empty vector-transfected counterparts. However, overexpression of N17-Rac abolished cortactin translocation to the cell periphery in response to SS (Figure 10), whereas EC transfected with empty vector revealed the same extent of cortactin as nontransfected cells exposed to SS. Thus, SS-induced cortactin translocation to the cortical cytoskeleton appears to be regulated via a Rac-dependent mechanism. One potential cytoskeletal target for the Rac-dependent cytoskeletal rearrangement is via p21-activated kinase (PAK), which is known to alter the cortical cytoskeleton in nonendothelial tissues (47) and in endothelium (48). To investigate the role of Rac and PAK in the sustained cytoskeletal response to SS, we analyzed HPAEC cultures exposed to 24 h of SS transiently transfected with either N17 Rac or dominant-negative PAK 1. We found that F-actin remodeling and cell reorientation was abolished in both N17-Rac overexpressing cells (Figure 11, upper panels) and cells overexpressing dominant negative PAK1 (Figure 11, middle panels) compared with control cells transfected with empty vector (Figure 11, lower panels). Thus, our results demonstrate novel Rac-PAK1-dependent regulation of acute and sustained SS-mediated cytoskeletal remodeling in bovine and human pulmonary endothelial cells.

View larger version (105K): [in this window] [in a new window]   Figure 10.   Overexpression of dominant negative Rac (N17-Rac) abolishes SS-induced cortactin translocation. Human pulmonary artery EC were transfected with plasmid encoding dominant negative Rac (HA-tagged N17-Rac) or empty vector 24 h before SS experiment. Intracellular cortactin localization using anti-cortactin antibody (lower row) and detection of transfected cells using anti-HA-tag antibody (upper row) was performed after EC exposure to SS, as described in MATERIALS AND METHODS. Under static conditions, N17-Rac overexpression does not affect the diffuse cortactin distribution (left panels). Prominent cortactin translocation to cell periphery in response to 15 min SS (10 dynes/cm2) is observed in nontransfected and mock-transfected EC, as shown by arrows on right panel, but not in N17-Rac overexpressing cell (middle panel ). N17-Rac- overexpressing cells (23 of 27 transfected cells) exhibited attenuation of cortactin translocation in response to acute SS. The results are representative of four independent experiments.

View larger version (153K): [in this window] [in a new window]   Figure 11.   Overexpression of dominant-negative Rac and PAK-1 abolishes SS-induced F-actin reorientation after 24 h SS. Human pulmonary artery EC were transfected with either dominant-negative Rac (upper panels), or dominant- negative PAK-1 (middle panels), or control vector (lower panels) 24 h before SS exposure. After 24 h SS at 10 dynes/ cm2, cells were stained for F-actin (left column), and cells overexpressing Rac or PAK-1 were detected by immunofluorescent counterstaining using anti-tag antibody (right column). N17-Rac1- (17 of 19 transfected cells) and dominant-negative PAK-1 (14 of 18 transfected cells) overexpression exhibited attenuation of F-actin reorientation in response to SS. The results are representative of five independent experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Although both pulmonary and systemic circulation are constantly exposed to varying levels of shear, lung endothelial cells experience variable flow patterns in vivo under physiologic and pathologic conditions that are dictated by unique features of the pulmonary circulation. For example, the distribution of blood flow throughout the human pulmonary vasculature is nonuniform and decreases from the base to the apex of the lung. In pathologic situations, such as severe hypovolemia or mechanical ventilation at excessive airway pressure, apical pulmonary arterial pressures may fall below alveolar pressures, resulting in capillary collapse and cessation of blood flow. Cessation of pulmonary flow through defined segments is observed after thromboembolism, or in response to hypoxic vasoconstriction, a condition unique to the pulmonary circulation, which may result in capillary collapse. Thus, the study of lung endothelial cells exposed to acute and chronic SS has important tissue-specific implications.

Analysis of cellular mechanisms of endothelial adaptation to flow suggests time-dependent activation of intracellular signaling pathways which culminate in cytoskeletal rearrangement (for review see Ref. 11). Morphologic (7) and morphodynamic (24) studies, although performed at different flow regimens, suggest three distinct phases of EC adaptation to flow. The initial phase of this response is characterized by a compensatory enhancement of EC cytoskeleton characterized by increased stress fiber formation, thicker intercellular junctions, and more apical actin filaments (7). In the secondary phase, EC exhibit characteristics of motility (7) and remodeling of intercellular junctions (9), whereas the final phase of SS-induced EC monolayer remodeling is characterized by EC orientation in the direction of flow, and re-establishment of both intercellular contacts and monolayer integrity (7, 9, 11, 24).

Although long-term effects of SS (several hours) on cytoskeletal reorientation (6, 11), focal adhesions (10, 16), and adherens junctions (9) have been well described, little is known about molecular mechanisms triggering the initial phase of SS-activated cytoskeletal remodeling. Our data highlight the importance of cortical cytoskeletal rearrangements in control of early steps of flow-induced EC remodeling and barrier function. Acute SS-induced a rapid increase in MLC phosphorylation consistent with previous reports (49). Surprisingly, inspection of intracellular accumulation of diphospho-MLC revealed preferential colocalization with the cortical rim of actin. This pattern was dramatically different from typical colocalization of diphospho-MLC, with central stress fibers observed in thrombin-stimulated EC in static culture (Figure 3B) that reflect a global actomyosin contraction, cell retraction, and gap formation (1).

A number of kinases, including Ca²⁺/calmodulin-dependent MLCK, RhoK, or Rac-activated PAK 1 are capable of directly phosphorylating MLC in vitro (39, 50, 51). Our results suggest a dual regulation of SS-induced MLC phosphorylation via MLCK- and RhoK-dependent mechanisms, as SS-induced F-actin rearrangement and MLC phosphorylation was attenuated by specific inhibitors of MLCK and RhoK. Although Ca²⁺/calmodulin is essential for MLCK activation, and an increase in cytosolic [Ca²⁺] in response to SS has been previously described (49), additional mechanisms of SS-mediated MLCK activation cannot be excluded. We recently described splice variant-specific regulation of the EC myosin light chain kinase MLCK-1 isoform by p60^Src (52). Although cortactin translocation is not dependent upon p60^Src, spatial distribution of MLC diphosphorylation, increased protein tyrosine phosphorylation, and stress fiber formation at the cell periphery in response to acute SS (Figures 5 and 6) may reflect local tyrosine kinase-mediated activation of EC MLCK-1, possibly via p60^Src. Although speculative, this notion is supported by the partial attenuation of SS-induced increase in stress fiber formation by p60^Src-specific inhibitor PP-2 (Figure 7). Further studies are aimed at elucidation of this potential mechanism.

Cortical actin cytoskeleton plays an important role in the maintenance of EC monolayer integrity by scaffolding intercellular adherens complexes and cell-substrate focal adhesions, allowing significant tethering of the EC monolayer as a whole (53). Although there is limited direct evidence for the role of cortical actin meshwork in the enhancement of EC barrier properties, we have found that enhancement of the cortical actin rim induced by diperoxovanadate (54) and sphingosine-1-phosphate (48), or loss of the cortical actin cyto- skeleton after treatment with actin depolymerizing agents cytocholasin D and latrunculin B, correlate well with alterations in transendothelial electrical resistance, a reflection of EC monolayer barrier properties. The essential elements of cortical actin regulation are complex; however, we and others postulate a significant role for cortactin, an 80-85 kD actin-binding protein, in the formation of the cortical actin cytoskeleton via activation of cortical actin polymerization and branching (27). Cortactin directly binds the Arp2/3 complex, a de novo actin nucleator which promotes nucleation and branching of actin filaments (26, 27). Our results demonstrate that SS stimulation induces rapid translocation of cortactin to the cell cortical layer, highly suggestive of a role for cortactin in rapid SS-induced cortical actin remodeling. Cortactin translocation to the cell periphery is regulated via diverse mechanisms including p60^Src-mediated tyrosine phosphorylation (32), and Rac-dependent mechanisms (31). The involvement of cortactin in the SS-induced cytoskeletal realignment remains unknown; however, our results demonstrate that in contrast to growth factor stimulation, cortactin translocation induced by SS does not require p60^Src activation, but is primarily regulated by Rac GTPase activity. A variety of pharmacologic inhibitors (MEK, p60^Src, tyrosine kinases, MLCK, RhoK) failed to alter SS-induced cortactin translocation. Although inhibition of MLC phosphorylation by ML-7 and Y27632 significantly abolished SS-induced stress fiber formation (Figure 7), this event did not affect cortactin translocation (Figure 6). Thus, early steps of SS-induced cortical cytoskeleton remodeling, such as actin polymerization activated by cortactin/Arp-2,3 complex, may occur independently of stress fiber assembly promoted by MLCK- and RhoK-dependent MLC phosphorylation. In contrast, we found that overexpression of the dominant-negative Rac 1, and its downstream effector PAK 1, abolished transient cortactin translocation to cell periphery as well as SS-induced cell reorientation, suggesting direct involvement of Rac GTPase-mediated pathways in the cortactin targeting to the cortical cytoskeleton and rearrangement in the late phase of SS-induced cytoskeletal remodeling. These results are consistent with the previously reported critical role of Rac 1 in cortactin translocation to the cell periphery, cortical cytoskeletal rearrangements, and formation of membrane ruffles in cultured fibroblasts upon growth factor stimulation (31). Furthermore, although Rac activation has been shown to be involved in SS-induced tyrosine phosphorylation and activation of Erk-1,2 MAP kinase via regulation of SS-induced production of reactive oxygen species (46), the role of SS-induced Rac activation in cytoskeletal remodeling remain unclear. Our results demonstrate, for the first time, the direct involvement of Rac GTPase in SS-induced lung EC cortical cytoskeletal changes. We recently reported that the potent barrier-protective effect of a platelet-derived phospholipid, sphingosine 1-phosphate, involves a critical role for Rac GTPase- and PAK-mediated enhancement of cortical actin rim (48). Taken together, these results demonstrate common mechanisms of sphingosine 1-phosphate- and SS-mediated cortical cytoskeletal remodeling consistent with enhancement of EC barrier properties. Our results do not exclude the involvement of other members of small G-proteins, such as Cdc42 and Rho, in endothelial responses to SS, as our results (Figure 6) and those of others (55) indicate that SS-induced cell alignment and stress fiber formation are inhibited by the pharmacologic RhoK inhibitors and dominant-negative mutants of Rho and its downstream target, a serine/ threonine kinase p160ROCK, but not by the dominant negative mutant of Cdc42.

Based on the combined results, we propose a model of EC cytoskeletal response to SS involving Rac, Rho, cortactin, and MLCK as key regulatory elements. In this model, SS initially activates Rac and Rho GTPases. Rac triggers cortactin translocation and activation of actin polymerization at the cell periphery, whereas Rho/RhoK and MLCK contributes to spatially defined MLC phosphorylation, assembly of actomyosin and cell orientation along the direction of flow. These two processes in concert lead to enhancement and strengthening of the EC cortical cytoskeleton, intercellular junctions, and thus pulmonary EC barrier properties, which can be considered an adaptive response of the endothelial cell monolayer to acute mechanical stimulation. However, more sustained flow stimulation (> 1-2 h) induces a second phase of EC monolayer remodeling associated with the temporal disassembly of adherens junctions (9), F-actin rearrangement (7, 9), and decreased EC barrier properties (56), events potentially regulated by p38 MAP kinase- (14), cdc42-, and/or Rho kinase-dependent mechanisms (55).

In conclusion, our studies demonstrate that pulmonary EC exhibit rapid cytoskeletal response to flow that may play an adaptive role in the maintenance of EC barrier properties under varying hemodynamic conditions. The results of this work suggest that potential barrier disruptive action of biophysical forces during early phase of flow readjustment may be counteracted by enhancement of the endothelial cortical cytoskeleton via cortactin- and Rac-dependent mechanisms.