RhoA and Rho-kinase dependent and independent signals mediate TGF-
-induced pulmonary endothelial cytoskeletal reorganization and permeability
Richard T. Clements,1
Fred L. Minnear,2
Harold A. Singer,1
Rebecca S. Keller,1 and
Peter A. Vincent1
1Center for Cardiovascular Sciences, Albany Medical College, Albany, New York; and 2West Virginia University, Morgantown, West Virginia
Submitted 8 June 2004
; accepted in final form 5 October 2004
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ABSTRACT
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Transforming growth factor (TGF)-
is a potent inflammatory mediator involved in acute lung injury. TGF-
directly increases pulmonary endothelial myosin light chain (MLC) phosphorylation, which is associated with increased endothelial stress fiber formation, gap formation, and protein permeability, all hallmarks of pulmonary endothelial responses during acute lung injury. We performed the following experiments in pulmonary endothelial monolayers to determine whether RhoA and Rho-kinase mediate these TGF-
-induced responses. TGF-
caused the sustained activation of RhoA 2 h posttreatment associated with increased MLC phosphorylation. Inhibition of either RhoA or Rho-kinase with either C3 exoenzyme or Y-27632 blocked MLC phosphorylation. In addition, both C3 and Y-27632 partially attenuated the maximal TGF-
-induced increase in permeability but did not affect the initial phase of compromised barrier integrity. Inhibition of Rho-kinase completely blocked the TGF-
-induced increase in the content of filamentous actin (F-actin) but only partially inhibited TGF-
-induced changes in actin reorganization. To assess the contribution of Rho-kinase in RhoA-mediated responses independent of additional TGF-
-induced signals, cells were infected with a constitutively active RhoA adenovirus (RhoAQ63L) with or without Y-27632. RhoAQ63L increased MLC phosphorylation, F-actin content, and permeability. Treatment with Y-27632 blocked these responses, suggesting that Rho-kinase mediates these RhoA-induced effects. Collectively, these data suggest the following: 1) the RhoA/Rho-kinase pathway is an important component of TGF-
-induced effects on endothelial MLC phosphorylation, cytoskeletal reorganization, and barrier integrity; and 2) additional signaling mechanisms independent of the RhoA/Rho-kinase signaling cascade contribute to TGF-
-induced changes in cytoskeletal organization and permeability.
myosin light chain; actin; stress fiber; transforming growth factor-
TRANSFORMING GROWTH FACTOR-
1 (TGF-
) is a multifunctional cytokine with a diverse array of effects on multiple cell types. A number of studies have demonstrated that TGF-
is an important mediator of acute lung injury. Adenoviral gene delivery of TGF-
to rat lungs mimics many of the pulmonary effects associated with acute respiratory distress syndrome (ARDS), including the development of increased pulmonary vascular permeability followed by severe pulmonary fibrosis (39). Recently, Laun et al. (27) found that increased levels of circulating TGF-
in trauma patients are associated with the development of sepsis and/or ARDS. In addition, in a mouse model of septic lung injury, Pittet et al. (33) showed that endotoxin-induced pulmonary edema and the subsequent fibrosis are inhibited by the addition of a soluble TGF-
1 receptor. Our laboratory has provided in vitro evidence that TGF-
can act directly on pulmonary endothelial monolayers to induce interendothelial gap formation and increased albumin permeability, an initiating event in acute lung injury (21). These studies establish TGF-
as a key mediator of increased pulmonary endothelial permeability in the development of pulmonary edema during acute lung injury.
Although it is known that TGF-
increases pulmonary endothelial permeability, little is known concerning the signaling events that mediate this process. We demonstrated previously that TGF-
increases stress fiber formation, interendothelial gap formation, and myosin light chain (MLC) phosphorylation, all of which temporally associate with TGF-
-induced endothelial permeability (19). A number of studies have demonstrated that increased MLC phosphorylation contributes to a loss of endothelial barrier function induced by inflammatory mediators such as thrombin (6, 17, 29, 42). In nonmuscle cell types such as fibroblasts and endothelial cells, phosphorylation of nonmuscle MLC II on Ser19 and/or Thr18 activates myosin Mg2+ ATPase activity, promotes the generation of actin- and myosin-containing stress fibers, and increases cellular tension (24, 25). In addition to regulating barrier function, TGF-
-induced development of cell tension has been implicated in the regulation of fibronectin matrix incorporation (49) and mesenchymal transformation (1, 3). Thus the mechanisms that regulate TGF-
-induced MLC phosphorylation and cytoskeletal reorganization may be important regulators of TGF-
-induced permeability, fibrosis, and cellular changes that contribute to the pathophysiology of acute lung injury.
Abundant evidence shows that the small GTPase RhoA is a critical mediator of changes in cytoskeletal organization, stress fiber formation, and MLC phosphorylation in a variety of cell types. Active GTP-bound RhoA can bind numerous effector proteins generally involved in regulating cytoskeletal dynamics (reviewed in Refs. 28 and 35). One of these effectors, Rho-kinase, can regulate the phosphorylation of MLC either through phosphorylation and subsequent inhibition of myosin phosphatase or direct phosphorylation of MLC on Ser19 and Thr18 (2, 16, 26, 41). Microinjection of constitutively active RhoA, RhoV14, causes the formation of actin stress fibers and MLC phosphorylation in fibroblasts and endothelial cells (36, 46). Also, thrombin-induced endothelial MLC phosphorylation, stress fiber formation, tension generation, and permeability are RhoA and Rho-kinase dependent and temporally associate with thrombin-induced increases in the levels of GTP-bound RhoA (12, 42, 45, 47). These studies demonstrate that activation of the RhoA/Rho-kinase pathway can simultaneously mediate increases in endothelial MLC phosphorylation and cytoskeletal reorganization that associate with increases in endothelial permeability.
Recent studies established that RhoA and Rho-kinase play a role in regulating the increase in stress fiber formation observed following treatment of fibroblasts with TGF-
. Indeed, Edlund et al. (11) demonstrated that TGF-
-induced stress fiber formation in fibroblasts is inhibited by either C3 or dominant negative RhoA adenovirus. In addition, TGF-
-induced stress fiber formation in fibroblasts is dependent on increased expression of NET1, a Rho guanine nucleotide exchange factor that facilitates the exchange of GDP for GTP, thereby activating RhoA (37, 38). Also, in transformed epithelial cells, TGF-
increases the level of active RhoA, which temporally associates with TGF-
-induced cytoskeletal reorganization (11). To date, no one has examined the role of the RhoA/Rho-kinase signaling cascade in TGF-
-induced pulmonary endothelial MLC phosphorylation and permeability. Therefore, we performed the following studies to determine whether the RhoA/Rho-kinase pathway mediates TGF-
-induced increases in endothelial MLC phosphorylation, stress fiber formation, and permeability. In addition, we determined whether active RhoA is sufficient to mimic these TGF-
-inducible responses in pulmonary endothelial monolayers and whether Rho-kinase contributes to RhoA-mediated effects.
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METHODS
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Antibodies and reagents.
All commonly used laboratory reagents unless otherwise specified were from Fisher or Sigma Chemical companies. Goat IgG anti-MLC (1:500), rabbit IgG anti-RhoA (1:200), and rabbit IgG anti-hemagglutinin (HA)-epitope (1:100) were from Santa Cruz Biotechnologies (Santa Cruz, CA). Mouse MAb IgG anti-
-actin (1:20,000) and mouse IgM anti-MLC (1:10,000) were from Sigma. For immunoblots, all secondary antibodies were from Jackson Labs (West Grove, PA). For immunofluorescent visualization, goat anti-rabbit IgG Alexa Fluor 488 was used to stain anti-Ser19/Thr18 phosphorylated MLC, and Alexa Fluor 594 phalloidin was used to stain filamentous actin (F-actin; Molecular Probes, Eugene, OR). Phosphospecific MLC antibodies were generated in rabbits (Strategic Biosolutions) against mono- (Ser19) and diphosphorylated (Ser19/Thr18) peptides corresponding to endogenous nonmuscle MLC. The peptides LLRPERATS*AVFC and LLRPERAT*S*AVFC (* indicates phosphorylated residue) were synthesized and provided by Dr. T. Andersen (Albany Medical College, Albany, NY). Antibody specificity was determined by ELISA before use in immunoblots.
Cell culture and treatments.
Bovine pulmonary artery endothelial (BPAE) cells were obtained from Vectec (Rensselaer, NY) or isolated from bovine pulmonary arteries. Isolated cells were verified for purity and endothelial phenotype by acylated low-density lipoprotein uptake and the presence of vascular endothelial cadherin. Cells were used at passage 3 until passage 12. Cells were grown in T-75 flasks in MEM supplemented with 20% FBS and penicillin/streptomycin. Cells were passed every 3 or 4 days. When passed, cells were washed three times in PBS, trypsinized, and reseeded at 3 x 106 cells in T-75 flasks. For experiments, BPAE monolayers were seeded at confluence. After 2 or 3 days, media were switched overnight to MEM with 5% FBS. The following day, BPAE monolayers were treated with 1 ng/ml of active TGF-
1 (R&D Systems) for the indicated times. In experiments examining Rho-kinase inhibition, Y-27632 (gift of Yoshitomi Pharmaceuticals) was added at the indicated concentrations 0.5 h before treatment with TGF-
. When Rho-kinase inhibition was examined in the presence of constitutively active RhoA, 1 µM Y-27632 was added 3 h postviral infection and every 15 h thereafter for 48 h. For constitutively active RhoA experiments, cells were infected with green fluorescent protein (GFP),
-galactosidase, or RhoAQ63L for the indicated times and dose in 5% FBS-MEM.
Adenoviral expression of recombinant proteins.
Constitutively active RhoAQ63L,
-galactosidase, and GFP adenoviruses were produced using the pAdEasy system described by He et al. (20). Recombinant adenoviruses were amplified in QBI-293A cells and purified using cesium chloride gradients. Multiplicity of infection (MOI) was determined using the method described by OCarroll et al. (32). All infections of RhoAQ63L were accompanied by control infection with either
-galactosidase (immunofluorescent studies) or GFP using a MOI equal to the greatest viral number used in the experimental groups.
Protein transfection.
C3 exoenzyme was transfected in confluent endothelial monolayers according to a method adapted from Tinsley et al. (40). This method has previously been shown to achieve
90% transfection efficiency in confluent endothelial cells. Transfections for 35-mm plates were carried out in 1 ml of 5% FBS-MEM. Briefly, 24 µl of Transit-LT-1 (Mirus) was incubated with 200 µl of MEM for 20 min. After incubation, C3 was added at the indicated concentration (for the final 1-ml volume) and incubated for another 20 min. After 20 min, the transfection mixture was brought up to 1 ml in 6.25% FBS-MEM to yield a final concentration of 5% MEM. The final transfection mixture was incubated with the cells for 8 h. Control experiments showed that an 8-h incubation with transit alone decreased the barrier integrity of endothelial monolayers; therefore, after 8 h, the transfection mixture was washed 5x with 1 ml of 5% MEM and incubated for another 8 h to recover. Control or TGF-
was added for 4 h following recovery. Cells were then lysed and processed for SDS-PAGE and immunoblot. For electric cell-substrate impedance sensor (ECIS) experiments, the transfection protocol was the same except all volumes were adjusted to achieve a final transfection and wash volume of 400 µl. For heat inactivation of C3 exoenzyme, C3 was boiled for 10 min before being added to the transfection mixture.
In vitro kinase reactions.
Mono- and diphosphorylated light chains were generated by incubation of purified rat smooth muscle MLC (a gift of P. de Lanerolle). The reaction consisted of the following components: 0.4 mg/ml MLC, 1 mM CaCl2, 10 mM MgCl2, 25 mM Tris·HCl, and 1 mM ATP. Myosin light chain kinase (MLCK) monophosphorylated reactions also contained 1.2 µM calmodulin and 0.5 µg/ml of purified MLCK (a gift of J. Stull). MLCK diphosphorylated reactions contained 50 µg/ml of MLCK. Phosphorylation of MLC by PKC contained the following additional components: 50 µg/ml phosphatidylserine, 1 µM PMA, and 2.5 µg/ml PKC
(Upstate Biotechnology, Lake Placid, NY). Reactions were initiated by the addition of kinase, and 10-µl samples were taken at 5 min (monophosphorylated), 45 min (diphosphorylated), and 1 h (PKC phosphorylated) and placed in 1 ml of 10% TCA on ice to terminate the reaction. Precipitated protein was then used in immunoblot analysis following native urea-PAGE.
Urea gel electrophoresis and immunoblot.
Assays to determine mono- and diphosphorylated MLC were performed as described by Garcia et al. (17). BPAE monolayers in 60-mm plates were scraped in 1 ml of 10% TCA and 0.05% DTT and allowed to precipitate for 30 min on ice. The precipitate was pelleted 10,000 g for 7 s. The supernatant was drawn off and replaced with ice-cold acetone. Samples were vortexed and placed on ice for 10 min. This was repeated four times. Pellets were dried at room temperature for 30 min and resuspended in urea buffer containing 6.7 M urea, 20 mM Tris base, 22 mM glycine, 9 mM DTT, 0.004% bromphenol blue, and 1% Triton X-100. Proteins were separated on a native gel (10% acrylamide, 0.5% bis-acrylamide, 40% glycerol, 20 mM Tris base, 22 mM glycine, and 0.22 µM ammonium persulfate) at 400 V for 1 h and transferred to nitrocellulose at 40 V for 14 h. MLCs were visualized with anti-MLC (1:10,000), anti-phospho-Ser19 MLC (1:100), or anti-phospho-Ser19/Thr18 (1:10,000), followed by the appropriate peroxidase-conjugated secondary antibody.
SDS-PAGE and immunoblot.
BPAE cells were seeded in 35-mm plates and grown to confluence. After treatment, cells were lysed in 200 µl of boiling-hot Laemmli sample buffer with 100 mM NaF. Cells were scraped, syringe-sheared, and boiled for 5 min. Lysates were loaded onto the appropriate percentage polyacrylamide gels and run at 150 V for the necessary times (12 h). All blots were transferred at 100 V for 1 h or overnight at 40 V for 14 h. For MLC, duplicate blots were probed with the phospho-Ser19/Thr18 MLC antibody (1:10,000) (described above) or a non-phosphospecific MLC antibody (1:500) to determine load. To determine viral expression, duplicate blots were probed with anti-hemagglutinin (HA)-epitope (1:100) or RhoA (1:200). To determine load, some blots were probed with
-actin.
Affinity precipitation of GTP-loaded Rho.
Activation of Rho was determined using an assay based on Ren et al. (34). Briefly, activated Rho was affinity precipitated with a rhotekin-Rho binding domain (Rhotekin-RBD; Upstate Biotechnology) that selectively binds GTP-bound Rho. Cells were seeded at confluence in 150-mm plates. After treatment, dishes were moved to the cold room, kept on ice, washed twice with ice-cold Tris-buffered saline (pH 7.4), and drained for 1 min. Cells were then incubated for 1 min with 1 ml of lysis/wash buffer containing 25 mM HEPES (pH 7.5), 150 mM NaCl, 1% Igepal CA-630, 10 mM MgCl, 1 mM EDTA, 2% glycerol, and Complete EDTA-free antiprotease tablets (Boehringer-Mannheim, according to manufacturers instructions). Cells were rapidly scraped and clarified at 14,000 g for 2 min at 4°C. A 50-µl sample of the supernatant was taken to later run on a gel to determine the starting quantity of RhoA. From the remaining supernatant, 800 µl was removed and incubated with 30 µl of rhotekin-RBD beads (2530 µg) in 50% slurry for 45 min with rotation. Beads were collected with centrifugation (500 g for 20 s) and washed four times with lysis/wash buffer. Beads were resuspended in 20 µl of 2x sample buffer with 100 mM DTT and boiled for 5 min. The entire contents of each tube were run on the gel. Blots were probed with a RhoA-specific polyclonal antibody. All steps preceding boiling were performed at 4°C and on ice where appropriate.
Assessment of electrical impedance.
Changes in electrical impedance were assessed using the ECIS from Applied Biophysics (Troy, NY), explained in detail by Giaever and Keese (18). Briefly, endothelial cells (1.2 x 105 cells/cm2) were plated in a well containing a small gold electrode and a larger counter electrode. Culture medium was used as the electrolyte, and a 1-V, 4,000-Hz alternate current signal was supplied by a lock-in amplifier through a 1-M
resistor to approximate a constant current of 1 µA to the cells during measurement. After attachment to ECIS, cells were allowed to equilibrate for 412 h. Once resistances were relatively constant, treatments were added directly to the wells at the indicated times. Resistance was measured every 10 min for the length of the experiments. The average baseline resistance value of electrodes alone was subtracted from all data points. Data were normalized to the mean of the resistance measurements taken for the course of 1 h immediately before treatment.
Immunofluorescence microscopy.
BPAE monolayers were seeded on glass coverslips coated with 0.2% gelatin. Cells were washed three times in ice-cold PBS+ (pH 7.4) and fixed with 3% formaldehyde in PBS. Cells were washed again in PBS+ and permeabilized for 20 min with Triton X-100 in HEPES+ containing 300 mM sucrose, 50 mM NaCl, 3 mM MgCl2, 20 mM HEPES (pH 7.4), and 0.5% Triton X-100. Monolayers were incubated with blocking buffer containing 2% BSA, 50 mM glycine, and 0.2% Tween 20 in PBS+. Slides were incubated overnight with anti-phospho-Ser19/Thr18 (1:400) at 4°C in dilution buffer containing 0.2% BSA, 50 mM glycine, and 0.2% Tween 20 in PBS+. Monolayers were then incubated with Alexa Fluor 488-conjugated goat anti-rabbit (1:1,000) and Alexa Fluor 594-conjugated phalloidin (1:800) for 2 h in dilution buffer. Between all steps, cells were washed three times with PBS+. Cells were visualized with an Olympus BX-50 microscope with a reflected light fluorescence attachment (Tokyo, Japan). Images were captured using an Optronics digital camera and software.
Quantification of F-actin content in endothelial monolayers.
Measurement of F-actin levels was performed similar to an assay by Chan et al. (8). Briefly, following treatment, confluent monolayers in 24-well plates were placed on ice and washed three times with buffer F containing 5 mM KCl, 138 mM NaCl, 4 mM NaHCO3, 0.4 mM KHPO4, 1.1 mM Na2HPO4, 2 mM MgCl2 hexahydrate, 2 mM EGTA, and 5 mM PIPES (pH 7.2). Cells were fixed with 3.7% formaldehyde in buffer F for 10 min and washed three times with buffer F. Monolayers were permeabilized in 0.5% Triton X-100 in buffer F for 20 min and washed three times for 5 min each in buffer F. Monolayers were blocked with 0.1 M glycine in buffer F for 20 min and washed with five quick rinses in buffer F. Monolayers were then incubated with 150 µl of 1 µM Alexa Fluor 594-conjugated phalloidin in buffer F for 60 min. Cells were washed five times for 5 min each in buffer F and incubated with 500 µl of methanol at 4°C for 90 min to extract phalloidin. Samples of the methanol supernatant were read on a Perkin-Elmer HTS-7000 Plus fluorescent plate reader at 595 nm excitation and 635 nm emission to determine the amount of extracted fluorescent-tagged phalloidin. Plates were then washed with buffer F five times for 5 min and incubated with 1 ml of bicinchoninic acid (BCA) protein assay reagent for 30 min at 37°C with shaking to determine relative protein content. Absorbance of the BCA reagent was read at 562 nm on a Versamax tunable plate reader (Molecular Devices). Emission measurements were normalized to protein levels for each monolayer.
Statistical analysis.
For the RhoA affinity precipitation assay (see Fig. 2), statistical analysis was performed with SigmaStat software. Because of normality, a Kruskal-Wallis one-way ANOVA followed by a Dunns post hoc analysis was used to determine significant differences from control. All other statistical analyses were performed using Statistica software. Two-way ANOVA was used followed by Neuman-Keuls post hoc analysis to determine significant differences between groups. Significance levels were set at P < 0.05.
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RESULTS
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To determine the identity of the MLC residues involved in TGF-
-induced MLC phosphorylation, antibodies were produced to peptides corresponding to MLC phosphorylated on Ser19 and MLC phosphorylated on both Ser19 and Thr18. Antibodies were tested against purified MLC phosphorylated by MLCK under conditions to produce Ser19 only and Ser19/Thr18-phosphorylated MLC (22). Phosphorylation of purified MLC by PKC (previously shown to phosphorylate MLC on Ser1, Ser2, or Thr9) was used as a control. The purified MLC was separated using native gel electrophoresis to show the different phosphorylated forms of the protein. As seen in Fig. 1A, MLC treated with concentrations of MLCK previously documented to monophosphorylate MLC on Ser19 or diphosphorylate MLC on Ser19/Thr18 resulted in a shift in migration consistent with the predicted change in phosphorylation. Phosphorylation of MLC with PKC also caused a shift from the native protein consistent with a change in phosphorylation. The antibody to Ser19 reacted only with the monophosphorylated MLC (Fig. 1A), whereas the antibody generated to Ser19/Thr18 reacted only with the band corresponding to the diphosphorylated form (Fig. 1A). Neither antibody reacted with MLC phosphorylated with PKC. Cell lysates from endothelial monolayers treated with or without TGF-
for 4 h and analyzed using native gel electrophoresis showed an increase in diphosphorylated MLC, but no change in the levels of monophosphorylated MLC (Fig. 1B). Duplicate gels probed with anti-phospho-Ser19 (Fig. 1B) and anti-phospho-Ser19/Thr18 (Fig. 1B) confirmed that monophosphorylated MLC and diphosphorylated MLC corresponded to MLC phosphorylated on Ser19 and Ser19 and Thr18, respectively. Pulmonary endothelial monolayers were treated with TGF-
for 0, 0.5, 1, 2, 3, and 4 h. The TGF-
-induced increase in Ser19/Thr18-phosphorylated MLC began at
2 h and continued until at least 4 h posttreatment (Fig. 1C). Relative differences in MLC levels were determined using a nonphosphorylated MLC antibody (Fig. 1C). Thus TGF-
caused a sustained increase in the phosphorylation of MLC on Ser19/Thr18 beginning at
2 h.
We affinity precipitated activated RhoA to determine whether TGF-
activates RhoA in a time course similar to the TGF-
-induced increase in MLC phosphorylation and decrease in monolayer barrier function. Figure 2A is a representative blot showing that GTP-bound RhoA increases within 2 h after the addition of TGF-
and remains elevated until 34 h posttreatment. Total RhoA levels in the samples before precipitation were unchanged with TGF-
(Fig. 2A) and were used to normalize changes in RhoA activity for analysis of multiple experiments using a scanning densitometer. This quantitative analysis confirmed that RhoA activity significantly increased above control levels at 2 and 3 h post-TGF-
treatment (Fig 2B). Thus, TGF-
activates RhoA in endothelial cells, and RhoA activity increases in a time frame similar to induction of MLC phosphorylation.
To assess the role of RhoA activation in TGF-
-induced endothelial MLC phosphorylation, we transfected exoenzyme C3 from Clostridium botulinum into confluent endothelial monolayers (see METHODS). Treatment of endothelial monolayers with C3 exoenzyme caused a dose-dependent inhibition of TGF-
-induced Ser19/Thr18 MLC phosphorylation (Fig. 3A). In addition, this effect was specific to C3 catalytic activity, as transfection of the heat-inactivated enzyme did not affect the TGF-
-induced MLC phosphorylation. Changes in the levels of Ser19/Thr18-phosphorylated MLC were normalized to nonphosphorylated MLC (Fig. 3A). The results of four independent experiments were quantified and showed that C3 significantly (P < 0.05) inhibited TGF-
-induced MLC phosphorylation and that there was no significant difference between TGF-
-treated cells and TGF-
-treated cells in the presence of 2 µg/ml of heat-inactivated C3.
Although there are a number of known kinases that phosphorylate MLC, Rho-kinase is activated by Rho-GTP and has been shown to play a prominent role in the regulation of endothelial barrier function (5, 48). We used the Rho-kinase inhibitor Y-27632 to determine whether Rho-kinase mediates the TGF-
-induced increase in pulmonary endothelial MLC phosphorylation (23). Confluent BPAE monolayers were treated with various doses of Y-27632 for 30 min and continuously during a 4-h treatment with TGF-
or vehicle alone. Nonphosphorylated, monophosphorylated, and diphosphorylated MLC were visualized by subjecting monolayer lysates to urea-PAGE. Y-27632 blocked TGF-
-induced MLC diphosphorylation (representative blot, Fig. 4A). Similar results were obtained with the anti-phospho-Ser19/Thr18 antibody (representative blot, Fig. 4A). Changes in the levels of the phosphorylated forms of MLC were expressed as a percentage of total MLC (nonphosphorylated + monophosphorylated + diphosphorylated) from five independent experiments and were quantitated in Fig. 4B. TGF-
did not increase monophosphorylated MLC (Fig. 4B) but did increase diphosphorylated MLC. Pretreatment with Y-27632 inhibited the TGF-
-induced increase in diphosphorylated MLC at doses of 0.5 µM and greater. Coupled with the findings above, these data demonstrate that TGF-
increases RhoA activity, which acts through Rho-kinase to increase MLC phosphorylation.
To determine whether RhoA activation contributes to the TGF-
-induced change in barrier integrity, electrical resistance was assayed using ECIS following TGF-
-induced effects with or without C3 exoenzyme. In agreement with previous data (21), TGF-
caused a decrease in resistance beginning between 1 and 2 h after treatment (Fig. 5A). No change in baseline resistance was observed following recovery of endothelial monolayers transfected with C3 or treated with control/vehicle. Interestingly, 2 µg/ml of C3 exoenzyme did not inhibit the initial TGF-
-induced decrease in endothelial resistance, but it did attenuate the severity of the TGF-
-induced decrease in resistance. Statistical analysis of individual time points showed that at 6 h, TGF-
plus 2 µg/ml of C3-treated cells differed from monolayers receiving TGF-
and heat-inactivated C3 (Fig. 5B).
To determine whether Rho-kinase also contributes to the TGF-
-induced change in barrier integrity, electrical resistance was assayed following TGF-
treatment with or without Y-27632. In agreement with Fig. 5, TGF-
caused a decrease in resistance beginning between 1 and 2 h following treatment (Fig. 6A). No change in baseline resistance was observed following treatment with Y-27632. Interestingly, doses of 0.5 and 1 µM Y-27632 also did not inhibit the initial TGF-
-induced decrease in endothelial resistance but did attenuate the severity of the TGF-
-induced decrease in resistance. Statistical analysis of individual time points showed that at 2 h, none of the TGF-
plus Y-27632 groups differed from TGF-
(data not shown); however, at 6 h, both 0.5 and 1 µM Y-27632 differed from TGF-
alone (Fig. 6B). These experiments (Figs. 5 and 6) suggest that there may be an early component to TGF-
-induced permeability (i.e., <2 h) that is independent of RhoA and Rho-kinase activation and/or MLC phosphorylation, but that induction of the RhoA/Rho-kinase signaling cascade contributes to the maintenance or late-term component (i.e., >2 h) of TGF-
-induced disruption of endothelial barrier integrity.
The ability of C3 exoenzyme or Y-27632 to completely inhibit TGF-
-induced MLC phosphorylation, but only partially inhibit the decrease in barrier integrity, suggested that rearrangement of the actin cytoskeleton might occur independently of RhoA/Rho-kinase activity. Immunofluorescence microscopy was performed to visualize TGF-
-induced actin reorganization (Alexa Fluor 594-tagged phalloidin, red) and Ser19/Thr18-phosphorylated MLC (Alexa Fluor 488, green) localization in the presence and absence of Rho-kinase inhibition. Control BPAE monolayers exhibited peripheral actin bands, with some smaller filaments that reached across the cell (Fig. 7A). Ser19/Thr18-phosphorylated MLC showed minimal staining that colocalized with the peripheral actin bands (Fig. 7A). TGF-
treatment caused the formation of gaps between cells. In conjunction, TGF-
decreased cortical actin staining and dramatically increased the number of cross-cellular stress fibers (Fig. 7A). The amount of actin filaments increased upon TGF-
treatment, as determined by extracting Alexa Fluor phalloidin and assessing fluorescence intensity on a fluorescence plate reader (Fig. 7B, see METHODS for details). Phospho-Ser19/Thr18 MLC staining colocalized with the central actin filaments (Fig. 7A) and was also found in thick bands that formed along the cell periphery. The intense Ser19/Thr18-phosphorylated MLC peripheral bands associated with the presence of gap borders adjacent to the cells (Fig. 7A). Inhibition of Rho-kinase with 1 µM Y-27632 caused a sharp decrease in TGF-
-induced phospho-Ser19/Thr18 MLC staining consistent with the decrease in total MLC phosphorylation observed in Fig. 4. Although Y-27632 decreased the number of interendothelial gaps, Y-27632 did not completely inhibit gap formation following TGF-
treatment (Fig. 7A). Interestingly, TGF-
in the presence of Y-27632 still caused a rearrangement of the actin cytoskeleton from peripheral actin bands to centrally located actin filaments, without the accompanying increase in MLC phosphorylation (Fig. 7A). Although the actin fibers were rearranged, there was no change in the amount of F-actin in TGF-
-treated cells in the presence of Y-27632 compared with control monolayers. The inability of Y-27632 to completely prevent the rearrangement of actin and the formation of interendothelial cell gaps following TGF-
treatment is consistent with the partial inhibition of electrical resistance observed in Fig. 6.

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Fig. 7. Rho-kinase inhibition decreases TGF- -induced formation of central actin stress fibers, interendothelial gaps, and increased filamentous (F)-actin content. A: BPAE monolayers were treated with (b, b', d, d') or without (a, a', c, c') Y-27632 for 30 min before 4-h treatment with control/vehicle (a, a', b, b') or TGF- (c, c', d, d'). The F-actin cytoskeleton (ad) and Ser19/Thr18-phosphorylated MLC (a'd') were visualized as described in METHODS. Arrows (c) show the TGF- -induced loss of peripheral actin bands and the formation of gaps between cells. Arrowheads (d) show that Y-27632 results in smaller gaps but does not affect the loss of peripheral actin induced by TGF- . Images are representative of a minimum of 4 independent experiments. B: confluent monolayers were treated as in A and measured for F-actin content as described in METHODS. Data are presented as means ± SE (n = 5, *significantly greater than all other groups, P < 0.05.)
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The small GTPase RhoA activates numerous effector molecules in addition to Rho-kinase that can alter cytoskeletal integrity. To determine whether the Rho-kinase-independent effects of TGF-
were also independent of RhoA, we compared the effects of Rho-kinase inhibition on TGF-
-induced changes in MLC phosphorylation, actin rearrangement, and permeability to changes in these parameters following the expression of constitutively active Rho (RhoAQ63L). Infection with increasing amounts of adenovirus produced a dose-dependent increase in the expression of RhoAQ63L as determined by an antibody to the HA-epitope tag (Fig. 8A). The increase in RhoAQ63L expression resulted in an increase in Ser19/Thr18 MLC phosphorylation. Infection for 48 h with RhoAQ63L at an MOI of 200 induced MLC phosphorylation that was similar in intensity to TGF-
-induced MLC phosphorylation when assessed on the same immunoblot (data not shown). Infection with the GFP adenovirus had no effect on MLC phosphorylation. The RhoA-induced increase in MLC phosphorylation was inhibited by treatment with Y-27632 when added every 15 h beginning 3 h after the addition of virus (Fig. 8A). In addition to preventing the increase in MLC phosphorylation, the addition of Y-27632 also prevented changes in barrier function found following expression of RhoAQ63L. As shown in Fig. 8B, expression of RhoAQ63L resulted in a time-dependent decrease in electrical resistance beginning 1618 h after adenoviral infection. Figure 8C shows the 48-h data in Fig. 8B in bar graph format. Infection with RhoAQ63L significantly decreased endothelial resistance similar to TGF-
. In contrast to TGF-
-mediated effects, Y-27632 treatment completely prevented the RhoA-induced decrease in endothelial resistance. Thus activation of RhoA is sufficient to induce MLC phosphorylation and decreased endothelial barrier integrity in a Rho-kinase-dependent manner. These results, coupled with those of Fig. 6A, demonstrate that signals independent of RhoA/Rho-kinase and the subsequent MLC phosphorylation contribute to the initial loss of barrier integrity following treatment with TGF-
.
To determine the role of Rho-kinase on RhoA-induced changes in cytoskeletal organization and endothelial morphology, we used immunofluorescent microscopy to visualize actin reorganization and diphosphorylated MLC localization with and without Rho-kinase inhibition. BPAE monolayers infected with control adenovirus for 48 h demonstrated characteristic staining of cortical actin that was colocalized with Ser19/Thr18-phosphorylated MLC along the periphery of the cell (Fig. 9A). Inhibition of Rho-kinase with Y-27632 had little effect on actin organization but did cause reductions in the basal amount of diphosphorylated MLC (Fig. 9A). Infection of BPAE monolayers with RhoAQ63L caused a dramatic increase in actin filament formation and reorganized actin from distinct cortical bands into densely arranged central F-actin filaments throughout the cell (Fig. 9A). The increased levels of Ser19/Thr18-phosphorylated MLC were colocalized with the central actin filaments (Fig. 9A). Quantification showed that activated RhoA increases the amount of F-actin
40% (Fig. 9B). In addition, RhoAQ63L induced the formation of interendothelial cell gaps (Fig. 9A) consistent with the decrease in electrical resistance observed by ECIS. As seen in Fig. 9A, inhibition of Rho-kinase with Y-27632 prevented the reorganization of actin into central stress fibers in most cells, as shown by the loss of actin staining and MLC phosphorylation. Some cells still had stress fiber formation (Fig. 9), suggesting an inability of Y-27632 to inhibit Rho-kinase-dependent effects when faced with an overwhelming RhoA response. Similar results were found when assessing the content of F-actin, as treatment with Y-27632 resulted in a trend to decrease RhoA-induced actin filaments; however, this did not reach statistical significance (Fig. 9B). We conclude that Rho-kinase is primarily responsible for the RhoA-induced reorganization of the actin cytoskeleton, MLC phosphorylation, and stress fiber formation observed upon expression of RhoAQ63L. In addition, these results, in conjunction with Fig. 7, suggest that TGF-
activates additional cytoskeletal modifying signals, independently of the RhoA/Rho-kinase cascade, that mediate decreases in barrier integrity.

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Fig. 9. RhoA-induced cytoskeletal reorganization, stress fiber assembly, and gap formation are Rho-kinase dependent. A: BPAE monolayers were infected with -galactosidase ( -gal; a, a', b, b') or RhoAQ63L adenovirus (c, c', d, d') for 48 h followed by treatment with (b, b', d, d') or without (a, a', c, c') 1 µM Y-27632 as described in METHODS. The F-actin cytoskeleton (ad) and Ser19/Thr18-phosphorylated MLC (a'd') were visualized as described in METHODS. Arrows (c) show interendothelial gaps. Asterisks (d, d') show inhibition of actin- and myosin-containing stress fibers with Y-27632, and arrowheads show cells that retain actin stress fibers. B: confluent monolayers were treated as in A and measured for F-actin content (see METHODS). Data are presented as means ± SE (n = 6, 2-way ANOVA shows that RhoAQ63L caused a significant increase, *P < 0.05. however, there was no statistically significant interaction with Y-27632).
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DISCUSSION
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The above studies investigated the role of the RhoA/Rho-kinase signaling pathway in the increase in pulmonary endothelial permeability by TGF-
, a cytokine that plays a central role in the development of acute lung injury and pulmonary edema. We have shown previously that TGF-
increases the levels of diphosphorylated MLC (21). In this study, using phosphospecific antibodies, we demonstrate that TGF-
increases MLC phosphorylation on Ser19 and Thr18. In addition, we show that TGF-
causes a sustained increase in endothelial Ser19/Thr18 MLC phosphorylation beginning
2 h posttreatment (Fig. 1C) that remains elevated for at least 6 h (data not shown). The time course of TGF-
-induced MLC phosphorylation is considerably different than that of thrombin and lysophosphatidic acid (LPA), which cause much more of an acute (within 510 min) and transient (<1 h) increase in MLC phosphorylation on Ser19/Thr18. We have previously demonstrated that the TGF-
-induced increase in MLC phosphorylation is dependent on new protein synthesis, suggesting that changes in gene expression mediate the TGF-
-induced endothelial MLC phosphorylation (19). This may account for the different time course between the rapid thrombin/LPA response and the slow increase in TGF-
-induced MLC phosphorylation. However, similarities do exist in the role of the Rho/Rho-kinase signaling pathway in the regulation of the increase in MLC phosphorylation on Ser19 and Thr18 by both acute agents (thrombin and LPA) and TGF-
.
Our findings show that TGF-
activates RhoA in endothelial cells in a manner distinct from other known mediators, such as thrombin, LPA, cytotoxic necrotizing factor (CNF), Pasteurella multocida toxin (PMT), and lipopolysaccharide (LPS). CNF and PMT directly cause the constitutive activation of Rho-family GTPases (13, 14). Induction of RhoA activity by the vasoactive mediators thrombin and LPA is acute within 210 min and transient, lasting
3060 min (4, 34, 42, 44). In contrast, TGF-
-induced pulmonary endothelial RhoA activation is relatively delayed and sustained, beginning
2 h posttreatment and persisting for at least 2 h (Fig. 2). A delay in RhoA activation has been observed in epithelial cells, where TGF-
causes the sustained activation of RhoA between 6 and 24 h post-TGF-
treatment (11). In endothelial cells, the time course of TGF-
-induced RhoA activation and MLC phosphorylation is similar in some respects to LPS-induced changes in these parameters. LPS increases RhoA/Rho-kinase-dependent MLC phosphorylation beginning
12 h posttreatment, which remains elevated for many hours (15). However, the actions of LPS are most likely mediated through autocrine or paracrine mechanisms, as LPS causes production of a multitude of cytokines and growth factors, and its effects on MLC phosphorylation are protein synthesis dependent (15). Although TGF-
-induced MLC phosphorylation is dependent on new protein synthesis and TGF-
activates multiple transcriptional responses in endothelial cells, TGF-
does not appear to activate RhoA through autocrine or paracrine mechanisms, as blockade of growth factor signaling with suramin, following TGF-
treatment, failed to block TGF-
-induced MLC phosphorylation (data not shown). Rather, TGF-
may regulate expression of specific proteins that lead to increases in RhoA activity. Indeed, a requirement for upregulation of specific intracellular signaling proteins for TGF-
-induced cytoskeletal responses has been demonstrated previously. Shen et al. (38) showed that TGF-
-induced stress fiber formation in fibroblasts (a RhoA-dependent process) is dependent on TGF-
-induced expression of the Rho guanine nucleotide exchange factor NET1. We were unable to document NET1 expression in endothelial cells (data not shown); however, it is possible that TGF-
-induced endothelial activation of RhoA is subject to a similar type of regulation.
One downstream effector of RhoA is the serine/threonine kinase Rho-kinase. Numerous studies have demonstrated that activation of RhoA, mediated by extracellular signals, results in Rho-kinase-dependent MLC phosphorylation (48). In endothelial cells, thrombin and LPA induce MLC phosphorylation through a RhoA and Rho-kinase-dependent mechanism. Thrombin- and LPA-induced RhoA activation is associated with increased MLC phosphorylation that can be blocked by C3 exoenzyme or transfection with dominant negative RhoA, RhoN19 (4, 12, 44). In addition, both thrombin- and LPA-induced increases in endothelial MLC phosphorylation are Rho-kinase dependent, as treatment with the specific Rho-kinase inhibitor Y-27632 completely blocks MLC phosphorylation (7, 42, 44). Similarly, in this study, we demonstrate that TGF-
-induced endothelial MLC phosphorylation is dependent on the RhoA/Rho-kinase signaling cascade, as TGF-
activates RhoA, and pretreatment of endothelial monolayers with either the RhoA inhibitor C3 exoenzyme or the Rho-kinase inhibitor Y-27632 completely blocks TGF-
-induced MLC phosphorylation. We also show that infection of BPAE monolayers with a constitutively active RhoA virus, RhoAQ63L, is sufficient to induce phosphorylation of MLC (Fig. 8). Similar to the TGF-
-induced MLC phosphorylation, RhoA-induced MLC phosphorylation is Rho-kinase dependent, as Y-27632 completely inhibits this response (Fig. 8). Therefore, similar to thrombin and LPA, TGF-
-induced activation of RhoA is sufficient to induce Rho-kinase-mediated phosphorylation of MLC. However, one should note that unlike thrombin and LPA, TGF-
-induced increases in RhoA activity and subsequent MLC phosphorylation require hours rather than minutes. This is consistent with previous findings showing that protein synthesis is required for the TGF-
-induced increase in MLC phosphorylation (19).
Increases in endothelial MLC phosphorylation and stress fiber formation occur in association with the formation of interendothelial gaps and subsequent increases in permeability of pulmonary endothelial monolayers (17). Similar to thrombin-induced endothelial MLC phosphorylation, thrombin-induced permeability is dependent on RhoA and Rho-kinase. Numerous studies have demonstrated that the thrombin-induced increase in permeability can be attenuated by inhibition of Rho-kinase with Y-27632 (4, 42). The LPA-induced increase in permeability, also associated with an increase in MLC phosphorylation, is Rho-kinase dependent, as Y-27632 completely blocks the LPA-induced permeability response (44). Recently, evidence has shown that at higher concentrations (>2 µM), Y-27632 can nonspecifically inhibit additional kinases in vitro (9). Preliminary studies showed that 10 µM Y-27632 increased baseline permeability over 4 h (data not shown). In the current study, we found that 1 µM Y-27632 was sufficient to consistently block TGF-
-induced Ser19/Thr18 MLC phosphorylation (indicating effective inhibition of Rho-kinase) without increasing baseline permeability. However, inhibition of either RhoA with C3 or Rho-kinase with Y-27632 did not completely inhibit the increase in endothelial permeability following TGF-
, suggesting that additional signals other than RhoA/Rho-kinase and/or Rho-kinase-mediated MLC phosphorylation mediate the TGF-
-induced increase in permeability.
Previous studies have suggested that MLC phosphorylation is not solely responsible for increases in permeability but is necessary for the maintenance and severity of agonist-induced increases in MLC-associated permeability. Moy et al. (30, 31) presented evidence that suggests thrombin-induced increases in permeability occur as a two-part process. The initial decrease in barrier function is thought to result from a loss of cell-cell adhesion and is independent of MLC phosphorylation and tension generation. Once barrier function is lost, MLC phosphorylation maintains the increase in permeability. Thrombin-induced phosphorylation of MLC temporally associates with the development of force within endothelial monolayers, which begins at an approximately similar time to the maximal decreases in endothelial monolayer resistance. Treatment with ML-7, which decreases cell tension and light chain phosphorylation, had no effect on the initial thrombin-induced permeability response but resulted in a much shorter time for recovery to baseline (31). Similarities to these findings exist with the TGF-
-induced increase in endothelial permeability. First, inhibition of MLC phosphorylation with either C3 or Y-27632 inhibits
50% of TGF-
-induced permeability. Second, inhibition of MLC phosphorylation does not affect the initial decrease in endothelial monolayer resistance. Interestingly, inhibition of Rho-kinase and subsequent MLC phosphorylation causes the decrease in permeability to stabilize after 2 h and not worsen. In contrast to thrombin-mediated effects, inhibition of MLC phosphorylation does not facilitate recovery of TGF-
-induced permeability effects. These findings suggest that phosphorylation of MLC is an important contributor to TGF-
-induced permeability; however, TGF-
activates additional signaling mechanisms independently of MLC phosphorylation that are involved in the early phases of increased permeability.
In addition to increasing MLC phosphorylation, activation of the RhoA/Rho-kinase signaling cascade results in massive cytoskeletal reorganization and stress fiber formation associated with the formation of interendothelial cell gaps. The RhoA- and Rho-kinase-dependent cytoskeletal reorganization of the actin cytoskeleton is dependent on the integration of multiple downstream signals (35). RhoA-induced activation of numerous proteins has been implicated in Rho-A-mediated regulation of cytoskeletal dynamics. In addition to Rho-kinase, these proteins include phospholipase C, phospholipase D, phosphatidylinositol 4-phosphate 5-kinase, Dia, Na+/H+ exchanger isoform 1 and 4, and others (reviewed in Refs. 43 and 48). Rho-kinase itself also modulates the activity of multiple cytoskeleton-associated proteins other than MLC, including adducins, ezrin/radixin/moesin proteins, myosin phosphatase, and the LIM-kinase/cofilin pathway (reviewed in Refs. 35 and 43). Indeed, we present evidence in this study that RhoA and Rho-kinase mediate TGF-
-induced actin cytoskeletal reorganization and gap formation. After treatment with TGF-
, endothelial cells reorganize F-actin from thick cortical actin bands to centrally located actin filaments or stress fibers. TGF-
-induced reorganization of actin is associated with an increase in Ser19/Thr18 MLC phosphorylation that is colocalized with central actin filaments as well as intense staining at the periphery of cells adjacent to endothelial gaps. We demonstrate that the TGF-
-induced formation of stress fibers and phosphorylation of MLC is dependent on Rho-kinase activity (Fig. 7).
Although inhibition of Rho-kinase completely blocked MLC phosphorylation, Y-27632 did not block TGF-
-induced relocalization of actin from the cell periphery to more centrally located actin filaments or completely prevent the formation of interendothelial cell gaps. Because RhoA can activate additional signaling cascades independently of Rho-kinase, we initially proposed that additional RhoA-dependent signaling cascades may account for the Rho-kinase-independent TGF-
-induced cytoskeletal reorganization. In contrast to TGF-
-induced responses, Rho-kinase was the primary effector of RhoA-induced reorganization of actin to central stress fibers and the formation of interendothelial cell gaps. This suggests that the Rho-kinase-independent signaling mechanisms of TGF-
-induced actin reorganization and gap formation are also RhoA independent. Therefore, we conclude that RhoA- and Rho-kinase-mediated increases in actin filaments and MLC phosphorylation are important contributors to TGF-
-induced endothelial cytoskeletal changes and gap formation. In addition, TGF-
induces alternative signaling mechanisms, independently of RhoA and Rho-kinase, possibly involving the organizational state of actin, that regulate gap formation (Fig. 10). As shown in Fig. 7, TGF-
causes a loss of peripheral actin in addition to increasing central stress fibers. Peripheral actin has been implicated in maintaining cell-cell adhesion through the regulation of cadherin-mediated junctions (reviewed in Ref. 10). Whether TGF-
alters cadherin-mediated adhesion is unknown; however, one may speculate that this is involved with the Rho-independent pathway(s) depicted in Fig. 10.
In summary, we have investigated signaling pathways relevant to the formation of decreased pulmonary endothelial barrier integrity in response to TGF-
, a protein believed to play a central role in acute lung injury and pulmonary edema in multiple disease models. We demonstrate, for the first time, that TGF-
activates the RhoA-signaling cascade in endothelial cells. The TGF-
-induced activation of the RhoA/Rho-kinase signaling cascade in endothelial cells increases the content of F-actin and phosphorylation of MLC on Ser19 and Thr18, contributing to TGF-
-induced increases in stress fiber formation, interendothelial gap formation, and the subsequent decrease in monolayer resistance. Interestingly, our data demonstrate that the initial decrease in barrier function produced by TGF-
is independent of RhoA/Rho-kinase-dependent signals and suggest alternative signaling mechanisms contribute to TGF-
-induced cytoskeletal reorganization and compromised barrier integrity.
 |
GRANTS
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-68079 (to F. L. Minnear), T32-HL-07194 (to R. T. Clements), and HL-004332 and HL-54206 (to P. A. Vincent).
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ACKNOWLEDGMENTS
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The authors thank Nina C. Martino for technical assistance and Wendy Hobb and Debbie Moran for secretarial assistance in the preparation of this manuscript. We also thank Dr. Joseph Mazurkiewicz for technical assistance and use of the immunofluorescence microscope and imaging software and Dr. Gang Liu for assistance with the assay for measurement of F-actin content.
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FOOTNOTES
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Address for reprint requests and other correspondence: P. A. Vincent, Center for Cardiovascular Sciences, Albany Medical College, 47 New Scotland Ave., Albany, NY 12208 (E-mail: vincenp{at}mail.amc.edu)
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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