Cyclosporin A Inhibits Flow-mediated Activation of Endothelial Nitric-oxide Synthase by Altering Cholesterol Content in Caveolae*

Fluid shear stress generated by blood flowing over the endothelium is a major determinant of arterial tone, vascular remodeling, and atherogenesis. Nitric oxide (NO) produced by endothelial NO synthase (eNOS) plays an essential role in regulation of vascular function and structure by blood flow. Although cyclosporin A (CsA), an inhibitory ligand of cyclophilin A, is a widely used immunosuppressive drug, it causes arterial hypertension in part by impairing eNOS-dependent vasodilation. Here we show that CsA inhibits fluid shear stress-medi-ated eNOS activation in endothelial cells via decreasing cholesterol content in caveolae. Exposure of cultured bovine aortic endothelial cells to 1 (cid:1) M CsA for 1 h significantly inhibited NO production and eNOS phosphorylation at Ser-1179 induced by flow (shear stress (cid:2) 12 dynes/cm 2 ). The effect of CsA was not related to inhibition of two known eNOS kinases, protein kinase B (Akt) and protein kinase A, because CsA did not affect Akt or protein kinase A activation. In rabbit aorta perfused ex vivo , CsA also significantly inhibited flow-induced eNOS phosphorylation at Ser-1179 but had no effect on Akt measured by phosphorylation at Ser-473. However, CsA treatment decreased Adenovirus-expressing (cid:1) -galactosidase (LacZ) was used as a control. Ex vivo Flow Experiments— Using isolated rabbit aortic segments, flow experiments were performed exactly as described previously (31). Statistical Analysis— All results are reported as mean (cid:3) S.E. Western blots were quantified using the NIH Image 1.6 software to yield arbitrary densitometry units. The significance of the results was as-sessed by a paired Student’s t test or by analysis of variance where appropriate.

Vascular endothelial cells, which form the inner lining of the blood vessel wall, are exposed to fluid shear stress, the dragging force generated by blood flow. Fluid shear stress modulates endothelial structure and function and is a major determinant of vascular remodeling, arterial tone, and atherogenesis (1,2). Physiologically, fluid shear stress is the most important stimulus for the continuous formation of nitric oxide (NO) 1 by endothelial nitric-oxide synthase (eNOS) in vessels (3,4), which plays an essential role in mediating many effects of fluid shear stress including regulation of vascular tone and diameter (5). Although flow-induced NO production appears to be both Ca 2ϩ -dependent and Ca 2ϩ -independent (6, 7), phosphorylation of eNOS by flow has been recognized as a critical regulatory mechanism controlling eNOS activity (6,8). In particular, we and others have showed that increased eNOS phosphorylation in response to flow occurs mainly at serine 1179 (pS1179-eNOS, serine 1177 in the human eNOS sequence) in bovine aortic endothelial cells (BAEC) (9 -11). Protein kinase B (Akt) and cAMP-dependent protein kinase A (PKA) are two upstream kinases shown to phosphorylate eNOS Ser-1179 in response to flow (9 -12).
Phosphorylation and activation of eNOS is not only dependent on upstream kinases but is also determined by its specific subcellular location (13). In particular, the localization of eNOS in caveolae affects the function of the enzyme, including the interaction of eNOS with proteins such as caveolin-1 and heat shock protein 90 (6,13). Whereas eNOS bound with caveolin-1 is inactive in caveolae, localization of eNOS to caveolae is required for its activation by stimuli because conditions that inhibit the localization of eNOS in caveolae (e.g. inhibition of eNOS myristoylation and cholesterol depletion) markedly decrease eNOS activity (14 -16). These data suggest that eNOS needs to be localized in caveolae to interact with proteins required for its activation (17). Cholesterol is one of the main constituents of caveolae and is essential for normal caveolae function (13). Incubating endothelial cells with agents that lower the cholesterol content of caveolae such as oxidized low density lipoprotein and ␤-cyclodextrin (␤-CD) cause eNOS to translocate from the caveolae and inhibit eNOS activation (16).
Cyclosporin A (CsA) is an immunosuppressive drug that acts by binding cyclophilin A (CyPA), thereby inhibiting the activity of calcineurin, which regulates responses necessary for T cell activation (18). Since its introduction in clinical practice, CsA has been linked to a major side effect, arterial hypertension (19,20). Although several mechanisms have been proposed including nephrotoxicity, sympathetic nervous system activation, and endothelin-1 secretion (21), much evidence points to endothelial dysfunction as an essential factor in the pathogen-esis of CsA-induced hypertension. Roullet et al. (22) found that CsA-induced hypertension was the consequence of a primary effect on resistance vessel relaxation, not increased vasoconstriction. On one hand, CsA is known to increase superoxide production (23) that should decrease NO bioavailability caused by peroxynitrite formation. On the other hand, CsA has been suggested to decrease NO production by eNOS (24,25). Most recently, Kuo et al. (26) showed that CsA inhibited VEGFinduced eNOS activation by blocking calcineurin-mediated eNOS dephosphorylation at serine 116 but not its phosphorylation at serine 1179. However, the mechanisms by which CsA inhibits flow-mediated eNOS activation remain unknown. Here we show that CsA treatment inhibits flow-mediated eNOS phosphorylation and NO production. We provide evidence that this inhibitory effect of CsA is caused by a decrease of caveolae cholesterol content resulting in the displacement of eNOS from caveolae without inhibition of kinases known to phosphorylate eNOS.
Cell Culture-BAEC purchased from Cambrex Bio Science Walkersville, Inc. (formerly Clonetics), were grown on gelatin-coated 60-mm tissue culture dishes in media containing 10% fetal bovine serum (Invitrogen). After confluence the cells were growth-arrested overnight using media with 0.1% fetal bovine serum for the flow experiments.
Flow Experiments for Cultured Cells-Before the experiment BAEC in culture dishes were rinsed twice with Hanks' balanced saline solution (130 mmol/liter NaCl, 5 mmol/liter KCl, 1.5 mmol/liter CaCl 2 , 1.0 mmol/ liter MgCl 2 , and 20 mmol/liter HEPES, pH 7.4) with l g/liter glucose and 140 mg/liter CaCl 2 at 37°C. The cells were then subjected to either steady laminar flow in a cone and plate viscometer or static conditions for the same amount of time at 37°C (27). For harvest, cells were washed with ice-cold phosphate-buffered saline containing 1 g/ml protease inhibitor mixture and 1 mM sodium orthovanadate (Sigma).
Immunoblot and Immunoprecipitation Analysis-BAEC were lysed in Triton/Nonidet P-40 lysis buffer (0.5% Triton X-100, 0.5% Nonidet P-40, 10 mM Tris, pH 7.5, 2.5 mM KCl, 150 mM NaCl, 20 mM ␤-glycerolphosphate, 50 mM NaF, 1 mM Na 3 VO 4 , 1 l/ml protease inhibitor (Sigma), scraped off the dish, sonicated (4 ϫ 10 s bursts, Ultrasonic Homogenizer 4710 Series, Cole-Palmer Instrument Co., Chicago IL), and then centrifuged for 10 min at 10,000 ϫ g. The supernatant was analyzed for protein concentration using the Bradford method (Bio-Rad), and equal amounts of protein (30 g/sample) were separated by SDS-PAGE and transferred to a nitrocellulose membrane (Hybond TM ECL, Amersham Biosciences). The membranes were blocked for 2 h with 5% bovine serum albumin, phosphate-buffered saline, pH 7.5, 0.1% Tween 20 and incubated with primary antibody for 2 h at room temperature or overnight at 4°C. The secondary antibody was horseradish peroxidase-conjugated and was visualized with the ECL system. For immunoprecipitation, lysates containing equal amounts of proteins were precleaned by incubating with protein A/G-Sepharose for 2 h and then incubated with anti-CyPA antibody and rocked overnight at 4°C. After incubation with protein A/G-Sepharose for 2 h, precipitates were washed three times in lysis buffer and resuspended in SDS-PAGE sample buffer. After denaturation the samples were separated by SDS-PAGE and immunoblotted with an anti-caveolin-1 antibody.
Detergent-free Purification of Caveolae-enriched Membrane Fractions-Caveolae-enriched membrane fractions were prepared following a protocol reported (28) with minor modifications. Cells were grown in gelatin-coated 150-mm tissue culture dishes. Upon confluence they were growth-arrested overnight. After stimulation, cells were washed twice with ice-cold phosphate-buffered saline and scraped in 10 ml of phosphate-buffered saline, pelleted, and resuspended in 2 ml of buffer 1 (500 mM Na 2 CO 3 pH 11, 25 mM MES, 150 mM NaCl, and 0.1% protease inhibitor mixture solution), homogenized (30 strokes in a Dounce homogenizer), and sonicated (3 ϫ 10-s bursts, Ultrasonic Homogenizer 4710 Series, Cole-Palmer Instrument Co., Chicago IL). The homogenate was placed in a 17-ml ultracentrifuge tube and was adjusted to 45% sucrose by adding 2 ml of buffer 2 (90% sucrose, 25 mM MES, pH 6.5, 150 mM NaCl, and 0.1% protease inhibitor mixture solution). 4 ml of each buffer 3 (35% sucrose, 250 mM sodium carbonate, pH 11, 25 mM MES, 150 mM NaCl, 0.1% protease inhibitor mixture solution) and buffer 4 (5% sucrose, 250 mM sodium carbonate, pH 11, 25 mM MES, 150 mM NaCl, 0.1% protease inhibitor mixture solution) were layered on top of it, and the samples were centrifuged at 28,000 rpm for 20 h in a SORVALL Discovery TM 100S Ultracentrifuge equipped with Surespin TM 630 (17 ml) rotor (Kendro Laboratory Products, Newton, CT). 12 ϫ 1-ml fractions were collected from the top to the bottom of each tube. An equal volume of each fraction was subjected to Western blot analysis and probed for eNOS, caveolin-1, and CyPA with corresponding antibodies.
NO Measurements-The media were collected after the flow experiments, and the NO content was quantified with a Sievers NOA280 nitric oxide analyzer (27).
PKA Activity Assay-PKA activity in caveolae-enriched membrane fractions was measured using a PKA activity assay kit from Stressgen Bioreagents (British Columbia, Canada) according to the manufacturer's instructions.
Cholesterol Measurements-Caveolae (represented by fractions 4, 5, and 6 from sucrose gradient separation) were pooled, and lipids were pelleted by centrifugation at 17,000 ϫ g for 15 min. The pellet was lipid-extracted (0.6 ml of chloroform, 0.3 ml of methanol and 150 l of saline). After mixing, the bottom layer containing chloroform with the lipids was left to evaporate under a hood. A Triton-chloroform extraction was then performed following a protocol described by De Hoff et al. (29). The amount of cholesterol was measured using a Cholesterol CII kit (Wako) and normalized to the total protein concentration.
Adenovirus Infection-For the infection of BAEC with recombinant adenovirus, BAEC were seeded on gelatin-coated dishes the day before infection. Cells were infected with recombinant adenovirus at 100 multiplicity of infection in serum-free Dulbecco's modified Eagle's medium for 2 h, incubated for 24 h in a growth medium, and then growtharrested overnight before the treatment. Construction of adenovirusexpressing PKA inhibitor (PKI) has been described previously (30). Adenovirus-expressing ␤-galactosidase (LacZ) was used as a control.
Ex vivo Flow Experiments-Using isolated rabbit aortic segments, flow experiments were performed exactly as described previously (31).
Statistical Analysis-All results are reported as mean Ϯ S.E. Western blots were quantified using the NIH Image 1.6 software to yield arbitrary densitometry units. The significance of the results was assessed by a paired Student's t test or by analysis of variance where appropriate.

CsA Inhibits Flow-induced NO Production and eNOS Phosphorylation in BAEC-In endothelial cells, flow increases NO
production proportional to the level of fluid shear stress applied (7). To evaluate the effect of CsA on NO production, we treated growth arrested BAEC with 1 M CsA or vehicle for 1 h and then exposed cells to either flow (an arterial level of laminar shear stress ϭ 12 dynes/cm 2 ) or static culture for 30 min. In response to flow for 30 min, NO released by BAEC increased by 2.2-Ϯ 0.5-fold relative to control cells maintained in static culture ( Fig. 1, p ϭ 0.02). In cells pretreated for 1 h with CsA, NO production stimulated by flow (1.2-Ϯ 0.2-fold) was significantly inhibited by 83% compared with control conditions (Fig.  1). CsA treatment had no significant effect on basal NO production ( Fig. 1).
CsA Does Not Inhibit Flow-stimulated Akt Activation-Akt has been shown to phosphorylate pS1179-eNOS in response to flow (9 -11). To measure Akt activation we used a phosphospecific antibody for Akt serine 473 (pS473-Akt) that correlates with Akt kinase activity (32). In response to flow, pS473-Akt increased with a time course very similar to pS1179-eNOS (Fig.  3A). There was, however, no inhibition of Akt phosphorylation by CsA pretreatment in contrast to the dramatic inhibition of pSer1179-eNOS (compare Figs. 2 and 3). Quantitation of the results showed that flow increased pS473-Akt maximally by 4.5-Ϯ 1.1-fold compared with static conditions (p ϭ 0.03, n ϭ 4). In cells pretreated with 1 M CsA, flow increased pS473-Akt maximally by 4.0-Ϯ 0.6-fold compared with static conditions (p ϭ 0.02, n ϭ 4). There were no significant differences between control and CsA-treated cells at any time point.
CsA Does Not Inhibit PKA-mediated eNOS Phosphorylation at Serine 1179 -In addition to Akt, PKA has been shown to mediate pS1179-eNOS in vitro and in vivo (33, 34). Boo et al. (34) recently showed that PKA inhibition by either its inhibitor H89 or adenovirus-expressing PKA inhibitor decreased flowinduced pS1179-eNOS with no effect on Akt activation. To evaluate the role of PKA in our system, we first characterized the inhibitory effect of PKI on forskolin-induced PKA activation and eNOS phosphorylation. PKA activation in cells was evaluated by phosphorylation of vasodilator-stimulated phosphoprotein (VASP), an established substrate for both PKA and cGMP-dependent protein kinase. VASP is a 46-kDa protein that is phosphorylated preferentially at Ser-239 by cGMP-dependent protein kinase and at Ser-157 by PKA. Phosphorylation of Ser-157 leads to a shift in the apparent molecular weight from 46 -50 kDa on SDS-PAGE, which is detectable by Western blot analysis using anti-VASP antibody (30,35). In BAEC infected with a control viral vector (adenovirus-expressing LacZ), forskolin induced VASP phosphorylation and associated mobility shift (Fig. 4A, upper panel). Infection with adenovirusexpressing PKI (100 multiplicity of infection) inhibited PKA as shown by the failure of forskolin to increase VASP phosphorylation and mobility shift (Fig. 4A, upper panel). PKI also inhibited pS1179-eNOS stimulated by forskolin (Fig. 4A, lower  panel). These data confirm that forskolin-induced VASP phosphorylation and pSer1179-eNOS are PKA-dependent. Next, we studied the effect of CsA on forskolin-induced PKA activation and pS1179-eNOS. Pretreatment with 1 M CsA did not inhibit forskolin-induced VASP phosphorylation and pS1179-eNOS (Fig. 4B). Taken together, these results suggest that CsA does not inhibit PKA-mediated pS1179-eNOS. Finally, we studied the effect of PKI on flow-mediated pS1179-eNOS. Infection with adenovirus-expressing PKI significantly inhibited (65%, n ϭ 3) flow-mediated pS1179-eNOS compared with infection with LacZ adenovirus (Fig. 4C, upper panel). These results indicate a significant role for PKA in flow-induced pS1179-eNOS. There was no significant effect of PKI on pS473-Akt (Fig. 4C, lower panel). To determine whether the inhibitory effect of CsA on flow-mediated pS1179-eNOS was caused by inhibition of PKA activation, we studied the effects of CsA on flow-mediated PKA activation. However, we did not observe any significant VASP phosphorylation in response to flow either in the absence or presence of CsA. This finding is consist- ent with previous reports documenting no change in cAMP level with flow measured in whole cells (36).
Because both PKA and eNOS have been shown to be compartmentalized and regulated by an interaction with caveolin in caveolae, we further examined the effect of CsA on PKA localization and activation in caveolae. To analyze PKA localization in BAEC, we used a sucrose density gradient method to separate cellular fractions and isolate caveolae (28). After centrifugation, 12 fractions were subjected to Western blot analysis and probed for PKA and caveolin-1 with corresponding antibodies. In control conditions without CsA treatment, caveolin-1 was found mainly in fractions 4, 5, and 6 that represent the caveolae fractions (28) (Fig. 4D, first panel). Although PKA mainly was detected in heavier fractions (Fig. 4D, second panel,  fractions 7-11), a small amount of PKA was found in caveolae as well. To our knowledge, this is the first demonstration that PKA is localized in caveolae of endothelial cells. However, exposure to 1 M CsA for 1 h did not induce any significant change in PKA distribution in caveolae (Fig. 4D, second panel  versus fourth panel). Furthermore, flow did not change the localization of PKA in caveolae (Fig. 4E), and CsA had no effect on PKA distribution in the presence of flow (Fig. 4E). Finally, neither CsA treatment nor flow changed PKA kinase activity in caveolae (Fig. 4F). These results indicate that CsA does not change PKA localization and activation in caveolae.
CsA Displaces eNOS from Caveolae in Endothelial Cells-The localization of eNOS in caveolae is important for its activation by physiologic stimuli (6,13,17). A possible explanation for CsA mediated inhibition of eNOS phosphorylation by flow is translocation of eNOS to a cellular site where it is no longer accessible for activation. As Exposure to 1 M CsA for 1 h induced a small redistribution of caveolin-1 from caveolae to heavier fractions (Fig. 5A, second panel, fractions 7-11), suggesting that CsA may slightly disrupts caveolae integrity. In contrast, although eNOS was concentrated in caveolae in control conditions (Fig. 5A, third panel), it showed a significant decrease in caveolae fractions after CsA treatment (Fig. 5A, fourth panel). Quantitation of eNOS distribution (Fig. 5B), showed a significant movement of eNOS from caveolae fractions (from 70 -46% of total) and an increase in heavier fractions (from 9 -31% in fractions 7-9) after CsA treatment.
CsA Diminishes the Cholesterol Content in Endothelial Caveolae-Cholesterol is essential for caveolae structure and function. Depleting cholesterol from cell membranes impairs caveolae structure, resulting in translocation of eNOS from caveolae and inhibition of eNOS activation (16). Recently, CsA has been shown to modify cholesterol trafficking. Ito et al. showed that CsA inhibited apolipoprotein A-I-mediated cholesterol trafficking (37). Also, Uittenbogaard et al. showed that CsA decreased cholesterol content in caveolae via blocking transport of newly synthesized cholesterol to caveolae (38). Therefore, we studied the effect of CsA on caveolae cholesterol content in endothelial cells. Using the protocol described in Fig.  5, we prepared caveolae from control and CsA-treated cells and measured caveolae cholesterol content. Treatment with 1 M CsA for 1 h significantly decreased cholesterol content in caveolae to 66% of control (Fig. 6, p Ͻ 0.01). As a positive control, we used 5 mM ␤-CD, a membrane-impermeable cholesterol-binding agent, which removes cell-surface cholesterol. We observed a similar decrease in cholesterol by ␤-CD (60% of control) (Fig.  6A). To further determine whether the effect of CsA on eNOS activation by flow is related to the decrease in cholesterol FIG. 4. CsA does not alter PKA localization and activation in BAEC. A, BAEC were infected with 100 multiplicity of infection PKI adenovirus or control LacZ and then exposed to 10 M forskolin for 30 min. Cell lysates were analyzed by Western blot with antibodies specific for VASP and the phosphorylated form of pS1179-eNOS. B, BAEC were pretreated with 1 M CsA or vehicle for 1 h and then exposed to 10 M forskolin for 30 min. Cell lysates were analyzed by Western blot with antibodies specific for VASP and the phosphorylated form of pS1179-eNOS. C, BAEC were transfected with LacZ or PKI adenovirus as in A and then exposed to flow for 30 min. Cell lysates were analyzed by Western blot with antibodies specific for VASP and the phosphorylated form of pS1179-eNOS. All blots are representative of three independent experiments. D, BAEC were treated with 1 M CsA for 1 h, and then the cell lysates were subjected to sucrose density gradient fraction analysis for caveolin-1 and PKA by Western blots with antibodies for caveolin-1 and PKA, respectively. E, BAEC were treated with 1 M CsA for 1 h and exposed to flow for 30 min or static conditions (time ϭ 0), and then caveolae-enriched membrane fractions were isolated. Samples from Fraction #5 were analyzed for caveolin-1 and PKA. The blots are representative of three independent experiments. F, isolated caveolae-enriched Fraction #5 was used for the assay of PKA activity, and the results are presented as mean Ϯ S.E. (n ϭ 3). content in caveolae, we asked whether ␤-CD (5 mM) could reproduce the effects of CsA, because we have shown that these two agents have a similar effect to decrease caveolae cholesterol content (Fig. 6). We pretreated BAEC with 5 mM ␤-CD for 1 h and then exposed the cells to flow for 30 min. Similar to the effect of CsA, ␤-CD markedly diminished flow-induced pSer1179-eNOS (Fig. 6B).
CsA Disrupts the Interaction between Caveolin-1 and CyPA-Caveolins have the ability to bind cholesterol and fatty acids and have been implicated in the regulation of cellular cholesterol metabolism in several cell types (39). Increasing evidence suggests that CsA affects caveolin-1 function by binding to CyPA (37,38,40). Thus, we studied the effect of CsA on CyPA localization and its interaction with caveolin-1. CyPA is mainly cytosolic, but we found a small portion of CyPA localized to caveolae fractions (Fig. 7A, Control Fraction #5). CsA treatment induced this portion of CyPA to translocate from caveolae fractions to heavier fractions (Fig. 7A, CsA). Moreover, exposure of BAEC to 1 M CsA significantly decreased the interaction of CyPA and caveolin-1 in a time-dependent manner with ϳ75% inhibition at 1 h as shown by co-precipitation with CyPA antibody (Fig. 7B, upper panel) and co-precipitation with caveolin-1 antibody (Fig. 7C, upper panel). CsA did not alter the levels of CyPA and caveolin-1 protein expression (Fig. 7, B and  C, lower panels).
Cholesterol Supplementation Restores Flow-induced eNOS Activation-To confirm that decreasing cholesterol in caveolae is important in CsA-mediated eNOS inhibition, we asked whether the CsA inhibitory effect could be reversed by cholesterol supplementation. We incubated BAEC with 30 g/ml cholesterol for 24 h before CsA treatment. Based on our previous report (41), this magnitude of cholesterol supplementation increases the caveolae number detected by electron microscopy. Although cholesterol supplementation did not increase flowmediated pS1179-eNOS, the CsA inhibitory effect was significantly diminished (Fig. 8A, compare the fourth and eighth  lanes). Quantitation of the results showed that CsA significantly inhibited flow-mediated pS1179-eNOS by 90% (Fig. 8B) in the absence of cholesterol supplementation. In contrast, cholesterol pretreatment completely blocked the inhibitory effect of CsA and restored pS1179-eNOS to the level observed in control cells (Fig. 8B).
CsA Inhibits Flow-induced eNOS Phosphorylation in Rabbit Aorta Perfused ex Vivo-To confirm that our findings in cell  (Fractions 4 -6) was measured using a cholesterol oxidase-based method and was reported relative to total protein concentration in the samples (mean Ϯ S.E. n ϭ 3), *, p Ͻ 0.05; **, p Ͻ 0.01. B, cells were treated with 5 mM ␤-CD for 1 h and then exposed to flow for 5, 10, and 30 min. Cell lysates were analyzed by Western blot with antibodies specific for the phosphorylated form of pS1179-eNOS and total eNOS. Blots are representative of three independent experiments.  3). B, BAEC were treated with 1 M CsA for indicated times, and then the cell lysates were subjected to immunoprecipitation with antibodies for CyPA and analyzed with antibodies for total caveolin-1 and CyPA (n ϭ 3). C, BAEC were treated with 1 M CsA for indicated times, and then the cell lysates were subjected to immunoprecipitation with antibodies for caveolin-1 and analyzed with antibodies for total CyPA and caveolin-1 (n ϭ 3).
culture are physiologically relevant, we used an ex vivo perfused vessel culture system characterized in our lab (31) to study the effect of CsA on flow-induced eNOS activation. The basal levels of pS1179-eNOS and pS473-Akt were very low in isolated rabbit aortas equilibrated with low flow (shear stress ϭ 0.4 dynes/cm 2 , data not shown) (31). Application of flow at physiological shear stress (12 dynes/cm 2 ) for 30 min strongly induced pS1179-eNOS (Fig. 9A) and pS473-Akt (Fig.  9B). CsA treatment for 1 h significantly inhibited flow-induced pS1179-eNOS (Fig. 9, A and C), but had no significant effect on pS473-Akt (Fig. 9, B and C). These results are consistent with our data in cultured BAEC, supporting the physiological relevance of our findings. DISCUSSION The major finding of the present study is that CsA decreases flow-mediated eNOS phosphorylation and generation of NO in endothelial cells. We believe that the most likely mechanism is that CsA reduces caveolae cholesterol content, causing translocation of eNOS from caveolae. The decrease in cholesterol content is probably induced by inhibition of cholesterol trafficking as shown by decreased interactions between CyPA and caveolin-1 as well as decreased CyPA in caveolae. This effect of CsA may be important in the pathophysiology of hypertension associated with CsA treatment. In addition, eNOS translocation from caveolae represents a novel mechanism for eNOS regulation and endothelial dysfunction.
It is well documented that pS1179-eNOS plays an important role in stimulation of eNOS activity in response to various physiological stimuli, including fluid shear stress (6,8). We found that CsA significantly decreased flow-induced pS1179-eNOS, which accounts for the inhibitory effect of CsA on flowstimulated eNOS activity. The finding that flow-mediated pS473-Akt was not affected by CsA suggests that the flow sensing mechanism was not impaired by CsA. Furthermore, data showing that CsA failed to inhibit eNOS activation by forskolin suggest that CsA does not act by inhibiting PKA. Thus inhibition of two well described eNOS kinases cannot explain the effect of CsA. Although our results with PKI indicate that PKA is involved in flow-mediated pS1179-eNOS, VASP phosphorylation in whole cell lysates was not increased in response to flow. Moreover, we showed for the first time the localization of PKA in caveolae of endothelial cells, but we found that both CsA and flow did not change PKA distribution and activation in caveolae. Future studies are needed to define the involvement of PKA in flow-stimulated eNOS phosphorylation.
Because CsA inhibits the protein phosphatase calcineurin (also named PP2B), it is possible that the inhibitory effect of CsA could be caused by the inhibition of phosphatases. At least three different classes of serine/threonine protein phosphatases (PP1, PP2A, and calcineurin) have been implicated in the regulation of eNOS dephosphorylation and activity (8). PP2A has been shown to dephosphorylate eNOS-S1179 (33,42). However, there is no evidence to suggest that CsA regulates PP2A activity. The Ca 2ϩ /CaM-dependent protein phosphatase calcineurin appears to mediate dephosphorylation of eNOS at serine 116 (26). Whereas calcineurin may be important in VEGF-stimulated eNOS activation, because CsA blocked VEGFdependent eNOS dephosphorylation at serine 116 (26), inhibition of calcineurin is unlikely to contribute to the effects of CsA on flow-mediated eNOS activation for two reasons. First, we found that CsA decreased flow-mediated pS1179-eNOS but not eNOS phosphorylation at serine 116. Second, calcineurin is a Ca 2ϩ /CaM-dependent enzyme and eNOS Ser-1179 phosphorylation by flow is Ca 2ϩ -independent. Thus eNOS protein phosphatases are not likely involved in the inhibitory effect of CsA on flow-mediated pS1179-eNOS.
In addition to regulation by protein kinases and phosphatases, phosphorylation of eNOS is largely determined by its specific subcellular location (6,8,13), especially localization in caveolae. For example, an eNOS mutant deficient in myristo- , and then CsA (1 M) or vehicle (methanol) was applied intraluminally for additional 1 h. Flow was increased to physiological level (12 dynes/cm 2 ) for 30 min. Endothelial cell lysates were specifically purified from rabbit aorta and used for immunoblotting for pS1179-eNOS and pSer473-Akt and total eNOS and Akt. C, phosphorylation of pS1179-eNOS and pSer472-Akt was determined by densitometry, and data are represented as mean Ϯ S.E. (n ϭ 2), *, p Ͻ 0.05. ylation shows no agonist-induced pS1179-eNOS due to failure to associate with plasma membrane (15). Oxidized low density lipoprotein displaces eNOS from caveolae and impairs eNOS activation (16). In this study, we show that CsA induces eNOS displacement from caveolae, suggesting a new mechanism for the CsA effect on endothelial cell function. The colocalization of the signal transduction molecules that comprise the "eNOS signaling complex" within the different membrane compartments facilitates enzyme activation. Thus, it is possible that Akt activated by flow cannot target eNOS because of eNOS translocation after CsA pretreatment. Also, phosphorylation and activation of eNOS by stimuli, including fluid shear stress, may determine eNOS subcellular localization (13,43). It should be noted that CsA treatment does not result in significant loss of caveolae structure because the majority of caveolin-1 is still in Triton X-100 insoluble fractions (Fig. 5A). In addition, our results showing that CsA did not increase basal eNOS activity (Fig. 1) suggest that the eNOS displaced from caveolae by CsA is inactive. In contrast, in caveolin-1-null mice that lack caveolae, eNOS activity is up-regulated (44,45) and plasma NO levels are greatly increased (46). A possible explanation for this discrepancy is that activation of "liberated" eNOS with a loss of caveolae in caveolin-1-null mice differs from CsA-induced eNOS redistribution without loss of caveolae.
Cholesterol in caveolae plays a key role in maintaining the integrity of caveolae structure and function. We previously reported that cholesterol supplementation increased caveolae numbers in BAEC (41). Depleting cholesterol with ␤-CD disrupts caveolae structure (47) and causes eNOS to redistribute to an intracellular membrane compartment impairing eNOS activation (16). We observed a similar decrease in cholesterol (60% of control) and in flow-mediated pS1179-eNOS by CsA compared with 5 mM ␤-CD (Fig. 6), suggesting that depletion of cholesterol in caveolae explains the CsA inhibitory effect. The fact that cholesterol supplementation restores pS1179-eNOS by flow in the presence of CsA (Fig. 9) supports an important role of cholesterol in maintaining functional caveolae required for flow-mediated eNOS activation. Consistent with our findings, plasma membrane cholesterol has been shown to play a key role in other flow-mediated signals (48 -50). In particular, Park et al. (49) showed that depleting cholesterol in caveolae by ␤-CD inhibited flow activation of extracellular signal-regulated kinase but not c-Jun NH 2 -terminal kinase.
Although the decrease of cholesterol content in caveolae by CsA is significant, the mechanisms are not clear. Ito et al. (37) showed that CsA binding to CyPA inhibited apolipoprotein A-I-mediated cholesterol trafficking via disrupting a cholesterol-caveolin complex. Cholesterol-caveolin-CyPA complex is also involved in transport of newly synthesized cholesterol to the plasma membrane in NIH 3T3 cells and L1210-JF lymphocyte cell line transfected with caveolin-1 (38) and in internalization of cholesteryl esters from caveolae in a Chinese hamster ovary cell line (51). In the present study, we found that CsA inhibited the interaction of CyPA and caveolin-1 in a time-dependent manner, suggesting that CsA alters cholesterol trafficking in BAEC. Further studies are needed to determine the role of CyPA and caveolin-1 in maintaining the cholesterol content in endothelial caveolae.