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Am J Physiol Lung Cell Mol Physiol 290: L405-L413, 2006. First published September 23, 2005; doi:10.1152/ajplung.00292.2005
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siRNA-induced caveolin-1 knockdown in mice increases lung vascular permeability via the junctional pathway

Kayo Miyawaki-Shimizu,* Dan Predescu,* Jun Shimizu, Michael Broman, Sanda Predescu, and Asrar B. Malik

Department of Pharmacology and Lung and Vascular Biology Center, University of Illinois College of Medicine, Chicago, Illinois

Submitted 5 July 2005 ; accepted in final form 8 September 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Caveolin-1, the principal integral membrane protein of caveolae, has been implicated in regulating the structural integrity of caveolae, vesicular trafficking, and signal transduction. Although the functions of caveolin-1 are beginning to be explored in caveolin-1–/– mice, these results are confounded by unknown compensatory mechanisms and the development of pulmonary hypertension, cardiomyopathy, and lung fibrosis. To address the role of caveolin-1 in regulating lung vascular permeability, in the present study we used small interfering RNA (siRNA) to knock down caveolin-1 expression in mouse lung endothelia in vivo. Intravenous injection of siRNA against caveolin-1 mRNA incorporated in liposomes selectively reduced the expression of caveolin-1 by ~90% within 96 h of injection compared with wild-type mice. We observed the concomitant disappearance of caveolae in lung vessel endothelia and dilated interendothelial junctions (IEJs) as well as increased lung vascular permeability to albumin via IEJs. The reduced caveolin-1 expression also resulted in increased plasma nitric oxide concentration. The nitric oxide synthase inhibitor L-NAME, in part, blocked the increased vascular albumin permeability. These morphological and functional effects of caveolin-1 knockdown were reversible within 168 h after siRNA injection, corresponding to the restoration of caveolin-1 expression. Thus our results demonstrate the essential requirement of caveolin-1 in mediating the formation of caveolae in endothelial cells in vivo and in negatively regulating IEJ permeability.

caveolae; interendothelial junction; nitric oxide

CAVEOLAE ARE 50- to 100-nm diameter subcellular organelles existing as omega-shaped plasma membrane invaginations and as apparently free vesicles in the cytoplasm that are abundant in endothelial cells, adipocytes, vascular smooth muscle cells, and fibroblasts (31, 45). In endothelial cells, caveolae have been implicated in the transport of molecules via transcytosis (30–32), cholesterol (29, 46, 47), and triglyceride homeostasis (5, 28, 37, 51), regulation of cell cycle (20, 23) and apoptosis (14), and integration of several cell signaling pathways (2, 19, 40). Although caveolae are referred to as stabilized plasma membrane rafts with a unique molecular composition (48), their physiological roles are cell specific (1). In vascular endothelial cells, a primary function of caveolae is to mediate the active processes of endocytosis and transcytosis (7, 31, 36), and thus they are believed to be important in the regulation of vascular permeability (32).

Biochemically, caveolae in all cell types studied, including endothelial cells, are defined by the presence of caveolin-1, the 22- to 24-kDa scaffold, and regulatory protein (1). Caveolin-1 and caveolin-2 are coexpressed in many cell types, whereas caveolin-3 is the specific isoform present in skeletal and cardiac muscle cells (39). Caveolin-1, the best characterized of the isoforms, has been shown to interact via its scaffold domain with caveola-associated signal proteins such as endothelial nitric oxide synthase (eNOS), Src family tyrosine kinases, G{alpha} proteins, diverse serine/threonine protein kinases, and insulin receptor (22, 43, 44, 52). The functional roles of caveolin-1 in vivo are beginning to emerge with the study of caveolin-1–/– mice. Genetic deletion of caveolin-1 resulted in impairment of cholesterol homeostasis (18), insulin sensitivity (9), increased nitric oxide (NO) production (10, 38, 53), alterations in Ca2+ signaling (10), and cardiac and lung dysfunction (8, 10, 25, 53). Studies also showed defects in the uptake and transport of albumin in vascular endothelia (41, 42). In addition, the knockout mice showed evidence of cellular hyperproliferation and severe vascular and extracellular matrix abnormalities (10, 38, 53). Because of the microvascular alterations occurring in caveolin-1-null mice and the finding that caveolin-2 expression also decreases in these mice (25), the precise role of caveolin-1 in the regulation of vascular permeability remains unclear. In the present study, we used the small interfering RNA (siRNA) approach to reduce specifically and transiently caveolin-1 expression in the mouse lung endothelium and studied the effects of suppression of caveolin-1 expression on lung vascular permeability as well as the time-dependent reversal of lung vascular permeability after restoration of caveolin-1 expression. We demonstrate herein changes in the number of caveolae in endothelial cells in situ that corresponded to the level of expression of caveolin-1 induced by siRNA. Lung microvascular permeability to albumin was increased after reduction in caveolin-1 expression in association with dilation of interendothelial junctions (IEJs). The increased vessel wall albumin permeability was prevented in part by treatment with NG-nitro-L-arginine methyl ester (L-NAME). We also observed time-dependent reversal of caveolin-1 expression coupled to restoration of vascular permeability to normal levels. Together, our studies show that caveolin-1 directly regulates caveolar formation in endothelial cells in vivo and that it has an effect on vascular permeability by controlling the tightness of IEJs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents and chemicals. Reagents were obtained as follows. PBS (pH 7.4), Hanks' balanced solution, protease inhibitor cocktail, dimethyldioctadecylammonium bromide (DDAB), and L-NAME were from Sigma-Aldrich (St. Louis, MO). SDS, 5% nonfat milk, and nitrocellulose membranes were from Bio-Rad (Hercules, CA). Cholesterol was from Calbiochem (La Jolla, CA). FuGENE 6 transfection reagent was from Roche Diagnostics (Indianapolis, IN). BCA protein assay reagent kit and Super Signal West Pico Chemiluminescent Substrate kit were from Pierce (Rockford, IL). Affinity-purified horseradish peroxidase (HRP)-coupled rabbit polyclonal anti-caveolin-1 IgG (N-20, sc-894) was from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal anti-caveolin-2 IgG1 antibody was from BD Biosciences (Franklin Lakes, NJ). Affinity-purified polyclonal antibody to mouse IgG heavy and light chains (whole IgG) raised in goat and labeled with HRP was from KPL (Gaithersburg, MD). LiposoFast-Basic was from Avestin (Ottawa, Canada). 2,4-Dinitrobenzene sodium sulfonate was from Aldrich Chemical (Milwaukee, WI). Bovine albumin was from ICN Biochemicals (Aurora, OH). BSA conjugated to 6- and 15-nm colloidal gold suspensions was from Electron Microscopy Science (Fort Washington, PA). siRNA and control siRNA sequences were synthesized by Integrated DNA Technologies (Coralville, IA).

Animals. Male CD1 mice (25–30 g) were kept under standard housing and feeding conditions, and animal protocols were approved by the University of Illinois Animal Care Committee. All experiments were made under anesthesia induced by intraperitoneal administration of a mixture of ketamine (60–100 mg/kg), xylazine (2–2.5 mg/kg), and acepromaxine (2–2.5 mg/kg) in saline.

siRNA constructs. We designed a single-stranded siRNA construct corresponding to mouse caveolin-1 (11–13): 5'-GAGAAGCAAGUGUAUGACG-3'. Another sequence in which four bases were altered, 5'-GAGAAGCAGUGAUACGACG-3', was used as a control construct.

Preparation of cationic liposome-siRNA complex and transfection. In vivo cationic liposomes were made using 50% M DDAB and 50% M cholesterol as described (49). Multilamellar liposomes were formed by rotary evaporation (Rotavapor R-124, Buchi) of a chloroform solution of the above mixture at 42°C, under vacuum, in a stream of argon for 20 min. A 5% glucose solution was added to the flask to dissolve the lipid film. The mixture was incubated for 60 min at 42°C with occasional vortexing (49). The suspension was extruded 15x through 50-nm polycarbonate filters mounted in LiposoFast-Basic, a mini-extruder device fitted with two 0.10-ml Hamilton syringes (26). The cationic liposome-siRNA complex was prepared by addition of 0.4–1.3 mg/kg of siRNA into 100–150 µl of liposome suspensions in 5% glucose (total 200 µl). The ratio of siRNA to liposome was 1:5 (wt/vol). The liposome-siRNA mixture was injected via the tail vein.

Mouse lung perfusion. For mouse lung perfusion, we required, for fixation of tissue used, a described protocol (36). Briefly, under anesthesia, an incision on the ventral line was made, and after removing the soft tissues from the neck region, the trachea was exposed and cannulated with a polyethylene tube (PE-50; Becton-Dickinson, Parsippany, NJ) for constant positive pressure ventilation at 120 breaths/min. The abdominal cavity was opened on the medium line, and the abdominal aorta was exposed and used as the outlet while an opening made in the vena cava caudalis provided the inlet. The vasculature was perfused with warm (37°C) Hanks' balanced solution, to which sodium bicarbonate was added for a final osmolality of 280 mosmol/kgH2O, using a peristaltic pump (Minipuls 3, Gilson) at a flow rate of 1.5 ml/min for 5 min (36).

Western blotting. The lungs perfused with Hanks' balanced solution, as described above, were removed from the animals, hand minced, mixed with 1% SDS in PBS containing protease inhibitor, and homogenized (3 x 30 s on ice) using a Polytron (Westbury, NY). The tissue lysate was cleared by centrifugation (15,000 g, 30 min) in a Beckman Avabti Centrifuge J-25 I using JA-25.50 rotor. The lysates from lungs were loaded on 12% SDS-PAGE gels, run for 1.5 h at constant voltage (150 V), and electrotransferred (2 h, 720 mA, at 4°C) onto nitrocellulose membranes. Nonspecific binding was blocked with 5% nonfat milk. Immunodetection was performed with an HRP-conjugated caveolin-1 monoclonal antibody (1:1,000) for 2 h at room temperature overnight at 4°C followed by incubation with HRP-coupled goat anti-mouse IgG (1:1,000). The antibody/antigen complexes were detected using the Super Signal West Pico Chemiluminescent Substrate kit.

Electron microscopy. The blood-free lungs obtained as described above were fixed in situ by 10-min perfusion with a mixture of freshly prepared 4% formaldehyde plus 2.5% glutaraldehyde plus 2 mM Ca2+ plus 1% tannic acid in 0.1 M PIPES (pH 7.2). The fixed tissues were cut into small pieces (3 mm x 3 mm) and further fixed by immersion in the same fixative (4°C overnight). The tissues were postfixed with 1% OsO4 (1 h on ice in absence of light), stained with 7.5% uranyl acetate, dehydrated in increasing concentrations of ethanol and propylene oxide, and embedded in Epon. Semithin sections (60 nm thick) were obtained with a Leica ultracut microtome, stained with lead citrate and uranyl acetate, and examined in a Jeol 1240 transmission electron microscope at 80 Kev.

Measurement of plasma NO concentration. Mouse blood was collected from the right ventricle and centrifuged (14,000 rpm, 15 min, 4°C). The total plasma NO was determined based on the enzymatic conversion of nitrate to nitrite by nitrate reductase, followed by spectrophotometric quantification of reaction product using the Griess reagent (50).

Lung vascular permeability to albumin tracer. Dinitrophenylated albumin (DNP-albumin) was prepared as described (21). Under anesthesia, 10 mg of DNP-albumin were injected via tail vein and allowed to circulate for 10 min. Then, unbound tracer was washed with Hanks' balanced solution as described in Mouse lung perfusion. The lungs were removed, weighed, homogenized (3 x 30 s on ice) with 1 ml of PBS by using a Polytron (Westbury, NY), and gently agitated at 4°C overnight for ELISA analysis. The lysates were cleared by centrifugation (45 min, 45,000 g) in a Beckman Avanti Centrifuge J-25 I using JA-25.50 rotor, and supernatants were used for ELISA. The accumulated DNP-albumin was measured by ELISA as described (33, 34). Anti-DNP HRP-coupled antibody (1:1,000) was used to quantify the amount of albumin tracer transported across the vessel wall. The data were expressed as nanograms of DNP-albumin per milligram of wet tissue per minute.

For morphological analysis of vascular permeability, the lungs were perfused with 6 or 15 nm of gold-conjugated BSA for 10 min. The unbound tracer was removed by 5-min perfusion, and the washed lungs were fixed and prepared for electron microscopy as described above.

Statistical analysis. Data were analyzed by Student's t-test or ANOVA for multiple sample comparisons (Bonferroni) wherever appropriate. Values were considered significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
siRNA-induced suppression of caveolin-1 expression and its time-dependent recovery in mouse lungs. The cationic liposome-siRNA complex was administrated by tail vein injection in CD1 mice, and the expression of caveolin-1 protein was evaluated at different times postinjection in lung lysates by Western blotting. Caveolin-1 expression in lungs was reduced in a concentration- and time-dependent manner in the siRNA-treated mice (Fig. 1, A–C). The inhibition of caveolin-1 protein expression was small at 24 h after caveolin-1 siRNA injection, whereas after 48 h, the caveolin-1 expression was reduced by >80% in the mice receiving the higher siRNA concentrations. The effect of the specific siRNA was marked at 72 h, but the decrease in expression reached a nadir at 96 and 120 h postinjection of construct (Fig. 1C). There were no significant differences between the 1.0 mg/kg and 1.3 mg/kg siRNA concentrations in silencing caveolin-1 expression at the 72-h time point (Fig. 1, A and B); thus we used the siRNA concentration of 1.0 mg/kg in all subsequent studies. Caveolin-1 expression was downregulated by 72 h, and its suppression lasted up to 144 h postinjection (Fig. 1C). Full recovery of caveolin-1 expression occurred at 168 h after siRNA administration (Fig. 1C).



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  		Fig. 1. Silencing and recovery of caveolin-1 in lungs after intravenous (iv) injection of small interfering RNA (siRNA) cationic liposome complex in mice. A: Western blots of caveolin-1 from mouse lung lysates show time- and dose-dependent reduction of the caveolin-1 protein expression. Caveolin-1 expression was decreased by 90% at 72 h after injection at the higher siRNA concentration. B: quantification of blots in A normalized to GAPDH. Differences are shown between control (Ctl) and 0.4, 0.7, 1.0, and 1.3 mg/kg of injected caveolin-1 siRNA (P < 0.05). C: caveolin-1 siRNA mice lung extracts show time-dependent decrease of caveolin-1 protein expression followed by its time-dependent recovery. Caveolin-1 silencing was evident as early as 48 h after systemic administration of siRNA and lasted until 144 h. At 168 h post-siRNA injection, the expression of caveolin-1 recovered to normal levels. All blots shown are representative of at least 5 experiments.

 
Specificity of caveolin-1 siRNA construct. We analyzed the specificity of caveolin-1 knockdown by Western blotting. Lung expression of the caveolae-targeted signaling protein eNOS expression was not changed in total lung homogenates obtained from caveolin-1 siRNA-treated mice (Fig. 2, A and B). Also, the expression of caveolin-2 was not altered (Fig. 2, A and B). Transfection of mismatched control siRNA construct did not affect the expression of caveolin-1 (Fig. 2, A and B).



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  		Fig. 2. Specificity of siRNA-induced caveolin-1 suppression in mouse lungs. A: siRNA did not interfere with the expression of caveolin-2 and endothelial nitric oxide synthase (eNOS) in mouse lungs. A control construct with 4 base alterations (MATERIALS AND METHODS) failed to silence caveolin-1 expression. B: mRNA expression data normalized to GAPDH. Differences between control and 24, 48, and 72 h after caveolin-1 siRNA injection (P < 0.05). All blots are representative of at least 3 experiments.

 
Decreased caveola number in lung endothelia of caveolin-1 siRNA-treated mice. The number of caveolae in mouse lung endothelia from different lung vascular segments was quantified from the electron micrographs after caveolin-1 knockdown (Table 1). Figure 3A shows electron micrographs of mouse lung capillary endothelium in the basal state. Many caveolae were opened luminal and abluminal as well as apparently free inside of endothelial cells, and the IEJ was sealed by a tight junction. Figure 3B shows the main alterations at 96 h after siRNA administration: 1) opening of IEJs allowing a direct communication between the vascular lumen and perivascular space and 2) disappearance of caveolae from endothelial cells (in comparison with Fig. 3A). Figure 3C illustrates the response at 192 h after siRNA, corresponding with the 1) reestablishment of caveola number with the same distribution within endothelial cells as in Fig. 3A, and 2) resealing of IEJ by tight junction (Fig. 3C, inset). The number of caveolae was significantly decreased at 96 h and fully restored by 192 h (Fig. 3, Table 1). Table 1 shows the quantification of the data in endothelia of mouse lung artery, capillary, and vein. Caveola numbers decreased in each segment as a function of time, with the maximal decrease occurring in capillary endothelia. The caveola numbers were fully restored at 192 h. Concomitant with the decrease in caveola number, we observed dilation of IEJs (Fig. 3B, Table. 2). This response was also reversed at 192 h with the time-dependent restoration of caveolin-1 expression (Fig. 3C). Table 2 quantifies the changes occurring in IEJs along the different lung vascular segments. The percent open IEJs was increased primarily in the venular endothelia at 96 h after siRNA administration, but we also found open IEJs in capillaries, and the responses in both segments were reversed at 192 h. The number of open IEJs was less in the artery and capillary endothelia compared with venous endothelia 96-h caveolin-1 knockdown mouse lungs (Table 2).


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  		Table 1. Time course in alterations of number of endothelial caveolae in different lung vascular segments after caveolin-1 siRNA injection

 


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  		Fig. 3. Caveolin-1 suppression induces dilation of interendothelial junctions (IEJs) and reduction in caveolar number. Electron micrographs show lung capillaries of control mice (A), mice at 96 h after siRNA injection (B), and mice at 192 h after siRNA injection (C). Caveolin-1 knockdown mice showed decreased number of caveolae and open IEJs. Caveolae reformed in mice after the restoration of caveolin-1 expression. C, inset: IEJ recover to establish a normally restrictive barrier. Thin arrows in A point to caveolar profiles in control cells. Thick arrows indicate IEJs in A and B. Scale bars: A, 250 nm; B, 175 nm; and C, 120 nm.

 

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  		Table 2. Opening of interendothelial junctions after caveolin-1 siRNA injection

 
Electron microscopic evaluation of albumin permeability in lung microvessels in mice treated with caveolin-1 siRNA. To define morphologically the alterations in lung vascular permeability observed in caveolin-1 knockdown mice, we used two sizes of gold particles (6 and 15 nm) to which BSA was absorbed (34). The two tracers were presented to the lung vascular beds of control and caveolin-1 knockdown mice by perfusion as described in MATERIALS AND METHODS. In control mice (Fig. 4A) and in mice in which caveolin-1 expression had recovered at 192 h (Fig. 4D), lung microvessel endothelial cells transported the albumin tracer only via caveolae when the IEJs were sealed by tight junctions. No tracer particles were found inside IEJs (Fig. 4A). However, at 96 h after injection of the siRNA, at a time of maximal suppression of caveolin-1 expression, we observed that the open IEJs (Fig. 4, B and C) were easily penetrated by the 6-nm gold-BSA tracer (Fig. 4B) but were impermeable to the 15-nm gold-BSA tracer (Fig. 4C). However, by 196-h administration of specific siRNA, we found sealed IEJs selectively excluding the 6-nm gold-BSA particles (Fig. 4D). It was clear that at 96 h after caveolin-1 siRNA treatment, the number of caveolae/endothelial cells was greatly reduced (Fig. 4, B and C).



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  		Fig. 4. Vascular hyperpermeability to albumin via IEJ in caveolin-1 knockdown mice. Electron micrographs with gold-conjugated BSA of lung capillaries in control mice (A), mice at 96 h after siRNA injection perfused with 6 nm of gold-BSA (B) and 15 nm of gold-BSA (C), and mice at 192 h after siRNA injection perfused with 6 nm of gold-BSA (D). The open IEJ in caveolin-1 knockdown mice was labeled by 6 nm of gold-BSA but restricted the passage of 15 nm of gold-BSA. D: IEJ recovers to establish a normally restrictive barrier impenetrable to 6 nm of gold-BSA. Scale bars: A, 250 nm; B, 100 nm; C, 120 nm; and D, 175 nm.

 
Increased plasma NO concentration in caveolin-1 siRNA-treated mice. The plasma NO concentration increased as the caveolin-1 expression decreased in response to caveolin-1-specific siRNA administration (Fig. 5A). Between 48 and 144 h after siRNA, plasma NO concentration was 2.5-fold greater in the siRNA-treated mice vs. the wild-type mice injected with control construct (i.e., 32.8 ± 2.3 µM vs. 12.4 ± 4.5 µM). At 168 h after siRNA administration, the concentration of NO recovered to normal values (i.e., 13.0 ± 1.1 µM vs. 12.4 ± 4.5 µM; Fig. 5A). L-NAME treatment reduced by 65% the increase in plasma NO concentration occurring at 96 h after siRNA injection (Fig. 5B).



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  		Fig. 5. siRNA-induced reduction in caveolin-1 expression is coupled to time-dependent and reversible increase in plasma concentration of nitric oxide (NO). A: plasma NO concentration in caveolin-1 knockdown mice. At 48 h post-siRNA injection, NO concentration in mice plasma was increased 2.5-fold over control wild-type mice value, whereas at 168 h, with reconstitution of caveolin-1 protein, NO concentration returned to the control level. Values are means ± SE, with n = 6 mice/group (*P < 0.05). B: increased NO production in knockdown of caveolin-1 is NG-nitro-L-arginine methyl ester (L-NAME) sensitive. Results show a 75% reduction in NO production in mice treated with L-NAME in which caveolin-1 expression had been reduced by siRNA (*P < 0.05). This did not happen in the control case or when caveolin-1 expression had recovered in the siRNA-treated mice. L-NAME itself increased NO concentration in caveolin-1 knockdown mice (**P < 0.05). Results are shown as means ± SE, n = 6 mice/group.

 
Increased lung microvascular permeability after caveolin-1 suppression and its partial rescue by L-NAME. To examine the role of caveolin-1 in regulating pulmonary vascular permeability to albumin in vivo, we injected intravenously DNP-albumin used as a tracer (MATERIALS AND METHODS). We observed an increase in pulmonary vascular albumin permeability in the caveolin-1 knockdown mice relative to controls (i.e., 10.5 ± 1.4 µg·mg–1·min–1 in knockdown mice vs. 4.9 ± 0.3 µg·mg–1·min–1 in control mice; Fig. 6A). The control siRNA construct, which failed to induce a decrease in caveolin-1 expression, had no effect in altering pulmonary vascular albumin permeability (data not shown). After the time-dependent recovery of caveolin-1 expression, albumin permeability was restored to the normal value (5.4 ± 0.2 µg·mg–1·min–1 vs. 4.9 ± 0.3 µg·mg–1·min–1 in the time-matched controls; Fig. 6A). We examined the possible role of increased NO production in mediating the hyperpermeability phenotype seen in caveolin-1 knockdown mouse lungs. L-NAME (30 mg/kg iv) (42) treatment of control mice increased the basal pulmonary vascular albumin permeability from 4.9 ± 0.3 ng·mg–1·min–1 to 7.2 ± 1.3 ng·mg–1·min–1. L-NAME also prevented 80% of the increase in lung vascular permeability observed at 96 h after siRNA administration (Fig. 6B).



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  		Fig. 6. siRNA-induced reduction in caveolin-1 expression results in increased lung microvascular permeability to albumin. A: increased microvascular permeability of dinitrophenylated albumin (DNP-albumin) tracer in caveolin-1 knockdown mouse lungs and its recovery after caveolin-1 reexpression. Values are means ± SE, n = 6 mice/group. *P < 0.05. B: treatment with L-NAME prevents the increase in lung vascular permeability in caveolin-1 knockdown mice. Caveolin-1 knockdown mice lung showed a significantly increased lung vascular DNP-albumin permeability, and this response expressed a % change from L-NAME alone that was significantly reduced in L-NAME-treated mice in which the caveolin-1 expressed were decreased. Data are means ± SEM, with n = 6 animals/group. *P < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The in vivo role of caveolin-1 in regulating vascular endothelial barrier function is not well understood. Recent studies in caveolin-1–/– mice have shown increased endothelial permeability (42), whereas other studies showed unchanged permeability (41). The interpretation of these results is complicated by the fact that genetic deletion of caveolin-1 results in cardiomyopathy, pulmonary hypertension and fibrosis, and severe microvascular pathology (8, 30, 53). In addition, caveolin-1–/– mice have a 90% decrease in the expression of caveolin-2 (7); thus the results cannot be ascribed solely to the absence of caveolin-1. Studies in endothelium-specific caveolin-1 transgenic mice have shown that overexpression of caveolin-1 did not change the caveola number (3), suggesting that the formation of caveolae in the endothelium in vivo may not be dependent on caveolin-1 protein expression. To explore the role of caveolin-1 in regulating endothelial barrier function in vivo, we used a caveolin-1-specific siRNA construct. We delivered the construct intravenously via liposomes to reduce caveolin-1 expression and focused on alterations in the lung vascular endothelium. We observed that caveolin-1 protein expression decreased in a time- and concentration-dependent manner, but importantly, the response was reversible. The siRNA sequence resulted in decreased protein expression within 48 h, and the response was fully restored within 7 days; thus the siRNA approach has the advantage that vascular barrier function could be studied during both downregulation and recovery phases. In contrast to the results in caveolin-1–/– mice (38), we observed that siRNA-induced suppression of caveolin-1 did not modify the expression of caveolin-2. The reason for this is not clear, but it might be that caveolin-2 was not degraded since there was some residual level of caveolin-1 after the siRNA treatment. Also, in an important control experiment, we showed that the caveolin-1 siRNA did not affect the expression of eNOS.

Caveolin-1 is believed to be responsible for caveola biogenesis in the endothelium in vivo because studies have shown that caveolae are absent in caveolin-1–/– mice (10, 38). Caveolin-1 expression in cells lacking it resulted in the formation of structures having morphology and function resembling caveolae (17, 24). However, studies in which caveolin-1 was overexpressed in endothelial cells of mice showed that caveola number did not increase upon its overexpression (3). To resolve whether caveolin-1 is required for caveolar formation, we quantified morphologically at the electron microscopic level the caveola number after siRNA-induced caveolin-1 suppression and its time-dependent restoration. The largest number of caveolae in the lung vasculature per unit of endothelial cell volume was found in the capillaries followed by the pulmonary vein and pulmonary artery. This could not be ascribed to a change in the volume of the cell since this value held constant. The largest decrease in caveolar number induced by siRNA occurring in the capillaries and veins were, strikingly, coupled to opening of IEJs in these vascular segments. Moreover, the time-dependent restoration of caveolin-1 expression resulted in the closure of IEJs and normal lung caveolar number. Thus we conclude that the level of caveolin-1 expression is an essential requirement for caveolar biogenesis in endothelial cells in vivo, and the reduction of caveolin-1 expression is associated with the opening of IEJs.

Previous studies have shown that caveolin-1 binds to and serves as a tonic inhibitor of eNOS activity, thereby limiting NO production in endothelial cells (6, 16, 27). Plasma NO concentration in caveolin-1–/– mice was fivefold greater than in wild-type mice (53). The high NO levels were most likely caused by increased eNOS activity since significant changes in eNOS protein expression were not observed by Western blotting (53). In caveolin-1 knockdown mice, we observed that the plasma NO concentration was 2.5-fold greater than wild-type mice. The increased NO production was rescued by the time-dependent normalization of caveolin-1 protein expression. Our results support the concept that the level of caveolin-1 expression is a key determinant of a physiological plasma NO concentration. We addressed whether L-NAME could reduce NO production in caveolin-1 knockdown mice; however, we observed that the inhibition of NO production was incomplete at concentrations of L-NAME that typically block eNOS activity. A possible explanation for the residual NO production in L-NAME-treated mice may be that the L-NAME concentration used may not have been sufficient to inhibit the caveolin-1-released eNOS. Alternatively, it may be that other NOS isoforms are upregulated in the caveolin-1 knockdown mice, but we do not think this is likely since we did not observe an increase in the expression of inducible NOS (not shown).

Previous studies have shown that the caveolar vesicle carriers are involved in the transcytosis of plasma proteins across endothelial cells (31–34). Recent studies in caveolin-1–/– mice showed that the absence of caveolin-1 increased microvascular permeability via the paracellular pathway (42), whereas the uptake of albumin was reduced in caveolin-1 siRNA-treated endothelial cells (22). Because it has been reported that IEJ are open in caveolin-1–/– mice, and capillary endothelial cells have defective adhesion to the basement membrane (42), it has been difficult to make a conclusion about the role of caveolin-1 in regulating endothelial permeability. Our results using siRNA to suppress caveolin-1 expression (and thereby avoiding the issue of preexisting microvascular pathology seen in caveolin-1–/– mice described above) showed that the decrease in caveolin-1 expression resulted in increased lung vascular permeability to albumin, and this response was reversed as the caveolin-1 expression returned to the basal value. We provide evidence that the cellular pathways responsible for increased ablumin permeability are the opened IEJs. Interestingly, the increased dimension of IEJs induced by caveolin-1 knockdown slowed the transport of 6-nm gold-albumin particles but not the 15-nm gold-albumin tracer particles. This firmly indicates that although IEJ permeability to albumin was increased, the junctional barrier still remained restrictive to molecules >15 nm.

An obvious concern with the present study is that the siRNA-induced suppression of caveolin-1 results in the pathology seen in caveolin-1–/– mice. However, it is unlikely that the 3-day period of decreased expression of caveolin-1 induced with siRNA would have resulted in the lung fibrosis seen in the caveolin-1–/– mice. Another important concern is whether a component of increased lung vascular albumin permeability could have been due to a convective flux. However, we do not believe this to be the case since the plasma NO concentration was increased in these mice, and pulmonary microvessel pressure would likely be decreased; therefore, this is not likely to induce an increase in the convective flux of albumin. Also, at the ultrastructural level, the IEJs were dilated in the caveolin-1 knockdown lung microvessels, in direct agreement with the observed increase in pulmonary vessel albumin permeability at the junctional level as demonstrated with the 6-nm gold-albumin tracer.

We also addressed whether the increased production of NO following the suppression of caveolin-1 expression was involved in mechanism of augmented IEJ permeability. This study was carried out using L-NAME, a general NOS inhibitor; however, we observed that L-NAME alone increased lung vascular permeability. L-NAME significantly reduced the increase in vascular permeability induced by siRNA that caveolin-1 expression, suggesting downregulation of the interplay of caveolin-1 with eNOS may be implicated in the regulation of microvascular permeability. The delivery of the scaffolding domain of caveolin-1 via a cell-permeable sequence also interfered with the NO-dependent increase in vascular permeability (4, 6), consistent with the endothelial barrier protective effect of L-NAME described above. Although the present data suggest that increased albumin transport via the paracellular pathway is partly the result of deinhibition of eNOS secondary to the reduced expression of caveolin-1, there are some caveats. The primary one is that the administration of L-NAME has an independent effect in increasing vascular permeability, perhaps because eNOS-derived NO production is required for normal vascular endothelial integrity (35). This makes it difficult to examine the effects of blocking eNOS activity to address the effects of the deinhibited eNOS in regulating vascular permeability in the caveolin-1 knockdown mice. Despite this uncertainty about the role of eNOS-derived NO in regulating the increase in lung vascular permeability in mice with reduced caveolin-1 expression, our data are clear in demonstrating a key role of caveolin-1 as a negative regulator of IEJ permeability in vivo.


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 MATERIALS AND METHODS
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This study was supported by National Heart, Lung, and Blood Institute Grants HL-060678, HL-045638, HL-046350, HL-064573-05, and T32-HL-007829.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. Predescu, Dept. of Pharmacology, Univ. of Illinois College of Medicine, 835 So. Wolcott Ave. (M/C868), Chicago, IL 60612 (e-mail: Predescu{at}uic.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.

* K. Miyawaki-Shimizu and D. Predescu contributed equally to this work. Back


   REFERENCES
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