Constitutive eNOS-derived nitric oxide is a determinant of endothelial junctional integrity
Dan Predescu,
Sanda Predescu,
Jun Shimizu,
Kayo Miyawaki-Shimizu, and
Asrar B. Malik
Department of Pharmacology, University of Illinois, College of Medicine, Chicago, Illinois
Submitted 17 May 2004
; accepted in final form 24 March 2005
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ABSTRACT
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Basal vascular endothelial permeability is normally kept low in part by the restrictiveness of interendothelial junctions (IEJs). We investigated the possible role of nitric oxide (NO) in controlling IEJ integrity and thereby regulating basal vascular permeability. We determined the permeability of continuous endothelia in multiple murine vascular beds, including lung vasculature, of wild-type mice, endothelial nitric oxide synthase (eNOS) null mice, and mice treated with NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME). Light and electron microscopic studies revealed that L-NAME treatment resulted in IEJs opening within a few minutes with a widespread response within 30 min. We observed a 35% increase in transendothelial transport of albumin, using as tracer dinitrophenylated albumin in mouse lungs and other organs studied. To rule out the involvement of blood cells in the mechanism of increased endothelial permeability, vascular beds were flushed free of blood, treated with L-NAME, and perfused with the tracer. The open IEJs observed in these studies indicated a direct role for NO in preserving the normal structure of endothelial junctions. We also used the electron-opaque tracer lanthanum chloride to assess vascular permeability. Lanthanum chloride was presented by perfusion to various vascular beds of mice lacking NO. Open IEJs were seen only in capillary and venular endothelial segments of mice lacking NO, and there was a concomitant increase in vascular permeability to the tracer. Together, these data demonstrate that constitutive eNOS-derived NO is a crucial determinant of IEJ integrity and thus serves to maintain the low basal permeability of continuous endothelia.
morphometric analysis; basal lung vascular permeability; interendothelial junctions; nitric oxide synthase inhibition; endothelial nitric oxide synthase knockout mice; transendothelial transport
THE ENDOTHELIAL CELL (EC) monolayer, separating the blood from parenchymal tissue, controls the movement of cells, liquid, and small and large solutes (e.g., plasma proteins) from the blood to interstitial space (54). The low permeability of the endothelium under basal conditions, the characteristic feature of the vascular endothelial barrier, is dependent on the unaltered structure of EC junctions that together with the underlying basement membrane form the "endothelial barrier" (55). ECs control vascular permeability by: 1) regulating the tightness of interendothelial junctions (IEJs), 2) their large number of transcytotic carriers involved in the active transendothelial transport named transcytosis (i.e., apical-to-basolateral vesicle traffic), and 3) formation of focal adhesion complexes that promote binding of ECs to the basement membrane (57). As ECs may also release factors capable of controlling the basal endothelial permeability, in the present study we investigated the possible role of nitric oxide (NO) that is constitutively produced by EC nitric oxide synthase (eNOS) in regulating endothelial permeability.
NO, a diatomic free radical with a half-life of a few seconds, is produced by eNOS in different cell types, most prominently ECs (24, 41, 42). The EC-generated NO controls the basal tone of arterioles and venules (67) and serves to prevent inappropriate platelet aggregation (19) and neutrophil adhesion to ECs (14). However, the role of constitutively generated NO in regulating vascular permeability is still a matter of debate. In some studies NO was shown to increase permeability in vascular beds (7, 8, 17, 2730, 40), whereas it decreased vascular permeability in other studies (31, 3537, 52, 70). Studies also showed that NOS inhibitors increased vascular permeability (1, 13, 32), raising the possibility that NO production may tonically regulate endothelial barrier function. In the present study, based on morphological observations of microvessels in multiple vascular beds, including the pulmonary circulation, we show that the absence of NO [as induced by eNOS inhibition with N-nitro-L-arginine methyl ester (L-NAME) or eNOS gene deletion in mice] causes the opening of IEJs in the capillary and venular segments of lung and other vascular beds. Structural alterations in the junctions were coupled to increased vascular permeability to tracer albumin. Thus our study demonstrates that eNOS-derived NO constitutively regulates basal vascular permeability.
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METHODS
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Animals
Wild-type (WT) C57BL/6, eNOS/ (strain B6.129P2-Nos3tm1Unc), and inducible nitric oxide synthase knockout (iNOS/, strain B6.129P2-Nos2tm1lan) mice were purchased from Jackson Labs (Bar Harbor, ME). Breeding colonies were maintained in the University Animal Facility. All experiments were made in accordance with policies of the institutional Animal Care Committee. All mice were fed a normal diet.
Reagents
We purchased from Sigma Chemical (St. Louis, MO) leupeptin, glycerol, benzamidine, phenylmethylsulfonyl fluoride (PMSF), 3-aminopropanolthriethoxysilane, 3% Monastral blue solution and Monastral blue, bovine serum albumin (BSA), lanthanum chloride (LaCl), L-NAME, and glycerol. We purchased from Amersham Pharmacia (Piscataway, NJ) Triton X-100, Nonidet P-40 (NP-40), sodium dodecyl sulfate (SDS), Tween 20, nitrocellulose (NC) membranes, and all chemicals for electrophoresis. We purchased from ElectronMicroscopy Science (Fort Washington, PA) paraformaldehyde (PFA), glutaraldehyde (GA), lanthanum nitrate, Surmount water base, Entellan, polyvinylpyrrolidone (PVP), and all electron microscopy (EM)-grade reagents. Protein A/G immunobeads were purchased from Calbiochem (San Diego, CA), and Vectashield mounting medium was from Vector Lab (Burlingame, CA). Bicinchoninic acid (BCA) kit and enhanced chemiluminescence (ECL) kit were from Pierce (Rockford, IL). Anti-eNOS and anti-iNOS antibodies (Ab) and horseradish peroxidase (HRP)-conjugated affinity-purified goat anti-rabbit and anti-mouse immunoglobulin G (IgG) were purchased from Transduction Laboratories (San Diego, CA). Rabbit anti-dinitrophenylated albumin (A-DNP) Ab and goat anti-rabbit HRP-coupled Ab were from Chemicon (Temecula, CA).
A-DNP, the albumin tracer used in these studies, was prepared as described in (44). Homogeneity, presence of BSA aggregates, and conformational changes induced by the derivatization procedure were checked for every plot. Only monomeric A-DNP, which is an adequate substitute for the native albumin, was used in these experiments.
Biochemical Procedures
Tissue lysates were prepared at 4°C using a solubilization buffer containing 0.5% SDS + 1% NP-40 in phosphate buffered-saline (PBS), plus protease inhibitors (1 mM leupeptin, 2 mM benzamidine, 1 mM PMSF, and 2 mM Na-pyrophosphate). Organs free of blood after perfusion with cold buffer (PBS or Hanks') containing 1% glucose and mixture of protease inhibitors were hand-minced, suspended in solubilization buffer at a ratio of 1:9 (wt/vol), and homogenized in a Warring blender set at maximum speed (4°C). After 1 h of being stirred at room temperature (RT), the resulting tissue lysates were clarified by centrifugation for 1 h at 40,000 rpm at 4°C in a Beckman Optima TLX ultracentrifuge with the TLA-50 rotor.
Protein concentrations were determined using the BCA method (61), with the Pierce kit and BSA as standard.
Immunoblotting.
Total protein (200 µg) from tissue lysates was loaded on a preparative minigel (1.5 mm, 520% SDS-PAGE gradient) run at 150 V, and transferred to NC as in Ref. 63 using an Idea Scientific blotting apparatus at 4°C and 750 mA of constant current for 90 min. Strips of NC membranes containing the transferred proteins were blocked for 2 h at RT with BSA 3% + 0.05% Tween 20 + 1% goat serum in PBS and incubated for 1 h at RT with anti-eNOS or anti-iNOS Ab diluted 1:1,000 in the same blocking buffer. NC membranes were washed (3 x 10 min) with washing buffer (PBS + 1% goat serum + 0.2% Triton X-100) and incubated for 1 h at RT with the reporter Ab (HRP-coupled affinity purified goat anti-rabbit or anti-mouse IgG). After another wash, the reaction was visualized using an ECL kit and X-Omat Blue film.
ELISA.
Enzyme-linked immunoassays were carried out as in Ref. 48 using 100 µg/ml (total protein concentration) as starting concentration and HRP-conjugated affinity-purified goat anti-rabbit IgG as the reporter Ab. The reaction was developed with 3,3',5,5'-tetramethylbenzidine and read at 450 nm in a Molecular Dynamics ELISA reader. In brief, to determine transendothelial albumin transport, the albumin tracer A-DNP was allowed to interact for 10 min with the vascular beds, after which they were flushed free of tracer, organs were removed, cleared of connective tissue, weighed, hand-minced, and homogenized with a Polytron at a 1:10 (wt/vol) ratio in PBS. The ensuing homogenates were centrifuged at 150,000 g in a Beckman TLX tabletop ultracentrifuge for 1 h using the TLA 55 rotor. The pellets were discarded and supernatants (representing the diluted interstitial fluid which included the tracer) were used to determine the total protein content and the concentration of A-DNP. The amount of transported tracer was quantified using A-DNP as standard. To calculate the concentrations of A-DNP in the final supernatants, a standard curve was generated using known concentrations of A-DNP tracer, and the amount of tracer transported was determined at a series of decreasing concentrations on the linear part of the standard curve. The amount of tracer transported (expressed as ng A-DNP transported per mg tissue wet weight over 5 min) was used to assess vascular permeability to albumin in different organs.
NO Measurement.
Blood collected by cardiac puncture was centrifuged, and serum concentrations of the NO metabolites, nitrite and nitrate, were measured using an NO analyzer (ENO-20, Eicom) as in Ref. 23.
Tissue Experiments
Effects of NOS inhibition using L-NAME.
Studies were initially carried out at the light microscopic level using the mouse cremaster preparation as described in Ref. 34 and a diaphragmatic muscle preparation by essentially the same technique. Subsequent studies employing detailed EM analysis addressed alterations in permeability of multiple vascular beds, including the lung vasculature. NOS activity was inhibited in two ways: 1) direct administration of a bolus dose of 30 mg/kg L-NAME iv with the mice studied in the first 4 h or 2) chronic administration of L-NAME (for 760 days) added to drinking water; intake of inhibitor was 3040 mg/day as in Ref. 51. The control groups received 0.2 ml of sterile saline solution administered intravenously for acute treatment and regular water for the chronic inhibition group. Systolic blood pressure measured by the tail-cuff method (26), and body weights were recorded biweekly. In the chronic inhibition group, the mice were used for morphological and biochemical analyses at the eighth week of treatment.
Tracer experiments.
Mouse perfusions were done under anesthesia induced with ketamine-xylazine (10 mg/ml ketamine + 0.5 mg/ml xylazine) injected intraperitoneally as in Ref. 46. For perfusion of all nonvascular beds, the abdominal aorta was exposed; an inlet catheter was inserted above the iliac artery bifurcation and secured in place while another catheter was inserted in the vena cava caudalis and used as outlet. For lung perfusion, the inlet catheter was inserted into pulmonary artery with the outflow catheter in the left atrium. The vasculature, via either route, was flushed blood-free for 3 min with oxygenated Hanks' solution warmed up to 37°C, supplemented with 4 g/l glucose, and perfused with a peristaltic pump (Pharmacia P1) at a flow rate of 3 ml/min. The A-DNP was added to the perfusate at a concentration of 5 mg/ml and presented to the vascular beds studied for 5 or 10 min (10, 11). The unbound tracer was flushed from the vasculature by 3 min perfusion with Hanks' solution. The organs were then fixed by in situ perfusion of a fixative mixture (4% PFA + 2% GA + 1 mM CaCl2 in cacodylate buffer, pH 7.4) for 10 min. The lungs, heart, diaphragm, and skeletal muscle were removed, cut into 3 x 3 x 3-mm sections, and processed for morphological surveys. For assessment of the permeability of different vascular beds, the organs were flushed blood free, weighed, and then homogenized as described above. The final supernatants of these homogenates were used in ELISA to quantify vascular permeability as described (44, 48).
Microscopy and Immunocytochemistry
Light microscopic studies.
eNOS/ and L-NAME-treated mice were injected intravenously with a 0.2-ml solution of the Monastral blue dye. At different time points, cremaster and diaphragm muscles were fixed in situ by injecting 5 ml of freshly prepared 8% PFA in 0.1 M Na-cacodylate buffer (pH 7.2) intraperitoneally for cremaster and 3 ml of the same fixative into the thoracic and abdominal cavities for the diaphragm. After 15 min at RT, the muscles were removed, dissected free of connective tissue, and postfixed in 2% PFA + 0.5% GA + 2.5% PVP in 0.1 M Na-cacodylate for 24 h at RT. Selected specimens (chosen under a stereomicroscope) were cleared for 96 h at RT in glycerol and mounted with Entellan. Micrographs were obtained with a Zeiss Axiophot 2 microscope.
EM studies.
The tissues were fixed in situ by perfusing a fixative mixture (3% PFA + 2.5% GA + 1% LaCl in 0.1 PIPES, pH 7.2) for 15 min, and selected specimens were further fixed with the same mixture (without LaCl) for 1 h at RT. All specimens were postfixed in 2% OsO4 in acetate veronal buffer, pH 6.8, for 1 h on ice, stained in the dark for 1 h with 7.5% uranyl-magnesium acetate (UA), dehydrated through increasing concentrations of ethanol (50, 70, 90, 100%), then in propylene oxide, and embedded in Epon 812. Specimens embedded in Epon were cured for 72 h at 90° C, and sections
60 nm thick obtained with a Leica microtome were examined and photographed in a Philips TM-10 electron microscope. For routine examinations, sections from WT and eNOS/ mice were stained with 7.5% UA for 5 min and saturated lead citrate for 3 min and then examined. When LaCl was added to the fixative as a tracer, the tissue samples were not stained with UA and lead citrate.
Immunocytochemistry at the EM level.
The specimens were collected from tissues perfused with A-DNP as described above. Collected specimens were further fixed by immersion in a triple fixative (58) for 1 h on ice, stained in block for 30 min with 7.5% UA, dehydrated slowly in graded ethanol solutions, and embedded in Epon. A-DNP was detected by a postembedding immunostaining procedure (10, 11). In brief, sections of
5060 nm obtained from blocks of embedded tissues were quenched for 45 min at RT in PBS + 1% BSA (A-PBS), incubated for 2 h at RT with anti-DNP IgG diluted 1:2,000 in A-PBS, washed 3 x 15 min with A-PBS, incubated with gold-tagged (6 nm) anti-rabbit IgG diluted 1:5,000 in A-PBS, fixed shortly (5 min) with 2.5% GA, examined, and micrographed in a Jeol 1220 electron microscope operated at 80 kV.
Morphometric Analysis
Light microscopy.
Sections (on average 30 slides for every organ and each condition; i.e., WT, L-NAME-treated, and eNOS/ mice) from lung, heart, skeletal muscle, diaphragm, and gut were analyzed using the ImagePro Morphometric program, which allowed us to perform automated analyses of the number of vessels in reference to the total area of section, mean vascular diameter, number of histochemical-positive ("tattooed") vessels. The images were acquired using an Axioplan 2 Carl Zeiss microscope equipped with a color digital camera, and the mentioned morphometric program was run on an Apple G4 computer.
EM.
We carried out extensive morphometric analysis of lungs from WT mice (C57BL) and eNOS/ mice. For lungs, four to six Epon blocks were used for thin sectioning, and six grids per block (every grid with 1525 sections) were examined. When lung tissues were used for morphometric analysis, vertical uniform random and isotropic uniform random sections were prepared from each left and right lung as in Ref. 16. In brief, the vertical axis of each lung was identified, and 4-mm-thick slices were cut perpendicularly to the axis starting from a randomly chosen point. The slices were cut into bars at 3-mm intervals, and every fifth tissue block was selected from a randomly chosen starting point. Sections (6070 nm) were obtained at random, and only vessels with a full circumference on the grid mesh were photographed. The images were acquired with a Gatan charge-coupled device camera coupled to an Apple G4 computer. A total of 255 images for each part of a vascular bed (arterial, capillary, and venular) and each organ studied at the final magnification of x28,000 were stacked as a queue, and grid no. 3 from the Stereology Toolbox program (Morphometrix) was used to quantify the main endothelial features.
Statistical Analysis
Data are expressed as means ± SD. Statistical analysis was performed using one-way analysis of variance and Student's t-test with a post hoc Bonferroni correction for multiple comparisons performed to identify group differences accounted for the significant overall ANOVA. Significance was set at P < 0.001.
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RESULTS
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Characteristics of L-NAME-treated and eNOS/ Mice
We compared eNOS/ and iNOS/ mice for the relative expressions of eNOS and iNOS. Immunoblotting using anti-eNOS Ab failed to detect the enzyme in any organ of eNOS/ mice, whereas there was iNOS immunoreactivity in these mice; the opposite was the case for eNOS/ mice (Fig. 1). We also determined mean arterial pressure with the tail-cuff method in L-NAME-treated and eNOS/ mice. Arterial pressure averaged 137 ± 22 mmHg (n = 47) in L-NAME-treated mice and 135 ± 24 mmHg (n = 172) in eNOS/ mice, compared with the value of 102 ± 29 mmHg in WT mice (n = 66) (P < 0.001). Blood NO level (expressed as NOx), which under the basal condition reflects the eNOS-derived production, was threefold less in L-NAME-treated and eNOS/ mice than WT mice (Table 1). Based on these results, we investigated the vascular permeability characteristics of both L-NAME-treated and eNOS/ mice.

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Fig. 1. Absence of compensatory inducible nitric oxide synthase (iNOS) expression in endothelial nitric oxide synthase knockout (eNOS/) mice. Homogenates of the identified organs (100 µg total protein/lane) were run on a 520% SDS-PAGE gel and probed after transfer to nitrocellulose membranes with antibodies to eNOS or iNOS. eNOS/ mice did not express the protein, and conversely eNOS was expressed in iNOS/ mouse tissue. Wild-type (WT) mice expressed both isoforms. Results are representative of 3 experiments.
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Vascular Leakage of Monastral Blue Dye in L-NAME-treated and eNOS/ Mice
In the initial studies carried out at the light microscopic level, we observed extensive tattooing1 (Monastral blue deposits) of postcapillary venules (1040 µm diameter), muscular venules (4060 µm diameter), and venules (>70 µm diameter) in cremaster and diaphragm muscles of L-NAME-treated and eNOS/ mice as illustrated in Fig. 2, AD. This finding was indicative of extensive dye leakage into the extravascular space. In WT mice, we observed no deposits of the Monastral blue dye. We also noted differences in density and frequency of tattooing in different vascular segments of L-NAME-treated and eNOS/ mice. Measurement of tattooed areas in 24 preparations from cremaster and 42 from diaphragm showed that the surface area of affected fields varied from <0.5 µm2 in regions where capillaries reside to 1.5 µm2 in postcapillary venules and to 23 µm2 in collecting venules. There was no significant difference between the effects of acute vs. chronic NOS inhibition with L-NAME (42% of total number of vessels were affected after acute L-NAME administration vs. 38% after chronic L-NAME administration). In the acute NOS inhibition protocol, open IEJs with extravascular deposits of the tracer were seen in the first minute after application of L-NAME and reached a plateau at
30 min after L-NAME administration.

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Fig. 2. Vascular leakage of Monastral blue dye induced by N-nitro-L-arginine methyl ester (L-NAME) treatment and deletion of eNOS in mice. Muscular venules (short arrows in B, C, D) and postcapillary venules (long arrows in A, C, D) show extensive vascular tattooing induced by Monastral blue dye in both the diaphragmatic and cremaster vascular beds. Less tattooing is seen in the capillaries (arrowheads in A, B, D). Results are from L-NAME-treated mice (A and B) and eNOS/ mice (C and D). Bar = 15 µm. Results are representative of 9 experiments.
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Tattooing densities were calculated in adjacent randomly selected fields of 0.035 mm2 (with an aggregated area of 9.2 mm2 for cremaster) and 0.041 mm2 (with an aggregated area of 12.1 mm2 for diaphragm). In cremaster and diaphragm muscles obtained from mice treated with L-NAME, open IEJs were seen in 42% of postcapillary venules, 43% of collecting venules, and 45% of large veins. Survey of Monastral blue leakage carried out in eNOS/ mice also showed the same findings; i.e., extensive tattooing in the identical postcapillary, collecting venular, and large vein segments. Because these measurements suggested IEJ defects as the result of L-NAME inhibition and deletion of eNOS, studies were carried out at the EM level in multiple vascular beds to define the endothelial structures involved in the dye leakage.
Morphological Analysis of IEJs at the EM Level
We observed upon extensive EM analysis of vascular beds from different organs open IEJs in vessels of L-NAME-treated mice as illustrated in Figs. 3 and 4. Figure 3 shows open IEJs from the murine lung vascular bed in a postcapillary venule (Fig. 3A), large muscular vein (Fig. 3B), and capillary (Fig. 3C). Insets in Fig. 3, B and C, show normal IEJ spaces (sealed by tight junction) from WT mice. The same findings obtained in other vascular beds are illustrated in Fig. 4. IEJs were open in heart capillaries (Fig. 4A), diaphragm capillaries (Fig. 4B), and mesenteric venules (Fig. 4C). Note a platelet migrating outward through the open IEJ of this mesenteric vessel (Fig. 4C). Insets in Fig. 4, B and C, illustrate normal IEJs from these vascular segments in control mice (with the interendothelial spaces sealed by tight junctions that are the most apical IEJ structure).

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Fig. 3. L-NAME disrupts the integrity of interendothelial junctions (IEJs) in murine lung endothelium. A: gap of 1012 nm in a postcapillary venule from mouse lung. The same gaps are found at the level of lung muscular venules (B) and capillaries (C). Larger gaps with a greater frequency were found in the venular end of the murine lung vascular bed. The insets in B and C are matching IEJs (muscular venule in B and capillary in C from the WT mouse lung vascular bed). Bar = 125 nm in A, 175 nm in B and in the accompanying inset, and 150 nm in C; the bar in the associated inset is 250 nm. Results are representative of 9 experiments. vl, vascular lumen; pvs, perivascular space.
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Fig. 4. Effects of L-NAME on integrity of IEJs in multiple continuous vascular endothelia. Shown are the interendothelial gaps found in vascular beds from murine heart (A) and diaphragm (B) at the level of capillaries, whereas C shows a gap of 2 µm in a postcapillary venule from the gut vascular bed. In this case a platelet is seen extravasating from the vascular lumina. The IEJ in A shows a tricellular junction where only 1 side is open. The insets in B and C show matching IEJs from the corresponding vascular beds (diaphragmatic capillary for B and gut postcapillary venule in C) in control mice. Bar = 125 nm. Results are representative of 12 experiments.
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To delineate the endothelial permeability pathway, we used the electron-opaque tracer LaCl, presented by in situ perfusion to lung vasculature, as well as heart, skeletal muscle, skin, gut, and diaphragmatic vascular beds. These studies were made in L-NAME-treated and eNOS/ mice. Figures 57 show results obtained in the lung vasculature. In Fig. 5, a representative EM micrograph of two full-profile capillaries from the mouse lung vasculature treated with L-NAME shows IEJs filled by a continuous column of the tracer. The higher magnifications of junctions (insets a and b for upper capillary and insets c and d for lower capillary) show the direct connection of the vascular lumina with the pericapillary space. Note the patchy distribution of tracer on the luminal surface of the two capillaries, which corresponds to the endothelial glycocalyx. Similar findings to those in Fig. 5 were made in the heart, skeletal muscle, diaphragm, skin, and gut vascular beds after L-NAME treatment (data not shown).

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Fig. 5. Efflux of LaCl in lung capillaries of L-NAME-treated mice. Murine lung capillaries of L-NAME-treated (acute administration) typically showed opened IEJs. The figure shows the opening of IEJs (insets a, b, c, d) in both capillaries along with an open IEJ (arrow) on the alveolar epithelial side of the alveolar-capillary barrier. Note the extensive labeling of the pericapillary spaces in the areas adjacent to open IEJs vs. the discreteness in labeling of the same space in the areas where caveolae are discharging their content (arrowheads). The insets are high magnifications of 4 IEJs showing continuous connection of the vascular lumina with the pericapillary spaces. Bar = 750 nm, for a and c = 300 nm, and for b and d = 250 nm. Results are representative of 12 experiments.
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Fig. 7. Lung postcapillary venules of eNOS/ mice. A: open IEJ in a lung postcapillary venule of eNOS/ mouse. The junctional space is filled with the tracer indicating a direct transendothelial pathway found in this phenotype. Note the heavy staining of the pvs and its presence in some caveolae from an epithelial cell. B: IEJ in a lung postcapillary venule of a WT mouse that is not penetrated by the tracer. Bar = 150 nm. Results are representative of 15 experiments.
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Figures 6B and 7A show results from eNOS/ mouse lung vessels perfused with LaCl. In both cases the IEJs (the two IEJs illustrated in Fig. 6B, the upper one from a capillary and lower one from a postcapillary vein, and the more complex IEJ shown in Fig. 7A from a muscular venule) are fully penetrated by the tracer. The tracer establishes direct connection between luminal and abluminal spaces as in the case of L-NAME treatment studies above. Note the continuous labeling of the surrounding perijunctional spaces (Figs. 6B and 7A). In Figs. 6A and 7B, IEJs from matching segments of WT murine lung vascular bed demonstrate that the tracer fails to penetrate the junctional space, and consequently no tracer leaves vascular lumina via IEJs and there is no labeling of perivascular spaces adjacent to the tissular opening of IEJs. Figures 57 also illustrate the continuous decoration of vascular luminal surface (reflecting the distribution of the endothelial glycocalyx) and of open caveolae by the tracer.

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Fig. 6. Efflux of LaCl in lung microvessels of eNOS/ mice. In A, LaCl labels the surface of capillaries (top) as well as postcapillary venules (bottom) in a WT mouse. Note that 1) tracer fails to penetrate the capillary at the level of the tight junction, 2) perivascular space is not stained, 3) patchy decoration of the luminal surface in both vessels, and 4) staining of open caveolae on the luminal surface as well as of caveolae apparently free in the endothelial cell. As a contrast, a segment from 2 vessels (capillary upper side and postcapillary venule lower side) from eNOS/ mice is shown in B. The panel illustrates the continuous column of LaCl filling the open IEJs in both capillaries and postcapillary venules. As in the case of L-NAME-treated mice, there is a direct and continuous communication between luminal and abluminal side of the endothelial barrier. Bar = 150 nm in A and 230 nm in B. Results are representative of 15 experiments.
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These findings in the murine lung vascular bed were also seen in murine heart and diaphragm beds of eNOS/ mice. In Figs. 810 we show representative data from eNOS/ mice. Figure 8A illustrates a complete profile of a capillary from an eNOS/ mouse heart vascular bed having both IEJs filled with a continuous column of LaCl. Figure 8B shows a junction from a heart capillary of WT mouse that is not penetrated by LaCl. Similar results, in the same vascular segment, were observed in all vascular beds studied, and Fig. 9 shows a capillary profile from the diaphragmatic vascular bed. In all vascular beds studied we encounter caveola-discharging tracer on the tissular front of capillaries along with apparently free vesicles inside EC labeled by LaCl (Figs. 5, 8A, and 9). Similar alterations were seen at the level of capillaries of skeletal muscle, gut, and skin vascular beds (data not shown). The same results were also obtained on the venular end of heart (Fig. 10) and diaphragmatic (data not shown) vessels. Figure 10 shows open IEJs in a postcapillary venule from the murine heart microvascular bed. The two insets are high magnification of the corresponding IEJs demonstrating the same continuous connection between vascular luminal and abluminal spaces.

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Fig. 8. Disruption of IEJs in heart capillaries of eNOS/ mice. Murine coronary capillaries of eNOS/ mice typically showed 1 or 2 open IEJs. The figure shows the opening of both IEJs along with extensive labeling of caveolar profiles by the LaCl tracer. The insets in A are high magnifications of the two IEJs showing the continuous column of tracer connecting the vascular lumina with the perivascular space. Note also that some open caveolae at the abluminal surface are filled with tracer as well as caveolae apparently free inside endothelial cells. B shows that under basal conditions, the tracer did not penetrate the IEJ in WT mice. Bar = 250 nm. Results are representative of 9 experiments.
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Fig. 10. Diaphragmatic microvessels of eNOS/ mice, As in the case of the heart, a continuous column of tracer in eNOS/ mice penetrated the IEJs. The 2 insets are high magnifications of the corresponding IEJs showing that the column of tracer is uninterrupted from 1 side (luminal) to the other (abluminal) of the endothelial barrier. Bar = 250 nm. Results are representative of 9 experiments.
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Fig. 9. Coronary venules of eNOS/ mice. Both IEJs (insets) shown are open allowing uninterrupted communication between the vascular lumina and tissue. The figure also shows caveolae discharging their content (LaCl tracer) on the abluminal side of the endothelium without any direct relation with the IEJs or the possibility of back filling. Bar = 300 nm. Results are representative of 9 experiments.
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Serial sections obtained from the murine lung vascular bed perfused with LaCl, for 10 min, showed that the only structures carrying the tracer across the endothelium were the caveolae. In this way, we were able to follow the same caveola from the moment when opened luminally, and labelled by LaCl, to the moment of being apparently free inside the EC, until at the stage of discharging its load into the perivascular space. This kind of analysis convincingly proves that the LaClis not able to penetrate the IEJs under basal conditions in WT mice outside of the 1020% normally opened IEJs in the postcapillary venules.
Based on these morphological findings, we conclude that: 1) tattooing of vessel walls induced by L-NAME observed by light microscopy (Fig. 2) is a reflection of open IEJs and of accumulation of tracer particles in the perivascular space, 2) capillaries, postcapillary venules, and muscular venules are the main segments affected in all vascular beds by the removal of NO, and 3) open IEJs are the essential hallmark of removing NO by pharmacological inhibition of NOS activity or genetic deletion of eNOS, in all vascular beds examined.
Transendothelial Albumin Permeability
We next used the A-DNP as a tracer to quantify changes in albumin transport due to the observed alteration of IEJs. The pathways followed by A-DNP from vascular lumen into the pericapillary space were identified by immunocytochemistry as in Refs. 11 and 44, and the permeability for A-DNP was quantified as described in METHODS. Figure 11 demonstrates the interactions of A-DNP (5 mg/ml) with the endothelial barrier when presented by perfusion. Figure 11, A and B, shows the A-DNP labeling pattern in ECs from the venular end of a lung microvessel in WT mice. Figure 11C illustrates the interactions of A-DNP with the ECs in venular microvessels of lungs from eNOS/ mice. The same findings were true for the labeling of lung capillaries as well as of microvessels from all organs studied. The IEJs are open and penetrated by the tracer particles when NO is removed from a vascular bed. In WT mice, under basal conditions, the tracer 1) binds to luminal surface, 2) labels all caveolae open at the luminal side, most of the caveolae free inside the cell, and caveolae discharging their content on the abluminal side, 3) is detected at sites of discharging caveolae, and 4) fails to penetrate the IEJ space. The major findings in the lung vascular bed of eNOS/ mice are open IEJs penetrated by tracer particles (Fig. 11C). The same findings were evident in L-NAME-treated mice injected intravenously with A-DNP (data not shown) and in all other vascular beds studied.

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Fig. 11. Increased IEJ permeability to albumin in the lung vascular bed of eNOS/ mice, revealed by postembedding immunocytochemistry. Upon perfusion of the monomeric tracer dinitrophenylated albumin (A-DNP) in the lung vascular bed of WT mice, we observed that within 10 min the tracer heavily labeled the capillaries (A) and postcapillary venules (B). The tracer also labeled the endothelial surface, the caveolae open to the vascular lumen or abluminal space, and caveolae apparently free inside of the endothelial cell. Note the presence of tracer particles in phase with caveolae (A and B) on both luminal and abluminal sides but the absence of tracer particles at the exit of the IEJ (B). As shown in B, the monomeric albumin tracer did not penetrate the IEJs. When the A-DNP was perfused in lungs of eNOS/ mice (C), the tracer particles are seen in phase with open IEJ, and there was also heavy labeling on the tissue side at sites of caveolae localized on the abluminal side. Bar = 175 nm. Results are representative of 10 experiments.
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The A-DNP vascular permeability values in different organs of WT mice (Table 2) are in agreement with published values (21, 25, 4850). The mean value for A-DNP permeability (in ng A-DNP·g wet tissue1·5 min1) in different vascular beds was 331 ± 25 in WT mice and 329 ± 22 in iNOS/ mice. The basal permeability values for A-DNP in different murine vascular beds of L-NAME-treated and eNOS/ mice were greater by 3540% (P < 0.001) compared with WT mice in all organs studied (Table 2).
Morphometric Analysis at the EM Level
The morphometric analysis of murine lung vascular bed is summarized in Table 3. The results indicate an opening of 34% of IEJs in capillaries and 48% of IEJs in venules of mice lacking NO, whereas in WT mice no open IEJs were found in capillaries and only 12% of venular IEJs were open. We found extremely rare (<0.5%) IEJs open on the arterial end of eNOS/ mice. The main structural characteristics of caveolae (average diameter of
70 nm, their surface and volume densities) open to the luminal or abluminal side or apparently free in the cytosol in both L-NAME-treated and eNOS/ mice were within the normal values published (56, 62). Most of the parameters shown in Table 3 were also in the normal range (56, 68).
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DISCUSSION
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In the present study, we addressed the possible role of eNOS-derived NO in regulating the structural integrity of the endothelial barrier and its effect on endothelial permeability. Previous studies have shown that inhibition of NO synthesis by L-NAME increases venular permeability in the rat mesentery (7), enhances the transendothelial leakage of albumin, and augments fluid permeability in mesenteric venules (1, 13). These changes appeared to result from alterations in the endothelial actin cytoskeleton. These studies suggested that NO has an important role in regulating microvascular permeability. This effect of constitutive NO release is different from that induced when large amounts of NO are produced by inflammatory mediators. NO released under these circumstances was also capable of increasing endothelial permeability (17, 18, 47).
In both eNOS/ and L-NAME-treated mice, the basal plasma levels of NO were low, consistent with the fact that NO is released constitutively by eNOS. In the present study, we reassessed the role of NO constitutively released by eNOS in tonically regulating basal endothelial permeability. We evaluated the morphological alterations of continuous type of endothelia from multiple vascular beds including the lung, and in addition we also measured endothelial permeability using A-DNP as a monomeric albumin tracer (11, 44) in both eNOS/ and L-NAME-treated mice. We were also able to map out the routes of albumin transport in microvessels from the same vascular beds, in the same experimental conditions by immunocytochemistry at the EM level.
We detected, in the initial series of studies at the light microscopic level, the leakage of Monastral blue dye from cremaster and diaphragmatic vascular bed of both eNOS/ and L-NAME-treated mice. This was not seen in microvessels from control mice. This dye, with molecular dimensions ranging from 0.021 µm, does not normally permeate the endothelial barrier (20). However, upon removal of NO (by L-NAME treatment or deletion of eNOS in eNOS/ mice), the leakage of Monastral blue was similar to the effects seen with permeability-increasing mediators such as platelet-activating factor (45) and histamine (34, 69). However, unlike these mediators, which induce junctional opening within minutes, the increase in permeability produced by L-NAME was delayed, reaching a plateau at
30 min after treatment. This finding suggests that different molecular mechanisms may account for the effect obtained after blocking NO production. Although the basis for the leakage of Monastral blue from venular segments was not evident from the light microscopy observations, one possibility is the formation of minute gaps between ECs at the level of IEJs (38).
To address cellular basis for the effect of removal of NO in increasing the vascular leakage of Monastral blue dye, we carried out a comprehensive EM analysis of the endothelial barrier under basal conditions in WT mice and of alterations induced by either L-NAME-treatment or eNOS deficiency. When the general morphological characteristics of mouse lung vasculature were analyzed (WT vs. L-NAME-treated and eNOS/ mice), the only statistically significant difference was the invariable presence of open IEJs in the mice lacking NO. This effect was seen in IEJs of capillaries and venules of the multiple vascular beds examined including the pulmonary circulation, but it was rarely seen in the arterial segments. This finding points to the key role of eNOS-derived NO as a determinant factor of basal endothelial permeability via its action in maintaining the integrity of IEJs.
We also used the electron-opaque tracer LaCl, which was presented to different vascular beds at the same time as the fixative to visualize the pathways and structures involved in the transport of the tracer from the vascular lumina and to resolve the dimensions of IEJ openings. Our results showed that in the absence of NO the tracer was cleared from the vascular lumina mainly via open IEJs. We also showed with another tracer, the monomeric A-DNP, that in the absence of NO this tracer leaked across the endothelial barrier via open IEJs while it is still transported by caveolae. In contrast, in control vessels, albumin did not penetrate the IEJs, and its transport occurred solely via caveolae as demonstrated in previous studies (464748, 5459). Our serial sectioning data showing that IEJs of continuous endothelia are normally impermeable to LaCl are in agreement with Wagner and Chen (65, 66), who, using the same technology applied on terbium-labeled microvessels, demonstrated that, under basal conditions, endothelial caveolae were the only subcellular structures participating in transendothelial transport. In control vessels, the LaCl or A-DNP tracer failed to penetrate IEJs beyond the tight junctions of lung vascular endothelia and the other vascular beds studied.
Using the A-DNP as tracer we determined that lung permeability for albumin increased by 3540% in mice lacking NO (eNOS/ and L-NAME treated) compared with WT mice. This augmented permeability can be explained by the increase in the number of leaky venular IEJs (from 12% under basal condition in WT mice to 45% in the absence of NO) and by an increased number of open junctions in capillaries (from 0 under basal condition to 34% in the absence of NO). The presence of open IEJs in mouse lung venules under basal condition is essentially similar to the observation in rat diaphragm and heart postcapillary venules made by Simionescu et al. (59). Thus our observations provide a strong argument for the crucial role of NO as a determinant factor of basal vascular permeability by regulating capillary and venular endothelial junctional integrity and by controlling the dimensions of IEJs. NO acts primarily at the level of the capillary and venular IEJs, which are tight enough to exclude molecules with molecular diameters
23 nm (48); therefore, albumin, because of its molecular dimensions (4 x 4 x 15 nm), never crosses the endothelial barrier via IEJs under basal conditions. However, in the absence of NO (as in eNOS/ and L-NAME-treated mice), the IEJs are opened (>12 nm) and the transport of albumin mainly takes place via this modified paracellular pathway.
The present results should be contrasted with data showing microvascular hyperpermeability when excessive amounts of NO are present in the vasculature (47) or when caveolin-1 is deleted in mice by homologous recombination (53) and large amounts of eNOS-derived NO are produced by ECs. Previous studies have shown that increased levels of NO such as induced by VEGF, which activates eNOS by Ca2+/calmodulin and Akt-mediated phosphorylation of eNOS (3, 4, 9, 33), promote the leakiness of the endothelial barrier (6, 22, 60), at least partially by opening IEJs. As caveolin-1 functions as an inhibitor of eNOS activity (5, 12), the increase in vascular permeability in caveolin-1/ mice was ascribed to the increased production of NO (53) and its prolonged half-life in the circulation (47). Thus open IEJs are also found when excessive production of NO as in inflammatory states (18) or when a more stable form of NO [NO attached to albumin by nitration or nitrosilation (47)] is present in circulation, and as a consequence its excess disturbs the paracellular junctional pathway (47, 64). This alteration of the endothelial paracellular pathway generates the recorded increase in vascular permeability. The increase in endothelial permeability in the above instances was prevented, to different degrees, by the NOS inhibitor L-NAME, indicating that the high levels of NO were responsible for the increase. However, it is likely that the molecular mechanisms responsible for the opening of IEJs when ECs are exposed to large amounts of NO are different from the increased endothelial permeability induced by the absence of NO as demonstrated by the present study. The molecular basis for the IEJ effect induced by the absence of NO remains unclear.
High levels of NO may modify actin polymerization and promote endothelial shape change, resulting in increased junctional permeability (2), whereas it is possible that basal production of eNOS-derived NO serves to stabilize IEJs. Studies have shown that NO induces the formation of endothelial gap junctions by promoting the incorporation of connexin-40 into the plasma membrane secondary to activation of protein kinase A (15); thus NO-mediated gap junction formation may favor cell-cell contact at the level of IEJs. Another possibility is that the constitutive production of NO may regulate the distribution of cortical actin and its interaction with endothelial junctions and thus contributes to maintenance of IEJ integrity as in the case of sphingosine-1-phosphate (39).
The question why eNOS/ mice are viable and their tissues are not grossly edematous, despite the leaky IEJs, is unresolved. It is possible compensatory mechanisms preventing edema formation are activated in these mice. These mechanisms may include an increase in the lung lymphatic drainage system (43) and an increase in interstitial hydrostatic pressure. Both of these would serve to limit or even resolve tissue edema formation in the unstressed mouse in the face of increased endothelial permeability induced by absence of NO.
In summary, the findings that open IEJs are the hallmark of NO deficiency as well as of NO excess give us the confidence to conclude that a restrictive endothelial barrier, in a given vascular bed, requires a physiological concentration of NO.
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FOOTNOTES
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Address for reprint requests and other correspondence: D. Predescu, Univ. of Illinois, College of Medicine, Dept. of Pharmacology, 835 S. Wolcott Ave., 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.
1 The term "tattooing" is used to describe tracer deposition (Monastral blue) in the microvessel wall as a consequence of open IEJs and trapping of tracer particles in the perivascular space; see Majno et al., J Biophys Biochem Cytol 11: 607611, 1961, and Predescu et al., Eur J Cell Biol 69: 8698, 1996. 
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