Pro-inflammatory endothelial cell dysfunction is associated with intersectin-1s down-regulation

Human lung microvascular ECs were purchased from Lonza (Walkersville, Inc., MD). LPS from Escherichia coli 011:B4, calmodulin from bovine testes, tetrahydrobiopterin (BH₄), protease inhibitors cocktail, L-arginine and primers for qPCR were from Sigma-Aldrich (St. Louis, MO). Superoxide dismutase (SOD) from bovine erythrocytes was purchased from MP Biomedicals. Freeze dried Hb was from ICN Biomedicals. The In Situ Cell Death Detection Kit, Fluorescein was from Roche (Indianapolis, IN). Prolong Antifade Kit and neutrAvidin Alexa Fluor 594 were from Molecular Probes (Eugene, OR). EZ-Link Sulfo NHS-SS-Biotin was from Fisher Scientific (Hanover Park, IL). Streptavidin-HRP conjugated, MicroBCA (bicinchoninic acid) Protein Assay Reagent and Enhanced Chemiluminescent (ECL) Western Blotting Substrate were from Pierce (Rockford, IL). Specific antibodies (Abs) were obtained from the following sources: anti-Bcl-X_L mAb, anti-NOS2 pAb from Santa Cruz Biotechnology (Santa Cruz, CA); anti-cyt c mAb from Calbiochem; anti-ITSN-1s mAb from BD Biosciences (San Jose, CA); anti-Bim pAbs from Millipore (Billerica, MA); anti-survivin pAbs from Novus Biologicals; Horseradish peroxidase (HRP)-conjugated reporters were from Cappel, Organon Teknika (Durham, NC).

ECs were cultured at 37°C in 5% CO2 in endothelial growth medium-2 obtained from Lonza and prepared according to manufacturer's instructions. Cells between the third and fifth passages were used for experiments. LPS (dry powder) was reconstituted in growth medium and applied over cells in a concentration of 1 μg/ml for a duration of up to 48 hours.

Control ECs or cells exposed to 1 μg/ml LPS in culture media were collected from culture dishes and washed with PBS. Cell pellets were lysed for 1 h at 4°C in 50 mM Tris HCl, pH 8.0, 150 mM NaCl, 1% NP-40, and protease inhibitors. For detection of phospho-Bim (Ser⁶⁹) levels, control and LPS-treated ECs were solubilized in kinase buffer (20 mM Tris/HCl, pH 7.4, 150 mM NaCl, 0.1% Nonidet P-40, 1% glycerol, 0.2 mM sodium vanadate, 0.83 mM benzamidine, 0.23 mM phenylmethylsulfonyl fluoride, 0.5 μg/ml aprotinin, and 0.5 μg/ml leupeptin). After centrifugation at 45000 rpm for 45 min at 4°C, the supernatants were collected and protein concentration determined by BCA with a bovine serum albumin standard. Equivalent protein amounts were subjected to SDS-PAGE and transferred to nitrocellulose membranes. The membranes were probed with anti-NOS2 pAb, anti-Bcl-X_L mAb, anti-ITSN-1 mAb, anti-Bim pAb, anti-survivin pAb, diluted in blocking buffer, (5% milk/TBS). Immunoreactive bands were visualized with the appropriate HRP-conjugated Abs and ECL detection. Densitometry was performed with ImageJ v1.37 software.

Control and LPS-treated ECs, grown on plastic Petri dishes or coverslips were washed with ice-cold PBS and then incubated with 0.5 mg/ml cleavable biotin reagent as in [15, 16]. Biotinylated cell surface proteins were internalized for 30 min, at 37°C. Biotinylated proteins still at the cell surface after 30 min were reduced with glutathione and the cells were further processed for morphological analysis or lysed for biochemical investigation. For biochemical studies, cells were lysed in TBS containing 2% Triton X-100, and the lysates were clarified by centrifugation for 30 min, at 4°C, 40,000 rpm in a TLA-45 Beckman rotor, to obtain a final supernatant expected to contain the internalized biotinylated proteins. The number of biotin molecules in the ECs lysates were quantified by ELISA as in [15]. Standard curves were generated using known concentrations of albumin-biotin. The average number of biotin molecules present in each cell lysate was determined at a series of decreasing concentrations from the linear part of the curve obtained by successively diluting a standard volume (100 μl) from each lysate and normalized per mg total protein. To obviate any interference between the biotinylated proteins internalized via caveolae and biotin present in mitochondria [17], we evaluated the biotin content of mitochondria by ELISA applied on lysate prepared from control ECs, not subjected to biotinylation of cell surface proteins. Mitochondrial proteins comprise only 0.25 × 10¹⁴ biotin molecule/mg total protein, a value without statistical significance, when compared to the extent of biotin internalization via caveolae.

ECs were grown to confluence on gold electrode array plates available from Applied Biophysics, Inc., Troy, NY. Transendothelial electrical resistance (TER) was measured by an electrical cell-substrate impedance sensing system (Applied Biophysics, Inc.) as described in [18]. Briefly, the gold electrode plates were connected to a phase-sensitive lock-in amplifier and a 1 volt 4000 Hz AC signal was applied through a 1 MΩ resistor. In-phase voltage was measured and used to determine TER which was normalized to initial values. A decrease in TER during the experiment reflects loss of cell-cell adhesion.

Transwell chambers with .4 μm pore filter inserts (BD Bioscience) were used. The inserts were coated overnight with 0.1% gelatin, at 37°C. Cells were seeded in the upper compartment and grown for additional 3 days post-confluency. Then, cell monolayers were subjected to 1 μg/ml LPS for 6 h, as an early time point, and 48 h, the time point used for biotin assay for caveolae internalization. The last hour of LPS treatment, 1 mg/ml dinitrophenylated (DNP)-BSA was added in the upper chamber. To quantify the transport, 500 μl medium from the lower chamber were removed and subjected to ELISA, via anti-DNP Ab, as previously described [19, 20]. All measurements were performed in triplicate and repeated three times. A standard curve was generated by diluting known concentration of DNP-BSA.

The full-length human ITSN-1s cDNA fragment (3,660 bp) was generated by PCR amplification from ITSN-1s cDNA (gift from Suzana la Luna, Center for Genomic Regulation, UPF, and Centro de Investigacion Biomedica en Red de Enfermedades Raras, Barcelona, Spain) with high-fidelity PCR enzyme (New England Biolabs), using the following primer pair: ITSN1s_F269-BstBI: 5' AGTA TTCGAA CC ATG GCT CAG TTT CCA ACA CCT T 3'and ITSN1s_R3929-NheI: 5' CGTA GCTAGC TTG CTG GCT TGG GTC CAT GTC TG 3'. The PCR products of the full-length ITSN-1s were digested with restriction enzyme BstBI and Nhe I (New England Biolabs) and purified (Qiagen purification kit), then cloned into a expression vector, pReiver-M10a (GeneCopoeia) at BstBI-NheI sites using T4 ligase (New England Biolabs). The ligation products were transformed into E. coli strain Top10 (Invitrogen). Dozens of transforming colonies were selected and subjected to PCR to identify the colonies with the ITSN-1s inserts using the same sequencing primer pair. The plasmid DNA was extracted from 6 selected growing clones bearing the ITSN-1s insert and was verified with correct restriction enzyme (BstBI and Nhe I) sites. Finally, one of them, CS-Z3999_ITSN1s #16 was selected for maxi-preparation of plasmid DNA, sequencing confirmation of the entire integrity of the full-length cDNA of ITSN-1s in frame in the vector and used for transfection of ECs. All transfections were performed using FuGENE 6, according to the manufacturer's instructions.

Control ECs and cells exposed to LPS (1 μg/ml) for up to 48 h were washed in PBS and then fixed and permeabilized in methanol for 7 min, at -20°C. The TUNEL reaction mixtures (label and enzyme solutions) were prepared as directed by the manufacturer. It was applied over EC monolayers for 60 min, at 37°C, under dark and humid conditions. Monolayers exposed to label solution only were used as negative controls. The cells were washed and coverslips mounted using the Prolong Antifade kit. Fluorescence microscopy with an excitation wavelength of 488 nm identified TUNEL-positive cells.

Cells were lysed using the Qiagen QIAShredder homogenizer and the RNA was isolated using the Qiagen RNeasy Mini RNA isolation kit as per the manufacturer's instructions. cDNA was generated with the high-capacity cDNA RT kit by Applied Biosystems following the manufacturer's protocol. Amplifications were carried out using the following primers: ITSN-1s (5'-CAGGCTTGAAAGTCCTCAAAG-3', 5'-GGTGATCATGCTGGAAGTCA-3'), Bim (5'-TCCCTGCTGTCTCGATCCTC-3', 5'-GGTCTTCGGCTGCTTGGTAA-3'), Bcl-X_L (5'-CTGTCGGTGGAAAGCGTAGA-3', 5'-TGCTGCATTGTTCCCATAGAG-3'), Survivin (5'-AAGAACTGGCCCTTCTT GGA-3', 5'-CAACCGGACGAATGCTTTT-3'). mRNA levels were quantified by real-time monitoring of the products of the cDNAs PCR with SYBR Green fluorescent dye. Cyclophilin was used as internal control.

Control and LPS-treated cells were subjected to standard EM procedure [15]. Briefly, EC monolayers were fixed in 1.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, containing 5% sucrose for 30 min at RT. After washing in the same buffer the cells were post-fixed in Palade's 1% osmium tetroxide for 1 h on ice, stained with Kellenberger buffer, dehydrated in graded ethanol, and embedded in Epon at 60°C.

It is well documented that ECs are active participants in and regulators of inflammatory processes such as severe bacterial infection [4, 23]. However, there is still significant debate on the extent of vascular dysfunction that is due to the direct effects of bacterial products such as LPS on the endothelium. Thus, in this study we evaluated by high-resolution EM the morphological changes caused by direct LPS exposure on human lung microvascular ECs. Confluent ECs monolayers were treated with 1 μg LPS/ml, for 48 h, and then subjected to standard EM procedure as in [7]. Detailed examination revealed disrupted interendothelial junctions (IEJs), as illustrated in Figure 1a, b, and increased accumulation of actin filaments at cell periphery (Figure 1c arrows), suggesting increased paracellular permeability, one of the first sign of endothelial activation in response to inflammation. Several cellular organelles such as Weibel-Palade bodies and Golgi show apparently normal morphology, but their number was significantly increased (Figure 1d, f, f1) by reference to control ECs. Golgi organelles were spread throughout the cell, while a swollen endoplasmic reticulum (Figure 1c), occupied the entire intracellular space, observations consistent with expression of pro-inflammatory/cytoprotective genes as well as the ER stress. Fragments of control ECs are shown for comparison, (Figure 2a, a1). Note that these cells maintain in culture their endothelial phenotype; in close proximity of the nucleus there are Golgi stacks and vesicles as well as two mitochondrial units showing well-organized cristae. A normal, unswollen endoplasmic reticulum is detected within the two mitochondrial units. Note also a Weibel-Palade body by the basolateral plasma membrane. Caveolae, the main vesicular carriers of ECs, involved in the transport of plasma proteins and nutrients across the endothelium are open apically or basolaterally, apparently charging or discharging their load, (Figure 2a1 arrows). In LPS-treated ECs we have also frequently noted numerous small mitochondrial units. Large vacuoles, increased frequency of tubular structures and membraneous rings, (Figure 2b) some of them opened to the apical front of the cell (Figure 2c, d) are detected, suggesting impaired endocytosis and activation of alternative, non-conventional, transport pathways [24]. Altogether, these observations are consistent with an activated and dysfunctional EC phenotype.

Since LPS signaling is regulated by caveolae endocytosis [25, 26] and since EM analysis revealed endocytic abnormalities in LPS-treated cells, we next used a biotin internalization assay as in [15, 16] to address if EC exposure to LPS affects caveolae endocytosis. This assay is a specific and reliable approach for addressing caveolae internalization in cultured ECs [15]. Caveolae-mediated uptake of biotinylated cell surface proteins is the dominant mechanism of internalization in ECs; the number of clathrin-coated vesicles in ECs is relatively small and their contribution to the internalization process is minor [27]. Briefly, control and LPS-exposed ECs were subjected to biotinylation of cell surface proteins, at 4°C, using a cleavable biotin reagent. Then, cells were transferred to 37°C, for 30 min, to allow internalization of biotinylated proteins. The biotinylated proteins, still on the cell surface after 30 min were reduced with glutathione. Then, the cells were either lysed for biochemical quantification of LPS effects on caveolae internalization by enzyme-linked immunosorbent assay (ELISA) or subjected to neutrAvidin-Alexa Fluor 594 staining for morphological surveys by fluorescence microscopy. Biochemical quantification of the number of biotin molecules internalized by control and LPS-exposed cells, Figure 3A indicated that in LPS-treated EC lysates, the number of biotin molecules was considerably smaller. A comparison of LPS-treated cells to controls shows an inhibition of 40%, (Figure 3B). Morphological analyses with NeutrAvidin-Alexa Fluor 594 revealed in control cells a fine punctate pattern throughout the cytoplasm, suggestive of vesicular association, (Figure 4c1). Frequently, biotin accumulation in the perinuclear area was detected. In LPS-exposed cells the staining was limited (Figure 4c2); large fluorescent puncta within the cytosol and no biotin accumulation in the perinuclear area were observed, suggestive of impaired biotin internalization. The intercellular spaces are large, consistent with disruption of IEJs. Highly magnified controlled and LPS-exposed cells (boxed areas), are shown, (Figure 4c1.1, c2.1).

Since 1 μg/ml LPS exposure downregulates ITSN-1s expression and as a result transcellular transport is impaired, we next evaluated the status of paracellular permeability of ECs monolayers by measurements of TER, under the same conditions of LPS exposure. Monolayers of ECs grown on gold microlectrodes, three days post-confluency, were first stabilized for two hours to establish a linear TER baseline. Then, The stabilized monolayers were stimulated with LPS (Figure 5A, arrow) and TER was monitored over 24 hours, Figure 5A. A gradual decrease in the baseline barrier resistance was recorded for the first hours after LPS stimulation with a maximal decrease after 8 h and remaining in a plateau for the next 16 h hours. These data strongly suggest that LPS disrupts endothelial barrier and significantly increases the paracellular permeability of lung microvascular ECs. To get more insights on the extent of endothelial barrier dysfunction, we quantified the paracellular transport of dinitrophenylated albumin (DNP-BSA) using a transwell assay, as described under Methods. In brief, cells were seeded in the upper chamber and 3 days post-confluency, endothelial monolayers were subjected to 1 μg/ml LPS for 6 h, as an early time point, and 48 h, the time point used for biotin assay for caveolae internalization. In the last hour of LPS treatment, BSA-DNP was layered on top of EC monolayers to a final concentration of 1 mg/ml. To quantify the transport, 500 μl medium from the lower chamber were removed and subjected to ELISA, via DNP Ab, as previously described [19, 20]. As shown in Figure 5B, the permeability of the endothelial monolayer for the DNP-BSA tracer were determined at a series of decreasing concentration from the linear part of the curve obtained by serial dilution of a standard volume of growth media removed from the lower chamber. In the presence of LPS, the amounts of DNP-BSA crossing the monolayer were considerably higher than control levels. Measurements of DNP-BSA amounts, using a series of concentrations on the straight part of the curve, indicated that at 6 hours after LPS exposure, the lower chamber contains 118.10 ± 7.3 ng DNP-BSA/100 μl medium, while at 48 h, 164.63 ± 12.4 ng DNP-BSA/100 μl medium, significant increase over the control levels estimated at 10.1 ± 0.9 ng DNP-BSA/100 μl medium, Figure 5C. The findings are consistent with endothelial barrier dysfunction, gap formation and DNP-BSA leakage, as result of LPS exposure.

Expression iNOS in response to LPS has been reported for several cell types such as epithelial cells, fibroblasts, hepatocytes, ECs [28, 29]. A mitochondrial NOS, mtNOS, constitutively expressed, continuously active and associated with the inner mitochondrial membrane has been reported [21, 30]. LPS-induced up-regulation of mitochondrial NO is one of the specific means, however still enigmatic, by which a dysfunctional and potentially pro-apoptotic environment is generated during the pro-inflammatory response in many cell types [31‐33]. Thus, to evaluate the expression of iNOS and mtNOS in LPS-exposed human lung microvascular ECs, we first investigated by immunoblotting the expression of iNOS in control and LPS-treated ECs (Figure 6A). We detected weak iNOS immuno-reactivity under control conditions, and it increases by 27%, at 24 h and by 75% at 48 hours post LPS exposure (Figure 6B). Next, we investigated the effects of LPS on mtNOS expression. To this intent, mitochondria-enriched fractions prepared from these cells were lysed and subjected to SDS-PAGE. Even if mtNOS is constitutively expressed, the levels seem to be low, since no iNOS immunoreactivity was detected in the absence of LPS and under experimental conditions used (Figure 6C). However, in the mitochondrial fraction prepared from LPS-exposed ECs, mtNOS protein expression is easily detected, at 48 hours of LPS exposure (Figure 6C). Next, we addressed if the increased expression of mtNOS resulted in increased NO production. mtNOS activity was assessed by exposing freshly prepared, unlysed mitochondrial fractions from ECs to oxyHb, as described under Methods. The rate of NO production was measured by the change in optical density of the reaction mixture as generated NO quickly oxidized oxyHb to metHb. As shown in Figure 6D, mitochondria from LPS-treated ECs exhibited a two-fold greater rate of NO formation than untreated controls (0.81 nmol/min mg total protein versus 0.40 nmol/min mg total protein).

We have recently shown that specific and efficient ITSN-1s knockdown via a specific siRNA duplex targeting ITSN-1 gene results in severe EC dysfunction and apoptotic death, events that can be reversed by overexpression of the anti-apoptotic Bcl-X_L[7]. Previous studies of LPS-induced endothelial dysfunction suggested the ability of LPS to induce pro-apoptotic pathways [34] as well as a role of Bcl-X_L in cyto-protection during inflammatory stress [3]. These observations led us to consider ITSN-1s as a participant in either of these events. We therefore evaluated the levels of ITSN-1s protein in lysates prepared from LPS-treated and control ECs by immunoblotting with ITSN-1 pAb. The 140 kDa band representing ITSN-1s begins to decrease in intensity at 24 h and remains significantly decreased at 48 h after LPS exposure (Figure 7A). We also evaluated the level of ITSN-1s mRNA expression by quantitative PCR in ECs exposed to 1 μg/ml LPS for 24 or 48 hours. ITSN-1s mRNA levels were dramatically reduced in LPS-exposed ECs compared to control cells (Figure 7B). ITSN-1s mRNA expression was reduced 4.3-fold at 24 hours and 3.2-fold at 48 hours of LPS treatment compared to untreated controls. These results are consistent with the idea that LPS down-regulates the expression of the pro-survival protein ITSN-1s; ITSN-1s deficiency may be relevant to ECs dysfunction induced by LPS.

Since LPS-exposed ECs show decreased mRNA levels for ITSN-1s and since ITSN-1s deficiency is expected to initiate intrinsic apoptosis, we evaluated the occurrence of apoptosis in cells treated with LPS for 48 h, by TUNEL, Figure 7C, c1. Morphological surveys and morphometric analyzes show that the number of TUNEL-positive ECs is extremely low, comparable to untreated ECs (Figure 7D). A positive control, in which cells were subjected to ITSN-1s knockdown by siRNA as in our previous work [7], is shown in Figure 7C, c2. A complementary strategy to evaluate if mitochondrial cell death occurred was the examination of cyt c efflux from mitochondria. An enriched-mitochondrial fraction and the post-mitochondrial supernatant prepared from LPS-treated ECs normalized for total protein content were subjected to SDS-PAGE and Western blot with cyt c mAb. No immunoreactivity for cyt c was detected in the post-mitochondrial supernatant, consistent with cyt c confinement to mitochondria (Figure 7E). The findings suggest that ITSN-1s down-regulation as caused by LPS exposure of ECs did not trigger the intrinsic apoptotic pathway and suggest that LPS induces a cyto-protective response.

The protective effects of Bcl-X_L overexpression against LPS and ITSN-1s deficiency-induced cell death were previously documented [3, 7]. However, whether LPS can directly induce Bcl-X_L expression has not been reported for human lung microvascular ECs. To determine whether LPS up-regulates the mRNA for Bcl-X_L, ECs were exposed to LPS for different time points (24 h, 48 h), and the levels of Bcl-X_L protein and mRNA were determined (Figure 8). At 24 h, the levels of Bcl-X_L are increased by 1.3-fold, while at 48 h a significant, 3.4 fold-increase, compared to controls is detected. Thus, LPS can up-regulate the mRNA for this cyto-protective protein. Levels of Bcl-X_L protein correspond to mRNA accumulation. Interestingly, ECs deficient in ITSN-1s show decreased Erk1/2 activation [7]. Erk1/2 activation is responsible for constitutive phosphorylation of the pro-apoptotic "BH3 only" protein Bim, at Ser⁶⁹ leading to proteasomal degradation [35‐37]. Thus, it is possible that down-regulation of Bim may be another possible mechanism by which ECs cyto-protection occurs in response to LPS. To determine whether LPS affects the levels of phospho-Bim (Ser⁶⁹), control and LPS-treated ECs were solubilized in kinase buffer; equal amounts of total protein were subjected to SDS-PAGE and Western blot with a specific phospho-Bim (Ser⁶⁹) Ab. Total Bim was detected with a specific anti-Bim pAb (Figure 8). Substantial decrease in both P-Bim (Ser⁶⁹) and total Bim was noted. Next, qPCR for mRNA levels were performed; in response to LPS exposure, Bim mRNA was dramatically reduced, 11-fold compared to untreated controls at 24 hours and recovered to 1.2-fold reduction at 48 hours (Figure 8). These studies are consistent with the idea that down-regulation of Bim, as well as up-regulation of Bcl-X_L serve as cyto-protective pathways against LPS-induced apoptosis in lung microvascular ECs.

Since Bim down-regulation was linked to the overexpression of survivin, an inhibitor of apoptosis protein, [38] and since LPS-induced NF-kB signaling may regulate survivin expression [39], we next examined by Western blot and qPCR the effects of LPS on survivin expression. As shown in Figure 8, survivin mRNA levels increased 5.2-fold in the first 24 h and remained increased by 4.2-fold at 48 h. The relative increase in survivin protein levels was confirmed by Western blot (Figure 8). The observation further documents that survivin up-regulation is another anti-apoptotic effect induced by LPS in ECs.

Since cav-1 is a potential molecular chaperone that directly inactivates all NOS isoforms [40], and since ITSN-1s down-regulation inhibits caveolae internalization and thereby, increases LPS signaling [25], we sought to investigate if restoring endocytosis by ectopic expression of myc-ITSN-1s can affect iNOS expression. To this intent, subconfluent ECs monolayers, exposed to LPS for 48 h, were transfected with myc tagged full-length ITSN-1s, as previously described [15]. Efficient ITSN-1s expression was detected at 48 h post-transfection by immunoblotting of ECs lysates with anti-myc Ab, Figure 9C. Next, ECs grown on coverslips and exposed to LPS for 48 h, were transfected with myc-ITSN-1s and subjected to the biotin internalization assay. Fluorescent microscopy analyses of internalized biotin via neutrAvidin AlexaFluor 594 staining indicated that most of the cells display a fine punctate pattern staining (Figure 9A, a2, a3) similar to control cells (Figure 9A, a1), suggestive of biotin internalization via caveolae. Biochemical quantification of biotin molecules in lysate prepared from LPS-treated/ITSN-1s-transfected cells shows that these ECs significantly recovered their ability to internalize biotin, when compared to untreated cells (Figure 9B). Less than 25% inhibition of biotin internalization, still detected after myc-ITSN-1s expression, can be explained by the incomplete transfection of the entire ECs population. For the same reason, evaluation of the effects of ITSN-1s rescue on paracellular permeability is methodologically limited.

To evaluate the effects of myc-ITSN-1s expression and effective endocytosis on iNOS expression, we prepared total cell lysates from control, LPS-treated and LPS-treated/ITSN-1s-transfected ECs; equivalents of total protein amounts were analyzed by SDS-PAGE, electrotransfer to nitrocellulose membranes and western blot using NOS2 Ab. LPS-treated/ITSN-1s-transfected ECs show lower immunoreactivity to NOS-2 Ab and thereby, lower expression of iNOS, by comparison to LPS-treated ECs (no ITSN-1s transfection), Figure 9C. Then, the effects of myc-ITSN-1s expression on mtNOS in LPS-exposed ECs, were analyzed on mitochondrial-enriched fractions prepared from control, LPS-treated and LPS-treated/ITSN-1s-transfected ECs; mitochondrial lysates were normalized for total protein content and analyzed by SDS-PAGE, electrotransfer to nitrocellulose membranes and western blot using NOS2 Ab. As shown in Figure 9D, mtNOS is detected in the mitochondria fraction prepared from LPS-treated ECs. No NOS2 immunoreactivity was detected in control or LPS-treated/ITSN-1s-transfected ECs. The observation is consistent with the idea that ITSN-1s deficiency and the resultant impaired caveolae internalization is relevant to ECs response to inflammatory stimuli.