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Am J Physiol Heart Circ Physiol 292: H893-H903, 2007. First published September 29, 2006; doi:10.1152/ajpheart.00498.2006
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Nitric oxide preconditioning regulates endothelial monolayer integrity via the heat shock protein 90-soluble guanylate cyclase pathway

Galina N. Antonova,1 Connie M. Snead,1 Alexander S. Antonov,1 Christiana Dimitropoulou,2 Richard C. Venema,1,3 and John D. Catravas1,2

1Vascular Biology Center, 2Department of Pharmacology and Toxicology, and 3Department of Pediatrics, Medical College of Georgia, Augusta, Georgia

Submitted 15 May 2006 ; accepted in final form 19 September 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Large (pathological) amounts of nitric oxide (NO) induce cell injury, whereas low (physiological) NO concentrations often ameliorate cell injury. We tested the hypotheses that pretreatment of endothelial cells with low concentrations of NO (preconditioning) would prevent injury induced by high NO concentrations. Apoptosis, induced in bovine aortic endothelial cells (BAECs) by exposing them to either 4 mM sodium nitroprusside (SNP) or 0.5 mM N-(2-aminoethyl)-N-(2-hydroxy-2-nitrosohydrazino)-1,2-ethylenediamine (spermine NONOate) for 8 h, was abolished by 24-h pretreatment with either 100 µM SNP, 10 µM spermine NONOate, or 100 µM 8-bromo-cGMP (8-Br-cGMP). Repair of BAECs following wounding, measured as the recovery rate of transendothelial electrical resistance, was delayed by 8-h exposure to 4 mM SNP, and this delay was significantly attenuated by 24-h pretreatment with 100 µM SNP. NO preconditioning produced increased association and expression of soluble guanyl cyclase (sGC) and heat shock protein 90 (HSP90). The protective effect of NO preconditioning, but not the injurious effect of 4 mM SNP, was abolished by either a sGC activity inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) or a HSP90 binding inhibitor (radicicol) and was mimicked by 8-Br-cGMP. We conclude that preconditioning with a low dose of NO donor accelerates repair and maintains endothelial integrity via a mechanism that includes the HSP90/sGC pathway. HSP90/sGC may thus play a role in the protective effects of NO-generating drugs from injurious stimuli.

bovine aortic endothelial cells; 8-bromoguanosine 3',5'-cyclic monophosphate; nitric oxide synthase; transendothelial electrical resistance

NITRIC OXIDE (NO) is generated by three isoforms of NO synthase (NOS). Endothelial NOS (eNOS or NOS3) and neuronal NOS (nNOS or NOS1) are expressed constitutively, whereas the inducible isoform (iNOS or NOS2) is expressed in response to inflammatory stimuli (36). NOS isoforms affect tissue damage differently: myocardial injury is dramatically enhanced in eNOS-deficient mice, whereas genetic overexpression of eNOS attenuates myocardial infarction (16, 17). Injury to the vascular endothelium is a critical event in acute (e.g., endotoxic shock and acute lung injury) and in chronic inflammatory diseases [e.g., atherosclerosis, transplant vasculopathy, etc. (9, 25, 45, 46)]. Large concentrations of NO, induced in blood vessels by inflammatory stimuli as a result of iNOS overexpression or by millimolar concentrations of NO donors, are cytotoxic to many cell types (33, 45, 46), whereas low concentrations of NO are protective against apoptosis induced by different stimuli, including TNF-{alpha}, oxidative stress, large amounts of NO, and serum or glucose deprivation (5, 6, 28, 49, 51).

Preconditioning, the phenomenon of adaptation by which a traumatic or stressful stimulus confers protection against subsequent injury (47), occurs in two phases: an early phase, which lasts 2–3 h, and a late phase, which begins 12–24 h later and lasts 3–4 days. Delayed preconditioning can protect endothelial cells (26, 27), via yet unclear mechanisms that include NO (3, 15, 26, 27, 29). NO donors are widely used clinically and may prove useful in mimicking the protective effect of preconditioning (10, 22, 29, 50).

The effects of NO can be either cGMP dependent, involving the production of cGMP following NO activation of soluble guanyl cyclase (sGC), or cGMP independent, most frequently mediated by reactive nitrogen species, which are products of interaction of NO with oxygen or superoxide radicals. At or below micromolar concentrations, NO selectively activates sGC; at higher concentrations, NO may produce a variety of effects in addition to activating sGC (23, 28).

High concentrations of NO signal and trigger apoptosis, which, in endothelial cells, is related to cell detachment and is termed anoikis. Anoikis is initiated by the action of antiadhesive substances, such as NO. NO interferes with focal adhesion kinase tyrosine phosphorylation and inhibits endothelial cell adhesion, spreading and formation of focal adhesions [reviewed in Ref. 33]. Interestingly, activation of sGC leading to the formation of cGMP is a proposed mechanism of the antiapoptotic effect of low concentrations of NO (35). A membrane-permeable analog of cGMP, 8-bromo-cGMP (8-Br-cGMP) can mimic protective effects of NO preconditioning against ischemic-reperfusion injury (12, 29, 43).

At least a fraction of eNOS, iNOS, and sGC exists in complex with heat shock protein 90 (HSP90) (8, 40, 48). The sGC/HSP90 complex appears to facilitate NO donor-induced cGMP production in endothelial cells (48). These complexes may also represent physiological mechanisms aimed at maximizing intracellular cGMP production in response to endogenous or drug-derived NO in endothelial cells and thus affecting permeability, proliferation, migration, and apoptosis, minimizing NO scavenging by superoxide and reducing the formation of peroxynitrite, which is injurious to cells. These complexes may also prolong sGC stability by retarding its degradation.

The ability of the endothelium to maintain optimal repair in response to injury is a critical event in its integrity. Cell death, spreading, and migration contribute to the rate of the repair process.

In the present study, we tested two hypotheses: 1) that NO preconditioning inhibits apoptosis and accelerates endothelial repair in response to toxic NO concentrations and 2) that NO preconditioning increases sGC expression and association with HSP90, leading to increased cGMP production and attenuation of endothelial injury.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. The anti-sGC antibody was purchased from Cayman Chemical (catalog no. 160897); the anti-HSP90 antibody was obtained from Transductions Laboratories (catalog no. 610419). Radicicol, 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), 8-Br-cGMP, sodium nitroprusside (SNP), tetramethylrhodamine isothiocynate (TRITC)-conjugated phalloidin, and IBMX were from Sigma Chemical. The cGMP enzyme immunoassay kit was from BioMol. N-(2-aminoethyl)-N-(2-hydroxy-2-nitrosohydrazino)-1,2-ethylenediamine (spermine NONOate) was purchased from Calbiochem. Geldanamycin was obtained from NCI. Hoechst 333342 and mounting medium (ProLong antifade kit, P-7481) were obtained from Molecular Probes. The protein bicinchoninic acid (BCA) assay kit was from Pierce Chemical. Secondary antibodies conjugated with fluorescent dye Cy2 (goat anti-rabbit IgG) and Cy3 (goat anti-mouse IgG) were obtained from Jackson ImmunoResearch Laboratories. Protein A/G agarose beads were from Santa Cruz, and SuperSignal chemiluminescence substrate was from Pierce Chemical. The anti-cleaved poly(ADP-ribose) polymerase (PARP) (Asp214) antibody was from Cell Signaling (catalog no. 9541). Igepal CA-630, nonionic detergent, was obtained from Sigma, and DOC (deoxycholic acid sodium salt) was purchased from Ampresco (Solon, OH).

Cell culture. Cells were grown as previously described (48). Briefly, in-house-harvested bovine aortic endothelial cells (BAECs) were subcultured from primary cultures and used in experiments during passages 2–5. Cultures were maintained in medium 199 supplemented with 10% fetal bovine serum, 5% iron-supplemented calf serum, 20 mg/ml L-glutamine, 1x MEM amino acid and vitamin solutions, 0.6 mg/ml thymidine, 500 IU/ml penicillin, and 500 mg/ml streptomycin. Before all treatments, media was changed to 2% serum growth media.

Wound-healing assays. After pretreatment with vehicle or 0.1 mM SNP for 24 h, conditioned media was removed and saved and new media was added. Cells were scratched with a cell scraper (1.5-mm-width blade) and washed (to avoid repopulation). The saved conditioned media, rather than fresh media, was returned (to avoid adding new variables to the protocol). Vehicle or 4 mM SNP was added as indicated, and cell migration was observed with phase-contrast microscopy (Olympus, IMT-2) after 4 and 24 h.

In separate experiments, wound healing/cell migration was studied through changes in electric cell-substrate impedance (19), monitored with an ECIS model 1600R and ECIS electrode arrays (8W1E, Applied Biophysics). Complete medium (200 µl) was placed in each well of an 8W1E array, equipped with a small gold film surface active electrode (250 mm diameter) and a large counter electrode, and was allowed to equilibrate in the incubator for 30 min. Cell suspension (200 µl; 1 x 105 cells) was added to each well, and experiments were conducted after electrical resistance reached steady state (>6,000 {Omega}). The endothelial monolayer was wounded by electroporation (3 V, 40 kHz, 10 s), and the rate of wound healing/cell migration was monitored as the change in transendothelial electrical resistance (TER), normalized to the TER at the time of wounding. It was not necessary to wash cells out after electrical wounding, because wounded cells are dead and do not repopulate, as previously shown (19).

Actin and nuclear fluorescent staining. BAECs were grown to confluence on glass coverslips, washed three times with PBS, fixed in freshly prepared 3.7% paraformaldehyde in PBS for 15 min, washed with PBS three times, and permeabilized with 1% Triton X-100 in PBS, pH 7.4 for 5 min. Actin was visualized by TRITC-labeled phalloidin (1 µM in PBS, with 0.1% BSA) and placed onto the coverslips for 30 min. To visualize nuclei or for chromatin condensation detection, DNA was stained with the chromatin dye Hoechst 333342 (0.1 µg/ml in PBS and 0.1% BSA) for 1 min. Coverslips were washed three times in PBS (0.1% BSA) and mounted onto glass slides with mounting medium (ProLong antifade kit), and cells were viewed under a ZEISS Axiophot-2 epifluorescence microscope equipped with SPOT camera (Diagnostic Instruments).

HSP90 and sGC immunofluorescent staining. BAECs were grown to confluence on glass coverslips in medium 199 supplemented with 15% serum; the medium was then changed to 2% serum, and, after all treatments, cells were washed with PBS, fixed and permeabilized in a pure methanol-acetone mix (1:1) at –20°C for 20 min, washed in PBS, blocked with 1% BSA, and incubated with specific rabbit polyclonal anti-sGC and mouse monoclonal anti-HSP90 antibodies, 1:100 dilution in blocking buffer, for 1 h at 37°C. Specimens were washed, exposed to a secondary antibody (1:300 dilution in blocking buffer for 1 h at room temperature), and conjugated with cyanine fluorescent dyes, either Cy2 (excitation: 492nm, emission: 510nm; laser source: Argon/2) or Cy3 (excitation: 550nm, emission: 570nm, laser source: HeNe1). Coverslips were mounted on slides with ProLong antifade mounting medium and analyzed under a laser-scanning confocal microscope (LSM 510, Meta 3.2, Zeiss). Fluorescent intensity is presented in arbitrary units. Nonspecific fluorescence was determined after specimen exposure to secondary antibody alone and was subtracted from all other measurements to arrive at specific fluorescence.

Western blotting and immunoprecipitation analyses. BAECs were grown in 100-mm dishes. After treatments, cells were rinsed with PBS and scraped and pelleted (500 g for 10 min), and the pellet was then lysed in 1 ml ice-cold modified radioimmunoprecipitation assay buffer, pH 7.5 [75 mM Tris·HCl, 50 mM NaCl, 1% Igepal CA-630 (nonionic detergent), 0.05 mM EDTA, 0.05 mM EGTA, 0.25% DOC (deoxycholic acid sodium salt)] containing a protease and phosphatase inhibitor cocktail (Sigma P-8340 and P-5726). Cell lysates were agitated for 30 min at 4°C and centrifuged at 16,000 g for 15 min. Protein was estimated with the BCA method (Pierce). For Western blotting analysis, 2x sample buffer was added to cell lysates, samples were boiled, and extracts were separated on SDS-PAGE, transferred to nitrocellulose membranes (30 V for 18 h), and reacted with the primary antibody of interest. For immunoprecipitation studies, cell lysates were precleared with 20 µl 50% protein A/G agarose by incubation with agitation for 1 h at 4°C. Beads were pelleted by centrifugation at 2,500 rpm (1,000 g) for 5 min at 4°C, and the supernatant was transferred to a fresh microcentrifuge tube on ice. Anti-HSP90 antibody (2.5 µg) or 1 µg IgG (for negative control) was added, mixed, and incubated overnight at 4°C; protein A/G agarose (30 µl) was then added and incubated for 2 h at 4°C, and the beads were washed three times with lysis buffer. Proteins were eluted from beads by boiling the samples in 2x SDS sample buffer. Agarose beads were pelleted by centrifugation, and protein supernatants were separated on SDS-PAGE, transferred to nitrocellulose (30 V for 18 h), and reacted with antibodies of interest. Immunoreactive proteins were visualized with an enhanced chemiluminescent detection system. The relative intensities of the protein bands were scanned and quantified using National Institutes of Health ImageJ software.

Statistical analysis. Values are reported as means ± SE. Comparisons between control and treated cells were performed utilizing one-way ANOVA, unpaired t-test, or Mann-Whitney test, as appropriate, with the aid of SigmaStat. Differences of P ≤ 0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
NO preconditioning stimulates endothelial wound healing. Confluent BAEC monolayers were pretreated for 24 h with either vehicle or 0.1 mM SNP (NO preconditioning), wounded by scraping, and exposed to either vehicle or 4 mM SNP. Under similar experimental conditions, 0.1 mM SNP releases a relatively constant amount of 50–150 nM NO, whereas 4 mM SNP releases 3–5 µM NO (13). At 4 h after scraping (Fig. 1A), cells treated with vehicle or 100 µM SNP exhibited partial migration from the wound edge, spreading by lamellopodia extention laterally and into the wounded area. Conversely, cells treated with 4 mM SNP without NO preconditioning did not demonstrate spreading activity. NO-preconditioned cells that were exposed to 4 mM SNP exhibited a near-normal migration pattern. This is better observed at 24 h after scraping (Fig. 1B), where NO-preconditioned and 4 mM SNP-exposed cells demonstrated resistance to injury compared with vehicle-preconditioned and 4 mM SNP-exposed cells. To address the question of whether preconditioning with NO donors selectively protects from NO-induced cytotoxicity, BAECs were treated with H2O2 (10–500 µM) or staurosporine (50 and 400 nM). Cell morphology was examined 4, 6, and 24 h after addition of the injurious stimulus. Preconditioning (24 h) with 0.1 mM SNP did not prevent the toxic effects of either H2O2 or staurosporine (data not shown).


Figure 1
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  		Fig. 1. Nitric oxide (NO) preconditioning stimulates endothelial wound healing in the presence of toxic concentrations of a NO donor. Bovine aortic endothelial cell (BAEC) monolayers were pretreated for 24 h with either vehicle or 0.1 mM sodium nitroprusside (SNP) (preconditioning), scraped, and exposed to either vehicle or 4 mM SNP. Representative photomicrographs from 4 experiments, with 10 fields examined in each experimental and treatment, observed at 4 h (A) or 24 h (B) after scraping.

 
In separate experiments, the rate of wound healing/cell migration was measured from the recovery of TER following injury (Fig. 2). Confluent BAEC monolayers grown on gelatin-covered gold electrodes were vehicle or NO preconditioned and then exposed to vehicle or 4 mM SNP for 8 h. Wounding was induced by applying an elevated electrical field (3 V, 40 kHz) for 10 s. TER was monitored in real time and normalized to its value at the time of wounding (Fig. 2A). Wounding produced an immediate and profound decrease in TER, which, in control cells, returned to normal within 4–6 h after wounding and was not affected by NO preconditioning. Conversely, exposure to 4 mM SNP significantly delayed wound repair; at 16 h after wounding, TER remained well below control values. NO preconditioning significantly increased the rate of wound healing in 4 mM SNP-exposed cells, and, by 12 h, TER had returned to control levels. When data are expressed as the rate of wound healing (between 3 and 4 h, Fig. 2B), NO preconditioning increased the speed of endothelial barrier repair in the presence of 4 mM SNP almost threefold compared with vehicle-preconditioned cells.


Figure 2
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  		Fig. 2. Protective effect of NO preconditioning on the rate of wound healing. A: BAECs were placed on gelatin-covered gold electrodes, grown to confluence, and preconditioned with vehicle or 0.1 mM SNP for 24 h. Cells were then exposed to vehicle or 4 mM SNP, and an elevated electrical field (3 V, 40 kHz) was applied for 10 s. Transendothelial electrical resistance (TER) was normalized to the value at the time of wounding and plotted as a function of time. B: same data expressed as the rate of wound healing (calculated between 3 and 4 h). ##P < 0.01 compared with the 0 mM SNP/control group; **P < 0.01 compared with the 4 mM SNP group (n = 11–18 experiments).

 
NO preconditioning prevents apoptosis and endothelial injury. Exposure of BAECs to 4 mM SNP for 8 h induced endothelial injury, reflected in denuded areas and rounded, detached cells (Fig. 3A). NO preconditioning maintained endothelial morphology comparable with control. Furthermore, BAECs exposed to 4 mM SNP exhibited standard morphological characteristics of apoptosis (33): membrane blebbing (Fig. 3B, top), chromatin condensation, nuclear fragmentation, and apoptotic bodies formation (Fig. 3B, middle). Apoptosis, following exposure to 4 mM SNP, was also reflected in increased amounts of cleaved PARP (Fig. 3C). NO-preconditioned cells were protected from apoptosis induced by 4 mM SNP as revealed by significantly lower amounts of cleaved PARP (Fig. 3C). Preconditioning with 0.1 mM SNP also protected BAECs from injury induced by large concentrations of another NO donor, spermine NONOate (Fig. 4A). Furthermore, preconditioning with low (0.005–0.01 mM) concentrations of spermine NONOate also protected BAECs from apoptosis (Fig. 4B) and injury (Fig. 4C), induced by toxic concentration of either spermine NONOate (0.5 mM) or SNP.


Figure 3
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  		Fig. 3. NO preconditioning prevents apoptosis and reduces endothelial injury. A: BAECs were exposed to vehicle or 0.1 mM SNP for 24 h, followed by 8 h of exposure to vehicle or 4 mM SNP. Denuded areas (arrows) and rounded, detached cells were observed after exposure to 4 mM SNP. Preconditioning with 0.1 mM SNP maintained endothelial morphology comparable with control. B: BAECs exposed to 4 mM SNP exhibited morphological characteristics of apoptosis: membrane blebbing (top), chromatin condensation, nuclear fragmentation, and apoptotic bodies formation (middle). Bar = 50 µm. C: BAECs preconditioned with 0.1 mM SNP for 24 h were protected from apoptosis induced by 4 mM SNP as revealed by Western blotting analysis of cleaved poly(ADP-ribose) polymerase (PARP).

 

Figure 4
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  		Fig. 4. N-(2-aminoethyl)-N-(2-hydroxy-2-nitrosohydrazino)-1,2-ethylenediamine [spermine NONOate (NONO)] mimics both the protective and toxic effects of SNP. A: BAECs were pretreated with either vehicle or 0.1 mM SNP for 24 h, followed by exposure to either vehicle or 0.5 mM of the NO donor spermine NONOate. Preconditioning with 0.1 mM SNP protected BAECs from injury induced by 0.5 mM spermine NONOate. B and C: BAECs were pretreated with vehicle or various low concentrations (0.005–0.01 mM) of spermine NONOate for 24 h, followed by exposure to vehicle, 0.5 mM spermine NONOate, or 4 mM SNP. Preconditioning with low concentrations of spermine NONOate protected endothelial cells from apoptosis (B) and injury (C), induced by toxic concentrations of either NO donor (n = 3–5 experiments).

 
NO preconditioning increases sGC and HSP90 expression and sGC-HSP90 association. Using two color immunofluorescent staining and confocal fluorescent microscopy, we examined HSP90 and sGC expression in BAECs. Cells preconditioned with 0.1 mM SNP for 24 h exhibited increased expression of HSP90, sGC, and HSP90/sGC association (Figs. 5, B and D) compared with vehicle-treated cells (Fig. 5A). Vehicle-preconditioned and 4 mM SNP-exposed BAECs exhibited increased HSP90 expression but normal sGC expression and sGC-HSP90 association (Fig. 5C). Figure 5E presents quantification of three separate experiments, each including observations from 10 fields per treatment. These findings were also confirmed by immunoprecipitation and Western blotting analysis (Fig. 5, F, G, and H).


Figure 5
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  		Fig. 5. NO preconditioning increases soluble guanyl cyclase (sGC) and heat shock protein 90 (HSP90) expression and sGC-HSP90 association. BAECs were preconditioned with either vehicle (A and C) or 0.1 mM SNP (B and D) for 24 h and then exposed to either vehicle (A and B) or 4 mM SNP (C and D). SNP (0.1 mM)-preconditioned cells revealed increased HSP90 (B and D, red) and sGC (B and D, green) expression and sGC-HSP90 association. SNP (4 mM)-exposed BAECs exhibited increased HSP90 expression (C, red) but normal sGC (C, green) expression and sGC/HSP90 association. NS, lack of fluorescence in the absence of primary antibodies. E: quantification of 3 separate experiments, each including observations from 10 visual fields per treatment (***P < 0.001 from corresponding 0 mM SNP value). These findings were also confirmed by immunoprecipitation (IP) and Western immunoblotting (IB) analysis experiments (F–H; n = 3–5 experiments/group; *P < 0.05 and **P < 0.01 from corresponding vehicle value). Bar = 20 µm. AU, arbitrary units; HC, heavy chain of IgG.

 
The protective effect of NO preconditioning against apoptosis is attenuated by sGC activity and HSP90 binding inhibitors and mimicked by a cGMP analog. To explore the contribution of cGMP in the protective effects of NO preconditioning, BAECs were exposed to vehicle or the sGC inhibitor ODQ (0.3 µM) during the 24-h NO preconditioning, followed by exposure to vehicle or 4 mM SNP for 8 h. Cells were then stained with rhodamine-conjugated phalloidin or with Hoechst 33342 and examined by fluorescence microscopy, and the degree of membrane blebbing and nuclear chromatin condensation was quantified. ODQ-treated cells exhibited resistance to the protective effects of NO preconditioning, as expressed in both higher number of cells with membrane blebbing (Fig. 6A) and chromatin condensation (Fig. 6B).


Figure 6
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  		Fig. 6. The protective effect of NO preconditioning against apoptosis induced by 4 mM SNP is attenuated by sGC inhibition. BAECs were treated with vehicle or the sGC inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ; 0.3 µM) during the 24-h preconditioning period with 0.1 mM SNP, followed by exposure to vehicle or 4 mM SNP for 8 h. Cells were then stained with rhodamine-conjugated phalloidin to visualize actin cytoskeleton and quantify membrane blebbing (A) or stained with Hoechst 33342 to visualize the nucleus and quantify chromatin condensation (B). Data are means ± SE (n = 3 different experiments with >9 fields counted per experiment). ***P < 0.001 from 0 SNP/vehicle.

 
In additional experiments, BAECs were preconditioned with either 0.1 mM SNP or 0.1 mM 8-Br-cGMP for 24 h, then exposed to vehicle or 4 mM SNP for 8 h, washed, fixed, and stained with Hoechst 33342 for analysis of chromatin condensation. The cGMP analog mimicked the protective effect of NO preconditioning (Fig. 7A). These results were confirmed by analysis of cleaved PARP (Fig. 7B). The protective effect of NO preconditioning against apoptosis was attenuated by the sGC activity inhibitor ODQ, and this attenuation was prevented by concomitant exposure to 8-Br-cGMP (Fig. 7, C and D). The protective effect of NO preconditioning was also attenuated by exposure to the HSP90 binding inhibitor geldanamycin (Fig. 7E) during the preconditioning period.


Figure 7
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  		Fig. 7. The protective effect of NO preconditioning against apoptosis induced by 4 mM SNP is mimicked by a cGMP analog and attenuated by an HSP90 binding inhibitor. BAECs were preconditioned with vehicle, 0.1 mM SNP, or 0.1 mM 8-bromo-cGMP (8-Br-cGMP) for 24 h and then exposed to vehicle or 4 mM SNP for 8 h, and the number of cells containing condensed chromatin was estimated. The cGMP analog mimicked the protective effect of NO preconditioning (A). These results were confirmed by analysis of cleaved PARP (B). The protective effect of NO preconditioning against apoptosis was attenuated by the sGC activity inhibitor ODQ, and this attenuation was prevented by concomitant exposure to 8-Br-cGMP (C and D). The protective effect of NO preconditioning was also attenuated by exposure to the HSP90 binding inhibitor geldanamycin (E) during the preconditioning period. Data are means ± SE (n = 3–6 experiments); ***P < 0.001 from 0 SNP/vehicle; ##P < 0.01 and ###P < 0.001 from 4 mM SNP.

 
NO preconditioning accelerates endothelial cell repair via the sGC/HSP90 pathway. To further elucidate the role of the sGC/HSP90 pathway in mediating the effect of NO preconditioning, BAECs were preconditioned with 0.1 mM SNP for 24 h in the absence or presence of the sGC activity inhibitor ODQ or a chemically distinct HSP90 binding inhibitor, radicicol (0.1 µg/ml), followed by exposure to vehicle or 4 mM SNP. Cells were then wounded by an elevated electrical field (3 V, 40 kHz) for 10 s, and TER was monitored continuously and normalized to its value at the time of wounding. Pretreatment with ODQ (0.3 µM) delayed the accelerated recovery of TER in NO-preconditioned and 4 mM SNP-exposed cells (Fig. 8A). Preconditioning with 0.1 mM 8-Br-cGMP for 24 h, before exposure to 4 mM SNP, accelerated the recovery in TER (Fig. 8B). Radicicol significantly inhibited the recovery of TER (Figs. 8C and 7D). Data from the experiments shown in Fig. 8C were calculated as the change in normalized resistance per min between 3 and 4 h and are presented in Fig. 8D as the speed of wound healing. Radicicol decreased the speed of wound healing by 2.4-fold. These results provide evidence that sGC-derived cGMP and HSP90/sGC complex activation play regulatory roles in the protective effect of NO preconditioning on the rate of endothelial wound repair.


Figure 8
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  		Fig. 8. NO preconditioning stimulates accelerated endothelial repair via the sGC/HSP90 pathway. BAECs were treated with vehicle or 0.1 mM SNP (A and C) or 8-Br-cGMP (B) for 24 h in the absence or presence of the sGC activity inhibitor ODQ (A) or the HSP90 binding inhibitor radicicol (RA; C), followed by exposure first to vehicle or 4 mM SNP for 8 h and then to an elevated electrical field (3 V, 40 kHz) for 10 s. TER was monitored continuously, normalized to its value at the time of wounding, and plotted as a function of time. Pretreatment with ODQ (0.3 µM) delayed the recovery of TER (*P < 0.05, n = 9 experiments). Preconditioning with 0.1 mM 8-Br-cGMP for 24 h, before exposure to 4 mM SNP, accelerated the recovery in TER (*P < 0.05, n = 8 experiments; B). RA significantly inhibited the recovery of TER (**P < 0.05–0.001, n = 6 experiments). D: data from the experiments shown in C are expressed as the speed of wound healing, calculated as the change in TER per minute between 3 and 4 h (***P < 0.001 from control/vehicle, n = 5–18 experiments). RA decreased the speed of wound healing by 2.4-fold. Bar on the x-axis indicates the times when difference in TER is significant (ANOVA).

 
Effects of SNP on the actin cytoskeleton. Resting endothelial cells in confluent monolayer contain a dense peripheral band of F-actin microfilaments bundles, important in cell-cell adhesion, with minimal central microfilament or stress fibers (Fig. 9). NO-preconditioned BAECs not stimulated to migration contain thin microfilaments of aligned F-actin indicative of cell activation. Exposure of BAECs to 4 mM SNP greatly increased the formation of stress fibers, which are known to be contractile and serve to develop strong substrate anchorage with focal adhesion complexes and motile fibrillar adhesions. NO-preconditioned cells, which were then exposed to 4 mM SNP, formed filopodia, required for spreading and motility.


Figure 9
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  		Fig. 9. Effects of SNP on F-actin cytoskeleton. Cells treated with 0.1 mM SNP for 24 h or with 4 mM SNP following pretreatment with 0.1 mM SNP (B and D), form thin-actin microfilaments (B, arrow) or filopodia (D, arrow), reflecting high potential for migration. A: resting BAECs in confluent monolayer contain dense peripheral bands of actin microfilaments and few central microfilament or stress fibers. C: BAECs treated with 4 mM SNP form contractile stress fibers (arrows) and blebs, which are indicative of defective cell adhesion under stress conditions. F-actin rearrangement was examined by rhodamine-labeled phalloidin fluorescence; DNA was stained with Hoechst 33342. Bar = 20 µm. Each experiment was reproduced at least 3 times.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This data suggest that, in an environment of high NO concentrations akin to that under inflammatory conditions or perhaps under treatment with NO donors, endothelial cells undergo apoptosis and endothelial repair is delayed. However, if endothelial cells are preconditioned with small concentrations of NO, then apoptosis and injury are ameliorated and the rate of endothelial repair in a high NO environment is significantly accelerated, by as much as 2.4-fold. NO stimulates both directed (chemotactic) and random (chemokinetic) motility of endothelial cells in a cGMP-dependent manner (14). Low doses of NO also stimulate adhesion of endothelial cells on type I collagen in a cGMP-dependent manner. In these experiments, BAECs were allowed to grow until TER stabilized (>6,000 {Omega}), indicating that cells had achieved confluence. As can be seen in Fig. 2A in control cells and cells treated with 0.1 mM SNP, TER completely recovered after 6 h and that, in cells preconditioned with 0.1 mM SNP before 4 mM SNP, TER was restored by 12 h after wounding. Average duplication time for BAECs is 24 h. It is thus unlikely that cell proliferation has a significant contribution to monolayer repair. However, increased cell survival after preconditioning (Fig. 3C) likely contributes to the accelerated wound healing.

SNP is a frequently used NO donor with well-established dynamics of NO release; it is often the most effective and only drug in some difficult clinical circumstances (7). We have shown that apoptosis, induced by high concentrations of SNP, may critically compromise endothelial repair and that 24 h preconditioning with 0.1 mM SNP not only prevents apoptosis but also preserves the ability of the cell to migrate. NO-preconditioned endothelial cells exposed to 4 mM SNP form filopodia, indicative of increased potential for migration. Conversely, BAECs treated with 4 mM SNP alone form contractile stress fibers and blebs, which indicate defective cell adhesion and loss of anchorage. These data agree with reports that NO can induce adhesion-related apoptosis or anoikis (33). It also agrees with findings in fibroblasts and airway macrophages (5, 51) that pretreatment with small (0.1–0.2 mM) concentrations of the NO donor SNP can prevent the damaging effect of high (1–4 mM) SNP concentrations. In vascular diseases, the bioavailability of NO can be impaired by various mechanisms, including decreased NO production by eNOS and/or enhanced NO breakdown due to increased oxidative stress (11). Impaired NO secretion can be corrected with NO generating drugs (24, 31, 42).

We have shown that, in endothelial cells, preconditioning with SNP prevented the damaging effects of not only 4 mM SNP but also of 0.5 mM spermine NONOate. These data indicate that the protective effects of preconditioning are due to NO itself. Furthermore, preconditioning with low concentrations of spermine NONOate protected BAECs from apoptosis and injury induced by toxic concentration of either spermine NONOate or SNP but not from injury induced by H2O2 or staurosporine. Interestingly, Kim et al. (20) have reported that NO exposure [100 mM S-nitroso-N-acetyl-L-penicillamine (SNAP)] induced resistance to injury from both reactive nitrogen and reactive oxygen species (2 mM SNAP and 5 mM H2O2, respectively) in isolated rat hepatocytes. It was also reported that NO protects cultured rat hepatocytes from TNF-{alpha}-induced apoptosis (21).

In endothelial cells, NO interacts with several components of the apoptotic pathway, including inhibition of caspases and interference with antiapoptotic protein kinases (41). The protective effects of NO on apoptosis rely on the stimulation of cGMP-dependent protein kinase, modulation of the members of the bcl-2/bax family, induction of HSP70, and interaction with the ceramide pathway (23, 42). Initially, activation of sGC by NO, leading to the formation of the second messenger cGMP, was proposed to mediate the antiapoptotic effect of NO. Membrane-permeable cGMP analogs reduced apoptosis in B-cells, eosinophils, and T lymphocytes. In contrast, other studies failed to demonstrate a protective effect for cGMP. Neither membrane-permeable cGMP analogs nor pharmacological activation of sGC mimicked the NO effect (6). This can be partly explained by the fact that NO does not only stimulate sGC, but that, at higher concentrations (500 µM–1 mM SNP), it also has the potential to modulate sGC levels by downregulating sGC {alpha}- and beta-subunit mRNA and protein (1). These inhibitory effects are not present at lower levels of NO donors (10–100 µM SNP) (39) and probably reflect the concentration-dependent ability of SNP to affect cellular cAMP-to-cGMP ratio. cGMP can modulate sGC expression in a PKA-sensitive manner. Increases in intracellular cAMP can lead to decreased sGC activity through reduction of sGC subunit mRNA and protein levels.

The long-term antiapoptotic effects of NO may also be mediated by transcriptional upregulation of antiapoptotic proteins. HSPs are highly conserved and play a major role in cytoprotection. Apoptosis resistance is associated with high expression of HSPs. In mammalian cells, the stress response involves the induction of five major classes of HSP families, namely, HSP27, HSP60, HSP70, HSP90, and HSP104 (18, 30, 34). NOS and sGC can exist in complex with HSP90 (8, 40, 48). Additional proteins participate in these complexes, including Akt, Raf-1, caveolin-1, G protein-coupled receptors, VEGF receptor 2, etc. To date, more than 100 reported client proteins are associated with HSP90 (2, 4, 32, 44). Interaction between sGC and HSP90 has been demonstrated in endothelial cells (40, 48). One role of the HSP90/sGC complex may be to optimize the mechanism of action of NO (48), maximizing intracellular cGMP production in endothelial cells in response to endogenous or drug-released NO (affecting permeability, proliferation, migration, apoptosis, etc.) and possibly to prolong sGC stability by retarding its degradation. In the present study, we demonstrated that NO preconditioning increases sGC and HSP90 expression and sGC-HSP90 association in endothelial cells and increased cGMP production. Additionally, the protective effect of NO preconditioning was attenuated by sGC inhibition and mimicked by a cGMP analog.

Endothelial cell apoptosis was estimated from the appearance of endothelial blebbing and chromatin condensation, as well as from the levels of cleaved PARP. This protein is one of the main cleavage targets of caspase-3 in vivo (37). Cleavage of PARP during apoptosis facilitates cellular disassembly and ensures the completion and irreversibility of the process (38). The protective, antiapoptotic effect of NO preconditioning, but not the proapoptotic effect of 4 mM SNP, was attenuated by pretreatment with the sGC inhibitor ODQ. The ODQ-mediated attenuation of reduced cleaved PARP expression induced by NO preconditioning was associated with decreased cGMP accumulation, suggesting that NO preconditioning protects endothelial cells against apoptosis via sGC activation. We have found that the protective effect of NO preconditioning against apoptosis was attenuated by HSP90-binding inhibitor geldanamycin, indicating an important role of HSP90 in sGC stabilization and activation. Another HSP90 inhibitor, radicicol, also inhibited cell migration, an effect that appeared to involve the NOS-sGC pathway. Pretreatment with either ODQ or radicicol significantly delayed the accelerated recovery of TER in NO-preconditioned cells exposed to 4 mM SNP, whereas preconditioning with 0.1 mM 8-Br-cGMP for 24 h, before exposure to 4 mM SNP, accelerated the recovery in TER. Taken together, these data indicate that NO preconditioning accelerates endothelial repair and inhibits apoptosis in response to toxic NO donor concentrations, via the sGC-HSP90 pathway (Fig. 10). These mechanisms may play a role in the vasoprotective effects of NO-generating drugs against injurious stimuli.


Figure 10
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  		Fig. 10. NO preconditioning inhibits apoptosis and accelerates endothelial repair in response to toxic NO concentrations. The sGC-HSP90 pathway is involved in the protective effects of NO preconditioning in endothelial cells by increasing sGC expression and association with HSP90, leading to increased cGMP production and attenuation of endothelial injury. GA, geldanamycin.

 

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This study was supported by National Heart, Lung, and Blood Institute Grants HL-66993 and HL-070214.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. Antonova, Vascular Biology Center, Medical College of Georgia, Augusta, GA, 30912-2500 (e-mail: gantonova{at}mail.mcg.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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