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Integrative Physiology |
From the Department of Pharmacology, Vascular Cell Signaling and Therapeutics Program, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Conn. The present address for R.D.R. is the Center for Experimental Therapeutics, University of Pennsylvania School of Medicine, Philadelphia, Pa.
Correspondence to William C. Sessa, Dept of Pharmacology, Vascular Cell Signaling and Therapeutics Program, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, CT 06536. E-mail william.sessa{at}yale.edu
| Abstract |
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Key Words: adenovirus endothelium vascular endothelial growth factor signal transduction nitric oxide
| Introduction |
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There is evidence that phosphorylation of this residue may be important for NO release in intact blood vessels. Indeed, inhibitors of phosphatidylinositol 3-kinase (PI3K), the upstream activator of Akt, inhibit endothelium-dependent responses in the rat cerebral circulation in vivo.7 Further evidence of the importance of this pathway are experiments showing that dominant-negative Akt attenuates endothelium-dependent relaxation in rat8 and mouse aorta9 ex vivo and acetylcholine (ACh)-induced blood flow changes in rabbit femoral arteries in vivo.9 Therefore, these studies suggest that the PI3K/Akt pathway is involved in NO-mediated regulation of vascular tone, and hence, blood flow. However, both PI3K and Akt can influence a variety of cellular metabolic and survival pathways that may influence NO release.10 To directly address the importance of eNOS phosphorylation in intact blood vessels, we have developed adenoviruses that encode for the constitutively active form of eNOS,1 a phosphomimetic form of eNOS, by mutating serine 1179 to an aspartate residue (S1179DeNOS), or a non-phosphorylatable form of eNOS, by mutating serine 1179 to an alanine residue (S1179AeNOS). In the present study, we show that S1179DeNOS, but not S1179AeNOS, augments basal and vascular endothelial cell growth factor (VEGF)stimulated NO production in endothelial cells (ECs) and completely restores endothelium-dependent vasodilatation in pressurized arteries from eNOS knockout mice. These data underscore the importance of this residue in regulation eNOS-dependent responses in vivo.
| Materials and Methods |
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Characterization of eNOS Adenoviruses
Bovine aortic endothelial cells (BAECs) were cultured in 100-mm dishes (for basal NO measurement) or C6-well plates (for stimulated NO measurement) and infected with adenoviruses for 4 hours. Viruses were washed off and cells were incubated for 18 hours in complete medium. For basal NO release, medium was collected 48 hours after infection. For comparisons between viruses, equal eNOS protein levels were confirmed by Western blotting. For measurement of stimulated NO release, cells were washed with serum-free medium and stimulated with VEGF (40ng/mL) for 30 minutes.
NO Release From ECs
After infection with adenovirus, media were processed for measurement of nitrite (NO2-), the stable product of NO, by NO-specific chemiluminescence.14
Adenoviral Gene Transfer Into the Endothelium of Mouse Carotid Arteries
Male C57Bl/6J or eNOS knockout mice15 (8- to 12-weeks old) were anesthetized with ketamine/xylazine and exsanguinated via transection of the inferior vena cava followed by perfusion of saline through the left ventricle. The common carotid was cannulated, flushed with a small amount (
2 µL) of virus, and tied off proximally. Approximately 3 µL of virus was injected into the common carotid to fill and distend the vessel. The cannula was then removed, and the vessel was tied off distally. The virus-filled vessel was incubated in situ at 37°C for 2 hours and then dissected free from surrounding tissue and rinsed in saline before overnight (18 hours) incubation in complete DMEM at 37°C, 5% CO2. Control vessels were filled with viral storage buffer and treated in an identical manner.
Assessment of Gene Transfer
Efficiency of gene transfer was assessed using ß-gal staining,16 visualizing GFP fluorescence in the vessel wall, or by Western blotting. After ß-gal staining, vessels were either cut longitudinally and mounted for imaging en face, or embedded in OCT for cryosections (4 µm thickness). To visualize GFP, vessels were fixed for 1 hour at 4°C in formal saline (10% formaldehyde) and cut longitudinally for imaging en face using a laser scanning confocal microscope. Black and white confocal images were used to assess the area viral-infected, GFP-expressing cells per field using ScnImage software. White was assigned an arbitrary value of 1 and black as 250. Mean intensity was calculated from 4 vessels and each vessel was measured 5 times. Transgene expression was also determined by Western blot analysis. Infected carotid arteries (2 vessels/sample) were frozen in liquid N2, crushed, and homogenized. Proteins (11 µg/sample) and control samples of purified bovine eNOS were separated on 10% gel, transferred onto a nitrocellulose membrane, and probed with monoclonal antibody against eNOS (1:1000, Transduction Laboratories) and ß-actin (1:5000, Sigma). Densitometry of each blot was used to calculate the relative expression of eNOS as a ratio of ß-actin expression.
Ex Vivo Assessment of Gene Transfer on Vessel Reactivity
After overnight incubation, carotids were placed in cold (4°C) Krebs physiological saline solution (PSS) of the following composition (mmol/L): NaCl 119, KCl 4.7, CaCl2 · 2H2O 2.5, MgSO4 · 7H2O 1.2, NaHCO3 2.1, KH2PO4 1.2, glucose 11, ibuprofen 0.01, and gassed with 5% CO2 in air. Vessels were cut into 2 rings for isometric tension studies or left intact and mounted onto glass cannulae for isobaric studies.
Isometric Studies
Rings of carotid artery were mounted in a 5-mL vessel myograph (Kent Scientific) on 2 tungsten wires (25-µm diameter), and basal tension was set at 0.5 g. After 45 minutes equilibration, rings were contracted with 125 mmol/L KCl substituted for NaCl in PSS (KPSS). Responses to KPSS were repeated until the contraction was reproducible. Concentration-response curves were constructed to prostaglandin F2
(PGF2
, 10-8 to 3x10-5 mol/L). To assess endothelial function, vessels were precontracted with a submaximal (
80%) concentration of PGF2
(0.5 to 1x10-5 mol/L) before application of endothelium-dependent dilator acetylcholine (ACh, 10-10 to 3x10-6mol/L). Basal NO production was measured by increasing tone by
50%, using PGF2
(3 to 5x10-6 mol/L), and applying L-NAME (3x10-4 mol/L, 20 minutes). Sensitivity to NO was tested by measuring relaxation to the NO donor sodium nitroprusside (SNP, 10-10 to 10-6 mol/L).
Isobaric Studies
Carotid arteries were mounted in a perfusion myograph (Living Systems), pressurized to 100 mm Hg, and superfused at 10 mL/min. Intraluminal pressure was maintained using a pressure servo system, and internal diameter was monitored using a dimension analyzer. Vessels were equilibrated for 45 minutes at an intraluminal flow rate of 0.13 mL/min and then primed with 10-6 mol/L phenylephrine (PE). After a 30-minute washout period, vessels were constricted with 10-7 to 10-6 mol/L PE to reduce internal diameter by
15% and ACh (10-10 to 10-6 mol) was applied directly into the organ bath (5 mL). Basal NO production was measured by applying L-NAME (3x10-4 mol/L, 30 minutes) after preconstriction with PE. Dilatation to NO donor was tested by applying SNP (10-6 mol/L). Passive diameter was determined by superfusing with Ca2+-free PSS containing 2 mmol/L EGTA at the end of each experiment. Passive diameter of S1179DeNOS-infected vessels was 503±17.8 µm (n=6) and S1179AeNOS was 509±13.1 µm (n=11). The passive diameter of wild-type vessels was significantly (P<0.001) greater than knockout vessels (590±9.3 µm, n=5).
Statistical Analysis
Results are expressed as mean±SEM. Responses to dilators were calculated as percentage reversal of induced tone or constriction. Contraction to L-NAME (isometric) was calculated as percentage increase above basal contraction to PGF2
. Constriction to L-NAME (isobaric) was measured as the additional decrease in diameter above constrictor response to PE. Comparisons between groups were made using ANOVA followed by Bonferronis multiple comparison test. Statistical significance was considered when P<0.05.
| Results |
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Previously, we1 and others2,3 have shown that Akt-dependent phosphorylation of eNOS on serine 1179 is necessary for VEGF-induced NO release. As seen in Figure 1C, VEGF induces a 2-fold increase in NO2- release from control and GFP-transduced cells. Infection of BAECs with WTeNOS augmented VEGF-stimulated NO production, whereas infection with S1179DeNOS further increased basal and VEGF-stimulated NO production during the 30-minute collection period. Interestingly, transduction with S1179AeNOS resulted in less basal NO release than seen in WTeNOS- or S1179DeNOS-transduced cells but more than control or GFP-infected cells.
Luminal Delivery of eNOS Adenovirus to eNOS Knockout Vessels Restores Endothelium-Dependent Relaxations
Previous approaches to infect isolated blood vessels with adenoviruses have relied on incubation of vascular rings with virus in organ culture (for example see Lake-Bruse et al17). Although efficient, one cannot selectively deliver the gene of interest to the endothelium or adventitia because both layers are bathed in the virus. In order to circumvent this issue, we developed a technique for luminal delivery of viruses to selectively infect the endothelium. As seen in Figures 3A and 3B, in situ cannulation and luminal transduction of the endothelium of the mouse carotid with a virus expressing ß-gal resulted in ample expression of the virus in the endothelial layer. The transduction efficiency using a viral titer of 1011 pfu/mL, was 40% to 60% as assessed by en face imaging of the EC surface. Viral infection with LacZ had no significant effect on endothelial function as assessed by relaxation to ACh (see Figure 2C). Similarly, infection with adenovirus had no significant effect on contractile responses to PGF2
(n=4); EC50 and maximum responses were 5.4±1.21 µmol/L, 0.4±0.04 g, and 3.9±0.06 µmol/L, 0.3±0.1 g without and after viral infection, respectively. To test the function of luminal gene transfer, carotid arteries from eNOS knockout mice were infected with WTeNOS virus and ACh-induced relaxations of mouse carotid arterial rings examined. The carotid artery was used because in our preliminary experiments, ACh responses in knockout vessels were absent and abolished by L-NAME in WT vessels (n=4). As seen in Figure 2D, luminal transduction of mouse carotid arteries with WTeNOS virus completely reconstituted ACh-induced relaxations in eNOS knockout vessels, an effect abolished by L-NAME. EC50 values and maximum responses were 46±13.9 nmol/L, 92±3.0% (n=4) and 66±15.5 nmol/L, 82±8.1% (n=5) in WT and infected knockout vessels, respectively. Furthermore L-NAME, which had no constrictor effect in control uninfected vessels, produced an increase in isometric contraction of 65.3±26.4% (n=7) of vessels transduced with WTeNOS. Therefore, using endothelium-selective, adenoviral-mediated gene transfer of eNOS, we were able to restore both endothelium-dependent and basal NO release in eNOS knockout arteries. Interestingly, despite differences in basal NO production, contractile responses to either PGF2
or KPSS were unaffected by eNOS gene transfer. Maximum responses to PGF2
were 0.3±0.06 g (n=9) and 0.3±0.06 g (n=9), and to KPSS were 0.1±0.02 g (n=9) and 0.1±0.02 g (n=9) with and without viral infection, respectively. These results support previous findings by Lamping and Faraci18 where hyperresponsiveness to constrictors in the absence of eNOS (eNOS knockout arteries) was demonstrated using serotonin but not a thromboxane-mimetic. Surprisingly, responses to the NO donor SNP in knockout arteries (EC50 of 5±2.1 nmol/L, maximum response of 95±5.7%, n=4) were also not significantly affected by eNOS gene transfer (EC50 of 2±0.5 nmol/L, maximum response of 92±4.9%, n=4).
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Phosphorylation of eNOS on Serine 1179 Is Important for ACh-Mediated Vasodilatation in Pressurized Vessels
Phosphorylation of bovine eNOS at serine 1179 by Akt is important for regulation of enzyme activity in vitro. Indeed, earlier work from our laboratory demonstrated that overexpression of dominant-negative Akt significantly attenuates relaxation to ACh in murine aortic rings,9 thereby suggesting the importance of phosphorylation of eNOS in isolated arteries. We hypothesized that transduction of vessels with S1179DeNOS would restore normal eNOS function in arteries from knockout animals, whereas S1179AeNOS would not. To test this hypothesis, carotid arteries of eNOS knockout mice were transduced with either S1179DeNOS or S1179AeNOS to approximately equal levels of eNOS expression (Figure 3) assessed by both en face imaging of GFP (Figure 3A for S1179DeNOS and Figure 3B for S1179AeNOS) and semiquantitative Western blotting for eNOS protein (see Figure 3C) in transduced vessel segments. Mean intensity of GFP (arbitrary units) was 51.6±4.4 and 55.1±0.4 in S1179DeNOS and S1179AeNOS-transduced vessels, respectively (n=4 in both), representing approximately 50% of the endothelium virally infected. Relative protein expression of eNOS/ß-actin was 1.1±0.1 (n=4) for vessels transduced with S1179DeNOS and 0.9±0.4 (n=4) for S1179AeNOS. Thus, using 2 independent quantitative measurements, the proteins were equally expressed. We then examined functional responses of the transduced vessels in a perfusion myograph system. We opted to use pressurized vessels because it is a more physiologically relevant preparation (ie, with constant transmural pressure throughout the vessel segment) and it permits evaluation of the time course of vasodilation. Carotid arteries from eNOS knockout mice infected with a virus encoding for ß-gal did not exhibit significant changes in diameter to ACh (n=4; Figure 3D, open circles). Transduction of eNOS knockout arteries with either S1179DeNOS (n=6) or S1179AeNOS (n=11) restored dilatation to ACh to
50% of responses seen in normal WT vessels infected with adenovirus encoding for ß-gal, responses that were abolished after pretreatment with L-NAME (not shown). Maximum responses to ACh were 43±3.5% (n=6) and 38±9.8% (n=11) in the presence of S1179DeNOS and S1179AeNOS, respectively, whereas responses in ß-galinfected WT vessels were 91±4.3% (n=5). However, as seen in Figure 3D, transduction with S1179AeNOS significantly (P<0.05) reduced the dilator responses to doses of ACh compared with S1179DeNOS-transduced vessels. This effect was greater at lower doses (10-9 mol) of ACh, suggesting that the sensitivity to ACh was modulated.
Our studies in infected BAECs show that S1179AeNOS releases considerably less basal NO than S1179DeNOS. Similarly, measurement of basal NO-dependent tone in pressurized carotid arteries (Figure 3E), assessed by constriction to L-NAME, was significantly (P<0.05) less in vessels transduced with S1179AeNOS (47±11.9 µm, n=11) than with S1179DeNOS (68±2.1 µm, n=6). Moreover, consistent with lower basal NO release, responses to NO donor SNP were significantly (P<0.05) greater in the presence of S1179AeNOS (81±3.2%, n=11) than S1179DeNOS (70±2.6%, n=6). However, this difference in basal NO release did not affect constrictor reactivity; application of 10-6mol/L PE resulted in decrease in diameter of 16.2±0.8% (n=6) and 16.3±1.4% (n=11) in S1179DeNOS- and S1179AeNOS-infected arteries, respectively.
To test whether the time course of eNOS activation was different between the two mutant forms of eNOS, we tested the duration of vasodilatation in response to doses of ACh given in pressurized vessels. Despite a trend toward shorter duration of endothelium-dependent responses to ACh in vessels transduced with S1179AeNOS, there was no significant difference between the two mutant forms of eNOS (Figure 3F).
| Discussion |
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VEGF is a potent angiogenic factor that utilizes NO as a second messenger leading to cell survival, proliferation, migration, and angiogenesis in vitro. Genetic evidence supporting this autocrine pathway shows that ischemia-triggered angiogenesis,19,20 wound healing,20 and VEGF-driven angiogenesis21 and permeability21 are reduced in eNOS knockout mice. VEGF promotes NO release by stimulation of a PLC
pathway,22 leading to an increase in intracellular calcium, and via parallel activation of the PI3 kinase/Akt pathway system.23,24 Akt can phosphorylate serine 1179 on eNOS,1,3 and this posttranslational modification accelerates electron flux through the protein, thus increasing NO release.6 Accordingly, mutation of serine to aspartate at 1179 increases basal NO release from transfected COS cells and mutation of serine to alanine abolishes phosphorylation and Akt-stimulated NO release.1,2 In the present study, adenoviral transduction of S1179DeNOS markedly enhanced basal NO release consistent with published data showing that adenoviral transduction of ECs with activated Akt promotes NO release.1 Conversely, transduction of ECs with S1179AeNOS, although active in broken cell lysates, resulted in less basal and VEGF-stimulated NO release. Thus, compared with WTeNOS, S1179DeNOS behaves as a constitutively active form of the enzyme, whereas S1179AeNOS behaves as a loss of function mutant because it cannot be phosphorylated by Akt or other kinases that phosphorylate this residue. However, when overexpressed in the context of endogenous eNOS in BAECs, S1179AeNOS does not appear to exert a significant dominant-negative effect on NO release, presumably because the enzyme is still catalytically active and responds to calcium, but cannot respond to upstream activation via phosphorylation of S1179.
A salient feature of this study is the development of a technique to completely restore endothelial-dependent vasomotion by luminal delivery of adenoviruses followed by organ culture and functional studies. The rationale for developing this technique was to repopulate eNOS and the corresponding phosphorylation mutants into the endothelium of eNOS knockout mice to assess the true function of mutants previously assessed exclusively in cultured cell systems. The advantage of this methodology is that adenoviral delivery serves as an endothelial specific knock-in in a null background, thus allowing for direct examination of the importance of any given mutation on endothelium-dependent vasodilatation. As seen in Figure 3, luminal transduction results in considerable gene transfer to approximately 50% of endothelium and the transgene is confined to the endothelium. Moreover, this procedure does not affect reactivity of the vessels per se and allows for restoration of eNOS function in arteries from eNOS knockout mice. Another advantage of this technique is that the relative transduction efficiency can be assessed in living vessels using en face imaging of GFP-tagged proteins. This becomes a critical issue when one compares different mutations of the same gene product because differences in titer, particle/virion ratio, and transduction efficiencies can exert an effect on vessel function. Overall, the reconstitution of eNOS mutants back into the endothelium of eNOS knockout mice is an attractive, stringent alternative to cell culture and permits documentation of the functional importance of posttranslational phosphorylation.
Previous studies have demonstrated that Akt is involved in NO-mediated endothelium-dependent dilatation of arteries. These studies used dominant-negative Akt to suppress responses to ACh9 or adrenomedullin8 and assume that these effects are due to impairment of phosphorylation of eNOS at serine 1179. As stated previously, other kinases can indeed phosphorylate this site and other sites too. In order to directly study the importance of phosphorylation of serine 1179 in isolated blood vessels, we used luminal delivery to selectively infect endothelium of arteries with adenoviruses encoding for S1179DeNOS or S1179AeNOS. We did not compare responses of eNOS mutants to WT enzyme because the phosphorylation state of S1179 in WTeNOS cannot be controlled in our experiments or any experiments. By comparing S1179DeNOS with S1179AeNOS, we directly assessed the effects of phosphorylation on this particular residue on endothelial function. Infection with either S1179DeNOS or S1179AeNOS restored the ability of ACh to elicit dilatation, indicating that both eNOS mutants are capable of releasing sufficient amounts of NO to mediate these responses. However, the sensitivity and magnitude of vasodilatation to receptor-mediated stimulation was reduced with S1179AeNOS compared with that of S1179DeNOS. The negative charge at serine 1179, due to phosphorylation or the presence of aspartate, improves the rate of electron flux and decreases the disassociation of eNOS/calmodulin complex at low Ca2+ concentrations.6 Hence, the apparent increase in sensitivity of S1179DeNOS to ACh may be due to enhanced stability of eNOS/calmodulin complex at low-dose stimulation. However, we cannot rule in or out the importance of threonine 495 dephosphorylation synergizing with serine 1179.25,26 A recent report has shown that dephosphorylation of theronine 495 increases the ability of calmodulin (CaM) to interact with eNOS and enhances eNOS activity in vitro.26 The dephosphorylation is transient in nature and may relate to the initial binding of CaM to the enzyme. In this context, threonine 495 may initiate eNOS activation, an effect sustained through serine 1179 phosphorylation. Consistent with an important role for eNOS phosphorylation on serine 1179 in regulating basal NO release, the ability of L-NAME to further enhance vessel tone, as an index of basal NO release,27,28 was also less in S1179AeNOS-transduced vessels compared with S1179DeNOS-transduced vessels. Concomitantly with the greater basal release of NO, we observed a decrease in sensitivity of S1179DeNOS-transduced vessels to the NO donor SNP. This downregulation is typically seen in vessels exposed to NO29 or in eNOS transgenic mice.30 Because neither S1179AeNOS nor S1179DeNOS can be phosphorylated/dephosphorylated at S1179, we anticipated that the duration of the ACh response may be regulated by cycles of phosphorylation/dephosphorylation; however, to our surprise, there was no difference in the temporal kinetics of ACh-mediated dilatation of vessels transduced with either virus. This implies that other mechanisms, most likely related to CaM binding and release from eNOS, are more important than S1179. Thus, in summary, phosphorylation of eNOS on S1179 influences endothelium-dependent responsiveness of mouse carotid arteries.
Previous studies have documented that gene therapy with eNOS is an attractive strategy for treatment of several cardiovascular diseases, including atherosclerosis,31 diabetes,32 and restenosis.33 Expression of the phosphomimetic S1179DeNOS in endothelium modulates VEGF-induced NO synthesis in vitro, and expression in arterial endothelium results in greater basal NO release and enhanced endothelium-dependent responsiveness. Therefore, S1179DeNOS may be more beneficial than conventional WTeNOS for use in gene therapy in disease states associated with reduced NO release and impaired angiogenesis. In particular, because Akt activity is inhibited by atherogenic stimuli,34,35 perhaps the phosphomimetic S1179DeNOS may be a novel treatment for endothelial dysfunction associated with hypercholesterolemia and atherosclerosis.
| Acknowledgments |
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Received November 2, 2001; revision received March 14, 2002; accepted March 15, 2002.
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