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CARDIOVASCULAR
Laboratory of Molecular Pharmacology, Department of Pharmacy, University of Patras, Patras, Greece (A.P., V.K., C.P., E.P., A.P.); Foundation for Biomedical Research of the Academy of Athens, Basic Research Center, Cell Biology, Athens, Greece (D.B.); and "G.P. Livanos and M. Simou" Laboratories, Evangelismos Hospital, Department of Critical Care and Pulmonary Services, University of Athens School of Medicine, Athens, Greece (Z.Z.)
Received June 2, 2006; accepted August 28, 2006.
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Abstract |
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; Conway et al., 2001). In the adult, angiogenesis is tightly regulated with vessel growth limited to a few tissues; deregulated angiogenesis has been proposed to contribute to several disease processes, including tumor growth, psoriasis, arthritis, and diabetic retinopathy (Carmeliet, 2003). During angiogenesis, EC integrate signals from various soluble and matrix-bound molecules to form new vessels (Bischoff, 1997; Conway et al., 2001). Among the endogenous mediators proposed to play an important role in neovascularization is the labile diatom molecule nitric oxide (NO) (Morbidelli et al., 2003). Exogenously applied NO donors have been shown to stimulate EC growth and migration in vitro (Ziche et al., 1994; Isenberg et al., 2005); moreover, endogenous NO mediates many of the effects of the prototype angiogenic factor, vascular endothelial growth factor (VEGF) (Papapetropoulos et al., 1997; Ziche et al., 1997). In vivo, ischemia-induced angiogenesis was attenuated in endothelial nitric-oxide synthase (NOS) knockout animals, and VEGF-stimulated vessel formation in the cornea could be blocked by administration of the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) (Ziche et al., 1997; Murohara et al., 1998). However, in spite of the wealth of information of the angiogenic effects of NO, the downstream pathways mediating its effects remain poorly characterized.
Soluble or NO-sensitive guanylyl cyclase is a ubiquitously expressed enzyme that acts as a "receptor" for NO (Hobbs, 1997). The low basal activity of sGC increases several hundred-fold upon activation with NO converting GTP to cGMP (Lucas et al., 2000). The most common form of the obligate sGC heterodimer is 
1/
1 that is present in large amounts in smooth muscle, nerve cells, and platelets (Hobbs, 1997). In the vascular system, the biological role of sGC has been mostly studied in the context of smooth muscle tone and platelet aggregation (Lucas et al., 2000; Friebe and Koesling, 2003). We have previously shown that sGC subunit mRNA and catalytic activity are also present in endothelial cells from different vascular beds (Papapetropoulos et al., 1996); however, the physiological role of sGC in vascular endothelium remains for the most part unexplored. With respect to angiogenesis, some evidence for the involvement of sGC exists as NO-stimulated EC proliferation is cGMP-dependent, and a cell-permeable analog of cGMP promotes EC migration (Parenti et al., 1998; Kawasaki et al., 2003). The aim of the present study was to characterize the contribution of sGC in neovessel formation; to this end, we studied the effects of pharmacological activators and inhibitors of sGC in two in vivo models; moreover, we sought to determine whether alterations in sGC levels or activity affect the migratory and proliferative potential of EC and to elucidate the pathway(s) involved.
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Materials and Methods |
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1 and 1 sGC subunit antibodies, isobutylmethylxanthine, L-NAME, sodium nitroprusside (SNP), bovine serum albumin, EDTA, ODQ, zaprinast, and all protease inhibitors were purchased from Sigma-Aldrich (St Luis, MO); BAY 41-2272 was obtained from Qbiogene-Alexis GmbH (Grunberg, Germany); ERK1/2 and p38-phosphospecific and total antibodies along with the secondary antibodies were obtained from Cell Signaling Technology (Beverly, MA); luciferase reporter assay system was purchased from Boehringer-Mannheim (Mannheim, Germany); and Leghorn fertilized eggs were from Pindos (Iperos, Greece). CAM Angiogenesis Assay. Fertilized White Leghorn chicken eggs were placed in an incubator and kept under constant humidity at 37°C. On day 4, a square window was opened in the shell and then sealed with adhesive tape. On day 9, an O-ring (1 cm2) was placed on the surface of the CAM, and the tested substance (ODQ, BAY 41-2272, or zaprinast) was placed inside this restricted area. After 48 h, CAMs were fixed in Carson's solution (saline-buffered formalin), and angiogenesis was evaluated using image analysis software as follows. CAM tissues were photographed using a digital camera attached to a stereoscope and then imported into the Scion Image software (Scion Image Release Beta 4.0.2 software; Scion Corporation, Frederick, MD). Photomicrographs were then transformed to binary files, thresholded, and skeletonized. Vessel length was measured in pixels and expressed as percentage of control.
Zebrafish Lines and Pharmacological Treatment. Zebrafish were raised under standard laboratory conditions at 28°C. We used embryos from an incross of double heterozygous carriers for the following transgenes: the panendothelian gfp line Tg(flk1:EGFP)s843 (Jin et al., 2005) and the erythrocyte-specific DsRed line Tg(gata1: DsRed) (Traver et al., 2003). Embryos were incubated with 5 or 10 µM ODQ in embryo water. Analyses shown here were carried out with 5 µM ODQ. Control embryos were incubated in 0.05% DMSO in embryo water. Embryos were fixed in 4% paraformaldehyde overnight and then imaged.
Luciferase Activity. African green monkey COSm6 cells (2 x 105 cells) were plated in six-well plates and grown overnight. Cells were then transfected using the jetPEI transfection reagent (3 µg of DNA and 6 µl of jetPEI per well). After 24 h, cells were collected, and approximately 1 x 104 cells were added inside each O-ring at 6, 9, and 12 days of embryogenesis. After a 48-h incubation, CAM tissues were removed and assayed for activity using the luciferase reporter assay system according to the manufacturer's instructions.
Western Blotting. Proteins from tissues or cells were extracted after homogenization in a lysis buffer containing 1% Triton X-100, 1% SDS, 150 mM NaCl, 50 mM NaF, 1 mM Na3VO4, 0.5% sodium deoxycholate, 1 mM EDTA, 0.1 mM EGTA, and protease inhibitors (10 µg/ml aprotinin, 10 µg/ml pepstatin, and 20 mM phenylmethylsulfonyl fluoride). Samples were subjected to SDS-polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and incubated with the primary and secondary antibodies. Immunoreactive proteins were detected using a chemiluminescent substrate. Bands on autoradiographs were quantified using the Scion Image Release Beta 4.0.2 software. In the case of double bands, e.g., ERK1/2, the intensity from the two individual bands was calculated as one.
cGMP Enzyme Immunoassay. CAM tissues were removed and washed in Hanks' balanced salt solution. They were then incubated with the NOS inhibitor L-NAME for 10 min; tissues were then stimulated with sodium nitroprusside (100 µM) in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine (1 mM) for 20 min. Media were then aspirated, and tissues were homogenized in 0.1 N HCl to extract cGMP. After 30 min, HCl extracts were collected, and cGMP was analyzed using a commercially available enzyme immunoassay kit following the manufacturer's instructions.
Construction of Adenoviral Plasmids and Production of Adenoviruses. The adenoviral plasmids used in our study were constructed using standard methodology. In brief, rat 1 and 1 cDNAs were subcloned into the pShuttle-cytomegalovirus vector and recombined with pAdeasy-1 in BJ5183 cells. Recombinants were identified via restriction analysis and transfected into human embryonic kidney cells (2 x 106) using the jetPEI reagent. Replication incompetent adenoviruses were then propagated in human embryonic kidney cells and titered using the cytopathic effect assay. Human umbilical vein endothelial cells (HUVEC) were infected with Ad-sGC1 (20 m.o.i.) and Ad-sGC1 (20 m.o.i. each) or Ad-GFP (40 m.o.i.) as control. After 20 to 48 h of infection, the infected cells were used for biochemical analyses or trypsinized and used in migration, proliferation, and tube-formation assays.
HUVEC Cell Culture. HUVEC were isolated from fresh cords and grown on 100-mm dishes in M199 supplemented with 15% bovine calf serum, 50 U/ml penicillin, 50 µg/ml streptomycin, 50 µg/ml gentamycin, 2.5 µg/ml amphotericin B, 5 U/ml sodium heparin, and 150 to 200 µg/ml endothelial cell growth supplement. HUVEC between passages 1 and 2 were used for all experiments.
Matrigel in Vitro Tube-Formation Assay. The formation of tube-like structures by HUVEC was assessed on growth factor-reduced Matrigel. HUVEC were plated at 15,000 cells/well in 96-well plates, precoated with 45 µl of Matrigel in the presence of ODQ (10 µM), BAY 41-2272 (1 µM), or DMSO as vehicle. After 24 h of incubation, tube formation was quantified. In brief, phase-contrast photomicrographs of endothelial cell cultures were recorded and imported into the freeware image analysis program Scion Image (Release Beta 4.0.2). Images were converted to a binary format, and the binary threshold was adjusted to obtain the best contrast of tubules. The images were then skeletonized, and total tubule length was measured in pixels. The area occupied by aggregates of cells was considered as noise. The total length calculated was then expressed as a percentage of control.
Cell Proliferation. HUVEC were seeded in 24-well plates at 6 x 103 cells/cm2 and incubated in M199 supplemented with fetal bovine serum and endothelial cell growth supplement for 24 h. Cells were incubated with the indicated concentration of BAY 41-2272 (0.1 and 1 µM) and allowed to proliferate for 48 h. After this time, they were trypsinized, and cell number was determined using a hemocytometer.
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Data Analysis. Data are expressed as means ± S.E.M. of the indicated number of observations. Statistical comparisons between groups were performed using ANOVA followed by a post hoc test or Student's t test as appropriate. Differences were considered significant when p < 0.05.
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Results |
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1/1) were detected (Fig. 1A); 1 and 1 levels were developmentally regulated, showing peak expression during days 9 through 12.
Using a heterologous system, we demonstrated that endogenous sGC protein levels correlate with 1 promoter activity (Fig. 1B). COSm6 cells were transfected with an empty luciferase vector (pGL3) or a vector in which luciferase was expressed under the control of the sGC 1 promoter (Vazquez-Padron et al., 2004); cells were then placed on the CAM. Growth factors and other mediators produced in CAM tissue diffuse and modulate 1 promoter activity in the COSm6 cells. In these experiments, we observed a time-dependent increase in luciferase activity that mirrored the protein expression of 1. Some of the regulatory sequences in the 1 promoter required for expression reside within the 5'-untranslated region (5'-UTR), because deletion of this region resulted in reduced luciferase activity at all of the time points tested. To determine whether the sGC present in the CAM is catalytically active, we measured cGMP accumulation in the presence and absence of the NO donor SNP. Indeed, stimulation of CAM tissues with SNP led to a robust increase in cGMP levels (Fig. 1C).
We next examined the impact of sGC inhibition in angiogenesis in the CAM. Incubation with the selective sGC inhibitor ODQ (Garthwaite et al., 1995) caused a dose-dependent inhibition of vascular length, suggesting that sGC is important in the formation of neovessels (Fig. 1D). On the other hand, treatment of CAMs with the NO-independent sGC activator BAY 41-2272 or the phosphodiesterase 5 inhibitor zaprinast stimulated angiogenesis as indicated by the increase in CAM vessel length (Fig. 1E). The increase in vessel length in the CAM in the presence of either BAY 41-2272 or zaprinast was approximately half of the response observed with 1 µg of VEGF (30 ± 9.1% increase over control).
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sGC Activation Promotes the Expression of an Angiogenic Phenotype in Cultured Endothelial Cells. To determine whether sGC activation affects EC properties related to angiogenesis, we treated HUVEC with BAY 41-2272. Such treatment resulted in a concentration-dependent increase in EC proliferation (Fig. 3A). In addition, incubation of HUVEC with BAY 41-2272 or the cell-permeable analog of cGMP, 8-Br-cGMP, stimulated EC migration in vitro (Fig. 3B). In a different series of experiments, we observed that HUVEC grown on Matrigel in the presence of the sGC inhibitor ODQ formed fewer capillary-like structures (Fig. 3C). To test whether sGC is part of the signaling cascade activated by angiogenic factors, we employed vascular endothelial growth factor and measured EC migration in response to this growth factor in the presence and absence of ODQ. VEGF stimulated migration approximately 4-fold in an ODQ-sensitive manner, suggesting that sGC mediates some of the angiogenic actions of this prototype-angiogenic growth factor (Fig. 3D).
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1 and 1 protein levels and sGC activity compared with uninfected or GFP-infected cells (data not shown). sGC-overexpressing cells exhibited higher proliferation rates than control cells (Fig. 4A). Similar results were obtained in migration assays where 1/1-overexpressing cells showed a 2-fold greater basal migration rate compared with cells infected with a GFP-expressing adenovirus and exhibited an augmented migratory response to VEGF (Fig. 4B). Increased sGC expression also correlated with an increase in the ability of EC to form tube-like networks on Matrigel; the increase in network formation (40%) was similar to that seen with naive EC stimulated with BAY 41-2272 (Fig. 4C).
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Mechanisms of sGC-Triggered Angiogenic Responses. We next sought to determine the pathways involved in sGC-regulated responses that are relevant to angiogenesis. Because MAPK members have been implicated in EC proliferation and migration, we tested whether sGC-overexpressing cells exhibit increased levels of ERK1/2 and p38 activation. Indeed, cells infected with viruses containing the sGC transgenes displayed a significantly higher pERK1/2/total ERK1/2 ratio, indicating that in these cells, ERK1/2 is activated (Fig. 5, A and B). Similar results were obtained for p38 (Fig. 5, A and B). Moreover, incubation of HUVEC with BAY 41-2272 stimulated ERK1/2 and p38 phosphorylation in a time-dependent manner (data not shown). Inhibition of sGC by ODQ resulted in a reduction in the phosphorylation of both MAPK members tested (ERK1/2 and p38) in cells infected with the 1 and 1 sGC subunits (Fig. 5, C and D); endogenous sGC inhibition by ODQ reduced only ERK1/2 activation in GFP-infected cells. Finally, to prove the functional relevance of p38 activation to sGC-triggered migration, we studied EC migration in the absence and presence of p38 inhibitor SB203580. Pharmacological inhibition of p38 attenuated the migration of both uninfected cells stimulated with BAY 41-2272, as well as the migration brought about by increased sGC expression (Fig. 5E).
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Discussion |
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1 promoter activity, 1/1 immunoreactivity, and sGC activity in the CAM were expressed in a manner that coincided with maximal angiogenic activity in this tissue (days 9–12) (Maragoudakis et al., 1988). Inhibition of endogenous sGC activity by ODQ resulted in an inhibition of angiogenesis, whereas incubation with the sGC activator BAY 41-2272 increased neovascularization. In line with what was observed with the sGC activator BAY 41-2272, phosphodiesterase 5 inhibition by zaprinast led to an increase in vessel length in the CAM. It should be noted that conflicting results exist regarding the role of the endogenous sGC activator NO in the CAM; older studies (Pipili-Synetos et al., 1994) suggested that NO synthase inhibition stimulates angiogenesis, whereas more recent observations (Polytarchou and Papadimitriou, 2004) favor a proangiogenic role for NO in this system, in line with the angiogenesis-stimulating properties of NO in all other systems studied so far. The initially reported antiangiogenic actions of NO in the CAM might relate to the fact that, in the older studies, the material (coverslip) used to deliver the NO synthase inhibitor itself caused an inflammatory response, thereby making the use of cortisone acetate necessary and complicating data interpretation.
The molecular mechanisms that govern vascular development are highly conserved across species. Common growth and transcription factors participate in blood vessel formation in fish, avians, and mammals. For example, VEGF, a growth factor that signals through NO/cGMP, is critical for angioblast formation and differentiation into arterial endothelium in zebrafish (Nasevicius and Ekker, 2000). Likewise, EC are formed during development from angioblasts/endothelial precursors in response to VEGF-induced flk-1 activation in mice (Shalaby et al., 1995; Carmeliet et al., 1996). We took advantage of the fact that zebrafish embryos are free-living in the external environment, transparent during development, and that their vascular system develops rapidly to study the effects of sGC inhibition in vascular development. To visualize the appearance of EC in vivo, we used transgenic animals that expressed GFP under the control of the zebrafish flk-1 promoter. The GFP expression pattern is specific to the vasculature and recapitulates the endogenous expression of flk-1 (Liao et al., 1997). Incubation of embryos with ODQ starting at 10 hpf did not have an overt effect on vasculogenesis, because GFP-positive cells appeared normally and formed vessels. It should be noted that cGMP is detectable in zebrafish embryos at 8 hpf and that its levels double after 20 hpf (Holmqvist et al., 2004). Interestingly, embryos that had their sGC inhibited 10 to 24 hpf displayed fewer cranial vessels, thinner intersegmental vessels, and a disorganized posterior cardinal vein, indicative of an abnormal angiogenic response.
To evaluate the direct effects of sGC activation on EC properties that are important for neovascularization, we used HUVEC, exposed them to BAY 41-2272, and determined their proliferation rate. BAY 41-2272 is a new NO-independent heme-dependent activator of sGC (Stasch et al., 2001). We chose to use this agent to enhance sGC activity and increase intracellular cGMP levels over NO-generating cGMP-elevating agents, because we wanted to eliminate any cGMP-independent actions of NO donors, like S-nitrosylation, tyrosine nitration, interaction with heme and nonheme iron containing proteins or interaction with lipids and free radicals that could contribute to or modify the cGMP-mediated responses (Davis et al., 2001). Exposure of endothelial cells to BAY 41-2272 increased EC number in a concentration-dependent manner. Our observations are in line with those of Isenberg et al., who demonstrated that NO-donors, when used at low concentrations, promote EC growth in a cGMP-dependent manner, whereas higher concentrations have cGMP-independent inhibitory effects on proliferation (Isenberg et al., 2005).
Another property of EC that is important for angiogenesis is the ability of these cells to organize into patent capillary structures. Using Matrigel to drive network-like formation in vitro, we observed that EC incubated with ODQ engaged less in network formation. In agreement with these results, we have previously shown that inhibition of endogenous NO production attenuates VEGF- and transforming growth factor 1-induced capillary-like structure formation in three-dimensional collagen gels (Papapetropoulos et al., 1997). Finally, we tested whether activation of sGC or incubation with a cell-permeable analog of cGMP affects the ability of EC to migrate. Both BAY 41-2272 and 8-Br-cGMP promoted a 4-fold increase in EC migration in the absence of a growth factor, suggesting that sGC activation per se is sufficient to promote EC mobilization. Taken together, the data presented so far indicate that sGC plays an important role in all of the EC properties examined that are linked to angiogenesis. This conclusion is further strengthened by the observation that overexpression of sGC in EC using recombinant adenoviruses increased the proliferation rate, migratory ability, and organization of EC into network-like structures, mimicking the responses obtained using pharmacological activators of the enzyme.
VEGF is among the best characterized angiogenic factors (Ferrara et al., 2003; Zachary, 2003). Exposure of EC to VEGF increases NO production; the NO released then acts in an autocrine manner to promote angiogenesis (Papapetropoulos et al., 1997; Ziche et al., 1997; Ferrara et al., 2003). However, the contribution of cGMP to the angiogenic properties of VEGF has not been fully explored. In one study, inhibition of sGC by ODQ blocked the increase in VEGF-stimulated EC proliferation (Parenti et al., 1998). To determine whether cGMP formation also mediates the action of VEGF with respect to migration, we treated EC with ODQ and determined the migratory response to VEGF. ODQ-treated cells exhibited a blunted migratory response, suggesting that sGC activation is required for this classic growth factor to transmit some of its angiogenic signals. Moreover, our observation that cells infected with sGC adenoviruses exhibit an increased migratory response to VEGF lends further credence to the hypothesis that sGC plays an important role in mediating the biological responses to VEGF.
Mitogen-activated protein kinase cascades have been linked to many biological responses associated with angiogenesis (Seger and Krebs, 1995). Members of this family of kinases become fully active after phosphorylation on both threonine and tyrosine residues (Johnson and Lapadat, 2002). EC proliferation in response to VEGF and other growth factors depends on ERK1/2 activation, whereas p38 has been shown to mediate the migration of EC (Zachary, 2003). Previous reports have shown that NO donors are capable of activating ERK1/2 (Parenti et al., 1998; Oliveira et al., 2003). NO released from pharmacological concentrations of nitrovasodilators causes S-nitrosylation of Cys118 of p21Ras, leading to the recruitment and activation of several downstream kinases (Lander et al., 1997). On the other hand, cell-permeable analogs of cGMP have been proposed to activate Ras using a different, yet unidentified pathway (Oliveira et al., 2003). To investigate whether the EC proliferation observed in HUVEC overexpressing sGC correlates with an increase in ERK activation, we determined the phosphorylated/total ratio for this kinase. Indeed, higher sGC levels resulted in increased ERK1/2 phosphorylation in an ODQ-sensitive manner. ODQ also inhibited basal ERK1/2 phosphorylation, indicating that tonic production of cGMP contributes to basal activity of ERK1/2 in EC. Our data are in line with the observations that the NO-stimulated EC proliferation can be blocked by the mitogen-activated protein kinase kinase inhibitor 2'-amino-3'-methoxyflavone (PD98059) (Parenti et al., 1998).
Although the role of ERK1/2 in proliferation is well established, the biological significance of p38 in EC has only recently started to be investigated (Parenti et al., 1998; Zachary, 2003; McMullen et al., 2005). Activation of the p38 MAPK by angiogenic growth factors has been shown to occur in vitro and proposed to play a role in their ability to stimulate migration (McMullen et al., 2004). In addition, overexpression of mitogen-activated protein kinase kinase 6, an upstream activator of p38, promotes EC migration (McMullen et al., 2005). To find out whether activation of p38 occurs in cells overexpressing sGC, we used a phosphospecific antibody for this kinase to determine the phosphorylation status of p38; indeed, higher ratios phospho-p38/p38 ratios were observed in cells infected with sGC adenoviruses. The finding that the sGC inhibitor ODQ blocks the increase in p38 phosphorylation offers additional support to the hypothesis that increased cGMP levels promote p38 activation. To provide a functional link between p38 activation and sGC-driven migration, we studied the effect of SB203580 on the migratory behavior of cells exposed to BAY 41-2272 or cells infected with sGC-expressing adenoviruses. In both cases, p38 inhibition attenuated the migratory response, suggesting that activation of p38 is at least in part responsible for this response.
In summary, we have shown that sGC contributes to several aspects of the angiogenic process by promoting EC proliferation, migration, and organization in network-like structures. Moreover, we have demonstrated that sGC inhibition leads to abnormal/reduced angiogenic responses in two different systems in vivo. These actions of sGC in EC correlate with activation of at least two members of the MAPK signaling cascade known to regulate angiogenic responses, ERK1/2 and p38. We conclude that stimulation of sGC and the concomitant increase in cGMP exert a permissive role on blood vessel formation.
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Acknowledgements |
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1 promoter constructs. We also thank Y. Capetanaki for support while setting up the zebrafish facility in Foundation of Biomedical Research of the Academy of Athens. |
Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: EC, endothelial cells; NO, nitric oxide; NOS, nitric-oxide synthase; ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; BAY 41-2272, 5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine; CAM, chicken chorioallantoic membrane; ERK, extracellular signal-regulated kinase; GFP, green fluorescent protein; hpf, hours post-fertilization; HUVEC, human umbilical vein endothelial cells; MAPK, mitogen-activated protein kinase; VEGF, vascular endothelial growth factor; SNP, sodium nitroprusside; sGC, soluble guanylyl cyclase; L-NAME, NG-nitro-L-arginine methyl ester; SB203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole; DMSO, dimethyl sulfoxide; Ad, adenovirus; GFP, green fluorescent protein; m.o.i., multiplicity of infection; UTR, untranslated region; PD98059, 2'-amino-3'-methoxyflavone.
The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. 
Address correspondence to: Dr. Andreas Papapetropoulos, Laboratory of Molecular Pharmacology, Department of Pharmacy, University of Patras, Patras 26504, Greece. E-mail: apapapet{at}upatras.gr
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5'-UTR. 5'-UTR, mouse 
