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Am J Physiol Lung Cell Mol Physiol 291: L369-L377, 2006. First published May 5, 2006; doi:10.1152/ajplung.00265.2004
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VEGF-induced relaxation of pulmonary arteries is mediated by endothelial cytochrome P-450 hydroxylase

Elizabeth R. Jacobs,1 Daling Zhu,1 Stephanie Gruenloh,1 Bernardo Lopez,2 and Meetha Medhora1

1Cardiovascular Center, Pulmonary and Critical Care Division, Department of Medicine and 2Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin

Submitted 13 July 2004 ; accepted in final form 29 March 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The cytochrome P-450 metabolite 20-HETE induces calcium-, endothelial-, and nitric oxide (NO)-dependent relaxation of bovine pulmonary arteries (PA). VEGF is an NO-dependent dilator of systemic arteries and plays a key role in maintaining the integrity of the pulmonary vasculature. We tested the effect of VEGF on PA diameter and tone and the contribution of cytochrome P-450 family 4 (CYP4) to vasoactive effects of VEGF. Bovine PA rings (1 mm in diameter) relaxed with VEGF (0.1–10 nM) in an endothelial- and eNOS-dependent manner. This response was blunted by pretreatment with the CYP4 inhibitor dibromododecynyl methyl sulfonamide (DDMS) as well as a mechanistically different CYP4 inhibitor N-hydroxy-N'-(4-butyl-2-methylphenyl)formamidine. PAs also increased in diameter by 6–12% in the presence of VEGF (10 nM), and this increase was attenuated by DDMS. In contrast to that shown in PAs, 20-HETE constricted bovine renal arteries and did not increase intracellular Ca2+ in renal artery endothelial cells as observed in bovine pulmonary artery endothelial cells (BPAECs). VEGF-evoked increases in intracellular Ca2+ concentration ([Ca2+]i) in BPAECs were blunted by treatment with DDMS. Both VEGF (10 nM) and 20-HETE (1–5 µM) stimulated NO release from cultured BPAECs, and once again VEGF-induced increases were attenuated by pretreating the cells with DDMS. We conclude that CYP4/20-HETE contributes to VEGF-stimulated NO release and vasodilation in bovine PAs. Given the unique expression of 20-HETE-forming CYP4 in BPAECs vs. systemic arterial endothelial cells, CYP4 may be an important mediator of endothelial-dependent vasoreactivity in PAs.

20-HETE; nitric oxide signaling; vasoreactivity; lung

VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) is vital for growth and development of the vasculature (4, 7, 31) and is best known for its role in angiogenesis (8, 9, 10), increases in vascular permeability (3, 28, 37), and systemic vasodilation responses (20, 22, 39), which may be mediated by increases in intracellular Ca2+ and nitric oxide (NO) (20, 21, 27, 32, 37, 38). In the lungs, VEGF has been reported to cause a decrease in pulmonary vascular resistance and increase flow in late-gestation ovine fetal lungs mediated by the release of NO (14), whereas inhibition of VEGF results in elevations of pulmonary artery (PA) pressures and resistance in the same model (13). Direct observations of the vasoactive effects of VEGF on PAs, however, have not been reported.

20-HETE, a lipid metabolite of the essential fatty acid arachidonic acid (AA) dilates PAs. We have recently demonstrated that vasodilation to 20-HETE, like that mediated by VEGF, occurs via release of NO (40), which is generated by eNOS (11, 30). Furthermore, 20-HETE elevates intracellular Ca2+, probably by increasing influx from the extracellular environment in pulmonary artery endothelial cells (PAEC). Our group (44) has previously detected cytochrome P-450 family 4 (CYP4) enzymes, which catalyze conversion of AA to 20-HETE, in bovine and rat pulmonary vascular endothelium by in situ hybridization, immunohistochemistry, and Western blotting. Therefore, PAECs have the capability to synthesize 20-HETE. Recent experiments suggest that 20-HETE acts downstream of VEGF during angiogenesis in rat skeletal muscle induced by electrical stimulation (2). These observations led us to hypothesize that VEGF-induced dilation of lung vasculature may be mediated in part by upregulation of CYP4 activity and subsequent release of NO.

In this study, we examined the effects of two mechanistically distinct CYP4 inhibitors on VEGF-mediated dilation of bovine PAs. Both compounds, dibromododecynyl methyl sulfonamide (DDMS) (1, 36) and N-hydroxy-N'-(4-butyl-2-methylphenyl)formamidine (16-HET) (24), were developed to block selective formation of 20-HETE from AA. Our results suggest that activation of endothelial CYP4 enzymes mediate VEGF-induced dilation in isolated PAs, as well as NO release from isolated bovine PAECs (BPAECs). This vasodilatory action is in contrast to the systemic circulation (e.g., in renal microvessels), in which CYP4 enzymes have not been detected in the vascular endothelium. Likewise, although VEGF dilates renal arteries, 20-HETE (17) potently vasoconstricts the same vessels, consistent with different signaling pathways within these two vascular beds.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Ring tension studies. We followed previous studies (42, 43). Cow lungs and kidneys were obtained from a local abattoir and transported to the laboratory on ice. PAs and renal arteries were microdissected from these tissues, and rings ~0.3–1 mm in diameter were prepared in ice-cold PSS (in mM: 130 NaCl, 2.5 CaCl2, 15 NaHCO3, 1.2 MgSO4, 1.2 NaH2PO4, 4.7 KCl, 5.5 glucose, 10 HEPES, and 0.026 EDTA; pH 7.4). Some rings were denuded by gently scraping the inner endothelial lining with blunt forceps, followed by rinsing the rings with PSS.

Rings were mounted on tungsten wires, one connected to a fixed holder and other to a force displacement transducer (model FT03E, Grass Instruments) to continuously measure isometric tension. The apparatus was immersed in pH-adjusted, oxygenated PSS solution (95% O2-5% CO2 unless otherwise mentioned) at 37°C. Tension data were relayed from transducers to a signal amplifier, and acquired and analyzed using CODAS software (DataQ Instruments). Rings were preloaded with 0.3–0.5 g of passive tension and then equilibrated for an additional 30 min before the studies began. Tensions were continuously recorded in the presence of experimental reagents such as VEGF or vehicle. Vessels were pretreated with vehicle or the CYP4 {omega}-hydroxylase inhibitors DDMS (10 µM) or 16-HET (1 µM) (24, 29) and then challenged with VEGF (10 nM) in some experiments. Separate sets of rings were pretreated with vehicle and inhibitors to control for the effects of time and carrier on PA tension. Details of these and other treatments are described for each experiment with the results presented later. Viability of all rings was confirmed by measuring the contractile response to the addition of 60 mM KCl in the bath. Data from rings that did not show at least twofold increase in tension to KCl were eliminated from the analysis. All experimental results have been averaged after pooling data were obtained from multiple rings dissected from three or more animals.

Isolated pressurized vessels. Small bovine PAs (100–250 µm internal diameter and 10–12 mm in length) were placed in a perfusion chamber, cannulated with glass micropipettes, and secured in place with 8-0 polyethylene suture (Ethicon, Somerville, NJ); side branches were tied off with 10-0 polyethylene suture using a stereomicroscope (Carl Zeiss). The artery was bathed in PSS equilibrated with a 95% O2-5% CO2 gas mixture at 37°C. The inflow cannula was connected in series with a volume reservoir and a pressure transducer (Gould Instruments Division, Cleveland, OH) so that intraluminal pressure could be controlled and monitored. After samples were mounted, the vessel segments were stretched to match in vivo lengths, first determined by an eyepiece scale and then approximated between the seeking pipettes. The outflow cannula was clamped off, and intramural pressure was adjusted to 15 mmHg, where it remained for the duration of the experiment. Internal diameters of the vessels were measured with a video system composed of a CCTV camera (KP-130AU; Hitachi), a television monitor (CVM-1271; Sony), and a videomicrometer system (model 305; Colorado Video, Boulder, CO). After an equilibration period of 30–45 min, PAs were treated with VEGF (10 nM), and the changes in diameter were determined. Vessels were also incubated for 30 min with an inhibitor for CYP4 {omega}-hydroxylase, DDMS (10 µM), which was added to the bath; vessels were then stimulated with VEGF to record changes in diameters. Finally, vessels were washed and constricted with KCl (60 mM) at the end of the protocol to confirm viability. In selected experiments, indicated in the text, the endothelium was removed by passing a bubble through the lumen of the vessel. The percent changes in diameter caused by stimulation with VEGF without and with pretreatment with DDMS were compared.

The effect of 20-HETE was also studied in vessels dissected from bovine kidneys. These vessels were preconstricted submaximally with phenylephrine (10–7 M), rinsed until they returned to basal diameter, and then treated with varying concentrations of 20-HETE. Changes in microvascular diameter were measured in vessels from three or more animals. The pooled results were averaged for each data point.

Endothelial cell cultures. Primary cultures of PAs and renal artery endothelial cells were prepared as previously reported (44). Dissected PA or renal arteries 0.5 to 2.0 mm in diameter were slit open along their lengths and washed with PBS to remove blood. The vessels were placed with the lumen side down onto a 100-mm tissue culture dish for 5 min. After they adhered, vessels were covered with medium (RPMI 1640 containing 20% FBS) and allowed to grow for 3 days in a tissue culture incubator. Tissue pieces were then lifted out of the medium, and adherent endothelial cells were allowed to grow. After several days, trypsinized cells were diluted and plated in a 96-well dish with one or two cells/well to select for a pure population of cells based on characteristic cobblestone appearance. Selected cell populations were pooled and probed with the endothelial cell marker platelet endothelial cell adhesion molecule 1 (PECAM-1) and {alpha}-smooth muscle actin, for verification of population purity. Cells staining positive for PECAM-1 and negative for {alpha}-smooth muscle actin were cultured for experimental use.

Immunohistochemical staining of endothelial cells. Cells were grown to ~80% confluency and lightly fixed for 5–10 min with 4% paraformaldehyde in PBS (Sigma). Cells were washed three times; after a blocking period of 4 h with 2% BSA in PBS and 0.1% Triton X-100, cells were incubated with a primary antibody against PECAM-1 (Alexa-488, at a dilution of 1:500 vol/vol) or with anti-smooth muscle {alpha}-actin (1:150) in 2% BSA overnight at 4°C. After samples were washed, the slides were exposed to goat anti-rabbit IgG conjugated to FITC (A-11008; Molecular Probes, Eugene, OR) for PECAM-1 or goat anti-mouse tetramethyl rhodamine isothiocyanate (TRITC) for {alpha}-smooth muscle actin for 1 h at room temperature. The slides were rinsed and mounted, and images were obtained with a Nikon E600 microscope equipped with epifluorescence and digital camera.

Ca2+ measurements. These experiments examined the effect of 20-HETE on intracellular calcium concentration ([Ca2+]i) in cultured bovine renal artery endothelial cells and the effect of inhibition of CYP4 on the increase in [Ca2+]i induced by stimulation of BPAECs with VEGF. [Ca2+]i was measured after cells were loaded with 5 µM fura 2-AM (Molecular Probes) in culture medium for 45 min at 37°C in a cell culture incubator. After samples were loaded, cells were transferred to a 1-ml perfusion chamber mounted on an inverted microscope and washed with PSS. The cells were maintained in a dark area for 15 min at 37°C. [Ca2+]i was measured by an InCyto Im2 imaging system (Intracellular Imaging, Cincinnati, OH) mounted on an inverted microscope (IMT-2; Olympus Optical, Tokyo, Japan). The cells were visualized with a x40 UV long-working distance fluorescence objective. [Ca2+]i results were calculated based on the fluorescence intensity ratios obtained by using excitation and emission wavelengths of 340/380 and 510 nm and a standard curve generated by measurements obtained from solutions with known Ca2+ concentrations. After a 2-min control period, 20-HETE (1 µM final concentration) or vehicle (ethanol) was added to the chamber, and the recording was continued for another 15 min.

Measurement of nitrogen oxide release. For measurement of nitrite (NOx) release (the breakdown product of NO in aqueous media, representing NO2–), primarily isolated BPAECs (passages 2–3) were grown to 90% confluence in sixwell clusters, washed three times with HBSS (Sigma), and incubated in the same buffer supplemented with L-arginine (40 µM). Vehicle, VEGF (10 nM), or 20-HETE (1–10 µM) was added to different wells, and the cells were incubated for 30 min at 37°C. The supernatants were collected for analysis by NO-specific chemiluminescence (Sievers, Boulder, CO) as described (30). Samples (100 µl) containing NOx were refluxed in a solution of KI (50 mg in 1.5 ml distilled water) mixed with 5 ml of glacial acetic acid. The released nitrite was then quantitatively converted to NO, which reacted with ozone. The detected chemiluminescent product from this reaction was compared with a linear range of known amounts of sodium nitrite analyzed during the same experiment. Cells in the assay wells were scraped and lysed with 1 N NaOH and quantitatively transferred for total protein determination (BioRad).

Measurement of intracellular NO by fluorescent microscopy. In these experiments, the generation of NO was measured by loading the cultured BPAECs (~50% confluent) with a fluorescent NO indicator, 4-amino-5 methylamino-2',7'-difluorescein diacetate (DAF, 1 µM; Molecular Probes) and a NO substrate, L-arginine (1 mM), in PSS (in mM: 130 NaCl, 2.5 CaCl2, 15 NaHCO3, 1.2 MgSO4, 1.2 NaH2PO4, 4.7 KCl, 5.5 glucose, 10 HEPES, and 0.026 EDTA; pH 7.4) for 30 min at 37°C. After they were loaded, cells were washed with PSS and transferred to the recording chamber. L-Arginine (1 mM) was added before the recording was started. Fluorescence intensity was recorded with an imaging system employing single excitation and emission wavelengths of 475 and 510 nm, respectively.

Baseline fluorescence was measured for ~5 min and then vehicle was added to the bath. DAF fluorescence was then measured for an additional 15 min. The experiment was repeated in separate sets of cells with VEGF (5 nM) added to the bath instead of vehicle, with DAF fluorescence measured for 15 min. Other cells were also pretreated with 10 µM DDMS (CYP4 inhibitor) or vehicle (ethanol) for 60 min before basal NO production was measured. Cells were then stimulated with VEGF or vehicle, and fluorescence was measured for another 15 min.

Fluorescent intensities recorded in cells representing more than three independent experiments were normalized for a unit area with the use of Incyt Im2 software. Background fluorescent intensity was obtained by measuring fluorescence from a region of the slide devoid of cells; this value was subtracted from readings measured in the cells. The rates of NO production after differing treatments were calculated by comparing the rate of increase in relative fluorescence over a fixed time (100 s). Changes in fluorescence intensity over time of different groups of cells were compared by t-test (for 2 groups) or ANOVA (for more than 2 groups).

Statistical analysis. Pooled data obtained in each experiment were used to calculate the means ± SE for control (vehicle treated) or experimental (treated with pharmacological agents) samples. The data were tested for significance by a Student's t-test (for comparison between 2 unpaired samples) or Mann-Whitney’s rank sum test or ANOVA using SigmaStat Software (Jandel) for repeated measures. Experiments with P < 0.05 and power of performed test above 0.8 were considered significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Bovine PAs relax when treated with VEGF. Dissected bovine PA rings that were mounted on a force displacement transducer were treated with vehicle or VEGF (10 nM). Relaxation to VEGF occurred within 2 min of addition of the growth factor to the bath; relaxation was sustained until washout 10–15 min later. There was a decrease in tension (mean of 73 ± 4% of baseline value) in all rings treated with VEGF compared with vehicle-treated controls (Fig. 1A). This response was observed from vessels dissected from at least five different animals and was endothelial dependent because the difference (P < 0.001) in reactivity induced by VEGF was abolished in rings denuded of endothelium (mean decrease in tension to 99 ± 2% of baseline). Rings that had been preconstricted with 10–7 M phenylephrine to approximately twice their baseline tension also relaxed when treated with VEGF (10 nM, P = 0.003; Fig. 1B). The relaxing effect of VEGF was attenuated by pretreatment with the NOS inhibitor NG-monomethyl-L-arginine (L-NMMA; 3 mM, 102 ± 2% of baseline tension) compared with rings pretreated with vehicle and then VEGF (84 ± 5% Fig. 1C). These experiments were carried out in PSS equilibrated with 95% O2-5% CO2 to maximize viability of the rings. However, the relaxation to VEGF (82 ± 2%; n = 12) was also observed in rings that were bathed in PSS equilibrated with 21% O2-5% CO2-74% N2 (data not shown). Depleting the buffer of Ca2+ by replacing it with Ca2+-free PSS containing the Ca2+-chelator EGTA (5 mM) reduced the tension in the rings to 75 ± 5% of baseline, which is significantly different from PSS (P < 0.001; n = 7; data not shown), supporting the generation of some spontaneous tone in these sized PAs in vitro.


Figure 1
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  		Fig. 1. VEGF-induced decrease in tension of bovine pulmonary artery (PA) rings is mediated by intact endothelium and functional eNOS. A: significant decrease in tension of PA rings (n = 10) after application of 10–8 M VEGF (*P < 0.001). Note: this relaxation is absent in rings that have been denuded of the endothelial cell lining (P = 0.932). B: 10–8 M VEGF decreases the tension of PA rings (n = 6; *P = 0.003) after preconstriction with phenylephrine (PE). C: decrease in tension of PA rings after application of 10–8 M VEGF (*P < 0.001 relative to vehicle control) is eliminated in rings (n = 10) that have been pretreated with the eNOS inhibitor (L-NMMA).

 
VEGF-induced dilation is blocked by the CYP4 hydroxylase inhibitors DDMS and 16-HET. The effect of VEGF was attenuated in rings that were pretreated (for ~30 min) with CYP4 inhibitors DDMS (10 µM, Fig. 2A) and 16-HET (1 µM, Fig. 2B) compared with vehicle-treated rings. These compounds selectively inhibit CYP4 hydroxylase activity, thus blocking endogenous formation of 20-HETE. Stimulation with VEGF after pretreatment with DDMS resulted in tension that was 100 ± 2% of baseline as opposed to 78 ± 6% (P < 0.01) for vessels pretreated with the vehicle that was used to dissolve DDMS (ethanol, Fig. 2A). We then repeated these experiments with a second, mechanistically dissimilar inhibitor of CYP4, 16-HET (24, 29). This compound selectively inhibits the formation of 20-HETE at concentrations <100 nM and has no effect on activity of epoxygenase, cyclooxygenase, or lipoxygenase enzymes at concentrations up to 1 µM. At this concentration, it also has minimal effects on the activity of other cytochrome P-450 isoforms (CYP2C9, 2D6, 3A4). 16-HET is the most specific inhibitor of the synthesis of 20-HETE presently available. The compound significantly reduced VEGF-induced relaxation (change from baseline was 96 ± 3.9%, compared with 63 ± 5.9%, P < 0.001, for vehicle-treated rings relaxed with VEGF; Fig. 2B). Relaxation after application of VEGF was not observed in denuded rings (Fig. 2B).


Figure 2
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  		Fig. 2. VEGF-induced decrease in tension of preconstricted bovine PA rings is dependent on cytochrome P-450 family 4 (CYP4) activity. A: decrease in basal tension of small PA rings after application of 10–8 M VEGF and pretreatment with vehicle is not observed in rings (n = 8, *P < 0.01 relative to VEGF alone) pretreated with the CYP4 inhibitor dibromododecynyl methyl sulfonamide (DDMS). B: decreases in basal tension of small PA rings after application of 10–8 M VEGF and pretreatment with vehicle are blunted in rings pretreated with a second CYP4 inhibitor N-hydroxy-N'-(4-butyl-2-methylphenyl)formamidine (16-HET). Rings that were denuded and pretreated with either vehicle or 16-HET also did not decrease their tension in response to VEGF (n = 7 each group, *P < 0.001 relative to basal tension).

 
The effect of VEGF on relaxation of small PAs was examined again by analogous experiments using isolated pressurized arterioles. We observed an increase in diameter in the arterioles ranging from 6 to 20% in seven independent experiments within 30 s of application of 10 nM VEGF (Fig. 3A). VEGF induced an increase in diameter of small pressurized PA rings in a manner that was eliminated by removal of the endothelium from these vessels (Fig. 3A), supporting the endothelial dependence of this effect in both small- and medium-sized PAs. Pretreatment of the arterioles with 10 µM DDMS (30 min) nearly obviated the vessel response to VEGF (Fig. 3B), suggesting that 20-HETE contributes to the relaxation induced by VEGF.


Figure 3
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  		Fig. 3. A: summary of means ± SE of increase in diameter of isolated pressurized PAs treated with VEGF. This response was not observed after removal of the endothelium (n = 4, *P < 0.05 relative to baseline diameter). B: like their larger counterparts, the increase in diameter of small pressurized PAs to VEGF depends on CYP4, as it is eliminated by DDMS (n = 4, *P = 0.03 relative to diameter changes in vessels treated with VEGF alone).

 
20-HETE constricts small bovine renal arteries. A number of investigators have demonstrated that 20-HETE constricts renal arteries (17, 23), but the response in bovine renal arteries has not been reported. Because there are species differences in the response to eicosanoids, we tested bovine renal arterial rings for response to 20-HETE. Rings constricted after exposure to 1 µM 20-HETE (114 ± 4% of baseline tension; P = 0.018), and this response was also observed in rings that were denuded of endothelium (111 ± 4% of baseline tension; Fig. 4A). We also compared 20-HETE-associated constriction in isolated pressurized renal arterioles (100–250 µm diameter vessels). Renal arteries of diameter <250 µm were dissected, pressurized, and treated with increasing concentrations of 20-HETE (Fig. 4B). The vessels constricted in a concentration-dependent manner (16% with 10–8 M 20-HETE, P = 0.002, and 21% with 10–6 M 20-HETE, P < 0.001) graphically depicted in Fig. 4B (y-axis represents decrease in diameter).


Figure 4
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  		Fig. 4. Effect of 20-HETE on renal arteries. A: statistically significant constriction of bovine renal artery rings after treatment with 10–6 M 20-HETE. Note: constriction is also observed in rings that were denuded of endothelium. This response was not observed in equilibrated deendothelized rings that were stimulated with vehicle instead of 20-HETE (results not shown). B: concentration-dependent constriction of bovine renal arteries after treatment with 20-HETE (arteries are below 250 µm in diameter; n = 5, *P = 0.002 for 10–8 M 20-HETE; n = 5, **P < 0.001 for 10–6 M 20-HETE). Note: y-axis depicts decreasing diameter.

 
20-HETE does not increase [Ca2+]i in primarily isolated bovine renal endothelial cells. To resolve the opposite effects of 20-HETE in small pulmonary vs. renal arteries, we hypothesized presence of alternate signaling pathways within the endothelial cells of the two vascular beds. Because our group (40) previously demonstrated that 20-HETE induced increases in [Ca2+]i in PA-derived vascular endothelial cells, we performed this experiment with renal endothelial cells. Our primarily isolated cells demonstrated presence of the endothelial marker PECAM-1 after immunohistochemical staining with a specific PECAM-1 antibody followed by a FITC-conjugated secondary antibody. As shown in Fig. 5A, >90% of the cells were positively stained for PECAM-1 (green) but did not show detectable fluorescence with {alpha}-smooth muscle actin, treated with secondary anti-TRITC-conjugated antibody (result not shown).


Figure 5
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  		Fig. 5. 20-HETE does not increase intracellular Ca2+ concentration ([Ca2+]i) in cultured renal artery endothelial cells. A: endothelial cells were cultured as described in METHODS and stained with anti-platelet endothelial cell adhesion molecule 1 antibody (right). B: cells were loaded with the Ca2+-sensitive indicator fura 2-AM and treated with vehicle or 20-HETE. No significant difference in measurement of [Ca2+]i was observed in cells treated with vehicle or 20-HETE (n = 30).

 
Once we characterized these cells as originating from the endothelium, we imaged them in the presence of Ca2+-sensitive probes (see METHODS) to study the effect of 20-HETE on [Ca2+]i. Unlike PAECs (40), 20-HETE (10–6 M) did not increase [Ca2+]i in cultured renal artery endothelial cells (n = 30, Fig. 5B).

VEGF-induced increase in [Ca2+]i in BPAECs is attenuated by the CYP4 hydroxylase inhibitors DDMS. Stimulation of BPAECs with VEGF (5 nM) resulted in an expected rise in [Ca2+]i to values above baseline within 1 min. Pretreatment of the cells (n > 63 using at least 3 independent batches of cells) with the CYP4 inhibitor DDMS (10 µM) significantly attenuated this increase (Fig. 6; P < 0.001). This increase reached a plateau and did not respond to VEGF as observed in the vehicle-treated cells, suggesting that long-term inhibition of CYP4 enzymes may slowly increment [Ca2+]i but that the action of VEGF was significantly blocked by DDMS (Fig. 6).


Figure 6
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  		Fig. 6. VEGF-induced increase of [Ca2+]i in BPAECs is mediated by CYP4 activity. Graphical representation is shown of increase in [Ca2+]i in cells pretreated with vehicle (ethanol) or CYP4-inhibitor DDMS (10 µM for 30 min), both followed by stimulation with VEGF. Note attenuation of increase in [Ca2+]i by exposure of BPAECs to DDMS; *P < 0.001 relative to cells pretreated with vehicle then VEGF. The numbers for each experiment are ≥63 cells from at least 3 separate isolations for both groups.

 
VEGF stimulates release of NO by BPAECs. We measured NO release as detected by nitrate/nitrite chemiluminescence in BPAECs after treatment with VEGF and 20-HETE. Both agonists increased NO production by BPAECs (Fig. 7), although 10 nM VEGF (164% of control, n = 5; P = 0.044) was more potent than 1–10 µM 20-HETE (129% of control, n = 7; P = 0.017). We estimated that unstimulated BPAECs generated an average of 90 pmol of NO2–/mg protein, which is consistent with that previously reported from bovine artery endothelial cells (11).


Figure 7
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  		Fig. 7. VEGF-induced release of nitric oxide (NO) in BPAECs is mediated by CYP4 activity. Graphical representation is shown of relative amounts of NO2– measured in BPAECs after treatment with 20-HETE (n = 7; *P = 0.017 relative to vehicle) or VEGF (n = 5; **P = 0.044 relative to 20-HETE-treated cells). Both compounds significantly increased release of NO from the cells, as described in RESULTS.

 
VEGF-induced increase in NO is blocked by the CYP4 hydroxylase inhibitor DDMS. In addition to chemiluminescence measurements, stimulation of production of NO by VEGF was also demonstrated by loading cells with the fluorescent NO indicator DAF (see METHODS). BPAECs produced NO at a basal rate, which was increased after addition of VEGF (Fig. 8). Thus the rate of increase in fluorescence vs. time (100 s) before stimulation with VEGF was 0.15 ± 0.03 relative fluorescent units/s (n = 51 cells) and reached an average of 0.29 ± 0.03 relative fluorescent units/s (n = 51, P < 0.05) in the same cells after application of 10 nM VEGF, as demonstrated in Fig. 8B.


Figure 8
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  		Fig. 8. CYP4 mediates VEGF-induced increase of NO in BPAECs. Aa: averaged fluorescent trace of BPAECs that were loaded with the NO indicator 4-amino-5 methylamino-2',7'-difluorescein diacetate (DAF) and pretreated with vehicle and stimulated with VEGF. Note increase in slope of the tracing after application of VEGF. Figure represents activity measured in 1 representative experiment (n = 8 cells). Ab: typical trace representing normalized fluorescence from DAF-loaded BPAECs (n = 13) that were pretreated with DDMS (compared with vehicle) recorded over time after stimulation with VEGF. NO donor sodium nitroprusside (SNP; 0.3 mM) was added to monitor functional recording and successful loading of the cells with DAF. B: relative slopes averaged from cells treated as labeled (see RESULTS for details). Note the decreased slope (expressed as change in relative normalized fluorescent units/time in seconds) in cells pretreated with DDMS and then stimulated with VEGF, in which baseline and VEGF-stimulated NO release were significantly blunted (*P < 0.05).

 
Addition of VEGF to BPAECs pretreated with DDMS for 30 min and also loaded with DAF did not increase NO production (Fig. 8Ab). In fact, the baseline increase in fluorescence was much diminished in these cells relative to that of cells pretreated with vehicle (Fig. 8, Ab and B). This is not unexpected if DDMS inhibits baseline as well as VEGF-induced generation of NO in BPAECs. The minimal loss in signal over time in cells treated with vehicle and VEGF is masked by basal and stimulated NO release in the absence of DDMS. In cells treated with DDMS, this signal is overcome by a very slowly decreasing signal secondary to photobleaching.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Our investigations provide the first direct evidence that VEGF is a vasodilator of small PAs. We observed dilation of isolated pressurized bovine PAs, as well as decrease in tension of preconstricted PA rings after stimulation with VEGF. VEGF has been observed to dilate coronary (20), arcuate (26), and internal mammary arteries (22) and to reduce the resistance of fetal PAs (14). Next, we tested whether the dilatory action of VEGF could be mediated by products catalyzed by CYP4 enzymes using inhibitors DDMS and 16-HET. These compounds attenuated the dilation by VEGF in isolated vessels and in PA rings, suggesting that CYP4 products mediate some of the vasoactive effects of VEGF in the pulmonary circulation.

In the past decade, cytochrome P-450 metabolites of AA have been recognized to play an important role in mediating cardiovascular function [reviewed by Roman (29)]. Although most investigators have reported 20-HETE to cause dilation of PAs (42), this lipid has also been described to constrict guinea pig PAs (41). 20-HETE has an established record of regulating myogenic tone in the brain (12, 15) and kidney (17, 23, 45). In the kidney, it mediates actions of vasoactive peptides such as ANG II, vasopressin, norepinephrine, and endothelin [reviewed by Roman (29)]. It is believed that these vasoactive peptides stimulate cellular lipases to trigger release of AA from phospholipid stores. Synthesis of 20-HETE from AA is substrate limited, especially in vascular smooth muscle cells of microvessels that are rich in CYP4 enzymes. Inhibitors of 20-HETE synthesis attenuate the constrictor response to ANG II, vasopressin, norepinephrine, and endothelin, whereas ANG II increases 20-HETE levels in preglomerular arterioles isolated from rat kidneys (6). The mechanisms through which 20-HETE relaxes PAs, in contrast to constriction of these systemic arteries, are unknown, although our previous study (40) has demonstrated that relaxation is endothelial and NOS dependent.

Increased NO production by VEGF has been described in endothelial cells of many sources (16, 33) derived from human, bovine, porcine, and rabbit vessels (19, 25, 34). Recombinant human VEGF caused up to a threefold elevation in blood flow and decrease in pulmonary vascular resistance when infused into the left PA of fetal lambs. This increase was completely inhibited by pretreatment with the NOS inhibitor nitro-L arginine or the phosphatidylinositol 3-kinase blocker LY-294002. These data provide indirect evidence that VEGF acts as a dilator of PAs. VEGF (121) also increased collateral blood flow in rat limb calf muscles, and this observation was dependent on production of NO (38). In addition, both 20-HETE (40) and VEGF increase intracellular Ca2+ in responsive cell types.

Our results demonstrate that both 20-HETE and VEGF enhance release of NO from BPAECs. This is consistent with the effect of 20-HETE that we reported in the same cells as detected by a fluorescent NO indicator DAF (40). We were interested to confirm that both agonists released NO under identical experimental conditions. We were unable to measure the effects of inhibitors of 20-HETE synthesis by assaying release of NOx, due to nonspecific increase in the levels of NO2– released by introducing the inhibitor to the reaction, independent of the presence of cells. However, we were able to demonstrate effective inhibition of NOS activity by VEGF after pretreatment of BPAECs with the CYP4 inhibitor DDMS, by monitoring NO production using the fluorescence indicator DAF. In our previous study (40), we demonstrated that the rise in NO after stimulation of BPAECs with 20-HETE was blunted by the eNOS inhibitor N{omega}-nitro-L-arginine methyl ester. The same inhibitor also attenuated the dilator effect of 20-HETE on PAs, clearly supporting the hypothesis that 20-HETE-induced relaxation of PAs is mediated by release of NO. Because VEGF and 20-HETE both induce NO release and VEGF-stimulated NO release from BPAECs is nearly completely blocked by CYP4 inhibition, we posit that CYP4 mediates in part VEGF-evoked stimulation of eNOS in these cells.

NO production is regulated by a number of extensively characterized molecular mechanisms, some of which are dependent on Ca2+. 20-HETE transiently raises [Ca2+]i within 5 min of application to PAECs (40). Therefore, we examined the response of endothelial cells derived from arteries that constrict rather than relax to 20-HETE, specifically bovine renal arteries. Our data demonstrate that bovine renal arteries, like rat renal and cat cerebral arteries, constrict in a concentration-dependent manner to 20-HETE. Both VEGF and 20-HETE can induce increases in intracellular Ca2+ in responsive cells, although the mechanisms mediating this increase by 20-HETE is unknown. However, renal artery endothelial cells stimulated with 20-HETE under identical conditions to those that evoked sustained increases in Ca2+ in BPAECs exhibit no changes in [Ca2+]i (Fig. 4B). Thus the increase in [Ca2+]i in PAs may reflect differences in receptors/signaling pathways between these two types of endothelial cells so that there is activation of eNOS in BPAECs but not in bovine renal artery endothelial cells. Also, consistent with this hypothesis is the observation that removal of extracellular Ca2+ blunts 20-HETE-induced NO release in BPAECs (40). Furthermore, the increase in [Ca2+]i induced by VEGF is attenuated in BPAECs treated with the CYP4 inhibitor DDMS (Fig. 5), supplementing evidence that VEGF-induced vasodilation may be mediated by CYP4 activity.

In summary, our results are the first to suggest and support a role for CYP4 activation by VEGF in pulmonary arterioles, a signaling pathway that appears to account in part for the relaxation of bovine PAs by VEGF. This polypeptide and 20-HETE (product of CYP4) share similar intermediates [see accompanying article (5)] that cause endothelial-dependent relaxation in bovine PAs, such as increase in release of NO. Together, our results impact on regulatory mechanisms within the pulmonary vasculature. Because VEGF and its receptors play a role in maintaining the structure of small vessels and capillaries in the lung (18, 35), they may also have a significant role in ensuring adequate pulmonary blood flow via stimulation of CYP4 enzymes in the endothelium.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
We are grateful for financial support from National Heart, Lung, and Blood Institute Grants HL-049294 (E. R. Jacobs), HL-068627 (E. R. Jacobs), and HL-069996 (M. Medhora).


   ACKNOWLEDGMENTS
 
We thank Ying Gao, Kristine Hoefert, and Peter Clark for the excellent technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. R. Jacobs, Cardiovascular Center; Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226 (e-mail: ejacobs{at}mcw.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.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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