(Circulation. 1997;95:1030-1037.)
© 1997 American Heart Association, Inc.
Articles |
Vascular Endothelial Growth Factor/Vascular Permeability Factor Augments Nitric Oxide Release From Quiescent Rabbit and Human Vascular Endothelium
the Departments of Biomedical Research and Medicine (Cardiology), St Elizabeth's Medical Center, Tufts University, School of Medicine, Boston, Mass. Current address for Dr van der Zee is Department of Cardiology, Academic Hospital Maastrict, Maastrict, Netherlands.
Correspondence to Jeffrey M. Isner, MD, St Elizabeth's Medical Center, 736 Cambridge St, Boston MA 02135-2997. E-mail vejeff@aol.com.
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Abstract |
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Background Vascular endothelial growth factor (VEGF)/vascular permeability factor (VPF) is an endothelial cell (EC) mitogen. This feature is considered central to the documented role of VEGF/VPF in promoting angiogenesis. More recent evidence suggests that VEGF/VPF may also serve a "maintenance" function, modulating various aspects of EC biology. In the present study, we sought to determine the extent to which VEGF/VPF may stimulate the release of NO from normal ECs.
Methods and Results VEGF/VPF produced a dose-dependent rise in NO concentration ([NO]) from vascular segments of rabbit thoracic aorta, pulmonary artery, and inferior vena cava. In comparison to stimulation with acetylcholine, the onset of increased [NO] after administration of VEGF/VPF was slower, reaching a maximum value after 8 minutes. Preincubation of the aortic segments with L-arginine raised by twofold both baseline [NO] and [NO] stimulated by addition of 2.5 µg/mL VEGF/VPF. Removal of CaCl2 from the Krebs solution, disruption of the endothelium, and administration of NG-monomethyl-L-arginine abrogated the stimulatory effect of 10 µg/mL VEGF/VPF. Similar findings were documented with an NO-specific polarographic electrode to measure NO released from cultured human umbilical vein ECs.
Conclusions VEGF/VPF stimulates production of NO from rabbit and human ECs. This finding (1) constitutes inferential evidence for the presence of functional VEGF/VPF receptors on quiescent endothelium of the adult rabbit as well as human ECs and (2) supports the notion that putative maintenance functions of VEGF/VPF may include regulation of baseline synthesis and/or release of EC NO.
Key Words: endothelium-derived factors • nitric oxide • endothelium
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The factors that govern the production of NO by ECs of the arterial wall have been shown to include physical factors, such as shear stress1 2 and cyclic stretching,3 4 and neurohumoral factors, most notably ACh and bradykinin.5 The extent to which cytokines elaborated by the arterial wall may further modulate EC production of NO has been less well studied.
VEGF/VPF constitutes a possible candidate for this role. VEGF/VPF is a 45-kD heparin-binding dimeric glycoprotein that is mitogenic for ECs.6 7 8 The possibility that the effects of VEGF/VPF on EC biology extend beyond mitogenesis and/or migration to involve a "maintenance role" has been previously suggested by Peters et al9 and others.10 11 12 Consistent with this notion, VEGF/VPF has been previously shown by Brock et al13 to increase cytosolic Ca2+ in human ECs and by Ku et al14 to cause dose-dependent relaxation of isolated canine coronary arteries that could be abolished by prior endothelium disruption and/or L-NMMA pretreatment. Recent studies in our own laboratory have demonstrated that VEGF/VPF promotes recovery of disturbed endothelium-dependent flow in the rabbit ischemic hindlimb,15 causes NO-dependent hypotension in two different animal species,16 and inhibits intimal thickening in two different animal models of arterial injury.17 18
Accordingly, in the present study we sought to determine more directly the extent to which VEGF/VPF may stimulate the release of NO from the normal arterial wall. We first used a spectrophotometric assay based on the reaction of nitrite with Griess reagent to demonstrate a dose-dependent rise in NO concentration in vascular segments freshly harvested from the thoracic aorta, pulmonary artery, and inferior vena cava of New Zealand White rabbits; this response was abrogated by endothelial denudation, L-NMMA pretreatment, and/or removal of CaCl2 from the organ bath solution. These findings were then reproduced with an NO-specific polarographic electrode to measure NO released from cultured HUVECs. The results of these experiments thus suggest a potential role for VEGF/VPF in the regulation of NO release from ECs in the vasculature and, parenthetically, confirm the presence of VEGF/VPF receptors in quiescent adult endothelium.
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Methods |
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Vessel Preparations
Experiments were performed on vessels isolated from male New Zealand White rabbits weighing 3 to 3.5 kg. Only male rabbits were used to avoid inconsistent outcomes attributable to sex alone.19 The experimental protocol described was conducted according to protocols approved by the St Elizabeth's Institutional Animal Care and Use Committee. After premedication with xylazine 2 mg/kg, rabbits were anesthetized with a mixture of ketamine (Fort Dodge Laboratories) 50 mg/kg IM and acepromazine 0.8 mg/kg IM. A small incision was made to expose the carotid artery, after which the uppermost level of the exposed artery was ligated. A 20-gauge catheter introduced into the carotid artery was used to infuse 500 mL of 0.9% saline mixed with 3 mL heparin (Elkins-Sinn) and 0.6 mL ketamine at 100 mm Hg. A second 20-gauge catheter introduced into one femoral artery was connected to a small plastic tube for exsanguination. The rabbits died after the larger part of their blood was replaced by saline. Immediately after cessation of respiration, the chest was opened and the thoracic aorta was excised from the aortic valve to the diaphragmatic hiatus. The IVC was harvested from the right atrium to the diaphragm. The PA was excised from the pulmonary valve to its secondary branching. Care was taken during this procedure not to damage or stretch the vessels. After the vessels were taken out, they were bathed in Krebs solution containing (in mmol/L) NaCl 118, KCl 4.6, NaHCO3 27.2, MgSO4 1.2, CaCl2 2.5, KH2PO4 1.2, and glucose 11.1 (pH 7.4), aerated with a 95% O2/5% CO2 gas mixture, and maintained at a constant temperature of 37°C. Connective and other adhesive tissue was completely removed because it contained blood. Any remaining blood was removed by placing the excised vascular segments in Krebs solution for 5 minutes. The segments were then cut at 5-mm intervals to allow each segment optimal time for exposure to oxygen and the administered drugs. One segment of aorta from each rabbit was used for analysis of vasomotor reactivity after administration of VEGF/VPF (see below). For certain experiments, the endothelium was removed by gentle rubbing of the interior of the vessel rings with a cotton swab prewetted with Krebs solution.
Measurement of NO From Vessel Segments
Vessel segments were allowed to float for 10 minutes in the organ chamber before its contents were replaced with 10 mL of fresh Krebs solution, to remove any remaining hemoglobin. Fifteen minutes later (t=0), reagents to be tested were administered. In our experience, 15 minutes was the minimal time necessary for the vascular segments to equilibrate with the Krebs solution in the organ chamber. Concentrated solutions were used to avoid any significant increase in volume and were administered immediately after replacement of the Krebs solution (ie, t=-15 minutes).
To measure [NO] at different points in time, 0.7-mL aliquots of organ chamber solution were removed and added to a 1-mL cuvette with an optical path of 1 cm, containing 0.07 mL of Griess's solution [1% sulfanilic acid, 0.1% N-(1-naphthyl)ethylenediamine in 5% H3PO4]. N-(1-naphthyl)ethylenediamine reacts with NO as well as with nitrite (NO2-) to yield a product that renders the solution pink; the resulting product yields a spectrophotometric peak at 548 nm.20 We found that a 1:10 ratio of Griess's solution to the sample volume yielded results that were equal to the more commonly used ratio of 1:1; reducing the volume of Griess's solution avoided diluting the [NO] of the sample volume in the cuvette and increasing the sensitivity of the measurement. Immediately after the last sample was removed from the organ chamber, the absorbance at 548 nm was measured with a diode array photospectrometer (8452A, Hewlett Packard). Absorbance was proportional to [NO] according to a standard curve that, in our hands, was linear from 0.01 to 0.25 µmol/L NO2-. The Griess reaction has been typically reported to yield a sensitivity between 1 and 5 µmol/L. By diluting a 1-µmol/L NO2- sample by a factor of 2 until concentrations of 0.015625 µmol/L were obtained, we registered absorbances in the different solutions as displayed in the Table
. The resulting standard curve, based on four independent procedures, was virtually linear, including the range from 1 to 0.01 µmol/L (Fig 1). The same cuvette and volumes were used for experiments involving vascular segments. For a blank, 0.7 mL of Krebs solution in which no vessel segment had been placed was added to 0.07 mL of Griess solution. The deviation obtained in the construction of the standard curve was much smaller than the variation in values obtained from processing the solutions derived from the organ bath experiments. Repetitive measurements per sample to ensure that the variability among measurements did not exceed 10% and the reproducibility of the documented results suggest that the sensitivity of the Griess reaction for this type of experiment is sufficiently accurate at least to concentrations as small as 0.03 µmol/L. It should be noted that the values for NO reported here represent measured NO divided by the intimal surface area of a vessel segment (typically 250 to 700 mm2) (see below); the product of NO reported here (0.02x10-2 to 
0.10x10-2 µmol/L per mm2) multiplied by 250 to 700 (mm2) yields values that consistently fall within the calibration range of the linear relationship shown in Fig 1.
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Calculations
Four measurements per sample were taken; these were used to compute the average absorbance for each time point. [NO] was determined in the manner described above. The increase in [NO] caused by the sampling-induced decrease in volume of the organ chamber in the presence of an NO-producing source was corrected for according to the formula Ct=Cx-2+(Ct-Cx-2)·[(V-
Vs)/V], in which Ct is [NO] at time=t minutes, x=15 minutes, V is the initial volume in the organ chamber (ie, 10 mL), is number of sampling times, and Vs is the sample volume (ie, 0.7 mL). The [NO] 2 minutes before drug administration was taken as the baseline value.
After completion of the above procedure, the surface area of the vascular segments was carefully measured. The [NO] per surface area in µmol/L per mm2 at given points in time was described, assuming that production of NO was equal across the endothelium of the excised vascular segment.
Analysis of Vasomotor Reactivity
Aortic rings 5 mm long were mounted with two L-shaped 30-gauge stainless steel hooks, one of which was immobile and the other of which was connected by a silk suture to a force displacement transducer (model 7D polygraph, Grass Instrument Co) for recording isometric tension development. The assay was performed with rings placed in Krebs buffer as described above. Vessels were passively stretched to 2.0 g isometric force. After 45 minutes of equilibration, the aortic rings were exposed to 70 mmol/L KCl solution to assess the effect of maximal depolarization. When the contractile response reached a plateau phase, the solution in the organ chamber was replaced by fresh Krebs buffer and again was allowed to equilibrate for 45 minutes in the presence of 5 µmol/L indomethacin for complete inhibition of cyclooxygenase and consequent production of vasoactive prostanoids. Norepinephrine was used to achieve submaximal tone, defined as 30% to 50% of maximal tone induced by KCl, after which VEGF/VPF was added in cumulative fashion to the organ bath solution. The effect of VEGF/VPF was measured as the percent change in norepinephrine-induced vascular tone.
Measurement of NO From HUVECs
HUVECs were isolated from human umbilical cords by collagenase dissociation and grown onto 1% gelatin-coated plates in medium 199 (Life Technologies) with 20% heat-inactivated FCS (Life Technologies), 100 mg/mL EC growth supplement, and 50 U/mL heparin (HUVEC medium).21 Cells were passaged at confluence after dissociation with 0.05% trypsin (Life Technologies). Cultured HUVECs were used between passages 5 and 6.
NO release from a cultured HUVEC monolayer was measured with an NO-specific polarographic electrode connected to an NO meter (Iso-NO, World Precision Instruments).22 Calibration of the NO electrode was performed daily before the experimental protocol was begun according to the equation 2KNO2+2KI+2H2SO4
2NO+I2+2H2O+2K2SO4. The standard calibration curve was obtained by adding graded concentrations of KNO2 (0, 5, 10, 25, 50, 100, 250, and 500 nmol/L) into the calibration solution containing KI and H2SO4. The specificity of the iso-NO electrode for NO was previously determined by measurement of NO from authentic NO gas.23 The culture medium was removed, and the HUVECs were washed twice with Dulbecco's PBS. The cells were then bathed in 5 mL filtered Krebs solution in six-well plates. Cell plates were kept on a slide warmer to maintain a temperature between 35°C and 37°C. For NO measurements, the sensor probe was inserted vertically into the wells; the tip of the electrode remained 2 mm under the surface of the solution. The wells containing the confluent HUVECs were randomly divided into five experimental groups: (1) VEGF/VPF 1 ng/mL, (2) VEGF/VPF 10 ng/mL, (3) VEGF/VPF 100 ng/mL, (4) the NO synthase inhibitor L-NAME 100 µmol/L+VEGF/VPF 100 ng/mL, and (5) sodium nitroprusside 100 µmol/L. NO release was expressed as picomoles per 1x106 ECs. Measurements of NO released from HUVECs represent mean values of five to eight measurements in each group (number of cell culture wells).
Reagents
All reagents except for VEGF/VPF were purchased from Sigma Chemical Co.
Heterodimeric recombinant human VEGF/VPF purified from Escherichia coli was the generous gift of Napoleone Ferrara, Bruce Keyt, and Stuart Bunting, Genentech, Inc, South San Francisco, Calif. All VEGF/VPF concentrations are reported in grams per liter. Previous analyses24 disclosed that E. coli–derived VEGF/VPF appeared as a single protein band on SDS gels with a molecular weight of 39.8 kDa; the concentrations of VEGF/VPF used in the present experiments (2.5 and 10 µg/mL) thus correspond to 62.8 and 251 nmol/L, respectively.
Krebs solution and Griess reagent were prepared daily. Stock solutions of VEGF/VPF and ACh dissolved in distilled water were refrigerated until they were used. Pellets of L-arginine and L-NMMA were added to the organ chamber at established concentrations of 0.2 mmol/L.
Statistical Analysis
All values are given as mean±SEM. The data were evaluated with a two-factorial (significance over time, significance of drug effects) ANOVA for repeated measurements. Statistical significance was inferred when P<.05. In all experiments, n equals the number of rabbits from which the vessels were taken.
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Results |
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Thoracic Aorta
Stimulation of vascular segments of thoracic aorta with ACh resulted in a nearly immediate and significant increase in [NO] (Fig 2
). Stimulation with 10-5 mol/L ACh yielded a somewhat higher [NO] than that observed with 10-6 mol/L ACh, but this difference was not statistically significant. No increase in [NO] was observed when 0.2 mmol/L L-NMMA was administered at t=0 minutes.
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, P<.05 vs ACh 10-5 mol/L L-NMMA. Difference between ACh 10-6 and 10-5 mol/L was not statistically significant.
In comparison with ACh, the onset of increased [NO] after administration of VEGF/VPF was slower, reaching a maximum value after 8 minutes (Fig 3). Both tested concentrations (2.5 and 10 µg/mL) caused a significant rise in a dose-dependent fashion. Preincubation of the aortic segments with L-arginine raised by twofold both baseline [NO] and [NO] stimulated by addition of 2.5 µg/mL VEGF/VPF (Fig 4). Removal of CaCl2 from the Krebs solution abrogated the stimulatory effect of 10 µg/mL VEGF/VPF, resulting in [NO] that did not differ significantly from baseline conditions or from unstimulated aortic rings (Fig 5). Similar results were observed after mechanical removal of the endothelium. Administration of 0.2 mmol/L L-NMMA at time t=0 minutes also prevented any rise in [NO] after stimulation with 10 µg/mL VEGF/VPF. Moreover, incubating with L-NMMA produced a tendency to diminished [NO] compared with the values obtained for the unstimulated aortic rings.
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Inferior Vena Cava
After stimulation with VEGF/VPF, vascular segments retrieved from the IVC yielded results similar to those obtained for aortic segments (data not shown). [NO] for the unstimulated IVC was nearly identical to that obtained for aorta; stimulation of IVC with 2.5 µg/mL and 10 µg/mL VEGF/VPF, however, yielded a lower [NO] than that recorded for the aorta.
In contrast to results obtained in the aorta with VEGF/VPF, ACh applied to the IVC failed to produce a significant increase in [NO] (Fig 6).
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Pulmonary Artery
For vascular segments retrieved from the PA, 2.5 µg/mL VEGF/VPF did not lead to an increase in [NO] compared with results obtained for the unstimulated segments (data not shown). Stimulation with 10 µg/mL VEGF/VPF, however, did yield a significant increase in [NO], although peak levels in the aorta and the IVC segments were higher. Preincubation with 0.2 mmol/L L-arginine increased [NO] as for the aorta and IVC.
Again, as in the case of the aorta, ACh did not result in a rise in [NO] (Fig 7).
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Vasomotor Reactivity
Fig 8A shows the representative vasomotor response obtained from an endothelium-intact ring of rabbit aorta after administration of VEGF/VPF. Norepinephrine was used to induce the initial vasomotor tone. VEGF/VPF produced slowly developing relaxation, reversible with L-NMMA. This effect was also abolished after mechanical removal of the endothelium and by removal of CaCl2 from the organ bath solution (Fig 8B). The time course observed for VEGF/VPF-induced vasomotor reactivity, similar to that originally described by Ku et al14 in isolated canine coronary arteries, is consistent with the time course of VEGF/VPF-induced release of NO from isolated rings of rabbit aorta described above.
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Human Umbilical Vein ECs
The iso-NO electrode was calibrated by a chemical titration method as described in "Methods" (Fig 9A). Stimulation of HUVECs with VEGF/VPF 1, 10, and 100 ng/mL resulted in a concentration-dependent increase in NO release (Fig 9B). Peak NO release was observed between 5 and 8 minutes after addition of VEGF. Treatment of HUVECs with the NO synthase inhibitor L-NAME 100 µmol/L attenuated the VEGF/VPF 100 ng/mL–stimulated release of NO under the same experimental conditions. Sodium nitroprusside 100 µmol/L significantly increased NO concentration.
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pA indicates differences expressed in picoamperes. |
Discussion |
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These experiments establish that VEGF/VPF causes a dose-dependent increase of [NO] in vascular endothelium. This increase in [NO] can be demonstrated in vascular segments harvested from different regions, including aorta, vena cava, and PA, as well as from cultured human ECs. Parenthetically, these experiments also establish that ACh fails to enhance production of NO in both IVC and PA.
A variety of techniques have been used to measure NO. NO production has been studied with a chemiluminescence assay25 ; this approach measures the intensity of fluorescent radiation emitted after oxidation of NO by ozone. The use of this method for measurement of NO production by vascular segments in an organ chamber requires, first, reduction of NO2- back to NO and second, transfer of NO from solution to gas. A limitation of this technique is that removal of NO from solution may introduce significant error at lower concentrations.
Kiechle and Malinski25 reported an electrochemical method based on registration of current secondary to oxidation of NO. This technique permits measurements to be made within short time intervals on the surface of the cell membrane with electrochemical sensors with a diameter between 0.2 and 1.0 µm. A modification of this method was used in the present study to measure NO release from cultured HUVECs treated with VEGF/VPF. The size of the sensor, however, restricts its use to measurements of NO release from cells, and the method is not applicable for NO registration from intact vascular segments.
In the present study, we therefore used an alternative method, UV-visible spectrophotometry, which is relatively easy to perform, yields reproducible results, and allows measurement of significant differences of [NO] in the micromolar range from intact vascular segments. This approach is based on the premise that in common solutions, [NO] is dependent on the content of nitrogen oxides and nitrite: NO
+NO2+H2O
2HNO22H++2NO2-. The relationship between nitrous acid and nitrite is defined by pH-pK=log([NO2-]/[HNO2]). In dilute solutions, pK3.4 at 37°C. The experiments took place at pH=7.4, implicating a 10 000-fold greater concentration of nitrite than nitrous acid. Assuming that no NO or NO2 is present in the Krebs solution before the experiments were begun, an equation can be obtained to relate NO and NO2-: log[NO]=constant+log[NO2-]. Thus, the nitrite concentration we measured in samples taken from the organ chamber runs parallel to the concentration of NO: [NO]=constant·[NO2-].
Since the fluid in the organ chamber is exposed to atmospheric oxygen, there is some loss of NO and NO2- to air and also via the glass walls of the organ chamber. Samples taken after removal of the NO-producing vessels of the organ chamber demonstrated a decrease in nitrite concentration of about 10% after 15 minutes (data not shown). This explains both the decrease in [NO] after NO production was blocked by L-NMMA and the horizontal curve produced by the vascular segments exposed to neither VEGF/VPF nor ACh.
NO is a colorless, free-radical, simple gas with a half-life of 10 to 30 seconds. The heme group of hemoglobin readily combines with NO, yielding iron-nitrosyl adducts and/or nitrate (NO3-).26 Misko and colleagues,27 using a fluorometric assay, found that as little as 10 µmol/L hemoglobin completely abolished any signal representing nitrite (NO2-). The methodology used in the present study likewise required removal of any blood that remained after the vessels were harvested. This and handling of the excised vascular segments in a manner that avoids damaging the endothelium appear to be the two factors most responsible for minimizing variability in these types of experiments.
The onset for [NO] increase (4 minutes) and the time required for VEGF/VPF to induce maximum release of NO (8 minutes) were considerably longer than were observed for ACh. It is interesting to note that a similar time frame (5 to 8 minutes) was required for maximal relaxation of rings of thoracic aorta in the present study and for isolated canine coronary arteries described previously by Ku et al.14 Furthermore, in vivo experiments recently performed in our own laboratory16 disclosed that the nadir of endothelium-dependent hypotension occurred 8 minutes after VEGF/VPF administration. The apparent reduction in NO production after 8 minutes may be a function of the brief half-life of VEGF/VPF, reported to be 3 minutes (N. Ferrara, MD, oral communication, August 1995).
The rapid onset of increased NO production after stimulation with ACh is inferred to represent the expedited consequence of a G protein that is coupled to the ACh receptor.28 In contrast, VEGF/VPF binds to a non–G-protein–coupled tyrosine kinase receptor Flt-1 and/or Flk-1/KDR,29 30 which must dimerize in order to activate downstream signaling. Tyrosine phosphate–mediated phosphorylation of phospholipase C-
1, release of inositol 1,4,5-triphosphate from phosphoinositide, and a consequent increase in intracellular [Ca2+] are presumed to constitute the basis for increased EC production of NO in response to VEGF/VPF.14 These different signaling pathways may account for the temporal differences observed for the onset of increased [NO] in response to ACh versus VEGF.
VEGF/VPF caused an increase in [NO] in vascular segments from thoracic aorta, IVC, and pulmonary artery, implicating the presence of Flt-1 and/or Flk-1/KDR on the ECs of all three vessel types. ACh, however, failed to augment [NO] in either the IVC or PA, a finding that to the best of our knowledge has not been reported previously. This observation might represent the explanation for findings recently reported by Gao et al31 regarding failure of ACh to induce endothelium-dependent relaxation in isolated pulmonary arteries of full-term fetal lambs; although ACh did induce relaxation in lambs 5 to 6 weeks in age, this effect could not be abolished by an inhibitor of NO synthase.31 32 Similar observations in isolated canine femoral veins were reported by Miller.33 These differences in response to ACh may reflect a difference in muscarinic receptor density among different types of vessels. Moreover, activation of different muscarinic receptor subtypes are known to cause the release of different endothelial vasoactive factors.34
VEGF/VPF receptor expression is widespread during vasculogenesis and angiogenesis in the developing embryo.30 Postnatally, both the Flt-1 and Flk-1/KDR receptors have been shown to be upregulated at sites of recurrent neovessel proliferation, such as the corpus lutea of the ovary,10 or in pathological tissues,35 36 37 particularly in conjunction with hypoxia.38 39 In contrast, expression of VGF/VPF receptors by quiescent endothelium in the adult has been considered to be typically reduced30 35 36 and in some organs, such as the human adult brain, has been reported to be altogether absent.37 Peters et al,9 however, observed expression of Flt-1 mRNA by quiescent endothelium of the adult mouse among multiple organs, including brain, corresponding to a similar pattern of 125I-rhVEGF binding described earlier by Jakeman et al10 ; these studies thus suggested that VEGF/VPF might have a function in mature vessels other than mediating vascular growth.
The observation in the present study that VEGF/VPF stimulates production of NO when applied to endothelium-intact segments retrieved from three different vascular districts constitutes inferential evidence for the presence of functional VEGF/VPF receptors on quiescent endothelium of the adult rabbit. The fact that this response is blocked by a competitive inhibitor of NOS suggests further that putative maintenance functions of VEGF/VPF may include regulation of baseline synthesis and/or release of EC NO and supports the notion of a "survival" or "maintenance/repair" role for VEGF/VPF.9 10 11 12 VEGF/VPF-induced recovery of disturbed endothelium-dependent flow in the rabbit ischemic hindlimb,15 for example, may reflect restored NO production by ECs initially damaged by protracted ischemia in the collateral-dependent limb.
NO has been implicated as an inhibitor of intimal thickening responsible for compromised arterial patency.40 41 42 43 44 De Meyer et al, for example, showed that the NO donor SPM-5185 could inhibit neointima formation and restore vascular reactivity in collared carotid arteries of rabbits.42 Von der Leyen et al43 likewise inhibited neointima formation in balloon-injured rat carotid arteries, and vascular reactivity in these vessels improved as well. More recently, Marks et al44 used a long-lived NO adduct to inhibit neointimal formation in injured rabbit arteries. VEGF/VPF, too, administered as the recombinant protein to the balloon-injured rat carotid artery17 or as the cDNA to the balloon-injured rabbit femoral artery,18 has been shown to inhibit intimal thickening. The results of these live animal studies in combination with the in vitro findings reported here may be interpreted to suggest that NO is at least in part responsible for mediating this inhibitory effect of VEGF/VPF on intimal thickening.
The extent to which NO release may contribute to the proliferative and migratory roles of VEGF/VPF believed to be responsible for stimulating angiogenesis45 46 remains speculative. Leibovich et al47 found that the angiogenic activity of monocytes stimulated with lipopolysaccharide was both L-arginine–dependent and inhibited by inhibitors of NO synthase. Recent work from our own laboratory has established that dietary supplements of L-arginine enhance angiogenesis in the rabbit model of hindlimb ischemia,48 consistent with a role for NO in promoting angiogenesis in vivo.
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Selected Abbreviations and Acronyms |
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-nitro-L-arginine methyl ester
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Acknowledgments |
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This study was supported in part by an Academic Award in Vascular Medicine (HL-02824) and grant HL-40518, both from the National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Md. Dr Murohara is a recipient of the Uehara Memorial Foundation Research Fellowship. Dr van der Zee is a recipient of the Wynand N. Pom Foundation Research Fellowship, Leusden, Netherlands.
Received June 26, 1996; revision received September 27, 1996; accepted September 30, 1996.
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 T. Wheeler, P.W. Evans, F.W. Anthony, K.M. Godfrey, D.T. Howe, and C. Osmond Relationship between maternal serum vascular endothelial growth factor concentration in early pregnancy and fetal and placental growth Hum. Reprod., June 1, 1999; 14(6): 1619 - 1623. [Abstract] [Full Text] [PDF]
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T. Murohara, B. Witzenbichler, I. Spyridopoulos, T. Asahara, B. Ding, A. Sullivan, D. W. Losordo, and J. M. Isner Role of Endothelial Nitric Oxide Synthase in Endothelial Cell Migration Arterioscler Thromb Vasc Biol, May 1, 1999; 19(5): 1156 - 1161. [Abstract] [Full Text] [PDF]
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W. G. Mayhan VEGF increases permeability of the blood-brain barrier via a nitric oxide synthase/cGMP-dependent pathway Am J Physiol Cell Physiol, May 1, 1999; 276(5): C1148 - C1153. [Abstract] [Full Text] [PDF]
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S. Fischer, M. Clauss, M. Wiesnet, D. Renz, W. Schaper, and G. F. Karliczek Hypoxia induces permeability in brain microvessel endothelial cells via VEGF and NO Am J Physiol Cell Physiol, April 1, 1999; 276(4): C812 - C820. [Abstract] [Full Text] [PDF]
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H. m. Wu, Y. Yuan, D. C. Zawieja, J. Tinsley, and H. J. Granger Role of phospholipase C, protein kinase C, and calcium in VEGF-induced venular hyperpermeability Am J Physiol Heart Circ Physiol, February 1, 1999; 276(2): H535 - H542. [Abstract] [Full Text] [PDF]
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A. Rivard, J.-E. Fabre, M. Silver, D. Chen, T. Murohara, M. Kearney, M. Magner, T. Asahara, and J. M. Isner Age-Dependent Impairment of Angiogenesis Circulation, January 12, 1999; 99(1): 111 - 120. [Abstract] [Full Text] [PDF]
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B.-Q. Shen, D. Y. Lee, H.-P. Gerber, B. A. Keyt, N. Ferrara, and T. F. Zioncheck Homologous Up-regulation of KDR/Flk-1 Receptor Expression by Vascular Endothelial Growth Factor in Vitro J. Biol. Chem., November 6, 1998; 273(45): 29979 - 29985. [Abstract] [Full Text] [PDF]
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J. Hood and H. J. Granger Protein Kinase G Mediates Vascular Endothelial Growth Factor-induced Raf-1 Activation and Proliferation in Human Endothelial Cells J. Biol. Chem., September 4, 1998; 273(36): 23504 - 23508. [Abstract] [Full Text] [PDF]
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V. Poppa, J. K. Miyashiro, M. A. Corson, and B. C. Berk Endothelial NO Synthase Is Increased in Regenerating Endothelium After Denuding Injury of the Rat Aorta Arterioscler Thromb Vasc Biol, August 1, 1998; 18(8): 1312 - 1321. [Abstract] [Full Text] [PDF]
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B. Witzenbichler, T. Asahara, T. Murohara, M. Silver, I. Spyridopoulos, M. Magner, N. Principe, M. Kearney, J.-S. Hu, and J. M. Isner Vascular Endothelial Growth Factor-C (VEGF-C/VEGF-2) Promotes Angiogenesis in the Setting of Tissue Ischemia Am. J. Pathol., August 1, 1998; 153(2): 381 - 394. [Abstract] [Full Text] [PDF]
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J. Folkman Therapeutic Angiogenesis in Ischemic Limbs Circulation, March 31, 1998; 97(12): 1108 - 1110. [Full Text] [PDF]
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T. Murohara, J. R. Horowitz, M. Silver, Y. Tsurumi, D. Chen, A. Sullivan, and J. M. Isner Vascular Endothelial Growth Factor/Vascular Permeability Factor Enhances Vascular Permeability Via Nitric Oxide and Prostacyclin Circulation, January 13, 1998; 97(1): 99 - 107. [Abstract] [Full Text] [PDF]
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 M. M. Höper, N. F. Voelkel, T. O. Bates, J. D. Allard, M. Horan, D. Shepherd, and R. M. Tuder Prostaglandins Induce Vascular Endothelial Growth Factor in a Human Monocytic Cell Line and Rat Lungs via cAMP Am. J. Respir. Cell Mol. Biol., December 1, 1997; 17(6): 748 - 756. [Abstract] [Full Text]
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J. R. Horowitz, A. Rivard, R. van der Zee, M. Hariawala, D. D. Sheriff, D. D. Esakof, G. M. Chaudhry, J. F. Symes, and J. M. Isner Vascular Endothelial Growth Factor/Vascular Permeability Factor Produces Nitric Oxide–Dependent Hypotension : Evidence for a Maintenance Role in Quiescent Adult Endothelium Arterioscler Thromb Vasc Biol, November 1, 1997; 17(11): 2793 - 2800. [Abstract] [Full Text]
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Z. Luo, M. Diaco, T. Murohara, N. Ferrara, J. M. Isner, and J. F. Symes Vascular Endothelial Growth Factor Attenuates Myocardial Ischemia-Reperfusion Injury Ann. Thorac. Surg., October 1, 1997; 64(4): 993 - 998. [Abstract] [Full Text]
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