Vascular Endothelial Growth Factor/Vascular Permeability Factor Produces Nitric Oxide–Dependent Hypotension
Evidence for a Maintenance Role in Quiescent Adult Endothelium
Abstract In vitro studies suggest that vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) may stimulate release of nitric oxide (NO) from endothelial cells. To investigate the hemodynamic consequences of recombinant VEGF/VPF administered in vivo, recombinant human VEGF/VPF was administered as a bolus dose of 500 μg to anesthetized (n=6) or conscious (n=5) New Zealand White rabbits, as well as anesthetized rabbits with diet-induced hypercholesterolemia (HC; n=7). Anesthetized Yorkshire farm pigs (no specific dietary pretreatment) were studied before and after receiving 500 μg intravenous (IV; n=5) or intracoronary (IC; n=5) VEGF/VPF. In anesthetized, normal rabbits, mean arterial pressure (MAP) fell by 20.5±1.4% (P<.05 versus baseline) within 3 minutes after IV VEGF/VPF. Pretreatment with Nω-nitro-l-arginine caused a significant inhibition of VEGF/VPF-induced hypotension. In conscious, normal rabbits, VEGF/VPF produced a consistent though lesser reduction in MAP. The fall in MAP induced by VEGF/VPF in anesthetized, HC rabbits (21.5±2.5% from baseline) was no different from that observed in normal anesthetized rabbits. In pigs, both IV and IC administration of VEGF/VPF produced a prompt reduction in MAP. Heart rate increased, while cardiac output, stroke volume, left atrial pressure, and total peripheral resistance all declined to a similar, statistically significant degree in both IV and IC groups. Epicardial echocardiography disclosed neither global nor segmental wall motion abnormalities in response to VEGF/VPF. We conclude that (1) VEGF/VPF-stimulated release of NO, previously suggested in vitro, occurs in vivo; (2) this finding suggests that functional VEGF/VPF receptors are present on quiescent adult endothelium, consistent with a maintenance function for VEGF/VPF, which may include regulation of NO; and (3) the preserved response of HC rabbits suggests that endothelial cell receptors for VEGF/VPF are spared in the setting of hypercholesterolemia.
- Received December 23, 1996.
- Accepted June 4, 1997.
Seminal reports by Leung,1 Keck,2 and Plouet3 identified VEGF/VPF as an endothelial cell mitogen. Because the high-affinity tyrosine kinase receptors for VEGF/VPF, flt4 and flk/KDR,5 6 are limited to endothelial cells, VEGF/VPF has been regarded as endothelial cell–specific. Studies performed in vivo have demonstrated that VEGF/VPF promotes angiogenesis in animal models of limb7 8 and myocardial9 10 ischemia and accelerates reendothelialization after arterial balloon injury.11
To elucidate the mechanisms by which VEGF/VPF may modulate endothelial cell biology, Brock et al12 used fura 2, a Ca2+-sensitive fluorescent dye, to monitor changes in [Ca2+]i in suspensions of human endothelial cells. They observed a threefold to fourfold increase in [Ca2+]i associated with an increase in inositol triphosphate in response to VEGF/VPF. While an influx of extracellular Ca2+ appeared to constitute the predominant source of [Ca2+]i, a blunted but reproducible increase in [Ca2+]i was obtained even in the absence of extracellular Ca2+, suggesting that VEGF/VPF-induced [Ca2+]i increase was due to release of Ca2+ from internal as well as extracellular pools.
Evidence that VEGF/VPF-induced increase in [Ca2+]i was sufficient to stimulate production of NO was subsequently published by Ku et al.13 They observed that VEGF/VPF-induced dose-dependent relaxation of isolated canine coronary arteries could be abolished by prior endothelium disruption, and/or pretreatment with L-NMMA. The findings of both Brock et al12 and Ku et al13 suggested that VEGF/VPF-receptor binding is associated with tyrosine kinase–mediated phosphorylation of phospholipase C-γ-1, release of inositol triphosphate from phosphoinositide, a consequent increase in [Ca2+]i, and ultimately increased endothelial cell production of NO. More recently, measurements performed in our own laboratory have confirmed that application of VEGF/VPF to freshly excised arterial segments with intact endothelium results in a dose-dependent increase in the production and/or release of NO.14
Systemic administration of VEGF/VPF has been considered as a potential alternative to gene therapy for promoting collateral vessel formation in patients with peripheral vascular disease.7 Delineation of the hemodynamic consequences associated with such therapy is therefore of interest. Accordingly, the current series of experiments was performed to investigate the hemodynamic response to VEGF/VPF in two mammalian species. Hemodynamic consequences were characterized specifically under conscious versus anesthetized conditions, after IV versus IC administration, and in a normal versus HC state.
All protocols were approved by the Institutional Animal Care and Use Committee of St Elizabeth’s Medical Center in accordance with the Guide for the Care and Use of Laboratory Animals (NIH publication 86-23, revised 1985).
New Zealand White rabbits, 3.0 to 3.5 kg in weight (Pine Acre Rabbitry, Norton, Mass) were fed either normal rabbit chow (n=11) or rabbit chow enriched with 2% cholesterol (ICN Biomedical, Cleveland, Ohio; n=7); in the latter case, the duration of the cholesterol-supplemented diet was 6 months and resulted in levels of total cholesterol of 1772±221 mg/dL (mean±SE). After premedication with xylazine, 2 mg/kg, all cholesterol-fed rabbits, as well as six of the non–cholesterol-fed rabbits, were anesthetized before hemodynamic study with a mixture of intramuscular ketamine, 50 mg/kg, and acepromazine, 0.8 mg/kg. All anesthetized rabbits (and pigs, vide infra) were allowed to stabilize hemodynamically after administration of anesthesia and before administration of VEGF/VPF. The remaining non–cholesterol-fed rabbits were studied in the conscious state; these rabbits were placed in a restrainer, and the left ear was anesthetized with 0.25 mL of 1% (vol/vol) lidocaine injected at the base of the ear before insertion of a 22-gauge catheter (Jelco; Critikon) into the central ear artery for direct intra-arterial blood pressure measurements.15 A 25-gauge butterfly needle was also inserted into the contralateral marginal vein of the right ear for IV administration of recombinant protein and/or drug.
Yorkshire farm pigs (Pine Acre Rabbitry), weighing an average of 30 kg with no specific dietary pretreatment, were all anesthetized before hemodynamic study with a mixture of ketamine, 22 mg/kg; acepromazine, 0.5 mg/kg; and atropine 0.05 mg/kg. All pigs were further maintained with an IV injection of sodium pentobarbital, 20 to 30 mg/kg, and 1% lidocaine in 1 L of normal saline and infused intravenously for the duration of the hemodynamic study. Cannulae (20-gauge) were placed in the marginal vein of the left ear and femoral artery for drug administration and hemodynamic measurements, respectively. After a left lateral thoracotomy was performed, the left pleura was opened, and a small incision was made in the pericardium. A 20-gauge Liddle left atrial silicone elastomer catheter (Research Medical, Inc.) was then directly inserted into the left atrium for LAP measurements.
After lysis of the E. coli cells by sonication, centrifugation yielded the 165–amino acid isoform of VEGF/VPF (VEGF165) protein in an insoluble pellet. The pellet was washed with 4 mol/L urea in 20 mmol/L Tris buffer at pH 8 with 5 mmol/L EDTA before solubilization with 25 mmol/L dithiothreitol. The extraction was allowed to continue for 2 hours with stirring at 4°C before centrifugation to remove insoluble bacterial components. The extract was then dialyzed overnight against 0.4 mol/L NaCl, 20 mmol/L Tris-HCl, pH 8, at 4°C, during which time the extracted protein was allowed to refold. The dialyzed, refolded VEGF165 was purified by absorption to S-Sepharose, and eluted with a gradient of 0.4 to 1.0 mol/L NaCl. Fractions containing dimeric VEGF165 as determined by SDS-polyacrylamide gel electrophoresis were pooled, and the protein was further purified by C4 reversed-phase chromatography in 0.1% trifluoroacetic acid with elution by an acetonitrile gradient. VEGF165 eluted in approximately 30% acetonitrile. All rhVEGF employed in this study was documented to be free of endotoxin contamination.
Administration of VEGF/VPF
Previous studies performed in our laboratory7 8 established that the efficacy of VEGF/VPF for stimulating angiogenesis in a rabbit model of hindlimb ischemia was most consistently achieved at a dose of 500 μg of VEGF165; we therefore sought to characterize the hemodynamic consequences of an apparently physiologically relevant dose of the recombinant protein in this species. Because previous experience with rhVEGF was limited to normal rabbits, we also studied animals with diet-induced hyperlipidemia (a common finding in patients with limb ischemia) to determine whether such a metabolic abnormality altered the hemodynamic response to VEGF/VPF. For all 18 rabbits, VEGF/VPF was administered as an IV bolus of 500 μg via the marginal ear vein.
An additional six rabbits were anesthetized as described above to address specific experimental conditions. Four of these rabbits received normal chow, while the other two were fed a 1% cholesterol diet. (Although all animals in this study, including those fed a 2% cholesterol diet, were outwardly healthy, the additional animals fed a 1% cholesterol diet were included to exclude pathology related to the higher-cholesterol diet.) In these six animals, blood pressure was measured continuously through a central catheter positioned in the abdominal aorta. A second catheter was used to inject albumin, VEGF, or phenylephrine.
Four animals (two normal, two HC) were submitted to the same protocol: first, rabbit serum albumin (0.1%) was injected, after which systemic blood pressure (MAP) was recorded for 15 minutes. VEGF was then injected in increasing doses of 150, 350, and 500 μg, and MAP was monitored continuously. MAP was allowed to return to baseline and stabilize for a minimum of 15 minutes between different injections. In addition, two normal rabbits were used to study the effect of VEGF on MAP after pretreatment with L-NNA or phenylephrine. In these rabbits, 80 mg of L-NNA or phenylephrine at a dose of 10 to 20 μg/min induced a similar 10% to 20% increase in MAP. After blood pressure stabilization, 500 μg of VEGF was administered intravenously with continuous MAP monitoring.
To determine whether there were species-dependent differences in VEGF/VPF-induced hemodynamic findings and to evaluate the effect of coronary versus systemic protein administration on hemodynamic outcomes, we administered VEGF/VPF to normal pigs. Of the 10 pigs, 5 received an IV bolus of 500 μg, administered via the ear vein. The remaining 5 pigs received an identical IC dose administered via the left main coronary artery.16 (Attempts to administer larger doses (≥1.0 mg) to the pigs produced irreversible hypotension.) For IC administration, a 6 Fr (JR4: Super Torque Plus; Cordis Corp) Judkins catheter was introduced into the left carotid artery and advanced to the left coronary ostium under fluoroscopic guidance. After VEGF/VPF administration, the syringe and catheter were washed with 3 mL of PBS containing 0.1% rabbit serum albumin (Sigma).
Administration of L-NNA
L-NNA (Sigma), a competitive inhibitor of NO synthase,17 was dissolved in 8 mL of PBS at 60°C in preparation for IV administration. For rabbits, 80 mg of L-NNA was administered intravenously either immediately before VEGF/VPF administration or after VEGF/VPF administration at the time that MAP had reached a nadir; for pigs, the dose of L-NNA was 240 mg.
In rabbits, MAP and HR were measured from the central artery of the left ear using an intra-arterial (22-gauge) cannula interfaced with a Gould TA-11 physiological recorder; for pigs, MAP and HR were measured directly from the femoral artery; LAP was measured directly from the left atrial catheter. All signals were digitized at 1 kHz and average values of each signal were written to the fixed disk of a microcomputer once per second for subsequent analysis. In pigs, CO was measured using a COM-2 Cardiac Output Computer (Baxter Healthcare Corp) with a 7.5 Fr Swan-Ganz thermodilution catheter advanced to the pulmonary artery under fluoroscopic guidance. SV was calculated as CO/HR. TPR was calculated as (MAP-LAP)/CO.
Epicardial echocardiography was performed in five pigs receiving IV VEGF/VPF. Short-axis images of the left ventricle at the midpapillary muscle level were obtained using a 5-MHz transducer within a sterile sheath, interfaced with a Sonos 500 ultrasound console (Hewlett Packard). A warm sterile saline bath provided a medium around the exposed heart for ultrasound transmission. Dynamic short-axis images were recorded continuously on videotape, beginning at baseline and proceeding through and post-VEGF/VPF administration.
In Vitro Experiments
To corroborate the in vivo findings, we investigated the vasomotor response of aortic rings excised at necropsy from New Zealand White rabbits fed a 1% cholesterol diet for 6 weeks. Thoracic aortic segments were harvested and prepared as described previously,14 and then placed in an organ chamber continuing Krebs buffer aerated with 95% O2/5% CO2 gas mixture, and maintained at a constant temperature of 37°C. The aortic rings, 5 mm in length, were mounted using two L-shaped 30-gauge stainless steel hooks, one of which was immobile and the other connected by a silk suture to force displacement transducers (model 7D polygraph, Grass Instrument Company) for recording isometric tension. Vessels were passively stretched to 2.0 g isometric force. After 45 minutes of equilibration, the aortic rings were exposed to 70 μmol/L KCl-solution to assess 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 subsequent production of vasoactive prostanoids. The effect of VEGF/VPF or ACh was determined after evoking submaximal tone (defined as approximately 30% to 50% of the maximal inducible tone with KCl) with norepinephrine before the cumulative addition of either VEGF/VPF or ACh into the organ bath solution. Data are expressed as percentage of change in norepinephrine-induced vascular tone.
All results are expressed as mean±SEM. Repeated measures ANOVA were used to test statistical significance of percent change over time as well as to compare the means of the two cholesterol groups. Student’s t tests were used to evaluate percent change at individual time points versus baseline with Bonferroni adjustments made for multiple comparisons when needed. For all tests, a value of P <.05 was inferred to represent statistical significance.
Fig 1⇓ illustrates the representative effects of VEGF/VPF and L-NNA on MAP and HR over time. For anesthetized but otherwise normal rabbits (n=6, Fig 2⇓), MAP measured at 3 minutes after IV administration of a 500 μg bolus injection of VEGF/VPF fell by 20.5±1.4% (P<.05 versus baseline); this fall in MAP was accompanied by a statistically significant (P<.05) increase in HR of 23.2±4.7% measured at the same time point. The nadir of MAP (−47.8±4.6%) occurred 18 minutes after IV VEGF/VPF, while peak HR (+37.9±9.1%) was observed at 13 minutes. In the absence of L-NNA (see below), MAP remained persistently reduced for >60 minutes.
The same dose of VEGF/VPF (500 μg) administered to conscious but otherwise normal rabbits (n=5) consistently produced a statistically significant reduction in MAP (Fig 2⇑), although the magnitude of this fall in MAP was at no time point as great for the conscious as the anesthetized rabbits.
MAP and HR were also recorded after administration of VEGF/VPF to seven unconscious, HC rabbits. Fig 3⇓ shows that within 3 minutes after a bolus injection of VEGF/VPF, MAP dropped by 21.5±2.5% from baseline (P<.05), while HR at this time point increased by 6.9±1.8% among HC rabbits. The nadir of MAP in HC rabbits (−38.1±4.2%) was again observed at 18 minutes post-VEGF/VPF, while peak HR (9.4±3.1%) occurred at 8 minutes. For MAP, no significant difference was observed between normal and HC rabbits groups at any time point. For HR, however, the difference was significant (P<.05) by 3 minutes (+23.2±4.7% versus +6.9±1.8% for normal versus HC, respectively). The observed difference in HR response between normal and HC rabbits is presumed to be secondary to HC-induced impairment of the baroreceptor reflex.18 As MAP and HR plateaued, a significant difference persisted for up to 20 minutes between groups (33.4±9.8% versus 8.1±4.4% [P<.05] for normal and HC, respectively at 20 minutes).
L-NNA administered post-VEGF/VPF returned MAP and HR to baseline within 12.5 minutes (Fig 1⇑). L-NNA pretreatment caused a significant inhibition of VEGF/VPF-induced hypotension. The small increase (≈10%) in MAP observed after L-NNA is consistent with previously reported hemodynamic consequences of NO synthase inhibitors administered systemically to normal rabbits.19 The magnitude of this effect would thus be unlikely to account for the reversal or inhibition of a nearly 50% reduction in MAP observed after VEGF/VPF in the absence of L-NNA.
An additional group of four rabbits, two normal and two fed with 1% cholesterol-supplemented diet, was used to determine the impact of certain other variables. The two rabbits fed a 1% cholesterol diet were employed to exclude the possibility that VEGF-induced hypotension was due to systemic toxicity related to the higher (2%) cholesterol-supplemented rabbits. Indeed, these rabbits too demonstrated systemic hypotension (mean reduction of 13.9%) after IV administration of 500 μg of rhVEGF165. Moreover, normal and HC rabbits experienced a similar reduction in MAP (mean=−16.1%) when VEGF was administered at two lower doses, 350 μg and 150 μg. In contrast, ACh (1.5 μg · kg−1 · min−1) reduced MAP by 1.8% and 2.1%, respectively, in normal and HC rabbits, while nitroprusside lowered MAP by 9.2% and 7.6%, respectively, in the same groups. Rabbit serum albumin (0.1%) produced no change in blood pressure or HR. Finally, administration of phenylephrine sufficient to achieve an increase in MAP similar to that observed with L-NNA pretreatment (Fig 4⇓) failed to inhibit VEGF-induced hypotension (mean=−35.5%).
IV or IC administration of VEGF/VPF (n=5 each) produced a prompt reduction in MAP after a bolus injection of VEGF/VPF (Fig 4⇑). The onset of hypotension occurred earlier (within 25 seconds) in the pigs than in the rabbits. Hemodynamic measurements recorded systematically at 300 seconds disclosed that MAP had dropped 45.8±1.1% from baseline in the IV group and 45.4±3.8% in the IC group (P<.05 for both versus baseline). Maximum reduction in MAP was 47.4±3.4% and 47.7±0.8% for IV and IC administration, respectively. The fall in MAP after IV and IC administration was similar at all time points (Fig 5⇓). MAP returned to baseline within 675 seconds regardless of the route of administration.
The reduction in MAP, as well as the remaining hemodynamic measurements recorded after IV and IC administration of VEGF/VPF, are summarized in the Table⇓ . After IV VEGF/VPF, the reduction in MAP recorded at 300 seconds was associated with an increase in HR (4.5±2.9%) and a decrease in CO (−15.3±10.7%), SV (−18.2±10.1%), LAP (−21.4±9.0%), and TPR (−35.0±5.1%) (all P<.05 versus baseline). Similarly, measurements recorded at the same time point (300 seconds) after IC VEGF/VPF disclosed that the 45.4±3.8% reduction in MAP was accompanied by increased HR (9.9±5.8%) and reduced CO (−8.3±7.2%), SV (−17.5±5.5%), LAP (−31.3±12.7%), and TPR (−39.7±6.1%) (all P<.05 versus baseline). There was no statistically significant difference in IV versus IC administration of VEGF/VPF on any of the aforementioned hemodynamic findings for any time points.
Continuous two-dimensional epicardial echocardiographic recording performed in five pigs disclosed neither global nor segmental wall-motion abnormalities on administration of VEGF/VPF (Fig 6⇓). These findings are consistent with the notion that VEGF/VPF-induced reduction in MAP was due to a primary reduction in peripheral resistance rather than a negative inotropic effect.
Fig 7⇓ depicts the effects of 240 mg of L-NNA administered intravenously at the nadir of MAP to both groups of pigs. By 135 seconds, MAP increased 44.2±10.6% for the IV group, and 39.6±5.5% for the IC group. A corresponding increase in TPR (IV=36.9±5.2%, IC=48.8±7.7%) was calculated for the same time point.
In Vitro Experiments
Previous studies from our laboratory14 and others’13 documented that VEGF/VPF causes vasorelaxation of aortic rings from normal rabbits, reversible with L-NMMA. In aortic rings from HC rabbits, VEGF/VPF also produced vasorelaxation, reversible with L-NMMA (Fig 8⇓), whereas ACh failed to induce vasorelaxation. Fig 8b⇓ summarizes the vasomotor responses to VEGF/VPF in aortic rings from normal and HC rabbits.
The experiments described in this report extend previous in vitro findings to establish: that (1) the administration of VEGF/VPF to live animals results in a predictable hypotensive response and (2) this hypotensive response can be reversed or prevented by administration of an inhibitor of NO synthase. Thus, these experiments establish that VEGF/VPF induction of NO, previously suggested in vitro, occurs in vivo and is sufficiently robust to have systemic hemodynamic consequences. Hypotension was documented with IC as well as IV administration of VEGF/VPF.
The dose of VEGF/VPF used for rabbits was selected to match the dose we had previously found to be optimal for promoting collateral vessel development in similar-sized rabbits with hindlimb ischemia.7 8 IV administration of this same dose of VEGF/VPF to both rabbits and pigs and IC administration in pigs established that VEGF/VPF-induced synthesis and/or release of NO is independent of both species and route of administration. The latter has potential implications for the use of VEGF/VPF in the treatment of lower extremity and myocardial ischemia. Epicardial echocardiography performed in the larger animal model suggests that the observed reduction in CO was not due to impaired myocardial contractility. Moreover, experiments performed in conscious rabbits demonstrated that while attenuated, VEGF/VPF-induced, NO-mediated hypotension did not result simply from anesthesia-induced abrogation of compensatory reflexes.
These findings are consistent with a preliminary report of similar experiments performed in conscious rats20 and conscious rats treated with fibroblast growth factor-1.15 Findings in the latter study illustrate the potential for a common NO-mediated hemodynamic response among endothelial cell growth factor receptors having intrinsic tyrosine kinase activity, according to the paradigm outlined above by Ku et al.13
The hemodynamic response observed in rabbits treated with VEGF/VPF after several months of documented hypercholesterolemia was similar to that observed in normal rabbits. This is consistent with previous work by Cohen et al,21 who established that hypercholesterolemia does not impair the ability of endothelium to elaborate vasodilators, but instead results in selective endothelial cell receptor-mediated dysfunction. Endothelium-dependent relaxations of isolated coronary arteries from HC pigs caused by 5-hydroxytryptamine and substance P, for example, were reduced, while relaxation in response to norepinephrine and the calcium ionophore A23187 were unaffected. Similar findings were subsequently described for the bradykinin receptor in human subjects with hypercholesterolemia.22 The hypotensive response of HC rabbits to VEGF/VPF documented in the present study suggests that the receptors responsible for transducing the NO-dependent effects of VEGF/VPF on endothelial cells are spared in the setting of hypercholesterolemia, although it must be acknowledged that impairment of the response to VEGF/VPF in the HC model cannot be ruled out for the lower (<500 μg) dose range.
The profound hypotensive response observed in both rabbits and pigs under the various conditions outlined above supports the notion of a “survival” or “maintenance/repair” role for VEGF/VPF.23 24 25 26 VEGF/VPF has a circulating half-life of 3 to 5 minutes (N. Ferrara, personal communication, August 29, 1997) and has been previously shown to be bound by both high-affinity (flt and flk/KDR) and low-affinity (heparan sulfate proteoglycans) receptors on endothelial cells. VEFG/VPF receptor expression is widespread during vasculogenesis and angiogenesis in the developing embryo.5 Postnatally, both the flt and flk/KDR receptors have been shown to be upregulated at sites of recurrent neovessel proliferation, such as the corpus lutea of the ovary,24 or in pathological tissues,27 particularly in conjunction with hypoxia.28 29
In contrast, expression of VEGF/VPF receptors by quiescent endothelium in the adult has been considered to be typically reduced,5 and in some organs, such as the human adult brain, has been reported to be altogether absent.27 Peters et al,25 however, observed expression of flt 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 al24 ; these studies thus suggested that VEGF might have a function in mature vessels other than mediating vascular growth. The hypotensive response to VEGF/VPF observed in the present study constitutes evidence for the presence of functional VEGF/VPF receptors on quiescent endothelium of the adult rabbit and pig. The fact that this hypotensive response is blocked by a competitive inhibitor of NO synthase suggests further that putative maintenance functions of VEGF/VPF may include regulation of baseline synthesis and/or release of endothelial cell NO. VEGF-induced recovery of disturbed endothelium-dependent flow in the rabbit ischemic hindlimb30 may reflect restored NO production by endothelial cells initially damaged by protracted ischemia in the collateral-dependent limb.
Cuevas et al15 successfully dissociated the mitogenic and hypotensive effects of aFGF, using a truncated form of the protein, mitogenically inactive due to loss of the nuclear translocation sequence, but nevertheless capable of producing hypotension equivalent to that observed with the full-length wild type. The results of the current study do not specifically address the extent to which NO release may contribute to the proliferative and migratory roles of VEGF/VPF, believed to be responsible for stimulating angiogenesis. Leibovich et al31 found that the angiogenic activity of monocytes stimulated with lipopolysaccharide was both l-arginine dependent and inhibited by inhibitors of NO synthase. Preliminary studies from our laboratory indicate that dietary supplementation of l-arginine augments angiogenesis in the rabbit ischemic hindlimb model.32 VEGF/VPF has also been shown to inhibit intimal thickening.11
Finally, as suggested by the designation vascular permeability factor,2 VEGF/VPF increases vascular permeability when assessed by the Miles assay, and as such may have contributed via VEGF/VPF-induced third space effects to hypotension observed in the current experiments. The mechanism responsible for such augmented permeability remains enigmatic. The possibility that NO, previously shown to promote microvascular leakage,33 contributes to this feature of VEGF/VPF deserves further study.
Selected Abbreviations and Acronyms
|LAP||=||left atrial pressure|
|MAP||=||mean arterial pressure|
|TPR||=||total peripheral resistance|
|VEGF||=||vascular endothelial growth factor|
|VPF||=||vascular permeability factor|
This study was supported in part by an academic award in vascular medicine (HL 02824) and grant HL53354 from the National Heart, Lung, and Blood Institute of the National Institutes of Health, Bethesda, Md; the Cora and John H. Davis Foundation, Washington, DC; and the E.L. Wiegand Foundation, Reno, Nev. The authors gratefully acknowledge the generous cooperation of Stuart Bunting, PhD, Bruce Keyt, PhD, and Napoleone Ferrara, MD, of Genentech, Inc, in making available the VEGF/VPF used in this study.
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