Recombinant VEGF121 infusion lowers blood pressure andimproves renal function in rats with placental ischemia-inducedhypertension

Jeffrey S Gilbert1,3, Joseph Verzwyvelt1, Drew Colson1, Marietta Arany1, S. AnanthKarumanchi2, and Joey P Granger1,*1Department of Physiology and the Center for Excellence in Cardiovascular-Renal Research,University of Mississippi Medical Center, Jackson, MS2Howard Hughes Medical Institute and Beth Israel Deaconess Medical Center, Harvard MedicalSchool, Boston, MA

AbstractAntagonism of vascular endothelial growth factor (VEGF) signaling by soluble fms-like tyrosinekinase-1 (sFlt-1) occurs during preeclampsia and is proposed to play an important role in thepathogenesis of preeclampsia. We recently reported that hypertension associated with chronicreductions in uteroplacental perfusion pressure (RUPP) is associated with increased sFlt-1 anddecreased free VEGF. Whether restoration of circulating VEGF can restore renal function andchronically decrease arterial pressure associated with placental ischemia remains unknown. Wehypothesized that chronic infusion of VEGF121 would attenuate hypertension, increase glomerularfiltration rate (GFR), and reverse the endothelial dysfunction associated with chronic RUPP.VEGF121 (at either 90 or 180ug/kg/day) was administered for five days via osmotic mini-pumpplaced intraperitoneal. Mean arterial pressure (MAP), renal function and tissues were obtained onday 19 of pregnancy from RUPP+VEGF, RUPP and normal pregnant (NP) dams. MAP (mm Hg)was increased in the RUPP (131±3) compared to the NP (102±1) and infusion of VEGF121 resolvedthe hypertension (105±5). GFR (ml/min) was decreased in the RUPP dams (1.5±0.3), and restoredto NP levels (3.1±0.5) by VEGF121 treatment (3.1±0.4). Effective renal plasma flow, decreased byRUPP was also increased by VEGF121 infusion. Relaxation to acetylcholine was enhanced by theVEGF treatment (P<0.05). These data demonstrate that chronic infusion of VEGF121 during lategestation restores GFR and endothelial function, and reduces high blood pressure associated withplacental ischemia. The present results suggest VEGF121 may be a candidate molecule formanagement of preeclampsia and its related complications.

Keywordspreeclampsia; gestation; VEGF; blood pressure; angiogenic

*Corresponding author: Joey P Granger, PhD, Department of Physiology and Biophysics, University of Mississippi Medical Center,2500 N State St, Jackson, MS 39216-4505. jgranger@physiology.umsmed.edu, Phone: (601) 984-1821, FAX: (601) 984-1817.3Present address for JSG: Department of Physiology and Pharmacology, University of Minnesota Medical School-Duluth, Duluth, MNConflict(s) of Interest/Disclosure(s) Statement: S.A.K. is a co-inventor on patents filed by the Beth Israel Deaconess Medical Centerfor the diagnosis and therapy of preeclampsia. S.A.K. has served as consultant to Scios, Inc.

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Published in final edited form as:Hypertension. 2010 February ; 55(2): 380–385. doi:10.1161/HYPERTENSIONAHA.109.141937.

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IntroductionPreeclampsia is a major obstetric problem that affects approximately 5% of all pregnanciesand is a significant source of maternal and neonatal morbidity and mortality 1. Moreover, theincidence of preeclampsia has seen a 40% increase in recent years2. Although the preeclampticsyndrome has been well characterized and many studies indicate that hypertension, proteinuria,endothelial cell dysfunction and insufficient placentation are key features of this disorder3, 4,the underlying pathophysiological mechanisms of this disorder remain unclear.

Recent studies have established the existence of an imbalance between pro- and anti-angiogenicfactors such as vascular endothelial growth factor (VEGF), placental growth factor (PlGF) andsoluble fms-like tyrosine kinase-1 (sFlt-1) in preeclamptic women5-10. Moreover, findingsfrom several clinical studies suggest that alterations in circulating sFlt-1 concentrations maypresage the clinical onset of preeclamptic symptoms5, 11-13. Viewed in concert with recentexperimental studies in animals, it appears that this dysregulation of angiogenic factors mayplay a key role in the pathogenesis of preeclampsia9, 10, 14-19.

Although the mechanisms underlying the overexpression of anti-angiogenic factors such assFlt-1 are currently unresolved, in vitro and in vivo evidence suggest that placental hypoxia/ischemia may initiate this imbalance of angiogenic factors14, 16, 20. Both Gilbert et al. andMakris et al. have shown in vivo that chronic placental ischemia results in elevated circulatinglevels of sFlt-1 and hypertension14, 16. In addition, previous studies have shown that reduceduterine perfusion pressure (RUPP) is associated with decreased endothelium-dependentvascular relaxation21 and decreased glomerular filtration rate (GFR) 22. In an elegantlydesigned study reported several years ago, Maynard et al. reported that exogenousadministration of sFlt-1 into pregnant rats via adenovirus mediated gene transfer resulted inincreased arterial pressure and proteinuria, and decreased plasma free VEGF and PlGFconcentrations similar to that observed in the preeclamptic patients10. More recently, Bridgeset al. demonstrated that chronic elevations of recombinant sFlt-1 results in endothelialdysfunction that is attenuated by the superoxide dismutase mimetic Tiron 18. Further, recentstudies by Li et al. have demonstrated that delivery of recombinant VEGF121, the most solubleof the VEGF isoforms, can rescue the symptoms generated by chronic elevations of sFlt-123. Although these findings are intriguing, the mechanisms by which the hypertensivephenotype is rescued remain unclear. Moreover, the efficacy of VEGF121 treatment to improvehypertension and renal function in a model of hypertension during preeclampsia withspontaneously occurring over-expression of sFlt-1 remains unknown.

Thus, the purpose of the present study was to test the hypothesis that chronic infusion of VEGFwill attenuate the endothelial dysfunction, impaired renal function and hypertension associatedwith reduced uterine perfusion pressure (RUPP) in the pregnant rat. To this end, we employedour established model of hypertension associated with placental ischemia in which chronicreductions of uterine perfusion pressure lead to endothelial dysfunction, decreased GFR andhypertension in the pregnant rat.

Methods and ApparatusAnimals

Studies were performed in timed pregnant Sprague-Dawley rats purchased from Harlan Inc.(Indianapolis Ind). Animals were housed in a temperature-controlled room (23°C) with a 12:12light:dark cycle. All experimental procedures executed in this study were in accordance withNational Institutes of Health guidelines for use and care of animals. All protocols wereapproved by the Institutional Animal Care and Use Committee (IACUC) at the University ofMississippi Medical Center. On day 14 of gestation, rat dams were randomly assigned to one

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of five experimental groups: 1) normal pregnant (NP) plus vehicle which served as the controlgroup (n=9); 2) RUPP plus vehicle (sterile phosphate buffered saline; PBS) group (n=11); 3)RUPP + VEGF (low dose, 90μg/kg/day; n=8); 4) RUPP + VEGF (high dose, 180 μg/kg/day;n=7) group.

Reduced Uterine Perfusion Pressure (RUPP) ProcedureThe RUPP procedure is a well established model for studying the links between placentalischemia and hypertension in the pregnant rat and has been described in detail previously 21,22, 24-28. In brief, surgical procedures were completed with rats under isoflurane anesthesia(Webster, Sterling, Mass) delivered by an anesthesia apparatus (Vaporizer for ForaneAnesthetic, Ohio Medical Products, Madison, Wis). Pregnant rats entering the RUPP groupsunderwent the following clipping procedure at 14 days of gestational age (dGA). After amidline incision, the lower abdominal aorta was isolated and a silver clip (0.203-mm ID) wasplaced around the aorta superior to the iliac bifurcation. Silver clips (0.100-mm ID) were alsoplaced on branches of both the right and left ovarian arteries that supply the uterus. Mini-pumpsfilled with VEGF121 in sterile phosphate buffered saline (PBS) or sterile PBS alone was placedintraperitoneal at the time of the RUPP surgeries and on day 14 of gestation in the NP rats.When the clipping procedure resulted in total reabsorption of the fetuses, rats were excludedfrom data analyses.

Measurement of Renal Hemodynamics in Conscious Rats With Use of Chronic ProtocolsA separate cohort of rats was selected for the determination of renal hemodynamics followingchronic VEGF infusion. Renal hemodynamics measurements were determined in consciouspregnant rats on day 19 of gestation. During isoflurane anesthesia, rats were surgicallyinstrumented with venous and arterial catheters of V-3 tubing (SCI, Lake Hayasu City, Ariz)for blood sampling and blood pressure monitoring. The bladder was cannulated with flare-tipped PE 90 tubing for urine collection. All catheters were tunneled to the back of the neckand exteriorized. Animals were allowed to recover for 3 days before renal functionmeasurements. On the day of renal function measurements, the rats were removed from theirmetabolic cages and placed in modified restraining cages. The venous catheter was connectedto an infusion pump that delivered isotonic saline containing [125I]iothalamate (IsotexDiagnostics; 0.05 mCi · kg-1 · min-1) and Para-aminohippuric acid at a fixed rate of 3 mL/h.Arterial pressure was monitored with a pressure transducer (Cobe III Transducer CDX Sema,Birmingham, Ala) for continuous recording. After a 1-hour equilibration period, 2 consecutive20-minute urine collections were obtained in each rat. Urine volume was determinedgravimetrically. GFR and effective renal plasma flow (ERPF) were calculated fromconcentrations of [125I] and [PAH] in plasma and urine

Measurement of Mean Arterial Pressure (MAP) in Chronically Instrumented Conscious RatsAnimals were instrumented and arterial pressure was determined in both groups of rats at day19 of gestation as described previously 26. Briefly, on day 17 of gestation rats wereinstrumented with carotid catheters of V-3 tubing (SCI, Lake Hayasu City, Ariz) while underisoflurane anesthesia (Webster) delivered by an anesthesia apparatus (Vaporizer for ForaneAnesthetic). Catheters were tunneled to the back of the neck and exteriorized after implantation.On day 19 of gestation, rat dams were placed in individual restraining cages for arterial pressuremeasurements using a pressure transducer (Cobe III Transducer CDX Sema, Birmingham,Ala). MAP was recorded continuously for a 2-hour period after 1-hour of stabilization.

Conceptus MeasurementsAfter the measurement of MAP, the dams were placed under isoflourane anesthesia and aventral midline incision was made to isolate the abdominal aorta for plasma and serum

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collection. The uterus was exteriorized and the number of viable and resorbed pups was countedand recorded and the pups and placentae were excised, blotted dry and weighed. The heaviestand the lightest placentae were collected from each uterine horn, snap frozen in liquid nitrogenand stored at -80°C until further analyses were performed.

Plasma/Serum Assays and Recombinant ProteinBlood was collected for subsequent assays into Corvac® sterile serum separator tubes(Sherwood Davis, St. Louis, MO) and plasma into BD Vacutainer® EDTA containing tubes.Circulating VEGF and sFlt-1 concentrations were measured using commercial kits availablefrom R&D systems (Minneapolis, MN) as reported previously 14, 18. Recombinant VEGF121has been previously described 23 and was a gift from Scios, Inc, Fremont, CA.

Vascular Wire Myography ExperimentsThe non-instrumented carotid was removed and prepared for vessel-reactivity studies in organ-chamber baths, as previously described 18, 29. Carotid arteries were chosen to be consistentwith our earlier work 29. Resting tension was adjusted stepwise to reach a final tension of 0.75g. For studies of vessel relaxation, carotid segments were precontracted with the thromboxanemimetic, U46619 (0.4 μg/mL). After the vessel reached stable tension, concentration responsesto acetylcholine (ACh) and sodium nitroprusside (SNP) (10−8 to 10−4 M) were performed toassess endothelial-dependent and smooth muscle-dependent relaxation, respectively. Toevaluate viability of vessel segments after the final relaxation curve, maximal contractileresponses to U46619 (10−8 to 10−4 M) were also tested.

Statistical Analysis and CalculationsAll data are presented as mean ± SEM and statistical significance was accepted when P < 0.05.A Grubb's test was applied to identify statistical outliers. Conceptus data were calculated asmean per pregnancy. sFlt-1:VEGF ratio data were log transformed to obtain normaldistributions for subsequent statistical analysis. Comparisons between groups were made withANOVA followed by post-hoc Newman-Keuls multiple comparison test. Myography datawere analyzed by non-linear regression best fit modeling followed by an F test for Log of thehalf maximal effective concentration (EC50), Hill slope and curve fitting. Statisticalcalculations were made with GraphPad Prism version 4.00 for Windows (GraphPad Software,San Diego, CA USA).

ResultsBlood pressure during late gestation

Figure 1 illustrates the effects of RUPP and VEGF121 on MAP during late gestation.VEGF121 infusion reduced MAP in the RUPP rats in a dose dependent manner with the highdose treatment abrogating the placental ischemia induced hypertension.

Renal HemodynamicsFigure 2A illustrates the effects of RUPP and VEGF121 on GFR during late gestation. Similarto the effects of observed with respect to MAP, VEGF121 infusion improved GFR in the RUPPrats in a dose dependent manner with the high dose treatment abrogating the placental ischemiainduced decline in renal function. Figure 2B shows that ERPF was restored by high doseVEGF121 infusion.

Endothelial functionBecause the high dose of chronic infusion of VEGF121 resulted in increased GFR and decreasedMAP, we sought to determine if this was due to enhanced vascular endothelial cell function.

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Figure 3 shows that endothelial dependent vascular relaxation of the carotid artery to ACh wasenhanced by chronic infusion of VEGF121 to RUPP rats (Figure 3). The log EC50 for AChwas decreased in the RUPP+VEGF121 compared to RUPP rats (log -6.998 vs. -10.29; P<0.05).

Plasma angiogenic factorsAs reported previously, RUPP resulted in a decrease of VEGF and an increase of sFlt-1 whencompared to NP rats (Figure 4A and B). Infusion of VEGF121 restored circulating VEGF toNP levels (Figure 4A). Interestingly, chronic infusion of VEGF121 decreased plasma sFlt-1levels to that of NP rats (Figure 4B). While this decrease in plasma sFlt-1 levels may be dueto the inability of the sFlt-1 ELISA assay to detect the complexed sFlt-1 bound to VEGF-121,it cannot be ruled out that VEGF121 infusion may negatively regulate sFlt-1 expression.

Conceptus morphometricsFigure 5A illustrates that RUPP fetuses were smaller than the NP fetuses (P<0.05). Chronicinfusion of VEGF121 at 90 μg/kg (low dose) in the RUPP group resulted in fetuses that werenot smaller than the NP groups. High dose infusion of VEGF121 (180 μg/kg) into RUPP ratshad no effect on fetal growth as fetuses remained smaller (P < 0.05) than those in the NP group.Placental size was not altered by RUPP or VEGF121 infusion in the present study (Figure 5B).Treatment with VEGF121 had no effect on resorption number (data not shown).

DiscussionThe present study reports several novel findings regarding the effects of recombinantVEGF121 infusion in a rat model of preeclampsia whereby sFlt-1 over-expression is generatedspontaneously following reduction of utero-placental perfusion pressure. As we have reportedpreviously, RUPP rats have increased arterial pressure, renal impairment and endothelialdysfunction in conjunction with elevated circulating sFlt-1 and decreased plasma VEGF15,21, 22, 28.

In the present work, we report several novel findings. Foremost, we report that chronic infusionof recombinant VEGF121 results in reductions of arterial pressure in a dose dependent manner.Secondly, we found renal hemodynamic improvement as determined by increased GFR andERPF. Third, we found that the reduction in AP and restoration of GFR in the high doseVEGF121 infusion group was associated with significant improvements in endothelial functionas determined by relaxation responses to ACh. Lastly, we found that five days of VEGF121infusion negatively regulates sFlt-1 expression in rats with placental ischemia and restoressFlt-1 back to normal pregnant levels by day 19 of pregnancy. Thus, the present study is thefirst to report on the efficacy of VEGF121 infusion to reduce AP, improve GFR and reducesFlt-1 levels in a robust, reproducible and well characterized animal model of placentalischemia induced hypertension in which plasma sFlt-1 concentrations are increased due toplacental ischemia.

We 14 and others16 have demonstrated placental ischemia results in hypertension that isassociated with increases in sFlt-1. In addition, other groups have shown that sFlt-1 over-expression results in hypertension during pregnancy 9, 10, 23. Our results are consistent withthose of Li et al. that report VEGF121 reverses the preeclamptic phenotype of the adenovirus-induced sFlt-1 over-expression model23. Moreover, both the present study and that of Li et al.23 show that VEGF121 infusion lowers arterial pressure in a dose dependent manner. Takentogether, these data illustrate the importance of sFlt-1 in the pathophysiology of hypertensionduring preeclampsia. In contrast to the work of Li et al.23, the present study is in a model inwhich the RUPP animals present with elevated levels of other factors (in addition to sFlt-1)that play important roles in preeclampsia such as tumor necrosis factor-α and soluble endoglin

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28, 30-32. Hence, the current findings demonstrate that chronic infusion of VEGF121 lowersblood pressure and improves renal function in the presence of multiple factors involved in thepreeclamptic syndrome.

Previous work in our laboratory22 has shown that the hypertension in this model is associatedwith a decline in renal function. Hence, we set out to determine if the improvement observedin arterial pressure was associated with restoration of GFR in VEGF121 infused rats. Althoughwe found that VEGF121 infusion resulted in dose dependent increases in GFR, only the highdose restored GFR to normal pregnant levels. These data suggest that VEGF antagonism bysFlt-1 play an important role in the decline in renal function and therefore hypertensionassociated with placental ischemia.

Prior work has shown that RUPP-hypertension is associated with endothelial dysfunction andimpaired ACh mediated nitric oxide production and vasorelaxation 21. Moreover, previousstudies have provided evidence that VEGF antagonism by sFlt-1 results in decreasedendothelial function 10, 18 in both renal microvessels and carotid arteries. In the presentinvestigation, we find that high dose VEGF121 infusion results in significant improvements inACh mediated vasorelaxation of the carotid artery. Hence, this data suggests one possiblemechanism, the restoration of endothelial function in the renal vasculature, for the increasedrenal function and attenuation of hypertension in this model.

In accord with our previous findings, we found that fetal weight was decreased in the presentcohort of RUPP dams15, 21, 22, 28. Although fetal weight was not significantly increased withVEGF121 infusion, the low dose regimen attenuated the growth restriction associated withchronic RUPP. The high dose VEGF121 infusion showed no effect on fetal growth. Thesefindings are similar to the previous report by Li and colleagues, who also reported no significantimprovement in fetal growth with the chronic administration of VEGF121 in the sFlt-1 over-expression model. The apparent differential effects observed between the low and high dosesof VEGF121 infusion may relate to the extent of arterial pressure change with each dose. Thehigh dose resulted in abrogation of RUPP-induced hypertension, whereas the low dose merelyattenuated the hypertension associated with placental ischemia. Thus, the higher pressure inconcert with the permeability effects of VEGF121 may have resulted in greater tissue perfusionand augmented fetal growth in the low dose VEGF121 group.

Previous work suggests the primary source of circulating sFlt-1 in preeclamptic humans derivesfrom the uteroplacental unit10, 33, 34 and that excessive placental ischemia plays a role in thedysregulation of sFlt-1 in pregnancy14, 20. Viewed together, these studies suggest that theincrease in circulating sFlt-1 observed in preeclampsia may be a consequence of aberrantplacental perfusion and that elevated sFlt-1 is likely responsible for the maternal syndrome.Whether there is a pathogenic role for sFlt-1 in early pregnancy remains unknown at the presenttime. Importantly, our studies suggest that antagonism of sFlt-1 with VEGF121 does appear toameliorate maternal hypertension without significant adverse effects to the fetus.

It is important to recognize that circulating VEGF levels are much different in the pregnant ratcompared to the pregnant woman. Hence, the relative contributions of VEGF compared withplacental growth factor (PlGF) may be species specific. Nevertheless, the present studies showthat restoration of the angiogenic balance has beneficial effects on blood pressure and renalfunction in hypertension associated with placental ischemia. Further studies are clearlyrequired to determine the relative contributions of VEGF and PlGF in this and other models.

PerspectivesWhile it appears clear that anti-angiogenic factors play a role in the pathogenesis ofpreeclampsia, the manner in which these factors result in the features of the preeclamptic

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syndrome remain unclear. The present study which relies on data gathered by employing a wellcharacterized and robust animal model of hypertension during pregnancy provides furtherevidence that antagonism of VEGF plays an important role in the link between placentalischemia, endothelial dysfunction and hypertension in the preeclamptic syndrome. It is difficultto directly extrapolate these findings to preeclampsia in humans largely because of thedifferences in the duration of hypertension and exposure to toxic factors such as sFlt-1. Whilethe present results suggest that sFlt-1 antagonists, such as VEGF121, may be appropriatecandidate molecules for further studies on the management of preeclampsia, careful studieswill be required to determine the therapeutic range in women.

AcknowledgmentsSources of Funding: This work was supported in part by National Institutes of Health grants HL38499, HL51971 toJ.P.G., and HL90269 to J.S.G. S.A.K is an investigator of the Howard Hughes Medical Institute.

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Figure 1.Mean arterial pressure (MAP) was increased (P<0.05) in the reduced uterine perfusion pressure(RUPP) compared to normal pregnant+vehicle (NP) rats on day 19 of gestation (term = 21days). VEGF121 infusion reduced MAP in the RUPP+VEGF121 rats in a dose dependentmanner with the high dose treatment abrogating the placental ischemia induced hypertension(P<0.05, RUPP+VEGF121 high dose vs. RUPP).

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Figure 2.Glomerular filtration rate (GFR; Figure 2, panel A) and effective renal plasma flow (ERPF;Figure 2, panel B) was decreased (P<0.05) in the reduced uterine perfusion pressure (RUPP)compared to normal pregnant (NP) rats on day 19 of gestation (term = 21 days). VEGF121infusion increased GFR (panel A) and ERPF (panel B) in the RUPP+VEGF121 rats in a dosedependent manner with the high dose treatment abrogating the placental ischemia-induceddecline in renal function (P<0.05, RUPP+VEGF121 high dose vs. RUPP).

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Figure 3.Endothelial dependent vascular relaxation of the carotid artery (preconstricted to U46619) toacetylcholine (ACh) was enhanced by chronic infusion of VEGF121 (180μg/kg) for five daysto reduced uterine perfusion pressure (RUPP) rats when compared to normal pregnant + vehiclecontrol (NP) rats. The log EC50 for ACh was decreased in the RUPP+VEGF121 compared toRUPP rats (log -6.998 vs. -10.29; P<0.05). *Indicates significantly different than RUPP treatedgroup.

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Figure 4.Reduced uterine perfusion pressure (RUPP) resulted in a decrease of VEGF and an increaseof sFlt-1 when compared to normal pregnant+vehicle (NP) rats at day 19 of pregnancy (Figure4A and B). Infusion of VEGF121 restored circulating VEGF to NP levels (Figure 4A).Interestingly, chronic infusion of VEGF decreased plasma sFlt-1 levels in the RUPP+VEGF121 group to that of NP rats (Figure 4B).

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Figure 5.Panel A illustrates that reduced uterine perfusion pressure (RUPP) fetuses were smaller thanthe normal pregnant+vehicle (NP) fetuses (P<0.05). Chronic infusion of VEGF121 at 90 μg/kg(low dose) in the RUPP+VEGF121 group resulted in fetuses that were not smaller than the NPgroups. High dose infusion of VEGF121 (180 μg/kg) into RUPP rats had no effect on fetalgrowth. Placental size was not altered by RUPP or VEGF121 infusion in the present study(Panel B).

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