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. Author manuscript; available in PMC: 2022 Dec 1.
Published in final edited form as: Hypertension. 2021 Nov 1;78(6):1888–1901. doi: 10.1161/HYPERTENSIONAHA.121.17713

An Elastin-like Polypeptide – VEGF-B Fusion Protein for Treatment of Preeclampsia

Jamarius P Waller 1, John Aaron Howell 2, Hali Peterson 3, Eric M George 4,5, Gene L Bidwell III 1,2,5,*
PMCID: PMC8585700  NIHMSID: NIHMS1745051  PMID: 34719237

Abstract

Preeclampsia is characterized by the development of elevated blood pressure during the second and third trimester of pregnancy that is accompanied by end organ dysfunction. The pathogenesis of preeclampsia is multifactorial but is commonly characterized by endothelial dysfunction and the overproduction of antiangiogenic factors, including the soluble Vascular Endothelial Growth Factor (VEGF) receptor sFlt-1. Previously, administration of exogenous VEGF-A, bound to a carrier protein called Elastin-like polypeptide (ELP), significantly reduced free sFlt-1 levels and attenuated the hypertensive response in a rodent model of preeclampsia. However, VEGF-A administration induces multifactorial effects mediated through its direct activation of the Flk-1 receptor. In response to this, we developed a therapeutic chimera utilizing ELP bound to VEGF-B, a VEGF isoform that binds to sFlt-1 but not to Flk-1. The purpose of this study was to evaluate the in vitro activity and pharmacological properties of ELP-VEGF-B and to test its efficacy in the reduced uterine perfusion pressure (RUPP) rat model of placental ischemia. ELP-VEGF-B was less potent than ELP-VEGF-A in stimulation of endothelial cell proliferation and matrix invasion, indicating that it is a weaker angiogenic driver. However, after repeated subcutaneous administration in pregnant rats, ELP-VEGF-B was maternally sequestered and reduced blood pressure when compared to saline treated animals following induction of placental ischemia (123.38 ± 11.4 versus 139.98 ± 10.56 mmHg, p=0.0129). Blood pressure reduction was associated with a restoration of the angiogenic capacity of plasma from rats treated with ELP-VEGF-B. ELP-VEGF-B is a non-angiogenic, maternally sequestered protein with potential efficacy for treatment of preeclampsia.

Keywords: Preeclampsia, Vascular Endothelial Growth Factor, sFlt-1, Elastin-like Polypeptide, Drug Development, Placental Transfer

Summary:

Overabundance of anti-angiogenic factors and the subsequent sequestration of certain pro-angiogenic growth factors, like VEGF and PLGF, has been shown to play an important role in the development of the preeclamptic phenotype. Exogenous administration of these pro-angiogenic factors has been proven to modulate levels of certain anti-angiogenic factors, such as s-Flt1, and increase the bioavailable levels of VEGF. Our work further illustrates the utility of VEGF administration by examining the efficacy of an isoform of VEGF, VEGF-B, in combination with the ELP drug carrier, as a potential treatment for preeclampsia.

Introduction

Preeclampsia is a disease that affects about 2–8% of pregnancies worldwide1. It is characterized by new onset hypertension and some form of end organ dysfunction, most commonly proteinuria, which is usually diagnosed after 20 weeks of gestation in otherwise normotensive patients1,2. More recently, preeclampsia has become one of the major causes of maternal and fetal morbidity and mortality in pregnant women3,4. The pathogenesis of preeclampsia is not well understood, but is known to be multifactorial2, with increased inflammation, angiogenic factor imbalance, and enhanced reactive oxygen species levels all contributing to the maternal syndrome. One consistent mechanism that exists in many preeclamptic patients is the presence of angiogenic imbalance centering around the overexpression of soluble Fms-like tyrosine kinase receptor 1 (sFlt-1).

sFlt-1 plays an important role in promoting endothelial dysfunction during pregnancy because of its ability to act as a sequestrant of both VEGF and PlGF, preventing them from interacting with their receptors and disrupting the homeostatic balance of these factors. In preeclampsia, sFlt-1 levels have been found to be significantly elevated in the later stages of pregnancy compared to normal pregnant women5,6. Maynard et al. discovered that the excess levels of plasma sFlt-1 in these women was accompanied by a significant decrease in free VEGF and PlGF, two factors crucial for proper endothelial and placental function. These studies postulated that, by reducing free circulating levels of VEGF and PlGF, excess circulating sFlt-1 may play a role in development of preeclampsia, and that reduction of sFlt-1 may be a viable therapeutic target for treatment of the disease.

Multiple techniques have been used to target sFlt-1in preeclamptic patients, including plasma apheresis, siRNA interference, as well as the administration of both VEGF and PlGF to serve as therapeutic sequestrants of sFlt-17–9. While none of these techniques have been shown to completely attenuate the clinical symptoms of preeclampsia, research has shown that reduction of free circulating sFlt-1 is strongly associated with reductions in proteinuria and blood pressure in both humans and non-human models of preeclampsia8,10,11.

Previously, our lab developed a therapeutic candidate to target preeclampsia utilizing a combination of an elastin like polypeptide (ELP) drug carrier and an isoform of VEGF, VEGF-A121 (hereafter referred to as ELP-VEGF-A). We tested this agent in a rodent model of preeclampsia, the reduced uterine perfusion pressure (RUPP) model of surgically induced placental ischemia. This model was chosen for its features that closely resemble the preeclamptic state in humans. Results from this study showed that ELP-VEGF-A was successful in reducing free circulating sFlt-1 levels as well as significantly reducing the increased blood pressure seen with the RUPP model9. However, ELP-VEGF-A is a potent stimulator of angiogenesis and vascular permeability. In our previous study, chronic intraperitoneal infusion of the protein resulted in a dose-dependent increase in ascites in the peritoneum of dams as well as uninhibited vascular tissue growth around the administration pump. While these side effects were most likely related to the administration route (continuous intraperitoneal infusion) and may not be relevant if the protein were given via a more clinically relevant route, these observations prompted the development of a second ELP-VEGF fusion using another isoform of VEGF, VEGF-B167 (ELP-VEGF-B). VEGF-B primarily interacts with the Flt-1 receptor, as well as sFlt-1, and it does not bind to the more active Flk-1 receptor that is responsible for the majority of the angiogenic effects and the increased vascular permeability. Because of this difference in receptor interaction, we theorized that ELP-VEGF-B could be efficacious for treatment of preeclampsia and have a wider safe-dosing window.

The angiogenic drive of ELP-VEGF-B was first tested in vitro in comparison to its predecessor, ELP-VEGF-A. Secondly, the pharmacokinetics and biodistribution of ELP-VEGF-B were determined following intravenous as well as subcutaneous dosing. Finally, the efficacy of ELP-VEGF-B was tested in the RUPP rat model of preeclampsia.

Methods

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Cloning, Purification, Labeling, and Characterization of ELP-VEGF-B.

A DNA fragment containing the coding sequence for an N-terminal peptide with a lysine residue for labeling, an SfiI restriction site, and the human VEGF-B167 coding sequence, all codon optimized for expression in E. coli, was synthesized and inserted into a cloning vector between NdeI and BamHI restriction sites (Invitrogen GeneArt). The cassette was removed from the cloning vector by restriction digestion and inserted into the pET25b expression vector between its NdeI and BamHI restriction sites. The coding sequence for an ELP (160 repeats of the pentapeptide VPGxG, where x is valine, glycine, or alanine in a 1:7:8 ratio) was removed from pUC19 and inserted into the SfiI site in the expression vector, creating an in-frame fusion. The clone was confirmed by DNA sequencing. The fusion protein containing ELP and human VEGF-B167 (ELP-VEGF-B) was purified via recombinant expression in E. coli as described previously12. Purification of ELP-VEGF-B was confirmed by SDS-PAGE and Western blot. For biodistribution experiments, ELP-VEGF-B was covalently labeled on exposed primary amines using an NHS-Rhodamine (5/6-carboxy-tetramethyl-rhodamine succinimidyl ester), mixed isomer (Thermo Fisher) by incubating the protein with the dye overnight at 4 °C. Unreacted dye was removed by ultrafiltration using an Amicon spin filter with a 3,000 dalton molecular weight cutoff. Labeling efficiency was determined spectrophotometrically13. Transition temperature of ELP-VEGF-B suspended in phosphate buffered solution (PBS) was detected by monitoring the optical density at 350 nm (OD350) while increasing the temperature at a rate of 0.5 °C / minute in a UV/Visible spectrophotometer with a temperature-controlled Peltier block (Cary). The transition temperature (Tt) of ELP-VEGF-B, defined as the temperature at which a maximum was observed in a plot of the first derivative of the aggregation curve, was 52.4 °C at 10 μM in PBS.

MTS Proliferation Assay.

Human glomerular microvascular endothelial cells (HGME) were plated at a density of 2000 cells/well in a 96 well plate (Costar) in supplemented media (Cell Systems, Complete Classic Media) and allowed to attach for 24 hours. After 24 hours, media was aspirated and replaced with 10% complete media (Cell Systems) supplemented with either ELP-VEGF-A or ELP-VEGF-B, at various concentrations. Cells were then incubated for 72 hours at 37 °C. Media was removed, and 40 μL of CellTiter 96 Aqueous Cell Proliferation Assay (Promega) was added to each well. Cells were then returned to the incubator for 1.5–4 hours, and absorbance was read at 490 nM. The data represent the mean ± standard deviation of 3 independent experiments.

Matrigel Invasion Assay.

HGME cells were seeded at a density of 30,000 cells/well onto growth factor reduced Matrigel invasion chambers (BioCoat) in 10% complete media (Cell Systems). Invasion chambers were added to a 24-well plate into wells that contained 10% complete media that was supplemented with either PBS, ELP-VEGF-A (100 nM), or ELP-VEGF-B (100 nM) and incubated for 24 hours. Chambers were then removed, washed with sterile PBS, stained with 1% crystal violet in 10% ethanol solution for 10 minutes, and air dried for 1 hour. Invasion chambers were imaged at 20x in 5 non-overlapping fields (Biotek Cytation). Cells per field were counted and averaged using the multi-point tool in ImageJ. The data represent the mean ± standard deviation of 3 independent experiments.

Animal Use.

All protocols were approved by the University of Mississippi Medical Center Institutional Animal Care and Use Committee and followed the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Female timed pregnant Sprague Dawley rats (Charles River SAS Sprague Dawley, strain code 400) were received on GD10 (with the observation of the ejaculatory plug defined as GD1), pair housed and acclimated until GD13, then single housed after initial surgical procedures. Rats were maintained on a 12:12 light:dark cycle, maintained at constant 23 °C, and provided food and water ad libitum.

Biodistribution and Pharmacokinetics of ELP-VEGF-B in Pregnant Rats.

Timed-pregnant Sprague Dawley rats (n=3) were anesthetized on GD13 and received a subcutaneous injection of either rhodamine-labeled ELP-VEGF-B or sterile saline. Blood samples were taken from the lateral tail veins at time 0, 1, 2, 4, 6, 8, 24, and 33 hours after injection. This process was repeated on GD15 and GD17. Animals were euthanized on GD18, and pups, placentae, and maternal organs were harvested into sterile PBS. Pups and placentae were excised from the amniotic sacs and imaged using the In Vivo Imaging System (IVIS, Perkin Elmer) in fluorescence mode (535 nm excitation, 580 nm emission, auto exposure). Maternal heart, liver, brain, lungs, spleen, and kidneys were also imaged using the IVIS. Regions of interest (ROIs) were drawn around organs, pups, and placentae using Living Image software (Perkin Elmer). Average radiant efficiency was determined for each organ or pup. Autofluorescence values were determined from imaging organs from a saline injected animal and were subtracted from the average radiant efficiency of each organ.

A second cohort of timed-pregnant Sprague Dawley rats (n=3) were injected intravenously on GD13 with rhodamine-labeled ELP-VEGF-B, and acute tail vein blood draws were taken from times 0–4 hours post injection. After 4 hours, animals were euthanized, and amniotic sacs and organs were harvested and analyzed via the same imaging procedure outlined above.

Blood samples were centrifuged, and plasma was transferred into 1.5mL centrifuge tubes. The fluorescence of plasma samples was directly measured using a Nanoquant plate (Tecan) and a fluorescence plate reader (Tecan, Excitation: 535, Emission: 580, Gain: 150) and fit to a standard curve of Rhodamine-labeled ELP-VEGF-B.

Reduced Uterine Perfusion Pressure Surgery.

Timed pregnant Sprague Dawley rats (n=10 for each treatment group) on GD13 were placed under anesthesia with 3% isoflurane and maintained at 2.5% isoflurane with 100% oxygen as a carrier gas. Abdominal fur was shaved, and the skin was sanitized with betadine and 70% ethanol. Carprofen (5mg/kg) was administered subcutaneously for analgesia. A 2-inch incision was made through the skin and through the rectus abdominis muscle. Both the left and right uterine horns were externalized and wrapped in gauze soaked with sterile saline. The abdominal aorta was isolated from the inferior vena cava using blunt dissection. For animals receiving the RUPP procedure, a silver clip with an internal opening of 0.203 mm was placed rostral to the bifurcation of the aorta, and second and third clips, with an internal opening of 0.1mm, were placed on the ovarian artery rostral to the first segmental artery. Sham surgeries were performed similarly without the placement of any clips. The uterine horns were returned to the abdominal cavity and both the muscle and skin layers were stitched using 4–0 suture. Either saline or ELP-VEGF-B (50mg/kg) was administered subcutaneously underneath the skin dorsally. Animals were returned to a clean cage, single housed, and given acetaminophen (75 mg/d PO, Bio-Serv). Animals received subsequent injections of saline or ELP-VEGF-B (50mg/kg) on GD15 and GD17, and acetaminophen tablets were administered daily. Additionally, body weights were taken daily on all animals until they were euthanized.

Blood Pressure Measurements.

On GD17, animals were anesthetized with 3% isoflurane and maintained at 2.5% isoflurane with 100% oxygen as a carrier gas. Fur was shaved ventrally on the neck and dorsally between the shoulder blades. The skin over these areas were sanitized with betadine and 70% ethanol. A 1-inch incision was made in the ventral neck area, and an indwelling catheter (V1 and V3 tubing, Scientific Commodities) was placed in the carotid artery, routed under the skin, externalized dorsally between the shoulder blades, and secured with VetBond. The ventral incision was also closed with VetBond. On GD18, rats were acclimated to restrainer cages for 30 minutes before 30 minutes of continuous mean arterial pressure (MAP) measurements were taken directly via the carotid catheters. MAP was taken via Cobe III pressure transducers (CDX Sema), and data were collected and analyzed using receivers, amplifiers, and Lab Chart 7 PowerLab software from AD Instruments. Carotid catheters were flushed once with 30% heparin in saline at the time of implantation and before blood pressure measurement. MAP for each animal was determined as the average of 1-minute averages over the 30-minute recording period. Blood pressure analysis was performed by J.P.W., who was blinded to the treatment groups of each animal. Performance of the RUPP surgery on GD13 and blood pressure measurement on GD18 represents a one-day difference than many other RUPP studies, which often span GD14 – GD19 or GD20. However, no differences in the primary endpoints (maternal blood pressure, fetal growth restriction, fetal reabsorption rate) were observed compared to published studies or our historical data.

Blood and Tissue Collection.

After MAP measurements were taken, animals were anesthetized, and blood was drawn via the abdominal aorta with a butterfly needle. Both plasma and serum were harvested into BD Vacutainer tubes. Animals were then exsanguinated before maternal heart, kidneys, aorta, and renal arteries were harvested, weighed, and stored in cryotubes that were then frozen in liquid nitrogen. Urine was also collected from the bladder and flash frozen in cryotubes. Viable pups and their placentae were removed from both uterine horns and weighed, while the number fetal resorptions were recorded. Placentae were placed into cryotubes and flash frozen with liquid nitrogen. “Pup Weight” and “Placental Weight” were calculated as the average weight of all viable pups per dam. Reabsorption rates were calculated by dividing the number of non-viable offspring by the total number of offspring for each dam.

Measurement of Plasma and Urine Markers.

At euthanasia on GD18, plasma and serum were collected in appropriate containers (BD Vacutainer), spun, and frozen in liquid nitrogen for storage at −80 °C. Urine was also collected from the bladder of pregnant dams and stored at −80 °C after being flash frozen with liquid nitrogen. Plasma levels of TBARS were measured using a commercially available colorimetric assay (Cayman). Total nitrate/nitrite and 15-isoprostane levels were measured in the urine using a commercially available colorimetric assay (Cayman) and ELISA kit (Oxford Biomedical Research Inc), respectively. Plasma or urine from 7–10 rats per group was used in these two assays due to the limited quantity of plasma or urine available to be assayed.

Renal and Placental Gene Expression.

RNA was extracted and purified from both the placenta and renal cortex of animals from each group (Renal cortex from 6 rats per group and placentae from 9–10 rats per group were used). RNA concentration was determined using a Nanoquant 2000c (Thermo Fisher). cDNA was synthesized using RevertAid First Strand cDNA Synthesis Kit (Thermos Scientific) and 300 ng of RNA. Quantitative real-time PCR was performed using a C1000 Touch Thermal Cycler and CFX 96 Optics Module Real-Time system head (Bio-Rad). Taqman HostStart (Thermo Scientific), nuclease-free water, and primers for GPX1, GPX2, or SOD1 (Thermo Scientific) were combined to make master mixes before being combined with cDNA and evaluated by qRT-PCR. All samples were measured in duplicate and normalized to their own β-actin expression (dCT). The average of the measurement of each sample was normalized to combined average expression of the control group (sham saline) (ddCT).

Tube Formation using HGME Cells.

A 24-well, sterile, and non–tissue-culture-treated plate was coated with 300 uL of growth factor reduced Matrigel (BD Biosciences) and incubated at 37 °C in a humidified incubator with 5% CO2 for 1hr. HGME cells (Cell Systems) were serum and growth factor starved for 2 to 3 hours before seeding them over Matrigel-coated wells at 50,000 cells/well in basal media containing 0.1 mg/mL of heparin supplemented with 3% (v/v) of plasma from pools of plasma from each treatment group. Pools of plasma from rats in each treatment group were generated by combining an equal volume of plasma from rats within each group (n=5 randomly chosen rats per group). The cells were incubated at 37 °C in a humidified incubator with 5% CO2 for 6 hours. After a 6-hour incubation, cells were imaged using a brightfield microscope at 2x magnification (Cytation 7, Biotek). Additionally, five nonoverlapping fields per well were imaged at 20x (Nikon) and the tubes between 2 cell nodes were counted for each field and averaged for each well. The data represent the mean ± standard deviation of 3 independent experiments.

Statistical Analysis.

Results from all experiments are expressed as mean ± standard deviation. HGME cell proliferation was analyzed using a 2-way ANOVA, with factors of protein treatment and dose, with post hoc Sidak correction for multiple comparison of each experimental mean to the control group. HGME cell Matrigel invasion was analyzed using a 1-way ANOVA with post hoc Turkey’s correction for multiple comparison of each experimental mean to the control group. In the efficacy experiments, preset removal criteria included any RUPP-treated rat with <10% fetal resorption (unsuccessful RUPP surgery) or >90% fetal resorption. Data from these animals were excluded from all assays, and the final n numbers reported do not include these excluded rats. HGME tube formation, mean arterial pressures, plasma MDA levels, urinary 15-isoprostane levels, placenta weight, maternal heart and kidney weight, urinary nitrate/nitrite levels, reabsorption rate, litter size, and pup weight were analyzed by 2-way ANOVA, with factors for sham or RUPP surgery and saline or ELP-VEGF-B treatment, with a post hoc Sidak multiple comparisons analysis of the relevant treatment groups. Maternal body weights were analyzed with a 2-way repeated-measures ANOVA with a post hoc Turkey’s multiple comparisons. Gene expression and plasma toxicology were analyzed by 2-way ANOVA with a post hoc Sidak multiple comparison analysis of the relevant treatment groups. Statistical significance was accepted for p < 0.05 for all analyses. Statistics were calculated using GraphPad Prism.

Results

In Vitro Assessment of ELP-VEGF-B.

Previously, we determined that ELP-VEGF-A administration was effective for attenuating increased blood pressure in the RUPP model. However, the positive outcomes were accompanied by some undesired side effects which were likely attributable to continuous intraperitoneal administration combined with ELP-VEGF-A’s activation of the Flk-1 (VEGFR2) receptor, which is responsible for stimulating endothelial cell proliferation, migration, and angiogenesis. The ELP-VEGF-B fusion protein was developed with the goal to bind sFlt-1, freeing endogenous VEGF-A from sFlt-1 sequestration, while not directly activating Flk-1 with the exogenous therapeutic. To compare the in-vitro angiogenic capacity of ELP-VEGF-A and ELP-VEGF-B, a series of assays was performed using human glomerular microvascular endothelial (HGME) cells. A Matrigel-invasion assay demonstrated the potent chemokine function of ELP-VEGF-A, but not ELP-VEGF-B. ELP-VEGF-A treatment caused a significant increase in HGME invasion through the Matrigel coated chamber, while ELP-VEGF-B had no effect on invasion when compared to non-treated media (Figure 1A & 1B). ELP-VEGF-A also stimulated proliferation of HGME cells in a dose dependent manner, similar to previous studies9,14,15. The mitogenic effect of ELP-VEGF-B, on the other hand, was weaker than ELP-VEGF-A and did not significantly stimulate proliferation at these same doses (Figure 1C).

Figure 1.

Figure 1.

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Comparative Analysis of ELP-VEGF-A and ELP-VEGF-B. HGME Matrigel invasion was assessed using ELP-VEGF-A or ELP-VEGF-B (100 nM) as a chemoattractant (A. & B.). HGME proliferation (C.) was measured to compare the mitogenic drive of ELP-VEGF-B to ELP-VEGF-A. Data points represent independent experiments, and lines and error bars represent mean +/− SD. * Statistically significant increase relative to control saline treatment, scale bar = 200 μm.

Biodistribution and Pharmacokinetics of ELP-VEGF-B in Pregnant Rodents.

Our prior work with ELP-VEGF-A utilized continuous intraperitoneal infusion to test the feasibility of the protein for treatment of preeclampsia. To examine more clinically relevant delivery routes, here ELP-VEGF-B was delivered intravenously (IV) on GD13 to determine its acute plasma clearance and biodistribution and by subcutaneous (SC) administration every other day beginning on GD13 to determine its bioavailability via this minimally invasive route. Following IV injection, ELP-VEGF-B plasma levels cleared with a biphasic pattern (Figure 2A), similar to previous reports with related ELPs9,16–18. Four hours after injection, ELP-VEGF-B accumulated predominantly in the maternal kidneys, with a lesser amount of protein present in the liver and low levels in the maternal lung, brain, and spleen (Figure 2B and C). ELP-VEGF-B was also detectable in the placentae, but was not detectable in the pups, confirming a lack of placental transfer (Figure 2B and C). In an attempt to attain a slower release profile and to facilitate repeated dosing, in a separate cohort of rats, ELP-VEGF-B was administered by repeated SC dosing every other day beginning on GD13. Plasma data revealed that the protein was rapidly absorbed into circulation, and it cleared via a similar two-compartment clearance mechanism (Figure 2D) with a terminal half-life of 5.2 – 6.4 hours. On GD18 following this SC dosing regimen, ELP-VEGF-B accumulated with a very similar biodistribution to the acute IV administered protein, primarily in the kidneys and liver (Figure 2E and F). Importantly, ELP-VEGF-B was present in the placenta, but there was little to no accumulation of ELP-VEGF-B in the pups (Figure 2E and F).

Figure 2.

Figure 2.

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Pharmacokinetics and Biodistribution of ELP-VEGF-B in Pregnant Rats. Plasma pharmacokinetics and biodistribution of ELP-VEGF-B were determined following IV injection on GD13 (A – C, 10 mg/kg dose) or SC injection on GD13, GD15, and GD17 (D – F, 50 mg/kg dose). Biodistribution was determined 4h after IV injection on GD13 (B,C) or on GD18 following repeated SC administration on alternating days (E,F) by whole-organ ex vivo fluorescence imaging. Representative maternal organs (heart, lungs, brain, spleen, kidneys, liver - top panels) and placenta and pups (bottom panels) are shown for IV injected rats on GD14 (C) and SC injected rats on GD18 (F). **, Levels undetectable over autofluorescence.

ELP-VEGF-B Attenuates Symptoms of Preeclampsia in the RUPP Model.

Since ELP-VEGF-B was bioavailable following SC administration, and since a dose of 50 mg/kg resulted in plasma levels expected to be efficacious based on our previous results with ELP-VEGF-A9, a SC dosing regimen administered on GD13, GD15, and GD17 was used in the RUPP model beginning immediately after induction of placental ischemia. Daily maternal body weights were tracked from GD13-GD18 in all groups. Body weights were significantly lower from GD15-GD18 in RUPP treated animals when compared to sham saline animals (Figure 3A). These results were consistent with our prior work in this model and mediated by the loss of fetal and placental mass following the RUPP surgery9. A hallmark of the RUPP model is an increase in blood pressure that occurs after placement of the ovarian and aortic clips19. The RUPP procedure induced a significant increase in MAP compared to sham saline animals (p<0.05, Figure 3B). Additionally, in RUPP animals treated with ELP-VEGF-B, there was a significant reduction in MAP (p<0.05) compared to RUPP animals that received saline, lowering MAP to levels that were not different from control rats. ELP-VEGF-B administration had no effect on MAP in sham animals.

Figure 3.

Figure 3.

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ELP-VEGF-B normalizes blood pressure in the RUPP model of PE. A. Body weight of rats following sham or RUPP surgery on GD13 and treatment with saline or ELP-VEGF-B on GD13, GD15, and GD17. B. Mean arterial pressure was measured on GD18. Pup weights (C), placental weights (D), fetal reabsorption rate, a surrogate marker for the degree of placental ischemia (E), and the number of viable pups (F) were determined at euthanasia on GD18. Points represent individual animals’ values, and lines and error bars represent mean +/− SD. * Statistically significant difference relative to sham saline group. ⍏ Statistically significant difference relative to RUPP saline group.

All viable pups and placentae were harvested and weighed on the day of sacrifice. Measurement of pup weights (Figure 3C) revealed a trend for reduced pup weight when comparing the sham saline group to the sham, RUPP group (p = 0.06), which is consistent with the intrauterine growth restriction that is a feature of the RUPP model. Interestingly, in the ELP-VEGF-B treated groups, there was no significant difference between pup weights in the sham and RUPP groups (p = 0.77). There was a significant increase in fetal reabsorption rate and corresponding decrease in viable pup numbers in RUPP animals compared to shams (Figure 3E and F), which is consistent with our previous work in the RUPP model9,16. Fetal reabsorption in this model is a result of the RUPP intervention and serves as a surrogate marker of placental ischemia. Importantly, ELP-VEGF-B treatment did not affect reabsorption rate or viable pup number in either sham or RUPP operated groups. Additionally, there were no significant differences in kidney (Figure S1A), heart (Figure S1B), or placental weights (Figure 3D) among the treatment groups.

A toxicology panel was performed on plasma collected from all animals on GD18. ELP-VEGF-B treatment had no significant toxicological effect on plasma markers of renal function, hepatic function, or tissue injury (Table 1). Plasma albumin was increased in RUPP saline animals compared to sham ELP-VEGF-B treated animals, but was not elevated in RUPP operated rats treated with ELP-VEGF-B.

Table 1.

Plasma Markers of Renal and Liver Function. Plasma samples collected on GD18 were assessed for markers of renal and hepatic function and kidney function using a Vet AXCEL chemistry analyzer. Data represents mean +/− SD. [Aspartate Transaminase (AST), Alanine Transaminase (ALT), Blood Urea Nitrogen (BUN, Gamma-Glutamyl Transferase (GGT), Lactate Dehydrogenase (LDH), Total Bilirubin (TBILI)]

Sham Saline Sham ELP-VEGF-B RUPP Saline RUPP ELP-VEGF-B
Albumin (g/dL) 2.94 ± 0.45 2.87 ± 0.40 3.36 ± 0.32* 3.14 ± 0.39
AST (U/L) 107.3 ± 39.28 100.7 ± 53.03 110.6 ± 51.53 123.8 ± 54.09
ALT (U/L) 48.00 ± 11.86 49.20 ± 10.24 49.00 ± 9.00 52.70 ± 18.72
BUN (mg/dL) 22.90 ± 7.6 19.70 ± 3.802 23.90 ± 5.86 23.80 ± 6.53
Creatinine (mg/dL) 0.56 ± 0.13 0.58 ± 0.15 0.55 ± 0.08 0.54 ± 0.14
GGT☨(U/L) 7.00 ± 0.00 7.00 ± 0.00 7.00 ± 0.00 7.00 ± 0.00
LDH (U/L) 389.3 ± 321.4 365.4 ± 484.9 365.9 ± 265.2 556.8 ± 614.4
TBILI (mg/dL) 1.00 ± 0.55 0.99 ± 0.68 0.66 ± 0.23 0.66 ± 0.30
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*

RUPP + Saline levels were significantly elevated relative to Sham + ELP-VEGF-B, p < 0.05, two-way ANOVA with post-hoc Sidak correction for multiple comparisons.

☨

GGT levels were at the lower detectable limit for all samples.

Effect of Placental Ischemia and ELP-VEGF-B on Markers of Oxidative Stress.

Oxidative stress has been demonstrated to be an important mechanism in promoting endothelial dysfunction and hypertension in the RUPP model20. In addition, VEGF-B has been shown to stimulate a host of antioxidant genes that play a critical role in the elimination of reactive oxygen species21,22. Select probes of key antioxidant genes including glutathione peroxidase 1 and 2 (GPX1, GPX2) and superoxide dismutase 1 (SOD1) were chosen based on previous literature showing their upregulation in response to VEGF-B in the retina of mice21. Renal cortex and placental expression of these target genes were examined due to the roles of these organs as either directly affected by placental ischemia or as the target of circulating factors produced during placental ischemia. Neither the RUPP procedure nor ELP-VEGF-B administration significantly affected expression of the chosen antioxidant genes in the placenta of these animals (Figure 4A). In the renal cortex, placental ischemia alone induced the expression of GPX1, GPX2, and SOD1 when compared to sham saline controls. ELP-VEGF-B treatment also increased expression of GPX1 and GPX2 in sham animals compared to sham saline controls, and GPX1 was increased to a similar level in RUPP animals treated with ELP-VEGF-B (Figure 4B). Thiobarbituric acid reactive substances (TBARS) and 15-isoprostane levels were also measured on the collected plasma and urine, respectively, as surrogate markers of oxidative stress. There were no significant differences among any of the experimental groups in both the TBARS and 15-isoprostane assays (Figure 4C and D). Since induction of antioxidant genes were similar in response to both placental ischemia and ELP-VEGF-B treatment, and since there were no differences in surrogate reactive oxygen species markers, the results were not sufficient to conclude that ELP-VEGF-B – induced antioxidant activity was responsible for the reduced blood pressure in this model.

Figure 4.

Figure 4.

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Effect of Placental Ischemia and ELP-VEGF-B on Markers of Oxidative Stress. Expression of three antioxidant genes, GPX1, GPX2, and SOD1, was measured in both placental lysate (A) and renal cortex lysate (B) from each treatment group. Plasma TBARS (C) and urinary 15-isoprostance (D) were measured in plasma and urine collected at sacrifice on GD18. Points represent individual animals’ values, and lines and error bars represent mean +/− SD. * Statistically significant difference relative to sham saline group.

Effect of Placental Ischemia and ELP-VEGF-B on Urinary Nitrate/Nitrite.

Certain isoforms of VEGF, particularly VEGF-A, stimulate eNOS phosphorylation, which contributes to the increase in nitric oxide observed with VEGF administration23,24. Nitric oxide plays an important role in vascular relaxation and overall endothelial cell health. Utilizing the RUPP model, previous experiments have shown that nitric oxide is reduced with the RUPP procedure and that administration of ELP-VEGF-A increases surrogate markers of nitric oxide production, specifically in the kidney9. While data suggest that the VEGF-B isoform does not possess the direct ability to stimulate nitric oxide production25, it is possible that VEGF-B can displace VEGF-A from both Flt-1 receptor and sFlt-1 which could lead to greater stimulation of the Flk-1 receptor, thus indirectly stimulating nitric oxide production. Total nitrate and nitrite were measured in the urine of all rats using a colorimetric ELISA, and neither the RUPP procedure nor ELP-VEGF-B caused any significant effects on total urinary nitrate and nitrite levels (Figure S2).

ELP-VEGF-B Treatment Attenuates Angiogenic Imbalance in the RUPP Model.

An imbalance in angiogenesis has been an important key to understanding the pathogenesis of preeclampsia. More recently, the balance between key factors like sFlt-1 and PlGF have been used as early markers for preeclampsia in pregnant women26. The RUPP model has been shown to also possess characteristics of this angiogenic imbalance27. To test whether ELP-VEGF-B altered this angiogenic imbalance, plasma from each treatment group was used in a tube formation assay performed using HGME cells plated on growth factor-reduced Matrigel. When cells were treated with basal media supplemented with 3% plasma pooled from rats in each group, plasma from RUPP animals caused a significant reduction in tube formation when compared to plasma from sham saline animals (Figure 5). Plasma from sham animals treated with ELP-VEGF-B caused no significant change in tube formation when compared to sham saline animals. However, plasma from RUPP animals treated with ELP-VEGF-B caused a significant increase in tube formation when compared to RUPP animals treated with saline (Figure 5). These data support the hypothesis that ELP-VEGF-B promotes a restoration of balance among pro- and anti-angiogenic factors in the plasma of placental ischemic rats.

Figure 5.

Figure 5.

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In-Vitro Evaluation of the Angiogenic Potential of Rat Plasma following Placental Ischemia and ELP-VEGF-B Treatment. Plasma harvested on GD18 was pooled using equal volumes of plasma from rats within each group. HGME cells plated on growth factor reduced Matrigel were treated with basal media supplemented with 3% rat plasma and analyzed for tube formation (A, quantified in B). Points represent three independent experiments, and lines and error bars represent mean +/− SD. * Statistically significant difference relative to sham saline group. ⍏ Statistically significant increase relative to RUPP saline group.

Discussion

The development of therapies for use during pregnancy is challenging due to the need to consider the health of both the mother and the baby. Treating preeclampsia is especially challenging because many of the most potent and effect anti-hypertensive drugs have undesired teratogenic effects on developing fetuses28. Even recently repurposed drugs like sildenafil, originally developed as an anti-hypertensive, have been shown to potentially induce adverse effects on pulmonary function after birth in babies exposed to the drug in utero29. This leaves the field in need of effective therapies to treat the preeclampsia syndrome that do not affect fetal growth or development and that minimize fetal exposure to the therapeutic.

The novel therapeutic, ELP-VEGF-B, was designed to antagonize one of the major pathways driving the maternal preeclampsia syndrome, the increase in circulating sFlt-1 levels. ELP-VEGF-B was maternally sequestered and reduced maternal MAP after multiple subcutaneous doses in a rodent model of placental ischemia. These encouraging results were further bolstered by a lack of toxicological side effects of ELP-VEGF-B administration in the mother and no adverse effects of ELP-VEGF-B on fetal survival or fetal weight. In fact, the fetal growth restriction that is a feature of the RUPP model was not observed in RUPP rats treated with ELP-VEGF-B. The efficacy observed in this study was consistent with other studies using recombinant VEGF-A10,30 as well as with our previously tested ELP-VEGF-A chimera9. The attenuation of blood pressure by ELP-VEGF-B was most likely due to one of two possible mechanisms. First, ELP-VEGF-B administration likely resulted in an occupation of the Flt-1 receptor on the endothelium and/or sequestered circulating sFlt-1, which displaced endogenous VEGF-A and restored the balance angiogenic and anti-angiogenic proteins. Secondly, ELP-VEGF-B administration may have directly stimulated an antioxidant or anti-inflammatory/immune response to counter the oxidative stress and/or the inflammation induced in the RUPP model19,20. These effects could be mediated by direct engagement of the Flt-1 receptor by ELP-VEGF-B.

In normal pregnancy, it is common to observe activation of various maternal immune cells31. However, in preeclampsia there is an increase in pro-inflammatory factors that lead to the overactivation of monocytes and other inflammatory cells that contribute to maternal syndrome of preeclampsia32,33. Given the increase in inflammation and different immune cell activation of the RUPP model34,35, it is possible that ELP-VEGF-B administration could be countering the inflammatory milieu of preeclampsia through activation of Flk-1 in different cell types, though future work is needed to address this hypothesis.

Oxidative stress also plays an important role in the promotion of maternal endothelial dysfunction in preeclampsia36. Increased oxidative stress is believed to be a response to hypoxia secondary to placental ischemia37, and this increase in oxidative stress contributes to the development of hypertension and renal dysfunction seen in preeclampsia. The RUPP model, a model of induced placental ischemia, also exhibits this increase in oxidative stress38–40. VEGF-B has been recently shown to serve as an antioxidant through stimulation of the Flt-1 receptor22. Within this study, we analyzed oxidative stress by both gene expression and the measurement of oxidative stress metabolites. Interestingly, our results revealed that neither the RUPP surgery or ELP-VEGF-B administration had any effect on the measured oxidative metabolites. In addition to measuring metabolic markers of oxidative stress, the gene expression of key antioxidant genes that are known to be upregulated by VEGF-B21 were also measured. These genes included glutathione peroxidase 1 and 2 (GPX1 and GPX2, respectively), two enzymes responsible for inhibition reactive oxygen species (ROS), as well as superoxide dismutase (SOD1), which converts superoxide to hydrogen peroxide and oxygen. Gene expression was measured in the placentae, the direct target of ischemia, and in the kidneys, because biodistribution studies revealed high accumulation of ELP-VEGF-B in the kidneys of pregnant rats and because of the kidneys’ role in regulating blood pressure. In the placenta, there were no significant differences in gene expression amongst the treatment groups. In the kidney, the expression of GPX1, GPX2, and SOD1 was increased in response to the RUPP procedure alone, presumably in response to a circulating factor produced as a product of placental ischemia or potentially as a direct consequence of the increased blood pressure. Also, ELP-VEGF-B treatment significantly increased expression of both GPX1 and GPX2 in the renal cortex of both sham and RUPP operated animals. Since these genes were upregulated by both ELP-VEGF-B and by placental ischemia, we could not conclude that ELP-VEGF-B induced antioxidant gene expression was responsible for the reduction in MAP seen in RUPP operated rats treated with ELP-VEGF-B.

Another possible mechanism for the reduction of MAP by ELP-VEGF-B treatment is centered around the imbalance of antiangiogenetic to angiogenic factors that exist in preeclampsia. Placental ischemia is known to directly induce circulating levels of the anti-angiogenic protein sFlt-1, which is associated with a corresponding reduction in angiogenic factors such as VEGF and PlGF5,41,42. Disruption of the angiogenic balance promotes the vascular dysfunction that is responsible for some of the systemic effects on blood pressure and organ function seen in preeclampsia43. Specifically, excess levels of sFlt-1 and subsequently reduced levels of VEGF and PlGF are responsible for contributing to this process. Moreover, VEGF supplementation is associated with attenuation of the hypertensive effects of preeclampsia in animal models of the disease10,44, and sFlt-1 removal by plasma apheresis has been shown to be associated with acute lowering of blood pressure in early clinical testing8. It is important to note however, that excessive VEGF-A administration it not without consequences. As a potent angiogenic and vascular permeability factor, VEGF-A administration has the potential to induce unwanted effects mediated by its activation of the Flk-1 receptor. Because VEGF-B does not promote activation of the Flk-1 receptor, we postulated that it may be a safer sequestrant of sFlt-1. Our initial hypothesis was that ELP-VEGF-B would reduce the levels of free sFlt-1and restore the balance of angiogenic factors. A similar strategy has been employed by exogenous administration of another Flt-1 agonist, PlGF. Recombinant PlGF administration reduced sFlt-1 levels and prevented hypertension in the RUPP rat model11, and PlGF supplementation in a baboon model of placental ischemia reduced blood pressure and placental sFlt-1 mRNA levels, but not circulating sFlt-1 levels45. Here, we found that plasma from rats subjected to the RUPP procedure had reduced angiogenic potential, as indicated by an in vitro endothelial cell tube formation assay. Furthermore, angiogenic potential was restored in plasma from RUPP rats treated with ELP-VEGF-B. From this we can postulate that this shift in angiogenic potential, most likely mediated by indirect increases in VEGF-A due to ELP-VEGF-B competing for sFlt-1 and/or Flt-1, is at least partially responsible for the attenuation of the hypertension observed in this group. However, we were not able to reliably measure circulating sFlt-1 levels in the plasma from rats in this study (commercially available rat sFlt-1 ELISAs routinely yielded undetectable or inconsistent results, and a mouse sFlt-1 ELISA that is often used in the field did not react with recombinant rat sFlt-1 in our limited testing), preventing the ability to directly confirm this mechanism. Additionally, the restoration of angiogenic balance by treatment with ELP-VEGF-B was not due to direct angiogenic activity by ELP-VEGF-B, as in vitro studies demonstrated that ELP-VEGF-B lacks the potent stimulatory effect of ELP-VEGF-A on endothelial cell proliferation and matrix invasion. Taken together, these results are consistent with our initial hypothesis that ELP-VEGF-B is less directly angiogenic than ELP-VEGF-A but is still able to ameliorate the maternal preeclampsia syndrome by competing with endogenous VEGF-A for sFlt-1 binding.

While we observed very promising efficacy with ELP-VEGF-B administration in this preclinical model of preeclampsia, there exist some limitations that merit future studies. First, this study utilized a rodent model of preeclampsia which utilizes mechanically induced placental ischemia. While the RUPP model effectively mimics the hypertension and maternal cardio-renal effects of midgestational placental ischemia, it is not possible to observe effects of interventions on the development of abnormal placentation and earlier stages of the disease using this model. Another limitation is the short term of treatment available with the RUPP procedure. Since the RUPP procedure was performed on GD13 and the animals were sacrificed on GD18, the treatment window was just 5 days. This may not have been sufficient time to observe all the effects of ELP-VEGF-B administration. Finally, while we collected strong data implicating restoration of angiogenic balance by ELP-VEGF-B and possibly ruled out an antioxidant effect of ELP-VEGF-B, there may be other pathways affected by this molecule that were not studied in this work, which accentuates the need for further analysis of the mechanism of action of this molecule.

In summary, ELP-VEGF-B was maternally sequestered and showed therapeutic efficacy in a rodent model of placental ischemia with no signs of toxicological side effects after repeated subcutaneous administration. Compared to our previously tested ELP-VEGF-A chimera, ELP-VEGF-B was similarly efficacious for attenuating hypertension in the rodent model of placental ischemia without inducing aberrant off-target angiogenesis or vascular permeability. Although blood pressure was reduced in RUPP treated animal, the drop in blood pressure did not produce a profound hypotension. Thus, it is unlikely that ELP-VEGF-B administration would cause deleterious effects on the developing fetus due to placental hypo-perfusion. In addition to this, there was a lack of fetal growth restriction in RUPP rats treated with ELP-VEGF-B. These results suggest that ELP-VEGF-B likely has beneficial effects on the developing fetus in this setting. Future work will involve further evaluation of the mechanism by which ELP-VEGF-B induces these effects as well as examining the interactions between ELP-VEGF-B and sFlt-1. We also plan to validate ELP-VEGF-B, as well as other ELP-fusion proteins, in other preclinical preeclampsia models, including large animal models. Overall, we believe that this protein as well as other ELP fusion proteins potentially represent a novel class of therapeutics to address many of the current issues with therapeutic treatment during pregnancy.

Perspectives

Preeclampsia is quickly becoming one of most prevalent causes of morbidity and mortality during pregnancy. This problem is compounded by the relative lack of effective therapies to address the symptoms of the disease. Currently, we understand that the etiology of preeclampsia is both complex and multifactorial. However, we know that the increase in certain anti-angiogenic factors, like s-Flt1, and the subsequent dysregulation of endothelial function play an important role in the progression of this disease in a large proportion of patients. In this study, we examined the efficacy of VEGF-B administration in addressing both the dysregulation of anti-angiogenic factors, like s-Flt1, and the hypertensive phenotype seen in preeclampsia. With this work we determined that VEGF-B administration, when given as a fusion with the Elastin-like Polypeptide (ELP) drug carrier, was: 1) bioavailable after subcutaneous administration, 2) sequestered to the maternal circulation and did not enter the fetus, 3) effective at significantly attenuating the hypertensive response seen in a rodent model of preeclampsia as well as 4) restoring the angiogenic environment in the plasma of rats with placental ischemia. Overall, ELP-VEGF-B has potential as a therapeutic candidate to address one of the many pathways that play an important role in the development of preeclampsia.

Supplementary Material

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Novelty and Significance.

What is new:

  • The creation of a novel chimeric fusion protein that combines an isoform of vascular endothelial growth factor, VEGF-B, with an elastin-like polypeptide (ELP) drug carrier.

  • An examination of the efficacy of this fusion protein for the treatment of preeclampsia utilizing the reduced uterine perfusion pressure (RUPP) model of preeclampsia.

What is relevant:

  • ELP-VEGF-B administration in a rodent model of placental ischemia was maternally sequestered and significantly attenuated the hypertensive response to placental ischemia.

Acknowledgements.

The authors thank Rowshan Begum for assistance purifying proteins for this study.

Sources of Funding.

This work was supported by NIH grants R01HL121527 and 1F31HL151180-01. Plasma chemistry analysis was performed by the UMMC Analytical, and Assay Core supported by NIH grants P01HL51971 and P20GM104357.

Footnotes

Disclosures. G.L.B. is owner of Leflore Technologies, a private company working to commercialize ELP-based drug delivery technologies. G.L.B. and E.M.G. are inventors on patents related to this work.

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