Elastin-Like Polypeptide: VEGF-B Fusion Protein for Treatment of Preeclampsia

Preeclampsia is characterized by the development of elevated blood pressure during the second and third trimesters 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 VEGF (vascular endothelial growth factor) receptor sFlt-1 (soluble Fms-like tyrosine kinase receptor 1). Previously, administration of exogenous VEGF-A, bound to a carrier protein called ELP (elastin-like polypeptide), 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 using 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 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 with saline treated animals following induction of placental ischemia (123.38±11.4 versus 139.98±10.56 mm Hg, 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 nonangiogenic, maternally sequestered protein with potential efficacy for treatment of preeclampsia.

Characterization of Mitochondrial Bioenergetics in Preeclampsia

Preeclampsia (PE) is characterized by new onset hypertension during pregnancy and is associated with oxidative stress, placental ischemia, and autoantibodies to the angiotensin II type I receptor (AT1-AA). Mitochondrial (mt) dysfunction in PE and various sources of oxidative stress, such as monocytes, neutrophils, and CD4 + T cells, have been identified as important players in the pathophysiology of PE. We have established the significance of AT1-AA, TNF-α, and CD4 + T cells in causing mitochondrial (mt) dysfunction in renal and placental tissues in pregnant rats. Although the role of mt dysfunction from freshly isolated intact placental mitochondria has been compared in human PE and normally pregnant (NP) controls, variations among preterm PE or term PE have not been compared and mechanisms contributing to mt ROS during PE are unclear. Therefore, we hypothesized PE placentas would exhibit impaired placental mt function, which would be worse in preterm PE patients than in those of later gestational ages. Immediately after delivery, PE and NP patient’s placentas were collected, mt were isolated and mt respiration and ROS were measured. PE patients at either < or >34 weeks gestational age (GA) exhibited elevated blood pressure and decreased placental mt respiration rates (state 3 and maximal). Patients delivering at >34 weeks exhibited decreased Complex IV activity and expression. Placental mtROS was significantly reduced in both PE groups, compared to NP placental mitochondria. Collectively, the study demonstrates that PE mt dysfunction occurs in the placenta, with mtROS being lower than that seen in NP controls. These data indicate why antioxidants, as a potential target or new therapeutic agent, may not be ideal in treating the oxidative stress associated with PE.

Progesterone Induced Blocking Factor Reduces Hypertension and Placental Mitochondrial Dysfunction with in Response to sFlt-1 during Pregnancy

Preeclampsia (PE) is characterized by new onset hypertension in association with placental ischemia, reduced fetal weight, elevated soluble fms-like tyrosine kinase-1 (sFlt-1), and placental mitochondrial (mt) dysfunction and oxidative stress (ROS). Progesterone induced blocking factor (PIBF) is a product of progesterone signaling that blocks inflammatory processes and we have previously shown PIBF to lower mean arterial blood pressure (MAP) and sFlt-1 in a rat model of PE. Infusion of sFlt-1 causes hypertension and many characteristics of PE in pregnant rodents, however, its role in causing mt dysfunction is unknown. Therefore, we hypothesize that PIBF will improve mt function and MAP in response to elevated sFlt-1 during pregnancy. We tested our hypothesis by infusing sFlt-1 via miniosmotic pumps in normal pregnant (NP) Sprague-Dawley rats (3.7 μg·kg−1·day−1) on gestation days (GD) 13–19 in the presence or absence of PIBF (2.0 µg/mL) injected intraperitoneally on GD 15 and examined mean arterial blood pressure (MAP) and placental mt ROS on GD 19. sFlt-1 increased MAP to 112 + 2 (n = 11) compared to NP rats (98 + 2 mmHg, n = 15, p < 0.05), which was lowered in the presence of sFlt-1 (100 + 1 mmHg, n = 5, p < 0.05). Placental mtATP was reduced in sFlt-1 infused rats versus NP controls, but was improved with PIBF. Placental mtROS was elevated with sFlt-1 compared to NP controls, but was reduced with PIBF. Sera from NP + sFlt-1 increased endothelial cell mtROS, which was attenuated with PIBF. These data demonstrate sFlt-1 induced HTN during pregnancy reduces placental mt function. Importantly, PIBF improved placental mt function and HTN, indicating the efficacy of improved progesterone signaling as potential therapeutics for PE.

Low Dose of IL-2 Normalizes Hypertension and Mitochondrial Function in the RUPP Rat Model of Placental Ischemia

IL-2 is a cytokine released from CD4+T cells with dual actions and can either potentiate the inflammatory response or quell a chronic inflammatory response depending on its circulating concentration. IL-2 is elevated in many chronic inflammatory conditions and is increased during preeclampsia (PE). PE is characterized by new-onset hypertension during pregnancy and organ dysfunction and increasing evidence indicates that proinflammatory cytokines cause hypertension and mitochondrial (mt) dysfunction during pregnancy. The reduced uterine perfusion pressure (RUPP) model of placental ischemia is a rat model of PE that we commonly use in our laboratory and we have previously shown that low doses of recombinant IL-2 can decrease blood pressure in RUPP rats. The objective of this study was to determine the effects of a low dose of recombinant IL-2 on multi-organ mt dysfunction in the RUPP rat model of PE. We tested our hypothesis by infusing recombinant IL-2 (0.05 ng/mL) into RUPP rats on GD14 and examined mean arterial pressure (MAP), renal, placental and endothelial cell mt function compared to control RUPP. MAP was elevated in RUPP rats (n = 6) compared to controls (n = 5) (122 ± 5 vs. 102 ± 3 mmHg, p < 0.05), but was reduced by administration of LD recombinant IL-2 (107 ± 1 vs. 122 ± 5 mmHg, n = 9, p < 0.05). Renal, placental and endothelial mt ROS were significantly increased in RUPP rats compared to RUPP+ IL-2 and controls. Placental and renal respiration rates were reduced in RUPP rats compared to control rats but were normalized with IL-2 administration to RUPPs. These data indicate that low-dose IL-2 normalized multi-organ mt function and hypertension in response to placental ischemia.

Abstract P225: Inhibiting B Cell Activating Factor Normalizes The Hypertension And Cytolytic Natural Killer Cells Associated With Placental Ischemia

Preeclampsia (PE) is new onset hypertension (HTN) during pregnancy and is associated with a pro-inflammatory state with increased T Cells, B Cells, cytolytic Natural Killer (cNK) Cells, and Agonistic Antibodies to the Angiotensin II Type 1 Receptor (AT1-AA) compared to Normal Pregnancy (NP). The Reduced Uterine Perfusion Pressure (RUPP) model of placental ischemia recapitulates these features. We have shown that blocking communication between T Cell CD40L and B Cell CD40 or B Cell depletion prevents HTN and AT1-AA in rat models of placental ischemia induced HTN. This suggests that T Cell dependent B Cell activation is involved in the HTN and AT1-AA seen in PE. B2 B cells maturing into antibody producing plasma cells are the product of T Cell Dependent B Cell Interactions. B Cell activating factor (BAFF) is an integral cytokine in the development of B2 cells specifically. Therapeutic antibodies against BAFF are used in treatment for Systemic Lupus Erythematosus with long-term treatment with Anti-BAFF antibodies reducing patient circulating B cells. Thus, we hypothesize that Anti-BAFF antibodies will selectively deplete B2 cells, therefore reducing HTN and cNK Cells in the RUPP rat model of PE. To test this hypothesis, Gestation Day (GD) 14 pregnant rats underwent the RUPP procedure and some were treated with 1 mg/kg Anti-BAFF antibodies by infusion via jugular catheters inserted 24 hours prior. On GD19, blood pressure (MAP) was measured and circulating, placental, and splenic B Cells and NK cells were measured by flow cytometry. RUPP rats had elevated MAP (119±2 mmHg, n=12) compared to NP (100±3 mmHg, n=9) which was normalized by Anti-BAFF treatment (100±3 mmHg, n=11)(p<0.05). RUPP fetuses were smaller at GD19 (1.97±0.08 g; n=12) compared to NP fetuses (2.207±0.1 g)(p<0.05), and this was unchanged with Anti-BAFF (1.82±0.1 g). RUPP rats had elevated cNK cells (9.1±2%, n=7) compared to NP (3.1±3%, n=3) which was attenuated with anti-BAFF therapy (3.6±1.3%, n=6). RUPP rats had elevated splenic B Cells (4±0.7%, n=8) compared to NP (2±0.7%, n=4) which was reduced by Anti-BAFF (2.8%±1.1%, n=4). In conclusion, Anti-BAFF treatment reduces B cells and cNK cells and normalizes the HTN associated with placental ischemia without worsening fetal weight in a rat model of PE.

Abstract 25: Inhibition Of The Agonistic Ang II Type 1 Receptor Autoantibody In A Rat Model Of Preeclampsia Improves Uteroplacental Perfusion And Fetal Growth In Late Gestation

Placenta ischemia, the initiating factor in preeclampsia (PE), is associated with intrauterine growth restriction (IUGR) and increased blood pressure (BP) in offspring. Yet, the only treatment for PE is delivery of the baby and placenta. The Reduced Uterine Perfusion Pressure (RUPP) rat model induced by placental ischemia at gestational day 14 (G14) mimics many facets of human PE including pregnancy-specific hypertension, an increase in the agonistic ANG II Type 1 receptor autoantibody (AT1-AA), IUGR and increased BP in the offspring. Inhibition of AT1-AA using an epitope-binding inhibitory peptide ('n7AAc') attenuates increased BP at gestational day 19 in the RUPP. Yet, whether use of ‘n7aac’ improves fetal growth and mitigates increased BP in the offspring is unknown. Thus, we tested the hypothesis that maternal administration of ‘n7aac’ improves fetal growth by attenuating reduced uterine blood flow and impaired placental remodeling. Sham or RUPP surgery was performed at G14 with administration of vehicle or ‘n7aac’ (144μg/day) via mini osmotic pump until gestational day 20 (G20). At G20 uterine artery resistance index was significantly elevated in vehicle RUPP (0.69±0.02 mm/s n=10) compared to vehicle Sham (0.48±0.02 mm/s n=8) (P<0.0001) and not increased in treated RUPP (0.49±0.02 mm/s n=10) or treated Sham (0.48±0.02 mm/s n=9). Fetal weight was significantly reduced in vehicle RUPP (3.24±0.2 g) compared to vehicle Sham (3.92±0.05 g) (P=0.013) and not decreased in treated RUPP (3.70±0.04 g) or Sham (3.98±0.10 g). Litter size of viable pups at G20 was only reduced in treated RUPP (5.3±1.4) compared to vehicle Sham (11.56±0.7) (P=0.003). Importantly, using in vivo imaging, little to no auto fluorescence of rhodamine-labeled peptide (480 μg/kg/day, n=4) was detectable in the pups at G20. Thus, our results demonstrate that maternal treatment with ‘n7aac’ in the RUPP rat model of PE improve UARI, which is associated with improved fetal weight at G20 in response to placental ischemia. Whether this benefit continues to birth and mitigates increased BP in IUGR offspring is unknown but is the focus of future studies. In conclusion, inhibition of the AT1AA during PE may not only provide benefit to the mother, but may also be associated with benefit in the offspring.

Abstract 19: Maternal B Cell Depletion Lowers Blood Pressure And Improves Fetal Weights In Offspring In A Rat Model Of Preeclampsia

Preeclampsia (PE), new onset hypertension during pregnancy, is the leading cause of death and morbidity for the mother and low birth weight in offspring. PE women have activated B cells producing agonistic autoantibodies to the angiotensin II type I receptor (AT1-AA). We have shown Rituximab (R), used clinically for B cell depletion, lowers mean arterial pressure (MAP), B cells, and AT1-AA in the RUPP rat model of PE. Clinical studies show no untoward effects on offspring of pregnant women maintained on R for treating lymphoma, however, R is not used during PE therefore, effects of maternal B cell depletion on offspring survival and growth in response to placental ischemia is unknown. We hypothesize that R will deplete maternal B cells in RUPP rats without worsening the effect of placental ischemia on pup growth and survival. To test this hypothesis, R (250 mcg/kg) was given on gestation day (GD) 14 via mini-osmotic pump. On GD 19 B cells were measured by flow cytometry, and MAP and pup weights were recorded. A separate group of dams were allowed to deliver, pup weights were recorded within 12 hours and weekly until 16 weeks, and B cells were analyzed. A one-way ANOVA was used for statistical analysis. MAP increased in RUPP 123±2 (n=19, p<0.05) compared to NP controls 101±1 (n=18) and was 106±3 mmHg in RUPP+R (n=8, p<0.05). On GD19, maternal circulating B cells were 16±2 % (n=14) in RUPPs, 8±2 % in NP rats, (n=7, p<0.05), and 5.5±1% gate in RUPP+R (n=5, p<0.05). RUPP male and female offspring were smaller 5.11±0.23 g, 5.19±0.14 g (n=4, n=4) at birth than NP offspring 6.09±0.15 g, 5.87±0.12 g (n=6, p<0.05; n=6, p<0.05) or RUPP+R offspring 5.75±0.24 g, 5.36±0.28 g (n=6, p=0.11; n=6, p=0.67). At 12 weeks, male and female RUPP offspring had elevated circulating B cells (21±3, 20±1 % (n=6; n=9)) compared to NP (1±0.23, 1.6±0.06 % (n=4, p<0.05; n=3, p<0.04)) which was normalized in RUPP+R offspring (0.4±0.1, 0.3±0.03 % gate (n=3, p<0.05; n=8, p<0.05)). At 16 weeks, B cells were comparable in male offspring from NP (0.78±0.09 % (n=10)) and RUPP+R rats (0.80±0.04 % gate (n=3)). Our findings indicate that R lowers maternal circulating B cells and MAP in RUPP rats and improves fetal weight and circulating B cells, indicating that R does not worsen adverse fetal outcomes in response to placental ischemia.

Abstract 31: Esomeprazole Improves Blood Pressure, Intrauterine Growth, Inflammation, And Vascular Function During Placental Ischemia Through Inhibition Of NLRP3

Preeclampsia (PE), a multisystem hypertensive disorder of pregnancy is characterized by intrauterine growth restriction (IUGR), inflammation, and vascular dysfunction. NLRP3 inflammasome is a cytoplasmic complex that mediates inflammation and is implicated in CVD. Clinical studies show an association between PE and increased placental NLRP3 expression. We hypothesized that inhibition of NLRP3 using (1) a specific NLRP3 small molecule inhibitor, MCC950 (M9, 20 mg/kg/d, i.p.) or (2) esomeprazole (ESO, 3.5 mg/kg/d, oral), a therapeutic that is safe in pregnancy, would improve MAP, inflammation, IUGR, and vascular dysfunction in the reduced uterine perfusion pressure (RUPP) rat model of placental ischemia. Sham (S) or RUPP surgery was performed in pregnant Sprague Dawley rats on gestation day (GD) 14. A subset of rats from both groups received either vehicle, M9, or ESO on GD14-19 (n=9/group). On GD18, Uterine Artery Resistance Index (UARI) was measured via Doppler Ultrasound. MAP, fetal, and placental weight were measured, and blood and tissues were processed for additional analyses on GD19. MAP (mmHg) was elevated in RUPP (133±1) vs S (108±2; p<0.05). Treatment with M9 or ESO in RUPP decreased MAP (111±1 and 115±3, respectively; p<0.05 vs RUPP). Fetal weight (g) was reduced in RUPP (2.1±0.04) vs S (2.4±0.05), and ESO normalized fetal weight (2.4±0.01; p<0.05 vs RUPP). Placental NLRP3 mRNA expression increased 5-fold in RUPP vs S (p<0.05); and was less than 2 fold in M9 and ESO treated RUPP rats (p<0.05 vs RUPP). Inflammatory T-helper 17 and cytolytic NK cells, evaluated by flow cytometry, were increased in the circulation, placenta, and kidney of RUPP vs S controls. Treatment with M9 or ESO normalized both cell populations in all tissues (p<0.05 vs RUPP). UARI was increased in RUPP (0.71±0.03) versus S (0.56±0.01; p<0.05) and was decreased to 0.48±0.01 in M9 and 0.61±0.03 in ESO-treated RUPPs (p<0.05 vs RUPP). Renal vascular resistance (mmHg/mL/min/g) was increased in RUPP (42±8) vs S (23±4, p<0.05) and was normalized to 26±3 after treatment with M9 (p<0.05 vs RUPP). These data implicate NLRP3 in mediating inflammation and vascular dysfunction to cause maternal HTN and IUGR in RUPP; and identify NLRP3 as a potential target and ESO as a potential therapeutic for PE.

Interferon Gamma Neutralization Reduces Blood Pressure, Uterine Artery Resistance Index, and Placental Oxidative Stress in Placental Ischemic Rats

Preeclampsia (PE) is characterized by maternal hypertension, intrauterine growth restriction, increased cytolytic natural killer cells (cNKs), which secrete interferon gamma (IFNγ). However, the precise role of IFNγ in contributing to PE pathophysiology remains unclear. Using the Reduced Uterine Perfusion Pressure (RUPP) rat model of placental ischemia, we tested the hypothesis that neutralization of IFNγ in RUPPs will decrease placental reactive oxygen species (ROS) and improve vascular function resulting in decreased MAP and improved fetal growth. On Gestation Day (GD) 14, the RUPP procedure was performed and on GDs 15 and 18, a subset of normal pregnant rats (NP) and RUPP rats were injected with 10μg/kg of an anti rat-IFNγ monoclonal antibody. On GD18, Uterine Artery Resistance Index (UARI) was measured via Doppler Ultrasound and on GD19, mean arterial pressure (MAP) was measured, animals were sacrificed, and blood and tissues were collected for analysis. Increased MAP was observed in RUPP rats compared to NP and was reduced in RUPP+anti-IFNγ. Placental ROS was also increased in RUPP rats compared to NP and was normalized in RUPP+anti-IFNγ. Fetal and placental weights were reduced in RUPP rats, but were not improved following anti-IFNγ treatment. However, UARI was elevated in RUPP compared to NP and was reduced in RUPP+anti-IFNγ. In conclusion, we observed that IFNγ neutralization reduced MAP, UARI, and placental ROS in RUPP recipients. These data suggest that IFNγ is a potential mechanism by which cNKs in contribute to PE pathophysiology and may represent a therapeutic target to improve maternal outcomes in PE.