Search

Purpose One of the most common, and most vexing, obstetric complications is preeclampsia—a major cause of maternal and perinatal morbidity. Hallmarked by new-onset hypertension and a myriad of other symptoms, the underlying cause of the disorder remains obscure despite intensive research into its etiology. Although the initiating events are not clear, one common finding in preeclamptic patients is failure to remodel the maternal arteries that supply the placenta, with resulting hypoxia/ischemia. Intensive research over the past 2 decades has identified several categories of molecular dysfunction resulting from placental hypoxia, which, when released into the maternal circulation, are involved in the spectrum of symptoms seen in these patients—in particular, angiogenic imbalance and the activation of innate and adaptive immune responses. Despite these new insights, little in the way of new treatments for the management of these patients has been advanced into clinical practice. Indeed, few therapeutic options exist for the obstetrician treating a case of preeclampsia. Pharmacologic management is typically seizure prophylaxis, and, in severe cases, antihypertensive agents for controlling worsening hypertension. Ultimately, the induction of labor is indicated, making preeclampsia a leading cause of premature birth. Here, the molecular mechanisms linking placental ischemia to the maternal symptoms of preeclampsia are reviewed, and several areas of recent research suggesting new potential therapeutic approaches to the management of preeclampsia are identified.

Introduction Preeclampsia is a hypertensive syndrome of pregnancy with few pharmacological treatment options. The risk of for adverse fetal effects is a major limitation for drug development. Our lab has developed a protein-based drug carrier called elastin-like polypeptide (ELP) that is capable of stabilizing therapeutic proteins or peptides in the maternal circulation while preventing their placental transfer. Objective/Hypothesis We hypothesize that ELP can be optimized for maternally sequestered drug delivery by altering the polymer length. We further hypothesize that ELP can be fused to vascular endothelial growth factor (VEGF) to restore angiogenic balance while preventing fetal exposure to the therapeutic. Methods A library of ELP carriers ranging in size from 25 to 110 kDa was created. The pharmacokinetics, biodistribution, and placental transfer of each agent were determined in a rat pregnancy model. A 60 kDa ELP domain was fused to human VEGF-A121, and the pharmacokinetics were determined following repeat dosing intravenous or subcutaneous administration. Results The plasma half-life of ELP was inversely related to its molecular weight. As the size of the molecule increased, the renal ELP deposition decreased and the placenta and other maternal organ levels increased. Within the placenta, ELP was localized broadly in the labyrinth and along the chorionic plate. No ELP crossed the placental barrier at any size tested. When fused to VEGF, daily IV dosing lead to a 2.1 +/- 1.1 h half-life after each injection. SC administration lead to an increase in plasma ELP-VEGF levels over the first three hours, followed by a slow clearance phase over 24 h that was accompanied by dispersal of the protein from the SC injection site. Discussion This work demonstrates the utility of ELP as a tunable, long-circulating, non-toxic, maternally sequestered drug carrier. The ELP-VEGF fusion protein has potential as a novel biologic for treatment of preeclampsia.

Introduction Preeclampsia (PE) is a multifactorial disease in which little progress has been made to develop adequate therapy. One hallmark of this disease is a rise in systemic maternal inflammatory factors, many of which signal through the NF-kB pathway. Hypothesis We hypothesize that administration of a novel NF-κB antagonist, ELP-p50i, will attenuate the hypertension and inflammation in the preclinical RUPP model of placental insufficiency. Methods Animals were received on GD11 and received the sham or RUPP surgery on GD14 along with drug or vehicle administration. On GD18, carotid catheters were placed for blood pressure measurement on GD19. Aortic ET-1 was measured by qRT-PCR using Taq primers specific for rat ET-1 and normalized to rat β-actin (Thermofisher). Placental TNF-α was measured using a commercial ELISA specific for rat TNF-α) (R&D). Results RUPP animals had significantly increased blood pressure (122.2 ± 2.56 mmHg vs 99.3 ± 3.83 mmHg, p Discussion Our data suggest that ELP-p50i is able to attenuate placental ischemia-induced hypertension and correct symptoms such as endothelial dysfunction and increased inflammation, potentially creating a therapeutic option for preeclampsia.

Introduction Placental ischemia appears to be a central causative factor in the maternal symptoms of preeclampsia. The maternal/fetal interface in the placenta is covered in a meshwork resembling the endothelial glycocalyx; consisting of proteoglycans like Syndecan-1 (SDC-1). This glycocalyx is essential for preventing immune cell adherence, and shed glycocalyx fragments are pro-inflammatory. Objective/Hypothesis We hypothesize that hypoxia/ischemia will cause degradation and shedding of the placental glycocalyx. Methods Placental ischemia was studied using the rodent RUPP model, which induces placental ischemia from GD14–19. Acute hypoxia utilized culture BeWo trophoblasts exposed at 8% or 1% oxygen for 24 h to simulate the normal or ischemic placenta respectively. SDC-1 was measured via western blot in placenta and by commercial ELISA in cell culture. Results RUPP rats showed a significant decrease in placental syndecan-1 compared to sham controls (100 ± 33% vs 67 ± 17%, p Discussion Placental ischemia and acute hypoxia both contribute to trophoblast shedding of the placental glycocalyx in a manner dependent on heparanase, MMP activity, and oxidative stress.

Introduction Preeclampsia (PE) is a multifactorial disease in which little progress has been made to develop adequate therapy. One of the etiologies of this maternal syndrome includes a rise in maternal anti-angiogenic factors; such as sFlt-1. Here we describe a novel therapeutic antagonist of sFlt-1 based on placental growth factor (PlGF). Hypothesis We hypothesize that our novel therapeutic, ELP-PlGF, will attenuate the hypertension and endothelial dysfunction in the preclinical RUPP model of placental insufficiency. Methods Animals were received on GD11 and received the sham or RUPP surgery on GD14 along with drug or vehicle administration. On GD18, carotid catheters were placed for blood pressure measurement on GD19. Western blots used serum samples with an antibody specific for PlGF (Abcam) and were normalized to the amount of serum loaded per well. Results Untreated HUVEC cells had significantly greater tube formation compared to cells treated with sFlt-1 (44.6 + 7.4 tubes/field vs 29.3 + 6.4 tubes/field, p + 4.3 tubes/field vs 44.6 + 7.4 tubes/field, p = 0.998). RUPP animals had significantly increased blood pressure compared to normal pregnant counterparts (116.7 + 1.96 mmHg vs 92.4 + 3.32 mmHg, p + 1.96 mmHg vs 105.6 + 4.70 mmHg, p = 0.051). Western blots showed a trend for decreased circulating PlGF (1.0 + 0.18 vs 0.73 + 0.11-fold change, p = 0.26, n = 4) in RUPP animals, with a near- significant trend towards increased endogenous free PlGF with ELP-PlGF administration (0.73 + 0.11 vs 1.79 + 0.35, p = 0.051, n = 4). Discussion Angiogenic imbalance is one of the best characterized contributors to the development of preeclampsia, largely lead by elevated sFlt-1. Our promising data suggest that our novel drug, ELP-PlGF, may work to correct this imbalance and potentially serve as a therapeutic for preeclampsia.

During pregnancy, there are a number of important changes to cardiovascular function which are necessary for progression of a successful pregnancy. Additionally, preexisting cardiovascular conditions can be exacerbated by the adaptations that occur during gestation. These can present serious therapeutic challenges in the management of the cardiology patient during pregnancy. Significantly, the number of pregnant women at risk of cardiovascular complications is on the rise, so identification of risk factors that predict cardiac outcomes is essential to proper screening of the obstetrical patient. In diagnosed preexisting conditions, such as pulmonary hypertension, counseling is important prior to pregnancy. In the case of underlying disorders unmasked by pregnancy, or new-onset complications like preeclampsia, appropriate monitoring and treatment of the cardiovascular complications is warranted. Ultimately, collaborative care by both obstetricians and cardiologists is essential for the successful resolution of cardiovascular dysfunction in the obstetrical patient.

Objectives Despite intensive research, little in the way of new therapeutics has been developed for the preeclampsia patient. Several in vitro reports have suggested that heme oxygenase (HO) and its metabolic byproducts could inhibit known pathological pathways active in preeclampsia; specifically sFlt-1 production, oxidative stress, and innate immune activation. Here, we investigated the potential of HO induction, or the administration of its metabolic byproducts carbon monoxide and bilirubin, to act as a palliative in preclinical models of pregnancy-induced hypertension and preeclampsia. Methods We utilized the rodent Reduced Uterine Perfusion Pressure (RUPP) model of placental ischemia-induced hypertension to assess the effects of HO induction by cobalt protoporphyrin (5 mg/kg i.p.) as well as the effects of a CO donor molecule (CORM-A1, 5 mg/kg/day i.v.) and bilirubin (30 mg/kg/q 48 h, i.p.). The effects of HO induction were also determined in both the sFlt-1 infusion model (7.4 μg/kg/day GD14–19) and TNF- α (50 ng/day GD14–19). On GD19, maternal blood pressure was measured via carotid catheter and tissues were collected for analysis. sFlt-1 and VEGF were measured via ELISA, placental ROS was measured via lucigenin chemilluminescence, and vascular production of endothelin-1 was assayed via qRT-PCR. The direct effects of HO and its metabolites on hypoxia-induced sFlt-1 and ROS production were also determined directly in placental explants ex vivo. Results RUPP induced hypertension was significantly attenuated by HO induction and administration of either CO or bilirubin. In addition, HO induction was associated with normalization of sFlt-1/VEGF balance, placental oxidative stress, and vascular production of endothelin-1 in both RUPP and sFlt-1 induced hypertension. Further, HO induction significantly attenuated both sFlt-1 and TNF- α induced hypertension. HO and its individual metabolites were shown to inhibit hypoxia-induced production of sFlt-1 and superoxide. Conclusions Pharmacological induction of HO or administration of its metabolites could be a useful therapeutic approach for the management of preeclampsia. Disclosures E.M. George: None. J.P. Granger: None.

Objectives Upregulation of placental soluble fms-like tyrosine kinase 1 (sFlt1) contributes to the pathogenesis of preeclampsia. Our aim was to evaluate novel upstream pathways that regulate placental sFlt1 production. Methods We screened a library of natural compounds ( N = 502) in primary cytotrophoblasts and placental villous cultures and characterized molecules that had biological activity in the nanomolar range. sFlt1 production was assayed by ELISA. Immunoblot analysis, Co-immunoprecipitation, RT-PCR were employed in Characterizing the sFlt1, HIF AND hsp27 proteins and RNA. Pregnancy-induced hypertension rats were used as animal models. Results Here we report three compounds in the cardiac glycoside family, ouabain, gitoxigenin and digitoxin that inhibited placental sFlt1 production at nanomolar concentrations in vitro. We further characterized ouabain and demonstrate that it inhibits sFlt1 mRNA and protein expression in human placental cytotrophoblasts and explant cultures in a dose and time dependent manner. Ouabain down-regulated sFlt1 production by inhibiting hypoxia inducible factor 1 (HIF-1 α ) protein expression in the placenta. Furthermore, we found that phosphorylation of heat shock protein 27 (HSP27) was necessary for ouabain to inhibit HIF-1 α translation. In a rat model of pregnancy-induced hypertension, ouabain reduced mean arterial pressure and enhanced placental HSP27 phosphorylation without any adverse effects on pups. Conclusions Collectively we show that ouabain inhibits placental sFlt1 production through a HIF-1 α / HSP27 dependent pathway. Further studies are needed to explore the usefulness of targeting HIF-1 α /HSP27 pathway in preeclampsia. Disclosures S. Rana: None. A. Rajakumar: None. C. Geahchan: None. S. Salahuddin: None. A. Cerdeira: None. S.D. Burke: None. E. George: None. J. Granger: None. S. Karumanchi: co-inventor on multiple patents for preeclampsia markers and reports service as a consultant to Siemens Diagnostics, and has financial interest in Aggamin LLC.

Objectives There are currently no effective interventions for preeclampsia short of induced delivery of the fetus. Improvements in preeclampsia management have been stifled due to deleterious effects of proposed small-molecule drugs on the fetus. Our overall goal is to generate novel, maternally sequestered therapeutics for preeclampsia. The progression of preeclampsia is driven by three major pathways, secretion of the VEGF antagonist sFlt-1 by the hypoxic placenta, induction of an inflammatory environment in the mother, and production of reactive oxygen species in the hypoxic placenta. We have generated novel biologics to target each of these pathways. Furthermore, these therapeutics are attached to a drug delivery vector called elastin-like polypeptide (ELP) that stabilizes them from rapid clearance and degradation in the maternal circulation while preventing them from crossing the placenta into the fetal circulation. Methods Biodistribution of ELP-fused therapeutics in pregnant rats was determined by ex vivo whole-organ fluorescence and quantitative fluorescence histology. Efficacy for reducing the maternal symptoms and improving markers of fetal health was assessed in a rat model of placental insufficiency. Results In vitro studies validated that each of these therapeutics are active against their desired target. The ELP carriers deposited at high levels in the placenta, but did not cross into the fetal circulation, even after continuous infusion for 5 days. In addition, attachment a therapeutic peptide to ELP increased its plasma half-life from minutes to hours, and placental levels of the ELP-delivered peptide were 35-fold higher than levels of the free peptide. Ongoing studies are evaluating the efficacy of each of these novel therapeutics for prevention of preeclampsia-like symptoms and improved markers of fetal health. Conclusions Our results have established ELP as a promising drug carrier for maternally sequestered therapy, and these novel agents have the potential to be useful new classes of therapeutics for preeclampsia. Disclosures G.L. Bidwell Commercial Interest: Owner and Manager, Leflore Technologies LLC. E.M. George: None.