Perinatal Micro-Bleeds and Neuroinflammation in E19 Rat Fetuses Exposed to Utero-Placental Ischemia.

Offspring of preeclampsia patients have an increased risk of developing neurological deficits and cognitive impairment. While low placental perfusion, common in preeclampsia and growth restriction, has been linked to neurological deficits, a causative link is not fully established. The goal of this study was to test the hypothesis that placental ischemia induces neuroinflammation and micro-hemorrhages in utero. Timed-pregnant Sprague Dawley rats were weight-matched for sham surgery (abdominal incision only) or induced placental ischemia (surgical reduction of utero-placental perfusion (RUPP)); n = 5/group on gestational day 14. Fetal brains (n = 1-2/dam/endpoint) were collected at embryonic day (E19). Placental ischemia resulted in fewer live fetuses, increased fetal demise, increased hematocrit, and no difference in brain water content in exposed fetuses. Additionally, increased cerebral micro-bleeds (identified with H&E staining), pro-inflammatory cytokines: IL-1ß, IL-6, and IL-18, eotaxin (CCL11), LIX (CXCL5), and MIP-2 (CXCL2) were observed in RUPP-exposed fetuses. Microglial density in the sub-ventricular zone decreased in RUPP-exposed fetuses, with no change in cortical thickness. Our findings support the hypothesis that exposure to placental ischemia contributes to microvascular dysfunction (increased micro-bleeds), fetal brain inflammation, and reduced microglial density in proliferative brain areas. Future studies will determine whether in utero abnormalities contribute to long-term behavioral deficits in preeclampsia offspring through impaired neurogenesis regulation.

Polymer size affects biodistribution and placental accumulation of the drug delivery biopolymer elastin-like polypeptide in a rodent pregnancy model.

INTRODUCTION: Fusion of therapeutic agents to Elastin-like Polypeptide (ELP) is a novel drug delivery strategy for prevention of placental drug transfer. Previous studies have used a 60 kDa ELP tag for this purpose. However, placental transfer of ELP may be size dependent. The goal of this study was to measure the effects of ELP polymer size on pharmacokinetics, biodistribution, and placental transfer of ELP. METHODS: Three ELPs ranging from 25 to 86 kDa (4.1-6.8 nm hydrodynamic radius) were fluorescently labeled and administered by i.v. bolus to pregnant Sprague Dawley rats on gestational day 14. Plasma levels were monitored for 4 h, organ levels and placental transfer determined by ex vivo fluorescence imaging, and placental localization determined by confocal microscopy. RESULTS: Increasing ELP size resulted in slower plasma clearance and increased deposition in all major maternal organs, except in the kidneys where an opposite effect was observed. Placental levels increased with an increase in size, while in the pups, little to no ELP was detected. DISCUSSION: Pharmacokinetics and biodistribution of ELPs during pregnancy are size dependent, but all ELPs tested were too large to traverse the placental barrier. These studies verify that ELP fusion is a powerful method of modulating half-life and preventing placental transfer of cargo molecules. The tunable nature of the ELP sequence makes it ideal for drug delivery applications during pregnancy, where it can be used to target drugs to the mother while preventing fetal drug exposure.

A Maternally Sequestered, Biopolymer-Stabilized Vascular Endothelial Growth Factor (VEGF) Chimera for Treatment of Preeclampsia.

BACKGROUND: Preeclampsia is a hypertensive syndrome that complicates 3% to 5% of pregnancies in the United States. Preeclampsia originates from an improperly vascularized and ischemic placenta that releases factors that drive systemic pathophysiology. One of these factors, soluble fms-like tyrosine kinase-1, is believed to sequester vascular endothelial growth factor (VEGF), leading to systemic endothelial dysfunction and hypertension. With the goal of targeting soluble fms-like tyrosine kinase-1 while simultaneously preventing fetal exposure to VEGF, we fused VEGF to elastin-like polypeptide, a biopolymer carrier that does not cross the placental barrier (ELP-VEGF). METHODS AND RESULTS: ELP-VEGF restored in vitro endothelial cell tube formation in the presence of plasma from placental ischemic rats. Long-term administered ELP-VEGF in pregnant rats accumulated in maternal kidneys, aorta, liver, and placenta, but the protein was undetectable in the pups when administered at therapeutic doses in dams. Long-term administration of ELP-VEGF in a placental ischemia rat model achieved dose-dependent attenuation of hypertension, with blood pressure equal to sham controls at a dose of 5 mg/kg per day. ELP-VEGF infusion increased total plasma soluble fms-like tyrosine kinase-1 levels but dramatically reduced free plasma soluble fms-like tyrosine kinase-1 and induced urinary excretion of nitrate/nitrite, indicating enhanced renal nitric oxide signaling. ELP-VEGF at up to 5 mg/kg per day had no deleterious effect on maternal or fetal body weight. However, dose-dependent adverse events were observed, including ascites production and neovascular tissue encapsulation around the minipump. CONCLUSIONS: ELP-VEGF has the potential to treat the preeclampsia maternal syndrome, but careful dosing and optimization of the delivery route are necessary.

A kidney-selective biopolymer for targeted drug delivery.

Improving drug delivery to the kidney using renal-targeted therapeutics is a promising but underdeveloped area. We aimed to develop a kidney-targeting construct for renal-specific drug delivery. Elastin-like polypeptides (ELPs) are nonimmunogenic protein-based carriers that can stabilize attached small-molecule and peptide therapeutics. We modified ELP at its NH2-terminus with a cyclic, seven-amino acid kidney-targeting peptide (KTP) and at its COOH-terminus with a cysteine residue for tracer conjugation. Comparative in vivo pharmacokinetics and biodistribution in rat and swine models and in vitro cell binding studies using human renal cells were performed. KTP-ELP had a longer plasma half-life than ELP in both animal models and was similarly accumulated in kidneys at levels fivefold higher than untargeted ELP, showing renal levels 15- to over 150-fold higher than in other major organs. Renal fluorescence histology demonstrated high accumulation of KTP-ELP in proximal tubules and vascular endothelium. Furthermore, a 14-day infusion of a high dose of ELP or KTP-ELP did not affect body weight, glomerular filtration rate, or albuminuria, or induce renal tissue damage compared with saline-treated controls. In vitro experiments showed higher binding of KTP-ELP to human podocytes, proximal tubule epithelial, and glomerular microvascular endothelial cells than untargeted ELP. These results show the high renal selectivity of KTP-ELP, support the notion that the construct is not species specific, and demonstrate that it does not induce acute renal toxicity. The plasticity of ELP for attachment of any class of therapeutics unlocks the possibility of applying ELP technology for targeted treatment of renal disease in future studies.

Therapeutic angiogenesis by vascular endothelial growth factor supplementation for treatment of renal disease.

PURPOSE OF REVIEW: Vascular endothelial growth factors (VEGFs) influence renal function through angiogenesis, with VEGF-A being the most potent inducer of vascular formation. In the normal glomerulus, tight homeostatic balance is maintained between the levels of VEGF-A isoforms produced by podocyte cells, and the VEGF receptors (VEGFRs) expressed by glomerular endothelial, mesangial, and podocyte cells. Renal disease occurs when this homeostatic balance is lost, manifesting in the abnormal autocrine and paracrine VEGF-A/VEGFR signaling, ultrastructural glomerular and tubular damage, and impaired filtration. RECENT FINDINGS: Preclinical disease models of ischemic renal injury, including acute ischemia/reperfusion, thrombotic microangiopathy, and chronic renovascular disease, treated with exogenous VEGF supplementation demonstrated therapeutic efficacy. These results suggest a therapeutic VEGF-A paracrine effect on endothelial cells in the context of acute or chronic obstructive ischemia. Conversely, renal dysfunction in diabetic nephropathy appears to occur through an upregulated VEGF autocrine effect on podocyte cells, which is exacerbated by hyperglycemia. Therefore, VEGF supplementation therapy may be contraindicated for treatment of diabetic nephropathy, but specific results will depend on dose and on the specific site of VEGF delivery. A drug delivery system that demonstrates cell specificity for glomerular or peritubular capillaries could be employed to restore balance to VEGF-A/VEGFR2 signaling, and by doing so, prevent the progression to end-stage renal disease. SUMMARY: The review discusses the preclinical data available for VEGF supplementation therapy in models of renal disease.

Preeclampsia and the brain: neural control of cardiovascular changes during pregnancy and neurological outcomes of preeclampsia.

Preeclampsia (PE) is a form of gestational hypertension that complicates ∼5% of pregnancies worldwide. Over 70% of the fatal cases of PE are attributed to cerebral oedema, intracranial haemorrhage and eclampsia. The aetiology of PE originates from abnormal remodelling of the maternal spiral arteries, creating an ischaemic placenta that releases factors that drive the pathophysiology. An initial neurological outcome of PE is the absence of the autonomically regulated cardiovascular adaptations to pregnancy. PE patients exhibit sympathetic overactivation, in comparison with both normotensive pregnant and hypertensive non-pregnant females. Moreover, PE diminishes baroreceptor reflex sensitivity (BRS) beyond that observed in healthy pregnancy. The absence of the cardiovascular adaptations to pregnancy, combined with sympathovagal imbalance and a blunted BRS leads to life-threatening neurological outcomes. Behaviourally, the increased incidences of maternal depression, anxiety and post-traumatic stress disorder (PTSD) in PE are correlated to low fetal birth weight, intrauterine growth restriction (IUGR) and premature birth. This review addresses these neurological consequences of PE that present in the gravid female both during and after the index pregnancy.

Alterations of functional properties of hippocampal networks following repetitive closed-head injury.

Traumatic brain injury (TBI) is the leading cause of death for persons under the age of 45. Military service members who have served on multiple combat deployments and contact-sport athletes are at particular risk of sustaining repetitive TBI (rTBI). Cognitive and behavioral deficits resulting from rTBI are well documented. Optimal associative LTP, occurring in the CA1 hippocampal Schaffer collateral pathway, is required for both memory formation and retrieval. Surprisingly, ipsilateral Schaffer collateral CA1 LTP evoked by 100 Hz tetanus was enhanced in mice from the 3× closed head injury (3× CHI) treatment group in comparison to LTP in contralateral or 3× Sham CA1 area, and in spite of reduced freezing during contextual fear conditioning at one week following 3× CHI. Electrophysiological activity of CA1 neurons was evaluated with whole-cell patch-clamp recordings. 3× CHI ipsilateral CA1 neurons exhibited significant increases in action potential amplitude and maximum rise and decay slope while the action potential duration was decreased. Recordings of CA1 neuron postsynaptic currents were conducted to detect spontaneous excitatory and inhibitory postsynaptic currents (sEPSCs/sIPSCs) and respective miniature currents (mEPSCs and mIPSCs). In the 3× CHI mice, sEPSCs and sIPSCs in ipsilateral CA1 neurons had an increased frequency of events but decreased amplitudes. In addition, 3× CHI altered the action potential-independent miniature postsynaptic currents. The mEPSCs of ipsilateral CA1 neurons exhibited both an increased frequency of events and larger amplitudes. Moreover, the effect of 3× CHI on mIPSCs was opposite to that of the sIPSCs. Specifically, the frequency of the mIPSCs was decreased while the amplitudes were increased. These results are consistent with a mechanism in which repetitive closed-head injury affects CA1 hippocampal function by promoting a remodeling of excitatory and inhibitory synaptic inputs leading to impairment in hippocampal-dependent tasks.

Corneal Penetrating Elastin-Like Polypeptide Carriers.

PURPOSE: Elastin-like polypeptide (ELP) is a bioengineered protein widely applied as a drug carrier due to its biocompatibility and amenability to modification with cell-penetrating peptides (CPPs) and therapeutic agents. The purpose of this study was to determine whether topically applied ELP or CPP-fused ELPs penetrate the corneal barrier. METHODS: In vitro binding and cytotoxicity to human corneal epithelial (HCE) cells were determined for ELP or CPP-ELPs. Corneal binding, clearance, and penetration were assessed in a rabbit model following topical application of the fluorescently labeled proteins by quantitative fluorescence imaging and histology. RESULTS: ELP bound to HCE cells in vitro, and binding/uptake was enhanced 2- to 3-fold by the addition of CPPs. When applied topically to rabbit eyes, ELP accumulated in the cornea at levels 7.4-fold higher than did an equivalent dose of immunoglobulin G. Both ELP and a CPP-ELP penetrated the corneal epithelium and were detectable in the stroma. Addition of CPPs to ELP, however, did not significantly enhance corneal uptake or penetration in vivo relative to ELP alone. The polypeptides cleared from the cornea over a period of 20-30 min after application, after which cornea levels reached a steady state of 15-30 µg/mL for up to 3 h. CONCLUSIONS: The ELP drug carrier can penetrate the corneal epithelium and accumulate in the stroma. Given its amenability for fusion to multiple types of therapeutic agents, ELP has the potential to serve as a drug carrier for topical ocular applications.