Ischemic strokes occur when a major cerebral artery or its branches are occluded, resulting in activation of inflammatory processes that cause secondary tissue injury, breakdown of the blood–brain ...

Preeclampsia is a hypertensive disorder of pregnancy that causes significant acute and long-term risk to the mother and the baby. The multifaceted maternal syndrome is driven by overproduction of circulating anti-angiogenic factors, widespread inflammation, and endothelial dysfunction. Nuclear factor-κB (NF-κB) is a transcription factor that plays a central role in the inflammatory response. Its activity is increased in the preeclamptic placenta, and it promotes the systemic endothelial dysfunction present in preeclampsia. There is an acute need for new therapeutics targeted to the causative pathways of preeclampsia. Our group has developed a drug delivery system based on the bioengineered protein ELP (elastin-like polypeptide) that is capable of stabilizing therapeutics in the maternal circulation and preventing their placental transfer. Here we used the ELP carrier system to deliver a peptide known to inhibit the NF-κB pathway. This polypeptide, containing a cell-penetrating peptide and an NF-κB inhibitory peptide derived from the p50 nuclear localization sequence (abbreviated SynB1-ELP-p50i), blocked NF-κB activation and prevented TNF-α (tumor necrosis factor alpha)–induced endothelin production in vitro. Fusion of the p50i peptide to the SynB1-ELP carrier slowed its plasma clearance and prevented its placental transfer in pregnant rats, resulting in increased deposition in the maternal kidney, liver, and placenta relative to the free peptide. When administered in a rat model of placental ischemia, SynB1-ELP-p50i partially ameliorated placental ischemia-induced hypertension and reduced placental TNF-α levels with no signs of toxicity. These data support the continued development of ELP-delivered NF-κB inhibitors as maternally sequestered anti-inflammatory agents for preeclampsia therapy.

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.

Receptor-interacting serine/threonine protein kinase 2 (RIPK2) is a kinase that is essential in modulating innate and adaptive immune responses. As a downstream signaling molecule for nucleotide-binding oligomerization domain 1 (NOD1), NOD2, and Toll-like receptors (TLRs), it is implicated in the signaling triggered by recognition of microbe-associated molecular patterns by NOD1/2 and TLRs. Upon activation of these innate immune receptors, RIPK2 mediates the release of pro-inflammatory factors by activating mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB). However, whether RIPK2 is essential for downstream inflammatory signaling following the activation of NOD1/2, TLRs, or both remains controversial. In this study, we examined the role of RIPK2 in NOD2- and TLR4-dependent signaling cascades following stimulation of microglial cells with bacterial muramyl dipeptide (MDP), a NOD2 agonist, or lipopolysaccharide (LPS), a TLR4 agonist. We utilized a highly specific proteolysis targeting chimera (PROTAC) molecule, GSK3728857A, and found dramatic degradation of RIPK2 in a concentration- and time-dependent manner. Importantly, the PROTAC completely abolished MDP-induced increases in iNOS and COX-2 protein levels and pro-inflammatory gene transcription of Nos2, Ptgs2, Il-1β, Tnfα, Il6, Ccl2, and Mmp9. However, increases in iNOS and COX-2 proteins and pro-inflammatory gene transcription induced by the TLR4 agonist, LPS, were only slightly attenuated with the GSK3728857A pretreatment. Further findings revealed that the RIPK2 PROTAC completely blocked the phosphorylation and activation of p65 NF-κB and p38 MAPK induced by MDP, but it had no effects on the phosphorylation of these two mediators triggered by LPS. Collectively, our findings strongly suggest that RIPK2 plays an essential role in the inflammatory responses of microglia to bacterial MDP but not to LPS.

Juveniles are at increased risk for mild traumatic brain injury (mTBI) due to recreational activities, domestic violence, and vulnerabilities of the developing skull. In industrialized nations, adolescents often consume high-sugar, high-fat diets (HFD), which can impair CNS health and exacerbate mTBI outcomes. Secondary injury following mTBI is characterized by prolonged neuroinflammation, with nuclear factor kappa B (NF-κB) acting as a central regulator of these inflammatory responses. We developed a polypeptide biologic, SynB1-ELP-p50i, consisting of an elastin-like polypeptide delivered NF-κB inhibitor (p50i) and the cell-penetrating peptide SynB1. This study investigates the interaction between diet and NF-κB inhibition in juvenile rats with mTBI by examining SynB1-ELP-p50i biodistribution, pharmacokinetics, therapeutic efficacy, and cell-specific effects. Long Evans rats were assigned to chow or HFD groups at postnatal day 20 (PND20), and mTBI or sham injury was induced at PND30 using the CHIMERA TBI model. Rats received 50 mg/kg SynB1-ELP-p50i or saline post-injury. Behavioral outcomes were assessed with the Morris water maze (MWM), and neuroinflammatory markers were measured using RT-qPCR and single-nucleus RNA sequencing (snRNA-seq). SynB1-ELP-p50i accumulated at injury sites (p < 0.05), and SynB1-ELP-p50i treated chow-fed rats showed improved MWM performance compared to vehicle controls and HFD subjects (p < 0.05). RT-qPCR confirmed increased Aif1 and Gfap expression post-injury (p < 0.05), and snRNA-seq revealed upregulation of markers of microgliosis in activated microglia. These findings indicate that mTBI triggers acute cognitive deficits and neuroinflammation, worsened by HFD. SynB1-ELP-p50i mitigated memory deficits in chow-fed animals, supporting NF-κB inhibition as a promising therapeutic strategy for juvenile mTBI.