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Background: After an acute stroke, highly reactive oxygen and nitrogen species (ROS/RNS) lead to microvessel injury and inhibit endogenous mediators to prevent the repair process of endothelial dysfunction and tissue injury. Among them, the peroxynitrite (ONOO−) generation as a result of nitric oxide (NO) and superoxide anion (O2·−) reaction contributes to ischemic brain injury. Purpose: To validate the targeted inhibition of ischemic injury's markers with the delivery of potent antioxidants such as β-carotene (BC) in lipid nanoparticles (LNPs) directly acting with ONOO− compound to prevent the breakdown of blood-brain barrier (BBB). Study design: In the current work, BC in polyethylene glycol (PEG) lipid nanoparticles (PEG-LNPs) with or without attached anti-nitrotyrosine antibody (3-NT) inhibits ONOO− generation in the transient middle cerebral artery occlusion (tMCAO) model. Transmission electron microscopy (TEM) was used to examine structural change in LNPs with or without 3-NT antibody attachment. Fluorescence imaging was employed to study drug encapsulated PEG-LNPs accumulation in mice brain following MCAO and spectrophotometry for the in-vitro drug release profile. Immunoblotting using antibodies specific for spectrin and calcineurin, occludin and ZO-1 and other targets was utilized. The NP3 fluorescent probe was used for the detection of ONOO−generation. Evans Blue was injected 24 h after tMCAO in vehicle and BC PEG-LNPs with 3-NT antibody treated animal groups to observe the tight junction breakdown. Results: The fluorescence's data reveals that attachment of 3-NT antibody has directed the delivery of BC to the ischemic ipsilateral region. BC, as well, is found effective to improve infarct volume and neurological deficit by neuronal protection by inhibiting spectrin and calcineurin (CaN) breakdown. Furthermore, inhibition of ONOO−generation is observed in NP3 probe incubation of brain slices and protects BBB from breakdown in Evans blue extravasation. Conclusion: The current work suggests that inhibiting ONOO−at ischemic site protects the brain microvessels from injury. 3-NT/BC/LNPs carrier system is potentially efficient in delivering BC at the site of ONOO−generation, and possibly suitable for ischemic region-targeted therapy.

Cross-presentation in dendritic cells (DC) requires the endosomal relocations of internalized antigens and the endoplasmic reticulum protein Sec61. Despite the fact that endotoxin-containing pathogen and endotoxin-free antigen have different effects on protein kinase B (Akt) and I-kappa B Kinase α/β (IKKα/β) activation, the exact roles of Akt phosphorylation, IKKα or IKKβ activation in endotoxin-containing pathogen-derived cross-presentation are poorly understood. In this study, endotoxin-free ovalbumin supplemented with endotoxin was used as a model pathogen. We investigated the effects of endotoxin-containing pathogen and endotoxin-free antigen on Akt phosphorylation, IKKα/β activation, and explored the mechanisms that the endotoxin-containing pathogen orchestrating the endosomal recruitment of Sec61 of the cross-presentation in bone marrow precursor cells (BMPC). We demonstrated that endotoxin-containing pathogen and endotoxin-free antigen efficiently induced the phosphorylation of Akt-IKKα/β and Akt-IKKα, respectively. Endotoxin-containing pathogen derived Akt+ IKKα/β+ Rab5+ signalosome, together with augmented the recruitment of Sec61 toward endosome, lead to the increased cross-presentation in BMPC. Importantly, the endosomal recruitment of Sec61 was partly mediated by the formation of Akt+ IKKα/β+ signalosome. Thus, these data suggest that Akt+ IKKα/β+ Rab5+ signalosome contribute to endotoxin-containing pathogen-induced the endosomal recruitment of Sec61 and the superior efficacy of cross-presentation in BMPC.

Abnormal angiogenesis and regression of the diseased retinal vasculature are key processes associated with ischemic retinopathies, but the underlying mechanisms that regulate vascular remodeling remain poorly understood. Here, we confirmed the specific expression of semaphorin 3G (Sema3G) in retinal endothelial cells (ECs), which was required for vascular remodeling and the amelioration of ischemic retinopathy. We found that Sema3G was elevated in the vitreous fluid of patients with proliferative diabetic retinopathy (PDR) and in the neovascularization regression phase of oxygen-induced retinopathy (OIR). Endothelial-specific Sema3G knockout mice exhibited decreased vessel density and excessive matrix deposition in the retinal vasculature. Moreover, loss of Sema3G aggravated pathological angiogenesis in mice with OIR. Mechanistically, we demonstrated that HIF-2α directly regulated Sema3G transcription in ECs under hypoxia. Sema3G coordinated the functional interaction between β-catenin and VE-cadherin by increasing β-catenin stability in the endothelium through the neuropilin-2 (Nrp2)/PlexinD1 receptor. Furthermore, Sema3G supplementation enhanced healthy vascular network formation and promoted diseased vasculature regression during blood vessel remodeling. Overall, we deciphered the endothelium-derived Sema3G-dependent events involved in modulating physiological vascular remodeling and regression of pathological blood vessels for reparative vascular regeneration. Our findings shed light on the protective effect of Sema3G in ischemic retinopathies. [ABSTRACT FROM AUTHOR]