Inflammatory pain resolution by mouse serum-derived small extracellular vesicles.

Chronic pain is a significant public health issue. Current treatments have limited efficacy and significant side effects, warranting research on alternative strategies for pain management. One approach involves using small extracellular vesicles (sEVs) to transport beneficial biomolecular cargo to aid pain resolution. Exosomes are 30-150 nm sEVs that can carry RNAs, proteins, and lipid mediators to recipient cells via circulation. Exosomes can be beneficial or harmful depending on their source and contents. To investigate the short and long-term effects of mouse serum-derived sEVs in pain modulation, sEVs from naïve control or spared nerve injury (SNI) model donor mice were injected intrathecally into naïve recipient mice. Basal mechanical thresholds transiently increased in recipient mice. This effect was mediated by opioid signaling as this outcome was blocked by naltrexone. Mass Spectrometry of sEVs detected endogenous opioid peptide leu-enkephalin. A single prophylactic intrathecal injection of sEVs two weeks prior to induction of the pain model in recipient mice delayed mechanical allodynia in SNI model mice and accelerated recovery from inflammatory pain after complete Freund's adjuvant (CFA) injection. ChipCytometry of spinal cord and dorsal root ganglion (DRG) from sEV treated mice showed that prophylactic sEV treatment reduced the number of natural killer (NK) and NKT cells in spinal cord and increased CD206+ anti-inflammatory macrophages in (DRG) after CFA injection. Further characterization of sEVs showed the presence of immune markers suggesting that sEVs can exert immunomodulatory effects in recipient mice to promote the resolution of inflammatory pain. Collectively, these studies demonstrate multiple mechanisms by which sEVs can attenuate pain.

Uptake of Fluorescent Labeled Small Extracellular Vesicles In Vitro and in Spinal Cord.

Small extracellular vesicles (sEVs) are 50-150 nm vesicles secreted by all cells and present in bodily fluids. sEVs transfer biomolecules such as RNA, proteins, and lipids from donor to acceptor cells, making them key signaling mediators between cells. In the central nervous system (CNS), sEVs can mediate intercellular signaling, including neuroimmune interactions. sEV functions can be studied by tracking the uptake of labeled sEVs in recipient cells both in vitro and in vivo. This paper describes the labeling of sEVs from the conditioned media of RAW 264.7 macrophage cells using a PKH membrane dye. It shows the uptake of different concentrations of labeled sEVs at multiple time points by Neuro-2a cells and primary astrocytes in vitro. Also shown is the uptake of sEVs delivered intrathecally in mouse spinal cord neurons, astrocytes, and microglia visualized by confocal microscopy. The representative results demonstrate time-dependent variation in the uptake of sEVs by different cells, which can help confirm successful sEVs delivery into the spinal cord.

Therapeutic and prophylactic effects of macrophage-derived small extracellular vesicles in the attenuation of inflammatory pain.

Small extracellular vesicles (sEVs) derived from antigen-presenting cells such as macrophages can induce therapeutically relevant immune responses. Anti-inflammatory miRNAs are elevated in sEVs secreted by RAW 264.7 mouse macrophages after lipopolysaccharide (LPS) stimulation. We observed uptake of these sEVs by primary mouse cortical neurons, microglia and astrocytes followed by downregulation of proinflammatory miRNA target genes in recipient cells. Pre-treating primary microglia with these sEVs decreased pro-inflammatory gene expression. A single intrathecal injection of sEVs derived from LPS stimulated RAW 264.7 cells attenuated mechanical hyperalgesia in the complete Freund's adjuvant (CFA) mouse model of inflammatory pain and formalin induced acute pain. Importantly, sEVs did not alter the normal pain threshold in control mice. RNA sequencing of dorsal horn of the spinal cord showed sEVs-induced modulation of immune regulatory pathways. Further, a single prophylactic intrathecal injection of sEVs two weeks prior, attenuated CFA-induced pain hypersensitivity and was ineffective in formalin model. This indicates that prophylactic sEVs administration can be beneficial in attenuating chronic pain without impacting responses to the protective physiological and acute inflammatory pain. Prophylactic administration of sEVs could form the basis for a safe and novel vaccine-like therapy for chronic pain or as an adjuvant, potentially reducing the dose of drugs needed for pain relief.

Stress Signal Regulation by Na/K-ATPase As a New Approach to Promote Physiological Revascularization in a Mouse Model of Ischemic Retinopathy.

Purpose: The identification of target pathways to block excessive angiogenesis while simultaneously restoring physiological vasculature is an unmet goal in the therapeutic management of ischemic retinopathies. pNaKtide, a cell-permeable peptide that we have designed by mapping the site of α1 Na/K-ATPase (NKA)/Src binding, blocks the formation of α1 NKA/Src/reactive oxygen species (ROS) amplification loops and restores physiological ROS signaling in a number of oxidative disease models. The aim of this study was to evaluate the importance of the NKA/Src/ROS amplification loop and the effect of pNaKtide in experimental ischemic retinopathy. Methods: Human retinal microvascular endothelial cells (HRMECs) and retinal pigment epithelium (ARPE-19) cells were used to evaluate the effect of pNaKtide on viability, proliferation, and angiogenesis. Retinal toxicity and distribution were assessed in those cells and in the mouse. Subsequently, the role and molecular mechanism of NKA/Src in ROS stress signaling were evaluated biochemically in the retinas of mice exposed to the well-established protocol of oxygen-induced retinopathy (OIR). Finally, pNaKtide efficacy was assessed in this model. Results: The results suggest a key role of α1 NKA in the regulation of ROS stress and the Nrf2 pathway in mouse OIR retinas. Inhibition of α1 NKA/Src by pNaKtide reduced pathologic ROS signaling and restored normal expression of hypoxia-inducible factor 1-α/vascular endothelial growth factor (VEGF). Unlike anti-VEGF agents, pNaKtide did promote retinal revascularization while inhibiting neovascularization and inflammation. Conclusions: Targeting α1 NKA represents a novel strategy to develop therapeutics that not only inhibit neovascularization but also promote physiological revascularization in ischemic eye diseases.

Inflammation potentiates miR-939 expression and packaging into small extracellular vesicles.

Extracellular RNA in circulation mediates intercellular communication in normal and pathological processes. One mode of circulating miRNA transport in bodily fluids is within 30-150 nm small extracellular vesicles (sEVs) or exosomes. Uptake of sEVs can regulate gene expression in recipient cells enabling circulating miRNAs to exert paracrine and systemic effects. Complex regional pain syndrome (CRPS) is a debilitating pain disorder characterized by chronic inflammation. Our previous investigations identified a significant decrease of hsa-miR-939 in whole blood from CRPS patients compared to control; we also observed that overexpression of miR-939 can negatively regulate several proinflammatory genes in vitro. Though downregulated in whole blood, miR-939 was significantly upregulated in sEVs isolated from patient serum. Here we investigated miR-939 packaging into sEVs in vitro under inflammation induced by monocyte chemoattractant protein-1 (MCP-1), a chemokine that is upregulated in CRPS patients. Stimulation of THP-1 monocytes by MCP-1 led to elevated levels of miR-939 in sEVs, which was abrogated using inhibitors of exosome secretion. miRNAs loaded into exosomes largely contain short miRNA sequence motifs called EXOmotifs. Mutation analysis of miR-939 showed that EXOmotif is one of the possible cellular mechanisms responsible for packaging miR-939 into sEVs. We confirmed gene expression changes in recipient cells following the uptake of sEVs enriched in miR-939 using RNA sequencing. Additionally, our data from primary immune cell-derived sEVs of CRPS patients and controls demonstrate that while the relative expression of miR-939 is higher in sEVs derived from B cells, T cells and NK cells relative to monocyte-derived sEVs in controls, only the B cell-derived sEVs showed a significantly higher level of miR-939 in CRPS patients. Differential miRNA sorting into exosomes and its functional impact on recipient cells may contribute to the underlying pathophysiology of CRPS.