Vepoloxamer improves functional recovery in rat after traumatic brain injury: A dose-response and therapeutic window study.

Traumatic brain injury (TBI) is a major cause of death and disability worldwide. There are no effective therapies available for TBI patients. Vepoloxamer is an amphiphilic polyethylene-polypropylene-polyethylene tri-block copolymer that seals membranes and restores plasma membrane integrity in damaged cells. We previously demonstrated that treatment of TBI rats with Vepoloxamer improves functional recovery. However, additional studies are needed to potentially translate Vepoloxamer treatment from preclinical studies into clinical applications. We thus conducted a study to investigate dose-response and therapeutic window of Vepoloxamer on functional recovery of adult rats after TBI. To identify the most effective dose of Vepoloxamer, male Wistar adult rats with controlled cortical impact (CCI) injury were randomly treated with 0 (vehicle), 100, 300, or 600 mg/kg of Vepoloxamer, administered intravenously (IV) at 2 h after TBI. We then performed a therapeutic window study in which the rats were treated IV with the most effective single dose of Vepoloxamer at different time points of 2 h, 4 h, 1 day, or 3 days after TBI. A battery of cognitive and neurological tests was performed. Animals were killed 35 days after TBI for histopathological analysis. Dose-response experiments showed that Vepoloxamer at all three tested doses (100, 300, 600 mg/kg) administered 2 h post injury significantly improved cognitive functional recovery, whereas Vepoloxamer at doses of 300 and 600 mg/kg, but not the 100 mg/kg dose, significantly reduced lesion volume compared to saline treatment. However, Vepoloxamer at 300 mg/kg showed significantly improved neurological and cognitive outcomes than treatment with a dose of 600 mg/kg. In addition, our data demonstrated that the dose of 300 mg/kg of Vepoloxamer administered at 2 h, 4 h, 1 day, or 3 days post injury significantly improved neurological function compared with vehicle, whereas Vepoloxamer administered at 2 h or 4 h post injury significantly improved cognitive function compared with the 1-day and 3-day treatments, with the most robust effect administered at 2 h post injury. The present study demonstrated that Vepoloxamer improves functional recovery in a dose-and time-dependent manner, with therapeutic efficacy compared with vehicle evident even when the treatment is initiated 3 days post TBI in the rat.

Treatment of stroke in aged male and female rats with Vepoloxamer and tPA reduces neurovascular damage.

Stroke is a leading cause of death and disability worldwide, mainly affecting the elderly. Unfortunately, current treatments for acute ischemic stroke warrant improvement. To date, tissue plasminogen activator (tPA) is of limited use in stroke patients mainly due to its narrow therapeutic window and potential for hemorrhagic complication. The adjuvant treatment with Vepoloxamer, a purified amphipathic polymer has been shown to enhance the thrombolytic efficacy of tPA treatment in young adult male rats after embolic stroke. However, most stroke patients are aged; therefore, the current study investigated the therapeutic effect of the combined tPA and Vepoloxamer treatment in aged male and female rats subjected to embolic stroke. Methods: Male and female Wistar rats at 18 months of age were subjected to embolic middle cerebral artery occlusion and treated either with monotherapy of tPA or Vepoloxamer, a combination of these two agents, or saline at 4 h after stroke onset. Neurological outcomes were evaluated with a battery of behavioral tests including adhesive removal, foot-fault, and modified neurological severity score tests at 1 and 7 days after stroke onset, followed by histopathological analysis of infarct volume. Residual clot size and vascular patency and integrity were analyzed. Results: The combination treatment with Vepoloxamer and tPA significantly reduced infarct volume and neurological deficits in male and female rats compared to rats treated with saline and the monotherapies of tPA and Vepoloxamer. While Vepoloxamer monotherapy moderately reduced neurological deficits, monotherapies with tPA and Vepoloxamer failed to reduce infarct volume compared to saline treatment. Furthermore, the combination treatment with tPA and Vepoloxamer accelerated thrombolysis, reduced ischemia and tPA-potentiated microvascular disruption, and concomitantly improved cerebrovascular integrity and perfusion in the male ischemic rats. Conclusion: Combination treatment with tPA and Vepoloxamer at 4 h after stroke onset effectively reduces ischemic neurovascular damage by accelerating thrombolysis and reducing ischemia and tPA potentiated side effects in the aged rats. This funding suggests that the combination treatment with tPA and Vepoloxamer represents a promising strategy to potentially apply to the general population of stroke patients.

Glymphatic transport is reduced in rats with spontaneous pituitary tumor.

Background and objective: Pituitary tumor in patients induces adverse alterations in the brain, accompanied by cognitive deficits. Dysfunction of glymphatic waste clearance results in accumulation of neurotoxic products within the brain, leading to cognitive impairment. However, the status of glymphatic function in the brain with pituitary tumor is unknown. Using magnetic resonance imaging (MRI) and an advanced mathematical modeling, we investigated the changes of glymphatic transport in the rats carrying spontaneous pituitary tumor. Methods: Rats (22-24 months, female, Wistar) with and without pituitary tumor (n = 7/per group) underwent the identical experimental protocol. MRI measurements, including T2-weighted imaging and dynamic 3D T1-weighted imaging with intracisternal administration of contrast agent, were performed on each animal. The contrast-induced enhancement in the circle of Willis and in the glymphatic influx nodes were observed on the dynamic images and verified with time-signal-curves (TSCs). Model-derived parameters of infusion rate and clearance rate that characterize the kinetics of glymphatic tracer transport were evaluated in multiple representative brain regions. Results: Our imaging data demonstrated a higher incidence of partially enhanced circle of Willis (86 vs. 14%; p < 0.033) and a lower incidence of enhancement in glymphatic influx nodes of pituitary (71 vs. 100%) and pineal (57 vs. 86%) recesses in the rats with pituitary tumor than in the rats with normal appearance of pituitary gland, indicating an intensification of impaired peri-vascular pathway and impeded glymphatic transport due to the presence of pituitary tumor. Consistently, our kinetic modeling and regional cerebral tissue quantification revealed significantly lower infusion and clearance rates in all examined regions in rats with spontaneous pituitary tumor than in non-tumor rats, representing a suppressed glymphatic transport in the brain with pituitary tumor. Conclusion: Our study demonstrates the compromised glymphatic transport in the rat brain with spontaneous pituitary tumor. The reduced efficiency in cerebral waste clearance increases the risk for neurodegeneration in the brain that may underlie the cognitive impairment commonly seen in patients with pituitary tumors.

Comparative proteomic analysis of exosomes derived from endothelial cells and Schwann cells.

Exosomes derived from endothelial cells and Schwann cells have been employed as novel treatments of neurological diseases, including peripheral neuropathy. Exosomal cargo plays a critical role in mediating recipient cell function. In this study, we thus performed a comprehensive proteomic analysis of exosomes derived from healthy mouse dermal microvascular endothelial cells (EC-Exo) and healthy mouse Schwann cells (SC-Exo). We detected 1,817and 1,579 proteins in EC-Exo and SC-Exo, respectively. Among them, 1506 proteins were present in both EC-Exo and SC-Exo, while 311 and 73 proteins were detected only in EC-Exo and SC-Exo, respectively. Bioinformatic analysis revealed that EC-Exo enriched proteins were involved in neurovascular function, while SC-Exo enriched proteins were related to lipid metabolism. Western blot analysis of 14 enriched proteins revealed that EC-Exo contained proteins involved in mediating endothelial function such as delta-like 4 (DLL4) and endothelial NOS (NOS3), whereas SC-Exo had proteins involved in mediating glial function such as apolipoprotein A-I (APOA1) and phospholipid transfer protein (PLTP). Collectively, the present study identifies differences in the cargo protein profiles of EC-Exo and SC-Exo, thus providing new molecular insights into their biological functions for the treatment of peripheral neuropathy.

Ablation of Argonaute 2 in Schwann cells accelerates the progression of diabetic peripheral neuropathy.

Schwann cells (SCs) form myelin and provide metabolic support for axons, and are essential for normal nerve function. Identification of key molecules specific to SCs and nerve fibers may provide new therapeutic targets for diabetic peripheral neuropathy (DPN). Argonaute2 (Ago2) is a key molecular player that mediates the activity of miRNA-guided mRNA cleavage and miRNA stability. Our study found that Ago2 knockout (Ago2-KO) in proteolipid protein (PLP) lineage SCs in mice resulted in a significant reduction of nerve conduction velocities and impairments of thermal and mechanical sensitivities. Histopathological data revealed that Ago2-KO significantly induced demyelination and neurodegeneration. When DPN was induced in both wild-type and Ago2-KO mice, Ago2-KO mice exhibited further decreased myelin thickness and exacerbated neurological outcomes compared with wild-type mice. Deep sequencing analysis of Ago2 immunoprecipitated complexes showed that deregulated miR-206 in Ago2-KO mice is highly related to mitochondrial function. In vitro data showed that knockdown of miR-200 induced mitochondrial dysfunction and apoptosis in SCs. Together, our data suggest that Ago2 in SCs is essential to maintain peripheral nerve function while ablation of Ago2 in SCs exacerbates SC dysfunction and neuronal degeneration in DPN. These findings provide new insight into the molecular mechanisms of DPN.

CLINICAL, MOLECULAR, AND EXOSOMAL MECHANISMS OF CARDIAC AND BRAIN DYSFUNCTION IN SEPSIS.

ABSTRACT: Sepsis is a complex disease resulting from a dysregulated inflammatory response to an infection. Initiation of sepsis occurs from a localized infection that disseminates to the bloodstream placing all organ systems at risk. Septic shock is classically observed to manifest itself as systemic hypotension with hyporesponsiveness to vasopressor agents. Myocardial dysfunction occurs resulting in an inability to perfuse major organ systems throughout the body. Most importantly, the brain is hypoperfused creating an ischemic and inflammatory state resulting in the clinical observation of acute mental status changes and cognitive dysfunction commonly known as sepsis-associated encephalopathy. This short review describes the inflammatory molecular mechanisms of myocardial dysfunction, discusses the evidence of the dual roles of the microglia resulting in blood-brain barrier disruption, and suggests that septic-derived exosomes, endosome-derived lipid bilayer spheroids released from living cells, influence cardiac and neurological cellular function.

Therapeutic Role of microRNAs of Small Extracellular Vesicles from Human Mesenchymal Stromal/Stem Cells in Treatment of Experimental Traumatic Brain Injury.

Mesenchymal stem/stromal cells (MSC)-derived small extracellular vesicles (sEVs) possess therapeutic potential for treatment of traumatic brain injury (TBI). The essential role of micro ribonucleic acids (miRNAs) underlying the beneficial effects of MSC-derived sEVs for treatment of TBI remains elusive. The present study was designed to investigate the role of microRNAs in sEVs from MSCs with Argonaute 2 knockdown (Ago2-KD) in neurological recovery, neuroinflammation, and neurovascular remodeling in TBI rats. Therapeutic effects of sEVs derived from naïve MSCs (naïve-sEV), MSCs transfected with a vector carrying scramble control short hairpin RNA (shRNA; vector-sEV), and MSCs transfected with a lentiviral vector-based shRNA against Ago2 to knock down Ago2 (Ago2-KD-sEV) were determined in adult male rats subjected to a moderate TBI induced by controlled cortical impact (CCI). sEVs (naïve-sEV, vector-sEV, and Ago2-KD-sEV) or vehicle (phosphate-buffered solution [PBS]) were given intravenously 1 day post-injury (PI). Multiple neurological functional tests were performed weekly PI for 5 weeks. The Morris water maze (MWM) test was performed for spatial learning and memory 31-35 days PI. All animals were euthanized 5 weeks PI and the brains were collected for analyses of lesion volume, cell loss, neurovascular remodeling, and neuroinflammation. Ago2-KD reduced global sEV miRNA levels. Compared with the vehicle treatment, both naïve-sEV and vector-sEV treatments significantly improved functional recovery, reduced hippocampal neuronal cell loss, inhibited neuroinflammation, and promoted neurovascular remodeling (angiogenesis and neurogenesis). However, Ago2-KD-sEV treatment had a significantly less therapeutic effect on all the parameters measured above than did naïve-sEV and vector-sEV treatments. The therapeutic effects of Ago2-KD-sEV were comparable to that of vehicle treatment. Our findings demonstrate that attenuation of Ago2 protein in MSCs reduces miRNAs in MSC-derived sEVs and abolishes exosome treatment-induced beneficial effects in TBI recovery, suggesting that miRNAs in MSC-derived sEVs play an essential role in reducing neuronal cell loss, inhibiting neuroinflammation, and augmenting angiogenesis and neurogenesis, as well as improving functional recovery in TBI. The findings underscore the important role of miRNAs in MSC-derived sEVs in the treatment of TBI.

Pharmacological Characterization of a Novel Neuroactive Steroid Prodrug, NTS-104, and Its Neuroprotective Activity in Experimental Stroke Models.

BACKGROUND: Ischemic stroke affects about 700 000 patients per year in the United States, and to date, there are no effective pharmacological agents that promote recovery. Here, we studied the pharmacokinetics, pharmacodynamics, and efficacy of NTS-105, a novel neuroactive steroid, and NTS-104, a prodrug of NTS-105, in 2 models of ischemic stroke. METHODS: The pharmacodynamics and pharmacokinetics of NTS-104/105 were investigated in naive and stroke rats, and models of embolic and transient middle cerebral artery occlusion were used to investigate the dose-related effects of NTS-104. All rats were randomly assigned into the experimental groups, and all outcome measurements were performed blindly. RESULTS: Blood plasma and brain pharmacokinetic analysis revealed that NTS-104 rapidly converted to NTS-105, which reached peak concentration at ≈1 hour after dosing and distributed similarly to normal and ischemic brains. NTS-104 administration 4 hours after embolic middle cerebral artery occlusion led to a dose-dependent improvement of neurological outcomes and a dose-dependent reduction of infarct volumes relative to vehicle-treated animals. A single dose level study confirmed that NTS-104 administered 4 hours after transient middle cerebral artery occlusion was also neuroprotective. Quantitative ELISA revealed that NTS-104 treatment resulted in time- and dose-dependent changes in AKT activation and cytokine levels within the ischemic brain, which included reductions of IL-6, VEGF, ICAM-1, IL-1ß, MCP-1, RAGE, and GM-CSF. Time- and dose-dependent reductions in IL-6 and GM-CSF were also observed in the plasma along with an elevation of galectin-1. CONCLUSIONS: NTS-104 is a novel prodrug that converts to a novel neuroactive steroid, NTS-105, which improves functional outcomes in experimental ischemic stroke models.

Cerebral endothelial cell derived small extracellular vesicles improve cognitive function in aged diabetic rats.

Small extracellular vesicles (sEVs) mediate cell-cell communication by transferring their cargo biological materials into recipient cells. Diabetes mellitus (DM) induces cerebral vascular dysfunction and neurogenesis impairment, which are associated with cognitive decline and an increased risk of developing dementia. Whether the sEVs are involved in DM-induced cerebral vascular disease, is unknown. Therefore, we studied sEVs derived from cerebral endothelial cells (CEC-sEVs) of aged DM rats (DM-CEC-sEVs) and found that DM-CEC-sEVs robustly inhibited neural stem cell (NSC) generation of new neuroblasts and damaged cerebral endothelial function. Treatment of aged DM-rats with CEC-sEVs derived from adult healthy normal rats (N-CEC-sEVs) ameliorated cognitive deficits and improved cerebral vascular function and enhanced neurogenesis. Intravenously administered N-CEC-sEVs crossed the blood brain barrier and were internalized by neural stem cells in the neurogenic region, which were associated with augmentation of miR-1 and -146a and reduction of myeloid differentiation primary response gene 88 and thrombospondin 1 proteins. In addition, uptake of N-CEC-sEVs by the recipient cells was mediated by clathrin and caveolin dependent endocytosis signaling pathways. The present study provides ex vivo and in vivo evidence that DM-CEC-sEVs induce cerebral vascular dysfunction and neurogenesis impairment and that N-CEC-sEVs have a therapeutic effect on improvement of cognitive function by ameliorating dysfunction of cerebral vessels and increasing neurogenesis in aged DM rats, respectively.

Aging-Related Alterations of Glymphatic Transport in Rat: In vivo Magnetic Resonance Imaging and Kinetic Study.

Objective: Impaired glymphatic waste clearance function during brain aging leads to the accumulation of metabolic waste and neurotoxic proteins (e.g., amyloid-ß, tau) which contribute to neurological disorders. However, how the age-related glymphatic dysfunction exerts its effects on different cerebral regions and affects brain waste clearance remain unclear. Methods: We investigated alterations of glymphatic transport in the aged rat brain using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and advanced kinetic modeling. Healthy young (3-4 months) and aged (18-20 months) male rats (n = 12/group) underwent the identical MRI protocol, including T2-weighted imaging and 3D T1-weighted imaging with intracisternal administration of contrast agent (Gd-DTPA). Model-derived parameters of infusion rate and clearance rate, characterizing the kinetics of cerebrospinal fluid (CSF) tracer transport via the glymphatic system, were evaluated in multiple representative brain regions. Changes in the CSF-filled cerebral ventricles were measured using contrast-induced time signal curves (TSCs) in conjunction with structural imaging. Results: Compared to the young brain, an overall impairment of glymphatic transport function was detected in the aged brain, evidenced by the decrease in both infusion and clearance rates throughout the brain. Enlarged ventricles in parallel with reduced efficiency in CSF transport through the ventricular regions were present in the aged brain. While the age-related glymphatic dysfunction was widespread, our kinetic quantification demonstrated that its impact differed considerably among cerebral regions with the most severe effect found in olfactory bulb, indicating the heterogeneous and regional preferential alterations of glymphatic function. Conclusion: The robust suppression of glymphatic activity in the olfactory bulb, which serves as one of major efflux routes for brain waste clearance, may underlie, in part, age-related neurodegenerative diseases associated with neurotoxic substance accumulation. Our data provide new insight into the cerebral regional vulnerability to brain functional change with aging.

Exosomes derived from bone marrow mesenchymal stromal cells promote remyelination and reduce neuroinflammation in the demyelinating central nervous system.

Injury of oligodendrocytes (OLs) induces demyelination, and patients with neurodegenerative diseases exhibit demyelination concomitantly with neurological deficit and cognitive impairment. Oligodendrocyte progenitor cells (OPCs) are present in the adult central nervous system (CNS), and they can proliferate, differentiate, and remyelinate axons after damage. However, remyelination therapies are not in clinical use. Multiple sclerosis (MS) is a major demyelinating disease in the CNS. Mesenchymal stromal cells (MSCs) have demonstrated therapeutic promise in animal models and in clinical trials of MS. Exosomes are nanoparticles generated by nearly all cells and they mediate cell-cell communication by transferring cargo biomaterials. Here, we hypothesize that exosomes harvested from MSCs have a similar therapeutic effect on enhancement of remyelination as that of MSCs. In the present study we employed exosomes derived from rhesus monkey MSCs (MSC-Exo). Two mouse models of demyelination were employed: 1) experimental autoimmune encephalomyelitis (EAE), an animal model of MS; and 2) cuprizone (CPZ) diet model, a toxic demyelination model. MSC-Exo or PBS were intravenously injected twice a week for 4 weeks, starting on day 10 post immunization in EAE mice, or once a week for 2 weeks starting on the day of CPZ diet withdrawal. Neurological and cognitive function were tested, OPC differentiation and remyelination, neuroinflammation and the potential underlying mechanisms were investigated using immunofluorescent staining, transmission electron microscopy and Western blot. Data generated from the EAE model revealed that MSC-Exo cross the blood brain barrier (BBB) and target neural cells. Compared with the controls (p < 0.05), treatment with MSC-Exo: 1) significantly improved neurological outcome; 2) significantly increased the numbers of newly generated OLs (BrdU+/APC+) and mature OLs (APC+), and the level of myelin basic protein (MBP); 3) decreased amyloid-ß precursor protein (APP)+ density; 4) decreased neuroinflammation by increasing the M2 phenotype and decreasing the M1 phenotype of microglia, as well as their related cytokines; 5) inhibited the TLR2/IRAK1/NFκB pathway. Furthermore, we confirmed that the MSC-Exo treatment significantly improved cognitive function, promoted remyelination, increased polarization of M2 phenotype and blocked TLR2 signaling in the CPZ model. Collectively, MSC-Exo treatment promotes remyelination by both directly acting on OPCs and indirectly by acting on microglia in the demyelinating CNS. This study provides the cellular and molecular basis for this cell-free exosome therapy on remyelination and modulation of neuroinflammation in the CNS, with great potential for treatment of demyelinating and neurodegenerative disorders.

Connectivity characterization of the mouse basolateral amygdalar complex.

The basolateral amygdalar complex (BLA) is implicated in behaviors ranging from fear acquisition to addiction. Optogenetic methods have enabled the association of circuit-specific functions to uniquely connected BLA cell types. Thus, a systematic and detailed connectivity profile of BLA projection neurons to inform granular, cell type-specific interrogations is warranted. Here, we apply machine-learning based computational and informatics analysis techniques to the results of circuit-tracing experiments to create a foundational, comprehensive BLA connectivity map. The analyses identify three distinct domains within the anterior BLA (BLAa) that house target-specific projection neurons with distinguishable morphological features. We identify brain-wide targets of projection neurons in the three BLAa domains, as well as in the posterior BLA, ventral BLA, posterior basomedial, and lateral amygdalar nuclei. Inputs to each nucleus also are identified via retrograde tracing. The data suggests that connectionally unique, domain-specific BLAa neurons are associated with distinct behavior networks.

Plasminogen deficiency causes reduced angiogenesis and behavioral recovery after stroke in mice.

Plasminogen is involved in the process of angiogenesis; however, the underlying mechanism is unclear. Here, we investigated the potential contribution of plasmin/plasminogen in mediating angiogenesis and thereby contributing to functional recovery post-stroke. Wild-type plasminogen naive (Plg+/+) mice and plasminogen knockout (Plg-/-) mice were subjected to unilateral permanent middle cerebral artery occlusion (MCAo). Blood vessels were labeled with FITC-dextran. Functional outcomes, and cerebral vessel density were compared between Plg+/+ and Plg-/- mice at different time points after stroke. We found that Plg-/- mice exhibited significantly reduced functional recovery, associated with significantly decreased vessel density in the peri-infarct area in the ipsilesional cortex compared with Plg+/+ mice. In vitro, cerebral endothelial cells harvested from Plg-/- mice exhibited significantly reduced angiogenesis assessed using tube formation assay, and migration, as evaluated using Scratch assays, compared to endothelial cells harvested from Plg+/+ mice. In addition, using Western blots, expression of thrombospondin (TSP)-1 and TSP-2 were increased after MCAo in the Plg-/- group compared to Plg+/+ mice, especially in the ipsilesional side of brain. Taken together, our data suggest that plasmin/plasminogen down-regulates the expression level of TSP-1 and TSP-2, and thereby promotes angiogenesis in the peri-ischemic brain tissue, which contributes to functional recovery after ischemic stroke.

MiR-17-92 Cluster-Enriched Exosomes Derived from Human Bone Marrow Mesenchymal Stromal Cells Improve Tissue and Functional Recovery in Rats after Traumatic Brain Injury.

Exosomes play an important role in intercellular communication by delivering microribonucleic acids (miRNAs) to recipient cells. Previous studies have demonstrated that multi-potent mesenchymal stromal cell (MSC)-derived exosomes improve functional recovery after experimental traumatic brain injury (TBI). This study was performed to determine efficacy of miR-17-92 cluster-enriched exosomes (Exo-17-92) harvested from human bone marrow MSCs transfected with a miR-17-92 cluster plasmid in enhancing tissue and neurological recovery compared with exosomes derived from MSCs transfected with an empty plasmid vector (Exo-empty) for treatment of TBI. Adult male rats underwent a unilateral moderate cortical contusion. Animals received a single intravenous injection of miR-17-92 cluster-enriched exosomes (100 µg/rat, approximately 3.75x1011 particles, Exo-17-92) or control exosomes (100 µg/rat, Exo-empty) or Vehicle (phosphate-buffered solution) one day after injury. A battery of neurological functional tests was performed weekly after TBI for five weeks. Spatial learning and memory were measured on days 31-35 after TBI using the Morris water maze test. All animals were sacrificed five weeks after injury. Their brains were processed for histopathological and immunohistochemical analyses of lesion volume, cell loss, angiogenesis, neurogenesis, and neuroinflammation. Compared with Vehicle, both Exo-17-92 and Exo-empty treatments significantly improved sensorimotor and cognitive function, reduced neuroinflammation and hippocampal neuronal cell loss, promoted angiogenesis and neurogenesis without altering the lesion volume. Moreover, Exo-17-92 treatment exhibited a significantly more robust therapeutic effect on improvement in functional recovery by reducing neuroinflammation and cell loss, enhancing angiogenesis and neurogenesis than did Exo-empty treatment. Exosomes enriched with miR-17-92 cluster have a significantly better effect on improving functional recovery after TBI compared with Exo-empty, likely by reducing neuroinflammation and enhancing endogenous angiogenesis and neurogenesis. Engineering specific miRNA in exosomes may provide a novel therapeutic strategy for management of unilateral moderate cortical contusion TBI.

Therapeutic effect of Cerebrolysin on reducing impaired cerebral endothelial cell permeability.

Cerebrolysin has been shown to promote neurovascular protection and repair in preclinical models of stroke and neural injury and is demonstrating promise for stroke and neural injury therapeutic application in the clinic. The effect of Cerebrolysin on the human cerebral endothelial cell function has not been investigated. Using an in-vitro cerebral endothelial cell permeability assay and western blot analyses of tight junction and proinflammatory and procoagulant proteins, the present study showed that tissue plasminogen activator (tPA) and fibrin substantially impaired human cerebral endothelial cell barrier function and increased permeability, which persisted for at least 24 h. western blot analysis revealed that tPA and fibrin significantly increased proinflammatory and procoagulation proteins of intercellular adhesion molecule 1, high mobility group box 1, tumor necrosis factor α and phosphorylated nuclear factor kappa B-p65, and significantly reduced tight junction proteins zonular 1, occludin and claudin. However, Cerebrolysin significantly diminished and reversed tPA- and fibrin-impaired endothelial cell permeability, which was associated with significant reductions of tPA- and fibrin-augmented proinflammatory and procoagulation proteins and significant elevations of tPA- and fibrin-decreased tight junction proteins. The beneficial effect of Cerebrolysin appears specific because cerebroprotein hydrolysate, with a distinct peptide composition, failed to show the reduction of tPA- and fibrin-impaired permeability. These data indicate that cererbrolysin has a therapeutic effect on tPA- and fibrin-impaired cerebral endothelial cell permeability by reducing proinflammatory and procoagulation proteins and by elevating tight junction proteins.

Treatment of diabetic peripheral neuropathy with engineered mesenchymal stromal cell-derived exosomes enriched with microRNA-146a provide amplified therapeutic efficacy.

Diabetic peripheral neuropathy (DPN) is one of the most prevalent chronic complications of diabetes mellitus with no effective treatment. We recently demonstrated that mesenchymal stromal cell (MSC)-derived exosomes (exo-naïve) alleviate neurovascular dysfunction and improve functional recovery. MicroRNA (miRNA), one of the exosomal cargos, downregulates inflammation-related genes, resulting in suppression of pro-inflammatory gene activation. In the present study, we developed engineered MSC-exosomes loaded with miR-146a (exo-146a) and compared the therapeutic effects of exo-146a with exo-naïve in diabetic (db/db) mice with DPN. Exo-146a possesses a high loading capacity, robust ability to accumulate in peripheral nerve tissues upon systemic administration, and evokes substantially enhanced therapeutic efficacy on neurological recovery compared with exo-naïve. Treatment of DPN in diabetic mice with exo-146a for two weeks significantly increased and decreased nerve conduction velocity, and thermal and mechanical stimuli threshold, respectively, whereas it took four weeks of exo-naive treatment to achieve these improvements. Compared with exo-naïve, exo-146a significantly suppressed the peripheral blood inflammatory monocytes and the activation of endothelial cells via inhibiting Toll-like receptor (TLR)-4/NF-κB signaling pathway. These data provide a proof-of-concept about both the feasibility and efficacy of the exosome-based gene therapy for DPN. The translation of this approach to the clinic has the potential to improve the prospects for people who suffer from DPN.

Small extracellular vesicles ameliorate peripheral neuropathy and enhance chemotherapy of oxaliplatin on ovarian cancer.

There are no effective treatments for chemotherapy induced peripheral neuropathy (CIPN). Small extracellular vesicles (sEVs) facilitate intercellular communication and mediate nerve function and tumour progression. We found that the treatment of mice bearing ovarian tumour with sEVs derived from cerebral endothelial cells (CEC-sEVs) in combination with a chemo-drug, oxaliplatin, robustly reduced oxaliplatin-induced CIPN by decreasing oxaliplatin-damaged myelination and nerve fibres of the sciatic nerve and significantly amplified chemotherapy of oxaliplatin by reducing tumour size. The combination therapy substantially increased a set of sEV cargo-enriched miRNAs, but significantly reduced oxaliplatin-increased proteins in the sciatic nerve and tumour tissues. Bioinformatics analysis revealed the altered miRNAs and proteins formed two distinct networks that regulate neuropathy and tumour growth, respectively. Intravenously administered CEC-sEVs were internalized by axons of the sciatic nerve and cancer cells. Reduction of CEC-sEV cargo miRNAs abolished the effects of CEC-sEVs on oxaliplatin-inhibited axonal growth and on amplification of the anti-cancer effect in ovarian cancer cells, suggesting that alterations in the networks of miRNAs and proteins in recipient cells contribute to the therapeutic effect of CEC-sEVs on CIPN. Together, the present study demonstrates that CEC-sEVs suppressed CIPN and enhanced chemotherapy of oxaliplatin in the mouse bearing ovarian tumour.

MicroRNA-214 enriched exosomes from human cerebral endothelial cells (hCEC) sensitize hepatocellular carcinoma to anti-cancer drugs.

Hepatocellular carcinoma (HCC) is the most common primary liver tumor worldwide. Current medical therapy for HCC has limited efficacy. The present study tests the hypothesis that human cerebral endothelial cell-derived exosomes carrying elevated miR-214 (hCEC-Exo-214) can amplify the efficacy of anti-cancer drugs on HCC cells. Treatment of HepG2 and Hep3B cells with hCEC-Exo-214 in combination with anti-cancer agents, oxaliplatin or sorafenib, significantly reduced cancer cell viability and invasion compared with monotherapy with either drug. Additionally, the therapeutic effect of the combination therapy was detected in primary tumor cells derived from patients with HCC. The ability of hCEC-Exo-214 in sensitizing HCC cells to anti-cancer drugs was specific, in that combination therapy did not affect the viability and invasion of human liver epithelial cells and non-cancer primary cells. Furthermore, compared to monotherapy with oxaliplatin and sorafenib, hCEC-Exo-214 in combination with either drug substantially reduced protein levels of P-glycoprotein (P-gp) and splicing factor 3B subunit 3 (SF3B3) in HCC cells. P-gp and SF3B3 are among miR-214 target genes and are known to mediate drug resistance and cancer cell proliferation, respectively. In conclusion, the present in vitro study provides evidence that hCEC-Exo-214 significantly enhances the anti-tumor efficacy of oxaliplatin and sorafenib on HCC cells.

Cerebral endothelial cell-derived small extracellular vesicles enhance neurovascular function and neurological recovery in rat acute ischemic stroke models of mechanical thrombectomy and embolic stroke treatment with tPA.

Treatment of patients with cerebral large vessel occlusion with thrombectomy and tissue plasminogen activator (tPA) leads to incomplete reperfusion. Using rat models of embolic and transient middle cerebral artery occlusion (eMCAO and tMCAO), we investigated the effect on stroke outcomes of small extracellular vesicles (sEVs) derived from rat cerebral endothelial cells (CEC-sEVs) in combination with tPA (CEC-sEVs/tPA) as a treatment of eMCAO and tMCAO in rat. The effect of sEVs derived from clots acquired from patients who had undergone mechanical thrombectomy on healthy human CEC permeability was also evaluated. CEC-sEVs/tPA administered 4 h after eMCAO reduced infarct volume by ∼36%, increased recanalization of the occluded MCA, enhanced cerebral blood flow (CBF), and reduced blood-brain barrier (BBB) leakage. Treatment with CEC-sEVs given upon reperfusion after 2 h tMCAO significantly reduced infarct volume by ∼43%, and neurological outcomes were improved in both CEC-sEVs treated models. CEC-sEVs/tPA reduced a network of microRNAs (miRs) and proteins that mediate thrombosis, coagulation, and inflammation. Patient-clot derived sEVs increased CEC permeability, which was reduced by CEC-sEVs. CEC-sEV mediated suppression of a network of pro-thrombotic, -coagulant, and -inflammatory miRs and proteins likely contribute to therapeutic effects. Thus, CEC-sEVs have a therapeutic effect on acute ischemic stroke by reducing neurovascular damage.

Extracellular vesicles derived from bone marrow mesenchymal stem cells enhance myelin maintenance after cortical injury in aged rhesus monkeys.

Cortical injury, such as stroke, causes neurotoxic cascades that lead to rapid death and/or damage to neurons and glia. Axonal and myelin damage in particular, are critical factors that lead to neuronal dysfunction and impair recovery of function after injury. These factors can be exacerbated in the aged brain where white matter damage is prevalent. Therapies that can ameliorate myelin damage and promote repair by targeting oligodendroglia, the cells that produce and maintain myelin, may facilitate recovery after injury, especially in the aged brain where these processes are already compromised. We previously reported that a novel therapeutic, Mesenchymal Stem Cell derived extracellular vesicles (MSC-EVs), administered intravenously at both 24 h and 14 days after cortical injury, reduced microgliosis (Go et al. 2019), reduced neuronal pathology (Medalla et al. 2020), and improved motor recovery (Moore et al. 2019) in aged female rhesus monkeys. Here, we evaluated the effect of MSC-EV treatment on changes in oligodendrocyte maturation and associated myelin markers in the sublesional white matter using immunohistochemistry, confocal microscopy, stereology, qRT-PCR, and ELISA. Compared to vehicle control monkeys, EV-treated monkeys showed a reduction in the density of damaged oligodendrocytes. Further, EV-treatment was associated with enhanced myelin maintenance, evidenced by upregulation of myelin-related genes and increases in actively myelinating oligodendrocytes in sublesional white matter. These changes in myelination correlate with the rate of motor recovery, suggesting that improved myelin maintenance facilitates this recovery. Overall, our results suggest that EVs act on oligodendrocytes to support myelination and improves functional recovery after injury in the aged brain. SIGNIFICANCE: We previously reported that EVs facilitate recovery of function after cortical injury in the aged monkey brain, while also reducing neuronal pathology (Medalla et al. 2020) and microgliosis (Go et al. 2019). However, the effect of injury and EVs on oligodendrocytes and myelination has not been characterized in the primate brain (Dewar et al. 1999; Sozmen et al. 2012; Zhang et al. 2013). In the present study, we assessed changes in myelination after cortical injury in aged monkeys. Our results show, for the first time, that MSC-EVs support recovery of function after cortical injury by enhancing myelin maintenance in the aged primate brain.