Background and Purpose Multipotent mesenchymal stromal cell (MSC) harvested exosomes are hypothesized as the major paracrine effectors of MSCs. In vitro, the miR-17-92 cluster promotes oligodendrogenesis, neurogenesis and axonal outgrowth. We therefore investigated whether the miR-17-92 cluster enriched exosomes (Exo-miR-17-92+) harvested from MSCs transfected with a miR-17-92 cluster plasmid enhance neurological recovery compared to control MSC derived exosomes (Exo-Con). Methods Rats subjected to 2 hours of transient middle cerebral artery occlusion (MCAO) were intravenously administered Exo-miR-17-92+, Exo-Con, or liposomes, and were sacrificed 28 days post MCAO. Histochemistry, immunohistochemistry and Golgi-Cox staining were used to assess dendritic, axonal, synaptic and myelin remodeling. Expression of phosphatase and tensin homolog (PTEN) and activation of its downstream proteins, protein kinase B (PKB or Akt), mechanistic target of rapamycin (mTOR), and glycogen synthase kinase 3 beta (GSK-3β) in the peri-infarct region were measured by means of Western blots. Results Compared with the liposome treatment, both exosome treatment groups exhibited significant improvement of functional recovery, but Ex-miR-17-92+ treatment had significantly more robust effects on improvement of neurological function, and enhancements of oligodendrogenesis, neurogenesis and neurite remodeling/neuronal dendrite plasticity in the ischemic boundary zone (IBZ) than the Ex-Con treatment. Moreover, Ex-miR-17-92+ treatment substantially inhibited PTEN, a validated miR-17-92 cluster target gene, and subsequently increased the phosphorylation of PTEN downstream proteins, Akt, mTOR and GSK-3β compared to Ex-Con treatment. Conclusions Our data suggest that treatment of stroke with tailored exosomes enriched with the miR-17-92 cluster increases neural plasticity and functional recovery after stroke, possibly via targeting PTEN to activate the PI3K/Akt/mTOR/GSK-3β signaling pathway.

Multipotent human bone marrow derived mesenchymal stem cells (hMSCs) improve functional outcome after experimental traumatic brain injury (TBI). The present study was designed to investigate whether systemic administration of cell-free exosomes generated from hMSCs cultured in 2-dimensional (2D) conventional conditions or in 3-dimensional (3D) collagen scaffolds promote functional recovery and neurovascular remodeling in rats after TBI. Wistar rats were subjected to TBI induced by controlled cortical impact; 24 hours later tail vein injection of exosomes derived from hMSCs cultured under 2D or 3D conditions or an equal number of liposomes as a treatment control were performed. The modified Morris water maze, neurological severity score and footfault tests were employed to evaluate cognitive and sensorimotor functional recovery. Animals were sacrificed at 35 days after TBI. Histological and immunohistochemical analyses were performed for measurements of lesion volume, neurovascular remodeling (angiogenesis and neurogenesis), and neuroinflammation. Compared with liposome-treated control, exosome-treatments did not reduce lesion size but significantly improved spatial learning at 33-35 days measured by the Morris water maze test, and sensorimotor functional recovery, i.e., reduced neurological deficits and footfault frequency, observed at 14-35 days post injury (p < 0.05). Exosome treatments significantly increased the number of newborn endothelial cells in the lesion boundary zone and dentate gyrus, and significantly increased the number of newborn mature neurons in the dentate gyrus as well as reduced neuroinflammation. Exosomes derived from hMSCs cultured in 3D scaffolds provided better outcome in spatial learning than exosomes from hMSCs cultured in the 2D condition. In conclusion, hMSC-generated exosomes significantly improve functional recovery in rats after TBI, at least in part, by promoting endogenous angiogenesis and neurogenesis and reducing neuroinflammation. Thus, exosomes derived from hMSCs may be a novel cell-free therapy for TBI, and hMSC-scaffold generated exosomes may selectively enhance spatial learning.

As neovascularization is essential for tumor growth and metastasis, controlling angiogenesis is a promising tactic in limiting cancer progression. Melatonin has been studied for their inhibitory properties on angiogenesis in cancer. We performed an in vivo study to evaluate the effects of melatonin treatment on angiogenesis in breast cancer. Cell viability was measured by MTT assay after melatonin treatment in triple-negative breast cancer cells (MDA-MB-231). After, cells were implanted in athymic nude mice and treated with melatonin or vehicle daily, administered intraperitoneally 1 hour before turning the room light off. Volume of the tumors was measured weekly with a digital caliper and at the end of treatments animals underwent single photon emission computed tomography (SPECT) with Technetium-99m tagged vascular endothelial growth factor (VEGF) C to detect in vivo angiogenesis. In addition, expression of pro-angiogenic/growth factors in the tumor extracts was evaluated by membrane antibody array and collected tumor tissues were analyzed with histochemical staining. Melatonin in vitro treatment (1 mM) decreased cell viability (p<0.05). The breast cancer xenografts nude mice treated with melatonin showed reduced tumor size and cell proliferation (Ki-67) compared to control animals after 21 days of treatment (p<0.05). Expression of VEGF receptor 2 decreased significantly in the treated animals compared to that of control when determined by immunohistochemistry (p<0.05) but the changes were not significant on SPECT (p>0.05) images. In addition, there was a decrease of micro-vessel density (Von Willebrand Factor) in melatonin treated mice (p<0.05). However, semiquantitative densitometry analysis of membrane array indicated increased expression of epidermal growth factor receptor and insulin-like growth factor 1 in treated tumors compared to vehicle treated tumors (p<0.05). In conclusion, melatonin treatment showed effectiveness in reducing tumor growth and cell proliferation, as well as in the inhibition of angiogenesis.

CONSPECTUS Responsive magnetic resonance imaging (MRI) contrast agents can change MR image contrast in response to a molecular biomarker. Quantitative detection of the biomarker requires an accounting of the other effects that may alter MR image contrast, such as a change in the agent’s concentration, magnetic field variations, and hardware sensitivity profiles. A second unresponsive MRI contrast agent may serve as an “internal control” to isolate the detection of the molecular biomarker. Chemical exchange saturation transfer (CEST) MRI contrast agents can be selectively detected, providing the opportunity to combine a responsive CEST agent and an unresponsive CEST agent during the same MRI scan session. When two CEST MRI contrast agents are used for molecular imaging applications, the CEST agents should be designed to maximize accurate quantification of the concentrations of the two agents. From a chemical perspective, CEST agents behave like enzymes that catalyze the conversion of an unsaturated water “substrate” into a saturated water “product”. The analysis of CEST agent kinetics parallels the Michaelis–Menten analysis of enzyme kinetics, which can be used to correlate the CEST effect with the concentration of the agent in solution. If the concentration of water “substrate” that is available to the CEST agent is unknown, which may be likely for in vivo MRI studies, then only a ratio of concentrations of the two CEST agents can be measured. In both cases, CEST agents should be designed with minimal T1 relaxivity to improve concentration quantifications. CEST agents can also be designed to maximize sensitivity. This may be accomplished by incorporating many CEST agents within nanoparticles to create a large number of exchangeable protons per nanoparticle. Finally, CEST agents can be designed with rapid detection in mind. This may be accomplished by minimizing T1 relaxivity of the CEST agent so that MRI acquisition methods have time to collect many MRI signals following a single selective saturation period. In this Account, we provide an example that shows the sensitive and rapid detection of two CEST agents in an in vivo MRI study of a mouse model of mammary carcinoma. The ratio of the concentrations of the two CEST agents was quantified with analysis methods that parallel Michaelis–Menten enzyme kinetic analysis. This example demonstrates current limitations of the method that require additional research, but it also shows that two CEST MRI contrast agents can be detected and quantitatively assessed during in vivo molecular imaging studies.