Microvascular Endothelial Cells-Derived Microvesicles Imply in Ischemic Stroke by Modulating Astrocyte and Blood Brain Barrier Function and Cerebral Blood Flow

Background Endothelial cell (EC) released microvesicles (EMVs) can affect various target cells by transferring carried genetic information. Astrocytes are the main components of the blood brain barrier (BBB) structure in the brain and participate in regulating BBB integrity and blood flow. The interactions between ECs and astrocytes are essential for BBB integrity in homeostasis and pathological conditions. Here, we studied the effects of human brain microvascular ECs released EMVs on astrocyte functions. Additionally, we investigated the effects of EMVs treated astrocytes on regulating BBB function and cerebral ischemic damage. Results EMVs prepared from ECs cultured in normal condition (n-EMVs) or oxygen and glucose deprivation (OGD-EMVs) condition had diverse effects on astrocytes. The n-EMVs promoted, while the OGD-EMVs inhibited the proliferation of astrocytes via regulating PI3K/Akt pathway. Glial fibrillary acidic protein (GFAP) expression (marker of astrocyte activation) was up-regulated by n-EMVs, while down-regulated by OGD-EMVs. Meanwhile, n-EMVs inhibited but OGD-EMVs promoted the apoptosis of astrocytes accompanied by up/down-regulating the expression of Caspase-9 and Bcl-2. In the BBB model of ECs-astrocytes co-culture, the n-EMVs, conversely to OGD-EMVs, decreased the permeability of BBB accompanied with up-regulation of zonula occudens-1(ZO-1) and Claudin-5. In a transient cerebral ischemia mouse model, n-EMVs ameliorated, while OGD-EMVs aggravated, BBB disruption, local cerebral blood flow (CBF) reduction, infarct volume and neurological deficit score. Conclusions Our data suggest that EMVs diversely modulate astrocyte functions, BBB integrity and CBF, and could serve as a novel therapeutic target for ischemic stroke

miR-132-3p Priming Enhances the Effects of Mesenchymal Stromal Cell-Derived Exosomes on Ameliorating Brain Ischemic Injury

Backgrounds/aims: Mesenchymal stromal cell-derived exosomes (MSC-EXs) could exert protective effects on recipient cells by transferring the contained microRNAs (miRs), and miR-132-3p is one of angiogenic miRs. However, whether the combination of MSC-EXs and miR-132-3p has better effects in ischemic cerebrovascular disease remains unknown. Methods: Mouse MSCs transfected with scrambler control or miR-132-3p mimics were used to generate MSC-EXs and miR-132-3p-overexpressed MSC-EXs (MSC-EXsmiR-132-3p). The effects of EXs on hypoxia/reoxygenation (H/R)-injured ECs in ROS generation, apoptosis, and barrier function were analyzed. The levels of RASA1, Ras, phosphorylations of PI3K, Akt and endothelial nitric oxide synthesis (eNOS), and tight junction proteins (Claudin-5 and ZO-1) were measured. Ras and PI3K inhibitors were used for pathway analysis. In transient middle cerebral artery occlusion (tMCAO) mouse model, the effects of MSC-EXs on the cerebral vascular ROS production and apoptosis, cerebral vascular density (cMVD), Evans blue extravasation, brain water content, neurological deficit score (NDS), and infarct volume were determined. Results: MSC-EXs could deliver their carried miR-132-3p into target ECs, which functionally downregulated the target protein RASA1, while upregulated the expression of Ras and the downstream PI3K phosphorylation. Compared to MSC-EXs, MSC-EXsmiR-132-3p were more effective in decreasing ROS production, apoptosis, and tight junction disruption in H/R-injured ECs. These effects were associated with increased levels of phosphorylated Akt and eNOS, which could be abolished by PI3K inhibitor (LY294002) or Ras inhibitor (NSC 23766). In the tMCAO mouse model, the infusion of MSC-EXsmiR-132-3p was more effective than MSC-EXs in reducing cerebral vascular ROS production, BBB dysfunction, and brain injury. Conclusion: Our results suggest that miR-132-3p promotes the beneficial effects of MSC-EXs on brain ischemic injury through protecting cerebral EC functions

Abstract P195: Overexpression of ACE2 Boosts the Therapeutic Effects of Endothelial Progenitor Cells Derived Exosomes on Hemorrhagic Stroke

We have previously demonstrated that angiotensin converting enzyme 2 (ACE2) could boost the therapeutic effects of endothelial progenitor cell (EPCs) on ischemic stroke. Here, we tested whether ACE2 could enhance the effects of EPC derived exosomes (EXs) on intracerebral hemorrhagic stroke (ICH). A bolus of EPC-EXs or ACE2-EPC-EXs (1 x 1011EXs/100 ul) labeled with PKH26 was intravenously administrated to ICH mice (C57BL/6) 24 hrs after collagenase (VII-S; 0.075 U/0.5 μl) injection. ACE2 blocker, DCX 600 was used to verify the effects of ACE2. The neurological deficit score (NDS), hemorrhage volume, brain water content, and blood brain barrier (BBB) permeability were measured at day 24 hrs after injection. The levels of ACE2, inflammatory factors in the brain were measured. We found (table): 1) Both EPC-EXs and ACE2-EPC-EXs were dominantly uptaken by the brain endothelial cells, neurons and astrocytes in peri-infarct area; 2) ACE2-EPC-EXswere more effective than EPC-EXs in decreasing hemorrhage volume, brain edema, BBB permeability and improving NDS; 3) As compared to EPC-EXs, ACE2-EPC-EXs resulted in an up-regulation of ACE2, and more down-regulated TNF-α, NFкB, IκBα expressions. 4) DCX 600 could block the protective effects of ACE2-EPC-EXs. The data suggest that infusion of ACE2-EPC-EXsexhibited protective role for anti-inflammatory effect of ACE2 mediating TNF-α/NFкB in mice after ICH

Antitumor function and mechanism of phycoerythrin from Porphyra haitanensis

The anti-tumor effect of R-Phycoerythrin (R-PE) from Porphyra haitanensis was studied using cell line HeLa as an in vitro model and Sarcoma-180 (S180) tumor-bearing mice as an in vivo model. The results showed that the combination treatment of R-PE and photodynamic therapy PDT) significantly inhibited the growth of HeLa cells up to 81.5%, with a fair dose-effect relationship, but did not inhibit endothelial cells. The annexin v-fitc/PI fluorescence staining experiments demonstrated that at doses between 0~60µg/mL, apoptosis cells and later stage apoptosis cells or necrosis cells increased significantly as the R-PE dosage increased. DNA electrophoresis showed that after R-PE+PDT treatment of HeLa cells for 24 hours, a light "smear" band between 100~400bp appeared to indicate the degradation of genomic DNA. The QRT-PCR results showed that R-PE+PDT treatment increased caspase-3 and caspase-10 gene expression and decreased the Bcl-2 gene expression level significantly as the R-PE dose increased, implying that R-PE promoted HeLa cell apoptosis. Compared with untreated S180 tumor-bearing mice, R-PE injection significantly inhibited the growth of S180 in tumor-bearing mice up to 41.3% at a dose of 300mg-kg-1. Simultaneously, the significant increase of superoxide dismutase (SOD) activity in serum (p < 0.01) and the decrease of the malondialdehyde (MDA) level in liver suggests that R-PE improved the anti-oxidant ability of the S180 tumor-bearing mice, which may related to its antitumor effect. In addition, the R-PE caused a significant increase (p < 0.05) in the spleen index and thymus index, and a significant increase (p < 0.01) in lymphocyte proliferation, NK cell kill activity and the TNF-&#945; level in the serum of S180 tumor-bearing mice. These results strongly suggest that the antitumor effect of R-PE from Porphyra haitanensis functioned by increasing the immunity and antioxidant ability of S180 tumor-bearing mice, promoting apoptosis by increasing protease gene expression and TNF-&#945; secretion

Overexpression of ACE2 Boosts the Therapeutic Effects of Endothelial Progenitor Cells Derived Exosomes on Hemorrhagic Stroke

We have previously demonstrated that angiotensin converting enzyme 2 (ACE2) could boost the therapeutic effects of endothelial progenitor cell (EPCs) on ischemic stroke. Here, we tested whether ACE2 could enhance the effects of EPC derived exosomes (EXs) on intracerebral hemorrhagic stroke (ICH). A bolus of EPC-EXs or ACE2-EPC-EXs (1 x 1011EXs/100 ul) labeled with PKH26 was intravenously administrated to ICH mice (C57BL/6) 24 hrs after collagenase (VII-S; 0.075 U/0.5 μl) injection. ACE2 blocker, DCX 600 was used to verify the effects of ACE2. The neurological deficit score (NDS), hemorrhage volume, brain water content, and blood brain barrier (BBB) permeability were measured at day 24 hrs after injection. The levels of ACE2, inflammatory factors in the brain were measured. We found (table): 1) Both EPC-EXs and ACE2-EPC-EXs were dominantly uptaken by the brain endothelial cells, neurons and astrocytes in peri-infarct area; 2) ACE2-EPC-EXswere more effective than EPC-EXs in decreasing hemorrhage volume, brain edema, BBB permeability and improving NDS; 3) As compared to EPC-EXs, ACE2-EPC-EXs resulted in an up-regulation of ACE2, and more down-regulated TNF-α, NFкB, IκBα expressions. 4) DCX 600 could block the protective effects of ACE2-EPC-EXs. The data suggest that infusion of ACE2-EPC-EXsexhibited protective role for anti-inflammatory effect of ACE2 mediating TNF-α/NFкB in mice after ICH

Multilayer Membranes of Glycosaminoglycans and Collagen I Biomaterials Modulate the Function and Microvesicle Release of Endothelial Progenitor Cells

Multilayer composite membrane of biomaterials can increase the function of adipose stem cells or osteoprogenitor cells. Recent evidence indicates endothelial progenitor cells (EPCs) and EPCs released microvesicles (MVs) play important roles in angiogenesis and vascular repair. Here, we investigated the effects of biomaterial multilayer membranes of hyaluronic acid (HA) or chondroitin sulfate (CS) and Collagen I (Col I) on the functions and MVs release of EPCs. Layer-by-layer (LBL) technology was applied to construct the multilayer composite membranes. Four types of the membranes constructed by adsorbing either HA or CS and Col I alternatively with different top layers were studied. The results showed that all four types of multilayer composite membranes could promote EPCs proliferation and migration and inhibit cell senility, apoptosis, and the expression of activated caspase-3. Interestingly, these biomaterials increased the release and the miR-126 level of EPCs-MVs. Moreover, the CS-Col I membrane with CS on the top layer showed the most effects on promoting EPCs proliferation, EPCs-MV release, and miR-126 level in EPCs-MVs. In conclusion, HA/CS and Collagen I composed multilayer composite membranes can promote EPCs functions and release of miR-126 riched EPCs-MVs, which provides a novel strategy for tissue repair treatment

Enrichment of miR-126 Boosts the Therapeutic Effects of Endothelial Progenitor Cells Derived Exosomes on Ischemic Stroke in Diabetic Mice

We have demonstrated that endothelial progenitor cell (EPCs) have therapeutic effects on ischemic stroke in diabetic mice and that microRNA (miR)-126 modulates the function of EPC through vascular endothelial growth factor receptor 2 (VEGFR2) pathway in cell culture studies. Here, we determined the effects of EPC-released exosomes (EPC-EXs) on ischemic stroke in diabetic mice and tested whether miR-126 enriched EPC-EXs (EPC-EXsmiR126) could have enhanced efficacy. Type 2 diabetic mice subjected to filament-induced focal ischemic stroke were intravenously administrated with vehicle, or PKH26 labelled EPC-EXs or EPC-EXsmiR-126. The neurological deficit score (NDS), cerebral blood flow (CBF), infarct volume, cerebral microvascular density (MVD), cell death, angiogenesis and neurogenesis, and levels of miR-126, VEGFR2 and cleaved caspase-3 were measured. We found: 1) Injected EPC-EXs merged with brain endothelial cells, neurons and astrocytes dominantly in the peri-infarct area; 2) EPC-EXsmiR126 were more effective than EPC-EXs in decreasing NDS, ischemic damage and cell death, and increasing CBF and MVD on both day 2 and 14, and in promoting angiogenesis and neurogenesis on day 14; 3) These effects were accompanied with down-regulated cleaved caspase-3 on day 2 and prolonged VEGFR2 up-regulation till day 14. The data suggest that transfusion of EPC-EXsmiR126 has enhanced therapeutic efficacy on ischemic stroke in diabetic mice by attenuating acute injury and promoting neurological function recovery

Enrichment of miR-126 Boosts the Therapeutic Effects of Endothelial Progenitor Cells Derived Exosomes on Ischemic Stroke in Diabetic Mice

We have demonstrated that endothelial progenitor cell (EPCs) have therapeutic effects on ischemic stroke in diabetic mice and that microRNA (miR)-126 modulates the function of EPC through vascular endothelial growth factor receptor 2 (VEGFR2) pathway in cell culture studies. Here, we determined the effects of EPC-released exosomes (EPC-EXs) on ischemic stroke in diabetic mice and tested whether miR-126 enriched EPC-EXs (EPC-EXsmiR126) could have enhanced efficacy. Type 2 diabetic mice subjected to filament-induced focal ischemic stroke were intravenously administrated with vehicle, or PKH26 labelled EPC-EXs or EPC-EXsmiR-126. The neurological deficit score (NDS), cerebral blood flow (CBF), infarct volume, cerebral microvascular density (MVD), cell death, angiogenesis and neurogenesis, and levels of miR-126, VEGFR2 and cleaved caspase-3 were measured. We found: 1) Injected EPC-EXs merged with brain endothelial cells, neurons and astrocytes dominantly in the peri-infarct area; 2) EPC-EXsmiR126 were more effective than EPC-EXs in decreasing NDS, ischemic damage and cell death, and increasing CBF and MVD on both day 2 and 14, and in promoting angiogenesis and neurogenesis on day 14; 3) These effects were accompanied with down-regulated cleaved caspase-3 on day 2 and prolonged VEGFR2 up-regulation till day 14. The data suggest that transfusion of EPC-EXsmiR126 has enhanced therapeutic efficacy on ischemic stroke in diabetic mice by attenuating acute injury and promoting neurological function recovery

Hypoxia/Aglycemia-Induced Endothelial Barrier Dysfunction and Tight Junction Protein Downregulation Can Be Ameliorated by Citicoline

This study explores the effect of citicoline on the permeability and expression of tight junction proteins (TJPs) in endothelial cells under hypoxia/aglycemia conditions. Hypoxia or oxygen and glucose deprivation (OGD) was utilized to induce endothelial barrier breakdown model on human umbilical vein endothelial cells (HUVECs) and mouse brain microvascular endothelial cells (bEnd.3s). The effect of citicoline on endothelial barrier breakdown models was determined at either low or high concentrations. FITC-Dextran flux was used to examine the endothelial permeability. The expression of TJPs was measured by immunofluorescence, Real-time PCR and Western Blot methods. Results showed that hypoxia or OGD increased the permeability of HUVECs accompanied with down-regulation of occludens-1 (ZO-1) and occludin at both mRNA and protein levels. Similarly in bEnd.3s, hypoxia increased the permeability and decreased the expression of ZO-1 and claudin-5. Citicoline treatment dose-dependently decreased the permeability in these two models, which paralleled with elevated expression of TJPs. The data demonstrate that citicoline restores the barrier function of endothelial cells compromised by hypoxia/aglycemia probably via up-regulating the expression of TJPs