Propofol inhibits myocardial injury induced by microvesicles derived from hypoxia-reoxygenated endothelial cells via lncCCT4-2/CCT4 signaling.

BACKGROUND: Ischemia-reperfusion (IR) induces increased release of extracellular vesicles in the heart and exacerbates myocardial IR injury. We have previously shown that propofol attenuates hypoxia/reoxygenation (HR)-induced injury in human umbilical vein endothelial cells (HUVECs) and that microvesicles derived from propofol-treated HUVECs inhibit oxidative stress in endothelial cells. However, the role of microvesicles derived from propofol post-treated HUVECs ((HR + P)-EMVs) in IR-injured cardiomyocytes is unclear. In this study, we aimed to investigate the role of (HR + P)-EMVs in cardiac IR injury compared to microvesicles derived from hypoxic/reoxygenated HUVECs (HR-EMVs) and to elucidate the underlying mechanisms. METHODS: Hypoxia/reoxygenation (HR) models of HUVECs and AC16 cells and a mouse cardiac IR model were established. Microvesicles from HR-injured HUVECs, DMSO post-treated HUVECs and propofol post-treated HUVECs were extracted by ultra-high speed centrifugation, respectively. The above EMVs were co-cultured with HR-injured AC16 cells or injected intracardially into IR mice. Flow cytometry and immunofluorescence were used to determine the levels of oxidative stress and apoptosis in cardiomyocytes. Apoptosis related proteins were detected by Western blot. Echocardiography for cardiac function and Evans blue-TTC staining for myocardial infarct size. Expression of lncCCT4-2 in EMVs and AC16 cells was analysed by whole transcriptome sequencing of EMVs and RT-qPCR. The molecular mechanism of inhibition of myocardial injury by (HR + P)-EMVs was elucidated by lentiviral knockdown of lncCCT4-2, plasmid overexpression or knockdown of CCT4, and actinomycin D assay. RESULTS: In vitro and in vivo experiments confirmed that HR-EMVs exacerbated oxidative stress and apoptosis in IR-injured cardiomyocytes, leading to increased infarct size and worsened cardiac function. Notably, (HR + P)-EMVs induced significantly less oxidative stress and apoptosis in IR-injured cardiomyocytes compared to HR-EMVs. Mechanistically, RNA sequencing of EMVs and RT-qPCR showed that lncCCT4-2 was significantly upregulated in (HR + P)-EMVs and cardiomyocytes co-cultured with (HR + P)-EMVs. Reduction of lncCCT4-2 in (HR + P)-EMVs enhanced oxidative stress and apoptosis in IR-injured cardiomyocytes. Furthermore, the anti-apoptotic activity of lncCCT4-2 from (HR + P)-EMVs was achieved by increasing the stability of CCT4 mRNA and promoting the expression of CCT4 protein in cardiomyocytes. CONCLUSIONS: Our study showed that (HR + P)-EMVs uptake by IR-injured cardiomyocytes upregulated lncCCT4-2 in cardiomyocytes and promoted CCT4 expression, thereby inhibiting HR-EMVs induced oxidative stress and apoptosis.

Enrichment of miR-17-5p enhances the protective effects of EPC-EXs on vascular and skeletal muscle injury in a diabetic hind limb ischemia model.

BACKGROUND/AIMS: Diabetes mellitus (DM) is highly susceptible to diabetic hind limb ischemia (DHI). MicroRNA (MiR)-17-5p is downregulated in DM and plays a key role in vascular protection. Endothelial progenitor cell (EPC)-released exosomes (EPC-EXs) contribute to vascular protection and ischemic tissue repair by transferring their contained miRs to target cells. Here, we investigated whether miR-17-5p-enriched EPC-EXs (EPC-EXsmiR-17-5p) had conspicuous effects on protecting vascular and skeletal muscle in DHI in vitro and in vivo. METHODS: EPCs transfected with scrambled control or miR-17-5p mimics were used to generate EPC-EXs and EPC-EXsmiR-17-5p. Db/db mice were subjected to hind limb ischemia. After the surgery, EPC-EXs and EPC-EXsmiR-17-5p were injected into the gastrocnemius muscle of the hind limb once every 7 days for 3 weeks. Blood flow, microvessel density, capillary angiogenesis, gastrocnemius muscle weight, structure integrity, and apoptosis in the hind limb were assessed. Vascular endothelial cells (ECs) and myoblast cells (C2C12 cells) were subjected to hypoxia plus high glucose (HG) and cocultured with EPC-EXs and EPC-EXsmiR-17-5p. A bioinformatics assay was used to analyze the potential target gene of miR-17-5p, the levels of SPRED1, PI3K, phosphorylated Akt, cleaved caspase-9 and cleaved caspase-3 were measured, and a PI3K inhibitor (LY294002) was used for pathway analysis. RESULTS: In the DHI mouse model, miR-17-5p was markedly decreased in hind limb vessels and muscle tissues, and infusion of EPC-EXsmiR-17-5p was more effective than EPC-EXs in increasing miR-17-5p levels, blood flow, microvessel density, and capillary angiogenesis, as well as in promoting muscle weight, force production and structural integrity while reducing apoptosis in gastrocnemius muscle. In Hypoxia plus HG-injured ECs and C2C12 cells, we found that EPC-EXsmiR-17-5p could deliver their carried miR-17-5p into target ECs and C2C12 cells and subsequently downregulate the target protein SPRED1 while increasing the levels of PI3K and phosphorylated Akt. EPC-EXsmiR-17-5p were more effective than EPC-EXs in decreasing apoptosis and necrosis while increasing viability, migration, and tube formation in Hypoxia plus HG-injured ECs and in decreasing apoptosis while increasing viability and myotube formation in C2C12 cells. These effects of EPC-EXsmiR-17-5p could be abolished by a PI3K inhibitor (LY294002). CONCLUSION: Our results suggest that miR-17-5p promotes the beneficial effects of EPC-EXs on DHI by protecting vascular ECs and muscle cell functions.

MiR-17-5p Mediates the Effects of ACE2-Enriched Endothelial Progenitor Cell-Derived Exosomes on Ameliorating Cerebral Ischemic Injury in Aged Mice.

Aging is one of the key mechanisms of vascular dysfunction and contributes to the initiation and progression of ischemic stroke (IS). Our previous study demonstrated that ACE2 priming enhanced the protective effects of exosomes derived from endothelial progenitor cells (EPC-EXs) on hypoxia-induced injury in aging endothelial cells (ECs). Here, we aimed to investigate whether ACE2-enriched EPC-EXs (ACE2-EPC-EXs) could attenuate brain ischemic injury by inhibiting cerebral EC damage through their carried miR-17-5p and the underlying molecular mechanisms. The enriched miRs in ACE2-EPC-EXs were screened using the miR sequencing method. EPC-EXs, ACE2-EPC-EXs, and ACE2-EPC-EXs with miR-17-5p deficiency (ACE2-EPC-EXsantagomiR-17-5p) were administered to transient middle cerebral artery occlusion (tMCAO)-operated aged mice or coincubated with hypoxia/reoxygenation (H/R)-treated aging ECs. The results showed that (1) the level of brain EPC-EXs and their carried ACE2 were significantly decreased in aged mice compared to in young mice, and (2) after tMCAO, aged mice displayed increases in brain cell senescence, infarct volume, and neurological deficit score (NDS) and a decrease in cerebral blood flow (CBF). (3) Compared with EPC-EXs, ACE2-EPC-EXs were enriched with miR-17-5p and more effective in increasing ACE2 and miR-17-5p expression in cerebral microvessels, accompanied by obvious increases in cerebral microvascular density (cMVD) and cerebral blood flow (CBF) and decreases in brain cell senescence, infarct volume, neurological deficit score (NDS), cerebral EC ROS production, and apoptosis in tMCAO-operated aged mice. Moreover, silencing of miR-17-5p partially abolished the beneficial effects of ACE2-EPC-EXs. (4) In H/R-treated aging ECs, ACE2-EPC-EXs were more effective than EPC-EXs in decreasing cell senescence, ROS production, and apoptosis and increasing cell viability and tube formation. In a mechanistic study, ACE2-EPC-EXs more effectively inhibited PTEN protein expression and increased the phosphorylation of PI3K and Akt, which were partially abolished by miR-17-5p knockdown. Altogether, our data suggest that ACE-EPC-EXs have better protective effects on ameliorating aged IS mouse brain neurovascular injury by inhibiting cell senescence, EC oxidative stress, apoptosis, and dysfunction by activating the miR-17-5p/PTEN/PI3K/Akt signaling pathway.

Hemolytic iron regulation in traumatic brain injury and alcohol use.

Hemorrhage is a major component of traumatic brain injury (TBI). Red blood cells, accumulated at the hemorrhagic site, undergo hemolysis upon energy depletion and release free iron into the central nervous system. This iron must be managed to prevent iron neurotoxicity and ferroptosis. As prior alcohol consumption is often associated with TBI, we examined iron regulation in a rat model of chronic alcohol feeding subjected to fluid percussion-induced TBI. We found that alcohol consumption prior to TBI altered the expression profiles of the lipocalin 2/heme oxygenase 1/ferritin iron management system. Notably, unlike TBI alone, TBI following chronic alcohol consumption sustained the expression of all three regulatory proteins for 1, 3, and 7 days post-injury. In addition, alcohol significantly affected TBI-induced expression of ferritin light chain at 3 days post-injury. We also found that alcohol exacerbated TBI-induced activation of microglia at 7 days post-injury. Finally, we propose that microglia may also play a role in iron management through red blood cell clearance.

Circulating endothelial microvesicles and their carried miR-125a-5p: potential biomarkers for ischaemic stroke.

BACKGROUND: Endothelial microvesicles (EMVs) are closely associated with the status of endothelial cells (ECs). Our earlier study has shown that EMVs could exert protective roles in ECs by transferring their carried miR-125a-5p. However, whether circulating EMVs and their carried miR-125a-5p can be used as biomarkers in ischaemic stroke (IS) are remain unknown. METHODS: We recruited 72 subjects with IS, 60 subjects with high stroke risk and 56 age-matched controls. The circulating EMVs and their carried miR-125a-5p (EMV-miR-125a-5p) levels were detected. We used microRNA (miR) array to study expression changes of miRs in plasma EMVs samples of three IS patients and three matched healthy controls. Transient middle cerebral artery occlusion (tMCAO) was used to establish IS mouse model. RESULTS: EMVs level was obviously elevated in IS patients, with the highest level in acute stage, and was positively related to carotid plaque, carotid intima-media thickness (IMT), National Institutes of Health Stroke Scale (NIHSS), infarct volume. On the contrary, we observed that EMV-miR-125a-5p level was obviously reduced in IS, with the lowest level in acute stage, and was negatively correlated with carotid plaque, IMT, NIHSS scores, infarct volume. EMVs and EMV-miR-125a-5p levels were closely related with large artery atherosclerosis subgroup. Importantly, EMVs and EMV-miR-125a-5p levels could serve as independent risk factors, and receiver operating characteristic curve achieved an area under curve (AUC) of 0.720 and 0.832 for IS, respectively, and elevated to 0.881 after their combination. In IS mouse model, control EMVs or n-EMVs administration could decrease the infarct volume and neurological deficit score, while increase the cerebral blood flow of IS mice compared with vehicle group, while IS EMVs or oxygen and glucose deprivation (OGD)-EMVs administration aggravated the tMCAO induced ischaemic injury. In addition, we observed that OGD EMVmiR-125a-5p could partially ameliorate the OGD EMVs induced brain injury after IS. CONCLUSIONS: These findings demonstrate that circulating EMVs and EMV-miR-125a-5p are closely related with the occurrence, progress, subtypes and severity of IS, and they can serve as innovative biomarkers and therapeutic targets for IS, especially when they are combined.

Combination of EPC-EXs and NPC-EXs with miR-126 and miR-210 overexpression produces better therapeutic effects on ischemic stroke by protecting neurons through the Nox2/ROS and BDNF/TrkB pathways.

BACKGROUNDS/AIMS: Neural progenitor cells (NPCs) and endothelial progenitor cell (EPCs) exhibit synergistical effects on protecting endothelial cell functions. MiR-126 and miR-210 can protect cell activities by regulating brain-derived neurotrophic factor (BDNF) and reactive oxygen species (ROS) production. Exosomes (EXs) mediate the beneficial effects of stem cells via delivering microRNAs (miRs). Here, we investigated the combination effects of EXs from EPCs (EPC-EXs) and NPCs (NPC-EXs), and determined whether these EXs with miR-126 (EPC-EXsmiR-126) and miR-210 overexpression (NPC-EXsmiR-210) had better effects on hypoxia/reoxygenation (H/R)-injured neurons and ischemic stroke (IS). METHODS: Cultured neurons were subjected to hypoxia for 6 h and then co-cultured with culture medium, NPC-EXs, EPC-EXs, NPC-EXs + EPC-EXs or NPC-EXsmiR-210 + EPC-EXsmiR-126 under normoxia for 24 h. Cell apoptosis, ROS production, neurite outgrowth and BDNF level were analyzed. Permanent middle cerebral artery occlusion (MCAO) was performed on C57BL/6 mice to build IS model. The mice were injected with PBS or various EXs via tail vein 2 h after MCAO operation. After 24 h, infarct volume and neurological deficits score (NDS), neuronal apoptosis, ROS production and spine density of dendrites, and brain BDNF level were analyzed. For mechanism study, NADPH oxidase 2(Nox2) and BDNF receptor tyrosine kinase receptor B (TrkB) were determined, and TrkB inhibitor k-252a was used in in vitro and in vivo study. RESULTS: 1) The level of miR-210 or miR-126 was increased after NPC-EXs or EPC-EXs treatment respectively. 2) In H/R-injured neurons, NPC-EXs or EPC-EXs decreased cell apoptosis and ROS production and promoted neurite outgrowth, which were associated with the downregulation of Nox2 and the increase of BDNF and p-TrkB/TrkB level. 3) In MCAO mice, NPC-EXs or EPC-EXs decreased infarct volume and NDS, reduced neural apoptosis and ROS production, and promoted the spine density of dendrites. The levels of Nox2, BDNF and p-TrkB/TrkB in mouse brain tissues changed in similar patterns as seen in the in vitro study. 4) In both cell and mouse models, combination of NPC-EXs and EPC-EXs was more effective than NPC-EXs or EPC-EXs alone on all of these effects. 5) EPC-EXsmiR-126 + NPC-EXsmiR-210 had better effects compared to NPC-EXs + EPC-EXs, which were inhibited by k-252a. CONCLUSION: EPC-EXsmiR-126 combined NPC-EXsmiR-210 further orchestrate the combinative protective effects of EPC-EXs and NPC-EXs on IS, possibly by protecting H/R-injured neurons through the Nox2/ ROS and BDNF/TrkB pathways.

Propofol inhibits myocardial injury induced by microvesicles derived from hypoxiareoxygenated endothelial cells via lncCCT4-2/CCT4 signaling

BACKGROUND: Ischemia-reperfusion (IR) induces increased release of extracellular vesicles in the heart and exacerbates myocardial IR injury. We have previously shown that propofol attenuates hypoxia/reoxygenation (HR)-induced injury in human umbilical vein endothelial cells (HUVECs) and that microvesicles derived from propofol-treated HUVECs inhibit oxidative stress in endothelial cells. However, the role of microvesicles derived from propofol post-treated HUVECs ((HR + P)-EMVs) in IR-injured cardiomyocytes is unclear. In this study, we aimed to investigate the role of (HR + P)-EMVs in cardiac IR injury compared to microvesicles derived from hypoxic/reoxygenated HUVECs (HR-EMVs) and to elucidate the underlying mechanisms. METHODS: Hypoxia/reoxygenation (HR) models of HUVECs and AC16 cells and a mouse cardiac IR model were established. Microvesicles from HR-injured HUVECs, DMSO post-treated HUVECs and propofol post-treated HUVECs were extracted by ultra-high speed centrifugation, respectively. The above EMVs were co-cultured with HR-injured AC16 cells or injected intracardially into IR mice. Flow cytometry and immunofluorescence were used to determine the levels of oxidative stress and apoptosis in cardiomyocytes. Apoptosis related proteins were detected by Western blot. Echocardiography for cardiac function and Evans blue-TTC staining for myocardial infarct size. Expression of lncCCT4-2 in EMVs and AC16 cells was analysed by whole transcriptome sequencing of EMVs and RT-qPCR. The molecular mechanism of inhibition of myocardial injury by (HR + P)-EMVs was elucidated by lentiviral knockdown of lncCCT4-2, plasmid overexpression or knockdown of CCT4, and actinomycin D assay. RESULTS: In vitro and in vivo experiments confirmed that HR-EMVs exacerbated oxidative stress and apoptosis in IR-injured cardiomyocytes, leading to increased infarct size and worsened cardiac function. Notably, (HR + P)-EMVs induced significantly less oxidative stress and apoptosis in IR-injured cardiomyocytes compared to HR-EMVs. Mechanistically, RNA sequencing of EMVs and RT-qPCR showed that lncCCT4-2 was significantly upregulated in (HR + P)-EMVs and cardiomyocytes co-cultured with (HR + P)-EMVs. Reduction of lncCCT4-2 in (HR + P)-EMVs enhanced oxidative stress and apoptosis in IR-injured cardiomyocytes. Furthermore, the anti-apoptotic activity of lncCCT4-2 from (HR + P)-EMVs was achieved by increasing the stability of CCT4 mRNA and promoting the expression of CCT4 protein in cardiomyocytes. CONCLUSIONS: Our study showed that (HR + P)-EMVs uptake by IR-injured cardiomyocytes upregulated lncCCT4-2 in cardiomyocytes and promoted CCT4 expression, thereby inhibiting HR-EMVs induced oxidative stress and apoptosis. Highlights Microvesicles from hypoxic/reoxygenated HUVECs (HR-EMVs) exacerbated oxidative stress and apoptosis in IR-injured cardiomyocytes. Microvesicles from propofol post-treated HUVECs ((HR + P)-EMVs) induced diminished oxidative stress and apoptosis in IR-injured cardiomyocytes compared with microvesicles from hypoxic/reoxygenated HUVECs (HR-EMVs). lncCCT4-2 was significantly highly expressed in (HR + P)-EMVs and cardiomyocytes co-cultured with (HR + P)-EMVs, and reduction of lncCCT4-2 in (HR + P)-EMVs enhanced oxidative stress and apoptosis in IR-injured cardiomyocytes. lncCCT4-2 inhibited HR-EMVs induced oxidative stress and apoptosis in HR-injured AC16 cells by increasing the stability of CCT4 mRNA and promoting the expression of CCT4 protein in AC16 cells.

Enrichment of miR-17-5p enhances the protective effects of EPC-EXs on vascular and skeletal muscle injury in a diabetic hind limb ischemia model

BACKGROUND/AIMS: Diabetes mellitus (DM) is highly susceptible to diabetic hind limb ischemia (DHI). MicroRNA (MiR)-17-5p is downregulated in DM and plays a key role in vascular protection. Endothelial progenitor cell (EPC)-released exosomes (EPC-EXs) contribute to vascular protection and ischemic tissue repair by transferring their contained miRs to target cells. Here, we investigated whether miR-17-5p-enriched EPC-EXs (EPC-EXsmiR-17-5p) had conspicuous effects on protecting vascular and skeletal muscle in DHI in vitro and in vivo. METHODS: EPCs transfected with scrambled control or miR-17-5p mimics were used to generate EPC-EXs and EPC-EXsmiR-17-5p. Db/db mice were subjected to hind limb ischemia. After the surgery, EPC-EXs and EPC-EXsmiR-17-5p were injected into the gastrocnemius muscle of the hind limb once every 7 days for 3 weeks. Blood flow, microvessel density, capillary angiogenesis, gastrocnemius muscle weight, structure integrity, and apoptosis in the hind limb were assessed. Vascular endothelial cells (ECs) and myoblast cells (C2C12 cells) were subjected to hypoxia plus high glucose (HG) and cocultured with EPC-EXs and EPC-EXsmiR-17-5p. A bioinformatics assay was used to analyze the potential target gene of miR-17-5p, the levels of SPRED1, PI3K, phosphorylated Akt, cleaved caspase-9 and cleaved caspase-3 were measured, and a PI3K inhibitor (LY294002) was used for pathway analysis. RESULTS: In the DHI mouse model, miR-17-5p was markedly decreased in hind limb vessels and muscle tissues, and infusion of EPC-EXsmiR-17-5p was more effective than EPC-EXs in increasing miR-17-5p levels, blood flow, microvessel density, and capillary angiogenesis, as well as in promoting muscle weight, force production and structural integrity while reducing apoptosis in gastrocnemius muscle. In Hypoxia plus HG-injured ECs and C2C12 cells, we found that EPC-EXsmiR-17-5p could deliver their carried miR-17-5p into target ECs and C2C12 cells and subsequently downregulate the target protein SPRED1 while increasing the levels of PI3K and phosphorylated Akt. EPC-EXsmiR-17-5p were more effective than EPC-EXs in decreasing apoptosis and necrosis while increasing viability, migration, and tube formation in Hypoxia plus HG-injured ECs and in decreasing apoptosis while increasing viability and myotube formation in C2C12 cells. These effects of EPC-EXsmiR-17-5p could be abolished by a PI3K inhibitor (LY294002). CONCLUSION: Our results suggest that miR-17-5p promotes the beneficial effects of EPC-EXs on DHI by protecting vascular ECs and muscle cell functions.

Exosomal miR-132-3p from mesenchymal stromal cells improves synaptic dysfunction and cognitive decline in vascular dementia.

BACKGROUND/AIMS: Vascular dementia (VD) results in cognition and memory deficit. Exosomes and their carried microRNAs (miRs) contribute to the neuroprotective effects of mesenchymal stromal cells, and miR-132-3p plays a key role in neuron plasticity. Here, we investigated the role and underlying mechanism of MSC EX and their miR-132-3p cargo in rescuing cognition and memory deficit in VD mice. METHODS: Bilateral carotid artery occlusion was used to generate a VD mouse model. MiR-132-3p and MSC EX levels in the hippocampus and cortex were measured. At 24-h post-VD induction, mice were administered with MSC EX infected with control lentivirus (EXCon), pre-miR-132-3p-expressing lentivirus (EXmiR-132-3p), or miR-132-3p antago lentivirus (EXantagomiR-132-3p) intravenously. Behavioral and cognitive tests were performed, and the mice were killed in 21 days after VD. The effects of MSC EX on neuron number, synaptic plasticity, dendritic spine density, and Aß and p-Tau levels in the hippocampus and cortex were determined. The effects of MSC EX on oxygen-glucose deprivation (OGD)-injured neurons with respect to apoptosis, and neurite elongation and branching were determined. Finally, the expression levels of Ras, phosphorylation of Akt, GSK-3ß, and Tau were also measured. RESULTS: Compared with normal mice, VD mice exhibited significantly decreased miR-132-3p and MSC EX levels in the cortex and hippocampus. Compared with EXCon treatment, the infusion of EXmiR-132-3p was more effective at improving cognitive function and increasing miR-132-3p level, neuron number, synaptic plasticity, and dendritic spine density, while decreasing Aß and p-Tau levels in the cortex and hippocampus of VD mice. Conversely, EXantagomiR-132-3p treatment significantly decreased miR-132-3p expression in cortex and hippocampus, as well as attenuated EXmiR-132-3p treatment-induced functional improvement. In vitro, EXmiR-132-3p treatment inhibited RASA1 protein expression, but increased Ras and the phosphorylation of Akt and GSK-3ß, and decreased p-Tau levels in primary neurons by delivering miR-132-3p, which resulted in reduced apoptosis, and increased neurite elongation and branching in OGD-injured neurons. CONCLUSIONS: Our studies suggest that miR-132-3p cluster-enriched MSC EX promotes the recovery of cognitive function by improving neuronal and synaptic dysfunction through activation of the Ras/Akt/GSK-3ß pathway induced by downregulation of RASA1.

Rab27a-dependent exosomes protect against cerebral ischemic injury by reducing endothelial oxidative stress and apoptosis.

INTRODUCTION: Multicellular crosstalk within the brain tissue has been suggested to play a critical role in maintaining cerebral vascular homeostasis. Exosomes (EXs) mediated cell-cell communication, but its role in cerebral ischemic injury is largely unknown. Rab27a is one of the major genes controlling EX release. Here, we explored the role of Rab27a in regulating brain EXs secretion, and the effects of Rab27a-mediated EXs on ischemia evoked cerebral vascular disruption and brain injury. METHODS: Cerebral ischemia was induced in Rab27a knockout (Rab27a-/- ) and wide type (WT) mice by transient middle cerebral artery occlusion (tMCAO). Differential gene expression analysis was performed in ischemic brain tissue by using mRNA sequencing. EXs isolated from brain tissue of Rab27a-/- and WT mice (EXWT or EXRab27a-/- ) were pre-administrated into tMCAO operated Rab27a-/- mice or oxygen and glucose deprivation (OGD) treated primary brain vascular endothelial cells (ECs). RESULTS: We demonstrated that Rab27a expression in the peri-infarct area of brain was significantly elevated, which was associated with local elevation in EXs secretion. Rab27a deficiency dramatically decreased the level of EXs in brain tissue of normal and tMCAO-treated mice, and Rab27a-/- mice displayed an increase in infarct volume and NDS, and a decrease in cMVD and CBF following tMCAO. Pre-infusion of EXWT increased the brain EXs levels in the tMCAO operated Rab27a-/- mice, accompanied with an increase in cMVD and CBF, and a decrease in infarct volume, NDS, ROS production, and apoptosis. The effects of EXRab27a-/- infusion were much diminished although in a dose-dependent manner. In OGD-treated ECs, EXRab27a-/- showed less effectivity than EXWT in decreasing ROS overproduction and apoptosis, paralleling with down-regulated expression of NOX2 and cleaved caspase-3. CONCLUSION: Our study demonstrates that Rab27a controls brain EXs secretion and functions, contributing to cerebral vascular protection from ischemic insult by preventing oxidative stress and apoptosis via down-regulating NOX2 and cleaved caspase-3 expression.

Case Report: Identification of Compound Heterozygous Mutations in a Patient With Late-Onset Glycogen Storage Disease Type II (Pompe Disease).

Pompe disease is an autosomal recessive hereditary lysosomal disorder and correlated with acid α-glucosidase enzyme (GAA) deficiencies, which lead to accumulation of glycogen in all tissues, most notably in skeletal muscles. Adult late-onset Pompe disease (LOPD) is a slowly progressive disease of proximal myopathy with later involvement of the respiratory muscles, resulting in respiratory failure. In this study, we reported a 22-year-old Chinese woman with inability to withstand heavy physical activity since childhood, who presented with respiratory and ambulation weakness in 2 months. On admission, her bilateral upper limbs strength was 4/5 and lower limbs strength was 3/5 according to Medical Research Council (MRC) score. The patient had compound heterozygotes containing a newly identified 4 nt deletion of coding sequence (deletion nt 1411_1414) in one of the acid α-glucosidase alleles and a c.2238G>C (p.Trp746Cys) missense mutation. This deletion has been reported in infant-onset Pompe disease (IOPD) but not LOPD. Intriguingly, this deletion mutation was not found in the patient's family and was considered as pathogenic. Muscle biopsy showed scattered vacuoles with basophilic granules inside the subsarcolemmal area, which were strongly stained by periodic acid-Schiff (PAS). Laboratory tests revealed a significant increase of creatine kinase MB isoenzyme (CK-MB) and lactate dehydrogenase (LDH). GAA level was 9.77 nmol/1 h/mg and was not sufficient for the diagnosis of GAA activity deficiency (0-3.78 nmol/1 h/mg). In summary, mutational analysis of GAA and muscle biopsy are crucial in the diagnosis of Pompe disease.

Patients with SERPINC1 rs2227589 polymorphism found to have multiple cerebral venous sinus thromboses despite a normal antithrombin level: A case report.

BACKGROUND: The hereditary antithrombin (AT) deficiency caused by SERPINC1 gene mutation is an autosomal dominant thrombotic disorder. An increasing number of studies have shown that mutations in the SERPINC1 rs2227589 polymorphic site are correlated with a risk of venous thromboembolism (VTE) at common sites, such as lower extremity deep venous thrombosis and pulmonary thromboembolism. Currently, there are no reports of cerebral venous sinus thrombosis (CVST), a VTE site with a low incidence rate and rs2227589 polymorphism. CASE SUMMARY: Here, we report a Chinese CVST case with a mutation of the SERPINC1 rs2227589 polymorphic site, which did not cause significant AT deficiency. In a 50-year-old male patient presenting with multiple cerebral venous sinus thromboses no predisposing factors were detected, although a relative had a history of lower extremity deep venous thrombosis. We performed sequencing of the SERPINC1 gene for the patient and his daughter, which revealed the same heterozygous mutation at the rs2227589 polymorphic site: c.41+141G>A. CONCLUSION: The results showed that more studies should be conducted to assess the correlation between rs2227589 polymorphism and CVST.

Biphasic Effects of Ethanol Exposure on Waste Metabolites Clearance in the CNS.

We have shown that the effects of low-dose ethanol promote the clearance of waste metabolites, such as amyloid-beta (Aß) proteins, from the brain through the perivascular space (PVS). We demonstrated that dilative reactivity of arterial smooth muscle and endothelial cells regulate this clearance. These findings indicate the importance of blood-brain barrier (BBB) transvascular clearance of large size metabolites from the central nervous system (CNS), where the lymphatic clearance system is absent. We next examined the contrasting effects of acute low-dose and chronic moderate ethanol exposure on BBB-associated perivascular clearance. We injected a high molecular weight fluorescent dye into the interstitial space or directly into the cerebrospinal fluid (CSF). Bio-distribution of this tracer was then examined in different brain regions by multiphoton imaging and whole brain tissue section scanning. Ethanol-induced molecular/cellular mechanisms that drive the increase or decrease in movement of the fluorescent tracer were correlated to BBB integrity and arterial vessel reactivity. We found that activation of endothelial nitric oxide synthase (eNOS) under low-dose ethanol conditions with a shift to activation of inducible NOS (iNOS) under chronic high ethanol exposure conditions, which appeared to regulate these contrasting effects. We validated these observations by qualitative and quantitative investigation of eNOS, iNOS, BBB integrity, and perivascular clearance of waste metabolites. We concluded that the effects of low-dose ethanol increased the diffusive movement of waste metabolites via eNOS-derived NO, which increased the arterial endothelial-smooth muscle cell dilative reactivity without affecting BBB integrity, whereas a prolonged induction of iNOS under chronic ethanol exposure conditions caused oxidative damage of the arterial endothelial-smooth muscle layers resulting in cerebral amyloid-like angiopathy. This led to dysfunction of the BBB, dilative reactivity, and impaired waste metabolites movement from the interstitial space or subarachnoid space (SAS) through perivascular clearance.

Exosomes and Exosomal microRNAs in Age-associated Stroke.

Aging has been considered to be the most important non-modifiable risk factor for stroke and death. Changes in circulation factors in the systemic environment, cellular senescence and artery hypertension during human ageing have been investigated. Exosomes are nanosize membrane vesicles that can regulate target cell functions via delivering their carried bioactive molecules (e.g. protein, mRNA, and microRNAs). In the central nervous system, exosomes and exosomal microRNAs play a critical role in regulating neurovascular function and are implicated in stroke initiation and progression. MicroRNAs are small non-coding RNAs that have been reported to play critical roles in various biological processes. Recently, evidence has shown that microRNAs are packaged into exosomes and can be secreted into the systemic and tissue environment. Circulating microRNAs participate in cellular senescence and contribute to age-associated stroke. Here, we provide an overview of current knowledge on exosomes and their carried microRNAs in the regulation of cellular and organismal ageing processes, demonstrating the potential role of exosomes and their carried microRNAs in age-associated stroke.

In vivo Neuroprotective Effect of a Self-assembled Peptide Hydrogel.

Traumatic brain injury (TBI) is associated with poor intrinsic healing responses and long-term cognitive decline. A major pathological outcome of TBI is acute glutamate-mediated excitotoxicity (GME) experienced by neurons. Short peptides based on the neuroprotective extracellular glycoprotein ependymin have shown the ability to slow down the effect of GME - however, such short peptides tend to diffuse away from target sites after in vivo delivery. We have designed a self-assembling peptide containing an ependymin mimic that can form nanofibrous matrices. The peptide was evaluated in situ to assess neuroprotective utility after an acute fluidpercussion injury. This biomimetic matrix can conform to the intracranial damaged site after delivery, due its shear-responsive rheological properties. We demonstrated the potential efficacy of the peptide for supporting neuronal survival in vitro and in vivo. Our study demonstrates the potential of these implantable acellular hydrogels for managing the acute (up to 7 days) pathophysiological sequelae after traumatic brain injury. Further work is needed to evaluate less invasive administrative routes and long-term functional and behavioral improvements after injury.

Stem Cell-Derived Insulin-Producing Cells in 3D Engineered Tissue in a Perfusion Flow Bioreactor.

To circumvent the lack of donor pancreas, insulin-producing cells (IPCs) derived from pluripotent stem cells emerged as a viable cell source for the treatment of type 1 diabetes. While it has been shown that IPCs can be derived from pluripotent stem cells using various protocols, the long-term viability and functional stability of IPCs in vitro remains a challenge. Thus, the principles of three-dimensional (3D) tissue engineering and a perfusion flow bioreactor were used in this study to establish 3D microenvironment suitable for long-term in vitro culture of IPCs-derived from mouse embryonic stem cells. It was observed that in static 3D culture of IPCs, the viability decreased gradually with longer time in culture. However, when a low flow (0.02 mL/min) was continuously applied to 3D IPC containing tissues, enhanced survival and function of IPCs were demonstrated. IPCs cultured under low flow exhibited a significantly enhanced glucose responsiveness and upregulation of Ins1 compared to that of static culture. In summary, this study demonstrates the feasibility and benefits of 3D engineered tissue environment combined with perfusion flow in vitro for culturing stem cell-derived IPCs. Impact statement This in vitro three-dimensional tissue system combined with the flow can be used to better understand the role of biophysical cues that facilitates improved function and maturation of stem cell-derived insulin-producing cells, which can ultimately advance the field of pancreatic tissue engineering as well as in diabetes treatment.

Plasma endothelial microvesicles and their carrying miRNA-155 serve as biomarkers for ischemic stroke.

Endothelial microvesicles (EMVs) could reflect the status of endothelial cells (ECs) which are involved in the pathogenesis of ischemic stroke (IS). MiR-155 could regulate EC functions. However, their roles in IS remain unclear. This study aimed to investigate the levels of plasma EMVs and EMVs carrying miRNA-155 (EMVs-miR-155) in IS patients to explore their potential roles as biomarkers. Ninety-three IS patients and 70 controls were recruited in this study. The levels of circulating EMVs and EMVs-miR-155 were detected by fluorescence nanoparticle tracking analysis and quantitative real-time PCR, respectively. The correlations between level of EMVs/EMVs-miR-155 and the onset time, severity, infarct volume, and subtypes of IS were analyzed. The severity and infarct volume were assessed by NIHSS and magnetic resonance imaging, respectively. Multivariate logistic regression analysis was used to investigate the risk factors of IS. The ROC curve and area under ROC curve (AUC) of EMVs and EMVs-miR-155 were determined. The levels of plasma EMVs and EMVs-miR-155 were increased significantly in acute and subacute stages of IS and remained unchanged in chronic stage, and were positively related to the infarct volume and NIHSS scores and were associated with large artery atherosclerosis and cardioembolism subtypes defined by Trial of Org 10 172 in acute stroke treatment (TOAST) classification. Multivariate logistic regression analysis demonstrated that plasma EMVs and EMVs-miR-155 were significant and independent risk factors of IS and their AUC were 0.778 and 0.851, respectively, and increased to 0.892 after combination. Our study suggests that plasma EMVs and EMVs-miR-155 are promising biomarkers for IS. The diagnostic value of EMVs-miR-155 is higher and their combination is the best.

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.

Implication of MicroRNA503 in Brain Endothelial Cell Function and Ischemic Stroke.

The role of miR-503 in brain endothelium and ischemic stroke (IS) remains unclear. We aimed to study the relationship between plasma miR-503 and the onset time, severity, subtypes, and von Willebrand Factor (vWF) level in IS patients and to investigate the roles and underlying mechanisms of miR-503 in middle cerebral artery occlusion (MCAO) mice and cultured cerebral vascular endothelial cells (ECs). In MCAO mice, the effects of plasma from acute severe IS patients (ASS) with or without miR-503 antagomir on brain and ECs damage were determined. In cultured human ECs, the effects of miR-503 overexpression or knockdown on the monolayer permeability, apoptosis, ROS, and NO generation were investigated. For mechanism study, the PI3K/Akt/eNOS pathway, cleaved caspase-3, and bcl-2 were analyzed. Results showed that plasma miR-503 was significantly increased in IS patients, especially in acute period and severe cases and subtypes of LAA and TACI, and was positively correlated with vWF. Logistic analysis indicated that miR-503 was an independent risk factor for IS, with the area under curve of 0.796 in ROC analysis. In MCAO mice, ASS pretreatment aggravated neurological injury, BBB damage, brain edema, CBF reduction, and decreased NO production while increased apoptosis and ROS generation in brain ECs, which were partly abolished by miR-503 antagomir. In cultured ECs, miR-503 overexpression and knockdown confirmed its effects on regulating monolayer permeability, cell apoptosis, NO, and ROS generation via PI3K/Akt/eNOS pathway or bcl-2 and cleaved caspase-3 proteins. These together indicate that miR-503 is a promising biomarker and novel therapeutic target for IS.

MicroRNA-34a-mediated death of acute myeloid leukemia stem cells through apoptosis induction and exosome shedding inhibition via histone deacetylase 2 targeting.

We investigated the role of leukemia stem cells in chemoresistance and recurrence of acute myeloid leukemia. Total RNA was isolated from cells or tissues using TRIzol reagent. Cell viability was assessed with the tetrazolium assay. MicroRNA-34a (miR-34a), which acts on cell death regulation pathways, was noticeably downregulated in non-M3 acute myeloid leukemia stem cells compared with normal hematopoietic stem cells. Furthermore, inhibition of miR-34a-mediated suppression in leukemia stem cells was associated with poor clinical outcomes and impaired treatment efficacy in acute myeloid leukemia. Transfection with a miR-34a mimic triggered leukemia stem cell death and prevented leukemia. Bioinformatics analysis and a dual-luciferase reporter assay showed that miR-34a targeted the 3'-untranslated region of histone deacetylase 2, and the reinforced expression of miR-34a remarkably stimulated the expression of histone deacetylase 2 in leukemia stem cells. Ectopic miR-34a expression triggered death of leukemia stem cells via pathways involving the Janus kinase 1-signal transducer and activator of transcription 2-p53 axis. Targeting leukemia stem cells to trigger cell death through upregulation of miR-34a expression could be used to diagnose and treat acute myeloid leukemia.