Role of miRNAs in neurovascular injury and repair.

MicroRNAs (miRNA) are endogenously produced small, non-coded, single-stranded RNAs. Due to their involvement in various cellular processes and cross-communication with extracellular components, miRNAs are often coined the "grand managers" of the cell. miRNAs are frequently involved in upregulation as well as downregulation of specific gene expression and thus, are often found to play a vital role in the pathogenesis of multiple diseases. Central nervous system (CNS) diseases prove fatal due to the intricate nature of both their development and the methods used for treatment. A considerable amount of ongoing research aims to delineate the complex relationships between miRNAs and different diseases, including each of the neurological disorders discussed in the present review. Ongoing research suggests that specific miRNAs can play either a pathologic or restorative and/or protective role in various CNS diseases. Understanding how these miRNAs are involved in various regulatory processes of CNS such as neuroinflammation, neurovasculature, immune response, blood-brain barrier (BBB) integrity and angiogenesis is of empirical importance for developing effective therapies. Here in this review, we summarized the current state of knowledge of miRNAs and their roles in CNS diseases along with a focus on their association with neuroinflammation, innate immunity, neurovascular function and BBB.

Exercise-Intervened Endothelial Progenitor Cell Exosomes Protect N2a Cells by Improving Mitochondrial Function.

We have recently demonstrated that exosomal communication between endothelial progenitor cells (EPCs) and brain endothelial cells is compromised in hypertensive conditions, which might contribute to the poor outcomes of stroke subjects with hypertension. The present study investigated whether exercise intervention can regulate EPC-exosome (EPC-EX) functions in hypertensive conditions. Bone marrow EPCs from sedentary and exercised hypertensive transgenic mice were used for generating EPC-EXs, denoted as R-EPC-EXs and R-EPC-EXET. The exosomal microRNA profile was analyzed, and EX functions were determined in a co-culture system with N2a cells challenged by angiotensin II (Ang II) plus hypoxia. EX-uptake efficiency, cellular survival ability, reactive oxygen species (ROS) production, mitochondrial membrane potential, and the expressions of cytochrome c and superoxide-generating enzyme (Nox4) were assessed. We found that (1) exercise intervention improves the uptake efficiency of EPC-EXs by N2a cells. (2) exercise intervention restores miR-27a levels in R-EPC-EXs. (3) R-EPC-EXET improved the survival ability and reduced ROS overproduction in N2a cells challenged with Ang II and hypoxia. (4) R-EPC-EXET improved the mitochondrial membrane potential and decreased cytochrome c and Nox4 levels in Ang II plus hypoxia-injured N2a cells. All these effects were significantly reduced by miR-27a inhibitor. Together, these data have demonstrated that exercise-intervened EPC-EXs improved the mitochondrial function of N2a cells in hypertensive conditions, which might be ascribed to their carried miR-27a.

The miR-210 Primed Endothelial Progenitor Cell Exosomes Alleviate Acute Ischemic Brain Injury.

BACKGROUND: Stem cell-released exosomes (EXs) have shown beneficial effects on regenerative diseases. Our previous study has revealed that EXs of endothelial progenitor cells (EPC-EXs) can elicit favorable effects on endothelial function. EXs may vary greatly in size, composition, and cargo uptake rate depending on the origins and stimulus; notably, EXs are promising vehicles for delivering microRNAs (miRs). Since miR-210 is known to protect cerebral endothelial cell mitochondria by reducing oxidative stress, here we study the effects of miR-210-loaded EPC-EXs (miR210-EPC-EXs) on ischemic brain damage in acute ischemic stroke (IS). METHODS: The miR210-EPC-EXs were generated from EPCs transfected with miR-210 mimic. Middle cerebral artery occlusion (MCAO) surgery was performed to induce acute IS in C57BL/6 mice. EPC-EXs or miR210-EPC-EXs were administrated via tail vein injection 2 hrs after IS. To explore the potential mechanisms, inhibitors of the vascular endothelial growth factor receptor 2 (VEGFR2)/PI3 kinase (PI3K) or tyrosine receptor kinase B (TrkB)/PI3k pathways were used. The brain tissue was collected after treatments for infarct size, cell apoptosis, oxidative stress, and protein expression (VEGFR2, TrkB) analyses on day two. The neurological deficit score (NDS) was evaluated before collecting the samples. RESULTS: 1) As compared to EPC-EXs, miR210-EPC-EXs profoundly reduced the infarct volume and improved the NDS on day two post-IS. 2) Fewer apoptosis cells were detected in the peri-infarct brain of mice treated with miR210-EPC-EXs than in EPC-EXs-treated mice. Meanwhile, the oxidative stress was profoundly reduced by miR210-EPC-EXs. 3) The ratios of p-PI3k/PI3k, p- VEGFR2/VEGFR2, and p-TrkB/TrkB in the ipsilateral brain were raised by miR210-EPC-EXs treatment. These effects could be significantly blocked or partially inhibited by PI3k, VEGFR2, or TrkB pathway inhibitors. CONCLUSION: These findings suggest that miR210-EPC-EXs protect the brain from acute ischemia- induced cell apoptosis and oxidative stress partially through the VEGFR2/PI3k and TrkB/PI3k signal pathways.

Emerging therapeutic role of gut microbial extracellular vesicles in neurological disorders.

Extracellular vesicles (EVs) serve as cell-to-cell and inter-organ communicators by conveying proteins and nucleic acids with regulatory functions. Emerging evidence shows that gut microbial-released EVs play a pivotal role in the gut-brain axis, bidirectional communication, and crosstalk between the gut and the brain. Increasing pre-clinical and clinical evidence suggests that gut bacteria-released EVs are capable of eliciting distinct signaling to the brain with the ability to cross the blood-brain barrier, exerting regulatory function on brain cells such as neurons, astrocytes, and microglia, via their abundant and diversified protein and nucleic acid cargo. Conversely, EVs derived from certain species of bacteria, particularly from gut commensals with probiotic properties, have recently been shown to confer distinct therapeutic effects on various neurological disorders. Thus, gut bacterial EVs may be both a cause of and therapy for neuropathological complications. This review marshals the basic, clinical, and translational studies that significantly contributed to our up-to-date knowledge of the therapeutic potential of gut microbial-derived EVs in treating neurological disorders, including strokes, Alzheimer's and Parkinson's disease, and dementia. The review also discusses the newer insights in recent studies focused on developing superior therapeutic microbial EVs via genetic manipulation and/or dietary intervention.

The protective effects of miR-210 modified endothelial progenitor cells released exosomes in hypoxia/reoxygenation injured neurons.

We have previously demonstrated that endothelial progenitor cells (EPCs) provide beneficial effects on ischemic stroke by reducing oxidative stress, which could be through EPCs-released exosomes (EPC-EXs). EXs are emerging as a bioagent for mediating cell-cell communications via their carried microRNAs (miR). miR-210 is shown to provide a neuroprotection effect against ischemic stroke. Here, we aimed to determine whether the combination of EPC-EXs and miR-210 would provide an enhanced protective effect on neurons. The hypoxia and reoxygenation (H/R) model were applied to neurons to mimic the ischemic injury of neurons. EPCs were transfected with miR-210 mimic to elevate the level of miR-210 in cells and EPC-EXs (miR210-EPC-EXs). For functional studies, EPC-EXs were co-incubated with H/R-injured neurons, then the cell viability and reactive oxygen species (ROS) production were determined. The results showed 1) H/R induced apoptosis and ROS overproduction in neurons; 2) miR-210 mimic increased the level of miR-210 in both EPCs and EPC-EXs; 3) EPCs cultured in serum-free medium released more exosomes in comparison with cells grown in complete growth media, suggesting serum starving induce the release of EXs; 4) After transfection, EPCs grown in complete media had almost 50 times higher miR-210 level than EPCs had in serum-free media, while the EPCs-EXs isolated from the complete media has lower miR-210 expression than from the serum-free media in a time-dependent manner, suggesting the transfer of miR-210 through EXs; 5) After co-incubation, EPC-EXs and miR210-EPC-EXs were uptaken by neurons, and the miR-210 level in neurons was elevated by miR210-EPC-EXs; 6) miR210-EPC-EXs were more effective in promoting cell viability and decreasing apoptosis and ROS production than EPC-EXs. The present study demonstrated that EPCs-carried miR-210 could be released and transferred to neurons in a time-dependent manner and that miR-210 loading can enhance the protective effects of EPC-EXs on H/R-induced neuron apoptosis, oxidative stress, and decreased viability.

EPC-EXs improve astrocyte survival and oxidative stress through different uptaking pathways in diabetic hypoxia condition.

BACKGROUND: Hyperglycemia contributes to cardiovascular complications in patients with type 2 diabetes. We confirmed that high glucose (HG) induces endothelial dysfunction and cerebral ischemic injury is enlarged in diabetic mice. Stem cell-released exosomes have been shown to protect the brain from ischemic stroke. We have previously shown that endothelial progenitor cells (EPCs)-released exosomes (EPC-EXs) can protect endothelial cells from hypoxia/reoxygenation (H/R) and HG-induced injury. Here, we aim to investigate the effects of EPC-EXs on astrocytes under H/R and HG-induced injury and whether miR-126 enriched EPC-EXs (miR126-EPC-EXs) have enhanced efficacy. METHODS: EPC-EX uptake and co-localization were measured by fluorescent microscopy using PKH26 and DAPI staining. miR-126 enrichment was achieved by transfecting with miR-126 mimics and quantified with real-time PCR. After co-incubation, cell death or injury was measured by using LDH (Lactate Dehydrogenase) assay. Oxidative stress/ROS (reactive oxygen species) generation was measured by DHE (Dihydroethidium) staining and lipid peroxidation assay. RESULTS: The EPC-EXs were effectively taken up by the astrocytes in a concentration as well as time-dependent manners and were co-localized within the nucleus as well as the cytoplasm. Pathway uptake inhibitors revealed that the EPC-EXs are effectively taken up by the clathrin-mediated, caveolin-dependent, and micropinocytosis via PI3K/Akt pathway. H/R and HG-induced a cell injury which could be protected by EPC-EXs evidenced by decreased cell cytotoxicity, oxidative stress, and lipid peroxidation. Moreover, miR-126 overexpression could increase the level of miR-126 in astrocytes and enhance the protective effects of EPC-EXs. CONCLUSIONS: These results collectively indicate that the EPC-EXs could protect astrocytes against the HG plus H/R-induced damage.

Role of Exosomes in Mediating the Cross-Talk Between Adipose Tissue and the Brain.

Adipose tissue is recognized as the largest endocrine organ by releasing secretory factors to exert systemic function on the brain. Exosomes are one type of extracellular vesicles that transport bioactive molecules between cells and organs. The cargo delivered by exosomes can alter a wide range of cellular responses in recipient cells and play an important pathophysiological role in human diseases. Emerging research showed that adipose tissue-released exosomes could be one of the mechanisms to mediate the function of the brain. Here, we review the modulatory function of adipose tissue-released exosomes in the brain. In particular, we emphasize the role of adipose tissue-released exosomes and their carried miRNAs in neurological disorder diseases. We provide an overview of advances in the understanding of adipose tissues in the regulation of brain function and offer a perspective on the potential therapeutic targets for neurological disorders.

Ultraviolet B Irradiation Alters the Level and miR Contents of Exosomes Released by Keratinocytes in Diabetic Condition.

Ultraviolet B (UVB) stimulates the generation of extracellular vesicles, which elicit systemic effects. Here, we studied whether UVB affects the release and microRNA (miR) content of keratinocyte exosomes (EXs) in diabetic conditions. In vitro, we examined the UVB effects on affecting EX release from keratinocyte HaCaT cells (HaCaT-EX) pretreated with high glucose. HaCaT-EX functions were evaluated on Schwann cells (SCs). In vivo, UVB-induced miR change in skin EXs of diabetic db/db mice was analyzed. The miRs of interest were validated in HaCaT-EXs. We found that: (1) UVB promoted HaCaT-EX generation in dose- and time-dependent manners; 100 and 1800 J m-2 of UVB had the most prominent effect and were selected as effective low- and high-fluence UVB in vitro. (2) A total of 13 miRs were differentially expressed >3-fold in skin EXs in UVB-treated db/db mice; miR-126 was the most up-regulated by low-fluence UVB. (3) Functional studies revealed that the SC viability was improved by low-fluence UVB HaCaT-EXs, while worsened by high-fluence UVB HaCaT-EXs. (4) MiR-126 inhibitor attenuated the effects induced by low-fluence UVB HaCaT-EXs. Our data have demonstrated that low- and high-fluence UVBs promote HaCaT-EX generation but differentially affect exosomal miR levels and functions under diabetic conditions.

The promise of exosome applications in treating central nervous system diseases.

Exosomes (EXs), a type of extracellular vesicles, are secreted from virtually all types of cells. EXs serve as cell-to-cell communicators by conveying proteins and nucleic acids with regulatory functions. Increasing evidence shows that EXs are implicated in the pathogenesis of central nervous system (CNS) diseases. Moreover, EXs have recently been highlighted as a new promising therapeutic strategy for in vivo delivery of nucleotides and drugs. Studies have revealed that infusion of EXs elicits beneficial effects on the CNS injury animal models. As compared to cell-based therapy, EXs-based therapy for CNS diseases has unique advantages, opening a new path for neurological medicine. In this review, we summarized the current state of knowledge of EXs, the roles and applications of EXs as a viable pathological biomarker, and EX-based therapy for CNS diseases.

Keratinocyte-derived microvesicle particles mediate ultraviolet B radiation-induced systemic immunosuppression.

A complete carcinogen, ultraviolet B (UVB) radiation (290-320 nm), is the major cause of skin cancer. UVB-induced systemic immunosuppression that contributes to photocarcinogenesis is due to the glycerophosphocholine-derived lipid mediator platelet-activating factor (PAF). A major question in photobiology is how UVB radiation, which only absorbs appreciably in the epidermal layers of skin, can generate systemic effects. UVB exposure and PAF receptor (PAFR) activation in keratinocytes induce the release of large numbers of microvesicle particles (MVPs; extracellular vesicles ranging from 100 to 1000 nm in size). MVPs released from skin keratinocytes in vitro in response to UVB (UVB-MVPs) are dependent on the keratinocyte PAFR. Here, we used both pharmacologic and genetic approaches in cells and mice to show that both the PAFR and enzyme acid sphingomyelinase (aSMase) were necessary for UVB-MVP generation. Our discovery that the calcium-sensing receptor is a keratinocyte-selective MVP marker allowed us to determine that UVB-MVPs leaving the keratinocyte can be found systemically in mice and humans following UVB exposure. Moreover, we found that UVB-MVPs contained bioactive contents including PAFR agonists that allowed them to serve as effectors for UVB downstream effects, in particular UVB-mediated systemic immunosuppression.

Inhibition of Ferroptosis Alleviates Early Brain Injury After Subarachnoid Hemorrhage In Vitro and In Vivo via Reduction of Lipid Peroxidation.

Subarachnoid hemorrhage (SAH) is a serious cerebrovascular disease with high mortality, and the mean age at morbidity is younger than in other types of stroke. Early brain injury (EBI) plays a key role in the poor prognoses of SAH. In EBI, multiple forms of cell death have been identified and well studied; however, the role of ferroptosis has not been elucidated. Hence, in this study, we developed an in vivo (SAH rat model) and in vitro model (SH-SY5Y oxyhemoglobin injury model) to understand the role of ferroptosis in EBI, then explored the protective mechanism of ferrostatin-1 (Fer-1). Firstly, we found that neurological scores, blood-brain barrier permeability, brain edema deteriorated after SAH in the in vivo model, cell viability was decreased after SAH in both cortex and SH-SY5Y cells. Further, iron content in cortex was increased after SAH, while transferrin receptor 1 and ferroportin (Fpn) were increased in oxyhemoglobin-treated in vitro model. Additionally, glutathione content and glutathione peroxidase 4 activity were reduced in SAH rats, and lipid peroxides were increased in the oxyhemoglobin-treated cells. Finally, administration of Fer-1 upregulated Fpn and decreased the iron content, then improved the lipid peroxidation and EBI. However, Fer-1 had no effect on the apoptosis. Our study indicated that the ferroptosis was involved in EBI of SAH, and the inhibitor Fer-1 provided neuroprotection against EBI by alleviating ferroptosis, the potential protective mechanism might be via suppressing lipid peroxidation.

Exosomes from miRNA-126-modified endothelial progenitor cells alleviate brain injury and promote functional recovery after stroke.

AIMS: We previously showed that the protective effects of endothelial progenitor cells (EPCs)-released exosomes (EPC-EXs) on endothelium in diabetes. However, whether EPC-EXs are protective in diabetic ischemic stroke is unknown. Here, we investigated the effects of EPC-EXs on diabetic stroke mice and tested whether miR-126 enriched EPC-EXs (EPC-EXsmiR126 ) have enhanced efficacy. METHODS: The db/db mice subjected to ischemic stroke were intravenously administrated with EPC-EXs 2 hours after ischemic stroke. The infarct volume, cerebral microvascular density (MVD), cerebral blood flow (CBF), neurological function, angiogenesis and neurogenesis, and levels of cleaved caspase-3, miR-126, and VEGFR2 were measured on day 2 and 14. RESULTS: We found that (a) injected EPC-EXs merged with brain endothelial cells, neurons, astrocytes, and microglia in the peri-infarct area; (b) EPC-EXsmiR126 were more effective than EPC-EXs in decreasing infarct size and increasing CBF and MVD, and in promoting angiogenesis and neurogenesis as well as neurological functional recovery; (c) These effects were accompanied with downregulated cleaved caspase-3 on day 2 and vascular endothelial growth factor receptor 2 (VEGFR2) upregulation till day 14. CONCLUSION: Our results indicate that enrichment of miR126 enhanced the therapeutic efficacy of EPC-EXs on diabetic ischemic stroke by attenuating acute injury and promoting neurological function recovery.

miR-137 boosts the neuroprotective effect of endothelial progenitor cell-derived exosomes in oxyhemoglobin-treated SH-SY5Y cells partially via COX2/PGE2 pathway.

BACKGROUND: We have previously verified the beneficial effects of exosomes from endothelial progenitor cells (EPC-EXs) in ischemic stroke. However, the effects of EPC-EXs in hemorrhagic stroke have not been investigated. Additionally, miR-137 is reported to regulate ferroptosis and to be involved in the neuroprotection against ischemic stroke. Hence, the present work explored the effects of miR-137-overexpressing EPC-EXs on apoptosis, mitochondrial dysfunction, and ferroptosis in oxyhemoglobin (oxyHb)-injured SH-SY5Y cells. METHODS: The lentiviral miR-137 was transfected into EPCs and then the EPC-EXs were collected. RT-PCR was used to detect the miR-137 level in EPCs, EXs, and neurons. The uptake mechanisms of EPC-EXs in SH-SY5Y cells were explored by the co-incubation of Dynasore, Pitstop 2, Ly294002, and Genistein. After the transfection of different types of EPC-EXs, flow cytometry and expression of cytochrome c and cleaved caspase-3 were used to detect the apoptosis of oxyHb-injured neurons. Neuronal mitochondrial function was assessed by reactive oxygen species (ROS) level, mitochondrial membrane potential (MMP) depolarization, and cellular ATP content. Cell ferroptosis was measured by lipid peroxidation, iron overload, degradation of glutathione, and glutathione peroxidase 4. Additionally, recombinational PGE2 was used to detect if activation of COX2/PGE2 pathway could reverse the protection of miR-137 overexpression. RESULTS: The present work showed (1) EPC-EXs could be taken in by SH-SY5Y cells via caveolin-/clathrin-mediated pathways and macropinocytosis; (2) miR-137 was decreased in neurons after oxyHb treatment, and EXsmiR-137 could restore the miR-137 levels; (3) EXsmiR-137 worked better than EXs in reducing the number of apoptotic neurons and pro-apoptotic protein expression after oxyHb treatment; (4) EXsmiR-137 are more effective in improving the cellular MMP, ROS, and ATP level; (5) EXsmiR-137, but not EXs, protected oxyHb-treated SH-SY5Y cells against lipid peroxidation, iron overload, degradation of glutathione, and glutathione peroxidase 4; and (6) EXsmiR-137 suppressed the expression of the COX2/PGE2 pathway, and activation of the pathway could partially reverse the neuroprotective effects of EXsmiR-137. CONCLUSION: miR-137 overexpression boosts the neuroprotective effects of EPC-EXs against apoptosis and mitochondrial dysfunction in oxyHb-treated SH-SY5Y cells. Furthermore, EXsmiR-137 rather than EXs can restore the decrease in miR-137 levels and inhibit ferroptosis, and the protection mechanism might involve the miR-137-COX2/PGE2 signaling pathway.

C6-ceramide treatment inhibits the proangiogenic activity of multiple myeloma exosomes via the miR-29b/Akt pathway.

BACKGROUND: The increased bone marrow angiogenesis is involved in the progression of multiple myeloma (MM) with the underlying mechanism poorly understood. Cancer-released exosomes could play an important role in the pathological angiogenesis through exosomal microRNAs (miRs) delivery. It is reported that miR-29b played an important role in regulating the tumor angiogenesis. METHODS: In this study, we explored the role of C6-ceramide (C6-cer, a Ceramide pathway activator) in the angiogenic effect of MM exosomes and its potential mechanism. MM cells (OPM2 and RPMI-8226) treated with C6-cer were studied for its effects on the endothelial cell (EC) functions. RESULTS: Our results showed that exosomes released from MM cells treated by C6-cer (ExoC6-cer) significantly inhibited the proliferation, migration and tube formation of ECs. For mechanism studies, we found that the level of miR-29b was increased in ECs treated by ExoC6-cer, while mRNA and protein expressions of Akt3, PI3K and VEGFA were decreased in ECs, indicating the involvement of Akt pathway. Furthermore, downregulation of miR-29b by inhibitor administration could prevent the ExoC6-cer-induced cell proliferation, migration and angiogenesis of ECs, accompanied with the increased expressions of Akt3, PI3K and VEGFA. CONCLUSIONS: Collectively, our data suggest that ExoC6-cer-mediated miR-29b expression participates in the progression of MM through suppressing the proliferation, migration and angiogenesis of ECs by targeting Akt signal pathway.

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.

Thermal Burn Injury Generates Bioactive Microvesicles: Evidence for a Novel Transport Mechanism for the Lipid Mediator Platelet-Activating Factor (PAF) That Involves Subcellular Particles and the PAF Receptor.

Thermal burn injuries are an important environmental stressor that can result in considerable morbidity and mortality. The exact mechanism by which an environmental stimulus to skin results in local and systemic effects is an area of active research. One potential mechanism to allow skin keratinocytes to disperse bioactive substances is via microvesicle particles, which are subcellular bodies released directly from cellular membranes. Our previous studies have indicated that thermal burn injury of the skin keratinocyte in vitro results in the production of the lipid mediator platelet-activating factor (PAF). The present studies demonstrate that thermal burn injury to keratinocytes in vitro and human skin explants ex vivo, and mice in vivo generate microvesicle particles. Use of pharmacologic and genetic tools indicates that the optimal release of microvesicles is dependent upon the PAF receptor. Of note, burn injury-stimulated microvesicle particles do not carry appreciable protein cytokines yet contain high levels of PAF. These studies describe a novel mechanism involving microvesicle particles by which a metabolically labile bioactive lipid can travel from cells in response to environmental stimuli.

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.

Exosome-Mediated Transfer of ACE2 (Angiotensin-Converting Enzyme 2) from Endothelial Progenitor Cells Promotes Survival and Function of Endothelial Cell.

Angiotensin-converting enzyme 2 (ACE2) is an emerging cardiovascular protective target that mediates the metabolism of angiotensin (Ang) II into Ang (1-7). Our group has demonstrated that ACE2 overexpression enhances the function of endothelial progenitor cells (EPCs). Here, we investigated whether ACE2-primed EPCs (ACE2-EPCs) can protect cerebral microvascular endothelial cells (ECs) against injury and dysfunction in an in vitro model, with focusing on their exosomal and cytokine paracrine effects on endothelial mitochondria. Human EPCs were transfected with lentivirus containing null or human ACE2 cDNA (denoted as Null-EPCs and ACE2-EPCs, respectively). Their conditioned culture media, w/wo depletion of exosomes (ACE2-EPC-CMEX-, Null-EPC-CMEX-, ACE2-EPC-CM, and Null-EPC-CM), were used for coculture experiments. EC injury and dysfunction model was induced by Ang II before coculture. Apoptosis, angiogenic ability, mitochondrion functions (ROS production, membrane potential, fragmentation), and gene expressions (ACE2, Nox2, and Nox4) of ECs were analyzed. The supernatant was collected for measuring the levels of ACE2, Ang II/Ang-(1-7), and growth factors (VEGF and IGF). Our results showed that (1) ACE2-EPC-CM had higher levels of ACE2, Ang (1-7), VEGF, and IGF than that of Null-EPC-CM. (2) Ang II-injured ECs displayed an increase of apoptotic rate and reduction in tube formation and migration abilities, which were associated with ACE2 downregulation, Ang II/Ang (1-7) imbalance, Nox2/Nox4 upregulation, ROS overproduction, an increase of mitochondrion fragmentation, and a decrease of membrane potential. (3) ACE2-EPC-CM had better protective effects than Null-EPC-CM on Ang II-injured ECs, which were associated with the improvements on ACE2 expression, Ang II/Ang (1-7) balance, and mitochondrial functions. (4) ACE2-EPC-CMEX- and Null-EPC-CMEX- showed reduced effects as compared to ACE2-EPCs-CM and Null-EPCs-CM. In conclusion, our data demonstrate that ACE2 overexpression can enhance the protective effects of EPCs on ECs injury, majorly through the exosomal effects on mitochondrial function.

Underlying Mechanisms and Potential Therapeutic Molecular Targets in Blood-Brain Barrier Disruption after Subarachnoid Hemorrhage.

Aneurysmal subarachnoid hemorrhage (aSAH) is a subtype of hemorrhagic stroke with significant morbidity and mortality. Aneurysmal bleeding causes elevated intracranial pressure, decreased cerebral blood flow, global cerebral ischemia, brain edema, blood component extravasation, and accumulation of breakdown products. These post-SAH injuries can disrupt the integrity and function of the blood-brain barrier (BBB), and brain tissues are directly exposed to the neurotoxic blood contents and immune cells, which leads to secondary brain injuries including inflammation and oxidative stress, and other cascades. Though the exact mechanisms are not fully clarified, multiple interconnected and/or independent signaling pathways have been reported to be involved in BBB disruption after SAH. In addition, alleviation of BBB disruption through various pathways or chemicals has a neuroprotective effect on SAH. Hence, BBB permeability plays an important role in the pathological course and outcomes of SAH. This review discusses the recent understandings of the underlying mechanisms and potential therapeutic targets in BBB disruption after SAH, emphasizing the dysfunction of tight junctions and endothelial cells in the development of BBB disruption. The emerging molecular targets, including toll-like receptor 4, netrin-1, lipocalin-2, tropomyosin-related kinase receptor B, and receptor tyrosine kinase ErbB4, are also summarized in detail. Finally, we discussed the emerging treatments for BBB disruption after SAH and put forward our perspectives on future research.