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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., Exosomes are implicated in the central nervous system (CNS) diseases by mediating cell‐cell communications in the brain and modulating cellular functions through their carried molecules. Due to the nanosize and easy to cross blood‐brain barrier, exosomes hold great clinical application potentials for the CNS diseases by serving as biomarkers and therapeutic approaches.

We have previously demonstrated that endothelial progenitor cell (EPC) derived exosomes (EPC-EXs) can protect endothelial cells (ECs) against hypoxia injury. Given that EX function varies upon the cellular status and EPC function is declined in hypertension, we speculate the function of EPC-EXs is altered in hypertension-ischemia conditions. Here, we studied the EPC-EX mediated communications of EPCs with ECs in hypertension-ischemia conditions. EPC-EXs were prepared from the bone marrow EPCs of wild-type (WT) and hypertensive renin transgene (R+) mice (WT-EPC-EXs and R-EPC-EXs, respectively). To mimic hypertension-ischemia injury, ECs were challenged with angiotensin II (Ang II; 10−6 M) plus hypoxia (1% O2 for 6 h) and reoxygenation (21% O2 for 24 h). To determine the function of EPC-EXs, ECs were co-cultured with EXs during the reoxygenation period. EX uptake efficiency, EC viability, and angiogenic function were assessed. We found that: (1) The incorporation efficiency of R-EPC-EXs by ECs was significantly decreased compared to the WT-EPC-EXs. (2) Ang II plus hypoxia reoxygenation-injured ECs displayed decreased cell viability, increased cell apoptosis, and compromised angiogenic ability, which were alleviated by R-EPC-EXs. (3) WT-EPC-EXs elicited better effects than R-EPC-EXs on protecting ECs from hypertension plus hypoxia injury. In conclusion, our data have demonstrated that EPC-EXs mediated communication of EPCs and ECs is compromised in hypertension-ischemia conditions, suggesting that impairment of EPC exosomal communication might contribute to the exaggerated cerebral ischemia injury in hypertension-associated ischemic stroke.

Background: Vascular dementia (VD) is a severe cognitive impairment syndrome, characterized by a progressive memory and cognitive skill decline. Approximately 40 million people have dementia worldwide, and this number is expected to increase up to 115 million by 2050 due to the aging population. To date, no proven interventions are available to prevent VD. Chronic exposure to risk factors for vascular diseases, such as hypertension, diabetes, are implicated in the pathogenesis of VD, but the mechanisms remain to be fully understood. Methods: In this study, we utilized a renin hypertensive transgene mice (R+) mouse model to explore how the elevated blood pressure affects the pathogenesis of VD. We analyzed the cognitive ability, cerebral microvascular density, and neurons in young R+ mice, middle-aged R+ mice, and the wild type (WT) mice. Results: We found that middle-aged R+ mice had increased blood pressure associated with cognitive impairment as revealed by prolonged latency to target in morris water maze as compared to the young R+ mice and age-matched WT mice did. Meanwhile, our data showed that the R+ mice had brain microvessel rarefaction in the cortex and hippocampus, associating with a significantly low number of neurons in the hippocampus region (subiculum) as compared to that in WT mice. Conclusion: Our data have demonstrated that middle-aged renin transgene mice have impaired cognitive ability, accompanying with a low density of microvessel and fewer neurons, suggesting that chronic cerebral hypoperfusion might further damage neuron which could contribute to the progress of VD.

Objective We have previously demonstrated that angiotensin-converting enzyme 2 (ACE2) could boost the therapeutic effects of endothelial progenitor cells (ACE2-EPCs) on stroke. However, where this effect comes from is still unclear. Here, we investigated whether the exosomes (EXs) released from ACE2-EPCs could provide the benefit for acute intracerebral hemorrhagic stroke (ICH). Methods: The C57BL/6 mice were induced ICH by collagenase injection, followed by intravenously administration of ACE2-EPC-EXs. ACE2 blocker, DX600 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 2 after injection. The levels of ACE2 and inflammatory factors/genes in the brain were also measured. Results: EPC-EXs decreased hemorrhage volume, brain edema, BBB permeability, and improved NDS, which were enhanced by ACE2-EPC-EXs treatment; 2) As compared to EPC-EXs, ACE2-EPC-EXs resulted in an up-regulation of ACE2 in the brain, associating with the down-regulated expressions of TNF-α and NFкB and up-regulated level of IκBα. 3) DX600 blocked the above mentioned protective effects of ACE2-EPC-EXs in ICH mice. Conclusion: These data suggest that the infusion of ACE2-EPC-EXs could provide the therapeutic effect on acute ICH by alleviating the post-stroke inflammation via transferring ACE2.

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.

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., Schematic shows the mechanism of EPC‐EXsmiR‐126 on treating ischemic stroke in diabetes. Overexpression of miR‐126 enhanced the therapeutic effects of EPC‐EXs on diabetic ischemic stroke by reducing cell apoptosis in acute phase and promoting angiogenesis and neurogenesis to promote neurological function recovery in chronic phase.

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.

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.

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.

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.

The mammalian target of rapamycin (mTOR) was reported to regulate cell autophagy and outcomes of several neurological diseases. Mitochondria, which serve as critical organelles in neurons. are also involved in the pathology of neurological diseases. However, the role of mTOR in mitochondrial morphology has not been clarified especially in subarachnoid hemorrhage (SAH). In this study, we established SAH models both in vivo and in vitro. Rapamycin and 3-methyl adenine (3-MA) were then administered to alter mTOR activity. Post-SAH assessment included SAH grading, neurological evaluation, blood-brain barrier (BBB) permeability, brain water content, mitochondrial membrane potential (MMP), mitochondrial morphology, ATP content, cell viability, cytotoxicity, and expression of proteins related to apoptosis and mitochondrial fission. The results showed that (1) neurological deficits, BBB permeability, and brain edema were increased after SAH and that cell viability was exacerbated in brain tissue. (2) Excessive mitochondrial fission was evident based on changes in mitochondrial morphology, while MMP and ATP content were decreased in neurons after SAH. (3) Administration of rapamycin improved the excessive mitochondrial fission and restored mitochondrial function, which subsequently reduced apoptosis. (4) 3-MA showed an adverse effect on mitochondria and aggravated excessive mitochondrial fission and dysfunction in SAH. Neurological deficits and neuronal viability were also exacerbated following the administration of 3-MA. Therefore, our study suggests that mTOR inhibition has neuroprotective effects against neuronal injury after SAH via alleviating excessive mitochondrial fission.

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.

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.

Perivascular adipose tissue (PVAT), a type of adipose tissue that surrounds the blood vessels, has been considered an active component of the blood vessel walls and involved in vascular homeostasis. Recent evidence shows that increased inflammation and oxidative stress in PVAT contribute to endothelial dysfunction in type 2 diabetes (T2D). Exercise is an important nonpharmacological approach for vascular diseases. However, there is limited information regarding whether the beneficial effects of exercise on vascular function is related to the PVAT status. In this study, we investigated whether exercise can decrease oxidative stress and inflammation of PVAT and promote the improvement of endothelial function in a T2D mouse model. Diabetic db/db (5-week old) mice performed treadmill exercise (10 m/min) or keep sedentary for 8 weeks. Body weight, fasting blood glucose levels, glucose, and insulin tolerance were determined. The cytokines (IL-6, IL-10, IFN-γ, and TNF-a) and adiponectin levels, macrophage polarization and adipocyte type in PVAT, oxidative stress, and nitric oxide (NO) expression in the vascular wall were evaluated. The adhesion ability of primary aorta endothelial cells was analyzed. Our data showed that (1) diabetic db/db mice had increased body weight and fasting blood glucose level, compromised glucose tolerance, and insulin sensitivity, which were decreased/improved by exercise intervention. (2) Exercise intervention increased the percentage of multilocular brown adipocytes, promoted M1 to M2 macrophage polarization, associating with an increase of adiponectin and IL-10 levels and decrease of IFN-γ, IL-6, and TNF-a levels in PVAT. (3) Exercise decreased superoxide production in PVAT and the vascular wall of diabetic mice, accompanied with increased NO level. (4) The adhesion ability of aorta endothelial cells to leukocytes was decreased in exercised db/db mice, accompanied by decreased intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) expressions. Of interesting, coculture with PVAT-culture medium from exercised db/db mice could also reduce ICAM-1 and VCAM-1 expressions in primary endothelial cells. In conclusion, our data suggest that exercise improved endothelial function by attenuating the inflammation and oxidative stress in PVAT.

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.

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.

Perivascular adipose tissue (PVAT), a type of adipose tissue which surrounds blood vessels, has been considered as an active component of blood vessel walls and involved in vascular homeostasis. Recent evidence shows that increased inflammation and oxidative stress in PVAT contribute to endothelial dysfunction in type 2 diabetes (T2D). Exercise is an important non-pharmacological approach for vascular diseases. However, there is limited information regarding whether the beneficial effects of exercise on vascular function is related to PVAT status. In this study, we investigated whether exercise can decrease oxidative stress and inflammation of PVAT and promote the improvement of endothelial function in a T2D mouse model. Diabetic db/db (5-wk old) mice performed treadmill exercise (10 m/min) or keep sedentary for 8 wks. Body weight, fasting blood glucose levels, glucose and insulin tolerance were determined. The cytokines (IL-6, IL-10, IFN-r, TNF-a) and adiponectin levels, macrophage polarization in PVAT, and oxidative stress and nitric oxide expression in vascular wall were evaluated. The leukocyte adhesion ability of aorta endothelial cells isolated from the db/db mice was analyzed. Our data showed that: 1) Exercise decreased body weight and the fasting blood glucose level as well as improved glucose tolerance and insulin sensitivity in T2D mice. 2) Exercise increased adiponectin and IL-10 levels, decreased IFN-r, IL-6, TNF-a levels and promoted M1 to M2 polarization in PVAT. 3) Exercise decreased superoxide production in the vascular wall of diabetic mice, accompanied with increased nitric oxide bioavailability. 4) Exercise decreased the adhesion of diabetic aorta endothelial cells to leukocytes, accompanying with decreased ICAM-1 and VCAM-1 expression. In conclusion, our data suggest that exercise improved endothelial function by attenuating the oxidative stress and inflammation in PVAT. Disclosure J. Wang: None. V. Polaki: None. S. Chen: None. J. Bihl: None. Funding American Diabetes Association (1-17-IBS-187 to J.B.)

Oxidative stress is one of the mechanisms of ageing‐associated vascular dysfunction. Angiotensin‐converting enzyme 2 (ACE2) and microRNA (miR)‐18a have shown to be down‐regulated in ageing cells. Our previous study has shown that ACE2‐primed endothelial progenitor cells (ACE2‐EPCs) have protective effects on endothelial cells (ECs), which might be due to their released exosomes (EXs). Here, we aimed to investigate whether ACE2‐EPC‐EXs could attenuate hypoxia/reoxygenation (H/R)‐induced injury in ageing ECs through their carried miR‐18a. Young and angiotensin II‐induced ageing ECs were subjected to H/R and co‐cultured with vehicle (medium), EPC‐EXs, ACE2‐EPCs‐EXs, ACE2‐EPCs‐EXs + DX600 or ACE2‐EPCs‐EXs with miR‐18a deficiency (ACE2‐EPCs‐EXsanti‐miR‐18a). Results showed (1) ageing ECs displayed increased senescence, apoptosis and ROS production, but decreased ACE2 and miR‐18a expressions and tube formation ability; (2) under H/R condition, ageing ECs showed higher rate of apoptosis, ROS overproduction and nitric oxide reduction, up‐regulation of Nox2, down‐regulation of ACE2, miR‐18a and eNOS, and compromised tube formation ability; (3) compared with EPC‐EXs, ACE2‐EPC‐EXs had better efficiencies on protecting ECs from H/R‐induced changes; (4) The protective effects were less seen in ACE2‐EPCs‐EXs + DX600 and ACE2‐EPCs‐EXsanti‐miR‐18a groups. These data suggest that ACE‐EPCs‐EXs have better protective effects on H/R injury in ageing ECs which could be through their carried miR‐18a and subsequently down‐regulating the Nox2/ROS pathway.

Cell-derived exosomes (EXs) can modulate target cell differentiation via microRNAs (miRs) that they carried. Previous studies have shown that miR126 is highly expressed in hematopoietic stem cells (HSCs) and plays a role in hematopoiesis via modulating the Notch pathway that participates in progenitors' cell fate decisions. In this study we investigated whether HSC-derived EXs (HSC-EXs) could affect the differentiation of mouse embryonic stem cells (ESCs) into HSCs. We prepared HSC-EXs(con), HSC-EXs(sc) and HSC-EXs(miR126) from control HSCs and the HSCs transfected with scramble control or miR126 mimics, respectively. HSC-EXs were isolated by ultracentrifugation and analyzed using nanoparticle tracking analysis. We incubated the collected EXs with mouse ESCs over a 10-d differentiation induction period, during which HSC-EXs and a Notch pathway activator (Jagged1, 100 ng/mL) were added to the cultures every 3 d. After the 10-d differentiation period, the expression levels of miR126, SSEA1, CD117, Sca1, Notch1 and Hes1 in ESCs were assessed. The generated HSCs were validated by flow cytometry using antibodies against HSC markers (CD117, CD34 and Sca1). Our results revealed that: (1) transfection with miR126 mimics significantly increased miR126 levels in HSC-EXs(miR126). (2) HSC-EX co-culture promoted mouse ESCs differentiation into HSCs with the most prominent effect found in the HSC-EXs(miR126) co-culture. (3) HSC differentiation was verified by reduced SSEA1 expression and increased CD117 and Sca1 expression. (4) All the effects caused by HSC-EXs were accompanied by significant reduction of Notch1 and Hes1 expression, thus inhibition of the Notch1/Hes1 pathway, whereas activation of Notch by Jagged1 abolished the effects of HSC-EXs(miR126). In conclusion, HSC-EXs promote hematopoietic differentiation of mouse ESCs in vitro by inhibiting the miR126/Notch1 pathway.

// Xu Sun 1, * , Xiaotang Ma 2, * , Jinju Wang 3 , Yuhui Zhao 4 , Yue Wang 3 , Ji C. Bihl 3 , Yanfang Chen 2, 3 , Chuanlu Jiang 1 1 Department of Neurosurgery, The Second Affiliated Hospital, Harbin Medical University, Harbin 150086, China 2 Guangdong Key Laboratory of Age-Related Cardiac and Cerebral Diseases, Institute of Neurology, Affiliated Hospital of Guangdong Medical University, Zhanjiang 524000, China 3 Department of Pharmacology & Toxicology, Boonshoft School of Medicine, Wright State University, Dayton, OH 45435, USA 4 Department of Neurology and Stroke Center, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510000, China * These authors equally contributed to this work Correspondence to: Xu Sun, email: suen0321@163.com Chuanlu Jiang, email: jcl6688@163.com Keywords: GSCs, exosomes, miR-21, VEGF, angiogenesis Received: January 16, 2017 Accepted: March 21, 2017 Published: March 29, 2017 ABSTRACT Glioma stem cells (GSCs) play an important role in glioblastoma prognosis. Exosomes (EXs) mediate cell communication by delivering microRNAs (miRs). Glioblastoma has a high level of miR-21 which could upregulate vascular endothelial growth factor (VEGF) expression. We hypothesized GSC-EXs can promote the angiogenic ability of endothelial cells (ECs) through miR-21/VEGF signal. GSCs were isolated from U-251 cells with stem cell marker CD133. GSCs transfected without or with scramble or miR-21 mimics were used to produce GSC- EXs con, GSC-EXs sc and GSC-EXs miR-21 . Human brain ECs were co-cultured with vehicle, GSC-EXs con , GSC-EXs sc or GSC-EXs miR-21 plus VEGF siRNAs (siRNA VEGF ). After 24 hours, the angiogenic abilities of ECs were evaluated. The levels of miR-21, VEGF and p-Flk1/VEGFR2 were determined. Results showed: 1) Over 90% of purified GSCs expressed CD133; 2) The levels of miR-21 and VEGF in GSCs and GSC-EXs were up-regulated by miR-21 mimic transfection; 3) Compared to GSC-EXs con or GSC-EXs sc , GSC-EXs miR-21 were more effective in elevating the levels of miR-21 and VEGF, and the ratio of p-Flk1/VEGFR2 in ECs; 4) GSC-EXsmiR-21 were more effective in promoting the angiogenic ability of ECs than GSC-EXs con or GSC-EXs sc , which were remarkably reduced by siRNA VEGF pretreatment. In conclusion, GSC-EXs can promote the angiogenic ability of ECs by stimulating miR-21/VEGF/VEGFR2 signal pathway.

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.

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 10 11 EXs/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.

Our recent study has revealed that moderate exercise upregulates the levels of miR-126 in circulating endothelial progenitor cell-derived exosomes (EPC-EXs), accompanying with enhanced protective function on endothelial cells. Whether exercise can regulate the function of circulating EPC-EXs on protecting neurons against hypoxia/reoxygenation (H/R) injury remains unclear. Here, we isolated the circulating EPC-EXs from exercise mice (10 m/min) and investigated their role in neurons subjected to H/R. EPC-EXs were characterized by nanoparticle tracking and western blot analyses. Mouse neurons (N2a) cultured in hypoxia condition for 6 hr followed by 24 hr reoxygenation. The H/R-injured N2a cells were treated with EPC-EXs (4 x 10 7 EPC-EXs/ml) isolated from exercise (EPC-EXs E ) or sedentary mice (EPC-EXs s ). For mechanism study, N2a cells were transfected with miR-126 inhibitor or pre-treated with PI3k inhibitor (LY294002) for 2 hr. After 24 hr treatment, apoptosis and axon growth ability of N2a cells were determined. The growth factors (vascular endothelial growth factor/VEGF and brain derived neurotrophic factor/BDNF), miR-126 level and its downstream signal genes (PI3k and Akt) were measured. Our data showed that: 1) H/R insult induced decrease of VEGF and BDNF secretion as well as axon growth ability, increase of apoptosis of N2a cells; 2) EPC-EX S treatment raised miR-126 level and rescued N2a cells from H/R-induced injury, with better effect elicited by EPC-EXs E . 3) EPC-EXs E had better effect than EPC-EX S on activating the PI3k/Akt signal pathway, which was significantly reduced by miR-126 and PI3k inhibitors. Altogether, exercise boosts the effects of EPC-EXs on protecting neurons against H/R insult through activating the miR-126/PI3k/Akt signal pathway.

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. Disclosure J. Wang: None. Q. Pan: None. H. Liu: None. B. Zhao: None. Y. Yang: None. X. Ma: None. J. Bihl: None.

Endothelial cells (ECs) released microvesicles (EMVs) could modulate the functions of target cells by transferring their microRNAs (miRs). We have reported that miR-125a-5p protected EC function. In this study, we determined whether EMVs provided beneficial effects on ECs by transferring miR-125a-5p. Human brain microvessel ECs were transfected with miR-125a-5p mimic or miR-125a-5p short hairpin RNA to obtain miR-125a-5p overexpressing ECs and miR-125a-5p knockdown ECs, and their derived EMVs. For the functional study, ECs or hypoxia/reoxygenation injured ECs were coincubated with various EMVs. The survival and angiogenic function of ECs were measured. Western blot and quantitative real time polymerase chain reaction (qRT-PCR) were used for measuring the levels of phosphoinositide 3-kinase (PI3K), phosphorylation-Akt (p-Akt)/Akt, p-endothelial nitric oxide synthase (p-eNOS), cleaved caspase-3, and miR-125a-5p. PI3K inhibitor was used for pathway analysis. EMVs promoted the proliferation, migration, and tube formation ability of ECs, and alleviated the apoptotic rate of ECs. These effects were associated by an increase in p-Akt/Akt and p-eNOS, and a decrease in cleaved caspase-3 could be abolished by LY294002. Overexpression or downregulation of miR-125a-5p in EMVs promoted or inhibited those effects of EMVs. EMVs could enhance the survival and angiogenic function of ECs via delivering miR-125a-5p to modulate the expression of PI3K/Akt/eNOS pathway and caspase-3.

Angiotensin-converting enzyme 2 (ACE2) is an emerging cardiovascular protective target which mediates the metabolism of angiotensin (Ang) II into Ang (1-7). Our group has demonstrated that ACE2 overexpression enhances the therapeutic efficacy of endothelial progenitor cells (EPCs) for ischemic stroke. 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, ACE2-EPCs, respectively). Their conditioned culture media, w/wo ultra-centrifugation for depletion of exosomes (ACE2-EPC-CM EX- , Null-EPC-CM EX- , ACE2-EPC-CM and Null-EPC-CM), were used for co-culture experiments. ECs injury and dysfunction model was induced by Ang II (10 -7 M) for 8 hrs before 16 hr co-culture. After that, ECs were collected for analyses of apoptosis, angiogenic ability, mitochondrion functions (ROS production, membrane potential, fragmentation), and gene expressions (ACE2, Nox2 and Nox4). The co-culture medium 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 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, increase of mitochondrion fragmentation and 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-CM EX- and Null-EPC-CM EX- had remarkably reduced effects by over 70% as respectively 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.

Angiotensin (Ang) II, the main effector of the renin-angiotensin system, has been implicated in the pathogenesis of vascular diseases. Ang-(1-7) binds to the G protein-coupled Mas receptor (MasR) and can exert vasoprotective effects. We investigated the effects and underlying mechanisms of Ang-(1-7) on Ang II-induced dysfunction and oxidative stress in human brain microvascular endothelial cells (HbmECs). The pro-apoptotic activity, reactive oxygen species (ROS) and nitric oxide (NO) productions in HbmECs were measured. The protein expressions of nicotinamide adenine dinucleotide phosphate oxidase 2 (Nox2), serine/threonine kinase (Akt), endothelial nitric oxide synthase (eNOS) and their phosphorylated forms (p-Akt and p-eNOS) were examined by western blot. MasR antagonist and phosphatidylinositol-3-kinase (PI3K) inhibitor were used for receptor/pathway verification. We found that Ang-(1-7) suppressed Ang II-induced pro-apoptotic activity, ROS over-production and NO reduction in HbmECs, which were abolished by MasR antagonist. In addition, Ang-(1-7) down-regulated the expression of Nox2, and up-regulated the ratios of p-Akt/Akt and its downstream p-eNOS/eNOS in HbmECs. Exposure to PI3K inhibitor partially abrogated Ang-(1-7)-mediated protective effects in HbmECs. Our data suggests that Ang-(1-7)/MasR axis protects HbmECs from Ang II-induced dysfunction and oxidative stress via inhibition of Nox2/ROS and activation of PI3K/NO pathways.

Vascular disease constitutes the leading health problem throughout the entire world. Current therapies for vascular disease mainly rely on comprehensive strategies including control of risk factors, vascular interventions and conventional supportive treatments. To improve the preventive and therapeutic efficacies of current approaches, novel combinational therapies are required. Microparticles (MPs) are small membrane vesicles derived from cells undergoing stress, activation or apoptosis. They carry the characteristics of their parent cells, enabling them to serve as potential biomarkers for various diseases. Of note, MPs also have been shown to mediate cell communications through transferring membrane proteins, phospholipids and RNAs from their parent cells to recipient cells. Recent novel approaches have started to reveal the functions of MPs. In this review, we summarize the general concepts and the latest research progress in MPs. And the potential of MPs as novel targets of combinational therapy for vascular disease will be discussed.

Stroke is one of the leading causes of death and disability worldwide. Various types of stem cells have been applied to treat stroke and have been shown promising potential. The principal mechanism of therapeutic action has been partially ascribed to their strong paracrine capacity. Exosomes are small vesicles released from all kinds of cells and mediate intercellular communication by transferring exosomal protein and microRNA (miRNA) cargoes between cells in the brain. Among these cargoes, miRNAs play a key role in mediating biological function due to their prominent roles in gene regulation. Emerging data suggest that stem cell-released exosomes have advantages over stem cells to treat stroke, because exosomes could cross the blood bran barrier and easily to be modified and handled. Here, we first review the biogenesis, cargoes, and detection of exosomes. Then, we discussed the role of miRNAs in stroke. At last, we highlight the use of stem cell-released exosomes as biomarkers and therapeutic avenues in stroke. Perspectives on the developing role of stem cell-released exosomes mediated transfer of miRNAs as a therapeutic approach will also be discussed.

Background/Aims: Stem cell-derived exosomes (EXs) offer protective effects on various cells via their carried microRNAs (miRs). Meanwhile, miR-210 has been shown to reduce mitochondrial reactive oxygen species (ROS) overproduction. In this study, we determined the potential effects of endothelial progenitor cell-derived EXs (EPC-EXs) on hypoxia/ reoxygenation (H/R) injured endothelial cells (ECs) and investigated whether these effects could be boosted by miR-210 loading. Methods: Human EPCs were used to generate EPC-EXs, or transfected with scrambler control or miR-210 mimics to generate EPC-EXssc and EPC-EXsmiR-210. H/R-injured human ECs were used as a model for functional analysis of EXs on apoptosis, viability, ROS production and angiogenic ability (migration and tube formation) by flow cytometry, MTT, dihydroethidium and angiogenesis assay kits, respectively. For mechanism analysis, the mitochondrion morphology, membrane potential (MMP), ATP level and the expression of fission/fusion proteins (dynamin-related protein 1: drp1 and mitofusin-2: mfn2) were assessed by using JC-1 staining, ELISA and western blot, respectively. Results: 1) Transfection of miR-210 mimics into EPCs induced increase of miR-210 in EPC-EXsmiR-210 without change of average size; 2) EPC-EXsmiR-210, but not EPC-EXs or EPC-EXssc, significantly elevated miR-210 level in ECs; 3) EPC-EXsmiR-210 were more effective than EPC-EXs and EPC-EXssc in reducing H/R-induced EC apoptosis, ROS overproduction and angiogenic dysfunction; 4) EPC-EXs decreased mitochondrial fragmentation, elevated MMP and ATP level, as well as improved mitochondrial mfn2 and drp1 dysregulation, which were more effective in EPC-EXsmiR-210. Conclusion: Our results suggest that EPC-EXs protect ECs against H/R injury via improving mitochondrial function and miR-210 enrichment could boost their effects.

Ultraviolet B radiation (UVB) exerts profound effects on human skin. Much is known regarding the ability of UVB to generate a plethora of bioactive agents ranging from cytokines and other bioactive proteins, lipid mediators and microRNAs. It is presumed that these agents are in large part responsible for the effects of UVB, which is only absorbed appreciably in the epidermis. However, the exact mechanism by which these bioactive agents can leave the epidermis are as yet unclear. This review addresses the potential role of microvesicle particles (MVP) as UVB signaling agents through transmitting biologic mediators. New data are provided that UVB treatment of human skin explants also generates MVP production. We hypothesize that UVB production of MVPs (UVB-MVP) could serve this important function of transmitting keratinocyte-derived bioactive agents. Moreover, we propose that UVB-MVP formation involves the lipid mediator platelet-activating factor. This novel pathway has the potential to be exploited pharmacologically to modulate UVB effects.

Accurate analysis of specific microvesicles (MVs) from biofluids is critical and challenging. Here we described novel methods to purify and detect MVs shed from endothelial cells (ECs) and endothelial progenitor cells (EPCs) by combining microbeads with fluorescence quantum dots (Q-dots) coupled nanoparticle tracking analysis (NTA). In the in vitro screening systems, we demonstrated that 1) anti-CD105 (EC marker) and anti-CD34 (EPC marker) conjugated-microbeads had the highest sensitivity and specificity for isolating respective MVs, which were confirmed with negative controls, CD41 and CD235a; 2) anti-CD144 (EC marker) and anti-KDR (EPC marker) conjugated-Q-dots exhibited the best sensitivity and specificity for their respective MV NTA detection, which were confirmed with positive control, anti-Annexin V (MV universal marker). The methods were further validated by their ability to efficiently recover the known amount of EC-MVs and EPC-MVs from particle-depleted plasma and to detect the dynamical changes of plasma MVs in ischemic stroke patients, as compared with traditional flow cytometry. These novel methods provide ideal approaches for functional analysis and biomarker discovery of ECs- and EPCs- derived MVs.

Exosomes (EXs) are cell-derived vesicles that mediate cell-cell communication and could serve as biomarkers. Here we described novel methods for purification and phenotyping of EXs released from endothelial cells (ECs) and endothelial progenitor cells (EPCs) by combining microbeads and fluorescence quantum dots (Q-dots®) techniques. EXs from the culture medium of ECs and EPCs were isolated and detected with cell-specific antibody conjugated microbeads and second antibody conjugated Q-dots by using nanoparticle tracking analysis (NTA) system. The sensitivities of the cell origin markers for ECs (CD105, CD144) and EPCs (CD34, KDR) were evaluated. The sensitivity and specificity were determined by using positive and negative markers for EXs (CD63), platelets (CD41), erythrocytes (CD235a), and microvesicles (Annexin V). Moreover, the methods were further validated in particle-free plasma and patient samples. Results showed that anti-CD105/anti-CD144 and anti-CD34/anti-KDR had the highest sensitivity and specificity for isolating and detecting EC-EXs and EPC-EXs, respectively. The methods had the overall recovery rate of over 70% and were able to detect the dynamical changes of circulating EC-EXs and EPC-EXs in acute ischemic stroke. In conclusion, we have developed sensitive and specific microbeads/Q-dots fluorescence NTA methods for EC-EX and EPC-EX isolation and detection, which will facilitate the functional study and biomarker discovery.

Our previous study showed that circulating microvesicles (cMVs) of diabetic mice have negative effects on the function of endothelial progenitor cells (EPCs). Whether this is true in diabetic patients deserves further study. In this study, the effects of cMVs and EPC-derived MVs (EPC-MVs) on EPC migration, apoptosis, and reactive oxygen species (ROS) production in healthy controls, well-controlled, and uncontrolled diabetic patients were investigated. The levels of miR-126 and vascular endothelial growth factor receptor 2 (VEGFR2) in cMVs, EPC-MVs, and/or EPCs were analyzed. Moreover, miR-126 inhibitor or mimic was applied to EPCs to modulate the miR-126 level in EPC-MVs. We found the following: 1) the circulating EPC level was reduced but the circulating EPC-MV level increased in uncontrolled diabetic patients; 2) the cMVs and EPC-MVs of healthy controls had beneficial effects on EPCs (migration, apoptosis, ROS), whereas the effects were reversely changed in the cMVs and EPC-MVs of uncontrolled diabetic patients; and 3) the cMVs and EPC-MVs of uncontrolled diabetic patients carried less miR-126 and had downregulated VEGFR2 expression in EPCs. Manipulating the miR-126 level in EPC-MVs with inhibitor or mimic changed their function. The effects of cMVs and EPC-MVs are compromised in diabetes due to the reduction of their carried miR-126, which might provide a therapy target for diabetic vascular complications.

The lipid mediator platelet-activating factor (PAF) and oxidized glycerophosphocholine PAF agonists produced by ultraviolet B (UVB) have been demonstrated to play a pivotal role in UVB-mediated processes, from acute inflammation to delayed systemic immunosuppression. Recent studies have provided evidence that microvesicle particles (MVPs) are released from cells in response to various signals including stressors. Importantly, these small membrane fragments can interact with various cell types by delivering bioactive molecules. The present studies were designed to test if UVB radiation can generate MVP release from epithelial cells, and the potential role of PAF receptor (PAF-R) signaling in this process. We demonstrate that UVB irradiation of the human keratinocyte-derived cell line HaCaT resulted in the release of MVPs. Similarly, treatment of HaCaT cells with the PAF-R agonist carbamoyl PAF also generated equivalent amounts of MVP release. Of note, pretreatment of HaCaT cells with antioxidants blocked MVP release from UVB but not PAF-R agonist N-methyl carbamyl PAF (CPAF). Importantly, UVB irradiation of the PAF-R-negative human epithelial cell line KB and KB transduced with functional PAF-Rs resulted in MVP release only in PAF-R-positive cells. These studies demonstrate that UVB can generate MVPs in vitro and that PAF-R signaling appears important in this process.

Background Protection of cerebral endothelial cells (ECs) from hypoxia/reoxygenation (H/R)-induced injury is an important strategy for treating ischemic stroke. In this study, we investigated whether co-culture with endothelial progenitor cells (EPCs) and neural progenitor cells (NPCs) synergistically protects cerebral ECs against H/R injury and the underlying mechanism. Results EPCs and NPCs were respectively generated from inducible pluripotent stem cells. Human brain ECs were used to produce an in vitro H/R-injury model. Data showed: 1) Co-culture with EPCs and NPCs synergistically inhibited H/R-induced reactive oxygen species (ROS) over-production, apoptosis, and improved the angiogenic and barrier functions (tube formation and permeability) in H/R-injured ECs. 2) Co-culture with NPCs up-regulated the expression of vascular endothelial growth factor receptor 2 (VEGFR2). 3) Co-culture with EPCs and NPCs complementarily increased vascular endothelial growth factor (VEGF) and brain-derived neurotrophic factor (BDNF) levels in conditioned medium, and synergistically up-regulated the expression of p-Akt/Akt and p-Flk1/VEGFR2 in H/R-injured ECs. 4) Those effects could be decreased or abolished by inhibition of both VEGFR2 and tyrosine kinase B (TrkB) or phosphatidylinositol-3-kinase (PI3K). Conclusions Our data demonstrate that EPCs and NPCs synergistically protect cerebral ECs from H/R-injury, via activating the PI3K/Akt pathway which mainly depends on VEGF and BDNF paracrine.

Angiotensin (Ang)-(1-7) is a potential vasoprotective peptide. In the present study, we investigated its counteractive effects to Ang II on vascular smooth muscle cells (VSMCs) and intracerebral hemorrhagic stroke (ICH) through inflammatory mechanism. In in vitro experiments, human brain VSMCs (HBVSMCs) were treated with vehicle, Ang II, Ang II + Ang-(1-7), Ang II + A-779 or Ang II + Ang-(1-7) + A-779 (Mas receptor antagonist). HBVSMC proliferation, migration and apoptosis were determined by methyl thiazolyltetrazolium, wounding healing assay and flow cytometry, respectively. In in vivo experiments,C57BL/6 mice were divided into vehicle, Ang II, Ang II + Ang-(1-7), Ang II + A-779 or Ang II + Ang-(1-7) + A-779 groups before they were subjected to collagenase-induced ICH or sham surgery. Hemorrhage volume and middle cerebral artery (MCA) remodeling were determined by histological analyses. Levels of NFκB, Inhibitor of κBα (IκBα), tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein 1 (MCP-1) and interleukin (IL-8) were measured by western blot or ELISA. We found that 1) Ang II increased HBVSMC migration, proliferation and apoptosis, and increased the blood pressure (BP), neurological deficit score, MCA remodeling and hemorrhage volume in ICH mice. 2) Ang-(1-7) counteracted these effects of Ang II, which was independent of BP, with the down-regulation of NFκB, up-regulation of IκBα, and decreased levels of TNF-α, MCP-1 and IL-8. 3) The beneficial effects of Ang-(1-7) could be abolished by A-779. In conclusion, Ang-(1-7) counteracts the effects of Ang II on ICH via modulating NFκB inflammation pathway in HBVSMCs and cerebral microvessels.

Background Platelet activation and aggregation are critical in the pathogenesis of acute ischemic stroke (AIS). Circulating platelet microparticles (PMPs) and platelet parameters are biologic markers of platelet function in AIS patients; however, their associations with stroke subtypes and infarct volume remain unknown. Methods We recruited 112 AIS patients including large-artery atherosclerosis (LAA) and small-artery occlusion [SAO] subtypes and 35 controls in this study. Blood samples were collected at admission and after antiplatelet therapy. The levels of circulating PMPs and platelet parameters (mean platelet volume [MPV], platelet count, plateletocrit, and platelet distribution width) were determined by flow cytometry and hematology analysis, respectively. Infarct volume was examined at admission by magnetic resonance imaging. Results (1) The levels of circulating PMPs and MPV were significantly elevated in AIS patients compared with healthy controls; (2) the level of circulating PMPs, but not platelet parameters, was decreased after antiplatelet therapy in AIS patients; (3) the infarct volume in LAA subtype was larger than that in SAO subtype. Notably, circulating PMP level was positively correlated with the infarct volume in LAA subtype. No association with infarct volume in either AIS subtype was observed for platelet parameters; and (4) according to the regression analysis, circulating PMP was an independent risk factor for the infarct volume in pooled AIS patients after adjustments of other impact factors (hypertension and diabetes). Conclusions Our results suggest that circulating PMP level is associated with cerebral injury of AIS, which offers a novel evaluation parameter for AIS patients.

Angiotensin-(1-7) [Ang-(1-7)] is a potential vascular protective peptide which counteracts the effects of Ang II. In this study, we investigated the role of Ang-(1-7) in hemorrhagic stroke. Adult male C57BL/6 mice implanted with telemetric probe for recording arterial blood pressure (BP) were divided into four minipump treatment groups: saline, Ang II, Ang II + Ang-(1-7), Ang II + Ang-(1-7) + A-779. After 2 weeks of treatment, mice were subjected to induction of hemorrhagic stroke by microinjection of collagenase (type VII; 0.075 U in 0.5 μL) in striatum. Mice were sacrificed at twenty-four hours after ICH induction. Brains were dissected and cut into coronal slices for histological analysis of middle cerebral artery (MCA)remodeling and hemorrhagic size. The plasma levels of tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein 1 (MCP-1) and interleukin (IL-8) were determined by ELISA and the levels of NFκB and inhibitor of kappa B (IκB) in cerebral microvasculature were determined by western blot. We found (Figure): 1) Ang II increased BP, MCA remodeling and hemorrhagic size. 2) Ang-(1-7) significantly decreased the effects of Ang II on MCA and hemorrhage without affecting the BP, which were abolished by A-779. 3) Ang-(1-7) induced a 26-32% reduction in plasma level of TNF-α, MCP-1 and IL-8 increased by Ang II. 4) Ang-(1-7) also counter-regulated Ang II-induced NFƙB up-regulation and IƙB down-regulation. In conclusion, Ang-(1-7) counteracts Ang II on vascular remodeling and hemorrhagic injury by alleviating NFκB-related inflammation.

BACKGROUND MATERIALS & METHODS RESULTS RESULTS Cont’d 1. UVB generates MVP in vitro in a process involving PAF-R signaling. 2. UVB also generates MVP in human skin. 3. As MVP are thought to be functional signaling agents, these studies could provide a mechanism whereby UVB can generate systemic effects. SUMMARY For in vitro experiments, the human keratinocyte-derived cell line HaCaT was used. HaCaT cells were treated with UVB or the PAF-R agonist Carbamoyl-PAF (CPAF) or ethanol vehicle [3,5,7]. In some experiments HaCaT cells were preincubated for one hour with 0.5mM each of the antioxidants N-acetylcysteine and ascorbic acid before UVB/CPAF treatment. In some experiments the PAF-R-negative human epidermoid cell line KB cells transduced with functional PAF-Rs (KBP) or MSCV2.1 retroviral vector alone (KBM) were used [9]. At various times posttreatment supernatants were removed and MVP isolated using differential centrifugation and quantified using a NanoSight NS300 instrument using our previously published protocol [8,10]. For ex vivo experiments, we used skin discarded from human abdominoplasty experiments. The skin was warmed to ~37-38 degrees C and suction blisters separating epidermis from dermis were created using vacuum pumps and syringes (see Fig 5). Following blister formation, the blisters were treated with either sham or UVB or topical CPAF in ethanol:DMSO (1:9 v/v). Blister fluid was obtained and weighed in tared tubes. MVP were quantified as above. For in vivo experiments, the volar forearm of human volunteers underwent suction blistering and blisters were treated either sham or UVB. Again, blister fluid was removed and weighed and MVP quantified as outlined above. ACKNOWLEDGEMENTS These studies were supported in part by grants from the NIH (R01HL062996) and Basic Science VA Merit Award Platelet activating factor (PAF) is a lipid-derived 1-alkyl-2-acetly-glycerophosphocholine, produced in response to various stimuli that produces pronounced pro-inflammatory effects. These include marked vasodilatory effects, increased vascular permeability, bronchoconstriction, and platelet activation. PAF has also been implicated in sepsis [1,2]. PAF acts by binding to a cellular heterotrimeric G-protein coupled receptor PAF-R. This receptor is found on many cell types including granulocytes, B-cells, epithelial cells (keratinocytes), and some mesenchymal cells. PAF-R activation is known to induce intracellular calcium mobilization and is expressed on keratinocytes. Recent studies have demonstrated that PAF can be produced both enzymatically and through non-enzymatic oxidation of membrane glycerophosphocholines (ox-GPCs) [3]. Figure 1Biosynthesis of PAF through enzymatic and nonenzymatic pathways We and others have shown that in addition to its pro-inflammatory effects, PAF-R mediate pro-oxidative stressors including UVB-induced acute inflammation as well as delayed systemic immunosuppression [4-6]. Regarding the early acute effects of UVB, our previous studies have demonstrated that the PAFR plays an important role in the pain associated with sunburn [6], and mediates the increased UVB sensitivity in the Xeroderma Pigmentosum Complementation group A (XPA) knockout mouse [7]. Microvesicle particles (MVP) are small membrane-bound vesicles released by numerous cell types and can be found in the circulation. MVP can contain both nuclear and cytoplasmic constituents and are thought to provide a mechanism by which cells transmit cells systemically [8]. MVP are 200-1000nm in diameter and differ from smaller (40-200 nm) exosomes. Given that PAF is involved in UVB-mediated acute effects, these studies were designed to define if MVP are released from keratinocytes following UVB, and if PAF was involved in this process. Figure 3. Effect of UVB and CPAF on MVP release in HaCaT cells. HaCaT cells were either A) control (0) or UVB-irradiated at various fluences or B) vehicle control or various doses of CPAF. Four hours after treatment, the supernatants were removed and MVPs quantified as outlined in Methods. C) HaCaT cells were treated with 3.6 KJ/m2 UVB or 100 nM CPAF or control (CON), and were harvested 1 or 4 hrs post treatment. D). HaCaT cells were preincubated with 0.5 mM of N-aceytcysteine and 0.5 mM Vitamin C for one hour before treatment with 3.6 KJ/m2 UVB or 100 nM CPAF. The supernatants were removed and MVP measured 4 h later. The data presented are the mean ± SD. MVP numbers of duplicate values from a representative experiment of at least three performed. *Statistically (p changes from control values. 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Augmentation of UVB radiation- mediated early gene expression by the epidermal platelet-activating factor receptor. Journal of Investigative Dermatology. 128, 455-460, 2008. 10. Wang J et al., Analyses of Endothelial and Progenitor Cell Released Extracellular Microvesicles and Exosomes by Using Nanoparticle Tracking Analysis Method in Combination with Microbead and Q-dot Techniques. Science Reports 2016 in press. Figure 2. Picture of MVP released from a cell following stress UVB TREATMENT (KJ/m) M VP x 1 0 9 0 1 2 3 4 0 0.9 NS * *

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. Electronic supplementary material The online version of this article (doi:10.1186/s13041-016-0243-1) contains supplementary material, which is available to authorized users.