Qunwen Pan

Gut dysbiosis is tightly linked to type 2 diabetes (T2D) arterial calcification (AC), but the mechanism of gut dysbiosis remains poorly defined. Here, we found that a T2D AC associated‐gut Bacteroides fragilis (BF) was activated by M1 macrophage‐derived extracellular vesicles (EVs). BF internalized macrophage EVs via membrane protein OprM‐Jup interaction. Upon uptake, Mef2d, which was rich in EVs, translocated to the BF nucleoid and suppressed ArsR family transcriptional regulator (ArsR) transcription and enhanced BF proliferation and trimethylamine N‐oxide production in diabetic mice. Mechanistically, Mef2d suppressed the transcriptional activity of ArsR, an arsenic resistance regulator in BF. Overexpression of ArsR inhibited BF growth and trimethylamine synthesis, whereas ArsR knockdown exacerbated these phenotypes. Critically, ArsR overexpression abolished macrophage EV‐driven BF activation. Our findings reveal a macrophage EV‐BF signaling axis in which Mef2d‐mediated ArsR suppression drives bacterial pathogenicity, offering a pharmacologically targetable axis for precision inhibitation of BF pathobiont virulence in diabetic complications.

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

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

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

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

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-EXs^miR-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-EXs^miR-132-3pwere 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-EXs^miR-132-3pwas 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.

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.

Endothelial progenitor cells (EPCs) have shown the potential for treating ischemic stroke (IS), while microRNA-126 (miR-126) is reported to have beneficial effects on endothelial function and angiogenesis. In this study, we investigated the effects of miR-126 overexpression on EPCs and explore the efficacy of miR-126-primed EPCs (EPC^miR-126) in treating IS. The effects of miR-126 overexpression on EPC proliferation, migratory, tube formation capacity, reactive oxygen species (ROS) production, and nitric oxide (NO) generation were determined. In in vivo study, the effects of EPC^miR-126 on the cerebral blood flow (CBF), neurological deficit score (NDS), infarct volume, cerebral microvascular density (cMVD), and angiogenesis were determined. Moreover, the levels of circulating EPCs (cEPCs) and their contained miR-126 were measured. We found (1) miR-126 overexpression promoted the proliferation, migration, and tube formation abilities of EPCs; decreased ROS; and increased NO production of EPCs via activation of PI3K/Akt/eNOS pathway; (2) EPC^miR-126 was more effective than EPCs in attenuating infarct volume and NDS and enhancing cMVD, CBF, and angiogenesis; and (3) infusion of EPC^miR-126 increased the number and the level of miR-126 in cEPCs. Our data indicate that miR-126 overexpression enhanced the function of EPCs in vitro and in vivo.

MicroRNA-125a-5p (miR-125a-5p) could participate in the pathogenesis of vascular diseases. In this study, we investigated the role of miR-125a-5p in oxidized low-density lipoprotein (ox-LDL)-induced functional changes in human brain microvessel endothelial cells (HBMEC). The reactive oxygen species (ROS) production, nitric oxide (NO) generation, senescence, apoptosis, and functions of HBMEC were analyzed. For mechanism study, the epidermal growth factor receptor (EGFR)/extracellular signal-regulated protein kinase (ERK)/p38 mitogen-activated protein kinase (p38 MAPK) pathway and phosphatidylinositol-3-kinase (PI3K)/serine/threonine kinase (Akt)/endothelial nitric oxide synthase (eNOS) pathway were analyzed. Results showed the following: 1) Expression of miR-125a-5p was reduced in ox-LDL-treated HBMEC. 2) Overexpression of miR-125a-5p protected HBMEC from ox-LDL-induced apoptosis, senescence, ROS production, and NO reduction. 3) Overexpression of miR-125a-5p increased HBMEC proliferation, migration, and tube formation, while decreasing HBMEC adhesion to leukocytes, as well as counteracting the effects of ox-LDL on those functions. 4) The levels of EGFR/ERK/p38 MAPK pathway, PI3K/Akt/eNOS pathway, cleaved caspase-3, and adherent molecular ICAM-1 and VCAM-1 were associated with the effects of ox-LDL on these HBMEC functions. In conclusion, miR-125a-5p could counteract the effects of ox-LDL on various HBMEC functions via regulating the EGFR/ERK/p38 MAPK and PI3K/Akt/eNOS pathways and cleaved caspase-3, ICAM-1, and VCAM-1 expression.