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For determining the implications of circulating endothelial progenitor cells (cEPCs) and cellular membrane microparticles (MPs) in diabetic stroke, levels of EPCs, EPC-MPs, and endothelium-derived MPs (EMPs) and their correlations with blood glucose concentration, cerebral microvascular density (cMVD), and ischemic damage were investigated in type 2 diabetic db/db and db/+ (wild-type control) mice. Therapeutic efficacy of EPC infusion (preincubated with MPs) was also explored. Ischemic stroke was induced by middle cerebral artery occlusion (MCAO) surgery. Ischemic damage and cMVD were determined using histological analyses. The levels of cEPCs and MPs were determined using flow cytometric analyses. EPC generation and functions were evaluated by in vitro cell cultures. Results showed the following. 1) In db/db mice, the basal level of cEPCs was less and cMVDs were lower, but the levels of circulating EPC-MPs and EMPs were more; 2) MCAO induced a larger infarct volume and less of an increase in cEPCs in db/db mice; 3) the level of cEPCs correlated with blood glucose concentration (negatively), cMVD (positively), and ischemic damage (negatively), but the levels of EPC-MPs and EMPs correlated inversely with those parameters; 4) EPCs were reduced and dysfunctional in db/db mice, and preincubation with db/db MPs impaired EPC functions; and 5) infusion of EPCs preincubated with db/+ MPs increased the level of cEPCs and reduced ischemic damage, and these beneficial effects were reduced or lost in EPCs preincubated with db/db MPs. These data suggest that reduced cEPCs, impaired EPC generation/function, and increased production of MPs might be the mechanisms responsible for increased ischemic damage seen in db/db mice.

Angiotensin converting enzyme 2 (ACE2) is a lately discovered enzyme catalyzing Angiotensin II into Angiotensin (1-7). Angiotensin II has been reported to impair endothelial progenitor cell (EPC) function and is detrimental to stroke. Here, we studied the role of ACE2 in regulating EPC function in vitro and in vivo. EPCs were cultured from human renin and angiotensinogen transgenic (R+A+) mice and their controls (R-A-). In in vitro experiments, EPCs were transduced with lentivirus-ACE2 (Lenti-ACE2) or Lenti-GFP. The effects of ACE2 over-expression on EPC function and endothelial oxide synthase (eNOS)/NADPH oxidase (Nox) expression were determined. ACE2, eNOS and Nox inhibitors were used for pathway validation. In in vivo studies, the therapeutic efficacy of EPCs over-expressing ACE2 was determined at day 7 after ischemic stroke induced by middle cerebral artery occlusion. We found that 1) Lenti-ACE2 transduction resulted in a four-fold increase of ACE2 expression in EPCs. This was accompanied with an increase in eNOS expression and nitric oxide production, and a decrease in Nox2, 4 expression and reactive oxygen species production. 2) ACE2 over-expression improved the abilities of EPC migration and tube formation which were impaired in R+A+ mice. These effects were inhibited by ACE2 or eNOS inhibitor and further enhanced by Nox inhibitor. 3) Transfusion of Lenti-ACE2 primed EPCs reduced cerebral infarct volume and neurologic deficits, increased cerebral microvascular density and angiogenesis. Our data demonstrate that ACE2 improves EPC function via regulating eNOS and Nox pathways and enhances the efficacy of EPC-based therapy for ischemic stroke.

The CCMD-3 Task Force has prepared and completed field trials for the Chinese Classification of Mental Disorders Version 3 (CCMD-3). It has also worked on the Treatment and Nursing of Mental Disorders Relevant to CCMD-3, and the Rating Test for Health Problems and Diseases as a set of relevant diagnostic scales and software. From 1996 to 2000, the CCMD-3 Task Force has been actively engaged in prospective follow-up field trials aimed at improving the classification and diagnostic criteria of mental disorders in China. A total of 114 psychiatrists from 41 mental health centers and psychiatric hospitals carried out the trials, considering for the 17 adult mental disorders, and 7 mental disorders with onset usually occurring in childhood and adolescence. Based on the results of the field trials, the Task Force has finalized the CCMD-3. Throughout the phase of initial drafting, each item in CCMD-3 was compared with the corresponding item in the CCMD-2R, and the descriptive definitions were based on the Clinical descriptions and diagnostic guidelines of ICD-10. The diagnostic criteria also refer to the Research Criteria of ICD-10, and the DSM-IV. Following the decision made by the Chinese Psychiatric Association, CCMD-3 has been published as diagnostic guidelines to be used by all psychiatrists across the country.

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.

Angiotensin (Ang) II exaggerates cerebral injury in ischemic damage. Angiotensin-converting enzyme type 2 (ACE2) converts Ang II into Ang (1–7) and thus, may protect against the effects of Ang II. We hypothesized that neuronal ACE2 over-expression decreases ischemic stroke in mice with Ang II overproduction. Human renin and angiotensinogen double transgenic (RA) mice and RA mice with neuronal over-expression of ACE2 (SARA) were used for the study. The mean arterial pressure (MAP) was calculated from telemetry-recorded blood pressure (BP). SARA mice were infused peripherally with Norepinephrine to “clamp” the BP, or intracerebroventricularly-infused with a Mas receptor antagonist (A-779). Middle cerebral artery occlusion (MCAO) surgery was performed to induce permanent focal ischemic stroke. Cerebral blood flow (CBF) and neurological function were determined. Two days after surgery, brain samples were collected for various analyses. Results showed: 1) When compared to chronically hypertensive RA mice, SARA mice had lower basal MAP, less MCAO-induced infarct volume, and increased CBF, neurological function and cerebral microvascular density in the peri-infarct area; 2) These changes in SARA mice were not altered after MAP “clamping”, but partially reversed by brain infusion of A-779; 3) Ang (1–7)/Ang II ratio, angiogenic factors, endothelial nitric oxide synthase (eNOS) expression and nitric oxide production were increased, whereas, NADPH oxidase subunits and reactive oxygen species were decreased in the brain of SARA mice. ACE2 protects brain from ischemic injury via the regulation of NADPH oxidase/eNOS pathways by changing Ang (1–7)/Ang II ratio, independently of MAP changes.

Background Platelet activation and aggregation are critical in the pathogenesis of acute ischemic stroke (AIS). Circulating platelet microparticles (PMPs) and platelet parameters are biological 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 (PC), plateletocrit (PCT) and platelet distribution width (PDW)] 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 when 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; (4) According to the regression analysis, circulating PMPs 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.

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.