Yusen Chen

Circulating endothelial progenitor cells and cellular membrane microparticles in db/db diabetic mouse: possible implications in cerebral ischemic damage

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.

MicroRNA-155 Regulates ROS Production, NO Generation, Apoptosis and Multiple Functions of Human Brain Microvessel Endothelial Cells Under Physiological and Pathological Conditions.

The microRNA‐155 (miR155) regulates various functions of cells. Dysfunction or injury of endothelial cells (ECs) plays an important role in the pathogenesis of various vascular diseases. In this study, we investigated the role and potential mechanisms of miR155 in human brain microvessel endothelial cells (HBMECs) under physiological and pathological conditions. We detected the effects of miR155 silencing on ROS production, NO generation, apoptosis and functions of HBMECs at basal and in response to oxidized low density lipoprotein (ox‐LDL). Western blot and q‐PCR were used for analyzing the gene expression of epidermal growth factor receptor (EGFR)/extracellular regulated protein kinases (ERK)/p38 mitogen‐activated protein kinase (p38 MAPK), phosphatidylinositol‐3‐kinase (PI3K) and serine/threonine kinase(Akt), activated caspase‐3, and intercellular adhesion molecule‐1 (ICAM‐1). Results showed that under both basal and challenge situations: (1) Silencing of miR155 decreased apoptosis and reactive oxygen species (ROS) production of HBMECs, whereas, promoted nitric oxide (NO) generation. (2) Silencing of miR155 increased the proliferation, migration, and tube formation ability of HBMECs, while decreased cell adhesion ability. (3) Gene expression analyses showed that EGFR/ERK/p38 MAPK and PI3K/Akt were increased and that activated caspase‐3 and ICAM‐1 mRNA were decreased after knockdown of miR155. In conclusion, knockdown of miR155 could modulate ROS production, NO generation, apoptosis and function of HBMECs via regulating diverse gene expression, such as caspase‐3, ICAM‐1 and EGFR/ERK/p38 MAPK and PI3K/Akt pathways. J. Cell. Biochem. 116: 2870–2881, 2015.

The Role of Circulating Platelets Microparticles and Platelet Parameters in Acute Ischemic Stroke Patients

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.

Endothelial progenitor cells and neural progenitor cells synergistically protect cerebral endothelial cells from Hypoxia/reoxygenation-induced injury via activating the PI3K/Akt pathway

BackgroundProtection 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.ResultsEPCs 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).ConclusionsOur 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-(1–7) counteracts the effects of Ang II on vascular smooth muscle cells, vascular remodeling and hemorrhagic stroke: Role of the NFкB inflammatory pathway

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, wound 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.

Analyses of Endothelial Cells and Endothelial Progenitor Cells Released Microvesicles by Using Microbead and Q-Dot Based Nanoparticle Tracking Analysis

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.

NPC-EXs Alleviate Endothelial Oxidative Stress and Dysfunction through the miR-210 Downstream Nox2 and VEGFR2 Pathways

We have demonstrated that neural progenitor cells (NPCs) protect endothelial cells (ECs) from oxidative stress. Since exosomes (EXs) can convey the benefit of parent cells through their carried microRNAs (miRs) and miR-210 is ubiquitously expressed with versatile functions, we investigated the role of miR-210 in the effects of NPC-EXs on oxidative stress and dysfunction in ECs. NPCs were transfected with control and miR-210 scramble/inhibitor/mimic to generate NPC-EXscon, NPC-EXssc, NPC-EXsanti-miR-210, and NPC-EXsmiR-210. The effects of various NPC-EXs on angiotensin II- (Ang II-) induced reactive oxygen species (ROS) overproduction, apoptosis, and dysfunction, as well as dysregulation of Nox2, ephrin A3, VEGF, and p-VEGFR2/VEGFR2 in ECs were evaluated. Results showed (1) Ang II-induced ROS overproduction, increase in apoptosis, and decrease in tube formation ability, accompanied with Nox2 upregulation and reduction of p-VEGFR2/VEGFR2 in ECs. (2) Compared to NPC-EXscon or NPC-EXssc, NPC-EXsanti-miR-210 were less whereas NPC-EXsmiR-210 were more effective on attenuating these detrimental effects induced by Ang II in ECs. (3) These effects of NPC-EXsanti-miR-210 and NPC-EXsmiR-210 were associated with the changes of miR-210, ephrin A3, VEGF, and p-VEGFR2/VEGFR2 ratio in ECs. Altogether, the protective effects of NPC-EXs on Ang II-induced endothelial injury through miR-210 which controls Nox2/ROS and VEGF/VEGFR2 signals were studied.

Circulating CD133+ CD34+ Progenitor Cells and Plasma Stromal-Derived Factor-1Alpha: Predictive Role in Ischemic Stroke Patients

Circulating progenitor cells and stromal-derived factor-1alpha (SDF-1α) have been suggested to participate in tissue repair after ischemic injury. However, the predictive role of circulating CD133+ CD34+ progenitors and plasma SDF-1α in ischemic stroke (IS) patients remains unknown. In this study, we recruited 95 acute IS patients, 40 at-risk subjects, and 30 normal subjects. The National Institutes of Health Stroke Scale (NIHSS), infarct volume, and carotid intima-media thickness (IMT) were determined at day 1 and the modified Rankin scale (mRS) of functional outcome was assessed at day 21. The levels of circulating CD133+ CD34+ cells and plasma SDF-1α were determined by flow cytometry and enzyme-linked immunosorbent assay, respectively. Our data showed that: (1) the levels of CD133+ CD34+ cells were lower in at-risk subjects and IS patients at admission (day 1) when compared with normal controls; (2) the day 1 level of CD133+ CD34+ cells varied in IS subgroups and inversely correlated with NIHSS and carotid IMT and the level of SDF-1α inversely correlated with NIHSS and infarct volume; (3) the increment rates of circulating CD133+ CD34+ cells and plasma SDF-1α within the first week were correlated; and (4) patients with a higher level of CD133+ CD34+ cells at day 7 had a low mRS. The increased rate of CD133+ CD34+ cells in the first week was inversely associated with mRS. In conclusion, our findings demonstrate that the circulating CD133+ CD34+ progenitor cells and plasma SDF-1α can be used as predictive parameters for IS severity and outcome.

Association between polymorphisms in the flanking region of the TAFI gene and atherosclerotic cerebral infarction in a Chinese population.

BackgroundAtherosclerosis is the leading etiologic factor of Atherosclerotic Cerebral Infarction (ACI). Previous studies have shown that thrombin activatable fibrinolysis inhibitor (TAFI) may play an important role in the occurrence of acute cerebral infarction, and the levels of TAFI are affected by several single nucleotide polymorphisms (SNPs) located in the regulatory and coding regions of the gene encoding TAFI. The present study aimed to determine whether polymorphisms (TAFI – 2345 2G/1G, –1690 A/G, –438 A/G, +1583 A/T) of the TAFI gene were associated with ACI in a Han Chinese population.MethodsThe variant genotypes were identified by restriction fragment length polymorphism (RFLP) and allele-specific polymerase chain reactions (AS-PCR) in 225 patients with ACI and 184 age-matched healthy individuals.ResultsThere was a significant difference in the genotype and allele frequencies of TAFI – 2345 2G/1G and −1690 A/G polymorphisms between the ACI and control subjects. Further stratification analysis by gender revealed that the presence of the –438 AA genotype and the A allele conferred a higher risk of developing ACI in male patients (p < 0.05). Haplotype analysis demonstrated that four haplotypes of TAFI are significantly associated with ACI.ConclusionsOur study provides preliminary evidence that the TAFI – 2345 2G/1G and –1690 A/G polymorphisms are associated with ACI susceptibility in a Han Chinese population.

Microvesicles-mediated communication between endothelial cells modulates, endothelial survival, and angiogenic function via transferring of miR-125a-5p: PAN et al.

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.