Shuzhen 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.

Effects of Endothelial Progenitor Cell-Derived Microvesicles on Hypoxia/Reoxygenation-Induced Endothelial Dysfunction and Apoptosis

Oxidative stress-induced endothelial dysfunction plays a key role in ischemia/reperfusion injury. Recent evidence indicates that endothelial progenitor cell-derived microvesicles (EPC-MVs) can promote angiogenesis of endothelial cells (ECs). Here, we investigated the potential effects of EPC-MVs on hypoxia/reoxygenation (H/R) injury in human brain microvascular ECs (hb-ECs). MVs were prepared from EPCs cultured in a serum deprivation (SD) medium (starving stress, sEPC-MVs) or SD medium containing tumor necrosis factor-α (TNFα) (apoptotic stress, aEPC-MVs). H/R injury model of hb-ECs was produced by 6 hr hypoxia (1% O2) and 24 hr reoxygenation. The H/R hb-ECs were co-cultured with EPC-MVs. Results showed that (1) H/R hb-ECs were dysfunctional and coupled with increased apoptosis and ROS overproduction; (2) under two different conditions, EPCs displayed remarkable difference in caspase 3 and miR126 expression, which were carried by the corresponsive EPC-MVs; (3) functionally, sEPC-MVs had beneficial effects on H/R hb-ECs, whereas aEPC-MVs had detrimental effects; (4) the diverse effects of sEPC-MVs and aEPC-MVs were associated with the changes in miR126 and eNOS expression and were abolished by PI3K inhibitor. In conclusion, sEPCs-MVs and aEPC-MVs are functionally different on hb-EC apoptosis and dysfunction via their carried RNAs associated with ROS production and PI3K/eNOS/NO pathway.

Angiotensin-Converting Enzyme 2 Priming Enhances the Function of Endothelial Progenitor Cells and Their Therapeutic Efficacy

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 or lentivirus-green fluorescence protein. The effects of ACE2 overexpression on EPC function and endothelial NO synthase (eNOS)/nicotinamide adenine dinucleotide phosphate oxidase (Nox) expression were determined. ACE2, eNOS, and Nox inhibitors were used for pathway validation. In in vivo studies, the therapeutic efficacy of EPCs overexpressing ACE2 was determined at day 7 after ischemic stroke induced by middle cerebral artery occlusion. We found that (1) lentivirus-ACE2 transduction resulted in a 4-fold increase of ACE2 expression in EPCs. This was accompanied with an increase in eNOS expression and NO production, and a decrease in Nox2 and -4 expression and reactive oxygen species production. (2) ACE2 overexpression 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 lentivirus-ACE2–primed EPCs reduced cerebral infarct volume and neurological deficits, and 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.

Transfusion of CXCR4-Primed Endothelial Progenitor Cells Reduces Cerebral Ischemic Damage and Promotes Repair in db/db Diabetic Mice

This study investigated the role of stromal cell-derived factor-1α (SDF-1α)/CXC chemokine receptor 4 (CXCR4) axis in brain and endothelial progenitor cells (EPCs), and explored the efficacy of CXCR4 primed EPCs in treating ischemic stroke in diabetes. The db/db diabetic and db/+ mice were used in this study. Levels of plasma SDF-1α and circulating CD34+CXCR4+ cells were measured. Brain SDF-1α and CXCR4 expression were quantified at basal and after middle cerebral artery occlusion (MCAO). In in vitro study, EPCs were transfected with adenovirus carrying null (Ad-null) or CXCR4 (Ad-CXCR4) followed with high glucose (HG) treatment for 4 days. For pathway block experiments, cells were pre-incubated with PI3K inhibitor or nitric oxide synthase (NOS) inhibitor for two hours. The CXCR4 expression, function and apoptosis of EPCs were determined. The p-Akt/Akt and p-eNOS/eNOS expression in EPCs were also measured. In in vivo study, EPCs transfected with Ad-null or Ad-CXCR4 were infused into mice via tail vein. On day 2 and 7, the cerebral blood flow, neurologic deficit score, infarct volume, cerebral microvascular density, angiogenesis and neurogenesis were determined. We found: 1) The levels of plasma SDF-1α and circulating CD34+CXCR4+ cells were decreased in db/db mice; 2) The basal level of SDF-1α and MCAO-induced up-regulation of SDF-1α/CXCR4 axis were reduced in the brain of db/db mice; 3) Ad-CXCR4 transfection increased CXCR4 expression in EPCs and enhanced EPC colonic forming capacity; 4) Ad-CXCR4 transfection prevented EPCs from HG-induced dysfunction (migration and tube formation) and apoptosis via activation of PI3K/Akt/eNOS signal pathway; 4) Ad-CXCR4 transfection enhanced the efficacy of EPC infusion in attenuating infarct volume and promoting angiogenesis and neurogenesis. Our data suggest that Ad-CXCR4 primed EPCs have better therapeutic effects for ischemia stroke in diabetes than unmodified EPCs do.

Ischemia-Induced Brain Damage is Enhanced in Human Renin and Angiotensinogen Double Transgenic Mice

To investigate the role of brain angiotensin II (ANG II) in the pathogenesis of injury following ischemic stroke, mice overexpressing renin and angiotensinogen (R+A+) and their wild-type control animals (R-A-) were used for experimental ischemia studies. Focal brain ischemia was induced by middle cerebral artery occlusion (MCAO). The severity of ischemic injury was determined by measuring neurological deficits and histological damage at 24 and 48 h after MCAO, respectively. To exclude the influence of blood pressure and local collateral blood flow, brain slices were used for oxygen and glucose deprivation (OGD) studies. The severity of OGD-induced damage was determined by measuring indicators of tissue swelling and cell death, the intensity of the intrinsic optical signal (IOS), and the number of propidium iodide (PI) staining cells, respectively. Results showed 1) R+A+ mice showed higher neurological deficit score (3.8 +/- 0.5 and 2.5 +/- 0.3 for R+A+ and R-A-, respectively, P < 0.01) and larger infarct volume (22.2 +/- 1.6% and 14.1 +/- 1.2% for R+A+ and R-A-, respectively, P < 0.01); 2) The R+A+ brain slices showed more severe tissue swelling and cell death in the cortex (IOS: 140 +/- 6% and 114 +/- 10%; PI: 139 +/- 20 cells/field and 39 +/- 9 cells/field for R+A+ and R-A-, respectively, P < 0.01); 3) treatment with losartan (20 micromol/l) abolished OGD-induced exaggeration of cell injury seen in R+A+ mice. The data indicate that activation of ANG II/AT(1) signaling is harmful to brain exposed to ischemia.

Angiotensin-(1-7) counteracts angiotensin II-induced dysfunction in cerebral endothelial cells via modulating Nox2/ROS and PI3K/NO pathways.

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.

Activation of the ACE2/Ang-(1–7)/Mas Pathway Reduces Oxygen–Glucose Deprivation-Induced Tissue Swelling, ROS Production, and Cell Death in Mouse Brain with Angiotensin II Overproduction

We previously demonstrated that mice which overexpress human renin and angiotensinogen (R+A+) show enhanced cerebral damage in both in vivo and in vitro experimental ischemia models. Angiotensin-converting enzyme 2 (ACE2) counteracts the effects of angiotensin (Ang-II) by transforming it into Ang-(1-7), thus reducing the ligand for the AT1 receptor and increasing stimulation of the Mas receptor. Triple transgenic mice, SARA, which specifically overexpress ACE2 in neurons of R+A+ mice were used to study the role of ACE2 in ischemic stroke using oxygen and glucose deprivation (OGD) of brain slices as an in vitro model. We examined tissue swelling, the production of reactive oxygen species (ROS), and cell death in the cerebral cortex (CX) and the hippocampal CA1 region during OGD. Expression levels of NADPH oxidase (Nox) isoforms, Nox2 and Nox4 were measured using western blots. Results show that SARA mice and R+A+ mice treated with the Mas receptor agonist Ang-(1-7) had less swelling, cell death, and ROS production in CX and CA1 areas compared to those in R+A+ animals. Treatment of slices from SARA mice with the Mas antagonist A779 eliminated this protection. Finally, western blots revealed less Nox2 and Nox4 expression in SARA mice compared with R+A+ mice both before and after OGD. We suggest that reduced brain swelling and cell death observed in SARA animals exposed to OGD result from diminished ROS production coupled with lower expression of Nox isoforms. Thus, the ACE2/Ang-(1-7)/Mas receptor pathway plays a protective role in brain ischemic damage by counteracting the detrimental effects of Ang-II-induced ROS production.

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.

Angiotensin converting enzyme 2/Ang-(1-7)/mas axis protects brain from ischemic injury with a tendency of age-dependence.

The angiotensin (Ang) converting enzyme 2 (ACE2)/Ang‐(1‐7)/Mas receptor pathway is an important component of the renin–angiotensin system and has been suggested to exert beneficial effects in ischemic stroke.