Wenfeng Zhang

DAPK1 Interaction with NMDA Receptor NR2B Subunits Mediates Brain Damage in Stroke

N-methyl-D-aspartate (NMDA) receptors constitute a major subtype of glutamate receptors at extrasynaptic sites that link multiple intracellular catabolic processes responsible for irreversible neuronal death. Here, we report that cerebral ischemia recruits death-associated protein kinase 1 (DAPK1) into the NMDA receptor NR2B protein complex in the cortex of adult mice. DAPK1 directly binds with the NMDA receptor NR2B C-terminal tail consisting of amino acid 1292-1304 (NR2B(CT)). A constitutively active DAPK1 phosphorylates NR2B subunit at Ser-1303 and in turn enhances the NR1/NR2B receptor channel conductance. Genetic deletion of DAPK1 or administration of NR2B(CT) that uncouples an activated DAPK1 from an NMDA receptor NR2B subunit in vivo in mice blocks injurious Ca(2+) influx through NMDA receptor channels at extrasynaptic sites and protects neurons against cerebral ischemic insults. Thus, DAPK1 physically and functionally interacts with the NMDA receptor NR2B subunit at extrasynaptic sites and this interaction acts as a central mediator for stroke damage.

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.

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.

Human Endometrial Stromal Stem Cells Differentiate into Megakaryocytes with the Ability to Produce Functional Platelets

Human endometrium is a high dynamic tissue that contains endometrial stromal stem cells (hESSCs). The hESSCs have been differentiated into a number of cell lineages. However, differentiation of hESSCs into megakaryocytes (MKs) has not yet been investigated. The aim of this study was to investigate the feasibility of MK generation from hESSCs and subsequent production of functional platelets (PLTs). In our study, hESSCs were cultured from endometrial stromal cells as confirmed by positive stromal cell specific markers (CD90 and CD29) and negative hematopoietic stem cell markers (CD45 and CD34) expression. Then, hESSCs were differentiated in a medium supplemented with thrombopoietin (TPO) for 18 days. The MK differentiation was analyzed by flow cytometry and confocal microscopy. The differentiation medium was collected for PLT production analysis by flow cytometry, transmission electron microscopy and functional measurements. Our results show: 1) MKs were successfully generated from hESSCs as identified by expression of specific markers (CD41a: 1±0.09% and 39±3.0%; CD42b: 1.2±0.06% and 28±2.0%, control vs. differentiation) accompanied with reduction of pluripotent transcription factors (Oct4 and Sox2) expression; 2) The level of PLTs in the differentiation medium was 16±1 number/µl as determined by size (2–4 µm) and CD41a expression (CD41a: 1±0.4% and 90±2.0%, control vs. differentiation); 3) Generated PLTs were functional as evidenced by the up-regulation of CD62p expression and fibrinogen binding following thrombin stimulation; 4) Released PLTs showed similar ultra-structure characteristics (alpha granules, vacuoles and dense tubular system) as PLTs from peripheral blood determined by electron microscopic analysis. Data demonstrate the feasibility of generating MKs from hESSCs, and that the generated MKs release functional PLTs. Therefore, hESSCs could be a potential new stem cell source for in vitro MK/PLT production.