Proliferation and Differentiation of Neural Stem Cells Co-Cultured with Cerebral Microvascular Endothelial Cells after Oxygen-glucose Deprivation

Various stem cells, including neural stem cells （NSCs）, have been extensively studied in stroke models, but how to increase neuronal differentiation rate of NSCs remains unresolved, particu- larly in a damaged environment. The purpose of this study was to investigate the effects of cerebral mi- crovascular endothelial cells （CMECs） on the neurogenesis of NSCs with or without oxygen-glucose deprivation （OGD）. The NSCs acquired from primary culture were immunostained to prove cell purity. Survival and proliferation of NSCs were determined after the co-culture with CMECs for 7 days. After removing the CMECs, NSCs were randomly divided into two groups as follows： OGD and non-OGD groups. Both groups were maintained in differentiation culture for 4 days to evaluate the differentiation rate. Mouse embryo fibroblast （MEF） cells co-cultured with NSCs served as control group. NSCs co-cultured with CMECs had an increase in size （on the 7th day： 89.80±26.12 μm vs. 73.08±15.01μm, P〈0.001） （n=12） and number [on the 7th day： 6.33±5.61/high power objective （HP） vs. 2.23±1.61/HP, P〈0.001] （n=12） as compared with those co-cultured with MEF cells. After further differentiation cul- ture for 4 days, NSCs co-cultured with CMECs had an increase in neuronal differentiation rate in OGD and non-OGD groups, but not in the control group （15.16% and 16.07% vs. 8.81%; both P〈0.001） （n=6） This study provided evidence that OGD could not alter the effects of CMECs in promoting the neuronal differentiation potential of NSCs. These findings may have important implications for the development of new cell therapies for cerebral vascular diseases.

Concepts and opportunities for repair in cerebral microvascular disease and white matter stroke

Diffuse changes in white matter resulting from cerebral microvascular disease contribute to cognitive impairment （Jokinen et al., 2011）, declines in global functionality （Inzitari et al., 2009）, and even death （Debette and Markus, 2010）. Twenty years ago, estimations of the clinical incidence of ce- rebral microvascular disease approached 11 million per year in the US alone （Leary and Saver, 2003）. More recent estima- tions suggest the prevalence of diffuse white matter disease and silent brain infarction approaches 20% and increases dramatically in the presence of cardiovascular risk factors （Fanning et al., 2014）.

The relationship among amyloid-βdeposition,sphingomyelin level,and the expression and function of P-glycoprotein in Alzheimer’s disease pathological process

In Alzheimer’s disease,the transporter P-glycoprotein is responsible for the clearance of amyloid-βin the brain.Amyloid-βcorrelates with the sphingomyelin metabolism,and sphingomyelin participates in the regulation of P-glycoprotein.The amyloid cascade hypothesis describes amyloid-βas the central cause of Alzheimer’s disease neuropathology.Better understanding of the change of P-glycoprotein and sphingomyelin along with amyloid-βand their potential association in the pathological process of Alzheimer’s disease is critical.Herein,we found that the expression of P-glycoprotein in APP/PS1 mice tended to increase with age and was significantly higher at 9 and 12 months of age than that in wild-type mice at comparable age.The functionality of P-glycoprotein of APP/PS1 mice did not change with age but was significantly lower than that of wild-type mice at 12 months of age.Decreased sphingomyelin levels,increased ceramide levels,and the increased expression and activity of neutral sphingomyelinase 1 were observed in APP/PS1 mice at 9 and 12 months of age compared with the levels in wild-type mice.Similar results were observed in the Alzheimer’s disease mouse model induced by intracerebroventricular injection of amyloid-β1-42 and human cerebral microvascular endothelial cells treated with amyloid-β1-42.In human cerebral microvascular endothelial cells,neutral sphingomyelinase 1 inhibitor interfered with the changes of sphingomyelin metabolism and P-glycoprotein expression and functionality caused by amyloid-β1-42 treatment.Neutral sphingomyelinase 1 regulated the expression and functionality of P-glycoprotein and the levels of sphingomyelin and ceramide.Together,these findings indicate that neutral sphingomyelinase 1 regulates the expression and function of P-glycoprotein via the sphingomyelin/ceramide pathway.These studies may serve as new pursuits for the development of anti-Alzheimer’s disease drugs.

Roles of cyclooxygenase-2 in microvascular endothelial cell proliferation induced by basic fibroblast growth factor

Background The level of basic fibroblast growth factor （bFGF） increases rapidly after cerebral ischemia. However, the molecular mechanisms for the effects of bFGF on cerebral microvascular endothelial cells （cMVECs） have not yet been fully elucidated. In this study, a murine cMVEC line, bEnd.3, was employed to study the effects of bFGF on cyclooxygenase （COX） expression and its downstream effects in cMVECs. Methods After treatment with bFGF, RT-PCR and Western blotting analyses were carried out to evaluate the changes in COX-2 mRNA and protein expression, respectively. MTT assays were performed to measure cell proliferation. The prostaglandin E2 （PGE2） and vascular endothelial growth factor （VEGF） concentrations in the culture medium were measured by enzyme-linked immunosorbent assay （ELISA）. Results COX-2 mRNA and protein expressions in bEnd.3 cells were induced by bFGF in time- and dose-dependent manners. The bFGF-induced COX-2 upregulation led to enhanced PGE2 production by bEnd.3 cells, and this effect was abolished by the selective COX-2 inhibitor NS-398. bFGF also increased VEGF production by bEnd.3 cells, and this effect was blocked by NS-398 and the EP1/2 （PGE2 receptors） antagonist AH6809. Furthermore, exogenous PGE2 increased VEGF production in bend.3 cells, and AH6809 blocked this effect. Conclusion bFGF increases VEGF production in an autocrine manner by increasing COX-2-generated PGE2 in cMVECs and subsequently stimulates MVEC proliferation and angiogenesis.