Junji Wei

Transplantation of human bone marrow-derived mesenchymal stem cells promotes behavioral recovery and endogenous neurogenesis after cerebral ischemia in rats

Mesenchymal stem cells (MSCs) have been successfully used for the treatment of experimental stroke. However, the neurorestorative mechanisms by which MSCs improve neurological functional recovery are not fully understood. Endogenous cell proliferation in the subventricular zone (SVZ) after stroke is well known, but most of newly formed cells underwent apoptosis. In the present study, we tested the hypothesis that neurotrophic factors secreted by human bone marrow-derived MSCs (hBMSCs) promote endogenous neurogenesis, reduce apoptosis, and improve functional recovery. Adult rats subjected to 2-h middle cerebral artery occlusion (MCAO) were transplanted with hBMSCs or saline into the ipsilateral brain parenchyma at 3days after ischemia. There was a significant recovery of behavior in the hBMSCs-treated rats beginning at 14days after MCAO compared with the control animals. Higher levels of brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and vascular endothelial growth factor (VEGF) were detected in the hBMSCs-treated rat brain than the control. Human BMSCs treatment also enhanced endogenous cell proliferation both in the SVZ and in the subgranular zone (SGZ) of the hippocampus. In addition, more neuronal progenitor cells migrated from the SVZ to the ischemic boundary zone (IBZ) and differentiated into mature neurons with less apoptosis in rats treated with hBMSCs. Overall, these data suggest an essential role for hBMSCs in promoting endogenous neurogenesis, protecting newly formed cells, and improving functional recovery after ischemia in rats.

Transplantation of Flk‐1+ human bone marrow‐derived mesenchymal stem cells promotes angiogenesis and neurogenesis after cerebral ischemia in rats

Transplantation of bone marrow‐derived mesenchymal stem cells (BMSCs) is a potential therapy for cerebral ischemia. Although BMSCs‐induced angiogenesis is considered important for neurological functional recovery, the neurorestorative mechanisms are not fully understood. We examined whether BMSCs‐induced angiogenesis enhances cerebral tissue perfusion and creates a suitable microenvironment within the ischemic brain, which in turn accelerates endogenous neurogenesis and leads to improved functional recovery. Adult female rats subjected to 2 h middle cerebral artery occlusion (MCAO) were transplanted with a subpopulation of human BMSCs from male donors (Flk‐1+ hBMSCs) or saline into the ipsilateral brain parenchymal at 3 days after MCAO. Flk‐1+ hBMSCs‐treated rats exhibited significant behavioral recovery, beginning at 2 weeks after cerebral ischemia compared with controls. Moreover, rats treated with Flk‐1+ hBMSCs showed increased glucose metabolic activity and reduced infarct volume. Flk‐1+ hBMSCs treatment significantly increased the expression of vascular endothelial growth factor and brain‐derived neurotrophic factor, promoted angiogenesis, and facilitated cerebral blood flow in the ischemic boundary zone. Further, Flk‐1+ hBMSCs treatment enhanced proliferation of neural stem/progenitor cells (NSPCs) in the subventricular zone and subgranular zone of the hippocampus. Finally, more NSPCs migrated toward the ischemic lesion and differentiated to mature neurons or glial cells with less apoptosis in Flk‐1+ hBMSCs‐treated rats. These data indicate that angiogenesis induced by Flk‐1+ hBMSCs promotes endogenous neurogenesis, which may cause functional recovery after cerebral ischemia.

Postacute ischemia vascular endothelial growth factor transfer by transferrin-targeted liposomes attenuates ischemic brain injury after experimental stroke in rats.

Our objective was to achieve the enhanced delivery of vascular endothelial growth factor (VEGF) to ischemically disordered brain through transferrin-coupled liposomes (Tf-PLs) via intravenous administration, and to observe the effect of Tf-VEGF-PLs on ischemic brain neuroprotection and angiogenesis. Cerebral VEGF overexpression was achieved with Tf-PLs by intravenous injection 48 hr after an acute stroke. β-Galactosidase expression was monitored; saline was injected as a control. The success of postischemic gene transduction was confirmed by β-galactosidase staining and by increased VEGF mRNA and protein in ischemic brain. Vascular density, neurological recovery, and ischemic area calculation were performed to evaluate the effect of Tf-VEGF-PLs. The positive expression of β-galactosidase indirectly indicated that VEGF was successfully delivered into brain by Tf-VEGF-PLs. VEGF mRNA in the Tf-VEGF-PL group 24 hr after injection was significantly higher than in the control group (p < 0.05). Western blot analysis showed that postischemic Tf-VEGF-PLs resulted in increased VEGF protein levels compared with VEGF-PLs and saline-administered rats (p < 0.05) 48 hr after administration. At 21 days after drug injection, we observed a significant decrease in infarct volume and better neurological function in the Tf-VEGF-PL-treated group, compared with the VEGF-PL group. FITC-dextran marking showed increased vascular density in the penumbra of Tf-VEGF-PL-treated hemispheres (245,873.9, number of microvessels per field) compared with that in VEGF-PL-treated hemispheres (139,801.3) or saline-treated hemispheres (102,175.5) (p < 0.05). The remainder of the cerebral blood flow after ischemia in the Tf-VEGF-PL group was significantly more than in the control groups (0.35 vs. 0.29, 0.21; p < 0.05). We conclude that the VEGF gene can be delivered noninvasively into the brain by Tf-VEGF-PLs. Postischemic treatment with Tf-VEGF-PLs effectively promoted neuroprotection and vascular regeneration in the chronic stage of cerebral infarction.

RNA interference-mediated silencing of iASPP induces cell proliferation inhibition and G0/G1 cell cycle arrest in U251 human glioblastoma cells

AbstractiASPP is an evolutionally conserved inhibitory member of the ASPP (apoptosis-stimulating protein of p53) protein family. Overexpression of iASPP was observed in several types of human tumors, however, its role in tumorigenesis has not been fully clarified. To investigate the role of iASPP in human glioblastoma multiforme (GMB) progression, the authors employed lentivirus-mediated shRNA to silence endogenous iASPP expression and elucidated iASPP function by analysis of viability, colony formation, DNA synthesis, and cell cycle in p53-mutant glioblastoma cell line U251. iASPP was significantly and sustainably knocked down by iASPP-specific shRNA in U251 cells. Stable down-regulation of iASPP expression-induced cell proliferation inhibition and G0/G1 cell cycle arrest by down-regulation of cyclin D1 and up-regulation of p21Waf1/Cip1. Thus, the findings not only provide a molecular basis for the role of iASPP in cell cycle progression of glioblastoma cells but also suggest a novel therapeutic target for the treatment of GBM.

Angiopoietin 1 counteracts vascular endothelial growth factor-induced blood–brain barrier permeability and alleviates ischemic injury in the early stages of transient focal cerebral ischemia in rats

Abstract Objective: To examine the effects of intraventricular pre-treatment with a combination of recombinant adeno-associated virus vectors encoding VEGF (rAAV-VEGF) and Ang1 (rAAV-Ang1) on early stroke in a rat model of transient middle cerebral artery occlusion (tMCAO). Methods: rAAV-VEGF/rAAV-Ang1 or rAAV-VEGF/rAAV-null vector was delivered into the lateral ventricles of 48 rats. Eight weeks later, the rats were subjected to tMCAO for 2 hours. During the early stages of ischemic reperfusion, VEGF and Ang1 expression levels, blood–brain barrier (BBB) permeability and cerebral microvessel density were determined and compared statistically between groups. Cerebral infarct volume and modified neurological severity scores (NSS) were also determined to evaluate the therapeutic efficacy of rAAV-VEGF/rAAV-Ang1. Results: Intraventricular application of rAAV-VEGF/rAAV-Ang1, 8 weeks before tMCAO, resulted in VEGF and Ang1 overexpression, and significantly decreased Evans blue permeability following ischemia (p<0.05). The microvessel density in the peri-infarct zone was significantly increased in the rAAV-VEGF/rAAV-Ang1 group compared with the rAAV-VEGF/rAAV-null group (p<0.05). Cerebral infarct volume and NSS in the rAAV-VEGF/rAAV-Ang1 group were significantly decreased compared with the rAAV-VEGF/rAAV-null group (p<0.05). Conclusion: In cerebral ischemia, the combination of Ang1 and VEGF could be used early to promote the formation of mature neovessels and protect the injured cells, without inducing the side effects on BBB permeability. Early intraventricular injection of mixed rAAV-VEGF and rAAV-Ang1 may be a favorable therapeutic strategy in gene therapy for experimental stroke.

Suppression of STIM1 inhibits human glioblastoma cell proliferation and induces G0/G1 phase arrest

BackgroundDepletion of calcium (Ca2+) from the endoplasmic reticulum (ER) activates the ubiquitous store-operated Ca2+ entry (SOCE) pathway which sustains long-term Ca2+ signals and is critical for cellular functions. Stromal interacting molecule 1 (STIM1) serves a dual role as an ER Ca2+ sensor and activator of SOCE. Aberrant expression of STIM1 could be observed in several human cancer cells. However, the role of STIM1 in regulating tumorigenesis of human glioblastoma still remains unclear.MethodsExpression of STIM1 protein in a panel of human glioblastoma cell lines (U251, U87 and U373) in different transformation level were evaluated by Western blot method. STIM1 loss of function was performed on U251 cells, derived from grade IV astrocytomas-glioblastoma multiforme with a lentvirus-mediated short harpin RNA (shRNA) method. The biological impacts after knock down of STIM1 on glioblastoma cells were investigated in vitro and in vivo.ResultsWe discovered that STIM1 protein was expressed in U251, U87 and U373 cells, and especially higher in U251 cells. RNA interference efficiently downregulated the expression of STIM1 in U251 cells at both mRNA and protein levels. Specific downregulation of STIM1 inhibited U251 cell proliferation by inducing cell cycle arrest in G0/G1 phase through regulation of cell cycle-related genes, such as p21Waf1/Cip1, cyclin D1 and cyclin-dependent kinase 4 (CDK4), and the antiproliferative effect of STIM1 silencing was also observed in U251 glioma xenograft tumor model.ConclusionOur findings confirm STIM1 as a rational therapeutic target in human glioblastoma, and also indicate that lentivirus-mediated STIM1 silencing is a promising therapeutic strategy for human glioblastoma.

Transplantation of neural stem cells modified by human neurotrophin-3 promotes functional recovery after transient focal cerebral ischemia in rats.

The study tested the hypothesis that transplantation of human neurotrophin-3 (hNT-3) over-expressing neural stem cells (NSCs) into rat striatum after a severe focal ischemia would promote functional recovery. Rat NSCs, transduced by Flag-tagged hNT-3 gene mediated by lentiviral vector (LV), were transplanted into the striatum ipsilateral to the injury of adult rats 7 days after 2-h occlusion of the middle cerebral artery (MCAO). From 3 days to 2 weeks after transplantation, the modified cells (NSCs-hNT3, as defined by Flag immunofluorencence staining) that survived the transplantation procedures could secrete significantly higher levels of neurotrophin-3 protein in the graft sites than controls (P<0.001). Furthermore, the rats that accepted NSCs-hNT3 exhibited enhanced functional recovery on neurological and behavioral tests, compared with controlled animals transplanted with saline or untransduced NSCs. This study suggests: (1) LV is an ideal vector to transduce foreign gene into the NSCs; (2) modified NSCs could carry therapeutic genes to disease tissues and express effectively; (3) modified cells could survive in the ischemic brains and continue to secrete neurotrophin-3 abundantly for over 2 weeks, which might have values for enhancing functional recovery after stroke.

Neuronal regeneration and protection by collagen-binding BDNF in the rat middle cerebral artery occlusion model

It has been well confirmed that brain-derived neurotrophic factor (BDNF) has therapeutic effects following stroke. However, it is difficult to be maintained at a sufficient concentration of BDNF in the infarcted hemisphere. We have shown in our previous work that BDNF fused with a collagen-binding domain (CBD-BDNF) could specifically bind to collagen. The ventricular ependyma of the brain is rich in collagen. Therefore, we have speculated that in the infarcted hemisphere, CBD-BDNF will bind to the collagen of the ventricular ependyma and stimulate the cell proliferation in the subventricular zone (SVZ). Using a rat middle cerebral artery occlusion model (MCAO), we injected CBD-BDNF into the lateral ventricle of MCAO rats. The results demonstrated that CBD-BDNF was retained at high levels in the infarcted hemisphere, promoted neural regeneration and angiogenesis, reduced cell loss, decreased apoptosis, and improved functional recovery. In addition, brain perfusion and metabolism, as evaluated by SPECT and PET, were improved in the CBD-BDNF treated group.

A Comparative Study of Transfection Efficiency between Liposomes, Immunoliposomes and Brain-specific Immunoliposomes

This study investigated the transfection ability and efficiency of liposomes and immunoliposomes for exogenous gene delivery into the brain via the venous system. Four groups of rats underwent tail vein injection with one of the following: liposomes encapsulating pCMV (human cytomegalovirus promoter)-LacZ plasmid 80 μg (low dose) or 300 μg (high dose); general immunoliposomes encapsulating 80 μg transferrin receptor antibodies (OX26)-pCMV-LacZ plasmid; or brain-specific immunoliposomes encapsulating 80 μg OX26-pGFAP (glial fibrillary acidic protein promoter)-LacZ plasmid. A control group received no injected agent. The LacZ mRNA levels (1 h post-injection) and β-galactosidase activity (48 h post-injection) in the brain and peripheral organs were assayed using real-time reverse transcription–polymerase chain reaction and histochemical staining, respectively. Both immunoliposomes delivered exogenous DNA containing the LacZ gene into the brain after venous injection, resulting in extensive LacZ expression in the brain. Furthermore, the brain-specific OX26-pGFAP-LacZ immunoliposome decreased the non-specific expression of LacZ in peripheral organs without affecting transfection efficiency in the brain. Thus, brain-specific immunoliposomes are an efficient and brain-specific targeting vector.

Screening for AIP gene mutations in a Han Chinese pituitary adenoma cohort followed by LOH analysis

OBJECTIVE The aryl hydrocarbon receptor interacting protein gene (AIP) is associated with pituitary adenoma (PA). AIP has not been sequenced in East Asian PA populations, so we performed this study in a Han Chinese cohort. DESIGN Our study included six familial PA pedigrees comprising 16 patients and 27 unaffected relatives, as well as 216 sporadic PA (SPA) patients and 100 unrelated healthy controls. METHODS AIP sequencing was carried out on genomic DNA isolated from blood samples. Multiplex ligation-dependent probe amplification and microsatellite marker analyses on DNA from the paired tumor tissues were performed for loss of heterozygosity analysis. RESULTS We identified three common and four rare single nucleotide polymorphisms (SNPs), one intron insertion, one novel synonymous variant, four novel missense variants, and a reported nonsense mutation in three familial isolated PA (FIPA) cases from the same family. Large genetic deletions were not observed in the germline but were seen in the sporadic tumor DNA from three missense variant carriers. The prevalence of AIP pathogenic variants in PA patients here was low (3.88%), but was higher in somatotropinoma patients (9.30%), especially in young adults (≤30 years) and pediatric (≥18 years) paients (17.24% and 25.00% respectively). All AIP variant patients suffered from macroadenomas. However, the AIP mutation rate in FIPA families was low in this cohort (16.67%, 1/6 families). CONCLUSION AIP gene mutation may not be frequent in FIPA or SPA from the Han Chinese population. AIP sequencing and long-term follow-up investigations should be performed for young patients with large PAs and their families with PA predisposition.