Inhibition of GSK-3 reduces infarct volume and improves neurobehavioral functions.

In the present study, we have investigated the effects of glycogen synthase kinase-3 (GSK-3) inhibition on infarct volume and neurobehavioral functions in a focal cerebral ischemia model. To achieve our goals, GSK-3 inhibitor II or VIII was injected at several time points and in varing dosages. GSK-3 inhibitor VIII was more effective than inhibitor II, and infarct volume and water content in the VIII group were significantly decreased 24h after the onset of ischemic stroke, as compared with the control group. These protective effects were associated with reductions of TUNEL-positive cells, neutrophil infiltration, glucose levels after ischemia, and GSK-3 enzyme activity. In addition, expressions of death and inflammation-related signals decreased and those of survival-related signals increased. Lastly, neurobehavioral functions were restored to a greater extent in the VIII group than in the control group. Together, these results suggest that GSK-3 inhibition reduces infarct volume and restores neurobehavioral functions.

ß-PIX is critical for transplanted mesenchymal stromal cell migration.

Bone marrow-derived mesenchymal stromal cells (MSCs) have been used successfully as a source of stem cells for treating neurodegenerative diseases. However, for reasons that are not clear, autologous MSC transplants have not yielded successful results in human trials. To test one possible reason, we compared the migratory ability of MSCs from amyotrophic lateral sclerosis (ALS) patients with those of healthy controls. We found that MSCs derived from ALS patients (ALS-MSCs) had a reduced ability to migrate, which may explain why autologous transplantation is not successful. We also found that expression of one of the intracellular factors implicated in migration, ß-PIX, was significantly reduced in ALS-MSCs compared with healthy stem cells. Restoration of ß-PIX expression by genetic manipulation restored the migratory ability of ALS-MSCs, and inhibition of ß-PIX expression with shRNA reduced the migration of healthy MSCs. We suggest that transplantation of allogeneic or genetically modified autologous stem cells might be a more promising strategy for ALS patients than transplantation of autologous stem cells.

Ambidextrous magnetic nanovectors for synchronous gene transfection and labeling of human MSCs.

The synchronization of gene expression and cell trafficking in transfected stem cells is crucial for augmentation of stem cell functions (differentiation and neurotropic factor secretion) and real time in vivo monitoring. We report a magnetic nanoparticle-based gene delivery system that can ensure simultaneous gene delivery and in vivo cell trafficking by high resolution MR imaging. The polar aprotic solvent soluble MnFe2O4 nanoparticles were enveloped using cationic polymers (branched polyethyleneimine, PEI) by the solvent shifting method for a gene loading. Using our magnetic nanovector system (PEI-coated MnFe2O4 nanoparticles), thus, we synchronized stem cell migration and its gene expression in a rat stroke model.

Inhibitory effects of cilostazol on proliferation of vascular smooth muscle cells (VSMCs) through suppression of the ERK1/2 pathway.

AIM: The abnormal proliferation of vascular smooth muscle cells (VSMCs) in arterial walls is an important pathogenic factor of vascular disorders such as atherosclerosis and restenosis after angioplasty. During atherogenesis or in response to vessel injury, VSMC proliferation is induced by a number of peptide growth factors released from platelets and VSMCs. Cilostazol is a phosphodiesterase (PDE) 3 inhibitor that increases intracellular cAMP levels and decreases intracellular Ca(2+) levels, inhibiting platelet aggregation and inducing vasodilatation. Cilostazol is also known to have an inhibitory effect on the proliferation of VSMCs, but the anti-proliferative mechanism of cilostazol in VSMCs has not yet been established. In the present study, we investigated whether the anti-proliferative mechanism of cilostazol is associated with the suppression of extracellular signal-regulated kinases (ERK) and phosphatidylinositol 3 kinase (PI3K) signaling pathways. METHODS: To confirm the anti-proliferative effects of cilostazol on VSMCs, VSMCs were induced to proliferate by serum-induced mitogenesis and then were treated with cilostazol for 24 h. And, to investigate whether the anti-proliferative mechanism of cilostazol in VSMCs involves the suppression of the ERK and PI3K pathways, expression of the phosphorylated forms of ERK1/2, Raf, Akt, and glycogen synthase kinase (GSK)-3 were evaluated by western blot. RESULTS: Cilostazol inhibited VSMC proliferation in a dose-dependent manner. Phosphorylated ERK1/2 and Raf were significantly reduced in a dose-dependent manner, whereas phosphorylated Akt and GSK-3 were not changed. CONCLUSION: These results suggest that suppression of the ERK pathway but not the PI3K pathway is an important mechanism in the anti-proliferative effect of cilostazol on VSMCs.

The neuroprotective effect of erythropoietin-transduced human mesenchymal stromal cells in an animal model of ischemic stroke.

Erythropoietin (EPO) exhibits diverse cellular functions, including neurotrophic, anti-oxidant, anti-apoptotic, and anti-inflammatory effects in non-hematopoietic tissues. This study evaluated whether bone marrow mesenchymal stromal cells (MSCs) transduced with the EPO gene (EPO-MSCs) promoted neural cell survival and improved neurological deficits caused by ischemic stroke. EPO-MSCs stably produced high levels of EPO (10IU/ml) without any alteration of their mesenchymal phenotype. Both EPO transduction and treatment with 10 international units (IU) of recombinant human EPO (rhEPO) provided protection from H(2)O(2)-induced oxidative injury in human bone marrow mesenchymal stromal cells and in SH-SY5Y cells. EPO-MSCs were more protected than were MSCs treated with 10IU rhEPO (10U-MSCs). We also found that the expression of the neurotrophic factors BDNF, PD-ECGF, HGF, SDF-1alpha, and TGF-1beta increased in EPO-MSCs, while only BDNF and TGF-1beta increased in 10U-MSCs. Implantation of EPO-MSCs in an animal model of ischemic stroke significantly improved neurological function and decreased infarct volumes without affecting hematocrit level. An evaluation of the brain tissue 21days after implantation showed that EPO and phosphorylated Akt (a downstream mediator of EPO) increased only in brains implanted with EPO-MSCs. Transduction of the EPO gene into MSCs induced secretion of EPO and various trophic factors that may provide excellent neuroprotective effects in both in vitro and in vivo models of ischemic stroke.