Rho-Kinase Inhibitor, Fasudil, Prevents Neuronal Apoptosis via the Akt Activation and PTEN Inactivation in the Ischemic Penumbra of Rat Brain

Recently, some studies suggested that inhibition of Rho-kinase (ROCK) prevented cerebral ischemia injury through inhibiting inflammatory reaction, increasing cerebral blood flow, modulating the neuronal actin cytoskeleton polymerization, and preventing tau hyperphosphorylation and p25/CDK5 increase. However, there is little information regarding the effects of ROCK inhibitor on the neuronal apoptosis in ischemic brain injury. In this study, we determined whether ROCK inhibitor, fasudil, inhibited ischemic neuronal apoptosis through phosphatase and tensin homolog deleted on chromosome10 (PTEN)/Akt/signal pathway in vivo. Adult male Sprague-Dawley rats were subjected to permanent middle cerebral artery occlusion. Rats received ROCK inhibitor, fasudil (10?mg/kg), at 30?min before middle cerebral artery occlusion. The infarct area, neuronal apoptosis and caspase-3 activity was significantly decreased by fasudil with improvement of neurological deterioration. However, the beneficial effects of fasudil were attenuated by the co-application of LY294002 (PI3K inhibitor). Fasudil maintained postischemic Akt activity at relatively proper level and decreased the augmentation of PTEN and ROCK activity in the penumbra area. Furthermore, fasudil inhibited attenuation of GSK-β and Bad phosphorylation in the penumbra area. In conclusion, the findings provide another consideration that fasudil protects the brain against ischemia injury through decreasing neuronal apoptosis and reveals the link between the ROCK inhibition and the PTEN/Akt pathway.     

Human umbilical cord blood-derived cells differentiate into hepatocyte-like cells in the Fas-mediated liver injury model

Human umbilical cord blood (HUCB) contains stem/progenitor cells, which can differentiate into a variety of cell types. In this study, we investigated whether HUCB cells differentiate into hepatocytes in vitro and in vivo. We also examined whether CD34 could be the selection marker of stem cells for hepatocytes. HUCB cells were obtained from normal full-term deliveries, and CD34(+/-) cells were further separated. For in vitro study, HUCB cells were cultured for 4 wk, and expressions of liver-specific genes were examined. For the in vivo study, nonobese diabetic/severe combined immunodeficient mice were subjected to liver injury by a Fas ligand-carried adenoviral vector or only radiated. Mice were treated simultaneously with or without cell transplantation of HUCB, CD34(+), or CD34(-) cells. After 4 wk, human-specific gene/protein expression was examined. In the in vitro study, human liver-specific genes were positive after 7 days of culture. The immunofluorescent study showed positive staining of alpha-fetoprotein, cytokeratin 19, and albumin in round-shaped cells. In the in vivo study, immunohistochemical analysis showed human albumin-positive, hepatocyte-specific antigen-positive cells in mouse livers of the Fas ligand/transplantation group. Fluorescence in situ hybridization analysis using the human Y chromosome also showed positive signals. However, no difference between transplanted cell types was detected. In contrast, immunopositive cells were not detected in the irradiated/transplantation group. The RT-PCR result also showed human hepatocyte-specific gene expressions only in the Fas ligand/transplantation group. HUCB cells differentiated into hepatocyte-like cells in the mouse liver, and liver injury was essential during this process. The differences between CD34(+) and CD34(-) cells were not observed in human hepatocyte-specific expression.     

Differentiation of cryopreserved human umbilical cord blood-derived stromal cells into cells with an oligodendrocyte phenotype

In this study, we examined the phenotypic characteristics of human umbilical cord blood-derived mesenchymal stromal cells (UCB-derived MSCs) differentiated along an oligodendrocyte pathway. We induced human UCB-derived MSCs to form floating neurospheres, and these neurospheres were then induced to differentiate into oligodendrocyte progenitor-like cells using multiple induction factors. Differentiated UCB-derived MSCs showed morphologic characteristics of an oligodendrocyte phenotype. The expression of cell surface markers characteristic of oligodendrocyte progenitor cells or oligodendrocytes was determined by immunocytochemical staining. These results suggest that human UCB-derived MSCs can be induced to differentiate into cells with an oligodendrocyte phenotype and that these cells may have potential in the future cellular therapy of central neurological disorders.     

Transplantation of human umbilical cord blood cells improves glycemia and glomerular hypertrophy in type 2 diabetic mice

Recent in vitro and in vivo studies have shown that either animal- or human-derived embryonic stem cells can differentiate into insulin-secreting cells and lower blood glucose levels. However, studies utilizing human umbilical cord blood (HUCB) mononuclear cells to improve blood glucose levels in diabetic animals have received little attention. In this study, we examined the effect of transplanted HUCB mononuclear cells on blood glucose levels, survival, and renal pathology in obese mice with spontaneous development of type 2 diabetes. The results show that injection of HUCB mononuclear cells into orbital plexus of mice caused improvement not only in blood glucose levels and survival rate but also normalization of glomerular hypertrophy and tubular dilatation. Thus, transplantation of HUCB mononuclear cells appears to be another modality of stem cell therapy in diabetes mellitus.     

PI3K/Akt 通路在电针预处理诱导脑缺血耐受中的机制研究

目的:通过观察电针预处理对磷脂酰肌醇3激酶/蛋白质丝氨酸苏氨酸激酶(PI3K/Akt)通路的变化以及该通路抑制剂对电针预处理的脑保护的影响,探讨电针预处理诱导脑缺血耐受的可能机制。方法:线栓法单侧阻断大脑中动脉120min,再灌注24h制备大鼠大脑局灶性缺血再灌注(I/R)模型;Western Blot检测Akt磷酸化水平的变化;侧脑室注射PI3K/Akt通路抑制剂LY294002;神经行为学评分(Garcia标准)及TTC染色检测脑梗死体积比评价脑损伤程度。结果:电针预处理使大鼠神经行为学评分增高,脑梗死体积比降低(P<0.05);可上调Akt磷酸化水平,I/R2h达高峰(P<0.05)。侧脑室注射PI3K/Akt抑制剂LY294002,拮抗电针预处理的脑保护作用(P<0.05)。结论:电针预处理增加Ak(tSer473)磷酸化水平,在缺血再灌注早期上调PI3K/Akt通路可能是诱导大鼠脑缺血耐受的产生的主要机制。     

Hydroxysafflor Yellow A Protects Against Cerebral Ischemia–Reperfusion Injury by Anti-apoptotic Effect Through PI3K/Akt/GSK3β Pathway in Rat

Hydroxysafflor yellow A (HSYA) is the major active chemical component of the flower of the safflower plant, Carthamus tinctorius L. Previously, its neuroprotection against cerebral ischemia–reperfusion (I/R) injury was reported by anti-oxidant action and suppression of thrombin generation. Here, we investigate the role of HSYA in cerebral I/R-mediated apoptosis and possible signaling pathways. Male Wistar rats were subjected to transient middle cerebral artery occlusion for 2 h, followed by 24 h reperfusion. HSYA was administered via tail-vein injection just 15 min after occlusion. The number of apoptotic cells was measured by TUNEL assay, apoptosis-related proteins Bcl-2, Bax and the phosphorylation levels of Akt and GSK3β in ischemic penumbra were assayed by western blot. The results showed that administration of HSYA at the doses of 4 and 8 mg/kg significantly inhibited the apoptosis by decreasing the number of apoptotic cells and increasing the Bcl-2/Bax ratio in rats subjected to I/R injury. Simultaneously, HSYA treatment markedly increased the phosphorylations of Akt and GSK3β. Blockade of PI3K activity by wortmannin dramatically abolished its anti-apoptotic effect and lowered both Akt and GSK3β phosphorylation levels. Taken together, these results suggest that HSYA protects against cerebral I/R injury partly by reducing apoptosis via PI3K/Akt/GSK3β signaling pathway.     

Anti-inflammatory Effect of Mesenchymal Stromal Cell Transplantation and Quercetin Treatment in a Rat Model of Experimental Cerebral Ischemia

Here, we have investigated the synergistic effect of quercetin administration and transplantation of human umbilical cord mesenchymal stromal cells (HUMSCs) following middle cerebral artery occlusion in rat. Combining quercetin treatment with delayed transplantation of HUMSCs after local cerebral ischemia significantly (i) improved neurological functional recovery; (ii) reduced proinflammatory cytokines (interleukin(IL)-1β and IL-6), increased anti-inflammatory cytokines (IL-4, IL-10, and transforming growth factor-β1), and reduced ED-1 positive areas; (iii) inhibited cell apoptosis (caspase-3 expression); and (iv) improved the survival rate of HUMSCs in the injury site. Altogether, our results demonstrate that combined HUMSC transplantation and quercetin treatment is a potential strategy for reducing secondary damage and promoting functional recovery following cerebral ischemia.     

Murine survival of lethal irradiation with the use of human umbilical cord blood.

We have found that human umbilical cord blood (HUCB) will routinely protect mice exposed to lethal levels of irradiation. At the end of 50 days, over seventy percent (70%) of mice injected with HUCB survived 900 cGy or irradiation, which produced 100% deaths in the uninjected control animals. Moreover, there was some evidence that human colony stimulating factors further improved survival. Anti-Natural Killer cell (NK) antibody was utilized along with HUCB in these studies, however, Anti-NK cell serum alone had no radioprotective effect in mice. The studies reported here suggest the possibility of utilizing HUCB for immediate protection of humans from lethal irradiation.     

Fat-derived factor omentin stimulates endothelial cell function and ischemia-induced revascularization via endothelial nitric oxide synthase-dependent mechanism

Obesity-related diseases are associated with vascular dysfunction and impaired revascularization. Omentin is a fat-derived secreted protein, which is down-regulated in association with obese complications. Here, we investigated whether omentin modulates endothelial cell function and revascularization processes in vitro and in vivo. Systemic delivery of an adenoviral vector expressing omentin (Ad-omentin) enhanced blood flow recovery and capillary density in ischemic limbs of wild-type mice in vivo, which were accompanied by increased phosphorylation of Akt and endothelial nitric oxide synthase (eNOS). In cultured human umbilical vein endothelial cells (HUVECs), a physiological concentration of recombinant omentin protein increased differentiation into vascular-like structures and decreased apoptotic activity under conditions of serum starvation. Treatment with omentin protein stimulated the phosphorylation of Akt and eNOS in HUVECs. Inhibition of Akt signaling by treatment with dominant-negative Akt or LY294002 blocked the stimulatory effects of omentin on differentiation and survival of HUVECs and reversed omentin-stimulated eNOS phosphorylation. Pretreatment with the NOS inhibitor also reduced the omentin-induced increase in HUVEC differentiation and survival. Omentin protein also stimulated the phosphorylation of AMP-activated protein kinase in HUVECs. Transduction with dominant-negative AMP-activated protein kinase diminished omentin-induced phosphorylation of Akt and omentin-stimulated increase in HUVEC differentiation and survival. Of importance, in contrast to wild-type mice, systemic administration of Ad-omentin did not affect blood flow in ischemic muscle in eNOS-deficient mice in vivo. These data indicate that omentin promotes endothelial cell function and revascularization in response to ischemia through its ability to stimulate an Akt-eNOS signaling pathway.     

Stem cells from umbilical cord blood differentiate into myotubes and express dystrophin in vitro only after exposure to in vivo muscle environment

BACKGROUND INFORMATION: Duchenne muscular dystrophy is a disease characterized by progressive and irreversible muscle degeneration for which there is no therapy. HUCB (human umbilical cord blood) has been considered as an important source of haematopoietic and mesenchymal stem cells, each having been shown to differentiate into distinct cell types. However, it remains unclear if these cells are able to differentiate into muscle cells. RESULTS: We have showed that stem cells from HUCB did not differentiate into myotubes or express dystrophin when cultured in muscle-conditioned medium or with human muscle cells. However, delivery of GFP (green fluorescent protein)-transduced mononucleated cells from HUCB, which comprises both haematopoietic and mesenchymal populations, into quadriceps muscle of mdx (mouse dystrophy X-chromosome linked) mice resulted in the expression of human myogenic markers. After recovery of these cells from mdx muscle and in vitro cultivation, they were able to fuse and form GFP-positive myotubes that expressed dystrophin. CONCLUSIONS: These results indicate that chemical factors and cell-to-cell contact provided by in vitro conditions were not enough to trigger the differentiation of stem cells into muscle cells. Nevertheless, we showed that the HUCB-derived stem cells were capable of acquiring a muscle phenotype after exposure to an in vivo muscle environment, which was required to activate the differentiation programme.     

Neural stem cells: plasticity and therapeutic potential

Previous studies have suggested that human umbilical cord blood (HUCB) may serve as a rich source of hematopoietic and nonhematopoietic stem cells and that conditions exist that can coax hematopoietic cells to express neural characteristics. In our laboratory, these cells were tested for several models of neurodegenerative diseases and spinal cord injuries. Through a series of transplantation studies we have begun to uncover the properties of HUCB‐derived cells in neuropoietic regions of the neonatal (1) and aging rodent brain. The systematic application of phenotyping approaches to characterize survival, migratory potential and morphologic properties of the differentiated HUCB progeny within normal/unaffected brain will serve as a base for understanding the potential effect of these cells in the diseased brain. Acknowledgements: Supported in part by R01 6155039 (TZ). HUCB cells were obtained from Saneron CCEL Therapeutics, Inc. and BioWhittaker, Inc.     

Enhancement of ischemia-induced tyrosine phosphorylation of Kv1.2 by vascular endothelial growth factor via activation of phosphatidylinositol 3-kinase

Our studies observed that, consistent with the literature, ischemic/hypoxic insults increased the expression of voltage-gated potassium channel (Kv) 1.2 potassium channel as well as elevating the endogenous level of vascular endothelial growth factor (VEGF) in neurons of adult rat brain following middle cerebral artery occlusion and in SH-SY5Y cells after hypoxia and glucose deprivation. Concomitantly, we also observed that ischemic injury increased the tyrosine phosphorylation of Kv 1.2 in in vivo and in vitro; the introduction of exogenous VEGF could attenuate cell death in in vitro models. Furthermore, we found that the protective effect of VEGF is mediated through its up-regulative actions on the tyrosine phosphorylation of Kv 1.2, which in turn has a direct influence on cell viability after ischemic insult. In substantiation of this result, we used anti-sense methodology to suppress the expression of endogenous VEGF, which significantly inhibited the tyrosine phosphorylation of Kv 1.2 and increased cell death elicited by ischemic/hypoxic injury. Finally, the enhancement of the tyrosine phosphorylation of the channel by VEGF in neuronal cells was significantly attenuated in the presence of wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-K), or genestin, an inhibitor of tyrosine kinase, thus suggesting that the phosphorylation of Kv 1.2 induced by VEGF is mechanistically linked to the PI3-K pathway.     

Grafting dopamine neurons in Parkinson's disease: do stem cells have a role in the future?

Previous studies have suggested that human umbilical cord blood (HUCB) may serve as a rich source of hematopoietic and nonhematopoietic stem cells and that conditions exist that can coax hematopoietic cells to express neural characteristics. In our laboratory, these cells were tested for several models of neurodegenerative diseases and spinal cord injuries. Through a series of transplantation studies we have begun to uncover the properties of HUCB-derived cells in neuropoietic regions of the neonatal (1) and aging rodent brain. The systematic application of phenotyping approaches to characterize survival, migratory potential and morphologic properties of the differentiated HUCB progeny within normal/unaffected brain will serve as a base for understanding the potential effect of these cells in the diseased brain.
Acknowledgements:  Supported in part by R01 6155039 (TZ). HUCB cells were obtained from Saneron CCEL Therapeutics, Inc. and BioWhittaker, Inc.     

Human umbilical cord blood cells transfected with VEGF and L(1)CAM do not differentiate into neurons but transform into vascular endothelial cells and secrete neuro-trophic factors to support neuro-genesis-a novel approach in stem cell therapy

Genetically modified mono-nuclear cell fraction from human umbilical cord blood (HUCB) expressing human vascular endothelial growth factor (VEGF) and mouse neural L₁ cell adhesion molecule (L₁CAM) were used for gene-stem cell therapy of transgenic ^G93^A mice adopted as an animal amyotrophic lateral sclerosis (ALS) model. We generated non-viral plasmid constructs, expressing human VEGF₁₆₅ (pcDNA-VEGF) and mouse neural L₁ cell adhesion molecule (pcDNA-mL₁CAM). Mono-nuclear fraction of HUCB cells were transiently transfected by electro-poration with a mixture of expression plasmids (pcDNA-VEGF + pcDNA-mL₁CAM). Sixteen transgenic female and male mice were randomly assigned to three groups: (1) transplantation of genetically modified HUCB cells expressing L₁ and VEGF (n = 6), (2) transplantation of un-transfected HUCB cells (n = 5), and (3) control group (n = 5). In first two experimental groups 1 × 10⁶ cells were injected retro-orbitally in pre-symptomatic 22–25-week-old ^G93^A mice. Our results demonstrate that HUCB cells successfully grafted into nervous tissue of ALS mice and survived for over 3 months. Therefore, genetically modified HUCB cells migrate in the spinal cord parenchyma, proliferate, but instead of transforming into nerve cells, they differentiate into endothelial cells forming new blood vessels. We propose that: (A) expression of mouse neural L₁CAM is responsible for increased homing and subsequent proliferation of transplanted cells at the site of neuro-degeneration, (B) expression of human VEGF directs HUCB cell differentiation into endothelial cells, and (C) neuro-protective effect may stem from the delivery of various neuro-trophic factors from newly formed blood vessels.     

Adiponectin stimulates angiogenesis by promoting cross-talk between AMP-activated protein kinase and Akt signaling in endothelial cells

Adiponectin is an adipocyte-specific adipocytokine with anti-atherogenic and anti-diabetic properties. Here, we investigated whether adiponectin regulates angiogenic processes in vitro and in vivo. Adiponectin stimulated the differentiation of human umbilical vein endothelium cells (HUVECs) into capillary-like structures in vitro and functioned as a chemoattractant in migration assays. Adiponectin promoted the phosphorylation of AMP-activated protein kinase (AMPK), protein kinase Akt/protein kinase B, and endothelial nitric oxide synthesis (eNOS) in HUVECs. Transduction with either dominant-negative AMPK or dominant-negative Akt abolished adiponectin-induced eNOS phosphorylation as well as adiponectin-stimulated HUVEC migration and differentiation. Dominant-negative AMPK also inhibited adiponectin-induced Akt phosphorylation, suggesting that AMPK is upstream of Akt. Dominant-negative Akt or the phosphatidylinositol 3-kinase inhibitor LY294002 blocked adiponectin-stimulated Akt and eNOS phosphorylation, migration, and differentiation without altering AMPK phosphorylation. Finally, adiponectin stimulated blood vessel growth in vivo in mouse Matrigel plug implantation and rabbit corneal models of angiogenesis. These data indicate that adiponectin can function to stimulate the new blood vessel growth by promoting cross-talk between AMP-activated protein kinase and Akt signaling within endothelial cells.     

New approach to isolate mesenchymal stem cell (MSC) from human umbilical cord blood

HUCB (human umbilical cord blood) has been frequently used in clinical allogeneic HSC (haemopoietic stem cell) transplant. However, HUCB is poorly recognized as a rich source of MSC (mesenchymal stem cell). The aim of this study has been to establish a new method for isolating large number of MSC from HUCB to recognize it as a good source of MSC. HUCB samples were collected from women following their elective caesarean section. The new method (Clot Spot method) was carried out by explanting HUCB samples in mesencult complete medium and maintained in 37°C, in a 5% CO2 and air incubator. MSC presence was established by quantitative and qualitative immunophenotyping of cells and using FITC attached to MSC phenotypic markers (CD29, CD73, CD44 and CD105). Haematopoietic antibodies (CD34 and CD45) were used as negative control. MSC differentiation was examined in neurogenic and adipogenic media. Immunocytochemistry was carried out for the embryonic markers: SOX2 (sex determining region Y-box 2), OLIG-4 (oligodendrocyte-4) and FABP-4 (fatty acid binding protein-4). The new method was compared with the conventional Rosset Sep method. MSC cultures using the Clot Spot method showed 3-fold increase in proliferation rate compared with conventional method. Also, the cells showed high expression of MSC markers CD29, CD73, CD44 and CD105, but lacked the expression of specific HSC markers (CD34 and CD45). The isolated MSC showed some differentiation by expressing the neurogenic (SOX2 and Olig4) and adipogenic (FABP-4) markers respectively. In conclusion, HUCB is a good source of MSC using this new technique.     

Mild Sensory Stimulation Completely Protects the Adult Rodent Cortex from Ischemic Stroke

Despite progress in reducing ischemic stroke damage, complete protection remains elusive. Here we demonstrate that, after permanent occlusion of a major cortical artery (middle cerebral artery; MCA), single whisker stimulation can induce complete protection of the adult rat cortex, but only if administered within a critical time window. Animals that receive early treatment are histologically and behaviorally equivalent to healthy controls and have normal neuronal function. Protection of the cortex clearly requires reperfusion to the ischemic area despite permanent occlusion. Using blood flow imaging and other techniques we found evidence of reversed blood flow into MCA branches from an alternate arterial source via collateral vessels (inter-arterial connections), a potential mechanism for reperfusion. These findings suggest that the cortex is capable of extensive blood flow reorganization and more importantly that mild sensory stimulation can provide complete protection from impending stroke given early intervention. Such non-invasive, non-pharmacological intervention has clear translational potential.