Human umbilical cord blood cells induce neuroprotective change in gene expression profile in neurons after ischemia through activation of Akt pathway

Abstract

Human umbilical cord blood (HUCB) cell therapies have shown promising results in reducing brain infarct volume and most importantly in improving neurobehavioral function in rat permanent middle cerebral artery occlusion, a model of stroke. In this study, we examined the gene expression profile in neurons subjected to oxygen-glucose deprivation (OGD) with or without HUCB treatment and identified signaling pathways (Akt/MAPK) important in eliciting HUCB-mediated neuroprotective responses. Gene chip microarray analysis was performed using RNA samples extracted from the neuronal cell cultures from four experimental groups: normoxia, normoxia+HUCB, OGD, and OGD+HUCB. Both quantitative RT-PCR and immunohistochemistry were carried out to verify the microarray results. Using the Genomatix software program, promoter regions of selected genes were compared to reveal common transcription factor-binding sites and, subsequently, signal transduction pathways. Under OGD condition, HUCB cells significantly reduced neuronal loss from 68% to 44% [one-way ANOVA, F(3, 16)=11, p=0.0003]. Microarray analysis identified mRNA expression of Prdx5, Vcam1, CCL20, Alcam, and Pax6 as being significantly altered by HUCB cell treatment. Inhibition of the Akt pathway significantly abolished the neuroprotective effect of HUCB cells [one-way ANOVA, F(3, 11)=8.663, p=0.0031]. Our observations show that HUCB neuroprotection is dependent on the activation of the Akt signaling pathway that increases transcription of the Prdx5 gene. We concluded that HUCB cell therapy would be a promising treatment for stroke and other forms of brain injury by modifying acute gene expression to promote neural cell protection.

Figure 1

HUCB cells rescued neurons from OGD. There was no significant difference in cell viability determined using the FDA-PI assay in cultures exposed to normoxia in the absence (A) or presence of HUCB cells (B). There were more dead cells (red, PI) in the culture exposed to OGD in the absence of HUCB cells (C). Conversely, there were more viable cells (green, FDA) in the culture exposed to OGD treated with HUCB cells (D). (E) The percent survival of neurons exposed to either normoxia or OGD conditions for 20 h assessed by FDA-PI staining (***p < 0.0001, OGD vs. normoxic conditions; *p < 0.05, OGD vs. OGD + HUCB). Scale bar: 100 μM.

Figure 2

HUCB cell-mediated neuroprotection depends on activation of the Akt signaling pathway. Significant neuronal loss was observed in neurons exposed to OGD (A). HUCB cell treatment rescued neuronal loss (B), while HUCB cells lose their neuroprotective effects after adding Akt inhibitor (Akti; Akt inhibitor IV) into the culture media (C). Addition of mitogen-activated protein kinase (MAPK) inhibitor (MAPKi; MAPK inhibitor U0126) failed to influence HUCB cell-mediated neuronal protection (D). When the percent survival of neurons under OGD conditions was calculated (E), Akt and MAPK inhibitors did not alter survival compared to OGD alone, but HUCB cells significantly increased survival. Akt inhibitor added to neuronal–HUCB cell cocultures significantly decreased cell survival. In normoxic cultures (F), the Akt inhibitor decreased cell survival in neuronal cultures with and without HUCB cells. *p < 0.05; **p < 0.01; ***p < 0.001. Scale bar: 100 μm.

Figure 3

The effects of HUCB cell treatment on gene expression in neurons. Under normoxic conditions, HUCB cells significantly upregulated gene expression of (A) peroxiredoxin 5 (Prdx5; ***p < 0.001), (B) vascular cell adhesion molecule 1 (Vcam1; *p < 0.05), and (C) chemokine C-C motif ligand 20 (CCL20; **p < 0.01) compared to normoxic cultures alone. Under OGD conditions, HUCB cells appeared to downregulate gene expression of (E) activated leukocyte cell adhesion molecule (Alcam1; #p < 0.05) and (F) paired box 6 (Pax6; ##p < 0.01) compared to OGD alone. There was a tendency to decrease expression of chemokine C-X-C motif ligand 1 (CXCL1) (D), but it did not reach significance. The results are expressed as mean fold change ± SEM.

Figure 4

Gene expression changes in the ipsilateral brain hemisphere from rats treated with HUCB cells 48 h after MCAO. HUCB cells induced Prdx5 protein (B, C) and reduced Cxcl1 (E, F) expression in the rat ipsilateral hemisphere compared to nontreated animals (A, D). Arrows indicate representative examples of positively labeled cells. Bar graphs show quantitative evaluation of Prdx5 (C) and Cxcl1 (F) expression. The results were expressed as mean number of positive cells ± SEM. *p < 0.05 compared with control group. Scale bar: 50 μm.

Figure 5

The Akt pathway plays a significant role in HUCB-mediated neuronal survival. In vitro expression of Prdx5 (abbreviated to Prx5 in figure) in neurons following OGD (A), OGD +HUCB (B), OGD + HUCB +Akti (C), and OGD + HUCB + MAPKi (D). There are significantly more Prdx5-positive cells in neurons following OGD alone and cocultured with HUCB, while inhibition of Akt negates the effects of HUCB [one-way ANOVA, F(3, 11) = 6.4, p = 0.0163]. Graphical representation of the number of Prdx5-positive neurons after HUCB treatment subsequent to OGD (E). Data are presented as mean Prdx5-positive cells ± SEM. *p < 0.5 OGD versus OGD + HUCB, #p < 0.05 OGD + HUCB versus OGD + HUCB +Akti. Confocal images showing Prdx5 colocalized with Akt (F–H). Scale bar: 50 μm (A–D); scale bar: 20 μm (F–H).