Tissue Plasminogen Activator and Plasminogen Activator Inhibitor 1 Contribute to Sonic Hedgehog-Induced In Vitro Cerebral Angiogenesis

The molecular mechanisms underlying cerebral angiogenesis have not been fully investigated. Using primary mouse brain endothelial cells (MBECs) and a capillary-like tube formation assay, we investigated whether the sonic hedgehog (Shh) signaling pathway is coupled with the plasminogen/plasmin system in mediating cerebral angiogenesis. We found that incubation of MBECs with recombinant human Shh (rhShh) substantially increased the tube formation in naïve MBECs. This was associated with increases in tissue plasminogen activator (tPA) activation and reduction of plasminogen activator inhibitor 1 (PAI-1). Blockage of the Shh pathway with cyclopamine abolished the induction of tube formation and the effect of rhShh on tPA and PAI-1. Addition of PAI-1 reduced rhShh-augmented tube formation. Genetic ablation of tPA in MBECs impaired tube formation and downregulated of vascular endothelial growth factor (VEGF) and angiopoietin 1 (Ang1). Addition of rhShh to tPA−/− MBECs only partially restored the tube formation and upregulated Ang1, but not VEGF, although rhShh increased VEGF and Ang1 expression on wild-type MBECs. Complete restoration of tube formation in tPA−/− MBECs was observed only when both exogenous Shh and tPA were added. The present study provides evidence that tPA and PAI-1 contribute to Shh-induced in vitro cerebral angiogenesis

tPA activity partially mediated Shh-enhanced capillary tube formation.

<p>Panels A to F show representative images of capillary-like tube formation of primary cerebral endothelial cells incubated with control medium (A), rhShh (B), tPA−/−cerebral endothelial cells (C), tPA−/−cerebral endothelial cells incubated with rhShh (D), tPA−/−cerebral endothelial cells incubated with htPA (E), and), tPA−/−cerebral endothelial cells incubated with rhShh and htPA (F). Panel G shows quantitative data of capillary-like tube lengths in mm/mm² among different experimental groups (n = 6/group). Panel H shows the effect of different doses of exogenous tPA on capillary-like tube formation on tPA−/− cerebral endothelial cell (n = 6/group). *p<0.05 versus the tPA−/−group, # p<0.05 versus the tPA−/−+tPA group, ∧p<0.05 versus the tPA−/−+Shh group, $ p<0.05 vs the tPA−/−+tPA+Shh group, & p<0.05 versus the WT group, and + p<0.05 versus the tPA−/−+tPA+Shh group. Bar = 100 µm. WT = wild-type. tPA−/− = tPA knockout.</p

MicroRNA profiling in subventricular zone after stroke: MiR-124a regulates proliferation of neural progenitor cells through Notch signaling pathway.

The Notch signaling pathway regulates adult neurogenesis under physiological and pathophysiological conditions. MicroRNAs are small non-coding RNA molecules that regulate gene expression. The present study investigated the effect of miR-124a on the Notch signaling pathway in stroke-induced neurogenesis.We found that adult rats subjected to focal cerebral ischemia exhibited substantial reduction of miR-124a expression, a neuron specific miRNA, in the neural progenitor cells of the subventricular zone (SVZ) of the lateral ventricle, which was inversely associated with activation of Notch signals. In vitro, transfection of neural progenitor cells harvested from the SVZ of adult rat with miR-124a repressed Jagged-1 (JAG1), a ligand of Notch, in a luciferase construct containing the JAG1 target site. Introduction of miR-124a in neural progenitor cells significantly reduced JAG1 transcript and protein levels, leading to inactivation of Notch signals. Transfection of neural progenitor cells with miR-124a significantly reduced progenitor cell proliferation and promoted neuronal differentiation measured by an increase in the number of Doublecortin positive cells, a marker of neuroblasts. Furthermore, introduction of miR-124a significantly increased p27Kip1 mRNA and protein levels, a downstream target gene of the Notch signaling pathway.Collectively, our study demonstrated that in vivo, stroke alters miRNA expression in SVZ neural progenitor cells and that in vitro, miR-124a mediates stroke-induced neurogenesis by targeting the JAG-Notch signaling pathway

The effect of rhShh on expression of VEGF and Ang1 in wild-type and tPA−/− MBECs.

<p>Real-time RT-PCR (A, B) and Western blot (C to F) analyses showed that rhShh robustly increased Ang1 (A, C, D) and VEGF (B, E, F) expression in wild-type (WT) MBECs. Knockout of tPA (tPA−/−) substantially reduced Ang1 expression compared with wild-type MBECs (A, C, D), whereas incubation of tPA−/− MBECs with rhShh completely rescued Ang1 expression (A, C, D). However, incubation of tPA−/− MBECs with rhShh did not elevate VEGF expression, although knockout tPA significantly reduced VEGF protein levels (B, E, F). * p<0.05 versus the WT group, # p<0.05 versus the tPA−/− group. (n = 3/group). MBECs = mouse brain endothelial cells.</p

Shh increases tPA activity and expression by upregulating tPA expression and downregulating PAI-1 expression.

<p>Real-time RT-PCR analysis (n = 3/group) revealed that incubation of endothelia cells with rhShh significantly increased tPA mRNA levels (A) and reduced PAI-1 mRNA (C), but did not alter uPA mRNA levels (B). Zymography and Western blot analysis (n = 3/group) showed that rhShh significantly increased PA activity (D and E), protein expression (F and G), and reduced PAI-1 proteins (H and I), respectively. *p<0.05 compared to the control group. WT = wild-type.</p

Shh increases capillary-like tube formation in primary cerebral endothelial cells isolated from wild-type mice.

<p>Panels A to C show representative images of capillary-like tube formation of primary cerebral endothelial cells incubated with control medium (A), rhShh (B), and rhShh in presence of cyclopamine (C). Panel D shows quantitative data of capillary-like tube lengths in mm/mm² (n = 6/group). Real-time RT-PCR analysis (n = 3/group) showed that rhShh substantially increased mRNA levels of Ptch (E), a Shh receptor, and Gli 1(F), a transcription factor in endothelial cells. *p<0.05 the control group, # p<0.05 versus the rhShh group. Bar = 100 µm. WT = wild-type. CY = cyclopamine.</p

Angiopoietin 2 Mediates the Differentiation and Migration of Neural Progenitor Cells in the Subventricular Zone after Stroke*

Ischemic stroke stimulates neurogenesis in the adult rodent brain. The molecules underlying stroke-induced neurogenesis have not been fully investigated. Using real-time reverse transcription-PCR, we found that stroke substantially up-regulated angiopoietin 2 (ANG2), a proangiogenic gene, expression in subventricular zone neural progenitor cells. Incubation of neural progenitor cells with recombinant human ANG2 significantly increased the number of β-III tubulin-positive cells, a marker of immature neurons, but did not alter the number of glial fibrillary acidic protein (GFAP)-positive cells, a marker of astrocytes, suggesting that ANG2 promotes neuronal differentiation. Blockage of the ANG2 receptor, Tie2, with small interference RNA (siRNA)-Tie2 attenuated recombinant human ANG2 (rhANG2)-increased β-III tubulin mRNA levels compared with levels in the progenitor cells transfected with control siRNA. Chromatin immunoprecipitation analysis revealed that CCAAT/enhancer-binding protein (C/EBPβ) up-regulated by rhANG2 bound to β-III tubulin, which is consistent with published data that there are several C/EBPβ binding sites in the promoter of β-III tubulin gene. In addition, rhANG2 enhanced migration of neural progenitor cells measured by single neurosphere assay. Blockage of Tie2 with siRNA-Tie2 and a Tie2-neutralizing antibody did not suppress ANG2-enhanced migration. However, inhibition of matrix metalloproteinases with GM6001 blocked ANG2-enhanced migration. Collectively, our data suggest that interaction of ANG2, a proangiogenic factor, with its receptor Tie2 promotes neural progenitor cell differentiation into neuronal lineage cells, whereas ANG2 regulates neural progenitor cell migration through matrix metalloproteinases, which do not require its receptor Tie2