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Tissue plasminogen activator and plasminogen activator inhibitor 1 contribute to sonic hedgehog-induced in vitro cerebral angiogenesis.

Teng H, Chopp M, Hozeska-Solgot A, Shen L, Lu M, Tang C, Zhang ZG - PLoS ONE (2012)

Bottom Line: 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).Addition of PAI-1 reduced rhShh-augmented tube formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Henry Ford Hospital, Detroit, Michigan, United States of America.

ABSTRACT
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.

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The effect of rhShh on expression of VEGF and Ang1 in wild-type and tPA−/− MBECs.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.
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pone-0033444-g005: The effect of rhShh on expression of VEGF and Ang1 in wild-type and tPA−/− MBECs.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.

Mentions: Previous studies have shown that Shh upregulates VEGF and Ang1 in fibroblasts and interstitial mesenchymal cells [10], [11]. We therefore examined the effect of rhShh on expression of these genes in cerebral endothelial cells. Both real-time RT-PCR analysis and Western blot showed that rhShh significantly upregulated Ang1 and VEGF expression on wild-type endothelial cells (Fig. 5A to F). Blockage of Tie2 with a neutralizing antibody against Tie2 significantly reduced rhShh-induced tube formation (3.9±1.1 vs 6.4±0.4 mm/mm2 in rhShh group, n = 5/group, p<0.05), while inhibition of VEGF receptor 2 (VEGFR2) with SU1498, a specific antagonist of VEGFR2, did not significantly reduce rhShh-induced tube formation (4.1±1.2 vs 6.4±0.4 mm/mm2 in rhShh group, n = 5, p = 0.09). Interestingly, tPA−/− endothelial cells exhibited significant reductions of Ang1 mRNA and protein (Fig. 5A, C, D), and a significant decrease in VEGF protein (Fig. 5B, E, F) compared to wild-type endothelial cells, suggesting that knockout of tPA affects Ang 1 and VEGF expression. Incubation of tPA−/− endothelial cells with rhShh robustly upregulated Ang 1, but did not alter VEGF expression (Fig. 5A to F). These data suggest that Shh-upregulated Ang1 is tPA independent, whereas upregulation of VEGF by Shh requires tPA.


Tissue plasminogen activator and plasminogen activator inhibitor 1 contribute to sonic hedgehog-induced in vitro cerebral angiogenesis.

Teng H, Chopp M, Hozeska-Solgot A, Shen L, Lu M, Tang C, Zhang ZG - PLoS ONE (2012)

The effect of rhShh on expression of VEGF and Ang1 in wild-type and tPA−/− MBECs.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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3303815&req=5

pone-0033444-g005: The effect of rhShh on expression of VEGF and Ang1 in wild-type and tPA−/− MBECs.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.
Mentions: Previous studies have shown that Shh upregulates VEGF and Ang1 in fibroblasts and interstitial mesenchymal cells [10], [11]. We therefore examined the effect of rhShh on expression of these genes in cerebral endothelial cells. Both real-time RT-PCR analysis and Western blot showed that rhShh significantly upregulated Ang1 and VEGF expression on wild-type endothelial cells (Fig. 5A to F). Blockage of Tie2 with a neutralizing antibody against Tie2 significantly reduced rhShh-induced tube formation (3.9±1.1 vs 6.4±0.4 mm/mm2 in rhShh group, n = 5/group, p<0.05), while inhibition of VEGF receptor 2 (VEGFR2) with SU1498, a specific antagonist of VEGFR2, did not significantly reduce rhShh-induced tube formation (4.1±1.2 vs 6.4±0.4 mm/mm2 in rhShh group, n = 5, p = 0.09). Interestingly, tPA−/− endothelial cells exhibited significant reductions of Ang1 mRNA and protein (Fig. 5A, C, D), and a significant decrease in VEGF protein (Fig. 5B, E, F) compared to wild-type endothelial cells, suggesting that knockout of tPA affects Ang 1 and VEGF expression. Incubation of tPA−/− endothelial cells with rhShh robustly upregulated Ang 1, but did not alter VEGF expression (Fig. 5A to F). These data suggest that Shh-upregulated Ang1 is tPA independent, whereas upregulation of VEGF by Shh requires tPA.

Bottom Line: 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).Addition of PAI-1 reduced rhShh-augmented tube formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Henry Ford Hospital, Detroit, Michigan, United States of America.

ABSTRACT
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.

Show MeSH