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The GPR 55 agonist, L-α-lysophosphatidylinositol, mediates ovarian carcinoma cell-induced angiogenesis.

Hofmann NA, Yang J, Trauger SA, Nakayama H, Huang L, Strunk D, Moses MA, Klagsbrun M, Bischoff J, Graier WF - Br. J. Pharmacol. (2015)

Bottom Line: To study underlying signal transduction, Western blot analysis was performed.Four target-specific siRNAs against GPR55 prevented these effects of LPI on angiogenesis.These pro-angiogenic effects of LPI were transduced by GPR55-dependent phosphorylation of ERK1/2 and p38 kinase.

View Article: PubMed Central - PubMed

Affiliation: Institute for Molecular Biology and Biochemistry, Medical University Graz, Graz, Austria.

No MeSH data available.


Related in: MedlinePlus

Pharmacological and siRNA inhibition of GPR55 prevents LPI-induced angiogenic activity of ECFCs in vitro. (A–C) Effect of vehicle, GPR55 inhibitor CID16020046 (20 μM; CID), LPI (10 μM) or LPI + CID on neonatal ECFC. (A) proliferation, shown in % as compared with vehicle control after 48 h in vitro proliferation assay. (B) Branch point formation, shown in % as compared with vehicle control in an in vitro angiogenesis assay after 16 h. (C) Closure of in vitro endothelial scratch area, shown in % as compared with vehicle control after 16 h. (D) Western blot analysis of GPR55 expression and β-actin in whole cell lysates of ECFCs transfected with control siRNA (sicontrol) or four selective siRNAs against GPR55 (siGPR55). (E) Proliferation increase of ECFCs transfected with control siRNA (sicontrol) or four selective siRNAs against GPR55 (siGPR55) in response to vehicle or 10 μM LPI (48 h). All n = 9; **P < 0.01, significantly different from vehicle sicontrol; #P < 0.001, significantly different from LPI-treated sicontrol ECFCs. anova followed by Bonferroni test.
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fig03: Pharmacological and siRNA inhibition of GPR55 prevents LPI-induced angiogenic activity of ECFCs in vitro. (A–C) Effect of vehicle, GPR55 inhibitor CID16020046 (20 μM; CID), LPI (10 μM) or LPI + CID on neonatal ECFC. (A) proliferation, shown in % as compared with vehicle control after 48 h in vitro proliferation assay. (B) Branch point formation, shown in % as compared with vehicle control in an in vitro angiogenesis assay after 16 h. (C) Closure of in vitro endothelial scratch area, shown in % as compared with vehicle control after 16 h. (D) Western blot analysis of GPR55 expression and β-actin in whole cell lysates of ECFCs transfected with control siRNA (sicontrol) or four selective siRNAs against GPR55 (siGPR55). (E) Proliferation increase of ECFCs transfected with control siRNA (sicontrol) or four selective siRNAs against GPR55 (siGPR55) in response to vehicle or 10 μM LPI (48 h). All n = 9; **P < 0.01, significantly different from vehicle sicontrol; #P < 0.001, significantly different from LPI-treated sicontrol ECFCs. anova followed by Bonferroni test.

Mentions: To identify a pharmacological inhibitor of LPI-mediated pro-angiogenesis, we tested specific antagonists of known LPI receptors such as the CB1, CB2 recptors and GPR 55 (Pineiro and Falasca, 2012). The GPR55 antagonist CID16020046 (Kargl et al., 2013) decreased LPI-induced ECFC proliferation in a concentration-dependent manner with an IC50 of 17.9 (17.3–18.5) μM (Supporting Information Fig. S3a). LPI-stimulated ECFC proliferation was most effectively inhibited with a CID16020046 concentration of 20 μM, without affecting basal ECFC proliferation (Figure 3A). In contrast, the LPI-stimulated effect was not significantly inhibited by addition of the CB1 receptor antagonist/GPR55 agonist (AM251) or by antagonism of CB2 receptors with SR144528 (Supporting Information Fig. S3b). Furthermore, CID16020046 totally suppressed the LPI-induced network formation (Figure 3B) and endothelial wound healing (Figure 3C), without affecting the basal angiogenic capacity of endothelial cells. To confirm that LPI activity was GPR55 dependent, GPR55 was genetically knocked down with a mix of four validated siRNAs in ECFCs (Figure 3D). In response to LPI, siGPR55-ECFCs showed significantly reduced proliferation as compared with ECFCs transfected with control siRNA (Figure 3E). Simultaneous treatment with the GPR55 inhibitor CID16020046 significantly reduced the LPI-stimulated angiogenesis in the in vivo CAM model (Figure 4). Neither CID16020046 nor silencing of GPR55 significantly affected basal angiogenic activities of ECFCs in vitro nor angiogenesis in the CAM assay in vivo (Figures 3 and 4; Supporting Information Fig. S3). Altogether, these results demonstrate that exogenous LPI stimulates the pro-angiogenic capacity of ECFCs in vitro and angiogenesis in vivo in a specifically GPR55-dependent manner.


The GPR 55 agonist, L-α-lysophosphatidylinositol, mediates ovarian carcinoma cell-induced angiogenesis.

Hofmann NA, Yang J, Trauger SA, Nakayama H, Huang L, Strunk D, Moses MA, Klagsbrun M, Bischoff J, Graier WF - Br. J. Pharmacol. (2015)

Pharmacological and siRNA inhibition of GPR55 prevents LPI-induced angiogenic activity of ECFCs in vitro. (A–C) Effect of vehicle, GPR55 inhibitor CID16020046 (20 μM; CID), LPI (10 μM) or LPI + CID on neonatal ECFC. (A) proliferation, shown in % as compared with vehicle control after 48 h in vitro proliferation assay. (B) Branch point formation, shown in % as compared with vehicle control in an in vitro angiogenesis assay after 16 h. (C) Closure of in vitro endothelial scratch area, shown in % as compared with vehicle control after 16 h. (D) Western blot analysis of GPR55 expression and β-actin in whole cell lysates of ECFCs transfected with control siRNA (sicontrol) or four selective siRNAs against GPR55 (siGPR55). (E) Proliferation increase of ECFCs transfected with control siRNA (sicontrol) or four selective siRNAs against GPR55 (siGPR55) in response to vehicle or 10 μM LPI (48 h). All n = 9; **P < 0.01, significantly different from vehicle sicontrol; #P < 0.001, significantly different from LPI-treated sicontrol ECFCs. anova followed by Bonferroni test.
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Related In: Results  -  Collection

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fig03: Pharmacological and siRNA inhibition of GPR55 prevents LPI-induced angiogenic activity of ECFCs in vitro. (A–C) Effect of vehicle, GPR55 inhibitor CID16020046 (20 μM; CID), LPI (10 μM) or LPI + CID on neonatal ECFC. (A) proliferation, shown in % as compared with vehicle control after 48 h in vitro proliferation assay. (B) Branch point formation, shown in % as compared with vehicle control in an in vitro angiogenesis assay after 16 h. (C) Closure of in vitro endothelial scratch area, shown in % as compared with vehicle control after 16 h. (D) Western blot analysis of GPR55 expression and β-actin in whole cell lysates of ECFCs transfected with control siRNA (sicontrol) or four selective siRNAs against GPR55 (siGPR55). (E) Proliferation increase of ECFCs transfected with control siRNA (sicontrol) or four selective siRNAs against GPR55 (siGPR55) in response to vehicle or 10 μM LPI (48 h). All n = 9; **P < 0.01, significantly different from vehicle sicontrol; #P < 0.001, significantly different from LPI-treated sicontrol ECFCs. anova followed by Bonferroni test.
Mentions: To identify a pharmacological inhibitor of LPI-mediated pro-angiogenesis, we tested specific antagonists of known LPI receptors such as the CB1, CB2 recptors and GPR 55 (Pineiro and Falasca, 2012). The GPR55 antagonist CID16020046 (Kargl et al., 2013) decreased LPI-induced ECFC proliferation in a concentration-dependent manner with an IC50 of 17.9 (17.3–18.5) μM (Supporting Information Fig. S3a). LPI-stimulated ECFC proliferation was most effectively inhibited with a CID16020046 concentration of 20 μM, without affecting basal ECFC proliferation (Figure 3A). In contrast, the LPI-stimulated effect was not significantly inhibited by addition of the CB1 receptor antagonist/GPR55 agonist (AM251) or by antagonism of CB2 receptors with SR144528 (Supporting Information Fig. S3b). Furthermore, CID16020046 totally suppressed the LPI-induced network formation (Figure 3B) and endothelial wound healing (Figure 3C), without affecting the basal angiogenic capacity of endothelial cells. To confirm that LPI activity was GPR55 dependent, GPR55 was genetically knocked down with a mix of four validated siRNAs in ECFCs (Figure 3D). In response to LPI, siGPR55-ECFCs showed significantly reduced proliferation as compared with ECFCs transfected with control siRNA (Figure 3E). Simultaneous treatment with the GPR55 inhibitor CID16020046 significantly reduced the LPI-stimulated angiogenesis in the in vivo CAM model (Figure 4). Neither CID16020046 nor silencing of GPR55 significantly affected basal angiogenic activities of ECFCs in vitro nor angiogenesis in the CAM assay in vivo (Figures 3 and 4; Supporting Information Fig. S3). Altogether, these results demonstrate that exogenous LPI stimulates the pro-angiogenic capacity of ECFCs in vitro and angiogenesis in vivo in a specifically GPR55-dependent manner.

Bottom Line: To study underlying signal transduction, Western blot analysis was performed.Four target-specific siRNAs against GPR55 prevented these effects of LPI on angiogenesis.These pro-angiogenic effects of LPI were transduced by GPR55-dependent phosphorylation of ERK1/2 and p38 kinase.

View Article: PubMed Central - PubMed

Affiliation: Institute for Molecular Biology and Biochemistry, Medical University Graz, Graz, Austria.

No MeSH data available.


Related in: MedlinePlus