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Identification and characterization of a mirror-image oligonucleotide that binds and neutralizes sphingosine 1-phosphate, a central mediator of angiogenesis.

Purschke WG, Hoehlig K, Buchner K, Zboralski D, Schwoebel F, Vater A, Klussmann S - Biochem. J. (2014)

Bottom Line: In stably transfected CHO (Chinese-hamster ovary) cell lines expressing the S1P receptors S1PR1 or S1PR3, NOX-S93 inhibits S1P-mediated β-arrestin recruitment and intracellular calcium release respectively, with IC50 values in the low nanomolar range.The pro-angiogenic activity of S1P, and of the growth factors VEGF-A (vascular endothelial growth factor-A), FGF-2 (fibroblast growth factor-2) and IGF-1 (insulin-like growth factor-1), was effectively blocked by NOX-S93 in a cellular angiogenesis assay employing primary human endothelial cells.These data provide further evidence for the relevance of extracellular S1P as a central mediator of angiogenesis, suggesting pharmacological S1P neutralization as a promising treatment alternative to current anti-angiogenesis approaches.

View Article: PubMed Central - PubMed

Affiliation: *NOXXON Pharma AG, Berlin, Germany.

ABSTRACT
The sphingolipid S1P (sphingosine 1-phosphate) is known to be involved in a number of pathophysiological conditions such as cancer, autoimmune diseases and fibrosis. It acts extracellularly through a set of five G-protein-coupled receptors, but its intracellular actions are also well documented. Employing in vitro selection techniques, we identified an L-aptamer (Spiegelmer®) to S1P designated NOX-S93. The binding affinity of NOX-S93 to S1P had a Kd value of 4.3 nM. The Spiegelmer® shows equal binding to dihydro-S1P, but no cross-reactivity to the related lipids sphingosine, lysophosphatidic acid, ceramide, ceramide-1-phosphate or sphingosine phosphocholine. In stably transfected CHO (Chinese-hamster ovary) cell lines expressing the S1P receptors S1PR1 or S1PR3, NOX-S93 inhibits S1P-mediated β-arrestin recruitment and intracellular calcium release respectively, with IC50 values in the low nanomolar range. The pro-angiogenic activity of S1P, and of the growth factors VEGF-A (vascular endothelial growth factor-A), FGF-2 (fibroblast growth factor-2) and IGF-1 (insulin-like growth factor-1), was effectively blocked by NOX-S93 in a cellular angiogenesis assay employing primary human endothelial cells. These data provide further evidence for the relevance of extracellular S1P as a central mediator of angiogenesis, suggesting pharmacological S1P neutralization as a promising treatment alternative to current anti-angiogenesis approaches.

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Inhibition of S1P induced ERK1/2 phosphorylation in HUVECs by NOX-S93HUVECs were cultured in 2D, serum-starved and stimulated with 100 nM S1P in the presence or absence of NOX-S93 or the non-functional control Spiegelmer® revNOX-S93 for 5 min. Activation of ERK1/2 was studied by Western blotting using a phosphospecific antibody. Whereas a 10-fold excess of NOX-S93 completely inhibited S1P-induced ERK1/2 phosphorylation, the control Spiegelmer® revNOX-S93 was inactive.
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Figure 5: Inhibition of S1P induced ERK1/2 phosphorylation in HUVECs by NOX-S93HUVECs were cultured in 2D, serum-starved and stimulated with 100 nM S1P in the presence or absence of NOX-S93 or the non-functional control Spiegelmer® revNOX-S93 for 5 min. Activation of ERK1/2 was studied by Western blotting using a phosphospecific antibody. Whereas a 10-fold excess of NOX-S93 completely inhibited S1P-induced ERK1/2 phosphorylation, the control Spiegelmer® revNOX-S93 was inactive.

Mentions: To show that S1P can exert direct effects on HUVECs which can be inhibited by NOX-S93 we performed Western blot assays and found a strong S1P-induced activation of the ERK1/2 signalling pathway. After 5 min, 100 nM S1P stimulated ERK1/2 phosphorylation in 2D-grown HUVECs. This S1P-induced phosphorylation was completely inhibited by a 10-fold excess of NOX-S93, whereas the non-functional Spiegelmer® revNOX-S93 was inactive (Figure 5).


Identification and characterization of a mirror-image oligonucleotide that binds and neutralizes sphingosine 1-phosphate, a central mediator of angiogenesis.

Purschke WG, Hoehlig K, Buchner K, Zboralski D, Schwoebel F, Vater A, Klussmann S - Biochem. J. (2014)

Inhibition of S1P induced ERK1/2 phosphorylation in HUVECs by NOX-S93HUVECs were cultured in 2D, serum-starved and stimulated with 100 nM S1P in the presence or absence of NOX-S93 or the non-functional control Spiegelmer® revNOX-S93 for 5 min. Activation of ERK1/2 was studied by Western blotting using a phosphospecific antibody. Whereas a 10-fold excess of NOX-S93 completely inhibited S1P-induced ERK1/2 phosphorylation, the control Spiegelmer® revNOX-S93 was inactive.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4109837&req=5

Figure 5: Inhibition of S1P induced ERK1/2 phosphorylation in HUVECs by NOX-S93HUVECs were cultured in 2D, serum-starved and stimulated with 100 nM S1P in the presence or absence of NOX-S93 or the non-functional control Spiegelmer® revNOX-S93 for 5 min. Activation of ERK1/2 was studied by Western blotting using a phosphospecific antibody. Whereas a 10-fold excess of NOX-S93 completely inhibited S1P-induced ERK1/2 phosphorylation, the control Spiegelmer® revNOX-S93 was inactive.
Mentions: To show that S1P can exert direct effects on HUVECs which can be inhibited by NOX-S93 we performed Western blot assays and found a strong S1P-induced activation of the ERK1/2 signalling pathway. After 5 min, 100 nM S1P stimulated ERK1/2 phosphorylation in 2D-grown HUVECs. This S1P-induced phosphorylation was completely inhibited by a 10-fold excess of NOX-S93, whereas the non-functional Spiegelmer® revNOX-S93 was inactive (Figure 5).

Bottom Line: In stably transfected CHO (Chinese-hamster ovary) cell lines expressing the S1P receptors S1PR1 or S1PR3, NOX-S93 inhibits S1P-mediated β-arrestin recruitment and intracellular calcium release respectively, with IC50 values in the low nanomolar range.The pro-angiogenic activity of S1P, and of the growth factors VEGF-A (vascular endothelial growth factor-A), FGF-2 (fibroblast growth factor-2) and IGF-1 (insulin-like growth factor-1), was effectively blocked by NOX-S93 in a cellular angiogenesis assay employing primary human endothelial cells.These data provide further evidence for the relevance of extracellular S1P as a central mediator of angiogenesis, suggesting pharmacological S1P neutralization as a promising treatment alternative to current anti-angiogenesis approaches.

View Article: PubMed Central - PubMed

Affiliation: *NOXXON Pharma AG, Berlin, Germany.

ABSTRACT
The sphingolipid S1P (sphingosine 1-phosphate) is known to be involved in a number of pathophysiological conditions such as cancer, autoimmune diseases and fibrosis. It acts extracellularly through a set of five G-protein-coupled receptors, but its intracellular actions are also well documented. Employing in vitro selection techniques, we identified an L-aptamer (Spiegelmer®) to S1P designated NOX-S93. The binding affinity of NOX-S93 to S1P had a Kd value of 4.3 nM. The Spiegelmer® shows equal binding to dihydro-S1P, but no cross-reactivity to the related lipids sphingosine, lysophosphatidic acid, ceramide, ceramide-1-phosphate or sphingosine phosphocholine. In stably transfected CHO (Chinese-hamster ovary) cell lines expressing the S1P receptors S1PR1 or S1PR3, NOX-S93 inhibits S1P-mediated β-arrestin recruitment and intracellular calcium release respectively, with IC50 values in the low nanomolar range. The pro-angiogenic activity of S1P, and of the growth factors VEGF-A (vascular endothelial growth factor-A), FGF-2 (fibroblast growth factor-2) and IGF-1 (insulin-like growth factor-1), was effectively blocked by NOX-S93 in a cellular angiogenesis assay employing primary human endothelial cells. These data provide further evidence for the relevance of extracellular S1P as a central mediator of angiogenesis, suggesting pharmacological S1P neutralization as a promising treatment alternative to current anti-angiogenesis approaches.

Show MeSH
Related in: MedlinePlus