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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 HUVEC spheroid sprouting by NOX-S93 in the cellular angiogenesis assay(A) HUVEC spheroids were embedded in a 3D collagen gel, stimulated with 100┬ánM S1P and incubated for 24┬áh with different concentrations of NOX-S93. The mean┬▒S.D. of the cumulative sprout length of ten randomly selected spheroids per data point is depicted. Ôłĺ, basal sprouting without S1P stimulation and NOX-S93 treatment as a negative control. (B) IC50 of NOX-S93 for the inhibition of HUVEC spheroid sprouting after stimulation with 100┬ánM S1P. (C) NOX-S93 IC50 values for the inhibition of HUVEC spheroid sprouting after stimulation by different growth factors. The control Spiegelmer┬« revNOX-S93 showed no inhibition.
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Figure 4: Inhibition of HUVEC spheroid sprouting by NOX-S93 in the cellular angiogenesis assay(A) HUVEC spheroids were embedded in a 3D collagen gel, stimulated with 100┬ánM S1P and incubated for 24┬áh with different concentrations of NOX-S93. The mean┬▒S.D. of the cumulative sprout length of ten randomly selected spheroids per data point is depicted. Ôłĺ, basal sprouting without S1P stimulation and NOX-S93 treatment as a negative control. (B) IC50 of NOX-S93 for the inhibition of HUVEC spheroid sprouting after stimulation with 100┬ánM S1P. (C) NOX-S93 IC50 values for the inhibition of HUVEC spheroid sprouting after stimulation by different growth factors. The control Spiegelmer┬« revNOX-S93 showed no inhibition.

Mentions: The ability of NOX-S93 to inhibit angiogenesis of human endothelial cells was demonstrated in a cellular angiogenesis assay in vitro. First, the sprouting of HUVEC spheroids was induced with 100 nM S1P in the presence of different concentrations of NOX-S93. As shown in Figure 4(A), NOX-S93 dose-dependently inhibited the cumulative sprouting length of HUVEC spheroids. The calculated IC50 of NOX-S93 for inhibition of S1P-induced sprouting is approximately 340 nM (Figure 4B). At 10000 nM NOX-S93 spheroid sprouting was reduced below negative control levels possibly due to neutralization of basal concentrations of S1P effective in the angiogenesis assay.


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 HUVEC spheroid sprouting by NOX-S93 in the cellular angiogenesis assay(A) HUVEC spheroids were embedded in a 3D collagen gel, stimulated with 100┬ánM S1P and incubated for 24┬áh with different concentrations of NOX-S93. The mean┬▒S.D. of the cumulative sprout length of ten randomly selected spheroids per data point is depicted. Ôłĺ, basal sprouting without S1P stimulation and NOX-S93 treatment as a negative control. (B) IC50 of NOX-S93 for the inhibition of HUVEC spheroid sprouting after stimulation with 100┬ánM S1P. (C) NOX-S93 IC50 values for the inhibition of HUVEC spheroid sprouting after stimulation by different growth factors. The control Spiegelmer┬« revNOX-S93 showed no inhibition.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Inhibition of HUVEC spheroid sprouting by NOX-S93 in the cellular angiogenesis assay(A) HUVEC spheroids were embedded in a 3D collagen gel, stimulated with 100┬ánM S1P and incubated for 24┬áh with different concentrations of NOX-S93. The mean┬▒S.D. of the cumulative sprout length of ten randomly selected spheroids per data point is depicted. Ôłĺ, basal sprouting without S1P stimulation and NOX-S93 treatment as a negative control. (B) IC50 of NOX-S93 for the inhibition of HUVEC spheroid sprouting after stimulation with 100┬ánM S1P. (C) NOX-S93 IC50 values for the inhibition of HUVEC spheroid sprouting after stimulation by different growth factors. The control Spiegelmer┬« revNOX-S93 showed no inhibition.
Mentions: The ability of NOX-S93 to inhibit angiogenesis of human endothelial cells was demonstrated in a cellular angiogenesis assay in vitro. First, the sprouting of HUVEC spheroids was induced with 100 nM S1P in the presence of different concentrations of NOX-S93. As shown in Figure 4(A), NOX-S93 dose-dependently inhibited the cumulative sprouting length of HUVEC spheroids. The calculated IC50 of NOX-S93 for inhibition of S1P-induced sprouting is approximately 340 nM (Figure 4B). At 10000 nM NOX-S93 spheroid sprouting was reduced below negative control levels possibly due to neutralization of basal concentrations of S1P effective in the angiogenesis assay.

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