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Indirubin-3'-monoxime exerts a dual mode of inhibition towards leukotriene-mediated vascular smooth muscle cell migration.

Blazevic T, Schaible AM, Weinhäupl K, Schachner D, Nikels F, Weinigel C, Barz D, Atanasov AG, Pergola C, Werz O, Dirsch VM, Heiss EH - Cardiovasc. Res. (2013)

Bottom Line: Induction of haem oxygenase 1 accounted for this anti-migratory activity of I3MO in VSMC.In cell-based and cell-free assays, I3MO selectively inhibited 5-lipoxygenase (5-LO), the key enzyme in LT biosynthesis, with an IC50 in the low micromolar range.These inhibitory actions on both migratory stimulus and response complement the previously demonstrated anti-proliferative properties of I3MO and may further promote I3MO as promising vasoprotective compound.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacognosy, University of Vienna, Althanstrasse 14, Vienna A-1090, Austria.

ABSTRACT

Aims: The small molecule indirubin-3'-monoxime (I3MO) has been shown to inhibit vascular smooth muscle cell (VSMC) proliferation and neointima formation in vivo. The influence of I3MO on VSMC migration and vascular inflammation, two additional key players during the onset of atherosclerosis and restenosis, should be investigated.

Methods and results: We examined the influence of I3MO on VSMC migration, with focus on monocyte-derived leukotrienes (LTs) and platelet-derived growth factors (PDGFs) as elicitors. Exogenous LTB4 and cysteinyl leukotrienes as well as LT-enriched conditioned medium of activated primary human monocytes induced VSMC migration, which was inhibited by I3MO. I3MO also blunted migration of VSMC stimulated with the PDGF, the strongest motogen tested in this study. Induction of haem oxygenase 1 accounted for this anti-migratory activity of I3MO in VSMC. Notably, I3MO not only interfered with the migratory response in VSMC, but also suppressed the production of pro-migratory LT in monocytes. Conditioned media from monocytes that were activated in the presence of I3MO failed to induce VSMC migration. In cell-based and cell-free assays, I3MO selectively inhibited 5-lipoxygenase (5-LO), the key enzyme in LT biosynthesis, with an IC50 in the low micromolar range.

Conclusion: Our study reveals a novel dual inhibitory mode of I3MO on LT-mediated VSMC migration: (i) I3MO interferes with pro-migratory signalling in VSMC and (ii) I3MO suppresses LT biosynthesis in monocytes by direct inhibition of 5-LO. These inhibitory actions on both migratory stimulus and response complement the previously demonstrated anti-proliferative properties of I3MO and may further promote I3MO as promising vasoprotective compound.

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LTB4, CysLT, and LT-enriched conditioned medium from activated monocytes induce VSMC migration. VSMCs were stimulated with vehicle (0.33% ethanol; –), increasing concentrations of LTB4 (A) or cys LT (B) as indicated, or with 10 ng/mL of PDGF-BB as a positive control for 21 h in a wound healing assay. (C) VSMCs were incubated for 21 h in a wound healing assay with either vehicle (0.33% ethanol; veh), LTB4 (1 µM), or conditioned medium from monocytes treated for 10 min with either vehicle (0.33% ethanol + 0.1% DMSO; veh), or AA (20 µM) + A23187 (2.5 µM). (A–C) Graphs indicate the change in area occupied by cells as fold induction of VSMC migration from 4 to 6 (A) and 3 (B and C) biological replicates with four technical replicates each; mean + SE; *P < 0.05; **P < 0.01; ***P < 0.001.
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CVT339F1: LTB4, CysLT, and LT-enriched conditioned medium from activated monocytes induce VSMC migration. VSMCs were stimulated with vehicle (0.33% ethanol; –), increasing concentrations of LTB4 (A) or cys LT (B) as indicated, or with 10 ng/mL of PDGF-BB as a positive control for 21 h in a wound healing assay. (C) VSMCs were incubated for 21 h in a wound healing assay with either vehicle (0.33% ethanol; veh), LTB4 (1 µM), or conditioned medium from monocytes treated for 10 min with either vehicle (0.33% ethanol + 0.1% DMSO; veh), or AA (20 µM) + A23187 (2.5 µM). (A–C) Graphs indicate the change in area occupied by cells as fold induction of VSMC migration from 4 to 6 (A) and 3 (B and C) biological replicates with four technical replicates each; mean + SE; *P < 0.05; **P < 0.01; ***P < 0.001.

Mentions: Monocytes are major players within the inflammatory processes of the vessel wall during the onset of atherosclerosis.20 They express LO enzymes and produce LTs, which are assumed to contribute to VSMC migration and proliferation in vitro and in vivo.3,4 We first confirmed migration of our VSMC population and stimulated VSMCs with increasing concentrations of LTB4 (1–1000 nM), the major pro-migratory LT. In a scratch assay, LTB4 stimulated VSMC migration concentration dependently, with a significant effect at 100 nM (Figure 1A). A mixture of cysLT also induced VSMC migration being significant at 100 nM (Figure 1B). Using a BrdU assay, we could show that neither LTB4 nor the cysLT mixture induced VSMC proliferation in our experimental setting (see Supplementary material online, Figure S2), ruling out a proliferative component of the observed scratch closure upon LT treatment. Compared with the PDGF, LTs were less potent motogens, an effect that was corroborated by matching results from the complementary Boyden chamber chemotaxis assay (see Supplementary material online, Figure S3). Prostaglandin E2 (PGE2), 5-HETE or 5-oxo-ETE, and other AA-derived eisosanoids did not stimulate VSMC migration up to a concentration of 1 µM (data not shown).Figure 1


Indirubin-3'-monoxime exerts a dual mode of inhibition towards leukotriene-mediated vascular smooth muscle cell migration.

Blazevic T, Schaible AM, Weinhäupl K, Schachner D, Nikels F, Weinigel C, Barz D, Atanasov AG, Pergola C, Werz O, Dirsch VM, Heiss EH - Cardiovasc. Res. (2013)

LTB4, CysLT, and LT-enriched conditioned medium from activated monocytes induce VSMC migration. VSMCs were stimulated with vehicle (0.33% ethanol; –), increasing concentrations of LTB4 (A) or cys LT (B) as indicated, or with 10 ng/mL of PDGF-BB as a positive control for 21 h in a wound healing assay. (C) VSMCs were incubated for 21 h in a wound healing assay with either vehicle (0.33% ethanol; veh), LTB4 (1 µM), or conditioned medium from monocytes treated for 10 min with either vehicle (0.33% ethanol + 0.1% DMSO; veh), or AA (20 µM) + A23187 (2.5 µM). (A–C) Graphs indicate the change in area occupied by cells as fold induction of VSMC migration from 4 to 6 (A) and 3 (B and C) biological replicates with four technical replicates each; mean + SE; *P < 0.05; **P < 0.01; ***P < 0.001.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3928003&req=5

CVT339F1: LTB4, CysLT, and LT-enriched conditioned medium from activated monocytes induce VSMC migration. VSMCs were stimulated with vehicle (0.33% ethanol; –), increasing concentrations of LTB4 (A) or cys LT (B) as indicated, or with 10 ng/mL of PDGF-BB as a positive control for 21 h in a wound healing assay. (C) VSMCs were incubated for 21 h in a wound healing assay with either vehicle (0.33% ethanol; veh), LTB4 (1 µM), or conditioned medium from monocytes treated for 10 min with either vehicle (0.33% ethanol + 0.1% DMSO; veh), or AA (20 µM) + A23187 (2.5 µM). (A–C) Graphs indicate the change in area occupied by cells as fold induction of VSMC migration from 4 to 6 (A) and 3 (B and C) biological replicates with four technical replicates each; mean + SE; *P < 0.05; **P < 0.01; ***P < 0.001.
Mentions: Monocytes are major players within the inflammatory processes of the vessel wall during the onset of atherosclerosis.20 They express LO enzymes and produce LTs, which are assumed to contribute to VSMC migration and proliferation in vitro and in vivo.3,4 We first confirmed migration of our VSMC population and stimulated VSMCs with increasing concentrations of LTB4 (1–1000 nM), the major pro-migratory LT. In a scratch assay, LTB4 stimulated VSMC migration concentration dependently, with a significant effect at 100 nM (Figure 1A). A mixture of cysLT also induced VSMC migration being significant at 100 nM (Figure 1B). Using a BrdU assay, we could show that neither LTB4 nor the cysLT mixture induced VSMC proliferation in our experimental setting (see Supplementary material online, Figure S2), ruling out a proliferative component of the observed scratch closure upon LT treatment. Compared with the PDGF, LTs were less potent motogens, an effect that was corroborated by matching results from the complementary Boyden chamber chemotaxis assay (see Supplementary material online, Figure S3). Prostaglandin E2 (PGE2), 5-HETE or 5-oxo-ETE, and other AA-derived eisosanoids did not stimulate VSMC migration up to a concentration of 1 µM (data not shown).Figure 1

Bottom Line: Induction of haem oxygenase 1 accounted for this anti-migratory activity of I3MO in VSMC.In cell-based and cell-free assays, I3MO selectively inhibited 5-lipoxygenase (5-LO), the key enzyme in LT biosynthesis, with an IC50 in the low micromolar range.These inhibitory actions on both migratory stimulus and response complement the previously demonstrated anti-proliferative properties of I3MO and may further promote I3MO as promising vasoprotective compound.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacognosy, University of Vienna, Althanstrasse 14, Vienna A-1090, Austria.

ABSTRACT

Aims: The small molecule indirubin-3'-monoxime (I3MO) has been shown to inhibit vascular smooth muscle cell (VSMC) proliferation and neointima formation in vivo. The influence of I3MO on VSMC migration and vascular inflammation, two additional key players during the onset of atherosclerosis and restenosis, should be investigated.

Methods and results: We examined the influence of I3MO on VSMC migration, with focus on monocyte-derived leukotrienes (LTs) and platelet-derived growth factors (PDGFs) as elicitors. Exogenous LTB4 and cysteinyl leukotrienes as well as LT-enriched conditioned medium of activated primary human monocytes induced VSMC migration, which was inhibited by I3MO. I3MO also blunted migration of VSMC stimulated with the PDGF, the strongest motogen tested in this study. Induction of haem oxygenase 1 accounted for this anti-migratory activity of I3MO in VSMC. Notably, I3MO not only interfered with the migratory response in VSMC, but also suppressed the production of pro-migratory LT in monocytes. Conditioned media from monocytes that were activated in the presence of I3MO failed to induce VSMC migration. In cell-based and cell-free assays, I3MO selectively inhibited 5-lipoxygenase (5-LO), the key enzyme in LT biosynthesis, with an IC50 in the low micromolar range.

Conclusion: Our study reveals a novel dual inhibitory mode of I3MO on LT-mediated VSMC migration: (i) I3MO interferes with pro-migratory signalling in VSMC and (ii) I3MO suppresses LT biosynthesis in monocytes by direct inhibition of 5-LO. These inhibitory actions on both migratory stimulus and response complement the previously demonstrated anti-proliferative properties of I3MO and may further promote I3MO as promising vasoprotective compound.

Show MeSH
Related in: MedlinePlus