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Heparanase and Syndecan-4 Are Involved in Low Molecular Weight Fucoidan-Induced Angiogenesis.

Haddad O, Guyot E, Marinval N, Chevalier F, Maillard L, Gadi L, Laguillier-Morizot C, Oudar O, Sutton A, Charnaux N, Hlawaty H - Mar Drugs (2015)

Bottom Line: The phenotypic tests of LMWF-treated and EXT2- or HPSE-siRNA-transfected cells indicated that EXT2 or HPSE expression significantly affect the proangiogenic potential of LMWF.In addition, LMWF increased SDC-1, but decreased SDC-4 expressions.The effect of LMWF depends on SDC-4 expression.

View Article: PubMed Central - PubMed

Affiliation: Inserm U1148, Laboratory for Vascular Translational Science, UFR SMBH, Université Paris 13, Sorbonne Paris Cité, Groupe Biothérapies et Glycoconjugués, 93000 Bobigny, France. haddad.oualid@univ-paris13.fr.

ABSTRACT
Induction of angiogenesis is a potential treatment for chronic ischemia. Low molecular weight fucoidan (LMWF), the sulfated polysaccharide from brown seaweeds, has been shown to promote revascularization in a rat limb ischemia, increasing angiogenesis in vivo. We investigated the potential role of two heparan sulfate (HS) metabolism enzymes, exostosin-2 (EXT2) and heparanase (HPSE), and of two HS-membrane proteoglycans, syndecan-1 and -4 (SDC-1 and SDC-4), in LMWF induced angiogenesis. Our results showed that LMWF increases human vascular endothelial cell (HUVEC) migration and angiogenesis in vitro. We report that the expression and activity of the HS-degrading HPSE was increased after LMWF treatment. The phenotypic tests of LMWF-treated and EXT2- or HPSE-siRNA-transfected cells indicated that EXT2 or HPSE expression significantly affect the proangiogenic potential of LMWF. In addition, LMWF increased SDC-1, but decreased SDC-4 expressions. The effect of LMWF depends on SDC-4 expression. Silencing EXT2 or HPSE leads to an increased expression of SDC-4, providing the evidence that EXT2 and HPSE regulate the SDC-4 expression. Altogether, these data indicate that EXT2, HPSE, and SDC-4 are involved in the proangiogenic effects of LMWF, suggesting that the HS metabolism changes linked to LMWF-induced angiogenesis offer the opportunity for new therapeutic strategies of ischemic diseases.

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LMWF and glycosaminoglycan chain level in HUVECs. (A) Glycosaminoglycan quantification. HUVECs were incubated with 10 µg/mL LMWF for 24 h and the amount of total GAGs, CS and HS chains were determined in the supernatant according to a dimethyl-methylene blue (DMMB) assay; (B) Exostosin-1 and -2 (EXT1 or EXT2) mRNA levels were determined by real-time RT-PCR in cells treated with or without 10 µg/mL LMWF. (C) EXT1 or EXT2 protein levels were determined by western blot in cells treated with or without 10 µg/mL LMWF. Right panel shows a representative image of the western blot assay. * p < 0.05 versus control untreated (UT) cells. A.U.: arbitrary unit.
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marinedrugs-13-06588-f002: LMWF and glycosaminoglycan chain level in HUVECs. (A) Glycosaminoglycan quantification. HUVECs were incubated with 10 µg/mL LMWF for 24 h and the amount of total GAGs, CS and HS chains were determined in the supernatant according to a dimethyl-methylene blue (DMMB) assay; (B) Exostosin-1 and -2 (EXT1 or EXT2) mRNA levels were determined by real-time RT-PCR in cells treated with or without 10 µg/mL LMWF. (C) EXT1 or EXT2 protein levels were determined by western blot in cells treated with or without 10 µg/mL LMWF. Right panel shows a representative image of the western blot assay. * p < 0.05 versus control untreated (UT) cells. A.U.: arbitrary unit.

Mentions: We first investigated whether LMWF could modify the GAG chain level expressed by HUVECs. For that purpose, the level of total GAGs, HS chains, and chondroitin sulfate (CS) chains were determined by DMMB assays in the lysate of endothelial cells after 24 h of 10 μg/mL LMWF treatment and compared to UT control cells. There was no significant difference in the level of GAGs, HS, and CS after LMWF incubation (Figure S1). Following LMWF treatment, the amount of total GAGs in the conditioned medium of LMWF-treated cells decreased by 28% ± 8% at 24 h, as compared to untreated control cells (p < 0.05, n = 3, Figure 2A). Further analysis revealed that HS amounts decreased by 25% ± 5% in the conditioned medium of the LMWF-treated cells, whereas there was no variation of CS chain amount (Figure 2A). These data suggests that LMWF may modify the HS and HSPG turnover (HS synthesis or cleavage and HSPG shedding).


Heparanase and Syndecan-4 Are Involved in Low Molecular Weight Fucoidan-Induced Angiogenesis.

Haddad O, Guyot E, Marinval N, Chevalier F, Maillard L, Gadi L, Laguillier-Morizot C, Oudar O, Sutton A, Charnaux N, Hlawaty H - Mar Drugs (2015)

LMWF and glycosaminoglycan chain level in HUVECs. (A) Glycosaminoglycan quantification. HUVECs were incubated with 10 µg/mL LMWF for 24 h and the amount of total GAGs, CS and HS chains were determined in the supernatant according to a dimethyl-methylene blue (DMMB) assay; (B) Exostosin-1 and -2 (EXT1 or EXT2) mRNA levels were determined by real-time RT-PCR in cells treated with or without 10 µg/mL LMWF. (C) EXT1 or EXT2 protein levels were determined by western blot in cells treated with or without 10 µg/mL LMWF. Right panel shows a representative image of the western blot assay. * p < 0.05 versus control untreated (UT) cells. A.U.: arbitrary unit.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4663543&req=5

marinedrugs-13-06588-f002: LMWF and glycosaminoglycan chain level in HUVECs. (A) Glycosaminoglycan quantification. HUVECs were incubated with 10 µg/mL LMWF for 24 h and the amount of total GAGs, CS and HS chains were determined in the supernatant according to a dimethyl-methylene blue (DMMB) assay; (B) Exostosin-1 and -2 (EXT1 or EXT2) mRNA levels were determined by real-time RT-PCR in cells treated with or without 10 µg/mL LMWF. (C) EXT1 or EXT2 protein levels were determined by western blot in cells treated with or without 10 µg/mL LMWF. Right panel shows a representative image of the western blot assay. * p < 0.05 versus control untreated (UT) cells. A.U.: arbitrary unit.
Mentions: We first investigated whether LMWF could modify the GAG chain level expressed by HUVECs. For that purpose, the level of total GAGs, HS chains, and chondroitin sulfate (CS) chains were determined by DMMB assays in the lysate of endothelial cells after 24 h of 10 μg/mL LMWF treatment and compared to UT control cells. There was no significant difference in the level of GAGs, HS, and CS after LMWF incubation (Figure S1). Following LMWF treatment, the amount of total GAGs in the conditioned medium of LMWF-treated cells decreased by 28% ± 8% at 24 h, as compared to untreated control cells (p < 0.05, n = 3, Figure 2A). Further analysis revealed that HS amounts decreased by 25% ± 5% in the conditioned medium of the LMWF-treated cells, whereas there was no variation of CS chain amount (Figure 2A). These data suggests that LMWF may modify the HS and HSPG turnover (HS synthesis or cleavage and HSPG shedding).

Bottom Line: The phenotypic tests of LMWF-treated and EXT2- or HPSE-siRNA-transfected cells indicated that EXT2 or HPSE expression significantly affect the proangiogenic potential of LMWF.In addition, LMWF increased SDC-1, but decreased SDC-4 expressions.The effect of LMWF depends on SDC-4 expression.

View Article: PubMed Central - PubMed

Affiliation: Inserm U1148, Laboratory for Vascular Translational Science, UFR SMBH, Université Paris 13, Sorbonne Paris Cité, Groupe Biothérapies et Glycoconjugués, 93000 Bobigny, France. haddad.oualid@univ-paris13.fr.

ABSTRACT
Induction of angiogenesis is a potential treatment for chronic ischemia. Low molecular weight fucoidan (LMWF), the sulfated polysaccharide from brown seaweeds, has been shown to promote revascularization in a rat limb ischemia, increasing angiogenesis in vivo. We investigated the potential role of two heparan sulfate (HS) metabolism enzymes, exostosin-2 (EXT2) and heparanase (HPSE), and of two HS-membrane proteoglycans, syndecan-1 and -4 (SDC-1 and SDC-4), in LMWF induced angiogenesis. Our results showed that LMWF increases human vascular endothelial cell (HUVEC) migration and angiogenesis in vitro. We report that the expression and activity of the HS-degrading HPSE was increased after LMWF treatment. The phenotypic tests of LMWF-treated and EXT2- or HPSE-siRNA-transfected cells indicated that EXT2 or HPSE expression significantly affect the proangiogenic potential of LMWF. In addition, LMWF increased SDC-1, but decreased SDC-4 expressions. The effect of LMWF depends on SDC-4 expression. Silencing EXT2 or HPSE leads to an increased expression of SDC-4, providing the evidence that EXT2 and HPSE regulate the SDC-4 expression. Altogether, these data indicate that EXT2, HPSE, and SDC-4 are involved in the proangiogenic effects of LMWF, suggesting that the HS metabolism changes linked to LMWF-induced angiogenesis offer the opportunity for new therapeutic strategies of ischemic diseases.

Show MeSH
Related in: MedlinePlus