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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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Effects of Low molecular weight fucoidan (LMWF) on endothelial cell abilities: migration and 2D-angiogenesis. Human vascular endothelial cells (HUVEC) were incubated with 10 µg/mL LMWF for 24 h and the migration (A), the lamellipodia formation (B) and the capillary tube formation (length and area) (C) were determined. (A) Migration chamber assay. HUVECs incubated with or without 10 μg/mL LMWF, were allowed to migrate through the porous fibronectin-coated membrane. They were stained with Mayer’s hemalum and counted. The results are expressed as cell number per field; (B) Lamellipodia formation. LMWF induced the formation of lamellipodia and ruffles (white arrows indicate lamellipodia/ruffle formation, DAPI-nucleus (blue), Phalloidin-F-actin (red)). Bar = 10 µm; (C) Capillary tube formation (2D-angiogenesis assay) on Matrigel. Left and right panels show the length (left) and area (right) of endothelial capillaries formed by HUVECs treated with or without 10 µg/mL LMWF. Lower right panel shows a representative image of capillary network, as photographed with phase contrast microscopy (magnification ×100). * p < 0.05 versus control untreated (UT) cells. A.U.: arbitrary unit.
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marinedrugs-13-06588-f001: Effects of Low molecular weight fucoidan (LMWF) on endothelial cell abilities: migration and 2D-angiogenesis. Human vascular endothelial cells (HUVEC) were incubated with 10 µg/mL LMWF for 24 h and the migration (A), the lamellipodia formation (B) and the capillary tube formation (length and area) (C) were determined. (A) Migration chamber assay. HUVECs incubated with or without 10 μg/mL LMWF, were allowed to migrate through the porous fibronectin-coated membrane. They were stained with Mayer’s hemalum and counted. The results are expressed as cell number per field; (B) Lamellipodia formation. LMWF induced the formation of lamellipodia and ruffles (white arrows indicate lamellipodia/ruffle formation, DAPI-nucleus (blue), Phalloidin-F-actin (red)). Bar = 10 µm; (C) Capillary tube formation (2D-angiogenesis assay) on Matrigel. Left and right panels show the length (left) and area (right) of endothelial capillaries formed by HUVECs treated with or without 10 µg/mL LMWF. Lower right panel shows a representative image of capillary network, as photographed with phase contrast microscopy (magnification ×100). * p < 0.05 versus control untreated (UT) cells. A.U.: arbitrary unit.

Mentions: LMWF at 10 μg/mL, but not high molecular weight fucoidan (HMWF) (600 kDa) increased HUVEC migration through fibronectin-coated 8 µm-porous membranes by 36% ± 8% (Figure 1A and data not shown). Confocal microscopy confirmed that LMWF induced the formation of lamellipodia and ruffles, which characterize a migration phenotype and reorganized actin cytoskeleton (Figure 1B). Using a 2D-angiogenesis assay, we demonstrated that LMWF induced the formation of capillary tubes in Matrigel by increasing their length by 4 fold and their area up to 84% ± 8%, as compared to untreated (UT) control cells (Figure 1C).


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)

Effects of Low molecular weight fucoidan (LMWF) on endothelial cell abilities: migration and 2D-angiogenesis. Human vascular endothelial cells (HUVEC) were incubated with 10 µg/mL LMWF for 24 h and the migration (A), the lamellipodia formation (B) and the capillary tube formation (length and area) (C) were determined. (A) Migration chamber assay. HUVECs incubated with or without 10 μg/mL LMWF, were allowed to migrate through the porous fibronectin-coated membrane. They were stained with Mayer’s hemalum and counted. The results are expressed as cell number per field; (B) Lamellipodia formation. LMWF induced the formation of lamellipodia and ruffles (white arrows indicate lamellipodia/ruffle formation, DAPI-nucleus (blue), Phalloidin-F-actin (red)). Bar = 10 µm; (C) Capillary tube formation (2D-angiogenesis assay) on Matrigel. Left and right panels show the length (left) and area (right) of endothelial capillaries formed by HUVECs treated with or without 10 µg/mL LMWF. Lower right panel shows a representative image of capillary network, as photographed with phase contrast microscopy (magnification ×100). * p < 0.05 versus control untreated (UT) cells. A.U.: arbitrary unit.
© Copyright Policy
Related In: Results  -  Collection

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

marinedrugs-13-06588-f001: Effects of Low molecular weight fucoidan (LMWF) on endothelial cell abilities: migration and 2D-angiogenesis. Human vascular endothelial cells (HUVEC) were incubated with 10 µg/mL LMWF for 24 h and the migration (A), the lamellipodia formation (B) and the capillary tube formation (length and area) (C) were determined. (A) Migration chamber assay. HUVECs incubated with or without 10 μg/mL LMWF, were allowed to migrate through the porous fibronectin-coated membrane. They were stained with Mayer’s hemalum and counted. The results are expressed as cell number per field; (B) Lamellipodia formation. LMWF induced the formation of lamellipodia and ruffles (white arrows indicate lamellipodia/ruffle formation, DAPI-nucleus (blue), Phalloidin-F-actin (red)). Bar = 10 µm; (C) Capillary tube formation (2D-angiogenesis assay) on Matrigel. Left and right panels show the length (left) and area (right) of endothelial capillaries formed by HUVECs treated with or without 10 µg/mL LMWF. Lower right panel shows a representative image of capillary network, as photographed with phase contrast microscopy (magnification ×100). * p < 0.05 versus control untreated (UT) cells. A.U.: arbitrary unit.
Mentions: LMWF at 10 μg/mL, but not high molecular weight fucoidan (HMWF) (600 kDa) increased HUVEC migration through fibronectin-coated 8 µm-porous membranes by 36% ± 8% (Figure 1A and data not shown). Confocal microscopy confirmed that LMWF induced the formation of lamellipodia and ruffles, which characterize a migration phenotype and reorganized actin cytoskeleton (Figure 1B). Using a 2D-angiogenesis assay, we demonstrated that LMWF induced the formation of capillary tubes in Matrigel by increasing their length by 4 fold and their area up to 84% ± 8%, as compared to untreated (UT) control cells (Figure 1C).

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