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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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Assessment of HPSE involvement in biological effects of LMWF in HUVECs. HUVECs were transfected with HPSE-siRNA or SNC-siRNA control. (A) HPSE mRNA levels were determined in HPSE-siRNA- or SNC-siRNA-transfected cells by real-time RT-PCR. HPSE mRNA level normalized to GAPDH mRNA level in SNC-siRNA-transfected control cells was arbitrary set to 1; (B) 2D-angiogenesis was assayed in cells treated with or without 10 µg/mL LMWF. The difference in the capillary network length between LMWF-treated and untreated cells in HPSE RNA interference condition was compared to that in SNC-siRNA-transfected cells; (C) Migration was assayed in cells treated with or without 10 µg/mL LMWF. Control LMWF induction was arbitrary set at 100% for SNC-siRNA-transfected cells. * p < 0.05, ** p < 0.005 versus SNC-siRNA-transfected control cells. A.U.: arbitrary unit.
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marinedrugs-13-06588-f007: Assessment of HPSE involvement in biological effects of LMWF in HUVECs. HUVECs were transfected with HPSE-siRNA or SNC-siRNA control. (A) HPSE mRNA levels were determined in HPSE-siRNA- or SNC-siRNA-transfected cells by real-time RT-PCR. HPSE mRNA level normalized to GAPDH mRNA level in SNC-siRNA-transfected control cells was arbitrary set to 1; (B) 2D-angiogenesis was assayed in cells treated with or without 10 µg/mL LMWF. The difference in the capillary network length between LMWF-treated and untreated cells in HPSE RNA interference condition was compared to that in SNC-siRNA-transfected cells; (C) Migration was assayed in cells treated with or without 10 µg/mL LMWF. Control LMWF induction was arbitrary set at 100% for SNC-siRNA-transfected cells. * p < 0.05, ** p < 0.005 versus SNC-siRNA-transfected control cells. A.U.: arbitrary unit.

Mentions: HPSE-siRNA-transfected cells were used for 2D-angiogenesis or migration assays. Quantitative RT-PCR showed that the expression of the mRNAs encoding for heparanase in HPSE-siRNA-transfected cells was reduced up to 74% ± 8%, as compared to the SNC-siRNA-transfected control cells (Figure 7A). Under basal conditions (in the absence of LMWF), HPSE-siRNA transfection decreased endothelial cell migration by 37% ± 5% (p < 0.05), but had no effect on 2D-angiogenesis (Figure S2A,B). However, upon LMWF stimulation and HPSE-siRNA transfection, the ability of HUVECs to form capillary network in Matrigel 2D-angiogenesis assay was altered. The capillary network length induced by LMWF treatment was largely decreased by 51% ± 11% in HPSE-siRNA-transfected cells, as compared to SNC-siRNA-transfected control cells (Figure 7B). In contrast, the cell migration induced by LMWF was significantly increased by 56% ± 9% (p < 0.05) in HPSE-siRNA-transfected, as compared to SNC-siRNA-transfected cells (Figure 7C).


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)

Assessment of HPSE involvement in biological effects of LMWF in HUVECs. HUVECs were transfected with HPSE-siRNA or SNC-siRNA control. (A) HPSE mRNA levels were determined in HPSE-siRNA- or SNC-siRNA-transfected cells by real-time RT-PCR. HPSE mRNA level normalized to GAPDH mRNA level in SNC-siRNA-transfected control cells was arbitrary set to 1; (B) 2D-angiogenesis was assayed in cells treated with or without 10 µg/mL LMWF. The difference in the capillary network length between LMWF-treated and untreated cells in HPSE RNA interference condition was compared to that in SNC-siRNA-transfected cells; (C) Migration was assayed in cells treated with or without 10 µg/mL LMWF. Control LMWF induction was arbitrary set at 100% for SNC-siRNA-transfected cells. * p < 0.05, ** p < 0.005 versus SNC-siRNA-transfected control cells. A.U.: arbitrary unit.
© Copyright Policy
Related In: Results  -  Collection

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

marinedrugs-13-06588-f007: Assessment of HPSE involvement in biological effects of LMWF in HUVECs. HUVECs were transfected with HPSE-siRNA or SNC-siRNA control. (A) HPSE mRNA levels were determined in HPSE-siRNA- or SNC-siRNA-transfected cells by real-time RT-PCR. HPSE mRNA level normalized to GAPDH mRNA level in SNC-siRNA-transfected control cells was arbitrary set to 1; (B) 2D-angiogenesis was assayed in cells treated with or without 10 µg/mL LMWF. The difference in the capillary network length between LMWF-treated and untreated cells in HPSE RNA interference condition was compared to that in SNC-siRNA-transfected cells; (C) Migration was assayed in cells treated with or without 10 µg/mL LMWF. Control LMWF induction was arbitrary set at 100% for SNC-siRNA-transfected cells. * p < 0.05, ** p < 0.005 versus SNC-siRNA-transfected control cells. A.U.: arbitrary unit.
Mentions: HPSE-siRNA-transfected cells were used for 2D-angiogenesis or migration assays. Quantitative RT-PCR showed that the expression of the mRNAs encoding for heparanase in HPSE-siRNA-transfected cells was reduced up to 74% ± 8%, as compared to the SNC-siRNA-transfected control cells (Figure 7A). Under basal conditions (in the absence of LMWF), HPSE-siRNA transfection decreased endothelial cell migration by 37% ± 5% (p < 0.05), but had no effect on 2D-angiogenesis (Figure S2A,B). However, upon LMWF stimulation and HPSE-siRNA transfection, the ability of HUVECs to form capillary network in Matrigel 2D-angiogenesis assay was altered. The capillary network length induced by LMWF treatment was largely decreased by 51% ± 11% in HPSE-siRNA-transfected cells, as compared to SNC-siRNA-transfected control cells (Figure 7B). In contrast, the cell migration induced by LMWF was significantly increased by 56% ± 9% (p < 0.05) in HPSE-siRNA-transfected, as compared to SNC-siRNA-transfected cells (Figure 7C).

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