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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 heparanase in HUVECs. (A) HPSE mRNA levels were determined by real-time RT-PCR in cells treated with or without 10 µg/mL LMWF; (B) HPSE protein levels were determined by western blot in the supernatant or in the lysate of cells treated with or without 10 µg/mL LMWF. Lower panel shows a representative image of the western blot assay. (C) Heparanase activity was checked in the lysate of LMWF-treated cells. HPSE activity in untreated cells was arbitrary set to 100%. * p < 0.05, *** p < 0.0005, LMWF-treated cells versus LMWF-untreated cells (UT). A.U.: arbitrary unit.
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marinedrugs-13-06588-f003: LMWF and heparanase in HUVECs. (A) HPSE mRNA levels were determined by real-time RT-PCR in cells treated with or without 10 µg/mL LMWF; (B) HPSE protein levels were determined by western blot in the supernatant or in the lysate of cells treated with or without 10 µg/mL LMWF. Lower panel shows a representative image of the western blot assay. (C) Heparanase activity was checked in the lysate of LMWF-treated cells. HPSE activity in untreated cells was arbitrary set to 100%. * p < 0.05, *** p < 0.0005, LMWF-treated cells versus LMWF-untreated cells (UT). A.U.: arbitrary unit.

Mentions: The HS-degrading enzyme heparanase (HPSE) is an endo-β-d-glucuronidase, which plays an important role in remodeling of the basement membrane and extracellular matrix during process of inflammation [13,14,15]. HPSE is synthesized as an inactive 65 kDa pro-form enzyme (pro-HPSE), can be transformed into active heterodimer consisting of 50 and 8 kDa subunits (active HPSE), and cleaves HS chains attached to proteoglycans, such as syndecans and perlecan [15,16]. HPSE mRNA expression, as assessed by quantitative RT-PCR, was increased by 2.4 fold in LMWF-treated cells, as compared to untreated control cells (Figure 3A). Active HPSE form (50 kDa) expression, as assessed by western blot, was significantly increased up to 2 fold in the supernatant or by 20% ± 5% in the lysate of the LMWF-treated cells (Figure 4B). HPSE activity was slightly but significantly increased up to 20% ± 3% in the lysate of LMWF-treated cells (Figure 3C), whereas it was not detected in the respective conditioned medium.


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 heparanase in HUVECs. (A) HPSE mRNA levels were determined by real-time RT-PCR in cells treated with or without 10 µg/mL LMWF; (B) HPSE protein levels were determined by western blot in the supernatant or in the lysate of cells treated with or without 10 µg/mL LMWF. Lower panel shows a representative image of the western blot assay. (C) Heparanase activity was checked in the lysate of LMWF-treated cells. HPSE activity in untreated cells was arbitrary set to 100%. * p < 0.05, *** p < 0.0005, LMWF-treated cells versus LMWF-untreated cells (UT). A.U.: arbitrary unit.
© Copyright Policy
Related In: Results  -  Collection

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

marinedrugs-13-06588-f003: LMWF and heparanase in HUVECs. (A) HPSE mRNA levels were determined by real-time RT-PCR in cells treated with or without 10 µg/mL LMWF; (B) HPSE protein levels were determined by western blot in the supernatant or in the lysate of cells treated with or without 10 µg/mL LMWF. Lower panel shows a representative image of the western blot assay. (C) Heparanase activity was checked in the lysate of LMWF-treated cells. HPSE activity in untreated cells was arbitrary set to 100%. * p < 0.05, *** p < 0.0005, LMWF-treated cells versus LMWF-untreated cells (UT). A.U.: arbitrary unit.
Mentions: The HS-degrading enzyme heparanase (HPSE) is an endo-β-d-glucuronidase, which plays an important role in remodeling of the basement membrane and extracellular matrix during process of inflammation [13,14,15]. HPSE is synthesized as an inactive 65 kDa pro-form enzyme (pro-HPSE), can be transformed into active heterodimer consisting of 50 and 8 kDa subunits (active HPSE), and cleaves HS chains attached to proteoglycans, such as syndecans and perlecan [15,16]. HPSE mRNA expression, as assessed by quantitative RT-PCR, was increased by 2.4 fold in LMWF-treated cells, as compared to untreated control cells (Figure 3A). Active HPSE form (50 kDa) expression, as assessed by western blot, was significantly increased up to 2 fold in the supernatant or by 20% ± 5% in the lysate of the LMWF-treated cells (Figure 4B). HPSE activity was slightly but significantly increased up to 20% ± 3% in the lysate of LMWF-treated cells (Figure 3C), whereas it was not detected in the respective conditioned medium.

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