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Regulation of pathologic retinal angiogenesis in mice and inhibition of VEGF-VEGFR2 binding by soluble heparan sulfate.

Nishiguchi KM, Kataoka K, Kachi S, Komeima K, Terasaki H - PLoS ONE (2010)

Bottom Line: Intraocular injection of heparan sulfate or heparin alone in these eyes resulted in reduced neovascularization.The binding of VEGF-A and HUVECs was reduced under a high concentration of heparin or ocular fluid compared to lower concentrations of heparin.The recognition that the high concentration of soluble heparan sulfate in the ocular fluid allows it to serve as an endogenous inhibitor of aberrant retinal vascular growth provides a platform for modulating heparan sulfate/heparin levels to regulate angiogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Nagoya University Graduate School of Medicine, Nagoya, Japan. kojinish@med.nagoya-u.ac.jp

ABSTRACT
Development of the retinal vascular network is strictly confined within the neuronal retina, allowing the intraocular media to be optically transparent. However, in retinal ischemia, pro-angiogenic factors (including vascular endothelial growth factor-A, VEGF-A) induce aberrant guidance of retinal vessels into the vitreous. Here, we show that the soluble heparan sulfate level in murine intraocular fluid is high particularly during ocular development. When the eyes of young mice with retinal ischemia were treated with heparan sulfate-degrading enzyme, the subsequent aberrant angiogenesis was greatly enhanced compared to PBS-injected contralateral eyes; however, increased angiogenesis was completely antagonized by simultaneous injection of heparin. Intraocular injection of heparan sulfate or heparin alone in these eyes resulted in reduced neovascularization. In cell cultures, the porcine ocular fluid suppressed the dose-dependent proliferation of human umbilical vein endothelial cells (HUVECs) mediated by VEGF-A. Ocular fluid and heparin also inhibited the migration and tube formation by these cells. The binding of VEGF-A and HUVECs was reduced under a high concentration of heparin or ocular fluid compared to lower concentrations of heparin. In vitro assays demonstrated that the ocular fluid or soluble heparan sulfate or heparin inhibited the binding of VEGF-A and immobilized heparin or VEGF receptor 2 but not VEGF receptor 1. The recognition that the high concentration of soluble heparan sulfate in the ocular fluid allows it to serve as an endogenous inhibitor of aberrant retinal vascular growth provides a platform for modulating heparan sulfate/heparin levels to regulate angiogenesis.

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HS GAG is increased in ocular fluid.(A) Profile of HS proteoglycans in the ocular fluid. Western blotting of the ocular fluid from P15 mice revealed multiple bands that presumably correspond to agrin, collagen XVIII, syndecan-3, syndecan-1, and syndecan -2 [14], [15]. (B) Time-course profile of HS concentration in ocular fluid. HS level was increased in younger mice (P7, P12, and P17; N = 6 each) compared to older animals (P60; N = 5). (C) Comparison of HS concentration among body fluids. HS level was higher in ocular fluid compared to plasma or urine at P17 (N = 7) and P60 (N = 5), respectively. (D) GS staining of retinal quadrants in the OIR model. At the beginning of O2 exposure (P7), retinal vessels are extending toward the periphery (asterisk). The retinal vessels obliterate and regress (outlined by arrows) after exposure to 80% oxygen (P12). Returning the mice to room air results in outgrowth of extra-retinal NV at P17 (NV tufts are outlined by arrowheads in inset). Note that clumps of extra-retinal NV show stronger GS staining compared to intra-retinal vessels. (E) Comparison of HS concentration between samples from OIR and wild-type (WT) mice (N = 7 each). No difference was detected. ON: optic nerve. All statistical data are expressed as mean ± standard error of the mean (S.E.M.).
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pone-0013493-g001: HS GAG is increased in ocular fluid.(A) Profile of HS proteoglycans in the ocular fluid. Western blotting of the ocular fluid from P15 mice revealed multiple bands that presumably correspond to agrin, collagen XVIII, syndecan-3, syndecan-1, and syndecan -2 [14], [15]. (B) Time-course profile of HS concentration in ocular fluid. HS level was increased in younger mice (P7, P12, and P17; N = 6 each) compared to older animals (P60; N = 5). (C) Comparison of HS concentration among body fluids. HS level was higher in ocular fluid compared to plasma or urine at P17 (N = 7) and P60 (N = 5), respectively. (D) GS staining of retinal quadrants in the OIR model. At the beginning of O2 exposure (P7), retinal vessels are extending toward the periphery (asterisk). The retinal vessels obliterate and regress (outlined by arrows) after exposure to 80% oxygen (P12). Returning the mice to room air results in outgrowth of extra-retinal NV at P17 (NV tufts are outlined by arrowheads in inset). Note that clumps of extra-retinal NV show stronger GS staining compared to intra-retinal vessels. (E) Comparison of HS concentration between samples from OIR and wild-type (WT) mice (N = 7 each). No difference was detected. ON: optic nerve. All statistical data are expressed as mean ± standard error of the mean (S.E.M.).

Mentions: Newborn mice lack vascular structures in their retinas. Developing retinal vessels grow radially from the optic nerve toward the periphery along the vitreo-retinal interface, followed by the formation of a deeper vascular plexus over the first 2 weeks after birth. We first assessed the composition of HS proteoglycans secreted in the ocular fluid from a mouse aged Post-natal day 15 (P15). An antibody that detects the urinate residues on the HS core proteins created by the treatment of heparinase III resulting in a specific degradation of HS GAGs was used (Figure 1A) [12]. As with the previous report on vitreous samples from chickens [13], multiple HS core proteins were observed. Soluble agrin, collagen XVIII, or syndecans 1, 2, and 3 were deduced based on their molecular weights from previous reports [14], [15]; many of these proteoglycans have been reported to be expressed in the retina [13], [16]–[18]. Next, we studied the concentrations of HS GAGs in the ocular fluid collected from mice ranging in age from P7 to P60 using an enzyme-linked immunosorbent assay (ELISA) that specifically detects HS polysaccharides (Figure 1B). The level of HS was much higher in younger mice compared to older mice; the level was 15.8-fold higher at P17 compared to P60. The HS concentration in ocular fluid appeared to be uniquely high compared to other body fluids such as plasma (178-fold and 71-fold higher at P17 and P60, respectively) or urine (236-fold and 30-fold higher at P17 and P60, respectively) from the same mice (Figure 1C).


Regulation of pathologic retinal angiogenesis in mice and inhibition of VEGF-VEGFR2 binding by soluble heparan sulfate.

Nishiguchi KM, Kataoka K, Kachi S, Komeima K, Terasaki H - PLoS ONE (2010)

HS GAG is increased in ocular fluid.(A) Profile of HS proteoglycans in the ocular fluid. Western blotting of the ocular fluid from P15 mice revealed multiple bands that presumably correspond to agrin, collagen XVIII, syndecan-3, syndecan-1, and syndecan -2 [14], [15]. (B) Time-course profile of HS concentration in ocular fluid. HS level was increased in younger mice (P7, P12, and P17; N = 6 each) compared to older animals (P60; N = 5). (C) Comparison of HS concentration among body fluids. HS level was higher in ocular fluid compared to plasma or urine at P17 (N = 7) and P60 (N = 5), respectively. (D) GS staining of retinal quadrants in the OIR model. At the beginning of O2 exposure (P7), retinal vessels are extending toward the periphery (asterisk). The retinal vessels obliterate and regress (outlined by arrows) after exposure to 80% oxygen (P12). Returning the mice to room air results in outgrowth of extra-retinal NV at P17 (NV tufts are outlined by arrowheads in inset). Note that clumps of extra-retinal NV show stronger GS staining compared to intra-retinal vessels. (E) Comparison of HS concentration between samples from OIR and wild-type (WT) mice (N = 7 each). No difference was detected. ON: optic nerve. All statistical data are expressed as mean ± standard error of the mean (S.E.M.).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2958111&req=5

pone-0013493-g001: HS GAG is increased in ocular fluid.(A) Profile of HS proteoglycans in the ocular fluid. Western blotting of the ocular fluid from P15 mice revealed multiple bands that presumably correspond to agrin, collagen XVIII, syndecan-3, syndecan-1, and syndecan -2 [14], [15]. (B) Time-course profile of HS concentration in ocular fluid. HS level was increased in younger mice (P7, P12, and P17; N = 6 each) compared to older animals (P60; N = 5). (C) Comparison of HS concentration among body fluids. HS level was higher in ocular fluid compared to plasma or urine at P17 (N = 7) and P60 (N = 5), respectively. (D) GS staining of retinal quadrants in the OIR model. At the beginning of O2 exposure (P7), retinal vessels are extending toward the periphery (asterisk). The retinal vessels obliterate and regress (outlined by arrows) after exposure to 80% oxygen (P12). Returning the mice to room air results in outgrowth of extra-retinal NV at P17 (NV tufts are outlined by arrowheads in inset). Note that clumps of extra-retinal NV show stronger GS staining compared to intra-retinal vessels. (E) Comparison of HS concentration between samples from OIR and wild-type (WT) mice (N = 7 each). No difference was detected. ON: optic nerve. All statistical data are expressed as mean ± standard error of the mean (S.E.M.).
Mentions: Newborn mice lack vascular structures in their retinas. Developing retinal vessels grow radially from the optic nerve toward the periphery along the vitreo-retinal interface, followed by the formation of a deeper vascular plexus over the first 2 weeks after birth. We first assessed the composition of HS proteoglycans secreted in the ocular fluid from a mouse aged Post-natal day 15 (P15). An antibody that detects the urinate residues on the HS core proteins created by the treatment of heparinase III resulting in a specific degradation of HS GAGs was used (Figure 1A) [12]. As with the previous report on vitreous samples from chickens [13], multiple HS core proteins were observed. Soluble agrin, collagen XVIII, or syndecans 1, 2, and 3 were deduced based on their molecular weights from previous reports [14], [15]; many of these proteoglycans have been reported to be expressed in the retina [13], [16]–[18]. Next, we studied the concentrations of HS GAGs in the ocular fluid collected from mice ranging in age from P7 to P60 using an enzyme-linked immunosorbent assay (ELISA) that specifically detects HS polysaccharides (Figure 1B). The level of HS was much higher in younger mice compared to older mice; the level was 15.8-fold higher at P17 compared to P60. The HS concentration in ocular fluid appeared to be uniquely high compared to other body fluids such as plasma (178-fold and 71-fold higher at P17 and P60, respectively) or urine (236-fold and 30-fold higher at P17 and P60, respectively) from the same mice (Figure 1C).

Bottom Line: Intraocular injection of heparan sulfate or heparin alone in these eyes resulted in reduced neovascularization.The binding of VEGF-A and HUVECs was reduced under a high concentration of heparin or ocular fluid compared to lower concentrations of heparin.The recognition that the high concentration of soluble heparan sulfate in the ocular fluid allows it to serve as an endogenous inhibitor of aberrant retinal vascular growth provides a platform for modulating heparan sulfate/heparin levels to regulate angiogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Nagoya University Graduate School of Medicine, Nagoya, Japan. kojinish@med.nagoya-u.ac.jp

ABSTRACT
Development of the retinal vascular network is strictly confined within the neuronal retina, allowing the intraocular media to be optically transparent. However, in retinal ischemia, pro-angiogenic factors (including vascular endothelial growth factor-A, VEGF-A) induce aberrant guidance of retinal vessels into the vitreous. Here, we show that the soluble heparan sulfate level in murine intraocular fluid is high particularly during ocular development. When the eyes of young mice with retinal ischemia were treated with heparan sulfate-degrading enzyme, the subsequent aberrant angiogenesis was greatly enhanced compared to PBS-injected contralateral eyes; however, increased angiogenesis was completely antagonized by simultaneous injection of heparin. Intraocular injection of heparan sulfate or heparin alone in these eyes resulted in reduced neovascularization. In cell cultures, the porcine ocular fluid suppressed the dose-dependent proliferation of human umbilical vein endothelial cells (HUVECs) mediated by VEGF-A. Ocular fluid and heparin also inhibited the migration and tube formation by these cells. The binding of VEGF-A and HUVECs was reduced under a high concentration of heparin or ocular fluid compared to lower concentrations of heparin. In vitro assays demonstrated that the ocular fluid or soluble heparan sulfate or heparin inhibited the binding of VEGF-A and immobilized heparin or VEGF receptor 2 but not VEGF receptor 1. The recognition that the high concentration of soluble heparan sulfate in the ocular fluid allows it to serve as an endogenous inhibitor of aberrant retinal vascular growth provides a platform for modulating heparan sulfate/heparin levels to regulate angiogenesis.

Show MeSH
Related in: MedlinePlus