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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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Heparin and ocular fluid inhibit proliferation of HUVECs.(A) Effect of heparin on the viability of HUVECs. Various concentrations of heparin were added to the cells in EG2 media supplemented with VEGF-A (10 ng/ml). Note viability of HUVECs is suppressed by soluble heparin in a dose-dependent manner. The experiment was done in quadruplicate and repeated. (B) Comparison of viability among HUVECs cultured with different medium. Note that viability of HUVECs is increased with addition of VEGF-A (10 ng/ml) when cultured with EG2 media but not with PBS or ocular fluid (N = 6 for each condition). Viability was low when cultured with PBS. The experiment was repeated. (C) Viability of HUVECs cultured in EG2 media or ocular fluid (OF) with increasing concentrations of VEGF-A. Note that a positive dose-response relationship was observed when cultured in EG2 media, while HUVECs were unresponsive to VEGF-A in the ocular fluid. OD measurements relative to a condition without heparin (A) or VEGF-A (C) are shown in percentages (%). Experiments were done in quadruplicate and repeated. (D,E) Effect of heparin and ocular fluid on scratch wound induced migration of HUVECs. Representative images of HUVECs 24 hrs after creating scratch wound (D). Groove was larger when cells were incubated with ocular fluid (lower panel) compared with PBS (upper panel). Dose-dependent inhibition of cell-migration by heparin was observed with or without co-administration of VEGF-A (E). The assay was done in triplicate and repeated. (F–K) Effects of heparin and ocular fluid on tube formation of HUVECs. Representative images of HUVECs cultured in media with 0 (F), 1,000 (G), and 10,000 (H)µg/ml heparin or in ocular fluid (I), all of which contained VEGF-A. Note that only 10,000 µg/ml heparin completely suppressed tube formation. The degree of tube formation was quantified by measuring tube length (J) and counting branch points (K). At the lowest dose tested, heparin promoted the tube formation while an opposite effect was observed at higher doses in culture media. Note that ocular fluid suppressed angiogenesis incompletely and completely with and without VEGF-A, respectively. The assays were conducted in triplicate and repeated. (L–N) Effects of heparin and ocular fluid on the binding of VEGF-A and HUVECs. Representative images of FACS analyses of fluorescein-labeled VEGF-A and HUVECs showing increased and decreased binding at 10 (M) and 10,000 (N) µg/ml heparin (purple) compared to condition without heparin (white), respectively. Analysis showed that presence of lower concentrations (10 and 100 µg/ml) of heparin promoted the bindings while showing inhibitory effect at the highest dose (10,000 µg/ml; N). Each data point is an average of two measurements. The experiment was repeated. Data presented in (A–C,) (E), (J), and (K) are expressed as mean ± S.E.M. Scale bars indicate 1 mm.
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pone-0013493-g004: Heparin and ocular fluid inhibit proliferation of HUVECs.(A) Effect of heparin on the viability of HUVECs. Various concentrations of heparin were added to the cells in EG2 media supplemented with VEGF-A (10 ng/ml). Note viability of HUVECs is suppressed by soluble heparin in a dose-dependent manner. The experiment was done in quadruplicate and repeated. (B) Comparison of viability among HUVECs cultured with different medium. Note that viability of HUVECs is increased with addition of VEGF-A (10 ng/ml) when cultured with EG2 media but not with PBS or ocular fluid (N = 6 for each condition). Viability was low when cultured with PBS. The experiment was repeated. (C) Viability of HUVECs cultured in EG2 media or ocular fluid (OF) with increasing concentrations of VEGF-A. Note that a positive dose-response relationship was observed when cultured in EG2 media, while HUVECs were unresponsive to VEGF-A in the ocular fluid. OD measurements relative to a condition without heparin (A) or VEGF-A (C) are shown in percentages (%). Experiments were done in quadruplicate and repeated. (D,E) Effect of heparin and ocular fluid on scratch wound induced migration of HUVECs. Representative images of HUVECs 24 hrs after creating scratch wound (D). Groove was larger when cells were incubated with ocular fluid (lower panel) compared with PBS (upper panel). Dose-dependent inhibition of cell-migration by heparin was observed with or without co-administration of VEGF-A (E). The assay was done in triplicate and repeated. (F–K) Effects of heparin and ocular fluid on tube formation of HUVECs. Representative images of HUVECs cultured in media with 0 (F), 1,000 (G), and 10,000 (H)µg/ml heparin or in ocular fluid (I), all of which contained VEGF-A. Note that only 10,000 µg/ml heparin completely suppressed tube formation. The degree of tube formation was quantified by measuring tube length (J) and counting branch points (K). At the lowest dose tested, heparin promoted the tube formation while an opposite effect was observed at higher doses in culture media. Note that ocular fluid suppressed angiogenesis incompletely and completely with and without VEGF-A, respectively. The assays were conducted in triplicate and repeated. (L–N) Effects of heparin and ocular fluid on the binding of VEGF-A and HUVECs. Representative images of FACS analyses of fluorescein-labeled VEGF-A and HUVECs showing increased and decreased binding at 10 (M) and 10,000 (N) µg/ml heparin (purple) compared to condition without heparin (white), respectively. Analysis showed that presence of lower concentrations (10 and 100 µg/ml) of heparin promoted the bindings while showing inhibitory effect at the highest dose (10,000 µg/ml; N). Each data point is an average of two measurements. The experiment was repeated. Data presented in (A–C,) (E), (J), and (K) are expressed as mean ± S.E.M. Scale bars indicate 1 mm.

Mentions: Next, we assessed the effect of ocular fluid and soluble heparin on the VEGF-A-mediated viability of human umbilical vein endothelial cells (HUVECs). Culture studies are suitable for reproducing the unique ocular environment in which acellular vitreous/ocular fluid overlays the plane of retinal vascular network. HUVECs were incubated with increasing concentrations of heparin in a standard endothelial culture media (EG2) supplemented with VEGF-A. The viability of HUVECs showed an inverse dose response to heparin, which revealed 31.2% reduction in optical density (OD) measurements at a heparin level (1,000 µg/ml) relevant to the HS concentrations in the ocular fluid from OIR models (Figure 4A).


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)

Heparin and ocular fluid inhibit proliferation of HUVECs.(A) Effect of heparin on the viability of HUVECs. Various concentrations of heparin were added to the cells in EG2 media supplemented with VEGF-A (10 ng/ml). Note viability of HUVECs is suppressed by soluble heparin in a dose-dependent manner. The experiment was done in quadruplicate and repeated. (B) Comparison of viability among HUVECs cultured with different medium. Note that viability of HUVECs is increased with addition of VEGF-A (10 ng/ml) when cultured with EG2 media but not with PBS or ocular fluid (N = 6 for each condition). Viability was low when cultured with PBS. The experiment was repeated. (C) Viability of HUVECs cultured in EG2 media or ocular fluid (OF) with increasing concentrations of VEGF-A. Note that a positive dose-response relationship was observed when cultured in EG2 media, while HUVECs were unresponsive to VEGF-A in the ocular fluid. OD measurements relative to a condition without heparin (A) or VEGF-A (C) are shown in percentages (%). Experiments were done in quadruplicate and repeated. (D,E) Effect of heparin and ocular fluid on scratch wound induced migration of HUVECs. Representative images of HUVECs 24 hrs after creating scratch wound (D). Groove was larger when cells were incubated with ocular fluid (lower panel) compared with PBS (upper panel). Dose-dependent inhibition of cell-migration by heparin was observed with or without co-administration of VEGF-A (E). The assay was done in triplicate and repeated. (F–K) Effects of heparin and ocular fluid on tube formation of HUVECs. Representative images of HUVECs cultured in media with 0 (F), 1,000 (G), and 10,000 (H)µg/ml heparin or in ocular fluid (I), all of which contained VEGF-A. Note that only 10,000 µg/ml heparin completely suppressed tube formation. The degree of tube formation was quantified by measuring tube length (J) and counting branch points (K). At the lowest dose tested, heparin promoted the tube formation while an opposite effect was observed at higher doses in culture media. Note that ocular fluid suppressed angiogenesis incompletely and completely with and without VEGF-A, respectively. The assays were conducted in triplicate and repeated. (L–N) Effects of heparin and ocular fluid on the binding of VEGF-A and HUVECs. Representative images of FACS analyses of fluorescein-labeled VEGF-A and HUVECs showing increased and decreased binding at 10 (M) and 10,000 (N) µg/ml heparin (purple) compared to condition without heparin (white), respectively. Analysis showed that presence of lower concentrations (10 and 100 µg/ml) of heparin promoted the bindings while showing inhibitory effect at the highest dose (10,000 µg/ml; N). Each data point is an average of two measurements. The experiment was repeated. Data presented in (A–C,) (E), (J), and (K) are expressed as mean ± S.E.M. Scale bars indicate 1 mm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2958111&req=5

pone-0013493-g004: Heparin and ocular fluid inhibit proliferation of HUVECs.(A) Effect of heparin on the viability of HUVECs. Various concentrations of heparin were added to the cells in EG2 media supplemented with VEGF-A (10 ng/ml). Note viability of HUVECs is suppressed by soluble heparin in a dose-dependent manner. The experiment was done in quadruplicate and repeated. (B) Comparison of viability among HUVECs cultured with different medium. Note that viability of HUVECs is increased with addition of VEGF-A (10 ng/ml) when cultured with EG2 media but not with PBS or ocular fluid (N = 6 for each condition). Viability was low when cultured with PBS. The experiment was repeated. (C) Viability of HUVECs cultured in EG2 media or ocular fluid (OF) with increasing concentrations of VEGF-A. Note that a positive dose-response relationship was observed when cultured in EG2 media, while HUVECs were unresponsive to VEGF-A in the ocular fluid. OD measurements relative to a condition without heparin (A) or VEGF-A (C) are shown in percentages (%). Experiments were done in quadruplicate and repeated. (D,E) Effect of heparin and ocular fluid on scratch wound induced migration of HUVECs. Representative images of HUVECs 24 hrs after creating scratch wound (D). Groove was larger when cells were incubated with ocular fluid (lower panel) compared with PBS (upper panel). Dose-dependent inhibition of cell-migration by heparin was observed with or without co-administration of VEGF-A (E). The assay was done in triplicate and repeated. (F–K) Effects of heparin and ocular fluid on tube formation of HUVECs. Representative images of HUVECs cultured in media with 0 (F), 1,000 (G), and 10,000 (H)µg/ml heparin or in ocular fluid (I), all of which contained VEGF-A. Note that only 10,000 µg/ml heparin completely suppressed tube formation. The degree of tube formation was quantified by measuring tube length (J) and counting branch points (K). At the lowest dose tested, heparin promoted the tube formation while an opposite effect was observed at higher doses in culture media. Note that ocular fluid suppressed angiogenesis incompletely and completely with and without VEGF-A, respectively. The assays were conducted in triplicate and repeated. (L–N) Effects of heparin and ocular fluid on the binding of VEGF-A and HUVECs. Representative images of FACS analyses of fluorescein-labeled VEGF-A and HUVECs showing increased and decreased binding at 10 (M) and 10,000 (N) µg/ml heparin (purple) compared to condition without heparin (white), respectively. Analysis showed that presence of lower concentrations (10 and 100 µg/ml) of heparin promoted the bindings while showing inhibitory effect at the highest dose (10,000 µg/ml; N). Each data point is an average of two measurements. The experiment was repeated. Data presented in (A–C,) (E), (J), and (K) are expressed as mean ± S.E.M. Scale bars indicate 1 mm.
Mentions: Next, we assessed the effect of ocular fluid and soluble heparin on the VEGF-A-mediated viability of human umbilical vein endothelial cells (HUVECs). Culture studies are suitable for reproducing the unique ocular environment in which acellular vitreous/ocular fluid overlays the plane of retinal vascular network. HUVECs were incubated with increasing concentrations of heparin in a standard endothelial culture media (EG2) supplemented with VEGF-A. The viability of HUVECs showed an inverse dose response to heparin, which revealed 31.2% reduction in optical density (OD) measurements at a heparin level (1,000 µg/ml) relevant to the HS concentrations in the ocular fluid from OIR models (Figure 4A).

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