Limits...
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.

Show MeSH

Related in: MedlinePlus

Soluble heparin-bound VEGF-A is inactivated but is also protected from proteolysis.(A) VEGF-A levels in the ocular fluid in OIR mice. VEGF-A concentration was determined at P8, P12, and P14 from wild-type control (WT) and OIR model, respectively (N = 6 each). (B,C) VEGF-A levels in the ocular fluid collected at P14 from OIR mice treated with heparinase III (HIII; N = 8) or heparin (Hep; N = 7) at P12. Intra-ocular heparin injection resulted in greatly increased VEGF-A levels (C) while levels were not affected by HIII treatment (B). (D,E) Kinetics of human VEGF-A in murine eyes at 6 and 48 hours after intraocular injection. Human isoform VEGF121 (5 ng/eye) was injected with heparin (20 µg/eye) in one eye and without in the other at P12. Ocular fluid was analyzed with human VEGF-specific ELISA at 6 and 48 hours later (N = 6 and N = 5, respectively). An identical experiment was conducted using human VEGF165 (5 ng/eye; N = 6 each). Only eyes co-injected with VEGF165 and heparin and analyzed at 6 hours post-injection showed increased human VEGF-A (D). The left and right vertical axes show concentration (ng/ml) and proportion (% relative to injected amount) of the remaining VEGF-A in the ocular fluid, respectively. Human VEGF-A was not detected in the uninjected control (N = 6). (F,G) The effect of heparin on in vitro degradation of VEGF-A by plasmin. VEGF165 (F), or VEGF121(G) was subjected to proteolysis by plasmin (400, 80, 16, and 3.2 ng for lanes 1 and 5, 2 and 6, 3 and 7, and 4 and 8, respectively). Undigested VEGF121 and VEGF165 were applied as references (arrowheads). All statistical data are expressed as mean ± S.E.M.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2958111&req=5

pone-0013493-g003: Soluble heparin-bound VEGF-A is inactivated but is also protected from proteolysis.(A) VEGF-A levels in the ocular fluid in OIR mice. VEGF-A concentration was determined at P8, P12, and P14 from wild-type control (WT) and OIR model, respectively (N = 6 each). (B,C) VEGF-A levels in the ocular fluid collected at P14 from OIR mice treated with heparinase III (HIII; N = 8) or heparin (Hep; N = 7) at P12. Intra-ocular heparin injection resulted in greatly increased VEGF-A levels (C) while levels were not affected by HIII treatment (B). (D,E) Kinetics of human VEGF-A in murine eyes at 6 and 48 hours after intraocular injection. Human isoform VEGF121 (5 ng/eye) was injected with heparin (20 µg/eye) in one eye and without in the other at P12. Ocular fluid was analyzed with human VEGF-specific ELISA at 6 and 48 hours later (N = 6 and N = 5, respectively). An identical experiment was conducted using human VEGF165 (5 ng/eye; N = 6 each). Only eyes co-injected with VEGF165 and heparin and analyzed at 6 hours post-injection showed increased human VEGF-A (D). The left and right vertical axes show concentration (ng/ml) and proportion (% relative to injected amount) of the remaining VEGF-A in the ocular fluid, respectively. Human VEGF-A was not detected in the uninjected control (N = 6). (F,G) The effect of heparin on in vitro degradation of VEGF-A by plasmin. VEGF165 (F), or VEGF121(G) was subjected to proteolysis by plasmin (400, 80, 16, and 3.2 ng for lanes 1 and 5, 2 and 6, 3 and 7, and 4 and 8, respectively). Undigested VEGF121 and VEGF165 were applied as references (arrowheads). All statistical data are expressed as mean ± S.E.M.

Mentions: VEGF-A, a heparin/HS-binding glycoprotein, is the major pro-angiogenic growth factor that controls the development of extra-retinal NV in OIR [21], [22], [25]. The dominant role of this protein in the OIR model was confirmed by the intraocular injection of soluble VEGF receptor 1 (VEGFR1), a potent antagonist of VEGF-A, which resulted in 98.0% reduction in NV (Figure S1). Since HS/heparin GAGs are known to influence VEGF-A signaling, it is important to study the interactions of the related molecules to further understand the mechanistic basis for the anti-angiogenic role of soluble GAGs in the eye. First, we analyzed the level of VEGF-A in the ocular fluid in an OIR model and in wild-type controls. VEGF-A concentrations in the ocular fluid were reduced with exposure to 80% oxygen at P8 and P12 (Figure 3A). The subsequent introduction of the mice to room air rapidly increased VEGF-A level, resulting in 9.7-fold higher concentration compared to that of controls at P14. These observations are consistent with the reported alteration of VEGF-A production in the retina of OIR mice [19], [21], [22]. Next, we studied VEGF-A levels in the ocular fluid at P14, two days after the administration of heparinase III at P12 (immediately after returning the mice to room air), where elevated VEGF-A levels in OIR was not affected by heparinase III treatment, i.e., no difference was detected compared with PBS-treated eyes (Figure 3B). However, the ocular fluid from the heparin-injected eyes revealed a further 11.3-fold increase in VEGF-A concentration compared with the control OIR eyes with high VEGF-A levels (Figure 3C). Similarly, VEGF-A levels in the ocular fluid were increased by 2.6-fold (P<0.001) in the eyes injected with high-dose HS (4,085±232 pg/ml: mean ± S.E.M.) compared with PBS-treated OIR eyes (1,550±94 pg/ml). These paradoxical observations showing greatly increased VEGF-A in the heparin- or HS-treated eyes with reduced NV indicated that GAGs inhibit the function of VEGF-A as a vascular endothelial mitogen.


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)

Soluble heparin-bound VEGF-A is inactivated but is also protected from proteolysis.(A) VEGF-A levels in the ocular fluid in OIR mice. VEGF-A concentration was determined at P8, P12, and P14 from wild-type control (WT) and OIR model, respectively (N = 6 each). (B,C) VEGF-A levels in the ocular fluid collected at P14 from OIR mice treated with heparinase III (HIII; N = 8) or heparin (Hep; N = 7) at P12. Intra-ocular heparin injection resulted in greatly increased VEGF-A levels (C) while levels were not affected by HIII treatment (B). (D,E) Kinetics of human VEGF-A in murine eyes at 6 and 48 hours after intraocular injection. Human isoform VEGF121 (5 ng/eye) was injected with heparin (20 µg/eye) in one eye and without in the other at P12. Ocular fluid was analyzed with human VEGF-specific ELISA at 6 and 48 hours later (N = 6 and N = 5, respectively). An identical experiment was conducted using human VEGF165 (5 ng/eye; N = 6 each). Only eyes co-injected with VEGF165 and heparin and analyzed at 6 hours post-injection showed increased human VEGF-A (D). The left and right vertical axes show concentration (ng/ml) and proportion (% relative to injected amount) of the remaining VEGF-A in the ocular fluid, respectively. Human VEGF-A was not detected in the uninjected control (N = 6). (F,G) The effect of heparin on in vitro degradation of VEGF-A by plasmin. VEGF165 (F), or VEGF121(G) was subjected to proteolysis by plasmin (400, 80, 16, and 3.2 ng for lanes 1 and 5, 2 and 6, 3 and 7, and 4 and 8, respectively). Undigested VEGF121 and VEGF165 were applied as references (arrowheads). All statistical data are expressed as mean ± S.E.M.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2958111&req=5

pone-0013493-g003: Soluble heparin-bound VEGF-A is inactivated but is also protected from proteolysis.(A) VEGF-A levels in the ocular fluid in OIR mice. VEGF-A concentration was determined at P8, P12, and P14 from wild-type control (WT) and OIR model, respectively (N = 6 each). (B,C) VEGF-A levels in the ocular fluid collected at P14 from OIR mice treated with heparinase III (HIII; N = 8) or heparin (Hep; N = 7) at P12. Intra-ocular heparin injection resulted in greatly increased VEGF-A levels (C) while levels were not affected by HIII treatment (B). (D,E) Kinetics of human VEGF-A in murine eyes at 6 and 48 hours after intraocular injection. Human isoform VEGF121 (5 ng/eye) was injected with heparin (20 µg/eye) in one eye and without in the other at P12. Ocular fluid was analyzed with human VEGF-specific ELISA at 6 and 48 hours later (N = 6 and N = 5, respectively). An identical experiment was conducted using human VEGF165 (5 ng/eye; N = 6 each). Only eyes co-injected with VEGF165 and heparin and analyzed at 6 hours post-injection showed increased human VEGF-A (D). The left and right vertical axes show concentration (ng/ml) and proportion (% relative to injected amount) of the remaining VEGF-A in the ocular fluid, respectively. Human VEGF-A was not detected in the uninjected control (N = 6). (F,G) The effect of heparin on in vitro degradation of VEGF-A by plasmin. VEGF165 (F), or VEGF121(G) was subjected to proteolysis by plasmin (400, 80, 16, and 3.2 ng for lanes 1 and 5, 2 and 6, 3 and 7, and 4 and 8, respectively). Undigested VEGF121 and VEGF165 were applied as references (arrowheads). All statistical data are expressed as mean ± S.E.M.
Mentions: VEGF-A, a heparin/HS-binding glycoprotein, is the major pro-angiogenic growth factor that controls the development of extra-retinal NV in OIR [21], [22], [25]. The dominant role of this protein in the OIR model was confirmed by the intraocular injection of soluble VEGF receptor 1 (VEGFR1), a potent antagonist of VEGF-A, which resulted in 98.0% reduction in NV (Figure S1). Since HS/heparin GAGs are known to influence VEGF-A signaling, it is important to study the interactions of the related molecules to further understand the mechanistic basis for the anti-angiogenic role of soluble GAGs in the eye. First, we analyzed the level of VEGF-A in the ocular fluid in an OIR model and in wild-type controls. VEGF-A concentrations in the ocular fluid were reduced with exposure to 80% oxygen at P8 and P12 (Figure 3A). The subsequent introduction of the mice to room air rapidly increased VEGF-A level, resulting in 9.7-fold higher concentration compared to that of controls at P14. These observations are consistent with the reported alteration of VEGF-A production in the retina of OIR mice [19], [21], [22]. Next, we studied VEGF-A levels in the ocular fluid at P14, two days after the administration of heparinase III at P12 (immediately after returning the mice to room air), where elevated VEGF-A levels in OIR was not affected by heparinase III treatment, i.e., no difference was detected compared with PBS-treated eyes (Figure 3B). However, the ocular fluid from the heparin-injected eyes revealed a further 11.3-fold increase in VEGF-A concentration compared with the control OIR eyes with high VEGF-A levels (Figure 3C). Similarly, VEGF-A levels in the ocular fluid were increased by 2.6-fold (P<0.001) in the eyes injected with high-dose HS (4,085±232 pg/ml: mean ± S.E.M.) compared with PBS-treated OIR eyes (1,550±94 pg/ml). These paradoxical observations showing greatly increased VEGF-A in the heparin- or HS-treated eyes with reduced NV indicated that GAGs inhibit the function of VEGF-A as a vascular endothelial mitogen.

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