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Binding inhibition of angiogenic factors by heparan sulfate proteoglycans in aqueous humor: potential mechanism for maintenance of an avascular environment¹

MICHAEL FANNON^*,2, KIMBERLY FORSTEN-WILLIAMS, CHRISTOPHER J. DOWD^¶, DEBORAH A. FREEDMAN^*, JUDAH FOLKMAN^* and MATTHEW A. NUGENT^¶

^* Department of Surgery, The Children’s Hospital, Boston, Massachusetts, USA;
Department of Chemical Engineering, Virginia Polytechnic Institute and State University, Blacksburg, Virginia, USA; and
^¶ Departments of Biochemistry and Ophthalmology, Boston University School of Medicine, Boston, Massachusetts, USA

²Correspondence: Department of Surgery, The Children’s Hospital, 300 Longwood Ave., Boston, MA 02115, USA. E-mail: michael.fannon{at}tch.harvard.edu

SPECIFIC AIMS

We hypothesized that there are components in the aqueous humor that play a role in maintaining the avascular environment of the cornea. We identified heparan sulfate proteoglycans in aqueous humor and demonstrated that they potently inhibit the binding of heparin binding growth factors to cell surfaces. We show with mathematical modeling that this inhibitory activity, combined with the rate of clearance of aqueous humor, can regulate bioavailability of angiogenic factors in the cornea.

PRINCIPAL FINDINGS

1. Aqueous humor inhibits binding of heparin binding growth factors
We found that bovine aqueous humor is a potent inhibitor of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) binding, both potent stimulators of angiogenesis in vitro and in vivo. These angiogenic growth factors bind to specific receptor proteins and cell surface heparan sulfate proteoglycans (HSPG) on the surface of endothelial cells. We investigated the binding of ¹²⁵I-bFGF and ¹²⁵I-VEGF to bovine aortic endothelial (BAE) cells in the presence and absence of aqueous humor. We observed potent inhibition of both ¹²⁵I-bFGF and ¹²⁵I-VEGF binding by aqueous humor in a dose-dependent manner. This suggests the possibility of potent inhibition of their tyrosine kinase signaling in an environment bathed exclusively in aqueous humor.

2. Heparan sulfate is required for inhibitory activity
We previously showed that the presence of heparin can either potentiate or inhibit bFGF binding in vitro depending on HSPG levels. Therefore, we hypothesized that the ability of aqueous humor to inhibit growth factor binding might depend on HSPG. Measurements of total glycosaminoglycan (GAG) and heparinase-sensitive GAG revealed significant quantities (10 µg/mL total GAG and 4 µg/mL heparan sulfate) of soluble proteoglycans in aqueous humor. Immunoblotting verified the presence of HSPG in aqueous humor. To evaluate the importance of HSPG, we treated aqueous humor with heparinase III, which specifically digests heparan sulfate. The heparinase was heat-inactivated before applying the aqueous humor to cells to prevent cleavage of cell surface HSPG. As shown in Fig. 1 , heat-treated aqueous humor at a concentration of 10% was a potent inhibitor of ¹²⁵I-bFGF binding to cell surfaces similar to untreated aqueous humor. However, heparinase III treatment resulted in complete ablation of the inhibitory activity of the aqueous humor. This suggested that the inhibition observed was due to or required heparan sulfate in the aqueous humor.

View larger version (65K): [in this window] [in a new window]   Figure 1. Effect of heparinase III on aqueous humor activity. Aqueous humor was treated with heparinase III or not. Treated and untreated aqueous humor samples were then heat-treated to destroy heparinase activity. The ability to inhibit 125I-bFGF binding (10 ng/mL) to BAE cells was tested. BAE cells were incubated at 4°C for 2 h with 125I-bFGF (10 ng/mL) in the presence of 10% aqueous humor. Cells were washed with cold buffer, then HSPG-bound 125I-bFGF was removed by a high salt wash. Error is shown as ± SD.

3. The inhibitory activity of aqueous humor is not endothelial cell specific
We speculated that the HSPG in aqueous humor required to inhibit bFGF and VEGF binding on BAE cells might function to inhibit the binding of other heparin binding growth factors in other cell systems, since nearly all cells express heparan sulfate. We evaluated the binding of ¹²⁵I-HB-EGF, which is heparin binding, and ¹²⁵I-EGF, which is not, to human foreskin fibroblasts and observed that aqueous humor inhibited HB-EGF binding but had little effect on EGF binding.

4. Purified proteoglycan from aqueous humor inhibits bFGF binding
To verify that proteoglycans are in aqueous humor and are responsible for the activity, we purified proteoglycans from aqueous humor. In ¹²⁵I-bFGF binding studies of bovine capillary endothelial (BCE) cells, the inhibitory activity of the purified proteoglycan was similar to that observed with the complete aqueous humor. In contrast, bovine corneal-derived keratan sulfate proteoglycan, a proteoglycan that does not bind bFGF or VEGF, showed no significant inhibitory activity.

5. Purified proteoglycan from conditioned medium of corneal endothelium inhibits bFGF binding
The presence of HSPG in aqueous humor does not indicate whether it is brought in through the blood filtration process, synthesized and secreted locally, or both. To determine whether corneal endothelial cells have the potential to produce HSPG capable of inhibiting bFGF binding, we purified proteoglycans from conditioned medium. ¹²⁵I-bFGF binding studies of BCE cells comparing the proteoglycans isolated from corneal endothelial cell conditioned medium with those isolated from aqueous humor showed similar dose response curves, suggesting that the corneal endothelial cells could, either in whole or in part, produce the HSPG found in aqueous humor.

7. A model for bFGF–HSPG interactions in solution
We developed a model to determine whether competitive binding of growth factors by aqueous humor HSPG in solution could eliminate growth factor binding to cells at 37°C using a time frame (4 h) that would allow for removal of bFG–-HSPG complexes through aqueous humor replenishment. We further evaluated how this activity would depend on the concentrations of growth factor and HSPG. At the relatively low bFGF concentration (1 ng/mL) measured in vivo in a nonpathological state, our model predicts that HSPG in aqueous humor, at the estimated concentration of 4 µg/mL, would potently inhibit bFGF binding to its receptors in a time frame that would allow clearance of soluble bFGF–HSPG complexes before bFGF–cell surface interactions could efficiently take place (Fig. 2 A). When the cumulative level of bFGF–FGF receptor complexes on the cell surface that formed during a 4 h period was evaluated as a function of aqueous humor HSPG level (Fig. 2B ), an IC₅₀ of 0.1 µg/mL is predicted. Thus, under these conditions the level of HSPG within aqueous humor (4 µg/mL) would be sufficient to inhibit bFGF binding over 4 h. If the concentration of bFGF increased, as occurs during certain pathological conditions, the IC₅₀ for HSPG would increase also.

View larger version (19K): [in this window] [in a new window]   Figure 2. Computational modeling of bFGF binding to cell surface receptors. Computational modeling was generated for bFGF binding to cell surface receptors in the absence () and presence (•) of aqueous humor HSPG. A) The number of FGFR–bFGF complexes on the cell surface as a function of time where both bFGF (1 ng/mL) and solution HSPG (4 µg/mL) are introduced at time 0. Values are scaled to the peak value (set at 100%) in the absence of solution HSPG. Peak value in the presence of aqueous humor HSPG is 1.6%. B) Cumulative FGFR–bFGF complexes over 4 h in the presence of solution HSPG. Cumulative FGFR–bFGF complexes represent the total number of FGFR–bFGF complexes formed over the 4 h period when the cells were exposed to bFGF. Values are scaled to the cumulative value in the absence of solution HSPG (set at 100% of control).

CONCLUSIONS

We have found that bovine aqueous humor can dramatically inhibit the binding of heparin binding growth factors to cell surface HSPG and receptors. We have also shown that aqueous humor contains a considerable amount (3–10 µg/mL) of heparan sulfate proteoglycans. Moreover, proteoglycans isolated from aqueous humor were shown to be potent inhibitors of the binding of bFGF, VEGF, and HB-EGF to cell surfaces. We did not determine whether these proteoglycans are produced locally in the anterior chamber, diffuse in through the normal circulatory process, or both, but we have shown that corneal endothelial cells grown in culture produce and secrete proteoglycans with activity similar to those within aqueous humor. We are currently designing an in vitro system to accurately reflect the effect of flow rate on growth factor binding to varying HSPG and growth factor concentrations at 37°C. Such studies will enable us to test the wide experimental parameters presented in the computer model. In vivo experiments using soluble heparin and heparan sulfate with concomitant administration of growth factors could enable clearance levels to be measured over an extended range of concentrations.

A well-established role of the circulatory system is to clear harmful agents and waste products from local environments. The replenishment of aqueous humor, coupled with the specific ability of the heparan sulfate within it to bind and sequester growth factors, offers the possibility of an active, constitutive regulatory process in the eye. This might suggest a more general process whereby HSPG could play important roles in regulating growth factors in blood and lymph as well.

There are some diseases, such as hemangioma and many cancers, in which elevated levels of bFGF and VEGF in the blood and urine are routinely present. Circulating HSPG might represent a strategy to sequester growth factors in blood and clear them, preventing their interaction with vessel walls. The effects of excess growth factors in disease, as well as studies using excess heparin in vivo, suggest that the balance can be shifted toward stimulation or inhibition of angiogenesis. This process might explain the inhibitory effect of heparin treatment on the growth of some tumors.

Some positive roles of HSPG on heparin binding growth factors have been documented. Heparin and heparan sulfate protect growth factors from degradation and increase their half-life in vitro. However, in vivo, the flow rate of aqueous humor from the eye is rapid, which would clear these molecules from the eye before they can interact with the cornea. We propose that the combination of high-capacity heparan sulfate sites in aqueous humor coupled with a rapid clearance rate constitutes a physiological regulatory process that aids in maintaining the avascular nature of the cornea. Exploitation of this balance might be an effective way to treat some diseases.

View larger version (26K): [in this window] [in a new window]   Figure 3. Model of the role of aqueous humor in corneal avascularity. Based on experimental parameters, our model predicts that soluble heparan sulfate in aqueous humor competes with cell surface interactions of heparin binding growth factors. The growth factors are sequestered in solution and cleared from the anterior chamber by normal aqueous humor circulation before they can form active complexes at cell surfaces.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0935fje; to cite this article, use FASEB J. (March 5, 2003) 10.1096/fj.02-0935fje

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