Hypoxia-Inducible Factor-Dependent Expression of Angiopoietin-Like 4 by Conjunctival Epithelial Cells Promotes the Angiogenic Phenotype of Pterygia.

Figures

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  Figure 1. HIF-1α expression is detected in conjunctival epithelium from surgically excised pterygia. (A) Immunohistochemical staining of the “apex” of a pterygium for CD34 highlighting vascular ECs in the fibrovascular stroma. VEGF and HIF-1α expression is detected in the overlying epithelium. IgG was used as a negative control. (B) Immunohistochemical staining of the “body” of a pterygium similarly demonstrates expression of VEGF and HIF-1α in the CjECs. Similar results were observed in 6/6 pterygia. (C) Immunohistochemical staining of VEGF and HIF-1α in “normal” conjunctiva of autopsy eyes with no known history of anterior segment disease. Similar results were observed in 4/4 autopsy eyes.

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  Figure 2. Accumulation of HIF-1α in cultured CjECs results in the secretion of angiogenic mediators. (A) Immunoblot for HIF-1α in ihCjECs exposed to hypoxia (1% O2) or a HIF inducer (100 μM DFO or 300 μM DMOG), in normoxia (20% O2) for 4 hours. One hundred nanomolar digoxin was used to inhibit HIF-1α accumulation. (B) Immunoblot for HIF-1α in prCjECs exposed to 1% O2 or 300 μM DMOG for 4 hours. (C) H&E stain of stratified prCjECs grown on vitrigel. (D) Immunoblot for HIF-1α in stratified prCjECs grown on vitrigel exposed to 1% O2 or 300 μM DMOG for 4 hours. (E–H) EC tubule formation by HMVECs treated with conditioned media from ihCjECs exposed to 1% O2 (E, F) or 100 μM DFO or 300 μM DMOG (G, H), in the absence (E, G) or presence (F, H) of 100 nM digoxin, compared to media conditioned by cells exposed to 20% O2 for 24 hours. Ten percent FBS was used as a positive control.

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  Figure 3. VEGF expression by cultured ihCjECs is not sufficient to explain the angiogenic potential of these cells. (A, B) VEGF mRNA expression in ihCjECs (A) or prCjECs (B) exposed to 1% O2, 100 μM DFO, or 300 μM DMOG for 8, 24, or 48 hours. (C) VEGF protein secretion in ihCjECs exposed to 1% O2, 100 μM DFO, or 300 μM DMOG for 24 hours. One hundred nanomolar digoxin was used to inhibit HIF-1α accumulation. (D, E) VEGF mRNA expression (D) and protein secretion (E) compared by ihCjECs transfected with RNAi targeting VEGF and exposed to 1% O2 for 24 hours. Mock transfected (Mock) or scrambled RNAi (Scr) transfected cells were used as a control. (F, G) EC tubule formation by HMVECs treated with conditioned media from ihCjECs transfected with VEGF RNAi and exposed to 1% O2 for 24 hours reported as fold induction (F) or percent reduction (G).

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  Figure 4. ANGPTL4 secretion from cultured CjECs is markedly increased in response to HIF-1α accumulation. (A–D) ANGPTL4 mRNA expression (A, C) and protein secretion (B, D) by ihCjECs exposed to 1% O2, 100 μM DFO, or 300 μM DMOG in the absence (A, B) or presence (C, D) of 100 nM digoxin. (E) ANGPTL4 mRNA expression by prCjECs exposed to 1% O2 or 300 μM DMOG for 8, 24, or 48 hours.

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  Figure 5. ANGPTL4 expression is detected in conjunctival epithelium from surgically excised pterygia. (A) Immunohistochemical staining of pterygia for ANGPTL4 and VEGF. IgG was used as a negative control. High-resolution insets are shown below. Similar results were obtained in 6/6 pterygia. (B) Immunohistochemical staining of ANGPTL4 in normal conjunctiva of autopsy eyes with no known history of anterior segment disease. A high-resolution inset is shown below. Similar results were observed in 4/4 autopsy eyes.

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  Figure 6. Inhibition of ANGPTL4 expression by CjECs reduces their angiogenic potential. (A, B) ANGPTL4 mRNA expression (A) and protein secretion (B) in ihCjECs transfected with RNAi targeting ANGPTL4 and exposed to 1% O2 for 24 hours. (C) VEGF mRNA expression in ihCjECs transfected with RNAi targeting ANGPTL4 and exposed to 1% O2 for 24 hours. (D, E) ANGPTL4 mRNA expression (D) and protein secretion (E) in ihCjECs transfected with RNAi targeting ANGPTL4 and exposed to 100 μM DFO for 24 hours. (F, G) EC tubule formation by HMVECs treated with conditioned media from ihCjECs pretreated with RNAi targeting ANGPTL4 and exposed to 1% O2 or 100 μM DFO for 24 hours reported as fold induction (F) or percent reduction (G).

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  Figure 7. Combined inhibition of both VEGF and ANGPTL4 expression by CjECs abolishes their angiogenic potential. (A–D) ANGPTL4 (A, C) and VEGF (B, D) mRNA expression (A, B) and protein secretion (C, D) in ihCjECs transfected with RNAi targeting ANGPTL4, VEGF, or both and exposed to 1% O2 for 24 hours. (E, F) EC tubule formation by HMVECs treated with conditioned media from ihCjECs pretreated with RNAi targeting ANGPTL4, VEGF, or both and exposed to 1% O2 for 24 hours reported as fold induction (E) or percent reduction (F).