|Molecular Vision 2006;
Received 30 September 2005 | Accepted 14 October 2006 | Published 4 December 2006
Effects of triamcinolone on the expression of VEGF and PEDF in human retinal pigment epithelial and human umbilical vein endothelial cells
Dennis SC Lam,2
Wai Man Chan,2
Kwong Wai Choy,3
Kwok Ping Chan,2
Chi Pui Pang2
(The first two authors contributed equally to this publication)
1Zheyi Eye Center, The First Affiliated Hospital, Medical College, Zhejiang University, Hangzhou, Zhejiang, 2Department of Ophthalmology & Visual Sciences, 3Department of Obstetrics & Gynaecology, The Chinese University of Hong Kong, Hong Kong, China
Correspondence to: Professor CP Pang, Department of Ophthalmology & Visual Sciences, The Chinese University of Hong Kong, Hong Kong Eye Hospital, 147K Argyle Street, Kowloon, Hong Kong, China; Phone: +852 27623169; FAX: +852 27159490; email: email@example.com
Purpose: To investigate whether triamcinolone acetonide (TA) affects the expression of vascular endothelial growth factor (VEGF) and pigment epithelial derived factor (PEDF) in human retinal pigment epithelium (ARPE19) and human umbilical vein endothelial (HUVE) cells. Study the time course of the effects of TA on VEGF and PEDF expressions in cultured cells.
Methods: ARPE19 and HUVE cells were grown to subconfluence and treated with TA (0.1 mg/ml, 1 mg/ml). The mRNA expressions of VEGF and PEDF were determined from 10 min to three days using real-time RT-PCR. Concurrently, the protein levels of VEGF and PEDF in ARPE cells were detected with ELISA.
Results: Real-time RT-PCR showed TA affected a 0.5 fold decrease in VEGF165 level and about a 2.5 fold increase in PEDF level at both TA concentrations. The effect was maintained at 12 h at 0.1 mg/ml TA and 24 h at 1 mg/ml TA. Similar changes were observed in the respective protein concentrations. The effects of TA on VEGF and PEDF transcript levels were similar in HUVE and ARPE19 cells. VEGF and PEDF protein productions in HUVE cells were too low for statistical analysis.
Conclusions: TA reduces the expression of VEGF but increases the expression of PEDF in ARPE19 and HUVE cells. These observations suggest TA may influence the inhibition of neovascularization and macular edema through differential VEGF and PEDF expressions.
Triamcinolone acetonide (9α-fluoro-16α-hydroxyprednisolone, TA) is an intermediate-acting corticosteroid in suspension form. It has been administered traditionally as a potent anti-inflammatory agent for treating ocular inflammation by retrobulbar and sub-Tenon's injections [1,2]. In recent years, intravitreal injection of TA (intravitreal TA) has become popular for treatment of exudative age-related macular degeneration (AMD) with choroidal neovascularization (CNV) and macular edema resulting from diabetic retinopathy and retinal vein occlusion resistant to laser photocoagulation [3-8]. In 1995, Penfold et al. first employed intravitreal TA to treat exudative AMD patients. They found intravitreal TA significantly slowed the progress of vision loss and stabilized the CNV membrane according to fluorescein angiograms . Results of this clinical study were subsequently confirmed and supplemented by other studies [3,4]. Some recent studies showed that intravitreal TA stabilized recurrent CNV and improved visual acuity in patients treated by photodynamic therapy [10-13]. In patients with clinically significant diabetic macular edema refractory to laser photocoagulation, the use of intravitreal TA was effective in reducing macular edema and improving visual acuity [7,8]. Our own previous work also indicated that intravitreal TA was a safe and effective treatment for managing diabetic patients with macular edema [5,6].
It is known that angiogenesis in the eye is tightly controlled by two counter-balancing systems: angiogenic stimulators and angiogenic inhibitors. There is evidence that vascular endothelial growth factor (VEGF) plays a critical role in the development of CNV in human exudative AMD . VEGF, a protein with multiple functions, is also a potent vascular permeability factor . Analyses of diabetic retina and vitreous indicate that the upregulation of VEGF is correlated with the presence of diabetic macular edema and proliferative diabetic retinopathy [16-18]. Meanwhile, pigment epithelial derived factor (PEDF) is among the most potent natural inhibitors of angiogenesis . Uchida et al. found retinoic acid, which modulates the anti-angiogenic functions of RPE cells, upregulated PEDF expression but not VEGF . In retinal glial Muller cells of guinea pigs, depletion of oxygen reduced PEDF production and thus enhanced the VEGF:PEDF ratio . Actually addition of VEGF decreased PEDF release in the Muller cells. Another steroid, dexamethasone, was observed to regulate PEDF expression in mouse Muller glial cells and rat glioma cells . A recent study shows that human umbilical venous endothelial (HUVE) cells expressed angiogenesis factors including VEGF and PEDF .
We have previously shown that TA caused stress responses of cultured ARPE19 cells . In this study we hypothesized that TA may affect the expressions of VEGF and PEDF in retinal pigment epithelium (RPE) cells and vascular endothelial cells to stabilize CNV and reduce macular edema. We therefore sought to investigate whether TA affects VEGF and PEDF in cultured human retinal pigment epithelium (ARPE19) and HUVE cells.
ARPE19 and HUVE cell lines were purchased from the American Type Culture Collection (Manassas, VA). Cell culture reagents, fetal bovine serum, and chemicals came from Invitrogen-Gibco (Rockville,MD), and containers from Corning Glass (Acton, MA).
Human ARPE19 cells were grown in 1:1 (vol/vol) mixture of Dulbecco's modified Eagle's and Ham's F12 medium (DF), containing 3 mM L-glutamine, 10% fetal bovine serum, and antibiotic mixtures of 100 U/ml penicillin G and 100 μg/ml streptomycin sulfate (Invitrogen-Gibco, Rockville, MD). HUVE cells were grown in F12K medium, containing 0.1 mg/ml heparin, 20% fetal bovine serum, 0.03 mg/ml endothelial cell growth supplement (BD Biosciences, San Jose, CA), and antibiotic mixtures of 100 U/ml penicillin G and 100 μg/ml streptomycin sulfate. Both types of cells were seeded onto 100 mmx20 mm plates. The cultures were maintained in a humidified 5% CO2 environment at 37 °C. All the cells within the same passages were grown to 70% confluence for TA treatment.
Treatment with triamcinolone
Cells were grown to 70% confluence, and cell cultures were adapted into fresh culture medium 12 h prior to addition of triamcinolone acetonide (TA, 9α-fluoro-16α-hydroxyprednisolone; kenacort-A, Bristol-Myers-Squibb, NY), which was serially diluted in culture medium to appropriate concentrations just before use. TA (0.1-1 mg/ml) and vehicle (benzyl alcohol, 0.025%) were added to the ARPE19 and HUVE cells. Treated and nontreated cells and their culture medium were collected at 0, 10 min, 30 min, 1 h, 3 h, 6 h, 12 h, 24 h, 2 d, and 3 d for RNA extraction, real-time RT-PCR and ELISA analysis. All experiments were performed at least twice and all time point experiments collected in triplicate. The culture medium used in these experiments was all freshly prepared and contained all ingredients as for culturing the cells.
The 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to examine the effects of TA on cell proliferation as previously described .
RNA isolation and real-time RT-PCR
Total RNA was extracted with a RNeasy kit (Qiagen GmbH, Hilden, Germany). Cells were lysed in lysis buffer containing 1% α-mercaptoethanol (Sigma, St.Louis, MO) and passed through a separation column (QIAShredder; Qiagen GmbH, Germany). Total RNA was obtained according to the supplier's protocol and quantified with a spectrophotometer (NanoDrop). For reverse-transcription 500 ng total RNA was used with 3 μg/μl random primer p[dN]6 (Roche Diagnostics GmbH, Mannheim, Germany) and a reverse transcriptase kit with RNase inhibitor (SuperscriptTM Reverse Transcriptase Kit and RNase OUT RNase inhibitor) purchased from Invitrogen (Carlsbad, CA).
The amount of cDNA corresponding to 25.0 ng RNA was selected and amplified with the following primer pairs: GAPDH forward, 5'-gaa ggt gaa ggt cgg agt-3', and reverse, 5'-gaa gat ggt gat ggg att tc-3'; VEGF165 forward, 5'-gac aag aaa atc cct gtg ggc-3', and reverse, 5'-aac gcg agt ctg tgt ttt tgc-3'; PEDF forward: 5'-cag aag aac ctc aag agt gcc-3', and reverse, 5'-ctt cat cca agt aga aat cct c-3'. We also measured the transcript expression of VEGF isoforms: VEGF165, VEGF121, VEGF189 with primer pair: forward 5'-atc ttc aag cca tcc tgt gtg cc-3', and reverse 5'-tca ccg cct cgg ctt gtc aca t-3'. Real-time RT-PCR analysis was performed using SYBR Green PCR master mix (Applied Biosystems, Foster City, CA). The relative quantification was normalized to the GAPDH gene expression level.
The ABI PRISM® 7000 Sequence Detection System was used for real-time detection of PCR and data analysis. The mean Ct value (threshold cycle; cycle at which the increase in signal associated with exponential growth of PCR product was first detected) of the stimulated sample was compared to that of the unstimulated control sample, using the Ct value of GAPDH as an internal control. ΔCt was the difference in Ct values derived from the target gene (in each sample assayed) and the GAPDH gene, while ΔΔCt represented the difference between the paired samples. The n-fold differential ratio was expressed as 2-ΔΔCt.
Enzyme-linked immunosorbent assay (ELISA) for VEGF165 and PEDF
Concentrations of VEGF165 and PEDF in the medium of the cultured ARPE19 cells were determined by an enzyme-linked immunosorbent assay (ELISA, ChemiKine, Chemicon International). Duplicate wells were used for all samples and standards.
Statistical and data analysis
The effects of TA on the expression of VEGF and PEDF between the control (vehicle) and TA-treated groups were analyzed with ANOVA. Significant differences were determined between control and TA-treated cells at the respective time points of sample collections. In all the experiments, p<0.05 was considered to indicate a statistically significant difference.
Effect of TA on VEGF165 and PEDF mRNA expression in RPE cells
TA was added to the culture medium of the ARPE19 cells at two concentrations (0.1 mg/ml and 1 mg/ml). Real-time RT-PCR measurements showed significant alterations in VEGF165 and PEDF mRNA expressions in triamcinolone-treated APRE19 cells (Figure 1). The maximum alterations occurred at approximately 1-3 h in all cases, a 0.5±0.05 fold decrease in levels of VEGF165 (p=0.00012) and 2.5±0.07 fold increase (p<0.0001) in levels of PEDF. The amplifications showed no significant difference between the two concentration of TA, but the effect was maintained till 12 h (p=0.00018) after 0.1 mg/ml TA stimulation and till 24 h (p=0.0007) after 1 mg/ml TA stimulation (Figure 1). Expression level of VEGF121 mRNA was similar to VEGF165, but only weak VEGF189 mRNA expression was obtained.
Effect of triamcinolone acetonide on VEGF165 and PEDF protein production in RPE cells
To examine whether the increased expression of VEGF165 and PEDF mRNA was accompanied by changes in protein production, expression of VEGF165 and PEDF in the culture medium were assayed by ELISA. There was a significant decrease in protein levels of VEGF165 (p=0.001) and an increase in PEDF (p=0.002) starting at 3 h after TA addition (Figure 2).
Expression studies of VEGF165 and PEDF in HUVE cells
The effects of TA on VEGF165 and PEDF transcript levels in HUVE cells were similar to those in ARPE19 cells. Quantitative RT-PCR measurements indicated a maximum reduction of 0.5 fold±0.05 in VEGF165 mRNA levels (p=0.0006) and a 2.2 fold±0.06 increase in PEDF mRNA levels (p=0.00005) 3 h after TA treatment in HUVE cells. The two different concentrations of TA (0.1 mg/ml and 1 mg/ml) had a similar effect (Figure 3). However, in contrast to real-time RT-PCR results, no difference in VEGF and PEDF protein expressions were observed in HUVE cells after TA treatments. The protein levels were too low for statistical analysis, less than 100 pg/ml for VEGF165 and undetectable (<1 ng/ml) for PEDF (data not shown).
Recently, intravitreal injection of TA has received much attention as a therapeutic modality for CNV and macular edema. Many clinical studies indicated that this approach is safe and effective [3-11]. Matsuda et al. showed that TA reduced VEGF expression and induced CTGF expression in ARPE19 cells exposed to oxidative stress . In the present study, VEGF mRNA expression was examined in normal cultured human ARPE19 cells. We also measured the transcript expression of the VEGF isoforms VEGF165, VEGF121, and VEGF189 in our preliminary experiments. RPE cells expressed strong VEGF165 and 121, and the expression patterns and levels were similar. Only VEGF189 was weakly expressed. VEGF165 is the major VEGF isoform and is the most abundant and biologically active . VEGF121 is biologically active in endothelial cells, but has lower potency than VEGF165. Therefore, we only measured VEGF165 transcript levels in our TA experiments. Our real-time quantitative analysis revealed that TA decreased the expression of VEGF in RPE cells at both the mRNA and protein levels (Figure 1 and Figure 2). This is consistent with the finding that glucocorticosteroids can reduce VEGF mRNA and protein expression in cultured eosinophils  as well as reduce VEGF in cultured aortic vascular smooth muscle cells . Countereffects of corticosteroids on VEGF have also been demonstrated in a recent rabbit study, in which intravitreal VEGF injections caused a time and dose-dependent breakdown of blood-retina and blood-aqueous barriers and led to vascular leakage. The breakdowns were blocked both by dexamethasone and TA, but not the nonsteroidal anti-inflammatory drug (NSAID) indomethacin .
It is possible that TA inhibits the angiogenesis effects of VEGF downstream from VEGF receptors or TA enhanced the expression of PEDF, which is a potent inhibitor of angiogenesis. VEGF has the ability to increase vascular permeability, which causes leakage of proteins and other molecules out of blood vessels . Enhanced PEDF production may disrupt VEGF induced vascular permeability. VEGF-driven angiogenesis likely plays a major role in the pathogenesis of CNV . Clinical trials of anti-VEGF therapy, such as anti-VEGF aptamer and anti-VEGF antibody fragment, have also shown their efficacy on CNV [31,32]. VEGF was originally identified as vascular permeability factor (VPF) as a result of its potent ability to increase vascular permeability, resulting in leakage of proteins and other molecules from blood vessels [15,30]. Such permeability change in blood vessels may be a cause of macular edema. This leakage effect by VEGF has been demonstrated in the breakdown of blood-retinal barrier in a rabbit model, which could be inhibited by corticosteroids . Although the exact effect of steroids on VEGF expression in RPE cells in vivo is still unclear, our results suggest that TA may reduce CNV and macular edema through regulating VEGF expression in RPE cells. Expressions of VEGF and PEDF have been detected in HUVE cells, and PEDF may help maintain the expression of VEGF-C under hypoxic conditions.  We also found that the effects of TA on HUVE cells were similar to those in ARPE19 cells (Figure 3), indicating the regulation of VEGF and PEDF expressions by TA on vascular endothelial cells.
Contrary to VEGF, TA increased the PEDF expression in cultured human ARPE19 cells (Figure 1 and Figure 2). A recent study reported dexamethasone can regulate the expression of PEDF in murine Muller glial cells and C6 rat glioma cells . Uchida et al. found that vitamin A upregulated the expression of PEDF in RPE cells . PEDF is one of the most potent inhibitors of angiogenesis so far described [33,34]. It also promotes cell differentiation in normal retinal cells, maintains morphological organization of the differentiated retina, and promotes photoreceptor outer segment formation and maturation [35-37]. It has been shown that VEGF secreted by RPE cells upregulated PEDF expression in an autocrine manner . In present study, TA downregulated the expression of VEGF and upregulated the expression of PEDF at the same time. Therefore, TA may prevent angiogenesis by both suppressing VEGF production and stimulating PEDF expression. This may also be one explanation for the beneficial clinical effects of TA, since high levels of PEDF can protect retinal photoreceptors and neurons and thus may result in favorable clinical outcome.
In conclusion, we demonstrated that TA reduces the expression of VEGF, but induces the expression of PEDF in APRE 19 cells. Similar effects were observed for their mRNA levels in HUVE cells. Although the detailed interactions between these molecules remain to be elucidated, our findings suggest VEGF and PEDF is involved in the mechanism by which TA influences the inhibition of CNV and macular edema.
We gratefully acknowledge Dr. Martine J. Jager for her expert advice on this paper. We also acknowledge financial support by (1) Dr Yu-Tung Cheng Eye Foundation, Hong Kong, (2) Scientific Research Grant, number 2006B036, of the Zhejiang Province Health Bureau (3) Scientific Research Grant, number 20061448, of the Zhejiang Province Education Bureau.
1. Tanner V, Kanski JJ, Frith PA. Posterior sub-Tenon's triamcinolone injections in the treatment of uveitis. Eye 1998; 12:679-85.
2. Gould ES, Bird AC, Leaver PK, McDonald WI. Treatment of optic neuritis by retrobulbar injection of triamcinolone. Br Med J 1977; 1:1495-7.
3. Jonas JB, Degenring RF, Kreissig I, Friedemann T, Akkoyun I. Exudative age-related macular degeneration treated by intravitreal triamcinolone acetonide. A prospective comparative nonrandomized study. Eye 2005; 19:163-70.
4. Gillies MC, Simpson JM, Luo W, Penfold P, Hunyor AB, Chua W, Mitchell P, Billson F. A randomized clinical trial of a single dose of intravitreal triamcinolone acetonide for neovascular age-related macular degeneration: one-year results. Arch Ophthalmol 2003; 121:667-73.
5. Lam DS, Chan CK, Tang EW, Li KK, Fan DS, Chan WM. Intravitreal triamcinolone for diabetic macular oedema in Chinese patients: six-month prospective longitudinal pilot study. Clin Experiment Ophthalmol 2004; 32:569-72.
6. Chan CK, Chan WM, Cheung BT, Yuen CY, Lee VY, Lam DS. Intravitreal injection of triamcinolone for diffuse diabetic macular edema. Arch Ophthalmol 2004; 122:1083-5;authorreply1086-8.
7. Ozkiris A, Evereklioglu C, Erkilic K, Tamcelik N, Mirza E. Intravitreal triamcinolone acetonide injection as primary treatment for diabetic macular edema. Eur J Ophthalmol 2004; 14:543-9.
8. Jonas JB, Kreissig I, Sofker A, Degenring RF. Intravitreal injection of triamcinolone for diffuse diabetic macular edema. Arch Ophthalmol 2003; 121:57-61.
9. Penfold PL, Gyory JF, Hunyor AB, Billson FA. Exudative macular degeneration and intravitreal triamcinolone. A pilot study. Aust N Z J Ophthalmol 1995; 23:293-8.
10. Chan WM, Lam DS, Wong TH, Lai TY, Kwok AK, Tam BS, Li KK. Photodynamic therapy with verteporfin for subfoveal idiopathic choroidal neovascularization: one-year results from a prospective case series. Ophthalmology 2003; 110:2395-402.
11. Lam DS, Chan WM, Liu DT, Fan DS, Lai WW, Chong KK. Photodynamic therapy with verteporfin for subfoveal choroidal neovascularisation of pathologic myopia in Chinese eyes: a prospective series of 1 and 2 year follow up. Br J Ophthalmol 2004; 88:1315-9.
12. Spaide RF, Sorenson J, Maranan L. Photodynamic therapy with verteporfin combined with intravitreal injection of triamcinolone acetonide for choroidal neovascularization. Ophthalmology 2005; 112:301-4.
13. Rechtman E, Danis RP, Pratt LM, Harris A. Intravitreal triamcinolone with photodynamic therapy for subfoveal choroidal neovascularisation in age related macular degeneration. Br J Ophthalmol 2004; 88:344-7.
14. Kwak N, Okamoto N, Wood JM, Campochiaro PA. VEGF is major stimulator in model of choroidal neovascularization. Invest Ophthalmol Vis Sci 2000; 41:3158-64.
15. Senger DR, Perruzzi CA, Feder J, Dvorak HF. A highly conserved vascular permeability factor secreted by a variety of human and rodent tumor cell lines. Cancer Res 1986; 46:5629-32.
16. Funatsu H, Yamashita H, Ikeda T, Mimura T, Eguchi S, Hori S. Vitreous levels of interleukin-6 and vascular endothelial growth factor are related to diabetic macular edema. Ophthalmology 2003; 110:1690-6.
17. Pe'er J, Folberg R, Itin A, Gnessin H, Hemo I, Keshet E. Upregulated expression of vascular endothelial growth factor in proliferative diabetic retinopathy. Br J Ophthalmol 1996; 80:241-5.
18. Mathews MK, Merges C, McLeod DS, Lutty GA. Vascular endothelial growth factor and vascular permeability changes in human diabetic retinopathy. Invest Ophthalmol Vis Sci 1997; 38:2729-41.
19. Dawson DW, Volpert OV, Gillis P, Crawford SE, Xu H, Benedict W, Bouck NP. Pigment epithelium-derived factor: a potent inhibitor of angiogenesis. Science 1999; 285:245-8.
20. Uchida H, Hayashi H, Kuroki M, Uno K, Yamada H, Yamashita Y, Tombran-Tink J, Kuroki M, Oshima K. Vitamin A up-regulates the expression of thrombospondin-1 and pigment epithelium-derived factor in retinal pigment epithelial cells. Exp Eye Res 2005; 80:23-30.
21. Eichler W, Yafai Y, Wiedemann P, Reichenbach A. Angiogenesis-related factors derived from retinal glial (Muller) cells in hypoxia. Neuroreport 2004; 15:1633-7.
22. Tombran-Tink J, Lara N, Apricio SE, Potluri P, Gee S, Ma JX, Chader G, Barnstable CJ. Retinoic acid and dexamethasone regulate the expression of PEDF in retinal and endothelial cells. Exp Eye Res 2004; 78:945-55.
23. Aparicio S, Sawant S, Lara N, Barnstable CJ, Tombran-Tink J. Expression of angiogenesis factors in human umbilical vein endothelial cells and their regulation by PEDF. Biochem Biophys Res Commun 2005; 326:387-94.
24. Yeung CK, Chan KP, Chiang SW, Pang CP, Lam DS. The toxic and stress responses of cultured human retinal pigment epithelium (ARPE19) and human glial cells (SVG) in the presence of triamcinolone. Invest Ophthalmol Vis Sci 2003; 44:5293-300.
25. Matsuda S, Gomi F, Oshima Y, Tohyama M, Tano Y. Vascular endothelial growth factor reduced and connective tissue growth factor induced by triamcinolone in ARPE19 cells under oxidative stress. Invest Ophthalmol Vis Sci 2005; 46:1062-8.
26. Houck KA, Leung DW, Rowland AM, Winer J, Ferrara N. Dual regulation of vascular endothelial growth factor bioavailability by genetic and proteolytic mechanisms. J Biol Chem 1992; 267:26031-7.
27. Horiuchi T, Weller PF. Expression of vascular endothelial growth factor by human eosinophils: upregulation by granulocyte macrophage colony-stimulating factor and interleukin-5. Am J Respir Cell Mol Biol 1997; 17:70-7.
28. Nauck M, Karakiulakis G, Perruchoud AP, Papakonstantinou E, Roth M. Corticosteroids inhibit the expression of the vascular endothelial growth factor gene in human vascular smooth muscle cells. Eur J Pharmacol 1998; 341:309-15.
29. Edelman JL, Lutz D, Castro MR. Corticosteroids inhibit VEGF-induced vascular leakage in a rabbit model of blood-retinal and blood-aqueous barrier breakdown. Exp Eye Res 2005; 80:249-58.
30. D'Amico DJ, Goldberg MF, Hudson H, Jerdan JA, Krueger DS, Luna SP, Robertson SM, Russell S, Singerman L, Slakter JS, Yannuzzi L, Zilliox P, Anecortave Acetate Clinical Study Group. Anecortave acetate as monotherapy for treatment of subfoveal neovascularization in age-related macular degeneration: twelve-month clinical outcomes. Ophthalmology 2003; 110:2372-83;discussin2384-5.
31. Krzystolik MG, Afshari MA, Adamis AP, Gaudreault J, Gragoudas ES, Michaud NA, Li W, Connolly E, O'Neill CA, Miller JW. Prevention of experimental choroidal neovascularization with intravitreal anti-vascular endothelial growth factor antibody fragment. Arch Ophthalmol 2002; 120:338-46.
32. Dvorak HF, Brown LF, Detmar M, Dvorak AM. Vascular permeability factor/vascular endothelial growth factor, microvascular hyperpermeability, and angiogenesis. Am J Pathol 1995; 146:1029-39.
33. Stellmach V, Crawford SE, Zhou W, Bouck N. Prevention of ischemia-induced retinopathy by the natural ocular antiangiogenic agent pigment epithelium-derived factor. Proc Natl Acad Sci U S A 2001; 98:2593-7.
34. Bouck N. PEDF: anti-angiogenic guardian of ocular function. Trends Mol Med 2002; 8:330-4.
35. Jablonski MM, Tombran-Tink J, Mrazek DA, Iannaccone A. Pigment epithelium-derived factor supports normal Muller cell development and glutamine synthetase expression after removal of the retinal pigment epithelium. Glia 2001; 35:14-25.
36. Jablonski MM, Tombran-Tink J, Mrazek DA, Iannaccone A. Pigment epithelium-derived factor supports normal development of photoreceptor neurons and opsin expression after retinal pigment epithelium removal. J Neurosci 2000; 20:7149-57.
37. Tombran-Tink J, Barnstable CJ. PEDF: a multifaceted neurotrophic factor. Nat Rev Neurosci 2003; 4:628-36.
38. Ohno-Matsui K, Yoshida T, Uetama T, Mochizuki M, Morita I. Vascular endothelial growth factor upregulates pigment epithelium-derived factor expression via VEGFR-1 in human retinal pigment epithelial cells. Biochem Biophys Res Commun 2003; 303:962-7.