Study On The Pathogenesis And Clinical Treatment Of Choroidal Neovascularization In Association With VEGF And PEDF

PURPOSETo investigate whether triamcinolone acetonide (TA) affects the expression of vascular endothelial growth factor (VEGF) and pigment epithelial derived factor (PEDF) in human retinal pigment epithelial (ARPE19) and human umbilical vein endothelial (HUVE) cells. DESIGNStudy the time course of the effects of TA on VEGF and PEDF expressions in cultured cells.METHODS Cell cultureHuman retinal pigment epithelial cell (ARPE19) and human umbilical venous endothelial cell (HUVE) 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 ug/ml streptomycin sulfate. HUVE cells were grown in F12K medium, containing 0.1 mg/ml heparin, 20% fetal bovine serum, 0.03mg/ml endothelial cell growth supplement (BD Biosciences, San Jose, CA) and antibiotic mixtures of 100 U/ml penicillin G and 100 ug/ml streptomycin sulfate (Invitrogen-Gibco, Rockville, MD). Both types of cells were seeded onto 100 mm X 20 mm plates. The cultures were maintained in a humidified 5% CO₂ environment at 37℃. All the cells within the same passages were grown to 70% confluence for TA treatment. Treatment with triamcinolone70% confluence cultures of cells were adapted into fresh culture medium 12 hours prior to addition of triamcinolone acetonide (TA, 9a-fluoro-16a-hydroxyprednisolone;kenacort-A, Bristol-Myers-Squibb, NY), which was serially diluted in culture medium to appropriate concentrations just before use. TA (0.1-lmg/ml) and vehicle (benzyl alcohol, 0.025%) were added to the ARPE19 and HUVE cells. Treated and non-treated 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. RNA Isolation and Real-time RT-PCRTotal RNA was extracted with a RNeasy kit (Qiagen GmbH, Germany). Cells were lysed in lysis buffer containing 1% p-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). 500ng total RNA was used for reverse-transcription with 3 ug/ul random primer p[dN]6 (Roche Diagnostics GmbH, Mannheim, Germany) and a reverse transcriptase kit with RNase inhibitor (Superscript IE 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 egg agt-3', and reverse, 5'-gaa gat ggt gat ggg art tc-3';VEGF165 forward, 5'- gac aag aaa ate cct gtg ggc-3', and reverse, 5'-aac gcgagt ctg tgt ttt tgc-3', PEDF forward : 5'-cag aag aac etc aag agt gcc-3', and reverse, 5'-ctt cat cca agt aga aat cct c-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 GAPDHgene expression level.The ABI PRISM? 7000 Sequence Detection System is used for real-time detection of PCR and data analysis, mean Ct (threshold cycle;cycle at which the increase in signal associated with exponential growth of PCR product is first detected) value of the stimulated sample was compared to that of the unstimulated control sample using the Ct value of GAPDH as an internal control. ACt was the difference in Ct values derived from the target gene (in each sample assayed) and the GAPDH gene, while AACt represented the difference between the paired samples. The n-fold differential ratio was expressed as 2"AACt. ELISAfor VEGF16S and PEDFConcentrations of VEGF]65 and PEDF in the medium of the cultured ARPE19 cells were determined by an enzyme-linked immunosorbent assay (ELISA) (ChemiKine, Chemicon International, USA). Duplicate wells were used for all samples and standards. Statistical and data analysisThe 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. RESULTS Effect ofTA on VEGFm and PEDF mRNA expression in RPE cellsTA was added to the culture medium of the ARPE19 cells at 2 concentrations (0.1 mg/ml and 1 mg/ml). Real-time RT-PCR measurements showed significant alterations in VEGF,65 and PEDF mRNA expressions in triamcinolone-treated APRE19 cells. The maximum alterations occurred at approximately lhr to 3hr in all cases, a 0.5 (±0.05) fold decrease in levels of VEGF,65 (p=0.00012) and 2.5 (± 0.07) fold increase (pO.OOOl) in levels of PEDF. The amplificationsshowed no significant difference between the two concentration of TA, but the effect was maintained till 12 hr (p=0.00018) after 0.1 mg/ml TA stimulation and till 24 hr (p=0.0007) after 1 mg/ml TA stimulation. Effect ofTA on VEGF165 and PEDF protein production in RPE cellsTo examine whether the increased expression of VEGF16S and PEDF mRNA was accompanied by the changes in protein production, expression of VEGF\65 and PEDF in the culture medium were assayed by ELISA. There were significant decrease in protein levels of VEGFl65 (p=0.001) and increase in PEDF (p=0.002) starting at 3 hr after TA addition. Expression studies of VEGFUs and PEDF in HUVE cellsThe effects of TA on VEGFus 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 hr after TA treatment in HUVE cells. The two different concentrations of TA (0.1 mg/mL and 1 mg/mL) had a similar effect. 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 lOOpg/ml for VEGFI65 and undetectable (0.05).All Subfoveal CNV lesionsFor the 24 eyes treated with PDT and IVTA, the mean logMAR BCVA changed from 0.88 (Snellen equivalent of 20/152) at baseline to 0.95 (Snellen equivalent of 20/178) at 1 year. The reduction in BCVA compared with baseline was not statistically significant at any of the follow-up visits (P > 0.05). For the PDT monotherapy group, the mean logMAR BCVA changed from 0.74 (Snellen equivalent of 20/110) at baseline to 1.09 (Snellen equivalent of 20/246) at 1 year. The reduction in visual acuity was statistically significant at all follow-up visits (PO.001).In terms of lines of BCVA changes, the mean number of lines reduced at 12 months for the PDT with IVTA and for the PDT monotherapy groups was 0.7 and 3.5 respectively. Patients who received combined treatment had significantly less number of lines lost compared with those who had monotherapy alone at all post-treatment visits (P<0.05).For the proportion of patients without moderate visual loss, 17 (70.8%) eyes in the combined PDT with IVTA group compared with eight (33.3%) eyes in the PDT monotherapy group lost fewer than three lines of BCVA at 12 months (chi-squared test, P=0.009).Although the combined PDT with IVTA group did receive less mean treatment sessions than the PDT monotherapy group, 1.50 and 1.96 treatments respectively in the first year, it did not have the statistically different (P=0.076). Subgroup analysis: predominately classic CNVThe mean logMAR BCVA of the combined therapy group improved slightly from 0.92 (Snellen equivalent of 20/166) at baseline to 0.89 (Snellen equivalent of 20/155) at 1 year, while that of PDT monotherapy declined from 0.78 (Snellen equivalent of 20/121) to 1.01 (Snellen equivalent of 20/205). The changes in logMAR BCVA were not statistically significant for the combined treatment group at all follow-up visits (P>0.05), whereas the logMAR BCVA was significantly worse at all post-PDT visits for the PDT monotherapy group (P<0.05). In terms ofthe mean line of BCVA changes at one year, the combined PDT with IVTA group improved by 0.3 lines, while the PDT monotherapy group decreased by 2.4 lines. Eyes with predominately classic CNV which had combined therapy had significantly less reduction in lines of BCVA at the 3 and 6-month visits (P=0.027 and P=0.019 respectively). Nine (75.0%) eyes in the combined PDT with IVTA compared with five (45.5%) eyes treated with PDT monotherapy lost fewer than three lines of BCVA one year after treatment (P=0.21). For the 12 eyes with predominately classic CNV which had combined PDT with IVTA and the 11 eyes which had PDT alone, the mean number of treatment was 1.67 and 1.91 respectively (P=0.55). Subgroup analysis: occult CNVThe mean logMAR BCVA declined from 0.85 (Snellen equivalent of 20/142) at baseline to 1.01 (Snellen equivalent of 20/205) at 12 month for the combined therapy group, and reduced from 0.71 (Snellen equivalent of 20/103) to 1.16 (Snellen equivalent of 20/289) for the monotherapy group. The reduction in logMAR BCVA was statistically significant at 9 month for the PDT with IVTA group (P=0.006) but not significant for all other visits, whereas for the PDT alone group, the changes in logMAR BCVA were significant at all visits (P < 0.05). Compared with the monotherapy group, eyes with occult CNV treated with combined PDT and IVTA had significantly less reduction in lines of BCVA at the 3 and 6-month visits (P=0.025 and P=0.033 respectively). Eight (66.7%) eyes in the combined PDT with IVTA group compared with three (23.1%) eyes treated with PDT monotherapy lost fewer than three lines of visual acuity one year after treatment (Fisher exact test, P=0.028). There were 12 and 13 eyes with occult CNV in the combined PDT with IVTA and PDT monotherapy groups respectively. The mean number of treatment for the combined and the monotherapy groups was 1.33 and 2.00 respectively (P=0.056). ComplicationsNone of the patients in this study developed ocular or systemic complications related to PDT. Eight (33.3%) of the 24 eyes in the combined treatment group developed transient increase in IOP more than 21 mmHg following IVTA injection and all were managed withanti-glaucomatous eye drops. Five (26.3%) of the 19 phakic patients in the combined therapy group also developed increase in cataract of which three (15.8%) finally required cataract surgery. No serious complications such as endophthalmitis and retinal detachment developed after IVTA injection. CONCLUSIONSCombined PDT with IVTA appeared more effective statistically at 12 months for stabilization of vision (<3 logMAR lines change) compared with PDT monotherapy.