Purpose
To investigate plasma and vitreous vascular endothelial growth factor (VEGF) levels after intravitreal bevacizumab (IVB) injection into eyes with proliferative diabetic retinopathy (PDR).
Design
Retrospective, interventional, nonrandomized, comparative study.
Methods
Fifty-six eyes of 56 patients with PDR and 13 eyes of 13 patients with nondiabetic ocular diseases were enrolled. Analysis included evaluation of basic clinical conditions and measurement of vitreous and plasma VEGF concentrations using enzyme-linked immunosorbent assays.
Results
PDR eyes without IVB had the highest vitreous VEGF levels; the levels were significant compared with those in the recent IVB group (previous injection within 1 week), the prolonged IVB group (injection more than 1 week previously), and the nondiabetic control group ( P = .001, P = .035, P < .001, respectively). The vitreous VEGF level in the recent IVB group was higher than that in prolonged IVB group ( P = .035). PDR eyes without IVB had the highest plasma VEGF level, and the level was significant compared with those in the recent IVB group, the prolonged IVB group, and the nondiabetic control group ( P < .001, P = .003, P < .001, respectively). The plasma VEGF level in the recent IVB group was lower than that in the prolonged IVB group ( P = .003). The vitreous VEGF level was associated significantly with the plasma VEGF level ( P = .002).
Conclusions
Vitreous and plasma VEGF levels were increased markedly in patients with PDR. VEGF concentrations in vitreous and plasma were decreased significantly after IVB into PDR eyes, and the effect lasted from 4.4 ± 2.2 days to 34.8 ± 33.7 days after injection.
Proliferative diabetic retinopathy (PDR), the serious, vision-threatening eye complication of diabetes, occurs when pathologic growth of new blood vessels branches out or proliferates in the eyes. The angiogenesis cascade occurs because of the disequilibrium between growth-promoting and growth-inhibiting mediators that interact in the internal environment of the eye. Increasing evidence indicates that vascular endothelial growth factor (VEGF) is considered to be the most important primary promoting factor contributing to the development of pathologic microvascular complications, including retinal angiogenesis, in PDR patients.
Panretinal photocoagulation (PRP) is the standard and traditional treatment for PDR patients. It takes a fair amount of time to cause regression of neovascularization and places the patient at risk of vitreous hemorrhage, tractional retinal detachment, and neovascular glaucoma. Previous researchers reported that VEGF concentrations in vitreous fluid declined after PRP.
In 1971, Folkman reported attempting to inhibit these angiogenic factors as a pathway in cancer treatment, where there was hope that an angiogenesis inhibitor would block the pathologic neovascularization mediators. The use of intravitreal anti-VEGF agents has greatly expanded since its introduction into ophthalmic care in 2005. In particular, bevacizumab (Avastin; Genentech, Inc, South San Francisco, California, USA), one of the VEGF inhibitors, has been used by means of intravitreal injections to treat neovascular ocular diseases, including PDR. Theoretically, intravitreal injection of bevacizumab avoids systemic complications by allowing for localized application of the drug and a very small dose.
VEGF levels clearly have been shown to be significantly reduced 7 days after intraocular bevacizumab injection. Recently, we reported that this administration route also would reduce the VEGF levels in blood circulation. However, the duration of the decline in VEGF levels with respect to PDR is less well known. To that end, this study was intended to expand on our knowledge of the duration of action of intraocular bevacizumab in PDR on both vitreous and plasma VEGF levels.
Methods
Subjects
Undiluted vitreous fluid and blood samples were harvested from 69 eyes of 69 patients at the start of pars plana vitrectomy for the treatment of retinal disease. The patients were divided into 4 groups: a control group of 27 consecutive PDR patients who underwent vitrectomy without preoperative IVB therapy (the blank control group), a second group of 19 consecutive PDR patients who underwent preoperative IVB within 7 days before vitrectomy (the recent IVB group), a third group of 10 consecutive PDR patients who underwent preoperative IVB operated more than 7 days before vitrectomy (the prolonged IVB group), and a fourth group of 13 consecutive patients who had nondiabetic ocular diseases (the nondiabetic control group), including idiopathic macular holes (8 eyes) or idiopathic preretinal membranes (5 eyes). As preoperative adjunctive therapy, 1.25 mg/0.05 mL bevacizumab was injected into the vitreous cavity a mean ± standard deviation 4.4 ± 2.2 days before vitrectomy in the recent IVB group and 34.8 ± 33.7 days in the prolonged IVB group. Exclusion criteria included (1) systemic and previous treatment with anti-VEGF therapy, (2) previous ocular surgery, (3) a history of tumor, and (4) a history of other neovascular ocular diseases.
Physical and Occular Examinations
Each patient underwent an ophthalmic and medical examination. The ophthalmic examinations included slit-lamp biomicroscopy, gonioscopy, ophthalmoscopy, fluorescein angiography, and fundus color photography, which were performed with a fundus camera (TRC-50EX; Tokyo Optical Co, Ltd, Tokyo, Japan). The severity of diabetic retinopathy was evaluated by standardized fundus color photographs and fluorescein angiograms, or by ocular ultrasound if a vitreous hemorrhage or lens opacity prevented an ophthalmoscopic examination of the ocular fundus. All diagnoses were confirmed by at least 2 doctors independently at the time of admission. Medical examinations included blood examinations for fasting plasma concentrations of blood glucose and glycosylated hemoglobin and measurements of body mass index and waist-to-hip ratio. Clinical histories of diseases and treatments were collected from the patients’ medical records.
Sample Collection
All surgeries were performed by the same surgeon (Y.R.J.) at the Peking University People’s Hospital. Approximately 1 mL undiluted vitreous sample was aspirated into a sterile tube with a vitreous cutter under simultaneous inflation of the vitreous cavity with air through the infusion cannula at the start of the removal of the vitreous body during pars plana vitrectomy. Blood samples also were collected from the patients. These samples immediately were placed on ice, centrifuged at 3000 rpm for 10 minutes at 4 C to remove cells and debris, and frozen at −80 C until analysis.
Assessment of Vascular Endothelial Growth Factor Level
The concentrations of VEGF in plasma and vitreous were measured by enzyme-linked immunosorbent assays using kits for human VEGF (human VEGF ELISA Kit; RapidBio Lab, Calabasas, California, USA). Each assay was performed according to the manufacturer’s instructions.
Statistical Analysis
Statistical analysis of the data was performed using a commercially available statistical software package (SPSS for Windows, version 17.0: SPSS, Inc, Chicago, Illinois, USA). Data are presented as frequencies or as the mean ± standard deviation. The 1-sample Kolmogorov-Smirnov test was performed to examine whether the samples distributed normally. Differences in gender, hypertension proportion, rubeosis, vitreous hemorrhage, and fibroneovascular membrane proportions were analyzed with the chi-square or Fisher exact test when appropriate. Differences in VEGF concentrations among the 4 groups were estimated with the 1-way analysis of variance, nonparametric Mann–Whitney U test, and Kruskal-Wallis test when appropriate. Data with a skewed distribution initially were transformed into a logarithmic scale. Correlation coefficients were determined by using the Pearson correlation test or nonparametric Spearman correlation test when appropriate. Two-tailed probabilities of less than .05 were considered to indicate statistical significance.
Results
General States of Patients
As shown in the Table , the general physical information, including age, gender, body mass index, waist-to-hip ratio, and hypertension proportion, did not vary significantly among the 4 groups ( P > .05). In particular, the timings of previous PRP, which may alter VEGF concentrations at the time of vitrectomy, were not significantly different among the blank control group, recent IVB group, and prolonged IVB group ( P = .171). In the PDR groups, PRP had been performed in 10 (37%), 9 (47%), and 7 (70%) patients in the blank control group, recent IVB group, and prolonged IVB group, respectively, which also showed no significant differences among the 3 groups ( P = .202). The numbers of individuals who had rubeosis in the blank control group, recent IVB group, and prolonged IVB group were 4 (15%), 3 (16%), and 0 (0%), respectively. In the blank control group, recent IVB group, and prolonged IVB group, respectively, 25 (93%), 17 (89%), and 8 (80%) patients had vitreous hemorrhage; 22 (81%), 18 (95%), and 10 (100%) patients had fibroneovascular membrane; 16 (59%), 15 (79%), and 8 (80%) patients had tractional retinal detachment. There were no significant differences among the 3 groups with regard to these features ( P > .05). The 13 nondiabetic patients included 8 (62%) with preretinal membranes and 5 (38%) with macular holes. None of these 13 patients had proliferative vitreoretinopathy.
| PDR without IVB before Vitrectomy | PDR with Recent IVB before Vitrectomy | PDR with Early IVB before Vitrectomy | Nondiabetic Ocular Disease | P Value | |
|---|---|---|---|---|---|
| No. | 27 | 19 | 10 | 13 | NA |
| Mean age ± SD, y | 55.3 ± 8.0 | 50.6 ± 15.5 | 51.7 ± 10.9 | 58.8 ± 13.7 | .235 |
| Female sex, no. (%) | 14 (52) | 14 (48) | 7 (70) | 16 (64.0) | .483 |
| Mean body mass index ± SD, kg/m 2 | 25.2 ± 2.9 | 24.5 ± 2.7 | 23.8 ± 0.9 | 23.7 ± 3.5 | .292 |
| Mean waist-to-hip ratio ± SD | 0.90 ± 0.06 | 0.88 ± 0.06 | 0.89 ± 0.07 | 0.89 ± 0.07 | .294 |
| Hypertension, no. (%) | 12 (44) | 5 (26) | 2 (20) | 5 (20.0) | .471 |
| Mean duration of diabetes ± SD, y | 12.2 ± 7.4 | 9.3 ± 6.3 | 13.1 ± 5.3 | NA | .235 |
| Mean fasting blood glucose ± SD, mmol/L | 8.5 ± 3.3 | 8.7 ± 4.8 | 6.7 ± 1.6 | NA | .339 |
| Mean glycosylated hemoglobin ± SD, % | 7.3 ± 1.4 | 7.6 ± 1.6 | 6.8 ± 1.3 | NA | .431 |
| Subgroups, no. (%) or no. (range) | |||||
| Period from last PRP to vitrectomy (mos) | 7.1 (0 to 108) | 7.1 (0 to 36) | 12.2 (0 to 48) | NA | .171 |
| PRP history | 10 (37) | 9 (47) | 7 (70) | NA | .202 |
| Rubeosis | 4 (15) | 3 (16) | 0 (0) | NA | .735 |
| Vitreous hemorrhage | 25 (93) | 17 (89) | 8 (80) | NA | .459 |
| Fibroneovascular membranes | 22 (81) | 18 (95) | 10 (100) | NA | .299 |
| Tractional retinal detachment | 16 (59) | 15 (79) | 8 (80) | NA | .264 |
| Idiopathic macular hole | NA | NA | NA | 5 (38) | NA |
| Idiopathic preretinal membrane | NA | NA | NA | 8 (62) | NA |
Vitreous Vascular Endothelial Growth Factor Level
The mean ± standard deviation vitreous VEGF concentration was 897.30 ± 496.91 pg/mL in the blank control group, 383.38 ± 245.65 pg/mL in the recent IVB group, 209.50 ± 182.11 pg/mL in the prolonged IVB group, and 82.92 ± 49.21 pg/mL in the nondiabetic control group. The vitreous VEGF level did not follow normal distribution through a 1-sample Kolmogorov-Smirnov test. Thus, a Mann–Whitney U test and Kruskal-Wallis test were performed to test the differences among the groups. Significant differences among the 4 groups were observed for the levels of VEGF in vitreous humors ( P < .001). As demonstrated in Figure 1 , the PDR eyes without IVB had the highest vitreous level of VEGF, and the level was statistically significant compared with that of PDR eyes with recent injection before surgery, that of PDR eyes with prolonged injection before surgery, and that of nondiabetic eyes ( P = .001, P = .035, P < .001, respectively). The vitreous VEGF concentration in the recent IVB group was significantly statistically higher than that in the prolonged IVB group (mean rank, 33.84 vs 21.00; P = .035).
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