Purpose
To examine vitreous succinate levels from proliferative diabetic retinopathy (PDR) patients and ascertain their association with PDR activity.
Design
Comparative case series.
Methods
A total of 81 eyes of 72 PDR patients were divided into active PDR (22 eyes), quiescent PDR (21 eyes), and active PDR with intravitreal bevacizumab injection (38 eyes). Twenty epiretinal membrane (ERM) patients (21 eyes) served as controls.
Results
Mean vitreous succinate levels were 1.27 μM in ERM and 2.20 μM in PDR, with the differences statistically significant ( P = .03). When comparing mean vitreous succinate levels (active PDR: 3.32 μM; quiescent PDR: 1.02 μM; active PDR with intravitreal bevacizumab injection: 1.20 μM), significant differences were found between active and quiescent PDR ( P < .01) and between active PDR and active PDR with intravitreal bevacizumab injection ( P < .01). Even though succinate levels were low, retinopathy activities were very high in patients with active PDR with intravitreal bevacizumab injection. Mean vitreous vascular endothelial growth factor (VEGF) levels (active PDR: 1696 pg/mL; quiescent PDR: 110 pg/mL; active PDR with intravitreal bevacizumab injection: n.d.) were similar to previous reports. Mean vitreous erythropoietin levels (active PDR: 703 mIU/mL; quiescent PDR: 305 mIU/mL; active PDR with intravitreal bevacizumab injection: 1562 mIU/mL) suggested very high retinopathy activities in patients with active PDR with intravitreal bevacizumab injection.
Conclusions
Succinate, like VEGF, may be an angiogenic factor that is induced by ischemia in PDR. Although succinate is reported to promote VEGF expression, VEGF inhibition decreases succinate. Thus, VEGF, via a positive feedback mechanism, may regulate succinate.
Intraocular neovascularization is the common final pathway in proliferative diabetic retinopathy (PDR), and it often results in vitreous hemorrhage, tractional retinal detachment, and neovascular glaucoma. Vascular endothelial growth factor (VEGF) has long been recognized as the primary mediator of retinal angiogenesis. Bevacizumab (Avastin; Roche, Reinach, Switzerland) is an anti-VEGF agent that was first used to treat metastatic colorectal cancer. Previous reports on the intravitreal injection of bevacizumab showed promise in targeting VEGF-implicated intraocular neovascularization that is associated with age-related macular degeneration and diabetic retinopathy. Recently, intravitreal bevacizumab injection has also gained popularity among vitreoretinal surgeons as an adjunct to vitrectomy in the management of severe PDR.
Other growth factors and cytokines, including angiopoietin-2, insulin-like growth factor-1, erythropoietin, hypoxia-inducible factor, platelet-derived growth factor, fibroblast growth factor-2, and hepatocyte growth factor, have also been demonstrated to be involved in ocular angiogenesis. However, there appear to be additional mediators that might be participating in ocular angiogenesis.
In the ischemic brain, Krebs cycle intermediates, such as succinate, have been found to accumulate during conditions linked to the insufficient oxygen supply. Recently, Previous studies report that succinate accumulates in the hypoxic retina of rodents and, via its cognate receptor, G protein-coupled receptor-91, induces VEGF expression and potently mediates vessel growth during both normal retinal development and proliferative ischemic retinopathy. We are unaware of previous reports on levels of succinate in the human vitreous fluid and aqueous humor.
Methods
Consecutive patients with PDR and idiopathic epiretinal membrane (ERM) who were treated by pars plana vitrectomy at Nagasaki University Hospital between July 1, 2008 and October 31, 2010 were enrolled in the study. Using a 30-gauge needle, some patients with active PDR received intravitreal injection of bevacizumab (1.25 mg, 0.05 mL) via the pars plana. Injections were performed at 3.5 mm posterior to the limbus and done on days 1 through 7 (2.69 ± 1.58) prior to the planned vitrectomy. This procedure was performed as an adjunct to the vitrectomy that was used to manage the severe proliferative diabetic retinopathy. Subsequently, samples of undiluted vitreous fluid and aqueous humor were harvested from the eyes of the participating PDR or ERM patients. While none of the patients with ERM had diabetes mellitus, all of the PDR patients had type 2 diabetes. The 9 female and 11 male patients with ERM (n = 9 and n = 12 eyes, respectively) were 61.7 ± 6.9 years of age (mean ± standard deviation). The 12 female and 27 male patients with PDR without intravitreal injection of bevacizumab (n = 14 and n = 29 eyes, respectively) were 65.0 ± 9.7 years of age. Age did not significantly differ between these 2 groups.
Based on a previously published method, we clinically classified PDR without intravitreal bevacizumab injection as being active PDR if there was evidence of perfused, multibranching iridic or preretinal capillaries. When active proliferation had fully regressed or if only nonperfused, gliotic vessels or fibrosis were present, patients were classified as having quiescent PDR without intravitreal bevacizumab injection. The 43 eyes of the 39 patients with PDR without intravitreal bevacizumab injection included 22 eyes from 21 patients with active PDR (51.2%) and 21 from 18 patients with quiescent PDR (48.8%). The indications for vitrectomy in patients with quiescent PDR included established vitreous hemorrhage, cystoids or diffuse macular edema, or tractional retinal detachment with inactive fibrovascular proliferation. The 7 female and 14 male patients with active PDR without intravitreal bevacizumab injection (n = 7 and n = 15 eyes, respectively) were 65.3 ± 8.4 years of age. The 5 female and 13 male patients with quiescent PDR without intravitreal bevacizumab injection (n = 7 and n = 14 eyes, respectively) were 64.8 ± 11.2 years of age. The 8 female and 26 male patients with active PDR after intravitreal bevacizumab injection (n = 10 and n = 28 eyes, respectively) were 55.4 ± 12.1 years of age. The subjects in the group with active PDR with intravitreal bevacizumab injection had a younger average age as compared with the group with active PDR without intravitreal bevacizumab injection and the group with quiescent PDR without intravitreal bevacizumab injection. The younger age of the active PDR with intravitreal bevacizumab injection group was intentional, as the study was also designed to examine the relationship between age and succinate concentration.
In order to remove debris, the vitreous fluid and aqueous humor samples were clarified by centrifugation in sterile tubes at 15 000 rpm for 5 minutes and then rapidly frozen at −80 C. Corpuscles were separated from blood samples in sterile tubes by centrifugation at 5000 rpm for 10 minutes, with the serum then also rapidly frozen at −80 C.
Succinate Quantitation Using High-Performance Liquid Chromatography–Mass Spectrometry
We quantified succinate levels in the vitreous fluid, aqueous humor, and serum using a previously described selective ion monitoring mode of high-performance liquid chromatography–mass spectrometry (HPLC/MS) with modifications. The study used ion exclusion column chromatography using 0.1% formic acid as the eluent and negative mode detection with electrospray ionization (ESI) mass spectrometry to ensure we achieved optimal performance during the quantification.
Quantitative Vascular Endothelial Growth Factor and Erythropoietin
We measured VEGF and erythropoietin levels in the vitreous fluid using enzyme-linked immunosorbent assays (ELISA) (R&D Systems, Minneapolis, Minnesota, USA).
Statistical Analysis
The primary objective of this study was to determine whether succinate is significantly increased in patients with PDR. Levels of succinate were compared in the groups with ERM and with PDR without intravitreal bevacizumab injection using a 2-tailed t test. A Dunn test was used to compare the levels of succinate, VEGF, and erythropoietin in active PDR without intravitreal bevacizumab injection, quiescent PDR without intravitreal bevacizumab injection, and active PDR with intravitreal bevacizumab injection. Levels of succinate were compared in the groups with and without systemic and ocular factors using a 2-tailed t test. Linear regression analysis (for the levels of succinate and age, and for the levels of succinate and hemoglobin A1c) along with tests of the regression were performed. All statistical analyses were carried out using Statflex ver. 6.0 software (Artech Co, Ltd, Osaka, Japan). Results are expressed as means ± standard deviation, unless otherwise indicated. P values < .05 were considered to be statistically significant.
Results
Succinate Increases in the Vitreous Fluid From Proliferative Diabetic Retinopathy
In the first part of our study, we compared succinate levels between the PDR patients and the nondiabetic patients. The mean succinate levels in the vitreous fluid were 1.27 ± 0.86 μM and 2.20 ± 2.20 μM in the 20 ERM patients (n = 21) and in the 39 PDR patients without intravitreal bevacizumab injection (n = 43), respectively. The mean vitreous succinate level was significantly elevated in the PDR without intravitreal bevacizumab injection group as compared to the ERM group ( P = .03, t test; Figure 1 , Top).
Succinate Concentrations in the Aqueous Humor and Serum
The mean succinate levels in the aqueous humor from patients with ERM (n = 17) and PDR without intravitreal bevacizumab injection (n = 31) were 1.94 ± 1.85 μM and 2.02 ± 1.63 μM, respectively. There were no significant differences noted among the groups ( Figure 1 , Middle). The mean succinate levels in the serum from patients with ERM (n = 10) and PDR without intravitreal bevacizumab injection (n = 15) were 1.37 ± 0.73 μM and 1.15 ± 0.54 μM, respectively. There were no significant differences noted among the groups ( Figure 1 , Bottom).
Comparison of Vitreous Succinate Levels
Next, we compared the succinate levels in active PDR without intravitreal bevacizumab injection, quiescent PDR without intravitreal bevacizumab injection, and active PDR with intravitreal bevacizumab injection in order to assess their association with PDR activity. The mean succinate levels in the vitreous fluid were 3.32 ± 2.43 μM, 1.02 ± 1.05 μM, and 1.20 ± 1.47 μM in the 21 patients with active PDR without intravitreal bevacizumab injection (n = 22), 18 patients with quiescent PDR without intravitreal bevacizumab injection (n = 21), and 34 patients with active PDR with intravitreal bevacizumab injection (n = 38), respectively. The mean vitreous succinate level was significantly elevated in active PDR without intravitreal bevacizumab injection when compared to both the quiescent PDR without intravitreal bevacizumab injection ( P < .01, Dunn test) and active PDR with intravitreal bevacizumab injection ( P < .01, Dunn test; Figure 2 , Top). Surprisingly, there were similar levels noted for quiescent PDR without intravitreal bevacizumab injection and active PDR with intravitreal bevacizumab injection, even though the retinopathy activities were very high for all of the active PDR patients with intravitreal bevacizumab injection.
Succinate Concentrations in the Aqueous Humor and Serum
The mean succinate levels in the aqueous humor from the active PDR without intravitreal bevacizumab injection (n = 13), quiescent PDR without intravitreal bevacizumab injection (n = 15), and active PDR with intravitreal bevacizumab injection (n = 31) patients were 2.10 ± 1.73 μM, 2.08 ± 1.51 μM, and 1.00 ± 0.93 μM, respectively. The aqueous humor succinate level in the active PDR without intravitreal bevacizumab injection patients tended to be higher than that seen in the active PDR with intravitreal bevacizumab injection, although this difference was not statistically significant ( Figure 2 , Middle).
The mean succinate levels in the serum of the active PDR without intravitreal bevacizumab injection (n = 9), quiescent PDR without intravitreal bevacizumab injection (n = 6), and active PDR with intravitreal bevacizumab injection (n = 20) patients were 1.30 ± 0.59 μM, 0.93 ± 0.42 μM, and 1.10 ± 1.02 μM, respectively. There were no significant differences found among these groups ( Figure 2 , Bottom).
Vitreous Fluid Vascular Endothelial Growth Factor and Erythropoietin Concentrations
To confirm the retinopathy activities of the PDR patients, we examined VEGF and erythropoietin concentrations in the vitreous fluid. While the VEGF vitreous levels were 1695 ± 1955 pg/mL in the active PDR patients without intravitreal bevacizumab injection (n = 22) and 110 ± 149 pg/mL in the quiescent PDR patients without intravitreal bevacizumab injection (n = 20), all of the active PDR patients with intravitreal bevacizumab injection (n = 10) had levels that were below the limits of detection, similar to that which has been previously reported. The mean vitreous VEGF level was significantly elevated in active PDR without intravitreal bevacizumab injection when compared to both quiescent PDR without intravitreal bevacizumab injection ( P < .01, Dunn test) and active PDR with intravitreal bevacizumab injection ( P < 0.01, Dunn test; Figure 3 , Top). Similar to the results reported in a previous study, the mean erythropoietin vitreous levels were 703 ± 669 mIU/mL and 305 ± 244 mIU/mL in active PDR without intravitreal bevacizumab injection (n = 19) and quiescent PDR without intravitreal bevacizumab injection (n = 20), respectively. In contrast, the levels in active PDR with intravitreal bevacizumab injection (n = 15) were 1562 ± 2167 mIU/mL ( Figure 3 , Bottom). This indicates that the retinopathy activities were very high in the active PDR patients with intravitreal bevacizumab injection. We evaluated correlations among succinate, VEGF, and erythropoietin, and determined that there was a significant correlation between the vitreous concentrations of succinate and the VEGF in the 32 eyes of the PDR patients without intravitreal bevacizumab injection (Spearman correlation coefficient, rS = 0.380, P = .0318). We found no significant relationships between the vitreous concentrations of succinate and erythropoietin in the 29 eyes of the PDR patients without intravitreal bevacizumab injection (Spearman correlation coefficient, rS = 0.045). However, the relationship between the vitreous concentration of VEGF and erythropoietin in the 29 eyes of the PDR patients without intravitreal bevacizumab injection was statistically significant (Spearman correlation coefficient, rS = 0.518, P < .01).
