Title Succinate Increases in the Vitreous Fluid of Patients With ActiveProliferative Diabetic Retinopathy

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=0.03). When comparing mean vitreous succinate levels (active PDR: 3.32, quiescent PDR: 1.02, and active PDR with intravitreal bevacizumab injection: 1.20 μM), significant differences were found between active and quiescent PDR (P < 0.01), and between active PDR and active PDR with intravitreal bevacizumab injection (P < 0.01). Even though succinate levels were low, retinopathy activities were very high in patients with active PDR with intravitreal bevacizumab injection. Mean vitreous VEGF levels (active PDR: 1696 pg/ml, quiescent PDR: 110 pg/ml, and active PDR with intravitreal bevacizumab injection: n.d.) were similar to previous reports. Mean vitreous erythropoietin levels (active PDR: 703, quiescent PDR: 305, 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. 246 words /250 words Page 2 Matsumoto et al. Succinate increases in vitreous fluid of active PDR patients. 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. 2 Vascular endothelial growth factor (VEGF) has long been recognized as the primary mediator of retinal angiogenesis. 4 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 vitreo-retinal 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. 22 Recently, Sapieha et al. reported 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. 24 We are unaware of previous reports on levels of succinate in the human vitreous fluid and aqueous humor. Page 3 Matsumoto et al. Succinate increases in vitreous fluid of active PDR patients. METHODS Consecutive patients with PDR and idiopathic epiretinal membrane (ERM) who were treated by pars plana vitrectomy at Nagasaki University Hospital between July 2008 and October 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 1-7 (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 two 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 non-perfused, 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 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 min and then rapidly frozen at 80°C. Corpuscles were separated from blood samples in sterile tubes by centrifugation at 5,000 rpm for 10 min, with the serum then also rapidly frozen at