From University of Massachusetts Medical School (ATT, JS), Department of Ophthalmology; Massachusetts Eye & Ear Infirmary (DVB, JGA); and Beth Israel Deaconess Medical Center (KDK, JGA), Harvard Medical School, Boston, Massachusetts.
Dr. Arroyo is a recipient of a K-23 Physician Training Award. This study was supported by funds from the Beth Israel Deaconess Medical Center Retina Service Research Fund.
The authors have no financial or proprietary interest in the materials presented herein.
Address correspondence to Jorge G. Arroyo, MD, MPH, Retina Service, Director, Division of Ophthalmology, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Shapiro 5th Floor, Boston, MA 02215.
The most severe ocular complication associated with diabetes mellitus is proliferative diabetic retinopathy (PDR), a disease process in which abnormal blood vessels grow on the surface of the retina, often leading to vitreous hemorrhaging, tractional retinal detachments, and loss of vision.1 The development of retinal neovascularization is associated with altered levels of endothelial cell mitogens, such as vascular endothelial cell growth factor (VEGF), basic fibroblast growth factor (bFGF), and insulin-like growth factor (IGF-1), in ischemic retina.2,3 PDR is initially treated with extensive laser panretinal photocoagulation (PRP),4,5 which destroys ischemic retina and lowers intraocular concentrations of VEGF, thereby causing the regression of abnormal blood vessels and the development of a fibrovascular membrane.2,5
Apoptosis is a regulated cell suicide pathway that is known to be mediated by both internal and exogenous stimuli in patients with PDR.6 Apoptosis has already been shown to be controlled and induced via exogenous stimuli at the retinal and optic nerve level.7 We studied surgically excised retinal fibrovascular membranes from patients with PDR with previous PRP treatment. Comparing apoptotic cell counts with the extent of prior laser treatment, we attempted to assess whether PRP treatment acted as an exogenous stimuli to initiate apoptosis in fibrovascular diabetic membranes.
Design and Methods
We obtained approval from the Massachusetts Eye & Ear Infirmary Institutional Review Board to perform this consecutive, interventional study. Pars plana vitrectomy and fibrovascular membrane delamination was performed on four patients who had undergone argon scattered laser treatment for PDR and one patient who had not undergone previous laser treatment. Laser burns were of moderate intensity following Early Treatment Diabetic Retinopathy Study protocol, and were not performed directly on the neovascular membrane before surgery. Each patient had a tractional retinal detachment, clinically significant macula edema, or both. The excised membranes were immediately fixed in 10% formalin and then embedded in paraffin at the time of surgery, and 6-micron paraffin sections were later deparaffinized and rehydrated using a standard method.
Apoptotic cells were identified using in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays, specifically the In Situ Cell Death Detection and Proteinase K kits (Roche Molecular Biochemicals, Indianapolis, IN). We created both positive and negative controls to ensure that the TUNEL reaction was successful. On completion, the tissues were counterstained with propidium iodide to allow us to detect false TUNEL-positive staining or autofluorescence, common in specimens previously fixed with paraffin. In addition, we performed hematoxylin–eosin staining of the membranes for histologic analysis to confirm apoptosis. We used a Hamamatsu camera (ORCA II ER; Hamamatsu Corporation, Bridgewater, NJ) connected to a Leica Digital Microwave Radio microscope (Leica Microsystems, Wetzlar, Germany) to capture the images, and OpenLab software (OpenLAB version 3.3.2; PerkinElmer, Inc.,Waltham, MA) on a Mac computer (Mac OS X 10.1; Apple Computer, Cupertino, CA) to view the figures and manually count the number of apoptotic cells per square millimeter of each membrane.
In situ DNA end labeling and propidium iodide staining demonstrated cells undergoing apoptosis and allowed us to distinguish them from false-positive staining due to autofluorescence (Fig. 1). Staining was observed in the positive control and not observed in the negative control. The hematoxylin–eosin and propidium iodide staining of the fibrovascular diabetic membranes revealed nuclei undergoing the various morphologic changes associated with apoptosis. We noted that cells undergoing apoptosis were usually located at the peripheral edges of the membrane in all samples (Fig. 1).
Figure 1. Photomicrographs of Fibrovascular Diabetic Membranes Excised at Surgery. (A) Arrows Indicate in Situ Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick End Labeling (TUNEL)-Positive Cells Revealed by Fluorescence. (B) Propidium Iodide-Red Fluorescence Reveals Cell Nuclei. Superimposition of (A) and (B), Arrows Indicate Positive Apoptotic Cells Revealed by Fluorescence (C).
The patients’ ages, gender, type and duration of diabetes mellitus, extent of previous PRP treatment, and interval between the last PRP and surgery were compared with the apoptotic cell count for each membrane (Table). The number of apoptotic cells per square millimeter of tissue qualitatively appeared to increase with an increase in the number of previously applied laser spots (Fig. 2). On the other hand, we saw no apparent relationships between number of apoptotic cells and duration of disease, interval between the last PRP and surgery, or patient age.
Table: Clinical Data and Apoptotic Cell Count
Figure 2. Number of Apoptotic Cells per Square Millimeter of Diabetic Fibrovascular Membrane Versus Number of Previously Applied Laser Spots. As the Number of Applied Laser Spots Increases, the Number of Apoptotic Cells per Square Millimeter also Appears to Increase.
This study demonstrates that apoptosis occurs in human diabetic fibrovascular membranes after PRP, and that the extent of apoptosis is associated with the extent of prior laser treatment. In general, we saw that an increased number of laser spots received before surgery led to a greater number of cells undergoing apoptosis in the fibrovascular membrane, although the small number of patients prevents us from forming definitive conclusions.
Besides PDR, apoptosis has been associated with various other proliferative eye diseases, including proliferative vitreoretinopathy and macular pucker.8,9 Apoptosis has also been induced in cultured human retinal pigment epithelial cells irradiated by laser.10
Previous studies have also demonstrated a strong association between retinal ischemia and increased levels of VEGF, which is known to induce retinal and iris neovascularization.2 Retinal laser photocoagulation has been shown to decrease the level of VEGF, resulting in regression of abnormal retinal blood vessels.11 The qualitative correlation between PRP treatment and apoptosis suggests that more extensive retinal laser photocoagulation may result in a greater decrease in extracellular levels of VEGF and a consequent regression of the retinal neovasculature.12 It is also possible that PRP treatment may induce apoptosis by altering the extracellular levels of other molecules, such as pigment epithelial-derived growth factor, bFGF, and IGF-1.13
We observed apoptotic cells predominantly at the edges of the fibrovascular membranes. This finding may have been due to the fact that the deeper layers of the membrane were more fibrous than the edges, which were relatively more cellular. Because of their position and their greater vascularization, the edges may also have been more susceptible to a sudden decrease in ambient mitogen.
This study identified cells undergoing apoptosis in diabetic fibrovascular membranes, replicating the results of two previous studies by Esser et al.8 and Zhang et al.9 However, we found that the number of apoptotic cells was related to the extent of previous laser treatments, a finding that, to our knowledge, has not been reported previously and one that is absent in the study by Esser et al. Apoptosis also appeared to be associated with patient age. However, age appeared to be only moderately associated with the number of PRP spots. This qualitative relationship may be due to the fact that older patients were more likely to have had PRP. In addition, this seeming relationship could also be due to the nature of aging, resulting in an increased sensitivity to the laser photocoagulation treatments. In contrast, no association between the number of apoptotic cells and duration of the disease was observed, supporting a conclusion of Esser et al.8 Our small sample size limits any further conclusions.
Our findings suggest that the number of neovascular cells undergoing apoptosis increases with the extent of prior PRP. Apoptotic cells were identified predominately at the edges of the fibrovascular membranes. Apoptosis in fibrovascular membranes may be triggered after PRP by falling extracellular levels of growth factors stimulating neovascular membrane formation. The limitations of our data set prevent any greater analysis, and further study is needed to better elucidate these findings.
- Kempen JH, O’Colmain BJ, Leske MC, et al. The prevalence of diabetic retinopathy among adults in the United States. Arch Ophthalmol. 2004;122:552–563. doi:10.1001/archopht.122.4.552 [CrossRef]
- Aiello LP, Avery RL, Arrigg PG, et al. Vascular endothelial growth factor in ocular fluid of patients with diabetic retinopathy and other retinal disorders. N Engl J Med. 1994;331:1480–1487. doi:10.1056/NEJM199412013312203 [CrossRef]
- Simo R, Lecube A, Segura RM, Garcia Arumi J, Hernandez C. Free insulin growth factor-I and vascular endothelial growth factor in the vitreous fluid of patients with proliferative diabetic retinopathy. Am J Ophthalmol. 2002;134:376–382. doi:10.1016/S0002-9394(02)01538-6 [CrossRef]
- The Diabetic Retinopathy Study Research Group. Preliminary report on effects of photocoagulation therapy. Am J Ophthalmol. 1976;81:383–396.
- The Diabetic Retinopathy Study Research Group. Photocoagulation treatment of proliferative diabetic retinopathy. Clinical application of Diabetic Retinopathy Study (DRS) findings, DRS Report Number 8. Ophthalmology. 1981;88:583–600.
- Kowluru RA. Diabetic retinopathy: mitochondrial dysfunction and retinal capillary cell death. Antioxid Redox Signal. 2005;7:1581–1587. doi:10.1089/ars.2005.7.1581 [CrossRef]
- Calandrella N, Scarsella G, Pescosolido N, Risuleo G. Degenerative and apoptotic events at retinal and optic nerve level after experimental induction of ocular hypertension. Mol Cell Biochem. 2007;301:155–163. doi:10.1007/s11010-006-9407-0 [CrossRef]
- Esser P, Heimann K, Bartz-Schmidt KU, et al. Apoptosis in proliferative vitreoretinal disorders: possible involvement of TGF-beta-induced RPE cell apoptosis. Exp Eye Res. 1997;65:365–378. doi:10.1006/exer.1997.0341 [CrossRef]
- Zhang X, Barile G, Chang S, et al. Apoptosis and cell proliferation in proliferative retinal disorders: PCNA, Ki-67, caspase-3, and PARP expression. Curr Eye Res. 2005;30:395–403. doi:10.1080/02713680590956306 [CrossRef]
- Barak A, Goldkorn T, Morse LS. Laser induces apoptosis and ceramide production in human retinal pigment epithelial cells. Invest Ophthalmol Vis Sci. 2005;46:2587–2591. doi:10.1167/iovs.04-0920 [CrossRef]
- Shinoda K, Ishida S, Kawashima S, et al. Clinical factors related to the aqueous levels of vascular endothelial growth factor and hepatocyte growth factor in proliferative diabetic retinopathy. Curr Eye Res. 2000;21:655–661.
- Armstrong D, Augustin AJ, Spengler R, et al. Detection of vascular endothelial growth factor and tumor necrosis factor alpha in epiretinal membrane of proliferative diabetic retinopathy, proliferative vitreoretinopathy and macular pucker. Ophthalmologica. 1998;212:410–414. doi:10.1159/000027378 [CrossRef]
- 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;98:2593–2597. doi:10.1073/pnas.031252398 [CrossRef]
Clinical Data and Apoptotic Cell Count
|Age at Surgery (Y)||Gender||Type of DM||Duration (Y)||Insulin||PRP (Spots)||Interval (Mo)a||TUNEL+ cells/mm2|