To investigate the changes in aqueous inflammatory and angiogenic cytokine levels after intravitreal injection of triamcinolone or bevacizumab for reducing foveal thickness in diabetic macular edema (DME).
Prospective, interventional case series.
Twenty-two eyes of 11 patients with bilateral DME and 6 eyes of 6 patients undergoing cataract surgery participated in this study. In each DME patient, 1 eye received an intravitreal injection of 4 mg triamcinolone acetonide and the other eye received 1.25 mg bevacizumab. Aqueous humor samples were obtained before and 4 weeks after the intravitreal injection in the DME group and before the surgery in the control group. Aqueous concentrations of interleukin (IL)-6, IL-8, interferon-induced protein (IP)-10, monocyte chemotactic protein (MCP)-1, platelet-derived growth factor (PDGF)-AA, and vascular endothelial growth factor (VEGF) were measured by multiplex bead assay.
Before the administration of the drugs, aqueous levels of IL-8, IP-10, MCP-1, and VEGF were significantly higher in the DME group than in the control group. After intravitreal injection, foveal thickness was more decreased in the triamcinolone acetonide (IVTA) group compared with the bevacizumab (IVBe) group. IL-6, IP-10, MCP-1, PDGF-AA, and VEGF were significantly decreased in the IVTA group, but only VEGF in the IVBe group. Aqueous levels of VEGF were more decreased in the IVBe group than in the IVTA group.
These findings suggest that the pathogenesis of DME is not only related to VEGF dependency, but also to other mechanisms suppressed by corticosteroids. We suppose that these cytokines would have an important role in both the pathogenesis of DME and the underlying mechanism of intravitreal injections.
Diabetic macular edema (DME) is the most common cause of visual impairment in patients with diabetic retinopathy (DR). DME, which is usually caused by the breakdown of the blood-retinal barrier and the leakage of intraretinal fluid from perifoveal abnormal capillary vessels or microaneurysms, is characterized by intraretinal and subretinal accumulations of fluid. However, the pathogenesis of DME remains unclear.
Vascular endothelial growth factor (VEGF) is a well-known potent angiogenic factor that is involved in the increased vascular permeability leading to macular edema and induces retinal neovascularization. Recent studies have shown that VEGF plays a major role in increasing vascular permeability in diabetic eyes and that vitreous levels of VEGF, interleukin (IL)-6, IL-8, and monocyte chemotactic protein (MCP)-1 are related to DME. There is evidence that the pathogenesis of DR is mediated by inflammatory responses including leukostasis and that DME has some features of chronic inflammation. Inflammatory features that characterize DR are increased expression of inflammatory mediators, macrophage infiltration, increased leukocyte adhesion, complement activation, and acute phase response protein expression.
Among recent treatments available for DME, intravitreal injections of triamcinolone acetonide and of bevacizumab (Avastin; Genentech, Inc, San Francisco, California, USA) have been shown to be safe, effective, and visually and anatomically beneficial in some patients with DME. The major effect of corticosteroids such as triamcinolone is to inhibit the synthesis of multiple inflammatory proteins. Bevacizumab is a full-length humanized monoclonal antibody directed against all isoforms of VEGF-A. Nonetheless, the exact mechanisms of such treatments and reasons for response to the treatment still remain unknown.
In our controlled study using an interventional case series design, we evaluated the changes in aqueous levels of inflammatory (IL-6, IL-8, interferon-induced protein [IP]-10, MCP-1, platelet-derived growth factor [PDGF]-AA) and angiogenic (VEGF) cytokines after intravitreal injection of corticosteroid or anti-VEGF agent in patients with DME, focusing on elucidating the exact role of intraocular cytokines in both the pathogenesis of DME and the underlying mechanisms of these treatments.
Patients and Methods
Inclusion criteria were 1) central foveal thickness (CFT) that was 250 μm or greater by optical coherence tomography (OCT), 2) type 2 diabetes, 3) clinically significant macular edema, and 4) DR. Exclusion criteria were 1) previous vitreous surgery, 2) previous retinal photocoagulation, 3) previous intravitreal injection of triamcinolone acetonide or bevacizumab, 4) vitreoretinal pathology other than diabetic retinopathy, and 5) signal strength (max 10) in OCT image less than 5.
Eleven consecutive patients with bilateral DME were recruited as a DME group at the eye center, Gachon University Gil Hospital, from June 3, 2008 through March 25, 2009. The control group was composed of 6 patients who had undergone cataract surgery without other ocular or systemic diseases.
Before and after the treatment, all patients underwent a comprehensive ophthalmic examination that included best-corrected visual acuity (BCVA) by Snellen chart recording, intraocular pressure (IOP) measurement, slit-lamp examination, fundus examination, and CFT measurement. The CFT measurements were taken with the STRATUS OCT3 version 4.0 software (Carl Zeiss Meditec, Dublin, California, USA) using the fast macular scan protocol. This protocol consists of 6 line scans that are 6.0 mm long, centered on fixation, and spaced 30 degrees apart around the circumference of a circle. The foveal or central 1-mm thickness is an average generated value based on central 21 scans of each of the 6 lines that pass through the patient’s fixation.
Intravitreal injections of 4 mg/0.1 mL triamcinolone acetonide (Kenalog; Dongkwang Pharm, Seoul, Korea) or 1.25 mg/0.05 mL bevacizumab (Avastin; Genentech, Inc, San Francisco, California, USA) were done with the generally used concentration within 7 days of baseline.
In each DME patient, the eye with thicker foveal thickness was defined as the primary eye and the other eye as the secondary eye. To make possible equal conditions of macular edema between bevacizumab- and triamcinolone-injected groups, bevacizumab and triamcinolone were injected into the primary eyes of consecutive patients by turn. For example, in the first patient, bevacizumab was injected into the primary eye and triamcinolone in the secondary eye in 1 patient, and subsequently in the next patient, triamcinolone was injected in the primary eye and bevacizumab in the secondary eye. Intravitreal injections for the second eye were performed within a week of the primary eye. Eyes assigned to intravitreal triamcinolone acetonide constituted the IVTA group and those assigned to bevacizumab constituted the IVBe group.
All injections were performed by a retinal specialist (H.J.S.) using topical anesthesia. Under sterile condition in the operation room, triamcinolone or bevacizumab was injected via 30-gauge needle through the pars plana at 3.5 mm posterior to the limbus. For the 2 weeks after injection, levofloxacin (Cravit; Santen, Osaka, Japan) ophthalmic solution was administered 4 times a day.
Undiluted aqueous humor samples (50-100 μL) were obtained just before an intravitreal injection of triamcinolone acetonide or bevacizumab and then 4 weeks after the injection in the DME group, and at the beginning of cataract surgery in the control group. All sample collections were performed by 3 physicians (H.J.S., D.Y.L., K.H.K.) under sterile conditions in the operating room. Aqueous humor was withdrawn through a limbal paracentesis site using a 30-gauge needle with a tuberculin syringe. Special care was taken to avoid touching intraocular tissues and to prevent mixing of aqueous samples with other fluids. The specimens were immediately transferred to a sterile plastic tube and stored at -70 C until assayed.
Measurement of Cytokines Using Multiplex Analysis
IL-6, IL-8, IP-10, MCP-1, PDGF-AA, and VEGF were measured in aqueous samples by the Luminex 100 multiplex array assay (Luminex Corporation, Austin, Texas, USA). This technique uses microspheres that are 5.5 μm in diameter as a vehicle for molecular reactions. The microspheres, which are available in 100 distinct sets, have a unique spectral domain by virtue of the unique orange/red emission profile of each set. Antibody specifically directed against the mediator of interest was covalently coupled to a set of color-coded microspheres. Antibody-conjugated microspheres are allowed to react with the sample (eg, the aqueous humor) and then with a biotinylated detection antibody (in principle, similar to a sandwich ELISA). The reaction mixture is detected by the addition of streptavidin-phycoerythrin, which binds to the biotinylated detection antibodies. The assay solution is drawn into the Luminex reader, which illuminates and reads the sample.
In the measurement of 7 factors in aqueous humor, instructions from the manufacturer were followed strictly (Lincoplex cytokine/chemokine multiplex kit, #HCYTO-60K; Millipore Corporation, Billerica, Massachusetts, USA). In brief, the desired number of wells on a 96-filter plate was blocked by pipetting 200 μL of the Lincoplex assay buffer and sealed and mixed on a plate shaker for 10 minutes at room temperature (20-25 C). The plate shaker was set at a speed of approximately 500 to 800 rpm. The buffer was removed by vacuum filtration. A serial dilution of standards (3.2-10 000 pg/mL) was used for processing a standard curve for each cytokine. The assay buffer (25 μL) was added to the sample plate and the 0 standard (background). Each standard or control (25 μL) was pipetted into the appropriate wells and the appropriate matrix diluent (25 μL) was added to the background, standards, and control wells. Aqueous humor (25 μL) was added to the appropriate wells. Mixed beads containing antibody-immobilized beads were added to each well and the plate was sealed and covered with aluminum foil, and incubated with agitation on a plate shaker for 1 hour at room temperature (20-25 C). Fluid was gently removed by vacuum and the plate was twice washed and vacuum-filtrated with 200 μL of Lincoplex wash buffer. Next, 25 μL of Lincoplex detection antibody cocktail was added to each well and the plate was sealed and covered with aluminum foil, and incubated with agitation on a plate shaker for 30 minutes at room temperature (20-25 C). Then, 25 μL of streptavidin-phycoerythrin solution was added to each well containing the 25 μL of Lincoplex detection antibody cocktail and the plate was sealed and covered with aluminum foil and incubated with agitation on a plate shaker for 30 minutes at room temperature (20-25 C). The samples/standards were gently removed by vacuum filtration, and were twice washed and vacuum-filtered with 200 μL/well of wash buffer. Then, 100 μL of sheath fluid was added to all wells and the plate was covered with aluminum foil and the beads in each well were resuspended on a plate shaker for 5 minutes. Next, the plate was run on a Luminex instrument. The median data using a 5-parameter logistic or spline curve-fitting method were saved and evaluated for calculation of the cytokine/chemokine concentrations in the samples.
All statistical analyses were carried out using SPSS 12.0 for Windows (SPSS, Inc, Chicago, Illinois, USA) by physician authors (H.J.S., D.H.H., D.H.N.). The aqueous levels of IL-6, IL-8, IP-10, MCP-1, PDGF-AA, and VEGF were expressed as a mean with standard deviation (SD). To analyze the statistical differences, the Wilcoxon signed rank test was used between preinjection and postinjection clinical data, and the Mann-Whitney U test was used both between control and DME groups and between IVTA and IVBe groups. A P value < .05 was judged as statistically significant.
Twenty-two eyes of 11 patients (4 male and 7 female) with bilateral DME and 6 eyes of 6 patients (2 male and 4 female) undergoing cataract surgery were included in this study. Mean age was 54.45 years (SD ± 10.22) in DME patients and 63.33years (SD ± 14.02) in controls ( P = .824). Ten of 11 patients (91%) had a history of hypertension and 1 patient (9%) had a history of dyslipidemia in the DME group. In the control group, 4 patients had a history of hypertension and 1 patient had a history of dyslipidemia. Four of 22 eyes (18%) were psedophakic in the DME group. All DME patients had type 2 diabetes.
In the IVTA and IVBe groups, BCVA (logMAR) improved from 0.414 ± 0.238 to 0.276 ± 0.214 ( P = .018) and 0.442 ± 0.320 to 0.301 ± 0.259 ( P = .011) respectively at 4 weeks after the injection. Between those 2 groups, there were no significant differences in both preinjection and postinjection BCVAs ( P = .699 and P = .748, respectively) ( Table 1 , Figure 1 ).
|Variable||Control Group (n=6)||IVTA Group (n=11)||IVBe Group (n=11)|
|Preinjection||Postinjection||P Value a||Preinjection||Postinjection||P Value a|
|Visual acuity (logMAR)||0.318 (0.0-1.00)||0.39 (0.0-0.82)||0.30 (0.0-0.52)||.018||0.52 (0.0-1.00)||0.22 (0.0-0.82)||.011|
|IOP (mm Hg)||12 (9-17)||12 (8-18)||16 (8-20)||.015||14 (8-19)||10 (6-17)||.079|
|CFT (um)||389 (253-559)||238 (150-285)||<.01||427 (251-547)||294 (197-454)||<.01|
Intraocular pressure (mm Hg) in the IVTA group increased from 12.7 ± 2.9 to 13.6 ± 3.6 at 4 weeks after the injection ( P = .015), whereas in the IVBe group there was no significant change of intraocular pressure (13.6 ± 3.6 to 12.0 ± 3.7) ( P = .079) ( Table 1 , Figure 2 ).
CFT (μm) in the IVTA group significantly decreased from 397.9 ± 114.0 to 228.4 ± 41.6 at 4 weeks after the injection ( P < .01). Likewise, in the IVBe group, CFT significantly decreased from 387.7 ± 111.3 to 311.3 ± 92.1 ( P < .01). Between those 2 groups, there was no significant difference in preinjection CFT ( P = .747); nevertheless, CFT in the IVTA group was more decreased than in the IVBe group after the injection ( P = .034) ( Table 2 , Figure 3 ).
|Variable||Control Group (n=6)||DME Group||P Value a|
|IVTA Group (n=11)||IVBe Group (n=11)||Control Group vs DME Group||IVTA Group vs IVBe Group|
|IL-6||11.7 (3.5-54.2)||29.9 (10.1-82.5)||26.7 (13.8-107.0)||.309||.478|
|IL-8||4.8 (3.0-16.7)||28.2 (6.2-77.5)||23.9 (11.1-39.7)||<.01||.401|
|IP-10||309 (104-328)||366.0 (171.0-1380)||401.0 (126.0-1990)||.024||.748|
|MCP-1||1060 (712-1940)||3850 (2060-4380)||3770 (2660-4490)||<.01||.949|
|PDGF-AA||71.0 (41.0-85.7)||68.7 (31.4-141.0)||81.0 (14.3-140.0)||.395||.847|
|VEGF||32.3 (12.9-69.7)||55.0 (36.0-262.0)||61.5 (31.8-200.1)||.039||.898|
Before the administration of the drugs, aqueous levels of IL-8, IP-10, MCP-1, and VEGF were significantly higher in the DME group than in the control group ( P < .01, P = .024, P < .01, and P = .039, respectively) ( Table 3 ).
|Variable||IVTA Group (n=11)||IVBe Group (n=11)|
|Preinjection||Postinjection||P Value a||Preinjection||Postinjection||P Value a|
|IL-6||29.9 (10.1-82.5)||13.8 (2.8-36.3)||<.01||26.7 (13.8-107.0)||24.0 (6.5-147.0)||.477|
|IL-8||28.2 (6.23-77.5)||25.3 (12.4-95.8)||.597||23.9 (11.1-39.7)||23.6 (11.0-74.2)||.374|
|IP-10||366.0 (171.0-1380)||249.0 (28.7-717.0)||.013||401.0 (126.0-1990)||433.0 (268.0-4570)||.110|
|MCP-1||3850 (2060-4380)||1090 (351-4150)||.010||3770 (2660-4490)||3840 (1790-4490)||.594|
|PDGF-AA||68.7 (31.4-141.0)||37.1 (10.9-89.7)||.016||81.0 (14.3-140.0)||72.7 (23.8-117.0)||.722|
|VEGF||55.0 (36.0-262.0)||10.5 (0.1-372.0)||.050||61.5 (31.8-200.1)||0.1 (0.1-28.3)||<.01|