Aqueous Humor Levels of Vascular Endothelial Growth Factor Before and After Intravitreal Bevacizumab in Type 3 Versus Type 1 and 2 Neovascularization. A Prospective, Case-Control Study


To determine the aqueous levels of vascular endothelial growth factor (VEGF) in patients with type 3 neovascularization (NV) secondary to age-related macular degeneration (AMD) and to compare the levels of those with type 1 and 2 NV secondary to AMD before and after administration of intravitreal bevacizumab (IVB).


Prospective, case-control study.


Aqueous samples were collected from 29 eyes of 29 patients with untreated wet AMD at baseline (day of the first IVB), month 1 (day of the second IVB), and month 2 (day of the third IVB). Among them, 10 eyes presented with type 1, 9 with type 2, and 10 with type 3 NV. A group of 14 aqueous samples from 14 patients who underwent cataract surgery without other ocular or systemic disease comprised the controls. Main outcome measures were concentration of VEGF at baseline and after IVB in the 3 NV groups; secondary outcome measures included best-corrected visual acuity (BCVA) and central macular thickness (CMT) changes after IVB. Levels of VEGF were determined by commercially available enzyme-linked immunosorbent assay kits.


VEGF concentrations in aqueous humor at baseline were higher in patients with type 3 NV when compared to controls ( P = .0001) and type 1 and 2 NV patients ( P = .002 and P = .0001 respectively). At month 1, levels of VEGF were significantly reduced compared to baseline ( P < .05) and significantly lower compared to the controls ( P < .005) in each NV group. These low levels were maintained at the 2-month interval. BCVA significantly improved in type 1 and 2 NV groups ( P < .05). CMT significantly reduced in each NV group compared to baseline ( P < .05).


In eyes with untreated wet AMD, aqueous levels of VEGF are significantly higher in type 3 NV than in type 1 or 2 NV. Regardless of the type of NV, aqueous VEGF levels significantly reduce 1 month after IVB as compared to both the baseline measurements and the values recorded in age-matched controls. These decreases are maintained at 2 months after administering a second IVB 30 days after the initial injection.

In 2001 Yannuzzi and associates coined the term retinal angiomatous proliferation (RAP) to describe a neovascular form of age-related macular degeneration (AMD) that appears to originate from the inner retinal circulation and is characterized by peculiar hemorrhagic, exudative, and retinal microangiopathic changes. Other investigators questioned this hypothesis, suggesting that occult choroidal vessels actually initiate the vasculogenesis, proliferating through atrophic retinal pigment epithelium (RPE) to infiltrate the neurosensory retina. Because of a lingering uncertainty as to the origin of these lesions, the term “type 3 neovascularization” (NV) has been recently proposed in reference to this vascular lesion.

Type 3 NV is a variant of wet AMD characterized by intraretinal proliferation of new vessels that may originate from both retinal and choroidal circulations. In type 3 NV, proliferating vessels harbor within and below the retina. This is different from type 1 NV, in which new vessels are located beneath the RPE, and from type 2 NV, in which new vessels have penetrated the RPE layer to proliferate in the subneurosensory space.

Type 3 lesions have a poor prognosis if left untreated and respond poorly to established treatments for wet AMD, including photodynamic therapy with verteporfin (PDT). However, type 3 NV seems to respond similarly to other NV subtypes when treated with anti–vascular endothelial growth factor (VEGF) therapy.

Among cytokines responsible for retinal and choroidal neovascularization, VEGF has been found to be strongly expressed in human neovascular membranes and to be temporally, spatially, and quantitatively associated with new vessel formation. Vitreous levels of secreted VEGF are presumed to correlate with retinal and choroidal levels; therefore they are used as a surrogate for determining VEGF expression in the retina and choroid. However, vitreous taps or biopsies are not risk-free and are usually obtained from patients undergoing surgery for other reasons. Conversely, aqueous humor samples are much easier and safer to collect, and there is a high correlation between aqueous and vitreous levels of VEGF.

We hypothesized that the more aggressive course of type 3 NV could be related to levels of VEGF higher than those of type 1 and type 2 NV. To test this hypothesis we performed a study to compare the levels of aqueous VEGF at baseline and following intravitreal bevacizumab (IVB) injections in patients affected by type 1, 2, and 3 NV secondary to AMD.


This was a prospective, case-control study investigating aqueous levels of VEGF in eyes with AMD-related NV treated with intravitreal bevacizumab at the Medical Retina Department, University of Molise, Campobasso, Italy between May 1, 2009 and September 30, 2010.

Primary goals of the study were to establish whether different types of wet AMD were associated with different concentrations of VEGF at baseline and to compare VEGF changes after IVB in different types of wet AMD. Secondary goals included comparing central macular thickness (CMT) and best-corrected visual acuity (BCVA) before and after treatment, and exploring the correlation among CMT, BCVA, and VEGF levels in each NV group.

Inclusion and Exclusion Criteria

Patients with previously untreated type 1, 2, and 3 wet AMD were eligible for inclusion in the present study. Classification of NV was based on its anatomic localization as ascertained by multimodal imaging employing fluorescein angiography (FA), indocyanine green angiography (ICGA), and optical coherence tomography (OCT) as recently proposed by Freund and associates.

In brief, type 1 NV is characterized by either late leakage of undetermined source or early stippled fluorescence followed by dye leakage and/or pooling, and is often associated with vascularized pigment epithelium detachment (PED) on FA, presence of plaque on ICGA, and new vessels localized between the hyperreflective Bruch membrane and the hyperreflective RPE band on OCT.

Type 2 NV is characterized by a well-defined or “classic” pattern of hyperfluorescence in the early phases of FA followed by extensive leakage in the late phases. With ICGA, type 2 vessels are more difficult to detect overlying the intense hyperfluorescence of the background choroidal circulation. Simultaneous FA/ICGA and spectral-domain OCT (SD-OCT) localizes the type 2 vessels above the RPE band and beneath the photoreceptor outer segments.

Since type 2 NV typically occurs in conjunction with type 1 vessels, in order to limit the bias, only eyes with FA evidence of “predominantly classic” lesions were included in the type 2 group in this study.

Type 3 NV is intraretinal NV previously referred to as RAP. FA definition of type 3 NV includes a focal or diffuse area of dye leakage associated with retinal anastomotic feeder or draining vessels. On ICGA a focal area of intense hyperfluorescence at the site of NV (the so-called hot spot), occasionally associated with an underlying placoid area of choroidal NV, is visible. OCT shows a hyperreflective structure within the retina in correspondence to the hot spot seen angiographically. This correspondence is confirmed by combining real-time angiography with an OCT. Additionally, adjacent cystic changes within the retina may be present and occasionally associated with subretinal fluid or PED.

Exclusion criteria included: BCVA at baseline less than 1.0 logarithm of the minimal angle of resolution (logMAR); any previous treatment of the neovascular lesion; previous vitrectomy; laser coagulation within the last 3 months; previous participation in any studies using investigational drugs within 3 months preceding day 0; intraocular surgery (including cataract surgery) in the study eye within 3 months preceding day 0; glaucoma in the study eye; diabetes mellitus; use of immunosuppressive drugs; or malignant tumors, regardless of their location.

Since, theoretically, VEGF levels in the vitreous and aqueous humor might be influenced by ocular fluid volume, and therefore by ocular axial length, only patients with a spherical equivalent refractive error between +3.00 and −3.00 were included in the study.

Diagnostic Procedures and Follow-up

At baseline all the patients underwent BCVA measurement using an early treatment diabetic retinopathy study (ETDRS) chart at 4 meters, fundus biomicroscopy, FA, ICGA, and SD-OCT. All examinations, with the exception of angiographic tests, were repeated at all follow-up appointments. Angiographic tests and OCT scans were recorded using Spectralis SD-OCT (Heidelberg Engineering, Heidelberg, Germany).

CMT was calculated after acquiring a sequence of 193 horizontal sections recorded in the high-resolution mode (1024 A-scans/30 degrees) and covering an area of 20 degrees (horizontal) × 20 degrees (vertical) with a distance of ∼30 μm between individual sections. On follow-up examinations, the imaging processing software allowed reevaluation at exactly the same location. The images were then processed by the “Thickness Map” analysis program. Field 1 of the map analysis protocol (central 1 mm) was used for central retinal thickness calculations.

Aqueous Sampling and Bevacizumab Injections

All patients with AMD-related NV received 3 intravitreal injections of 1.25 mg/0.05 mL of bevacizumab (Avastin; Genentech Inc, South San Francisco, California, USA) at baseline, month 1, and month 2.

Prior to injection, topical anesthesia was induced by tetracaine (1%) eye drops. Povidone-iodine was applied to the eyelid margins, the lashes, the conjunctiva bulbi, and the fornices. After application of a sterile drape, a lid speculum was inserted.

Immediately before each intravitreal injection, aqueous sampling was performed by aspirating 0.05 to 0.1 mL of aqueous using a 30-gauge needle connected to a tuberculin syringe at the temporal limbus. IVB injection was then performed using a 30-gauge needle in the inferotemporal quadrant at 3.5 to 4 mm posterior to the limbus. All the samples were collected in the operating theater using an operating microscope. The undiluted aqueous samples were transferred into sterile containers and immediately stored in a −80C freezer until analysis.

Control Group

Reference samples were obtained from 14 age-matched patients undergoing cataract surgery. Exclusion criteria for the control group were any type of retinal disease, glaucoma, previous vitrectomy, laser coagulation, diabetes mellitus, use of immunosuppressive drugs, malignant tumors at any location, and participation in any study of investigational drugs within 3 months preceding inclusion.

Aqueous humor samples were obtained in the same fashion described above for eyes with wet AMD. The undiluted aqueous samples were transferred into sterile containers and immediately stored in a −80C freezer until analysis.

Vascular Endothelial Growth Factor Assay

Collected samples were gradually equilibrated to room temperature before beginning the assay and diluted 1:1 with the sample diluent provided by the manufacturer.

The VEGF content was determined in 50 μL of diluted sample with a human VEGF ELISA kit (EHVEGF, Pierce Biotechnology, Rockford, Illinois, USA) according to the manufacturer’s instruction and using an extended standard curve including the concentrations at 16, 8, and 4 pg/mL. This VEGF kit permits the detection of VEGF165 and VEGF121 isoforms. All assays were performed in duplicate. The minimum detectable concentration of VEGF was 3.5 pg/mL. Values inferior to 3.5 pg/mL were considered equal to 1 for statistical analysis.

Statistical Analysis

Standard descriptive statistics were used to summarize the variables studied. Data were assessed using nonparametric tests (Mann-Whitney, Kruskal-Wallis, and Friedman tests). To assess the relationship between VEGF, CMT, and BCVA, Spearman rank-order correlation coefficients (ρ) were calculated.

P < .05 was considered significant. Statistical calculations were performed with commercial software (MedCalc Software, Version 11.5.1, Mariakerke, Belgium).


The mean ± standard deviation (SD) age of patients was 72.3 ± 8.6 years in type 1, 73.1 ± 8.4 in type 2, and 76.8 ± 5.7 in type 3 NV group and 71.7 ± 7.1 years in the control group. Age did not differ among groups ( P = .4). Male-to-female ratio was 4:6, 5:4, 5:5, and 6:8 in type 1 NV, type 2 NV, type 3 NV, and control group respectively. There were 6 phakic patients in type 1, 5 in type 2, and 6 in type 3 NV groups. IVB was administered exclusively to the patients presenting with AMD-related NV, specifically 10 with type 1, 9 with type 2, and 10 with type 3 NV. In total, 101 aqueous samples were collected (87 from eyes with AMD-related NV and 14 from controls). No ocular or systemic complications associated with IVB injections were recorded during the follow-up period.

Changes in Vascular Endothelial Growth Factor Level

At baseline the mean ± SD aqueous concentration of VEGF was 54.5 ± 28.8 in type 1, 53.7 ± 12.4 in type 2, and 93.9 ± 18.4 in type 3 NV groups and 39.5 ± 25.5 pg/mL in the control group. VEGF levels were significantly higher in type 3 NV as compared to controls ( P = .0001) and type 1 ( P = .002) and type 2 NV groups ( P = .0001). Neither type 1 nor type 2 NV groups were significantly different from the control group.

After IVB injection, the mean ± SD aqueous concentration of VEGF was significantly reduced ( P < .05) at month 1 and month 2 in all 3 experimental groups. Aqueous VEGF levels in all 3 experimental groups were also significantly lower than the control group ( P < .005).

Within each NV group the difference between VEGF concentration recorded at month 1 and month 2 was not statistically significant. Additionally, VEGF levels at months 1 and 2 were not statistically different among type 1, 2, and 3 NV. These results are summarized in Table 1 .


Vascular Endothelial Growth Factor Levels, Central Macular Thickness, and Best-Corrected Visual Acuity Before and After Intravitreal Injection of Bevacizumab in Type 1, 2, and 3 Neovascularization Groups

Type 1 NV Type 2 NV Type 3 NV
VEGF (pg/mL)
Baseline 54.5 ± 28.8 53.7 ± 12.4 93.9 ± 18.4
Month 1 11.0 ± 13.4 6.0 ± 2.9 11.3 ± 8.3
Month 2 12.5 ± 10.8 5.5 ± 4.4 9.1 ± 5.7
CMT (μm)
Baseline 433.3 ± 125.7 459.5 ± 93.6 439.6 ± 170.1
Month 1 297.3 ± 63.5 368.7 ± 101.9 347.9 ± 146.9
Month 2 280.4 ± 65.4 349.3 ± 92.8 330.2 ± 116.5
Baseline 0.59 ± 0.28 0.62 ± 0.21 0.63 ± 0.36
Month 1 0.46 ± 0.32 0.38 ± 0.18 0.58 ± 0.39
Month 2 0.41 ± 0.32 0.4 ± 0.20 0.56 ± 0.36

BCVA = best-corrected visual acuity; CMT = central macular thickness; logMAR = logarithm of the minimal angle of resolution; NV = neovascularization; VEGF = vascular endothelial growth factor.

Changes in Central Macular Thickness

Values of CMT at baseline, month 1, and month 2 are reported in Table 1 . The values at baseline did not differ among the NV groups ( P = .69). In both type 1 NV and type 2 NV the reduction in CMT was significant at months 1 and 2 as compared to baseline and at month 2 compared to month 1 ( P < .05). In type 3 NV, the reduction in CMT was significant at months 1 and 2 as compared to baseline ( P < .05); however, there was no difference between month 1 and month 2 values.

Changes in Best-Corrected Visual Acuity

Values of logMAR BCVA at baseline, month 1, and month 2 are reported in Table 1 . The values at baseline did not differ among the NV groups ( P = .9). In the type 1 and type 2 NV groups, BCVA significantly increased at months 1 and 2 as compared to baseline ( P < .05). No difference was noted between month 1 and month 2 values. In type 3 NV, BCVA also increased at months 1 and 2, as compared to baseline; however, this increase was not significant ( P = .07).

Correlation Among Aqueous VEGF Levels, CMT, and BCVA

The results of correlations among VEGF levels, CMT, and BCVA are summarized in Table 2 . A statistically significant correlation was found between reduction of CMT and improvement of logMAR BCVA in type 2 and 3 NV.

Jan 12, 2017 | Posted by in OPHTHALMOLOGY | Comments Off on Aqueous Humor Levels of Vascular Endothelial Growth Factor Before and After Intravitreal Bevacizumab in Type 3 Versus Type 1 and 2 Neovascularization. A Prospective, Case-Control Study

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