Oscillation of angiogenesis with vascular dropout in diabetic retinopathy by VESsel GENeration analysis (VESGEN)

Abstract

Purpose: Vascular dropout and angiogenesis are hallmarks of the progression of diabetic retinopathy (DR). However, current evaluation of DR relies on grading of secondary vascular effects, such as microaneurysms and hemorrhages, by clinical examination instead of by evaluation of actual vascular changes. The purpose of this study was to map and quantify vascular changes during progression of DR by VESsel GENeration Analysis (VESGEN).

Methods: In this prospective cross-sectional study, 15 eyes with DR were evaluated with fluorescein angiography (FA) and color fundus photography, and were graded using modified Early Treatment Diabetic Retinopathy Study criteria. FA images were separated by semiautomatic image processing into arterial and venous trees. Vessel length density (L(v)), number density (N(v)), and diameter (D(v)) were analyzed in a masked fashion with VESGEN software. Each vascular tree was automatically segmented into branching generations (G(1)...G(8) or G(9)) by vessel diameter and branching. Vascular remodeling status (VRS) for N(v) and L(v) was graded 1 to 4 for increasing severity of vascular change.

Results: By N(v) and L(v), VRS correlated significantly with the independent clinical diagnosis of mild to proliferative DR (13/15 eyes). N(v) and L(v) of smaller vessels (G(> or =6)) increased from VRS1 to VRS2 by 2.4 x and 1.6 x, decreased from VRS2 to VRS3 by 0.4 x and 0.6 x, and increased from VRS3 to VRS4 by 1.7 x and 1.5 x (P < 0.01). Throughout DR progression, the density of larger vessels (G(1-5)) remained essentially unchanged, and D(v1-5) increased slightly.

Conclusions: Vessel density oscillated with the progression of DR. Alternating phases of angiogenesis/neovascularization and vascular dropout were dominated first by remodeling of arteries and subsequently by veins.

Figures

Figure 1.

Mild and moderate stages of NPDR. Arterial and venous trees extracted from FA images of retinas diagnosed as having mild NPDR (A) and moderate NPDR (C) are displayed as overlapping vascular patterns (B, D). Although the trees are shown in red and blue for illustration, the image of a single isolated tree is imported into VESGEN as a binary (black/white) image. To preserve visualization of the critically important small blood vessels, the images are presented in two figures (see Fig. 2 for later stages of diabetic retinopathy). The FAs of Figures 1 and 2 were selected as illustrations because their VESGEN results and clinical ranking are median values for their groups (and close to mean values of vascular density by Nv and Lv; Figs. 5, 6).

Figure 2.

Severe NPDR and early PDR. Arterial and venous trees extracted from FA images of retinas diagnosed as having severe NPDR (A) and PDR (C) are displayed as overlapping vascular patterns (B, D). As for Figure 1, the FAs were selected because VESGEN results and clinical rankings are median values (Figs. 5, 6).

Figure 3.

Oscillation of arterial density. Eight branching generations (G1–G8) were mapped within arterial patterns by VESGEN (Figs. 1, 2). Vessel density increased from mild NPDR (A) to moderate NPDR (B), decreased at severe NPDR (C), and increased again at PDR (D) stages of DR. The arterial maps, therefore, display an oscillation between the opposing vascular phenotypes of angiogenesis (or neovascularization) and vascular dropout. Imaging fields for the FAs vary slightly with each photograph, but normalizing vessel density parameters by the ROI corrects for this small variation. In these illustrations, diameters of smaller vessels (G≥4) were enlarged by two pixels to increase visibility.

Figure 4.

Oscillation of venous density. Eight or nine branching generations (G1–G8 or G9) were mapped within venous patterns by VESGEN (Figs. 1, 2). Vessel density increased from mild NPDR (A) to moderate NPDR (B), decreased at severe NPDR (C), and again increased at PDR (D) stages of DR. Venous maps appear to correlate positively with results for arterial maps (Fig. 3). The diameters of small veins (G≥4) were enlarged by two pixels for improved visibility.

Figure 5.

Grouping of VRS by ranking of clinical diagnosis and VESGEN results. To determine appropriate analysis groups of progressive vascular remodeling for subsequent VESGEN quantification, the FAs were ranked by clinical diagnosis from 1 to 15, in order of increasing severity of diabetic retinopathy. Diagnosis was based on modified ETDRS clinical criteria that included density and location of microaneurysms, hemorrhagic leakage, exudates, ischemic areas, neovascularization, and vascular arcades. The density of all vessels (overall density) determined by VESGEN was plotted and compared with clinical ranking. Vessel number density (Nv) and vessel length density (Lv) are excellent measures of the space-filling capacity of tree-branching patterns. Results clearly reveal oscillation between angiogenesis and vascular dropout as a direct, positive function of clinically diagnosed progression of diabetic retinopathy. The plots show good agreement for these oscillatory trends between arterial and venous trees and between Nv and Lv. According to arterial results for Nv (A), the highest-ranking patients for the clinically diagnosed moderate and severe groups (eyes 8 and 13) were reclassified by vascular remodeling status as VRS3 (correlated to severe NPDR) and VRS4 (correlated to very severe NPDR/PDR). Because of the clear binning of clinically ranked grading and dominance of arterial remodeling compared with venous remodeling during the first angiogenic phase (mild to moderate), arterial results for Nv were used to define the four analysis groups, VRS1 to VRS4. Black vertical lines indicate this grouping of arterial and venous trees by vascular remodeling status into VRS1 to VRS4.

Figure 6.

By VESGEN analysis, angiogenesis and vascular dropout oscillate with progressive vascular remodeling of smaller arteries and veins. By Nv and Lv, the oscillation between angiogenesis and vascular dropout during diabetic retinopathy were restricted to smaller blood vessels (G≥6), as quantified by changes in vessel density during progression of vascular remodeling status from VRS1 to VRS4. Relative increases in vessel density by Nv and Lv were greater for arteries than for veins in the first phase of angiogenesis (VRS1-VRS2) but greater for veins in the second phase of angiogenesis (VRS3-VRS4). Data are plotted as mean ± SE. *P ≤ 0.05 and **P ≤ 0.01, one-tailed t-test, for confidence estimation of either increased or decreased vessel density for G1–5 and G≥6 from VRS1 to VRS2, VRS2 to VRS3, and VRS3 to VRS4. By a two-tailed t-test (for estimation of confidence in differences), P-values of Nv≥6 and Lv≥6 for all arterial and venous transitions were ≤0.01, except arterial and venous Lv≥6 for VRS3 to VRS4, which were 0.02.

Figure 7.

By VESGEN analysis, the diameters of large and medium-sized vessels increase with progressive vascular remodeling. By measurements of Dv, the diameters of large and medium-sized arteries and veins (G1–5) increased slightly, but consistently, with progressive vascular remodeling. The diameters of smaller vessels did not appear to vary greatly with disease progression. Data are plotted as mean ± SE. *P ≤ 0.05 and **P ≤ 0.01, one-tailed t-test, for confidence estimation of increased diameter during vascular remodeling of VRS2, VRS3, and VRS4 compared with VRS1.