Loss of CD34 expression in aging human choriocapillaris endothelial cells

Abstract

Structural and gene expression changes in the microvasculature of the human choroid occur during normal aging and age-related macular degeneration (AMD). In this study, we sought to determine the impact of aging and AMD on expression of the endothelial cell glycoprotein CD34. Sections from 58 human donor eyes were categorized as either young (under age 40), age-matched controls (> age 60 without AMD), or AMD affected (>age 60 with early AMD, geographic atrophy, or choroidal neovascularization). Dual labeling of sections with Ulex europaeus agglutinin-I lectin (UEA-I) and CD34 antibodies was performed, and the percentage of capillaries labeled with UEA-I but negative for anti-CD34 was determined. In addition, published databases of mouse and human retinal pigment epithelium-choroid were evaluated and CD34 expression compared between young and old eyes. Immunohistochemical studies revealed that while CD34 and UEA-I were colocalized in young eyes, there was variable loss of CD34 immunoreactivity in older donor eyes. While differences between normal aging and AMD were not significant, the percentage of CD34 negative capillaries in old eyes, compared to young eyes, was highly significant (p = 3.8×10(-6)). Endothelial cells in neovascular membranes were invariably CD34 positive. Published databases show either a significant decrease in Cd34 (mouse) or a trend toward decreased CD34 (human) in aging. These findings suggest that UEA-I and endogenous alkaline phosphatase activity are more consistent markers of aging endothelial cells in the choroid, and suggest a possible mechanism for the increased inflammatory milieu in the aging choroid.

Figure 1. Loss of CD34 in a subset of aging capillaries.

Eyes from two donors are depicted, ages 85 (A–C) and 88 (D–F). Large vessel endothelial cells showed retention of CD34 expression. Arrows in D–F indicate choriocapillaris vessels with greatly reduced CD34 reactivity. Large vessels (arrowheads) retained expression. Green fluorescence, anti-CD34 labeling; red fluorescence, UEA-I; blue fluorescence, DAPI. Scalebar = 50 µm.

Figure 2. Percentage of CD34(−) capillaries in human choriocapillaris.

(A) Average values segregated by disease phenotype. Y = young (mean age  = 31.1); CTL = age-matched controls; ARM = early/dry AMD; GA = geographic atrophy; CNV = choroidal neovascularization. Error bars indicate the standard error of the mean. (B) All donors plotted as percent capillaries without CD34 versus age. Note the age related increase in CD34(−) capillaries (p<10⁻⁵).

Figure 3. Expression of CD34 in pathologic blood vessels in choroidal neovascular membranes (CNVMs) from 3 donors with neovascular AMD.

Abnormal vessels within the CNVMs were invariably strongly positive (arrows). RPE, dystrophic retinal pigment epithelium; BlamD, layer of basal laminar deposit; CC, choriocapillaris. Scalebar in C, 50 µm.

Figure 4. Tandem triple labeling of CD34/UEA-I and endogenous alkaline phosphatase.

Following epifluorescence photomicrography (A, B,C), coverslips were removed and sections reacted with NBT/BCIP. Note the good correspondence between alkaline phosphatase activity (D) and UEA-I binding (A) that includes capillaries that do not react with CD34 antibody (B). CC, choriocapillaris; scalebar = 50 µm.