Oxidative stress and the regulation of complement activation in human glaucoma

Abstract

Purpose: As part of ongoing studies on proteomic alterations during glaucomatous neurodegeneration, this study focused on the complement system.

Methods: Human retinal protein samples obtained from donor eyes with (n = 10) or without (n = 10) glaucoma were analyzed by a quantitative proteomic approach using mass spectrometry. Cellular localization of protein expression for different complement components and regulators were also determined by immunohistochemical analysis of an additional group of human donor eyes with glaucoma (n = 34) compared with age-matched control eyes without glaucoma (n = 20). In addition, to determine the regulation of complement factor H (CFH) by oxidative stress, in vitro experiments were performed using rat retinal cell cultures incubated in the presence and absence of an oxidant treatment.

Results: Proteomic analysis detected the expression and differential regulation of several complement components in glaucomatous samples, which included proteins involved in the classical and the lectin pathways of complement activation. In addition, several complement regulatory proteins were detected in the human retinal proteome, and glaucomatous samples exhibited a trend toward downregulation of CFH expression. In vitro experiments revealed that oxidative stress, which was also prominently detectable in the glaucomatous human retinas, downregulated CFH expression in retinal cells.

Conclusions: These findings expand the current knowledge of complement activation by presenting new evidence in human glaucoma and support that despite important roles in tissue cleaning and healing, a potential deficiency in intrinsic regulation of complement activation, as is evident in the presence of oxidative stress, may lead to uncontrolled complement attack with neurodestructive consequences.

Figure 1.

Integration of the identified proteins into canonical complement activation pathways using bioinformatics analysis tools (Ingenuity Pathways Knowledge Base; Ingenuity Systems). Blue: proteins identified by the LC-MS/MS analysis of human retinal proteins. Yellow: additional proteins detected in the human retina by immunohistochemical analysis using specific antibodies.

Figure 2.

Differential regulation of CFH expression in human glaucoma. CFH expression was determined by Western blot analysis of retinal protein samples obtained from 10 human donor eyes with glaucoma and compared with 10 age-matched control eyes without glaucoma. After normalization to β-actin, average band intensities were compared between control and glaucomatous samples. This comparison detected a significant decrease in CFH expression in glaucomatous retinas compared with controls (Mann-Whitney rank sum test; P < 0.001). When the average normalized intensity value obtained from control samples was used to calculate the fold change in CFH expression, 7 of 10 glaucomatous samples exhibited a greater than two-fold decrease.

Figure 3.

Immunohistochemical analysis of the cellular localization of complement components in the human retina. Consistent with proteomic findings, histologic sections of the glaucomatous human retina exhibited prominent immunoperoxidase labeling for the complement components C1q and C3b and the membrane attack complex C5b-9. However, a decrease was detectable in immunolabeling of the glaucomatous retina for the complement regulatory protein, CFH. Immunolabeling for different complement components was most prominent in the inner retina, including primarily the RGCs and inner plexiform layers. The bottom panels show negative controls, in which the first antibody was replaced with serum. gc, retinal ganglion cells layer; in, inner nuclear layer; on, outer nuclear layer. Scale bar, 100 μm.

Figure 4.

Immunohistochemical analysis of the cellular localization of CFH expression in the human retina. Images show double-immunofluorescence labeling of the RGC layer in human retina sections. Used antibodies were a specific antibody to CFH (red) and antibodies to different cell markers (green), GFAP (an astrocyte marker), Brn-3 (a RGC marker), or NeuN (a nonspecific neuronal marker). (A) In the control retina, CFH immunolabeling was detectable in both GFAP-positive astrocytes and GFAP-negative neurons in the RGC layer. (B) In glaucomatous eyes, colocalization of CFH and GFAP was similar; however, neuronal CFH immunolabeling exhibited a prominent decrease. Note that the red corresponding to GFAP-negative neurons in the merged image in (A) decreased in (B). (C, D) CFH and Brn-3 double immunolabeling. Lower magnification images in these panels support a prominent decrease in CFH immunolabeling of multiple Brn-3–positive RGCs in glaucoma. Consistently, in the higher magnification image in (E), NeuN-positive neurons in the RGC layer (arrow) exhibit prominent immunolabeling for CFH in the control retina. However, no CFH immunolabeling is detectable in the NeuN-positive RGC (F), whereas NeuN-negative glial cells in the same glaucomatous retina still exhibit immunolabeling for CFH. (G, H) Double-immunofluorescence labeling of the RGC layer in glaucomatous eyes using antibodies to CD35 or CD59 (red) and a neuronal marker, NeuN (green). Although both neuronal and nonneuronal cells exhibited CD59 immunolabeling, CD35 immunolabeling was detectable primarily on NeuN-negative nonneuronal cells. Scale bars: 50 μm (A, B, E–H); 150 μm (C, D).

Figure 5.

Bioinformatics analysis of complement regulation. Bioinformatics analysis of the mass spectrometric data (using the Ingenuity Pathways Analysis System) established extended networks of signaling molecules associated with complement regulation in glaucoma. In this extended high-probability network, blue shows the proteins out of thousands of proteins identified in the human retinal proteome. Detailed information for abbreviated proteins is available at http://www.ncbi.nlm.nih.gov/protein.

Figure 6.

In vitro experiments determining the regulation of CFH expression by oxidative stress. (A) Phase-contrast image of the cocultured RGCs and macroglia. (B) Both GFAP-positive macroglia and NeuN- and Brn-3-positive RGCs exhibited CFH immunolabeling in these cocultures. (C) Treatment of cocultures with staurosporine (100 nM) or H₂O₂ (50 μM) for 24 hours resulted in a significant decrease in the number of surviving cells (Mann-Whitney rank sum test; P = 0.003 and P = 0.01, respectively). The survival rate was expressed as the percentage of the total cell number in control wells. (D) Quantitative Western blot analysis. Retinal cells exposed to H₂O₂-induced oxidative stress exhibited a significant decrease in CFH expression (Mann-Whitney rank sum test; P < 0.01), which was parallel to a prominent increase in HNE adducts. However, CFH expression did not prominently change in staurosporine-treated cells that exhibited no prominent HNE modifications. Data represent at least three independent experiments and are presented as mean ± SD.

Figure 7.

Oxidative stress in the glaucomatous human retina. Western blot analysis of human retinal protein samples obtained from 10 donors with glaucoma and 10 age-matched controls without glaucoma detected a prominent increase in HNE immunolabeling of glaucomatous samples compared with controls (Mann-Whitney rank sum test, P < 0.001). When the average β-actin–normalized intensity value obtained from control samples was used to calculate the fold change in HNE immunolabeling, all glaucomatous samples exhibited an over two-fold increase.