Phospholipase A2 in chamber angle of normal eyes and patients with primary open angle glaucoma and exfoliation glaucoma

Abstract

Purpose: Phospholipase A2 (PLA2) is a growing family of lipolytic enzymes that play a key role in various biological processes including general lipid metabolism, membrane homeostasis, and in diseases such as atherosclerosis, arthritis, and acute pancreatitis. Oxidative stress as well as inflammation may be associated with glaucoma pathogenesis. Therefore, our aim was to examine the expression of group IIA secretory PLA2 (sPLA2-IIA), group V secretory PLA2 (sPLA2-V), calcium-independent PLA2 (iPLA2), and cytosolic PLA2 (cPLA2) type in the trabecular meshwork (TM) and the canal of Schlemm in normal eyes and in juxtacanalicular tissue samples from patients with primary open angle glaucoma (POAG) or exfoliation glaucoma (ExG).

Methods: TM tissues were isolated from healthy donor eyes for corneal transplantation. Specimens of inner wall of the Schlemm's canal and the juxtacanalicular tissue were collected during deep sclerectomy from the eyes of patients who had POAG or ExG. Antibodies against PLA2s (sPLA2-IIA, sPLA2-V, iPLA2, and cPLA2) and a standard immunohistochemical procedure were used for the analysis. Quantification of immunoreactions was provided using a Photoshop-based image analysis. Double-staining immunofluorescence of macrophages and sPLA2-IIA was performed by using confocal microscopy.

Results: sPLA2-IIA was not present in normal TM. In contrast, sPLA2-IIA levels were significantly higher in glaucoma patients than in controls. Furthermore, sPLA2-IIA expression was much higher in POAG when compared to ExG. iPLA2 was found to predominate in normal human TM, and it demonstrated strong labeling in the uveal and corneoscleral meshwork. The staining of juxtacanalicular meshwork was only moderate in density. In contrast, expression of the enzyme was significantly decreased in glaucoma patients, especially in ExG, when compared to normal controls or to POAG. In addition, strong regional differences were detected in sPLA2-IIA and iPLA2 levels in POAG, whereas immunostaining of these enzymes was much lower and rather uniform throughout ExG sample. In POAG, sPLA2-IIA staining was restricted to certain parts of the trabecular samples where sPLA2-IIA positive macrophages were also present. Immunostaining of sPLA2-V or cPLA2 was low, and no significant changes were found in levels of these enzymes between normal and glaucomatous samples.

Conclusions: sPLA2-IIA, an oxidative stress marker in atherosclerosis, is overexpressed especially in POAG. This result supports the hypothesis that oxidative stress may play a significant role in the pathogenesis of POAG. In ExG, a dramatic decrease in the expression level of iPLA2, a housekeeping enzyme in phospholipid remodeling, may indicate imbalance in phospholipid turnover and also inhibition of normal physiological functions in the TM. These findings may contribute to understanding the pathogenesis of POAG and ExG and may be important for the development of novel therapeutic strategies to different glaucomas.

Figure 1

Immunohistochemical localization of PLA₂s in normal human trabecular meshwork. Immunostaining for sPLA₂-IIA (A) or sPLA₂-V (B) was negative in the trabecular meshwork. Intense staining for sPLA₂-IIA or sPLA₂-V was evident in a few inflammatory-like cells (arrow). iPLA₂ immunolabeling was strong (C). Labeling of the uveal and corneoscleral meshwork was stronger compared to the staining of the juxtacanalicular meshwork. Low positive staining was seen in the apical parts of the cells lining Schlemm's canal (arrowhead) as well as in nearby extracellular regions. Positive staining was also seen in a few macrophages (arrow). Inset: A portion of trabecular meshwork lamellae at higher magnification. Uveal trabecular meshwork cells covering the lamellae were more intensely labeled compared to connective tissue core. cPLA₂ was weakly positive (D) and staining was slightly higher in uveal and corneoscleral meshwork compared to juxtacanalicular meshwork. The cells lining Schlemm's canal showed weak staining. Positive staining was seen in a few macrophages (arrow). AC, anterior chamber; SC, Schlemm's canal. The scale bar is equal to 50 μm.

Figure 2

Surgical site. Shown in this schematic line drawing of the chamber angle is the location of the surgical site (dashed line). SC, Schlemm's canal

Figure 3

Immunostaining for PLA₂s in POAG and ExG. Immunoreaction for sPLA₂-IIA is heavier in POAG (A) compared to ExG (B). In trabecular meshwork tissue, immunoreactivity was found near the stained macrophages. PLA₂-V staining was weak in POAG (C) and ExG (D). In POAG (E) trabecular meshwork staining for iPLA₂ is near positively stained macrophages. In ExG samples (F) iPLA₂ staining is in macrophages. Cellular staining of cPLA₂ was low in POAG (G) and ExG (H) samples. The scale bar was equal to 50 μm.

Figure 4

Photoshop-based image analysis of sPLA₂-IIA, PLA₂-V, iPLA₂, and cPLA₂ levels in TM. Cryostat sections were obtained from POAG patients (red bars), ExG patients (blue bars), or from healthy donor eyes (green bars). The results are mean (arbitrary units)±SEM of six POAG patients, six ExG patients or three healthy donor eyes. Each set of experiments was performed in triplicate.

Figure 5

Immunostaining of macrophages. The number of macrophages is increased in POAG (A) when compared to ExG (B) or control samples (C). The scale bar is equal to 50 μm.

Figure 6

Colocalization of sPLA₂-IIA and macrophages in POAG and ExG. The first column shows nuclear staining (far red shown in blue) with SYTO 62. The second column shows sPLA₂-IIA staining (green), and the third column shows macrophages (red). Combined image (sPLA₂-IIA + macrophage) in the fourth column shows the colocalization of sPLA₂-IIA and macrophage (yellow). The scale bar is equal to 50 μm.

Figure 7

Western blot analysis. Human normal TM samples (10 μg protein) were subjected to SDS-PAGE followed by Western blot analysis using sPLA₂-IIA, sPLA₂-V, iPLA₂, or cPLA₂ antibody. Recombinant human sPLA₂-IIA (rh-sPLA₂-IIA; 200 ng) and sPLA₂-V (rh-sPLA₂-V; 200 ng) were used as positive controls. Molecular mass markers are indicated on the right. Results are representative of at least three separate experiments.