Abstract
purpose. To determine whether the cell adhesion molecule CD44, the principal
receptor of hyaluronan, is altered in the aqueous humor and the
anterior segment of patients with primary open-angle glaucoma (POAG).
methods. The trabecular meshwork (TM), iris, ciliary body, and sclera of POAG
and age-matched control eyes preserved in ethanol were microdissected
and subjected to 1% Triton X-100 solubilization at 4°C. Western blot
analysis was performed using monoclonal antibodies that recognize
either CD44H (hematopoietic; extracellular domain) or CD44S (soluble
ectodomain). The concentration of soluble CD44S in aqueous and
microdissected tissues was measured by enzyme-linked immunosorbent
assay (ELISA).
results. ELISA of soluble CD44S of aqueous from eyes of patients with POAG
indicated that the concentration of soluble CD44S is increased in
comparison with that of aqueous from normal eyes (P < 0.0003). Western blot analysis and densitometry of POAG iris and
ciliary body revealed a statistically significant increase in the
Triton X-100 extraction of CD44H. The predominant increases were in the
180-kDa (P < 0.001) and the 85-kDa
(P < 0.001) forms. ELISA of soluble CD44S
indicated that the concentration is statistically decreased in iris
(P < 0.05), ciliary body (P <
0.001), and TM (P < 0.005) of POAG eyes.
conclusions. Increased amounts of soluble CD44S in POAG aqueous and Triton
X-100–solubilized CD44H characterized POAG in the iris and ciliary
body. These soluble CD44 isoforms may influence the activity of the
transmembrane CD44H by acting as inhibitors of CD44H and, thereby,
adversely influence the cell survival of TM and retinal ganglion cells
in POAG.
Primary open-angle glaucoma (POAG) is a major blinding
disease affecting approximately 67 million persons
worldwide.
1 It is likely that several biochemical and
cellular factors influence the glaucoma process. A variety of cellular
insults or molecular defects
2 3 may intersect, leading
individually or collectively to cell death in the trabecular meshwork
(TM)
4 or retinal ganglion cells.
5 Moreover,
evidence suggests that activated immunity
6 and alterations
in the extracellular matrix
7 8 9 10 may be etiologic factors
in POAG. The extracellular matrix is a diverse group of macromolecules
that assemble to form a functional network. The majority of aqueous
outflow resistance in both normal and POAG eyes is within the TM,
especially within the extracellular matrix of the juxtacanalicular
connective tissue, which is a glycosaminoglycan-enriched
area.
10 11 12 Biochemical studies
10 and
computer-aided image analysis
11 indicate that POAG is
associated with a marked decrease in hyaluronan (HA) and an increase in
chondroitin sulfate in the TM. HA is a key factor in promoting cell
motility, adhesion, and proliferation.
13 14 These cell
events are orchestrated by three HA
15 cell
receptors—CD44, receptor for HA-mediated motility (RHAMM), and
intercellular adhesion molecule (ICAM)-1—all of which have been
identified in the human TM.
16
One receptor for HA, CD44, is a multifunctional receptor and
cell-adhesion molecule that increases with the aging of T
lymphocytes.
17 18 CD44H is an integral cell membrane
glycoprotein with postulated roles in a wide variety of biological
processes, including cell adhesion,
19 inflammation,
20 autoimmunity,
21 and
apoptosis.
22 CD44 exists as both an 80- to 90-kDa type 1
transmembrane glycoprotein, CD44 hematopoietic (CD44H), and a 30- to
50-kDa soluble form, soluble CD44S, generated by the release of
the extracellular domain by hydrolytic cleavage.
23 Hydrolysis of CD44H involves either the cleavage of
glycosylphosphatidylinositol (GPI)-anchor and/or the limited
proteolysis of the extracellular domain. These soluble CD44 isoforms
may regulate the effects of cognate ligands of CD44H by acting as
soluble inhibitors of CD44H. In addition, the proteolytic cleaved
soluble CD44S and the loss of the GPI-anchor of CD44H probably
constitute, at least in part, a turnover mechanism for downregulating
the CD44 receptor.
24
CD44H has diverse functional properties owing to sequence differences
arising from alternate splicing of mRNA, as well as extensive
posttranslational glycosylation.
25 CD44H is expressed on a
variety of ocular cells, including retinal ganglion
cells
26 and axons.
27 CD44S is present in
serum
28 and aqueous humor.
29 In vitro, axon
growth of retinal ganglion cells is inhibited by the presence of
CD44H.
30 Aging leads to replacement of virgin T cells by
memory T cells and to the accumulation of cells with signal
transduction defects.
31 Aged CD44H-positive cells are less
responsive to antigenic stimuli.
32 Yonemura et
al.
33 demonstrated that CD44H is the membrane-binding
partner for ezrin-radixin-moesin (ERM) proteins. The CD44-ERM complex
acts as a regulatable protein scaffold that anchors actin filaments to
the plasma membrane. Consequently, increased turnover of CD44H in POAG
may disrupt several normal cell functions and adversely affect TM cell
function
34 and survival. Therefore, we determined the
concentration of soluble CD44S in the aqueous humor of normal subjects
and patients with POAG in comparison with CD44H and soluble CD44S in
donor eyes, both of which appear to characterize POAG. Our data support
the hypothesis that CD44H may represent a protein marker of the
disease.
34
Samples of aqueous humor were obtained from normal subjects
(n = 41; age range, 46–85 years; mean, 72.5 ±
8.6) who were undergoing elective cataract surgery, patients with
glaucoma (n = 29; age range, 46–85 years; mean,
71.5 ± 9.8) who were undergoing elective cataract surgery
(n = 9) or filtration surgery (n = 20),
and one patient with ocular hypertension who was undergoing elective
cataract surgery. None of the subjects had had incisional ocular
surgery. Seventy microliters of aqueous humor was aspirated by limbal
paracentesis. Patients provided informed consent after the nature and
consequences of the study were explained, in accordance with the
Declaration of Helsinki, and the research was approved by institutional
review boards.
Donor Eyes and Triton X-100 Extraction: CD44H in the Iris and in
the Ciliary Body