Immunohistochemical Detection of CTGF in the Human Eye

<p><i>Purpose/Aim of the study</i>: Connective tissue growth factor (CTGF) is a key player in the control of extracellular matrix remodeling, fibrosis, and angiogenesis. It is also involved in the modification of the trabecular meshwork, thus potentially modulating outflow facility and intraocular pressure (IOP). As a consequence, CTGF might be relevant for the development of elevated IOP, a major risk factor in glaucoma-pathogenesis. While comprehensive information on the origins of CTGF in the human eye is not available, the goal of this study is to identify ocular sources of CTGF using morphological methods.</p> <p><i>Materials and Methods</i>: Human donor eyes were prepared for immunohistochemical analysis of CTGF, α-smooth muscle-actin (ASMA), and CD31. Confocal laser scanning microscopy was used for documentation.</p> <p><i>Results</i>: In the cornea, CTGF-immunoreactivity (CTGF-IR) was detected in the epithelium, mainly in basal layers, stromal keratinocytes, and endothelial cells. Adjacent conjunctiva showed also CTGF-IR in epithelial cells. In the iris, both, the sphincter and dilator muscles displayed CGTF-IR, as did iris and ciliary body vessels, deriving at this location from the vascular endothelium, as detected with CD31, but not from vascular smooth muscle cells, as detected with ASMA. In the ciliary body, CTGF-IR was detected in smooth-muscle cells of the ciliary muscle and further in the non-pigmented epithelium. In the retina, CTGF-IR was detected in the NFL and weakly in the IPL/OPL. In the choroid, the choriocapillaris and blood vessels displayed CTGF-IR. Further, few cells in the optic nerve head and the lamina cribrosa were CTGF-positive.</p> <p><i>Conclusion</i>: CTGF was detected in various structures of the human eye. Since CTGF has been also described in aqueous humor, the identified structures might be the sources of CTGF in the aqueous humor. By means of aqueous flow, CTGF is transported into the trabecular meshwork, where it could change outflow facility and therefore affecting IOP homeostasis.</p

Corneas of scAAV2(triple)- and scAAV8(Y733F)- injected animals.

<p>GFP expression was detected in (A) scAAV(triple) and (B) scAAV8(Y733F)- injected mouse as well as in (C) scAAV2(triple)- and (D) scAAV8(Y733F)- injected rat corneas. Arrows indicate GFP- positive keratinocytes.</p

Localization of the GFP signal in the anterior section of eyes injected with similar titers of scAAVs.

<p>The transduction efficiency (grading:-,-/+, +, ++) is based on the intensity and distribution of the GFP signal in tissues of the anterior section. Unless otherwise noted (see asterisks) the term cornea represents GFP-expression in the corneal endothelium.</p><p>* One out of five showed GFP-positivity in the corneal stroma in addition to the corneal endothelium.</p><p>** One out of five showed GFP-positivity in the corneal stroma.</p><p>Localization of the GFP signal in the anterior section of eyes injected with similar titers of scAAVs.</p

GFP expression in scAAV-injected rat eyes.

<p>In A-D, representative sections of the region around the chamber angle are shown. The small inserts illustrate the cornea, the iris and the NPE. Nuclei are stained with DAPI. Arrows indicate GFP signals in the cornea, the iris, the chamber angle and/or the NPE. Arrowheads mark the region of the TM.</p

Injection procedure in mice.

<p>(A) The cornea is punctured closely anterior to the iridocorneal angle. (B) Before vector administration, an air bubble is created to seal the puncture site. (C-D) scAAV or vehicle dyed with fluorescein is injected. The air bubble seals the puncture site and reflux is minimized. The air bubble is absorbed within 24 hrs.</p

Co-staining of GFP (green, arrows) and TSP-1 (red, arrowheads) in chamber angle of injected rat eyes.

<p>(A) scAAV2(Y444F) and (B) scAAV2(triple) mediate efficient transgene expression in the region of the trabecular meshwork. Nuclei are stained with DAPI. Asterisk indicates Schlemm‘s canal.</p

GFP expression in scAAV-injected mouse eyes.

<p>In A-D, representative sections of the region around the chamber angle are shown. The small inserts illustrate the cornea, the iris and the NPE. Nuclei are stained with DAPI. Arrows indicate GFP signals in the cornea, the iris, the chamber angle, the ciliary body and/or the NPE. Arrowheads mark the region of the TM.</p

AAV vectors and titers.

<p>AAV vectors and titers.</p

Experimental groups of titer-matched scAAV-injected and mock-injected eyes.

<p>A sample size of 5 eyes per group was analyzed by immunohistochemistry for GFP-expression. The experimental protocol was performed in C57BL/6 mice and Sprague Dawley rats.</p><p>Experimental groups of titer-matched scAAV-injected and mock-injected eyes.</p

Comparison of age-related macular degeneration case-control studies in the literature with the present study.

a<p>mtDNA = mitochondrial DNA.</p>b<p>According to PhyloTree.org <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030874#pone.0030874-vanOven1" target="_blank">[21]</a>.</p>c<p>P-values: present study: adjusted for age and sex. Jones et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030874#pone.0030874-Jones1" target="_blank">[12]</a>: adjusted for age, sex and current smoking. Canter et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030874#pone.0030874-Canter1" target="_blank">[13]</a>: adjusted for sex and three nuclear polymorphisms (CFH-Complement Factor H gene, rs1061170; LOC387715, rs10490924; APOE, ApoE2 allele). Udar et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030874#pone.0030874-Udar1" target="_blank">[14]</a>: no adjustment. SanGiovanni et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030874#pone.0030874-SanGiovanni1" target="_blank">[15]</a>: adjusted for age, sex and smoking.</p>d<p>CI = confidence interval.</p