Conclusions. Our results support our hypothesis and show that PRG4 is transcribed in human corneal andconjunctival epithelial cells, as well as in mouse lacrimal and meibomian glands. We are currently testing ourhypothesis that PRG4 mRNA is translated, secreted onto the ocular surface, and serves to protect the corneaand conjunctiva against significant shear forces generated during an eyelid blink.

Acknowledgments. The authors thank Ms. Sandra Michaud and Dr. Pablo Argueso for their assistance.

PROTEOGLYCAN 4 mRNA EXPRESSION IN HUMAN CORNEAL AND CONJUNCTIVALEPITHELIAL CELLS. Sullivan BD1, Richards SM2, Talbot D2, Schmidt TA1, and Sullivan DA2.1Department of Bioengineering, University of California at San Diego, La Jolla, CA, and 2Schepens EyeResearch Institute and Harvard Medical School, Boston, MA, USA

1. Introduction

This research was supported by NIH grant EY05612. Benjamin Sullivan is a consultant to OcuSense., Inc.David A. Sullivan is on the Scientific Advisory board of OcuSense., Inc.

5. References

1. Grodzinsky, A and Eisenberg, S. Bioelectromechanics, Course Notes: Fields Forces and Flows in BiologicalTissues and Membranes. Departments of Electrical Engineering and Computer Science, MechanicalEngineering, and Harvard-MIT Division of Health Sciences Technology, Department of BiomedicalEngineering, Boston University, 1995

2. Ikegawa S, Sano M, Koshizuka Y, Nakamura Y. Isolation, characterization and mapping of the mouse andhuman PRG4 (proteoglycan 4) genes. Cytogenet Cell Genet. 90(3-4):291-7. 2000

PURPOSE: Proteoglycan 4 (PRG4) may play a critical role as a boundarylubricant in articulating joints. This secreted glycoprotein, which is alsocalled lubricin and superficial zone protein, protects cartilaginous surfacesagainst frictional forces, cell adhesion and protein deposition. Wehypothesize that PRG4 may serve an analogous role on the ocular surfaceand protect the cornea and conjunctiva against significant shear forcesgenerated during an eyelid blink. We also hypothesize that PRG4 issynthesized by corneal and conjunctival epithelial cells and secreted ontothe ocular surface. The purpose of this study was to determine whetherocular surface cells express the gene for PRG4. For comparative purposes,we also examined whether PRG4 mRNA is expressed by mouse lacrimaland meibomian glands.

MATERIALS & METHODS: Human corneal epithelial cells wereisolated from the corneoscleral rims of male and female donors. Cellswere processed either directly (n = 8), or first cultured in phenol red-freekeratinocyte serum free media (n = 2). We also obtained bulbar

6. Support & Disclosures

conjunctivae during surgical procedures (n = 2), conjunctival impressioncytology samples (n = 9), immortalized human conjunctival epithelialcells after culture (n = 1), NOD mouse lacrimal glands (n = 5 adult mice/sex, 10 glands/sample) and BALB/c mouse meibomian glands (n = 7adult mice/sex, glands from 28 lids/sample). Samples were processed forthe analysis of PRG4 mRNA by using primarily RT-PCR (n = 18 human,all mouse), but also Affymetrix GeneChips (n = 4 human corneas). ThePRG4 primers for PCR spanned over 1 kbp of intron sequences, in orderto suppress amplification of contaminating chromosomal DNA (Table1). Amplified samples were screened for the presence of PRG4 productsby using agarose gel electrophoresis and an Agilent 2100 Bioanalyzer. Toconfirm the identity of amplicons, PCR products from cornea samples (n= 2), conjunctival epithelial cells (n = 1) and a human liver standard (n =1) were sequenced with a 3100 Genetic Analyzer at the MassachusettsEye and Ear Infirmary DNA Sequencing Center for Vision Research(Boston, MA) and resulting data were analyzed with BLASTn searchesof GenBank databases.

3. ResultsRESULTS. As shown in Figures 1, 2, 3 and 4, our RT-PCR results demonstrate that PRG4 mRNA is present

in human corneal epithelial cells, conjunctival epithelial cells and conjunctival impression cytology samples.

Similarly, our analysis of human female (n = 1) and male (n = 3) corneal epithelial cells from corneoscleral rims

with Affymetrix GeneChips also showed the presence of PRG4 mRNA. The identity of PRG4 PCR products

was confirmed by DNA sequence analysis (Table 2). The presence of PRG4 is not limited to human tissues.

As illustrated in Figure 5, PRG4 mRNA also occurs in mouse lacrimal and meibomian glands.

4. Conclusions

1 2 3

4

The loading of cornea and conjunctiva is likely dominated by shear forces.Eyelid blinking, as well as contact lens wear, generates significant stressupon ocular surface epithelial cells, and this is especially true in the presenceof a compromised tear film. As shown in Figure 1, increased shear stressleads to tear film instability, evaporative tear loss, hyperosmolarity, changesin swelling pressure and a feedback elevation in shear stress. Increasedshear stress also promotes inflammation, androgen deficiency and decreasedexpression of proteoglycans, all of which further increase shear stress. Overtime, increased shear stress and its sequelae may lead to a loss of boundarylubrication at the ocular surface.

2. Theory - the role of osmolarity in the swelling pressure of PRG4

5

Table 1. Oligonucleotide primers designed for RT- PCR analysis of PRG4 mRNA

Species Orientation Nucleotide sequence (5’ - 3’) Exons Amplicon size (bp)

Human Sense GAT-GCA-GGG-TAC-CCC-AAA 9-12 526Antisense CAG-ACT-TTG-GAT-AAG-GTC-TGC-C

Mouse Sense TGT-CAA-AGG-CTT-TGG-AGG-AC 9-11 367Antisense TCG-CTG-AAG-TAA-CAA-CAT-TTG

Table 2. Identification of amplicon sequences from human cornea, conjunctival and liver samples

Sequencing Direction Aligned Base Pairs Total Base Pairs BLASTn Searchto Human PRG4 from Amplicon Identity

Human Liver StandardA Forward 495 500 Human PRG4A Reverse 488 491 Human PRG4B Forward 496 499 Human PRG4B Reverse 498 500 Human PRG4

Human Cornea (24 year old female)A Forward 497 499 Human PRG4A Reverse 490 492 Human PRG4B Forward 500 504 Human PRG4B Reverse 498 501 Human PRG4

Human Cornea (51 year old female)A Forward 498 499 Human PRG4A Reverse 474 489 Human PRG4B Forward 496 498 Human PRG4B Reverse 490 491 Human PRG4

Human Conjunctival Epithelial CellsA Forward 496 499 Human PRG4A Reverse 490 492 Human PRG4B Forward 495 499 Human PRG4B Reverse 491 491 Human PRG4

Two different samples (A & B) of each preparation were sequenced in forward and reverse directions. Thehuman cornea samples were epithelial cells from the corneoscleral rims of female donors. The gene accessionnumber for human PRG4 is NM_005807