Claire L. Kublin

Differential roles for two gelatinolytic enzymes of the matrix metalloproteinase family in the remodelling cornea.

We have documented changes in collagenolytic/gelatinolytic enzymes of the matrix metalloproteinase family (MMP) in remodelling rabbit cornea. MMP-2 (65 kDa gelatinase) in the proenzyme form is synthesized by the cells of the normal corneal stroma. After keratectomy the level of MMP-2 is increased in the stroma and enzyme appears in both pro- and activated forms. In addition, corneal cells synthesize MMP-9 (92 kDa gelatinase) in the proenzyme form after keratectomy; expression occurs in both the epithelial as well as stromal corneal layers. Changes in expression of both enzymes are precisely localized to the repairing portion of cornea, but demonstrate important differences in timing that correlate with the timing of specific events of matrix remodelling. Our data suggest that each of the gelatinases plays a different role in tissue remodelling after injury. We hypothesize that MMP-2 performs a surveillance function in normal cornea, catalyzing degradation of collagen molecules that occasionally become damaged. After wounding, this enzyme appears to participate in the prolonged process of collagen remodelling in the corneal stroma that eventually results in functional regeneration of the tissue. MMP-9 expression does not correlate with stromal remodelling, but we suggest that the enzyme might play a part in controlling resynthesis of the epithelial basement membrane.

A Simple Method for the Removal of Rabbit Corneal Epithelium Utilizing n-Heptanol

A new method for removing corneal epithelial cells, using n-heptanol, is faster, more precise, and less damaging than scraping, which is the usual method of removing surface epithelium. We compared the results of n-heptanol removal with those of epithelial scraping, using scanning and transmission electron microscopy. Heptanol, unlike scraping, does not damage either the basement membrane or the extracellular stromal components. The rate of healing of corneal epithelium after heptanol treatment is similar to published observations of scraped corneas. Both scraping and heptanol destroy keratocytes, which underlie the basement membrane.

Regeneration of corneal tissue

Abstract Penetrating wounds in rabbit corneas heal to form an opaque tissue that eventually becomes transparent. DNA content, dry weight, water content, and collagen content of the tissue gradually become more like that of normal cornea. The healing tissues also synthesize low-sulfated keratan sulfate, hyaluronic acid, and heparan sulfate. These glycosaminoglycans are not found in normal adult corneas but have been reported in fetal corneas. Previous studies have shown that collagen from healing corneal wounds and collagen from fetal corneas have very similar cross-linking patterns, but these patterns are different from those in normal adult collagen. The similarities between collagen and glycosaminoglycans in healing corneal wounds and in fetuses suggest some recapitulation of ontogenetic processes. The biochemical sequence and eventual return of transparency to the rabbit cornea indicate a capability for true regeneration of stromal tissue in the rabbit.

Corneal scar formation

Abstract The present study is concerned with scar formation in rabbit cornea after removal of a centrally-located 2·0 mm full thickness corneal “button”. Simplicity in wounding, reproducibility in healing with minimum complications, and ease of recovery of adequate amounts of “pure” scar tissue make this experimental model suitable for quantitative biochemical analyses. Quantification of hexosamine in purified glycosaminoglycan fraction from corneal excision wounds indicates that the amount of glycosaminoglycan progressively increases to 74% of that in the normal stroma by the fourth week of healing. This observation is not consistent with previous finding in corneal incision wounds. Hydroxyproline determinations in corneal wounds strongly suggest that collagen accumulation is very rapid during the first two weeks of healing. This is followed by a gradual increase in collagen, approaching the level in normal corneal stroma by the ninth week of healing.

Mechanisms of Murine Lacrimal Gland Repair after Experimentally Induced Inflammation

PURPOSE The authors recently reported that a severe inflammatory response resulting in substantial loss of acinar cells was induced by a single injection of interleukin-1alpha into the lacrimal gland and that this effect was reversible. The purpose of the present study was to determine the mechanisms involved in lacrimal gland injury and repair. METHODS Inflammation was induced by direct injection of recombinant human interleukin-1alpha (IL-1alpha, 1 microg in 2 microL) into the exorbital lacrimal glands of anesthetized female BALB/c mice. Animals were killed 1, 2, 3, 4, 5, 6, or 7 days after injection. Exorbital lacrimal glands were then removed and processed for measurement of protein secretion, histology, immunohistochemistry, and Western blotting. RESULTS The results show that lacrimal gland acinar cells are lost through programmed cell death (apoptosis) and autophagy. They also show that the number of nestin (a stem cell marker)-positive cells increased 2 to 3 days after injury and that some of these cells were also positive for Ki67 (a cell proliferation marker) and alpha-smooth muscle actin (a marker of myoepithelial cells). Finally, they show that the amount of phosphorylated Smad1/5/8 (effector molecules of bone morphogenetic protein 7 [BMP7]) increased 2 to 3 days after injury and could also be detected in nestin-positive cells. CONCLUSIONS The lacrimal gland contains stem/progenitor cells capable of tissue repair after injury. Programmed cell death after injury triggers proliferation and differentiation of these cells, presumably through activation of the BMP7 pathway.

Isolation and Propagation of Mesenchymal Stem Cells From the Lacrimal Gland

PURPOSE Previously, it was reported that the murine lacrimal gland is capable of repair after experimentally induced injury and that the number of stem/progenitor cells was increased during the repair phase (2-3 days after injury). The aim of the present study was to determine whether these cells can be isolated from the lacrimal gland and propagated in vitro. METHODS Lacrimal gland injury was induced by injection of interleukin (IL)-1, and injection of saline vehicle served as control. Two and half days after injection, the lacrimal glands were removed and used to prepare explants or acinar cells for tissue culture. Cells derived from the explants and the acinar cells were grown in DMEM supplemented with 10% fetal bovine serum. Cells were stained for the stem cells markers, nestin, vimentin, ABCG2, and Sca-1. Cell proliferation was measured using an antibody against Ki67 or a cell-counting kit. The adipogenic capability of these cells was also tested in vitro. RESULTS Results show that nestin-positive cells can be isolated from IL-1-injected, but not saline-injected, lacrimal glands. A population of nestin-positive cells was also positive for vimentin, an intermediate filament protein expressed by mesenchymal cells. In addition, cultured cells expressed two other markers of stem cells, ABCG2 and Sca-1. These cells proliferated in vitro and can be induced to form adipocytes, attesting to their mesenchymal stem cell property. CONCLUSIONS Murine lacrimal glands contain mesenchymal stem cells that seem to play a pivotal role in tissue repair.

c-Jun NH2-terminal kinase mediates interleukin-1beta-induced inhibition of lacrimal gland secretion.

Sjögrens syndrome, an inflammatory disease affecting the lacrimal and salivary glands, is the leading cause of aqueous tear‐deficient type of dry eye. We previously showed that interleukin‐1β (IL‐1β) protein is up regulated in the lacrimal gland of a murine model of Sjögrens syndrome and that exogenous addition of this cytokine inhibits neurotransmitter release and lacrimal gland protein secretion. In the present study we investigated the role of c‐Jun NH2‐terminal kinase (JNK) in IL‐1β‐mediated inhibition of lacrimal gland secretion and tear production. In vitro, IL‐1β induced a time‐dependent activation of JNK with a maximum 7.5‐fold at 30 min. SP600125, a JNK inhibitor, inhibited, in a concentration‐dependent manner, IL‐1β‐induced activation of JNK with a maximum of 87% at 10−4 m. In vivo, IL‐1β stimulated JNK and the expression of the inducible isoform of nitric oxide synthase (iNOS). IL‐1β inhibited high KCl and adrenergic agonist induced protein secretion by 85% and 66%, respectively. SP600125 alleviated the inhibitory effect of IL‐1β on KCl‐ and agonist‐induced protein secretion by 79% and 47%, respectively, and completely blocked the expression of iNOS. Treatment for 7 days with SP600125 increased tear production in a murine model of Sjögrens syndrome dry eye. We conclude that JNK plays a pivotal role in IL‐1β‐mediated inhibition of lacrimal gland secretion and subsequent dry eye.

Role of Epithelial-Mesenchymal Transition in Repair of the Lacrimal Gland after Experimentally Induced Injury

PURPOSE Ongoing studies demonstrate that the murine lacrimal gland is capable of repair after experimentally induced injury. It was recently reported that repair of the lacrimal gland involved the mobilization of mesenchymal stem cells (MSCs). These cells expressed the type VI intermediate filament protein nestin whose expression was upregulated during the repair phase. The aim of the present study was to investigate the roles of vimentin, a type III intermediate filament protein and a marker of epithelial-mesenchymal transition (EMT) in repair of the lacrimal gland. METHODS Injury was induced by direct injection of interleukin (IL)-1 into the exorbital lacrimal gland. MSCs were prepared from injured glands using tissue explants. Expression of vimentin and the transcription factor Snai1, a master regulator of EMT, was determined by RT-PCR, Western blotting analysis, and immunofluorescence. RESULTS These data show that vimentin expression, at both the mRNA and the protein levels, was upregulated during the repair phase (2-3 days postinjury) and returned to the control level when repair ended. Temporal expression of Snai1 mirrored that of vimentin and was localized in cell nuclei. Cultured MSCs isolated from injured lacrimal glands expressed Snai1 and vimentin alongside nestin and alpha smooth muscle actin (another biomarker of EMT). There was a strong positive correlation between Snai1 expression and vimentin expression. CONCLUSIONS It was found that EMT is induced during repair of the lacrimal gland to generate MSCs to initiate repair, and that mesenchymal-epithelial transition is then activated to form acinar and ductal epithelial cells.

Morphogenesis of rabbit corneal endothelium

We studied ultrastructurally the development of rabbit corneal endothelium from the 13th day of gestation to 3 days after birth. Precursor corneal endothelial cells, stromal cells, and a vascular network migrate in close association with each other between the developing corneal and lens epithelia. During development, newly deposited extracellular fibrous matrices separate the prospective endothelium from the capillaries and corneal stroma. The extracellular matrix between the apical endothelial surface and the vascular network loses its fibrous appearance early in development. Simultaneously, randomly organized fibrils are deposited on the basal endothelial surface facing the stroma. These fibrils, gradually obscured by the deposition of a nonfibrous component, eventually become part of Descemets membrane. Early in development, prospective endothelial cells cannot be distinguished morphologically from the overlying corneal stromal cells. Morphologic differentiation of the endothelial cell is characterized by the formation of sinuous lateral borders that interdigitate with those of adjacent cells to form a continuous single-cell layer of tissue. The basal endothelial membrane forms a pitted surface, distinguishing it from the apical cell membrane. Intercellular junctions between lateral membranes, a cilium projecting into the anterior chamber, and deposition of Descemets membrane on the basal endothelial surface contribute to the polarization of the endothelium. Throughout most of corneal development the vascular pupillary membrane maintains a close association with the apical surface of the differentiating endothelium. We conclude that fetal corneal endothelium develops within a complex extracellular matrix environment and in proximity to the underlying vascular network. These structures play an important role in the morphogenesis of corneal endothelium.

Impaired Neurotransmission in Lacrimal and Salivary Glands of a Murine Model of Sjögren’s Syndrome

The epithelium of the conjunctiva is a non-keratinizing stratified squamous epithelium1 Goblet cells, highly specialized epithelial cells, are located in the apical surface of the conjunctiva between the layers of stratified epithelium.2 These cells are primarily responsible for the secretion of the inner mucous layer of the tear film, thereby providing a physical and chemical barrier that hydrates the conjunctiva and protects it from exposure to other injurious agents. Abnormal mucin secretion (either overproduction or underproduction) by goblet cells can eventually lead to deterioration of the ocular surface.