keratan-sulfate and Eye-Injuries

An important aspect of wound healing is the recruitment of neutrophils to the site of infection or tissue injury. Lumican, an extracellular matrix component belonging to the small leucine rich proteoglycan (SLRP) family, is one of the major keratan sulfate proteoglycans (KSPGs) within the corneal stroma. Increasing evidence indicates that lumican can serve as a regulatory molecule for several cellular processes, including cell proliferation and migration. In the present study, we addressed the role of lumican in the process of extravasation of polymorphonuclear leukocytes (PMNs) during the early inflammatory phase present in the healing of the corneal epithelium following debridement. We used Lum(-/-) mice and a novel transgenic mouse, Lum(-/-),Kera-Lum, which expresses lumican only in the corneal stroma, to assess the role of lumican in PMN extravasation into injured corneas. Our results showed that PMNs did not readily invade injured corneas of Lum(-/-) mice and this defect was rescued by the expression of lumican in the corneas of Lum(-/-),Kera-Lum mice. The presence of lumican in situ facilitates PMN infiltration into the peritoneal cavity in casein-induced inflammation. Our findings are consistent with the notion that in addition to regulating the collagen fibril architecture, lumican acts to aid neutrophil recruitment and invasion following corneal damage and inflammation.

Topics: Animals; Chondroitin Sulfate Proteoglycans; Cornea; Corneal Diseases; Corneal Injuries; Epithelium, Corneal; Eye Injuries; Flow Cytometry; Humans; Immunohistochemistry; Keratan Sulfate; Lumican; Mice; Neutrophils; Peritoneal Lavage; Wound Healing

We examined the effect of adenovirus-mediated transient expression of Smad7, an inhibitory Smad in TGFbeta/activin signaling, on injury-induced epithelial-mesenchymal transition (EMT) of lens epithelium in mice. A volume of 3 microl of adenoviral solution was injected into the right lens of adult male C57BL/6 mice (n=56) at the time of capsular injury made using a hypodermic needle under general anesthesia. A mixture of recombinant adenovirus carrying CAG promoter-driven Cre (Cre adv) and mouse Smad7 complementary DNA (Smad7 adv) was administered to induce Smad7 expression, while control lenses were treated with Cre adv alone. After healing intervals of 2, 3, 5, and 10 days, animals were killed 2 h after labeling with bromodeoxyuridine (BrdU) and eyes were processed for histology. During healing, marked expression of Smad7 was observed in lens epithelial cells in the Smad7 adv group with loss of nuclear translocation of Smads2/3, while little Smad7 and abundant nuclear Smads2/3 were seen in cells in the Cre adv group. Lens epithelial cells in the Cre adv control group exhibited a fibroblastic appearance at days 5 and 10 and the capsular break was sealed with fibrous tissue, while Smad7 adv-treated cells around the capsular break retained their epithelial morphology and the break was not sealed. Expression of snail mRNA, and alpha-smooth muscle actin, lumican, and collagen VI proteins, markers of EMT, was observed in control-treated eyes, but not in cells of the Smad7 adv group at day 5 with minimal expression at day 10. Additionally, cell proliferation increased in epithelium infected with Smad7 adv consistent with suppression of injury-induced upregulation of TGFbeta1 in epithelium. We conclude that gene transfer of Smad7 in mice prevents injury-induced EMT of lens epithelial cells and sealing of the capsular break with fibrous tissue.

Topics: Actins; Adenoviridae; Animals; Cell Division; Chondroitin Sulfate Proteoglycans; Collagen Type VI; Disease Models, Animal; DNA-Binding Proteins; Epithelial Cells; Eye Injuries; Gene Expression Regulation; Genetic Therapy; Genetic Vectors; Keratan Sulfate; Lens Capsule, Crystalline; Lens, Crystalline; Lumican; Male; Mesoderm; Mice; Mice, Inbred C57BL; RNA, Messenger; Smad7 Protein; Trans-Activators; Transduction, Genetic; Transforming Growth Factor beta; Wound Healing

Keratocytes synthesize keratan-sulfate proteoglycans (KSPG), lumican and keratocan, to develop and maintain proper collagen interfibrillar spacing and fibril diameter characteristics of the transparent cornea. The purposes of this study are to compare the expression patterns of KSPGs and keratin 12 (K12) respectively by corneal keratocytes and epithelial cells after three different types of injuries; partial and total epithelial debridement and alkali burn.. Corneas of 8-12 week old C57Bl/6J or FVBN mice were wounded by partial epithelial (2 mm in diameter) and total epithelial debridement, and alkali burn (0.1 M NaOH, 30 s) and were allowed to heal for various periods of time, from 1 to 84 days. The corneas were then subjected to light microscopy, in situ and Northern hybridization and RT-PCR for examining the expression of K12 and KSPG in the corneal epithelium and stroma, respectively. Immunohistochemistry with anti-alpha-smooth muscle actin (alpha-SMA) was used to identify myofibroblasts in the stroma of injured cornea.. In 2-3 days, partial epithelial denuded corneas were resurfaced by corneal epithelium positive for K12, and stromal edema caused by debridement disappeared. Total epithelial debridement wounded corneas were resurfaced by conjunctival epithelial cells in 2 weeks. Stromal edema in the total epithelial debridement corneas began to subside after 6 weeks. Corneal epithelial cells resurfaced alkali burned corneas within 3-5 days. In situ and Northern hybridization showed a decrease in keratocan and lumican expression at 6 weeks and increased at 12 weeks post-injury in all wound types. Alpha-SMA positive myofibroblasts in the cornea were detected via immunostaining at the time point when KSPG expression was lowest, 6 weeks post-injury.. The results suggest keratocan and lumican are down-regulated during wound healing at 6 weeks and returned to higher levels at 12 weeks post-injury; implicating that the cells repopulating the injured corneal stroma regained the characteristic function of keratocytes independent of the wound types. However, complete epithelial removal results in irreversible loss of K12 expression.

Topics: Animals; Blotting, Northern; Burns, Chemical; Chondroitin Sulfate Proteoglycans; Cornea; Corneal Injuries; Corneal Stroma; Down-Regulation; Epithelium, Corneal; Eye Burns; Eye Injuries; Eye Proteins; Fibroblasts; Immunoenzyme Techniques; In Situ Hybridization; Keratan Sulfate; Keratin-12; Keratins; Lumican; Mice; Mice, Inbred C57BL; Proteoglycans; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sodium Hydroxide; Wound Healing