Sensory nerve supports epithelial stem cell function in healing of corneal epithelium in mice: the role of trigeminal nerve transient receptor potential vanilloid 4.

In order to understand the pathobiology of neurotrophic keratopathy, we established a mouse model by coagulating the first branch of the trigeminal nerve (V1 nerve). In our model, the sensory nerve in the central cornea disappeared and remaining fibers were sparse in the peripheral limbal region. Impaired corneal epithelial healing in the mouse model was associated with suppression of both cell proliferation and expression of stem cell markers in peripheral/limbal epithelium as well as a reduction of transient receptor potential vanilloid 4 (TRPV4) expression in tissue. TRPV4 gene knockout also suppressed epithelial repair in mouse cornea, although it did not seem to directly modulate migration of epithelium. In a co-culture experiment, TRPV4-introduced KO trigeminal ganglion upregulated nerve growth factor (NGF) in cultured corneal epithelial cells, but ganglion with a control vector did not. TRPV4 gene introduction into a damaged V1 nerve rescues the impairment of epithelial healing in association with partial recovery of the stem/progenitor cell markers and upregulation of cell proliferation and of NGF expression in the peripheral/limbal epithelium. Gene transfer of TRPV4 did not accelerate the regeneration of nerve fibers. Sensory nerve TRPV4 is critical to maintain stemness of peripheral/limbal basal cells, and is one of the major mechanisms of homeostasis maintenance of corneal epithelium.

Impaired healing of cornea incision injury in a TRPV1-deficient mouse.

The present study attempts to elucidate the role of TRPV1 cation channel receptor on primary repair in an incision-wounded mouse cornea in vivo. Previous study revealed that blocking TRPV1 suppressed myofibroblast formation and expression of transforming growth factor ß1 (TGFß1) in cultured keratocytes or ocular fibroblasts. Male C57BL/6 (wild-type; WT) mice and male C57BL/6 Trpv1-null (KO) mice incurred a full-thickness incision injury (1.8 mm in length, limbus to limbus) in the central cornea of one eye with a surgical blade under general and topical anesthesia. The injury was not sutured. On days 0, 5, and 10, the eyes were enucleated, processed for histology, immunohistochemistry, and real-time RT-PCR gene expression analysis to evaluate the effects of the loss of TRPV1 on primary healing. Electron microscopy observation was also performed to know the effect of the loss of TRPV1 on ultrastructure of keratocytes. The results showed that the loss of Trpv1 gene delayed closure of corneal stromal incision with hindered myofibroblast transdifferentiation along with declines in the expression of collagen Ia1 and TGFß1. Inflammatory cell infiltration was not affected by the loss of TRPV1. Ultrastructurally endoplasmic reticulum of TRPV1-null keratocytes was more extensively dilated as compared with WT keratocytes, suggesting an impairment of protein secretion by TRPV1-gene knockout. These results indicate that injury-related TRPV1 signal is involved in healing of stromal incision injury in a mouse cornea by selectively stimulating TGFß-induced granulation tissue formation.

Lack of plakoglobin impairs integrity and wound healing in corneal epithelium in mice.

We generated cornea-specific plakoglobin (Jup; junctional plakoglobin) knockout mice in order to investigate the function of plakoglobin on the maintenance of the homeostasis of corneal epithelium in mice. Cornea epithelium-specific conditional knockouts (JupCEΔ/CEΔ) (cKO) were obtained by breeding keratin12-Cre (Krt12-Cre) mice to Jup-floxed (Jupf/f) mice. Light and transmission electron microscopic and immunohistochemical analyses were carried out to determine consequence of the loss of plakoglobin on maintaining corneal epithelium integrity under mechanical stress, e.g., brushing and wound healing. Immunohistochemistry analysis demonstrated that, although Jup ablation did not affect BrdU incorporation, basal cell-like cells labeled for keratin 14 were ectopically present in the supra-basal layer in mutant corneal epithelium, suggestive of altered cell differentiation. Plakoglobin-deficient epithelium exhibits increased fragility against mechanical intervention when compared to wild-type controls under identical treatment. Closure of an epithelial defect was significantly delayed in JupCEΔ/CEΔ epithelium. Our findings indicate that the lack of plakoglobin significantly affects corneal epithelium differentiation, as well as its structural integrity. Plakoglobin is essential to the maintenance of the structure of the corneal epithelium and its wound healing.

Meibomian gland morphogenesis requires developmental eyelid closure and lid fusion.

BACKGROUND AND PURPOSE: Meibomian glands (MGs) play an important role in the maintenance of ocular surface health, but the mechanisms of their development are still poorly understood. The MGs arise from the epithelium at the junction of eyelid fusion, raising the possibility that defective eyelid fusion disturbs the formation of MGs. METHODS: We examined, histologically and functionally, the development of MGs in mice with either normal or defective eyelid fusion, displaying eye-closed at birth (ECB) or eye-open at birth (EOB) phenotypes, respectively. RESULTS: The Meibomian anlage was detected in the epithelium at the eyelid fusion junction immediately after birth at postnatal day 0 (PD0), and it extended into the eyelid stroma at PD1 and started to branch and produce meibum at PD7 in the ECB mice. In contrast, few if any MG structures were detectable in the EOB mice in the early postnatal periods. The Meibomian gland ductile system was seen aligned along the eyelid margin in the adult ECB mice, but was absent or scarce in that of the EOB mice. While MG abnormalities were found in all EOB mice, the severity varied and corresponded to the position and the size of eye opening but not the genetic defects of the mice. CONCLUSION: Proper Meibomian gland formation and development require eyelid closure and fusion.

Corneal Epithelial Wound Healing.

The cornea is important for a clear vision by refracting light onto the lens, which in turn focusing on the retina. To maintain a smooth optical surface, corneal epithelium has to continuously renew itself to function as a barrier so that it protects the eye from various environmental insults. The adult corneal epithelium is maintained homeostatically by an integrated process of cell proliferation, migration, differentiation, stratification, and desquamation/apoptosis. Impairment of this process results in persistent corneal defect, leading to the blindness. Researches throughout the years revealed that appropriate integration and coordination of cell signaling events are responsible for corneal epithelial renewal and wound healing. In this chapter, we will review works done on cell culture, animal models, and human trials to focus on the signaling network during corneal wound healing process which will have potential for the discovery of novel drug to improve corneal wound healing.

Lumican promotes corneal epithelial wound healing.

Lumican regulates collagenous matrix assembly as a keratan sulfate proteoglycan in the cornea and is also present in the connective tissues of other organs and embryonic corneal stroma as a glycoprotein. In normal unwounded cornea, lumican is expressed by stromal keratocytes. Interestingly, injured mouse corneal epithelium ectopically and transiently expresses lumican during the early phase of wound healing, suggesting a potential lumican functionality unrelated to regulation of collagen fibrillogenesis, e.g., modulation of epithelial cell adhesion or migration. Healing of a corneal epithelial injury in lumican knockout (Lum(-/-)) mice was significantly delayed compared with Lum(+/-) mice. Addition of purified lumican to cultured medium promoted re-epithelialization and enhanced cell proliferation of wild-type mouse corneal epithelial cells in an organ culture. Therefore, administration of lumican may be beneficial for treating epithelial defects in the cornea and other tissues.

The development of meibomian glands in mice.

PURPOSE: The purpose of this study was to characterize the natural history of meibomian gland morphogenesis in the mouse. METHODS: Embryonic (E) and post natal (P) C57Bl/6 mouse pups were obtained at E18.5, P0, P1, P3, P5, P8, P15, and P60. Eyelids were fixed and processed for en bloc staining with Phalloidin/DAPI to identify gland morphogenesis, or frozen for immunohistochemistry staining with Oil red O (ORO) to identify lipid and antibodies specific against peroxisome proliferator-activated receptor gamma (PPARgamma) to identify meibocyte differentiation. Samples were then evaluated using a Zeiss 510 Meta laser scanning confocal microscope or Nikon epi-fluorescent microscope. Tissues from adult mice (2 month-old) were also collected for western blotting. RESULTS: Meibomian gland morphogenesis was first detected at E18.5 with the formation of an epithelial placode within the fused eyelid margin. Invagination of the epithelium into the eyelid was detected at P0. From P1 to P3 there was continued extension of the epithelium into the eyelid. ORO and PPARgamma staining was first detected at P3, localized to the central core of the epithelial cord thus forming the presumptive ductal lumen. Ductal branching was first detected at P5 associated with acinar differentiation identified by ORO and PPARgamma staining. Adult meibomian glands were observed by P15. Western blotting of meibomian gland proteins identified a 50 kDa and a 72 kDa band that stained with antibodies specific to PPARgamma. CONCLUSIONS: We have demonstrated that meibomian gland development bears distinct similarities to hair development with the formation of an epithelial placode and expression of PPARgamma co-incident with lipid synthesis and meibocyte differentiation.

Corneal wound healing in an osteopontin-deficient mouse.

PURPOSE: To investigate the effects of loss of osteopontin (OPN) in the healing of the injured cornea in mice. Cell culture study was also conducted to clarify the effects of OPN in fibroblast behaviors. METHODS: Ocular fibroblasts from wild-type (WT) and OPN-null (KO) mice were used to study the role of OPN on cell behavior. The effect of the lack of OPN on corneal wound healing was evaluated in mice. RESULTS: In cell culture, OPN is involved in cell adhesion and in the migration of ocular fibroblasts. Adhesion of the corneal epithelial cell line was not affected by exogenous OPN. OPN was upregulated in a healing, injured mouse cornea. Loss of OPN did not affect epithelial healing after simple epithelial debridement. Healing of an incision injury in cornea was delayed, with less appearance of myofibroblasts and transforming growth factor beta1 expression in a KO mouse than in a WT mouse. The absence of OPN promoted tissue destruction after an alkali burn, resulting in a higher incidence of corneal perforation in KO mice than in WT mice. CONCLUSIONS: OPN modulates wound healing-related fibroblast behavior and is required to restore the physiological structure of the cornea after wound healing.

Endogenous TNFalpha suppression of neovascularization in corneal stroma in mice.

PURPOSE: To examine the role of tumor necrosis factor alpha (TNFalpha) in stromal neovascularization in injured cornea in vivo and in cytokine-enhanced vessel-like endothelial cell tube formation in vitro. METHODS: An in vitro model of angiogenesis was used to examine the roles of TNFalpha on tube formation by human umbilical vein endothelial cells (HUVECs) cocultured with fibroblasts on induction by transforming growth factor beta1 (TGFbeta1) and vascular endothelial growth factor (VEGF). Central cauterization was used to induce stromal neovascularization in corneas of wild-type (WT) and TNFalpha-null (Tnfalpha(-/-)) mice. At 7, 14, or 21 days of injury, experimental mice were killed, and the eyes were enucleated and subjected to histologic and immunohistochemical examination and real-time reverse transcription-polymerase chain reaction. RESULTS: HUVECs formed a vessel-like tube structure on the fibroblast feeder layer. Adding TGFbeta1, VEGF, or both augmented vessel-like tube formation by HUVECs cocultured with fibroblasts. Adding TNFalpha (5 ng/mL) completely abolished the formation of tube-like structures despite the presence or absence of TGFbeta1 or VEGF in coculture. In vivo, cauterization of the central cornea induced the formation of CD31(+) new vessels surrounding the limbus in WT mice. More prominent central stromal neovascularization accompanied by increased expression of TGFbeta1 and VEGF was found in Tnfalpha(-/-) mice compared with WT mice. CONCLUSIONS: In addition to inhibiting TGFbeta1 and VEGF expression by fibroblasts, endogenous TNFalpha may counter the induction effects of TGFbeta1 and VEGF on vascular endothelial cells and may block neovascularization.

Loss of osteopontin perturbs the epithelial-mesenchymal transition in an injured mouse lens epithelium.

We previously reported that osteopontin (OPN), a matrix structural glycophosphoprotein, is upregulated in the injured mouse lens prior to the epithelial-mesenchymal transition (EMT). Here, we investigated the role of this protein in EMT of the lens epithelium during wound healing. The crystalline lens was injured by needle puncture in OPN-null (KO, n=40) and wild-type (WT, n=40) mice. The animals were killed at day 1, 2, 5, and 10 postinjury. Immunohistochemistry was employed to detect alpha-smooth muscle action (alphaSMA), a marker of EMT, collagen type I, transforming growth factor beta1 (TGFbeta1), TGFbeta2, and phospho-Smad2/3. Cell proliferation was assayed by examining uptake of bromodeoxyuridine (BrdU). The results showed that injury-induced EMT of mouse lens epithelium, as evaluated by histology, expression pattern of alphaSMA and collagen I, was altered in the absence of OPN with reduced phospho-Smad2/3 signaling. Upregulation of TGFbeta1 and TGFbeta2 in the epithelium was also inhibited. Cell proliferation was more active in KO mice as compared with WT mice at day 1 and 2, but not at day 5 and 10. An in vitro experiment shows OPN facilitates cell adhesion of lens epithelial cell line. OPN is required for activation of Smad2/3 signal in an injured lens epithelium and lens cell EMT.

Excess biglycan causes eyelid malformation by perturbing muscle development and TGF-alpha signaling.

Tissue morphogenesis during development is regulated by growth factors and cytokines, and is characterized by constant remodeling of extracellular matrix (ECM) in response to signaling molecules, for example, growth factors, cytokines, and so forth. Proteoglycans that bind growth factors are potential regulators of tissue morphogenesis during embryonic development. In this study, we showed that transgenic mice overexpressing biglycan under the keratocan promoter exhibited exposure keratitis and premature eye opening from noninfectious eyelid ulceration due to perturbation of eyelid muscle formation and the failure of meibomian gland formation. In addition, in vitro analysis revealed that biglycan binds to TGF-alpha, thus interrupting EGFR signaling pathways essential for mesenchymal cell migration induced by eyelid epithelium. The defects of TGF-alpha signaling by excess biglycan were further augmented by the interruption of the autocrine or paracrine loop of the EGFR signaling pathway of HB-EGF expression elicited by TGF-alpha. These results are consistent with the notion that under physiological conditions, biglycan secreted by mesenchymal cells serves as a regulatory molecule for the formation of a TGF-alpha gradient serving as a morphogen of eyelid morphogenesis.

Fibroblast growth factor 2: roles of regulation of lens cell proliferation and epithelial-mesenchymal transition in response to injury.

PURPOSE: To examine the role of fibroblast growth factor 2 (FGF2) in regulating lens cell proliferation and epithelial-mesenchymal transition (EMT) in response to injury. METHODS: The amount of FGF2 protein was determined in healing, injured rat lenses by enzyme immunoassay. The effects of FGF2 and transforming growth factor beta2 (TGFbeta2) on cell proliferation of alphaTN4 cells (a mouse lens epithelial cell line) were determined. FGF2-knockout mice were used to further examine the role of endogenous FGF2 on injury-induced epithelial cell proliferation and EMT. The anterior lens capsule was injured by a hypodermic needle under both general and topical anesthesia in one eye of 34 fgf2+/+ mice and 42 fgf2-/- mice. At days 2, 5, and 10 post-injury the mice were sacrificed following a 2 h labeling period with bromo-deoxyuridine (BrdU). The number of BrdU-positive cells in each specimen was determined. RESULTS: A capsular break caused a 10 fold increase of FGF2 protein accumulated in rat lens 14 days after injury. Addition of 3.43 ng/ml FGF2 enhanced proliferation of alphaTN4 cells. This occurred in the presence or absence of exogenous TGFbeta2, that has an inhibitory effect on alphaTN4 cell proliferation. Significantly fewer BrdU-labeled cells were found in fgf2-/- mice than in fgf2+/+ mice during healing post-injury. However, lacking FGF2 did not alter the expression patterns of alpha-smooth muscle actin and collagen type I, markers of EMT in lens cells. CONCLUSIONS: Endogenous FGF2 is required for increased cell proliferation but not essential for EMT during the lens response to injury.

Epithelial repair: roles of extracellular matrix.

PURPOSE: To review studies of the roles of extracellular matrix (ECM) metabolism in corneal epithelium during wound repair. Methods. 1) Alterations in the structure and composition of epithelial basement membrane during corneal epithelial healing were examined histologically and immunohistochemically. 2) The effects of procollagen and hyaluronan synthesis inhibitors on the spread of rabbit corneal epithelium were determined in organ culture. 3) Expression of keratan sulfate proteoglycan (KSPG) proteins in corneal epithelium was examined during repair after injury in wild-type and lumican-null mice. RESULTS: 1) Corneal epithelial basement membrane was transiently degraded and reassembled during tissue repair. Patterns of type IV collagen immunoreactivity were also transiently altered. The system of matrix metalloproteinase-tissue inhibitors of metalloproteinases may play an important role in the disassembly and reorganization of epithelial basement membrane. 2) Inhibitors of procollagen secretion and hyaluronan biosynthesis disrupted the spread of a corneal epithelial sheet in situ. 3) Among the corneal KSPG proteins examined, lumican was transiently expressed in migrating murine corneal epithelial cells. Anti-lumican antibody inhibited corneal epithelial resurfacing in organ culture. The absence of lumican was found to delay corneal epithelial wound healing in mice. CONCLUSION: Extracellular matrix metabolism by the injured corneal epithelium is important in the repair process.