Corneal epithelial basement membrane: Structure, function and regeneration.

Basement membranes are highly specialized extracellular matrices. More than providing scaffolds, basement membranes are recognized as dynamic and versatile structures that modulate cellular responses to regulate tissue development, function, and repair. Increasing evidence suggests that, in addition to providing structural support to adjacent cells, basement membranes serve as reservoirs and modulators of growth factors that direct and fine-tune cellular functions. Since the corneal stroma is avascular and has a relatively low keratocyte density, it's likely that the corneal BM is different in composition from the BMs in other tissues. BMs are composed of a diverse assemblage of extracellular molecules, some of which are likely specific to the tissue where they function; but in general they are composed of four primary components-collagens, laminins, heparan sulfate proteoglycans, and nidogens-in addition to other components such as thrombospondin-1, matrilin-2, and matrilin-4 and fibronectin. Severe injuries to the cornea, including infection, surgery, and trauma, may trigger the development of myofibroblasts and fibrosis in the normally transparent connective tissue stroma. Ultrastructural studies have demonstrated that defective epithelial basement membrane (EBM) regeneration after injury to the cornea underlies the development of myofibroblasts from both bone marrow- and keratocyte-derived precursor cells. Defective EBM permits epithelium-derived and tear-derived transforming growth factor beta (TGF-ß), platelet-derived growth factor (PDGF), and possibly other modulators, to penetrate the stroma at sustained levels necessary to drive the development and persistence of vimentin + alpha-smooth muscle actin + desmin+ (V + A + D+) mature myofibroblasts. A recent discovery that has contributed to our understanding of haze development is that keratocytes and corneal fibroblasts produce critical EBM components, such as nidogen-1, nidogen-2 and perlecan, that are essential for complete regeneration of a normal EBM once laminin secreted by epithelial cells self-polymerizes into a nascent EBM. Mature myofibroblasts that become established in the anterior stroma are a barrier to keratocyte/corneal fibroblast contributions to the nascent EBM. These myofibroblasts, and the opacity they produce, often persist for months or years after the injury. Transparency is subsequently restored if the EBM is fully regenerated, myofibroblasts are deprived of TGF-ß and undergo apoptosis, and keratocytes reoccupy the anterior stroma and reabsorb the disordered extracellular matrix.

The Impact of Photorefractive Keratectomy and Mitomycin C on Corneal Nerves and Their Regeneration.

PURPOSE: To determine how photorefractive keratectomy (PRK) and mitomycin C (MMC) affect corneal nerves and their regeneration over time after surgery. METHODS: Twenty-eight New Zealand rabbits had corneal epithelial scraping with (n = 3) and without (n = 3) MMC 0.02% or -9.00 diopter PRK with (n = 6) and without (n = 16) MMC 0.02%. Corneas were removed after death and corneal nerve morphology was evaluated using acetylcholinesterase immunohistochemistry and beta-III tubulin staining after 1 day for all groups, after 1 month for PRK with and without MMC, and 2, 3, and 6 months after PRK without MMC. Image-Pro software (Media Cybernetics, Rockville, MD) was used to quantitate the area of nerve loss after the procedures and, consequently, regeneration of the nerves over time. Opposite eyes were used as controls. RESULTS: Epithelial scraping with MMC treatment did not show a statistically significant difference in nerve loss compared to epithelial scraping without MMC (P = .40). PRK with MMC was significantly different from PRK without MMC at 1 day after surgery (P = .0009) but not different at 1 month after surgery (P = .90). In the PRK without MMC group, nerves regenerated at 2 months (P < .0001) but did not return to the normal preoperative level of innervation until 3 months after surgery (P = .05). However, the morphology of the regenerating nerves was abnormal-with more tortuosity and aberrant innervation compared to the preoperative controls-even at 6 months after surgery. CONCLUSIONS: PRK negatively impacts the corneal nerves, but they are partially regenerated by 3 months after surgery in rabbits. Nerve loss after PRK extended peripherally to the excimer laser ablated zone, indicating that there was retrograde degeneration of nerves after PRK. MMC had a small additive toxic effect on the corneal nerves when combined with PRK that was only significant prior to 1 month after surgery. [J Refract Surg. 2018;34(12):790-798.].

The Corneal Basement Membranes and Stromal Fibrosis.

Purpose: The purpose of this review was to provide detailed insights into the pathophysiology of myofibroblast-mediated fibrosis (scarring or late haze) after corneal injury, surgery, or infection. Method: Literature review. Results: The epithelium and epithelial basement membrane (EBM) and/or endothelium and Descemet's basement membrane (BM) are commonly disrupted after corneal injuries, surgeries, and infections. Regeneration of these critical regulatory structures relies on the coordinated production of BM components, including laminins, nidogens, perlecan, and collagen type IV by epithelial, endothelial, and keratocyte cells. Whether a cornea, or an area in the cornea, heals with transparency or fibrosis may be determined by whether there is injury to one or both corneal basement membranes (EBM and/or Descemet's BM) and delayed or defective regeneration or replacement of the BM. These opaque myofibroblasts, and the disordered extracellular matrix these cells produce, persist in the stroma until the EBM and/or Descemet's BM is regenerated or replaced. Conclusions: Corneal stromal fibrosis (also termed "stromal scarring" or "late haze") occurs as a consequence of BM injury and defective regeneration in both the anterior (EBM) and posterior (Descemet's BM) cornea. The resolution of fibrosis and return of stromal transparency depends on reestablished BM structure and function. It is hypothesized that defective regeneration of the EBM or Descemet's BM allows key profibrotic growth factors, including transforming growth factor beta-1 (TGF-ß1) and TGF-ß2, to penetrate the stroma at sustained levels necessary to drive the development and maintenance of mature opacity-producing myofibroblasts from myofibroblast precursors cells, and studies suggest that perlecan and collagen type IV are the critical components in EBM and Descemet's BM that bind TGF-ß1, TGF-ß2, platelet-derived growth factor, and possibly other growth factors, and regulate their bioavailability and function during homeostasis and corneal wound healing.

Fibrocyte migration, differentiation and apoptosis during the corneal wound healing response to injury.

The aim of this study was to determine whether bone marrow-derived fibrocytes migrate into the cornea after stromal scar-producing injury and differentiate into alpha-smooth muscle actin (αSMA) + myofibroblasts. Chimeric mice expressing green fluorescent protein (GFP) bone marrow cells had fibrosis (haze)-generating irregular phototherapeutic keratectomy (PTK). Multiplex immunohistochemistry (IHC) for GFP and fibrocyte markers (CD34, CD45, and vimentin) was used to detect fibrocyte infiltration into the corneal stroma and the development of GFP+ αSMA+ myofibroblasts. IHC for activated caspase-3, GFP and CD45 was used to detect fibrocyte and other hematopoietic cells undergoing apoptosis. Moderate haze developed in PTK-treated mouse corneas at 14 days after surgery and worsened, and persisted, at 21 days after surgery. GFP+ CD34+ CD45+ fibrocytes, likely in addition to other CD34+ and/or CD45+ hematopoietic and stem/progenitor cells, infiltrated the cornea and were present in the stroma in high numbers by one day after PTK. The fibrocytes and other bone marrow-derived cells progressively decreased at four days and seven days after surgery. At four days after PTK, 5% of the GFP+ cells expressed activated caspase-3. At 14 days after PTK, more than 50% of GFP+ CD45+ cells were also αSMA+ myofibroblasts. At 21 days after PTK, few GFP+ αSMA+ cells persisted in the stroma and more than 95% of those remaining expressed activated caspase-3, indicating they were undergoing apoptosis. GFP+ CD45+ SMA+ cells that developed from 4 to 21 days after irregular PTK were likely developed from fibrocytes. After irregular PTK in the strain of C57BL/6-C57/BL/6-Tg(UBC-GFP)30Scha/J chimeric mice, however, more than 95% of fibrocytes and other hematopoietic cells underwent apoptosis prior to the development of mature αSMA+ myofibroblasts. Most GFP+ CD45+ αSMA+ myofibroblasts that did develop subsequently underwent apoptosis-likely due to epithelial basement membrane regeneration and deprivation of epithelium-derived TGFß requisite for myofibroblast survival.

Femtosecond Lasers and Corneal Surgical Procedures.

Our purpose is to present a broad review about the principles, early history, evolution, applications, and complications of femtosecond lasers used in refractive and nonrefractive corneal surgical procedures. Femtosecond laser technology added not only safety, precision, and reproducibility to established corneal surgical procedures such as laser in situ keratomileusis (LASIK) and astigmatic keratotomy, but it also introduced new promising concepts such as the intrastromal lenticule procedures with refractive lenticule extraction (ReLEx). Over time, the refinements in laser optics and the overall design of femtosecond laser platforms led to it becoming an essential tool for corneal surgeons. In conclusion, femtosecond laser is a heavily utilized tool in refractive and nonrefractive corneal surgical procedures, and further technological advances are likely to expand its applications.

Injury and defective regeneration of the epithelial basement membrane in corneal fibrosis: A paradigm for fibrosis in other organs?

Myofibroblast-mediated fibrosis is important in the pathophysiology of diseases in most organs. The cornea, the transparent anterior wall of the eye that functions to focus light on the retina, is commonly affected by fibrosis and provides an optimal model due to its simplicity and accessibility. Severe injuries to the cornea, including infection, surgery, and trauma, may trigger the development of myofibroblasts and fibrosis in the normally transparent connective tissue stroma. Ultrastructural studies have demonstrated that defective epithelial basement membrane (EBM) regeneration after injury underlies the development of myofibroblasts from both bone marrow- and keratocyte-derived precursor cells in the cornea. Defective EBM permits epithelium-derived transforming growth factor beta, platelet-derived growth factor, and likely other modulators, to penetrate the stroma at sustained levels necessary to drive the development of vimentin+ alpha-smooth muscle actin+ desmin+ (V+A+D+) mature myofibroblasts and promote their persistence. Defective versus normal EBM regeneration likely relates to the severity of the stromal injury and a resulting decrease in fibroblasts (keratocytes) and their contribution of EBM components, including laminin alpha-3 and nidogen-2. Corneal fibrosis may resolve over a period of months to years if the inciting injury is eliminated through keratocyte-facilitated regeneration of normal EBM, ensuing apoptosis of myofibroblasts, and reorganization of disordered extracellular matrix by repopulating keratocytes. We hypothesize the corneal model of fibrosis associated with defective BM regeneration and myofibroblast development after epithelial or parenchymal injury may be a paradigm for the development of fibrosis in other organs where chronic injury or defective BM underlies the pathophysiology of disease.

Epithelial basement membrane injury and regeneration modulates corneal fibrosis after pseudomonas corneal ulcers in rabbits.

The purpose of this study was to investigate whether myofibroblast-related fibrosis (scarring) after microbial keratitis was modulated by the epithelial basement membrane (EBM) injury and regeneration. Rabbits were infected with Pseudomonas aeruginosa after epithelial scrape injury and the resultant severe keratitis was treated with topical tobramycin. Corneas were analyzed from one to four months after keratitis with slit lamp photos, immunohistochemistry for alpha-smooth muscle actin (α-SMA) and monocyte lineage marker CD11b, and transmission electron microscopy. At one month after keratitis, corneas had no detectible EBM lamina lucida or lamina densa, and the central stroma was packed with myofibroblasts that in some eyes extended to the posterior corneal surface with damage to Descemet's membrane and the endothelium. At one month, a nest of stromal cells in the midst of the SMA + myofibroblasts in the stroma that were CD11b+ may be fibrocyte precursors to myofibroblasts. At two to four months after keratitis, the EBM fully-regenerated and myofibroblasts disappeared from the anterior 60-90% of the stroma of all corneas, except for one four-month post-keratitis cornea where anterior myofibroblasts were still present in one localized pocket in the cornea. The organization of the stromal extracellular matrix also became less disorganized from two to four months after keratitis but remained abnormal compared to controls at the last time point. Myofibroblasts persisted in the posterior 10%-20% of posterior stroma even at four months after keratitis in the central cornea where Descemet's membrane and the endothelium were damaged. This study suggests that the EBM has a critical role in modulating myofibroblast development and fibrosis after keratitis-similar to the role of EBM in fibrosis after photorefractive keratectomy. Damage to EBM likely allows epithelium-derived transforming growth factor beta (TGFß) to penetrate the stroma and drive development and persistence of myofibroblasts. Eventual repair of EBM leads to myofibroblast apoptosis when the cells are deprived of requisite TGFß to maintain viability. The endothelium and Descemet's membrane may serve a similar function modulating TGFß penetration into the posterior stroma-with the source of TGFß likely being the aqueous humor.

Regeneration of Defective Epithelial Basement Membrane and Restoration of Corneal Transparency After Photorefractive Keratectomy.

PURPOSE: To study regeneration of the normal ultrastructure of the epithelial basement membrane (EBM) in rabbit corneas that had -9.00 D photorefractive keratectomy (PRK) and developed late haze (fibrosis) with restoration of transparency over 1 to 4 months after surgery and in corneas that had incisional wounds. METHODS: Twenty-four rabbits had one of their eyes included in one of the two procedure groups (-9.00 D PRK or nearly full-thickness incisional wounds), whereas the opposite eyes served as the unwounded control group. All corneas were evaluated with slit-lamp photographs, transmission electron microscopy, and immunohistochemistry for the myofibroblast marker alpha-smooth muscle actin and collagen type III. RESULTS: In the -9.00 D PRK group, corneas at 1 month after surgery had dense corneal haze and no evidence of regenerated EBM ultrastructure. However, by 2 months after surgery small areas of stromal clearing began to appear within the confluent opacity (lacunae), and these corresponded to small islands of normally regenerated EBM detected within a larger area of the excimer laser-ablated zone with no evidence of normal EBM. By 4 months after surgery, the EBM was fully regenerated and the corneal transparency was completely restored in the ablated zone. In the incisional wound group, the two dense, linear corneal opacities were observed at 1 month after surgery and progressively faded by 2 and 3 months after surgery. The EBM ultrastructure was fully regenerated at the site of the incisions, including around epithelial plugs that extended into the stroma, by 1 month after surgery in all eyes. CONCLUSIONS: In the rabbit model, spontaneous resolution of corneal fibrosis (haze) after high correction PRK is triggered by regeneration of EBM with normal ultrastructure in the excimer laser-ablated zone. Conversely, incisional wounds heal in rabbit corneas without the development of myofibroblasts because the EBM regenerates normally by 1 month after surgery. [J Refract Surg. 2017:33(5):337-346.].

EBM regeneration and changes in EBM component mRNA expression in stromal cells after corneal injury.

PURPOSE: To investigate the production of the epithelial basement membrane (EBM) component mRNAs at time points before lamina lucida and lamina densa regeneration in anterior stromal cells after corneal injury that would heal with and without fibrosis. METHODS: Rabbit corneas were removed from 2 to 19 days after -4.5D or -9.0D photorefractive keratectomy (PRK) with the VISX S4 IR laser. Corneas were evaluated with transmission electron microscopy (TEM) for full regeneration of the lamina lucida and the lamina densa. Laser capture microdissection (LCM) based quantitative real-time (RT)-PCR was used to quantitate the expression of mRNAs for laminin α-3 (LAMA3), perlecan, nidogen-1, and nidogen-2 in the anterior stroma. RESULTS: After -4.5D PRK, EBM was found to be fully regenerated at 8 to 10 days after surgery. At 4 days after PRK, the nidogen-2 and LAMA3 mRNAs levels were detected at statistically significantly lower levels in the anterior stroma of the -9.0D PRK corneas (where the EBM would not fully regenerate) compared to the -4.5D PRK corneas (where the EBM was destined to fully regenerate). At 7 days after PRK, nidogen-2 and LAMA3 mRNAs continued to be statistically significantly lower in the anterior stroma of the -9.0D PRK corneas compared to their expression in the anterior stroma of the -4.5D PRK corneas. CONCLUSIONS: Key EBM components LAMA3 and nidogen-2 mRNAs are expressed at higher levels in the anterior stroma during EBM regeneration in the -4.5D PRK corneas where the EBM is destined to fully regenerate and no haze developed compared to the -9.0D PRK corneas where the EBM will not fully regenerate and myofibroblast-related stromal fibrosis (haze) will develop.

Phototherapeutic Keratectomy: Science and Art.

PURPOSE: To describe, with videos, the principles of excimer laser phototherapeutic keratectomy (PTK) for the treatment of corneal scars, corneal surface irregularity, and recurrent corneal erosions. METHODS: Depending on the pathology in a treated cornea, the epithelium is removed either by transepithelial PTK ablation with the excimer laser or thorough scraping with a scalpel blade. Stromal PTK can be performed with or without photorefractive keratectomy (PRK), depending on the refractive status of both eyes. Residual surface irregularity is treated with masking-smoothing PTK. Typically, 0.02% mitomycin C treatment is applied for 30 seconds to corneas treated with PTK for scars and surface irregularity. RESULTS: Transepithelial PTK with masking-smoothing typically improves corrected distance visual acuity in the eye even if the entire stromal opacity cannot be removed and can be used to debulk surface irregularity to facilitate subsequent therapeutic customized wavefront-guided or optical coherence tomography-guided PTK or PRK. PTK for recurrent erosion is performed after thorough mechanical epithelial debridement of redundant epithelial basement membrane (EBM) with a scalpel and should only include a dusting of excimer laser to remove residual EBM without inducing central irregular astigmatism or damaging limbal tissues. Meta-analyses are provided for PTK treatment for corneal scars, corneal dystrophies, and recurrent corneal erosions. CONCLUSIONS: Excimer laser PTK is a highly effective treatment for superficial corneal scars, central corneal irregular astigmatism, and recurrent corneal erosions unresponsive to medical treatment or mechanical epithelial debridement alone. [J Refract Surg. 2017;33(3):203-210.].

OCT Study of the Femtosecond Laser Opaque Bubble Layer.

PURPOSE: To characterize the location and regularity of the opaque bubble layer (OBL) in the corneal stroma after femtosecond laser-assisted LASIK (FS-LASIK) flap generation. METHODS: In this prospective study, 30 eyes of 15 patients who had FS-LASIK surgery for myopia, astigmatism, and/or hyperopia were included. Screen captures were obtained at the end of the flap creation and the eyes with hard type OBL were immediately imaged with anterior segment optical coherence tomography. RESULTS: The mean age of the 9 men and 6 women was 40 ± 11.3 years (range: 22 to 60 years). Seven eyes (23.3%) developed hard type OBL that was typically localized in the central cornea beneath the LASIK flap and, in the majority of cases, located close to the hinge of the flap. Three of the seven eyes had OBL only within the laser cut, whereas the four other eyes had OBL in a spotty distribution within the stromal bed beneath the flap. None of the eyes had an accumulation of OBL within the flap itself. CONCLUSIONS: The excimer laser ablation of a stroma with OBL may be different from that of a stroma without OBL. Management of OBL when it occurs due to flap production, including allowing the bubble to dissipate when they overlie the pupil, is important to obtain the best outcomes with femtosecond laser-assisted LASIK. [J Refract Surg. 2017;33(1):18-22.].

Corneal Collagen Cross-linking in Advanced Keratoconus: A 4-Year Follow-up Study.

PURPOSE: To analyze the safety and efficacy of standard corneal collagen cross-linking (CXL) in advanced cases of progressive keratoconus after 4 years of follow-up. METHODS: A retrospective case series of patients with advanced progressive keratoconus (stages 3 and 4 of Amsler-Krumeich classification) underwent standard CXL treatment. The parameters examined were changes in uncorrected visual acuity (UDVA), corrected visual acuity (CDVA), keratometry values (mean, flat, steep, and apical), pachymetry, and endothelial cell count at the baseline and at 12, 24, and 48 months postoperatively. RESULTS: Forty eyes of 40 patients were enrolled in the study. The mean patient age was 22.5 years (range: 15 to 37 years). Both mean UDVA and CDVA remained stable during the time points; no statistically significant change was noted. Although a slight reduction was observed in all keratometric readings, a statistically significant reduction was only reached in the apical keratometry (P = .037) at 4 years after CXL. A significant reduction in the corneal thickness was also found (ultrasonic: 388 ± 49 to 379 ± 48 µm; slit-scanning: 362 ± 48 to 353 ± 51 µm); however, this change was likely not clinically meaningful. Endothelial cell count was not significantly different at the end of the study. Treatment failure or progression was noted in two patients (5%) over the follow-up period. CONCLUSIONS: Standard CXL treatment was safe and able to stabilize both visual acuity and topographic parameters at 4 years of follow-up in eyes with advanced keratoconus. [J Refract Surg. 2016;32(7):459-464.].

Corneal Molecular and Cellular Biology for the Refractive Surgeon: The Critical Role of the Epithelial Basement Membrane.

PURPOSE: To provide an overview of the recent advances concerning the corneal molecular and cellular biology processes involved in the wound healing response after excimer laser surface ablation and LASIK surgery. METHODS: Literature review. RESULTS: The corneal wound healing response is a complex cascade of events that impacts the predictability and stability of keratorefractive surgical procedures such as photorefractive keratectomy and LASIK. The generation and persistence of corneal myofibroblasts (contractile cells with reduced transparency) arise from the interaction of cytokines and growth factors such as transforming growth factor beta and interleukin 1 produced by epithelial and stromal cells in response to the corneal injury. Myofibroblasts, and the opaque extracellular matrix they secrete into the stroma, disturb the precise distribution and spacing of collagen fibers related to corneal transparency and lead to the development of vision-limiting corneal opacity (haze). The intact epithelial basement membrane has a pivotal role as a structure that regulates corneal epithelial-stromal interactions. Thus, defective regeneration of the epithelial basement membrane after surgery, trauma, or infection leads to the development of stromal haze. The apoptotic process following laser stromal ablation, which is proportional to the level of attempted correction, leads to an early decrease in anterior keratocyte density and the diminished contribution of these non-epithelial cells of components such as perlecan and nidogen-2 required for normal regeneration of the epithelial basement membrane. Haze persists until late repair of the defective epithelial basement membrane. CONCLUSIONS: Defective regeneration of the epithelial basement membrane has a critical role in determining whether a cornea heals with late haze after photorefractive keratectomy or with scarring at the flap edge in LASIK.

Femtosecond Laser-Assisted LASIK Flap Complications.

PURPOSE: To discuss intraoperative and postoperative femtosecond laser-assisted LASIK flap complications and their management. METHODS: Review of published literature. RESULTS: Flap creation is a critical step in LASIK. The femtosecond laser has improved the overall predictability and safety of the lamellar incision, but complications can still occur during or after flap creation. Although many complications (eg, epithelial ingrowth and flap striae) were reduced with the femtosecond laser application, other specific complications have emerged, such as vertical gas breakthrough, opaque bubble layer, and transient light-sensitivity syndrome. CONCLUSIONS: The application of femtosecond laser technology to LASIK flap creation has increased greatly since its introduction. These lasers have improved the safety and predictability of the lamellar incision step. The majority of the femtosecond laser-assisted flap complications can be well managed without significant effects on refractive outcomes.

Accelerated Corneal Collagen Cross-linking for Postoperative LASIK Ectasia: Two-Year Outcomes.

PURPOSE: To evaluate the effectiveness and safety of accelerated corneal collagen cross-linking for postoperative LASIK ectasia after 2 years. METHODS: A prospective, single-center case series was performed with patients treated for postoperative LASIK ectasia. All eyes underwent accelerated corneal collagen cross-linking (CCL-Vario Crosslinking; Peschke Meditrade GmbH, Zurich, Switzerland) at 9 mW/cm(2) for 10 minutes. The main outcome measures were changes in uncorrected distance visual acuity, corrected distance visual acuity, central corneal thickness, corneal topography, and endothelial cell density. These parameters were assessed at baseline and at the 6-month and 1- and 2-year follow-up visit. RESULTS: The study enrolled 40 eyes of 24 patients (15 male and 9 female) with a mean age of 33.8 ± 7.5 years (range: 24 to 52 years) that attained at least 2 years of follow-up. The surgical procedure was uneventful in all cases. All eyes stabilized after treatment without any further signs of progression and no statistically significant changes in the mean uncorrected distance visual acuity (P = .649), corrected distance visual acuity (P = .616), mean keratometry (P =.837), steep keratometry (P = .956), ultrasonic pachymetry (P = .135), slit-scanning pachymetry (P = .276), and endothelial cell density (P = .523). In addition, 72.5% of the patients presented stable or gains of Snellen lines over time. CONCLUSIONS: Accelerated corneal collagen cross-linking seems to be safe and effective in halting postoperative LASIK ectasia progression after 2 years of follow-up. However, a longer follow-up period with a larger cohort is needed to validate these findings.

Epithelial basement membrane proteins perlecan and nidogen-2 are up-regulated in stromal cells after epithelial injury in human corneas.

The epithelial basement membrane (BM) is a specialized extracellular matrix that has been shown to have a critical role in corneal development, wound healing, and disease. Although the epithelial BM contributes to corneal homeostasis, relatively little is know about non-epithelial production of its components that may be important in defective regeneration of the epithelial basement membrane associated with opacity after photorefractive keratectomy. The purpose of the current study was to investigate stromal production of corneal epithelial BM proteins in wounded human corneas using immunohistochemistry. A total of five unwounded control eyes and five 30-min epithelial-wounded corneas were obtained from fresh corneoscleral buttons removed from human eyes enucleated due to choroidal melanoma with normal anterior segments. In the wounded corneas, an eight mm patch of central corneal epithelium and epithelial BM was removed with a Beaver blade when the patient was under general anesthesia. Immunohistochemical analyses were performed to detect perlecan and nidogen-2 proteins-important components of the epithelial BM lamina lucida and lamina densa zones. Perlecan and nidogen-2 proteins were detected in the BM itself and at low levels in keratocytes in all unwounded corneas. After epithelial injury, both perlecan and nidogen-2 were expressed at high levels in stromal keratocytes, including superficial keratocytes in the early phases of apoptosis. Thus, after epithelial and epithelial BM injury, stromal keratocytes contribute important perlecan and nidogen-2 components to the regenerating epithelial BM.

Differential expression of epithelial basement membrane components nidogens and perlecan in corneal stromal cells in vitro.

PURPOSE: The purpose of this study was to examine the expression of corneal epithelial basement membrane (EBM) components in different corneal stromal cell types. In vitro model systems were used to explore the expression of EBM components nidogen-1, nidogen-2, and perlecan that are the primary components in the lamina lucida and the lamina densa that defectively regenerate in corneas with stromal opacity after in -9.0 D photorefractive keratectomy (PRK). METHODS: Primary rabbit corneal stromal cells were cultured using varying serum concentrations and exogenous growth factors, including fibroblast growth factor (FGF)-2 and transforming growth factor (TGF)-ß1, to optimize the growth of each cell type of interest. The expression of the keratocyte-specific marker keratocan and the myofibroblast-specific marker α-smooth muscle actin (α-SMA) were analyzed with real-time PCR, western blot, and immunocytochemical staining to evaluate the specificity of the cell types and select optimal conditions (high keratocan and low α-SMA for keratocytes; low keratocan and high α-SMA for myofibroblasts; low keratocan and low α-SMA for corneal fibroblasts). The expression of the EBM components nidogen-1, nidogen-2, and perlecan was evaluated in each corneal cell type using real-time PCR, immunostaining, and western blotting. In agreement with previous studies, serum-free DMEM was found to be optimal for keratocytes, DMEM with 10% serum and 40 ng/ml FGF-2 yielded the best marker profile for corneal fibroblasts, and DMEM with 1% serum and 2 ng/ml TGF-ß1 was found to be optimal for myofibroblasts. RESULTS: Nidogen-1 and nidogen-2 mRNAs were highly expressed in keratocytes, whereas perlecan was highly expressed in myofibroblasts. In keratocytes, nidogen-2 and perlecan proteins were expressed predominantly in intracellular compartments, whereas in myofibroblasts expression of both EBM components was observed diffusely throughout the cell. Although the perlecan mRNA levels were high in the myofibroblasts, the qualitative protein expression was different from that of the keratocytes. Corneal fibroblasts produced a low amount of each EBM component. CONCLUSIONS: We have demonstrated qualitative and quantitative differences in the expression of nidogen-1, nidogen-2, and perlecan by keratocytes compared to myofibroblasts that may contribute to defective regeneration of the lamina lucida and the lamina densa of the EBM associated with late stromal haze after high-correction PRK.