Topical Losartan Inhibition of Myofibroblast Generation in Rabbit Corneas With Acute Incisions.

PURPOSE: The purpose of this study was to study whether deep central corneal incisions close during topical losartan treatment and the effect of topical losartan on myofibroblast generation after incisions in rabbit corneas. METHODS: Rabbits (12) had a 0.35-mm deep radial incision from the center of the cornea into the limbus in 1 eye that was approximated with a single 10-0 nylon suture 1 mm inside the limbus. The incision was treated with 50 µL of topical 0.8 mg/mL losartan or 50 µL of balanced salt solution vehicle 6 times per day for 1 month. Standardized slitlamp photographs of the central incisions were analyzed for opacity with ImageJ before euthanasia. Triplex IHC was performed on cryofixed corneas for myofibroblast marker alpha-smooth muscle actin, mesenchymal cell marker vimentin, and basement membrane marker laminin alpha-5. Stromal α-SMA-positive myofibroblasts surrounding the incisions were quantitated with ImageJ. RESULTS: Topical losartan compared with vehicle did not affect closure of the radial incisions or the opacity that developed surrounding the incisions at 1 month after injury. Topical losartan compared with vehicle did significantly decrease the average density of stromal myofibroblasts surrounding the incisions. CONCLUSIONS: Topical losartan, a known inhibitor of transforming growth factor beta signaling, did not affect closure of deep corneal incisions. Losartan decreased myofibroblast generation surrounding nearly full-thickness radial corneal incisions compared with vehicle. The opacity at the incisions was not significantly affected by losartan-likely because corneal fibroblasts that develop in the stroma adjacent to the incisions were not changed by the losartan compared with the vehicle.

Topical Losartan Decreases Myofibroblast Generation But Not Corneal Opacity After Surface Blast-Simulating Irregular PTK in Rabbits.

Purpose: To evaluate the efficacy of topical losartan after blast injury-simulating irregular phototherapeutic keratectomy (PTK) in rabbits. Methods: Twelve NZW rabbits underwent 100 pulse 6.5 mm diameter PTK over a metal screen to generate severe surface irregularity and inhibit epithelial basement membrane regeneration. Corneas were treated with 0.8 mg/mL losartan in balanced salt solution (BSS) or BSS 50 µL six times per day for six weeks after PTK. All corneas had slit lamp photography, with and without 1% fluorescein at two, four, and six weeks after PTK, and were analyzed using immunohistochemistry for the myofibroblast marker α-smooth muscle actin (α-SMA), keratocyte marker keratocan, mesenchymal cell marker vimentin, transforming growth factor (TGF)-ß1, and collagen type IV. Results: Topical 0.8 mg/mL losartan six times a day significantly decreased anterior stromal α-SMA intensity units compared to BSS at six weeks after anterior stromal irregularity-inducing screened PTK (P = 0.009). Central corneal opacity, however, was not significantly different between the two groups. Keratocan, vimentin, TGF-ß1, or collagen type IV levels in the anterior stroma were not significantly different between the two groups. Conclusions: Topical losartan effectively decreased myofibroblast generation after surface blast simulation irregular PTK. However, these results suggest initial masking-smoothing PTK, along with adjuvant topical losartan therapy, may be needed to decrease corneal stromal opacity after traumatic injuries that produce severe surface irregularity. Translational Relevance: Topical losartan decreased scar-producing stromal myofibroblasts after irregular PTK over a metal screen but early smoothing of irregularity would also likely be needed to significantly decrease corneal opacity.

Transforming growth factor beta-3 localization in the corneal response to epithelial-stromal injury and effects on corneal fibroblast transition to myofibroblasts.

The purpose of this study was to evaluate the localization of TGF beta-3 in situ in unwounded rabbit corneas and corneas that had epithelial-stromal injuries produced by photorefractive keratectomy (PRK) in rabbits and to evaluate the in vitro effects of TGF beta-3 compared to TGF beta-1 on alpha-smooth muscle actin (α-SMA) protein expression and myofibroblast development in corneal fibroblasts. Forty-eight New Zealand white rabbits underwent either -3 diopter (D) or -9D PRK and were studied from one to eight weeks (four corneas in each group at each time point) after surgery with immunohistochemistry for TGF beta-3, laminin alpha-5, and alpha-smooth muscle actin (α-SMA). Rabbit corneal fibroblasts were treated with activated TGF beta-1 and/or TGF beta-3 at different concentrations and duration of exposure and studied with immunocytochemistry for myofibroblast development and the expression of α-SMA using Jess automated Western blotting. TGF beta-3 was detected at high levels in the stroma of unwounded corneas and corneas at one to eight weeks after -3D or -9D PRK, as well as in the epithelium and epithelial basement membrane (EBM). No difference was noted between corneas that healed with and without myofibroblast-mediated fibrosis, although TGF beta-3 was commonly associated with myofibroblasts. TGF beta-3 effects on corneal fibroblasts in vitro were similar to TGF beta-1 in stimulating transition to α-SMA-positive myofibroblasts and promoting α-SMA protein expression. The corneal stromal localization pattern of TGF beta-3 protein in unwounded corneas and corneas after epithelial-stromal injury was found to be higher and different from TGF beta-1 and TGF beta-2 reported in previous studies. TGF beta-3 had similar effects to TGF beta-1 in driving myofibroblast development and α-SMA expression in corneal fibroblasts cultured in medium with 1% fetal bovine serum.

Corneal epithelial basement membrane assembly is mediated by epithelial cells in coordination with corneal fibroblasts during wound healing.

Purpose: To understand which cell types, either alone or in combination, contribute to the assembly of the epithelial basement membrane (BM) during corneal wound healing. Methods: A 3D corneal organotypic model and an in situ rabbit photorefractive keratectomy (PRK) model were used in this study. The 3D corneal organotypic model was established by culturing the rabbit corneal epithelial cells with either corneal fibroblasts or myofibroblasts embedded in collagen type I for 18 days. Corneal fibroblasts were isolated from fresh rabbit corneas, and the myofibroblasts were derived either directly from bone marrow or differentiated from corneal fibroblasts. Immunocytochemistry for alpha-smooth muscle actin (SMA), vimentin, desmin, and vinculin markers confirmed well-differentiated myofibroblasts. Immunohistochemistry was performed in cryofixed sections for BM markers, including laminin alpha-5, laminin beta-3, perlecan, nidogen-1, and collagen type IV. Specimens were also examined with transmission electron microscopy (TEM). Corneas were collected from rabbits after -3 diopter (D) PRK at different time points after surgery, with four corneas at each time point in each group. Cryofixed corneal sections were stained for vimentin, alpha-SMA, and nidogen-1. Results: The formation of an epithelial BM with expression of laminin alpha-5, laminin beta-3, perlecan, nidogen-1, and collagen IV was observed at the interface between the corneal epithelial cells and corneal fibroblasts. TEM images further confirmed the presence of epithelial BM in organotypic cultures of epithelial cells and corneal fibroblasts. No epithelial BM was observed in cultures of corneal epithelial cells and myofibroblasts (cornea or bone marrow derived), corneal epithelial cells alone, or corneal fibroblasts alone. In rabbit corneas after -3D PRK, a strong association was observed between the regenerating epithelial BM and the presence of corneal fibroblasts at the site of epithelial BM generation. Conclusions: The corneal epithelial BM assembly is mediated by epithelial cells in coordination with corneal fibroblasts during wound healing.

Cell Biology of Spontaneous Persistent Epithelial Defects After Photorefractive Keratectomy in Rabbits.

Purpose: To evaluate wound healing in rabbit corneas that developed a spontaneous persistent epithelial defect (PED) after photorefractive keratectomy (PRK). Methods: Forty-eight 10- to 15-week-old female New Zealand White rabbits weighing 2.5 to 3.0 kg underwent either -3 diopter (D) or -9 D PRK to generate a series of corneas to study wound healing after injury. During that series, seven corneas developed a PED detected with 1% fluorescein staining at a slit lamp that either did not have epithelial closure by 1 week after surgery or subsequently had the closed epithelium break down to form a PED 2 to 3 weeks after surgery. The corneas had slit-lamp photography, with and without 1% fluorescein, and were removed from the normal PRK series. Each PED cornea was evaluated using immunohistochemistry for the myofibroblast marker α-smooth muscle actin (α-SMA), keratocyte marker keratocan, and mesenchymal cell marker vimentin, as well as basement membrane components perlecan and collagen type IV. Results: All seven corneas that had PRK with a PED, even the two evaluated at only 1 week after PRK, had α-SMA-positive myofibroblasts populating the anterior stroma within the PED, along with comingled α-SMA-negative cells that were likely corneal fibroblasts and possibly bone marrow-derived fibrocytes. Both perlecan and collagen type IV accumulated in the anterior stroma of the epithelial defects without an epithelial basement membrane, likely produced by corneal fibroblasts to modulate transforming growth factor-ß entering the stroma from the tears and peripheral epithelium. Conclusions: Corneas with a PED that occurred following PRK (a procedure that produces a transient neurotropic state in the cornea) had myofibroblasts populating the superficial stroma within the epithelial defect as early as 1 week after the surgery. Translational Relevance: Pharmacologic treatments that trigger myofibroblast apoptosis, including topical losartan, could facilitate decreased scarring fibrosis in corneas with a PED.

Standardization of corneal alkali burn methodology in rabbits.

Alkali burns are one of the most common injuries used in corneal wound healing studies. Investigators have used different conditions to produce corneal alkali injuries that have varied in sodium hydroxide concentration, application methods, and duration of exposure. A critical factor in the subsequent corneal healing responses, including myofibroblast generation and fibrosis localization, is whether, or not, Descemet's membrane and the endothelium are injured during the initial exposure. After exposures that produce injuries confined to the epithelium and stroma, anterior stromal myofibroblasts and fibrosis are typical, with sparing of the posterior stroma. However, if there is also injury to Descemet's membrane and the endothelium, then myofibroblast generation and fibrosis is noted full corneal thickness, with predilection to the most anterior and most posterior stroma and a tendency for relative sparring of the central stroma that is likely related to the availability of TGF beta from the tears, epithelium, and the aqueous humor. A method is described where a 5 mm diameter circle of Whatman #1 filter paper wetted with only 30 µL of alkali solution is applied for 15 s prior to profuse irrigation in rabbit corneas. When 0.6N, or lower, NaOH is used, then the injury, myofibroblasts, and fibrosis generation are limited to the epithelium and stroma. Use of 0.75N NaOH triggers injury to Descemet's membrane and the corneal endothelium with fibrosis throughout the stroma, but rare corneal neovascularization (CNV) and persistent epithelial defects (PED). Use of 1N NaOH with this method produces greater stromal fibrosis and increased likelihood that CNV and PED will occur in individual corneas.

Losartan Inhibition of Myofibroblast Generation and Late Haze (Scarring Fibrosis) After PRK in Rabbits.

PURPOSE: To study the effect of topical losartan compared to vehicle on the generation of myofibroblasts and development of late haze scarring fibrosis after photorefractive keratectomy (PRK) in rabbits. METHODS: Twelve rabbits had -9.00 diopter (D) PRK in one eye followed by 50 µL of topical 0.2 mg/mL losartan or 50 µL of vehicle six times per day for 1 month. Standardized slit-lamp photographs were obtained prior to death. Duplex immunohistochemistry was performed on cryofixed corneas for myofibroblast marker alpha-smooth muscle actin (α-SMA) and keratocyte marker keratocan or collagen type IV and transforming growth factor (TGF)-ß1. ImageJ software (National Institutes of Health) was used for quantitation. RESULTS: Topical losartan compared to vehicle significantly decreased corneal opacity (P = .04) and anterior stromal myofibroblast generation (P = .01) at 1 month after PRK. Topical losartan compared to vehicle also decreased anterior stromal non-basement membrane collagen type IV at 1 month after PRK (P = .004). CONCLUSIONS: Topical angiotensin converting enzyme II receptor inhibitor losartan, a known inhibitor of TGF-ß signaling, decreased late haze scarring fibrosis and myofibroblast generation after -9.00 D PRK in rabbits compared to vehicle. It also decreases TGF-ß-modulated, corneal fibroblast-produced, non-basement membrane stromal collagen type IV-likely also through inhibition of TGF-ß signaling. [J Refract Surg. 2022;38(12):820-829.].

Topical Losartan and Corticosteroid Additively Inhibit Corneal Stromal Myofibroblast Generation and Scarring Fibrosis After Alkali Burn Injury.

Purpose: To evaluate the efficacy of losartan and prednisolone acetate in inhibiting corneal scarring fibrosis after alkali burn injury in rabbits. Methods: Sixteen New Zealand White rabbits were included. Alkali injuries were produced using 1N sodium hydroxide on a 5-mm diameter Whatman #1 filter paper for 1 minute. Four corneas in each group were treated six times per day for 1 month with 50 µL of (1) 0.8 mg/mL losartan in balanced salt solution (BSS), (2) 1% prednisolone acetate, (3) combined 0.8 mg/mL losartan and 1% prednisolone acetate, or (4) BSS. Area of opacity and total opacity were analyzed in standardized slit-lamp photos with ImageJ. Corneas in both groups were cryofixed in Optimal cutting temperature (OCT) compound at 1 month after surgery, and immunohistochemistry was performed for alpha-smooth muscle actin (α-SMA) and keratocan or transforming growth factor ß1 and collagen type IV with ImageJ quantitation. Results: Combined topical losartan and prednisolone acetate significantly decreased slit-lamp opacity area and intensity, as well as decreased stromal myofibroblast α-SMA area and intensity of staining per section and confined myofibroblasts to only the posterior stroma with repopulation of the anterior and mid-stroma with keratocan-positive keratocytes after 1 month of treatment. Corneal fibroblasts produced collagen type IV not associated with basement membranes, and this production was decreased by topical losartan. Conclusions: Combined topical losartan and prednisolone acetate decreased myofibroblast-associated fibrosis after corneal alkali burns that produced full-thickness injury, including corneal endothelial damage. Increased dosages and duration of treatment may further decrease scarring fibrosis. Translational Relevance: Topical losartan and prednisolone acetate decrease myofibroblast-mediated scarring fibrosis after corneal injury.

Corneal fibroblast collagen type IV negative feedback modulation of TGF beta: A fibrosis modulating system likely active in other organs.

Collagen type IV (COL IV) is a major component of basement membranes (BM) in all organs. It serves functions related to BM organization and modulates the passage of growth factors from one tissue compartment to another. COL IV binds transforming growth factor (TGF) beta-1 and TGF beta-2 and, therefore, is a major modulator of TGF beta pro-fibrotic functions. After fibrotic corneal injury, TGF beta enters into the stroma from the tears, epithelium, endothelium and/or aqueous humor and markedly upregulates COL IV production in corneal fibroblasts in the adjacent stroma far removed from BMs. It is hypothesized this non-BM stromal COL IV binds TGF beta-1 (and likely TGF beta-2) in competition with the binding of the growth factors to TGF beta cognate receptors and serves as a negative feedback regulatory pathway to mitigate the effects of TGF beta on stromal cells, including reducing the developmental transition of corneal fibroblasts and fibrocytes into myofibroblasts. Losartan, a known TGF beta signaling inhibitor, when applied topically to the cornea after fibrotic injury, alters this COL IV-TGF beta pathway by down-regulating COL IV production by corneal fibroblasts. Non-BM COL IV produced in response to injuries in other organs, including the lung, skin, liver, kidney, and gut, may participate in similar COL IV-TGF beta pathways and have an important role in controlling TGF beta pro-fibrotic effects in these organs.

Corneal Opacity: Cell Biological Determinants of the Transition From Transparency to Transient Haze to Scarring Fibrosis, and Resolution, After Injury.

Purpose: To highlight the cellular, matrix, and hydration changes associated with opacity that occurs in the corneal stroma after injury. Methods: Review of the literature. Results: The regulated transition of keratocytes to corneal fibroblasts and myofibroblasts, and of bone marrow-derived fibrocytes to myofibroblasts, is in large part modulated by transforming growth factor beta (TGFß) entry into the stroma after injury to the epithelial basement membrane (EBM) and/or Descemet's membrane. The composition, stoichiometry, and organization of the stromal extracellular matrix components and water is altered by corneal fibroblast and myofibroblast production of large amounts of collagen type I and other extracellular matrix components-resulting in varying levels of stromal opacity, depending on the intensity of the healing response. Regeneration of EBM and/or Descemet's membrane, and stromal cell production of non-EBM collagen type IV, reestablishes control of TGFß entry and activity, and triggers TGFß-dependent myofibroblast apoptosis. Eventually, corneal fibroblasts also disappear, and repopulating keratocytes reorganize the disordered extracellular matrix to reestablish transparency. Conclusions: Injuries to the cornea produce varying amounts of corneal opacity depending on the magnitude of cellular and molecular responses to injury. The EBM and Descemet's membrane are key regulators of stromal cellularity through their modulation of TGFß. After injury to the cornea, depending on the severity of the insult, and possibly genetic factors, trace opacity to severe scarring fibrosis develops. Stromal cellularity, and the functions of different cell types, are the major determinants of the level of the stromal opacity.

Epithelial Basement Membrane Regeneration After PRK-Induced Epithelial-Stromal Injury in Rabbits: Fibrotic Versus Non-fibrotic Corneal Healing.

PURPOSE: To study epithelial basement membrane (EBM) regeneration in non-fibrotic and fibrotic corneas after photorefractive keratectomy (PRK). METHODS: Rabbits (120 total) had either epithelial scrape alone, -4.50 diopters (D) PRK, -9.00 D PRK, or no surgery. Immunohistochemistry was performed on cryofixed corneas at time points from unwounded to 8 weeks (four corneas at each time point in each group). Multiplex immunohistochemistry was performed for EBM components, including collagen type IV, laminin beta-3, laminin alpha-5, perlecan, and nidogen-1. Stromal cellular composition was studied by triplex immunohistochemistry for keratocan, vimentin, and alpha-smooth muscle actin (SMA). RESULTS: PRK-injured EBM significantly regenerated by 4 days after surgery. However, early TGF-beta-regulating perlecan incorporation into the nascent EBM declined 4 to 7 days after surgery in fibrotic corneas. Non-fibrotic corneas that had fully regenerated EBM (with all five components incorporated into the EBM) were transparent and had few SMA-positive myofibroblasts in the stroma. Conversely, corneas with defective nascent EBM that lacked perlecan developed many anterior stromal myofibroblasts and fibrosis at 3 to 4 weeks after surgery and had large amounts of collagen type IV in the nascent EBM and anterior stroma. Myofibroblasts synthesized perlecan but were incompetent to incorporate the heparin sulfate proteoglycan into the nascent EBM. Corneal transparency was restored over several months even in fibrotic corneas, and this was associated with a return of EBM perlecan, myofibroblast disappearance, and reabsorption of disordered extracellular matrix. CONCLUSIONS: Defective incorporation of perlecan into the regenerating EBM by subepithelial myofibroblasts, and likely their precursor cells, underlies the development and persistence of stromal fibrosis after PRK corneal injury. [J Refract Surg. 2022;38(1):50-60.].

Topical losartan inhibits corneal scarring fibrosis and collagen type IV deposition after Descemet's membrane-endothelial excision in rabbits.

The purpose of this study was to examine the effect of topical and/or oral angiotensin converting enzyme II inhibitor and TGF-beta signaling blocker losartan on corneal stromal fibrosis that developed in rabbit corneas after Descemetorhexis removal of central Descemet's membrane and corneal endothelium. Twenty-eight New Zealand white rabbits were included and either had 8 mm central Descemetorhexis or sham control surgery without Descemetorhexis in one eye. Groups of 4 eyes without Descemetorhexis were treated for one month with no medications, topical losartan or oral losartan. Groups of 4 eyes with Descemetorhexis were treated with topical and oral vehicle, topical losartan, oral losartan, or both topical losartan and oral losartan for one month. Standardized slit lamp photos were obtained with central opacity intensity measured with ImageJ. The posterior fibrotic zone of corneas was measured on immunohistochemistry for alpha-smooth muscle actin (SMA) and keratocan using QuPath analysis. Collagen type IV expression in the posterior cornea was quantitated with ImageJ and duplex immunohistochemistry for collagen type IV and TGF beta-1. After Descemetorhexis, topical, but not oral, losartan decreased the intensity of central stromal opacity, reduced peripheral corneal scarring, and decreased alpha-smooth muscle actin myofibroblast fibrosis area compared to corneas that had Descemetorhexis and treatment with vehicles alone. Topical losartan decreased posterior stromal cellular, non-Descemet's membrane, collagen type IV production, that is likely stimulated by TGF beta as part of a negative regulatory feedback mechanism, compared to vehicle treatment at one month after Descemetorhexis. Topical losartan is likely to be effective in reducing corneal scarring fibrosis produced by traumatic injury, microbial infection, and some corneal diseases and surgeries.

Descemet's membrane injury and regeneration, and posterior corneal fibrosis, in rabbits.

The purpose of this investigation was to study Descemet's membrane and corneal endothelial regeneration, myofibroblast generation and disappearance, and TGF beta-1 localization after Descemet's membrane-endothelial excision (Descemetorhexis) in rabbits. Thirty-six rabbits had 8 mm Descemetorhexis and standardized slit lamp photos at 1, 2 and 4 days, 1, 2 and 4 weeks, and 2, 4 and 6 months, as well as multiplex IHC for stromal cell markers keratocan, vimentin, and alpha-smooth muscle actin (SMA); basement membrane (BM) components perlecan, nidogen-1, laminin alpha-5, and collagen type IV; and corneal endothelial marker Na,K-ATPase ß1, and TGF beta-1, with ImageJ quantitation. Stromal transparency increased from the periphery beginning at two months after injury and progressed into the central cornea by six months. At six months, central transparency was primarily limited by persistent mid-stromal neovascularization. Stromal myofibroblast zone thickness in the posterior stroma peaked at one month after injury, and then progressively decreased until to six months when few myofibroblasts remained. The regeneration of a laminin alpha-5 and nidogen-1 Descemet's membrane "railroad track" structure was accompanied by corneal endothelial closure and stromal cell production of BM components in corneas from four to six months after injury. TGF beta-1 deposition at the posterior corneal surface from the aqueous humor peaked at one day after Descemetorhexis and diminished even before regeneration of the endothelium and Descemet's membrane. This decrease was associated with collagen type IV protein production by corneal fibroblasts, and possibly myofibroblasts, in the posterior stroma. Descemet's membrane and the corneal endothelium regenerated in the rabbit cornea by six months after eight mm Descemetorhexis. Real-time quantitative RT-PCR experiments in vitro with marker-verified rabbit corneal cells found that 5 ng/ml or 10 ng/ml TGF beta-1 upregulated col4a1 or col4a2 mRNA expression after 6 h or 12 h of exposure in corneal fibroblasts, but not in myofibroblasts. Stromal cells produced large amounts of collagen type IV that likely decreased TGF beta-1 penetration into the stroma and facilitated the resolution of myofibroblast-generated fibrosis.

Fibroblastic and bone marrow-derived cellularity in the corneal stroma.

The unwounded, normal corneal stroma is a relatively simple, avascular tissue populated with quiescent keratocytes, along with corneal nerves and a few resident dendritic and monocyte/macrophage cells. In the past, the resting keratocytes were thought of as a homogenous cellular population, but recent work has shown local variations in vimentin and nestin expression, and responsiveness to transforming growth factor (TGF)-ß1. Studies have also supported there being "stromal stem cells" in localized areas. After corneal wounding, depending on the site and severity of injury, profound changes in stromal cellularity occur. Anterior or posterior injuries to the epithelium or endothelium, respectively, trigger apoptosis of adjacent keratocytes. Many contiguous keratocytes transition to keratocan-negative corneal fibroblasts that are proliferative and produce limited amounts of disorganized extracellular matrix components. Simultaneously, large numbers of bone marrow-derived cells, including monocytes, neutrophils, fibrocytes and lymphocytes, invade the stroma from the limbal blood vessels. Ongoing adequate levels of TGFß1, TGFß2 and platelet-derived growth factor (PDGF) from epithelium, tears, endothelium and aqueous humor that penetrate defective or absent epithelial barrier function (EBF) and epithelial basement membrane (EBM) and/or Descemet's basement membrane (DBM) drive corneal fibroblasts and fibrocytes to differentiate into alpha-smooth muscle actin (SMA)-positive myofibroblasts. If the EBF, EBM and/or DBM are repaired or replaced in a timely manner, typically measured in weeks, then corneal fibroblast and fibrocyte progeny, deprived of requisite levels of TGFß1 and TGFß2, undergo apoptosis or revert to their precursor cell-types. If the EBF, EBM and/or DBM are not repaired or replaced, stromal levels of TGFß1 and TGFß2 remain elevated, and mature myofibroblasts are generated from corneal fibroblasts and fibrocyte precursors that produce prodigious amounts of disordered extracellular matrix materials associated with scarring fibrosis. This fibrotic stromal matrix persists, at least until the EBF, EBM and/or DBM are regenerated or replaced, and keratocytes remove and reorganize the affected stromal matrix.

TGFß1 and TGFß2 proteins in corneas with and without stromal fibrosis: Delayed regeneration of apical epithelial growth factor barrier and the epithelial basement membrane in corneas with stromal fibrosis.

The purpose of this study was to investigate the expression and localization of transforming growth factor (TGF) ß1 and TGFß2 in rabbit corneas that healed with and without stromal fibrosis, and to further study defective perlecan incorporation in the epithelial basement membrane (EBM) in corneas with scarring fibrosis. A total of 120 female rabbits had no surgery, -4.5D PRK, or -9D PRK. Immunohistochemistry (IHC) was performed at time points from unwounded to eight weeks after surgery, with four corneas at each time point in each group. Multiplex IHC was performed for TGFß1 or TGFß2, with Image-J quantitation, and keratocan, vimentin, alpha-smooth muscle actin (SMA), perlecan, laminin-alpha 5, nidogen-1 or CD11b. Corneas at the four-week peak for myofibroblast and fibrosis development were evaluated using Imaris 3D analysis. Delayed regeneration of both an apical epithelial growth factor barrier and EBM barrier function, including defective EBM perlecan incorporation, was greater in high injury -9D PRK corneas compared to -4.5D PRK corneas without fibrosis. Defective apical epithelial growth factor barrier and EBM allowed epithelial and tear TGFß1 and tear TGFß2 to enter the corneal stroma to drive myofibroblast generation in the anterior stroma from vimentin-positive corneal fibroblasts, and likely fibrocytes. Vimentin-positive cells and unidentified vimentin-negative, CD11b-negative cells also produce TGFß1 and/or TGFß2 in the stroma in some corneas. TGFß1 and TGFß2 were at higher levels in the anterior stroma in the weeks preceding myofibroblast development in the -9D group. All -9D corneas (beginning two to three weeks after surgery), and four -4.5D PRK corneas developed significant SMA + myofibroblasts and stromal fibrosis. Both the apical epithelial growth factor barrier and/or EBM barrier functions tended to regenerate weeks earlier in -4.5D PRK corneas without fibrosis, compared to -4.5D or -9D PRK corneas with fibrosis. SMA-positive myofibroblasts were markedly reduced in most corneas by eight weeks after surgery. The apical epithelial growth factor barrier and EBM barrier limit TGFß1 and TGFß2 entry into the corneal stroma to modulate corneal fibroblast and myofibroblast development associated with scarring stromal fibrosis. Delayed regeneration of these barriers in corneas with more severe injuries promotes myofibroblast development, prolongs myofibroblast viability and triggers stromal scarring fibrosis.