Salzmann's Nodular Degeneration in Refractive Surgery: The Earlier Hit Hypothesis of EBM Injury-Related Fibrosis of the Subepithelial Space and Deeper Corneal Extension.

PURPOSE: To review the atypical development of Salzmann's nodular degeneration (SND) after two cases of laser in situ keratomileusis (LASIK) and one case of photorefractive keratomileusis (PRK), and to highlight the pathophysiology of SND and its treatment. METHODS: Three cases of SND (two following LASIK performed with microkeratomes and one following PRK) were reviewed and Pubmed.gov and internet searches were performed. RESULTS: SND is myofibroblast-generated fibrosis in the subepithelial space between the epithelium and Bowman's layer that develops years or decades after traumatic, surgical, infectious, or inflammatory injuries to the cornea in which the epithelial basement membrane is damaged in one or more locations and does not fully regenerate. It is hypothesized based on these cases, and the previous immunohistochemistry of other investigators, that myofibroblast precursors, such as fibrocytes or corneal fibroblasts, that enter the subepithelial space are driven to develop into myofibroblasts, which slowly proliferate and extend the fibrosis, by transforming growth factor-beta from epithelium and tears that passes through the defective epithelial basement membrane. These myofibroblasts and the disordered collagens, and other extracellular matrix components they produce, make up the subepithelial opacity characteristic of SND. Nodules are larger accumulations of myofibroblasts and disordered extracellular matrix. If the injury is associated with damage to the underlying Bowman's layer and stroma, as in LASIK flap generation, then the myofibroblasts and fibrosis can extend into Bowman's layer and the underlying anterior stroma. CONCLUSIONS: SND fibrosis often extends into Bowman's layer and the anterior stroma if there are associated Bowman's defects, such as incisions or lacerations. In the latter cases, SND frequently cannot be removed by simple scrape and peel, as typically performed for most common SND cases, but can be trimmed to remove the offending tissue. This condition is more accurately termed Salzmann's subepithelial fibrosis. [J Refract Surg. 2024;40(5):e279-e290.].

Two-phase mechanism in the treatment of corneal stromal fibrosis with topical losartan.

Recent studies in rabbits and case reports in humans have demonstrated the efficacy of topical losartan in the treatment of corneal scarring fibrosis after a wide range of injuries, including chemical burns, infections, surgical complications, and some diseases. It is hypothesized that the effect of losartan on the fibrotic corneal stroma occurs through a two-phase process in which losartan first triggers the elimination of myofibroblasts by directing their apoptosis via inhibition of extracellular signal-regulated kinase (ERK)-mediated signal transduction, and possibly through signaling effects on the viability and development of corneal fibroblast and fibrocyte myofibroblast precursor cells. This first step likely occurs within a week or two in most corneas with fibrosis treated with topical losartan, but the medication must be continued for much longer until the epithelial basement membrane (EBM) is fully regenerated or new myofibroblasts will develop from precursor cells. Once the myofibroblasts are eliminated from the fibrotic stroma, corneal fibroblasts can migrate into the fibrotic tissue and reabsorb/reorganize the disordered extracellular matrix (ECM) previously produced by the myofibroblasts. This second stage is longer and more variable in different eyes of rabbits and humans, and accounts for most of the variability in the time it takes for the stromal opacity to be markedly reduced by topical losartan treatment. Eventually, keratocytes reemerge in the previously fibrotic stromal tissue to fine-tune the collagens and other ECM components and maintain the normal structure of the corneal stroma. The efficacy of losartan in the prevention and treatment of corneal fibrosis suggests that it acts as a surrogate for the EBM, by suppressing TGF beta-directed scarring of the wounded corneal stroma, until control over TGF beta action is re-established by a healed EBM, while also supporting regeneration of the EBM by allowing corneal fibroblasts to occupy the subepithelial stroma in the place of myofibroblasts.

Prevention and Treatment of Persistent Epithelial Defects After Common Refractive Surgery Procedures.

PURPOSE: To discuss the prevention and treatment of persistent epithelial defects after the most common refractive surgeries-photorefractive keratectomy, laser in situ keratomileusis, or small incision lenticule extraction. METHODS: PubMed was used to search the medical literature. RESULTS: Persistent epithelial defects are infrequent after photorefractive keratectomy, laser in situ keratomileusis, or small incision lenticule extraction. In the authors' opinion, any persistent epithelial defect present at 1 week or beyond after surgery should be treated aggressively with a properly fit bandage contact lens, lubrication with non-preserved artificial tears, and treatment of any eyelid abnormalities, including nocturnal lagophthalmos. Consideration should be given for presumptive treatment for herpes simplex virus or varicella zoster virus infection. If the persistent epithelial defect does not close within 2 weeks, then other measures should be considered, such as autologous serum drops, topical losartan, amniotic membranes, and topical human recombinant nerve growth factor to limit corneal scarring fibrosis and microbial infection. CONCLUSIONS: Persistent epithelial defects are among the most feared complications of refractive surgery. Timely and aggressive treatment should be instituted to close the epithelium prior to the development of scarring fibrosis and/or microbial corneal infection. [J Refract Surg. 2024;40(2):e117-e124.].

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.

Corneal stromal localization of TGF beta isoforms in spontaneous persistent epithelial defects after PRK in rabbits.

The purpose of this study was to evaluate transforming growth factor beta (TGFß) isoform localization in rabbit corneas with spontaneous persistent epithelial defects (PEDs) after photorefractive keratectomy (PRK). Four cryofixed corneas from a previously reported series of PEDs in rabbits that had PRK were evaluated with triplex immunohistochemistry (IHC) for TGFß3, myofibroblast marker alpha-smooth muscle actin (α-SMA) and mesenchymal marker vimentin. One cornea had sufficient remaining tissue for triplex IHC for TGFß1, TGFß2, or TGFß3 (each with α-SMA and vimentin) using isoform-specific antibodies. All three TGFß isoforms were detected in the subepithelial stroma at and surrounding the PED. Some of each TGFß isoform co-localized with α-SMA of myofibroblasts, which could be TGFß isoform autocrine production by myofibroblasts or TGFß-1, -2, and -3 binding to these myofibroblasts.

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.

The Cornea: No Difference in the Wound Healing Response to Injury Related to Whether, or Not, There's a Bowman's Layer.

Bowman's layer is an acellular layer in the anterior stroma found in the corneas of humans, most other primates, chickens, and some other species. Many other species, however, including the rabbit, dog, wolf, cat, tiger, and lion, do not have a Bowman's layer. Millions of humans who have had photorefractive keratectomy over the past thirty plus years have had Bowman's layer removed by excimer laser ablation over their central cornea without apparent sequelae. A prior study showed that Bowman's layer does not contribute significantly to mechanical stability within the cornea. Bowman's layer does not have a barrier function, as many cytokines and growth factors, as well as other molecules, such as EBM component perlecan, pass bidirectionally through Bowman's layer in normal corneal functions, and during the response to epithelial scrape injury. We hypothesized that Bowman's layer represents a visible indicator of ongoing cytokine and growth factor-mediated interactions that occur between corneal epithelial cells (and corneal endothelial cells) and stromal keratocytes that maintain the normal corneal tissue organization via negative chemotactic and apoptotic effects of modulators produced by the epithelium on stromal keratocytes. Interleukin-1 alpha, produced constitutively by corneal epithelial cells and endothelial cells, is thought to be one of these cytokines. Bowman's layer is destroyed in corneas with advanced Fuchs' dystrophy or pseudophakic bullous keratopathy when the epithelium becomes edematous and dysfunctional, and fibrovascular tissue commonly develops beneath and/or within the epithelium in these corneas. Bowman's-like layers have been noted to develop surrounding epithelial plugs within the stromal incisions years after radial keratotomy. Although there are species-related differences in corneal wound healing, and even between strains within a species, these differences are not related to the presence or absence of Bowman's layer.

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.

The corneal fibroblast: The Dr. Jekyll underappreciated overseer of the responses to stromal injury.

PURPOSE: To review the functions of corneal fibroblasts in wound healing. METHODS: Literature review. RESULTS: Corneal fibroblasts arise in the corneal stroma after anterior, posterior or limbal injuries and are derived from keratocytes. Transforming growth factor (TGF) ß1 and TGFß2, along with platelet-derived growth factor (PDGF), are the major modulators of the keratocyte to corneal fibroblast transition, while fibroblast growth factor (FGF)-2, TGFß3, and retinoic acid are thought to regulate the transition of corneal fibroblasts back to keratocytes. Adequate and sustained levels of TGFß1 and/or TGFß2, primarily from epithelium, tears, aqueous humor, and corneal endothelium, drive the development of corneal fibroblasts into myofibroblasts. Myofibroblasts have been shown in vitro to transition back to corneal fibroblasts, although apoptosis of myofibroblasts has been documented as a major contributor to the resolution of fibrosis in several in situ corneal injury models. Corneal fibroblasts, aside from their role as a major progenitor to myofibroblasts, also perform many critical functions in the injured cornea, including the production of critical basement membrane (BM) components during regeneration of the epithelial BM and Descemet's membrane, production of non-basement membrane-associated stromal collagen type IV to control and downregulate TGFß effects on stromal cells, release of chemotactic chemokines that attract bone marrow-derived cells to the injured stroma, production of growth factors that modulate regeneration and maturation of the overlying epithelium, and production of collagens and other ECM components that contribute to stromal integrity after injury. CONCLUSIONS: Corneal fibroblasts are major contributors to and overseers of the corneal response to injuries.

Topical Losartan: Practical Guidance for Clinical Trials in the Prevention and Treatment of Corneal Scarring Fibrosis and Other Eye Diseases and Disorders.

Losartan is an angiotensin II receptor blocker (ARB) that impedes transforming growth factor (TGF) beta signaling by inhibiting activation of signal transduction molecule extracellular signal-regulated kinase (ERK). Studies supported the efficacy of topical losartan in decreasing scarring fibrosis after rabbit Descemetorhexis, alkali burn, and photorefractive keratectomy injuries, and in case reports of humans with scarring fibrosis after surgical complications. Clinical studies are needed to explore the efficacy and safety of topical losartan in the prevention and treatment of corneal scarring fibrosis, and other eye diseases and disorders where TGF beta has a role in pathophysiology. These include scarring fibrosis associated with corneal trauma, chemical burns, infections, surgical complications, and persistent epithelial defects, as well as conjunctival fibrotic diseases, such as ocular cicatricial pemphigoid and Stevens-Johnson syndrome. Research is also needed to explore the efficacy and safety of topical losartan for hypothesized treatment of transforming growth factor beta-induced (TGFBI)-related corneal dystrophies (Reis-Bu¨cklers corneal dystrophy, lattice corneal dystrophy type 1, and granular corneal dystrophies type 1 and type 2) where deposited mutant protein expression is modulated by TGF beta. Investigations could also explore the efficacy and safety of topical losartan treatments to reduce conjunctival bleb scarring and shunt encapsulation following glaucoma surgical procedures. Losartan and sustained release drug delivery devices could be efficacious in treating intraocular fibrotic diseases. Dosing suggestions and precautions that should be considered in trials of losartan are detailed. Losartan, as an adjuvant to current treatments, has the potential to augment pharmacological therapeutics for many ocular diseases and disorders where TGF beta plays a central role in pathophysiology.

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 for Treating Corneal Fibrosis (Haze): First Clinical Experience.

PURPOSE: To report the first clinical experience with topical losartan for treating a case of severe corneal haze after complicated laser in situ keratomileusis (LASIK). METHODS: A 36-year-old woman presented with corneal haze in the left eye after femtosecond laser-assisted LASIK. The left eye had flap dislocation and significant striae, which had been re-lifted. Uncorrected distance visual acuity (UDVA) was 20/200 and corrected distance visual acuity was 20/30 in the left eye at the first presentation, 52 days after the first procedure. A dense layer of subepithelial opacity (haze) was noted in the left cornea. The patient elected to start the off-label treatment with topical losartan 0.8 mg/mL six times per day. RESULTS: Four and one-half months after initiating topical losartan, UDVA improved to 20/30 and CDVA improved to 20/25 in the left eye. A significant reduction of corneal haze was observed at the slit lamp and using Scheimpflug corneal tomography (Pentacam AXL; Oculus Optikgeräte GmbH) and anterior segment optical coherence tomography (Revo NX 130; Optopol). CONCLUSIONS: Losartan is an inhibitor of transforming growth factor-ß signaling. Topical treatment is promising to treat corneal haze formation after corneal injuries, chemical burns, and surgeries. Further clinical studies are needed to optimize losartan dosages and treatment durations. [J Refract Surg. 2022;38(11):741-746.].

Magic Bullets: The Coming Age of Meaningful Pharmacological Control of the Corneal Responses to Injury and Disease.

Corneal injuries from chemical burns, mechanical trauma, infections, immunological rejections, surgical complications, and some diseases are commonly associated with persistent epithelial defects (PED), neurotrophic epitheliopathy, scarring fibrosis, corneal neovascularization (CNV), and/or corneal endothelial damage that lead to vision loss. Several Food and Drug Administration (FDA) approved medications have recently become available, are currently in clinical trials, or are likely to enter clinical trials in the near future. For example, a 2-week course of topical human recombinant nerve growth factor is frequently an effective treatment for corneal neurotrophic epitheliopathy associated with PEDs. Topical losartan, an angiotensin converting enzyme II receptor antagonist that also inhibits TGF beta signaling, has been shown to effectively decrease myofibroblast generation and scarring fibrosis in alkali burn injury and Descemetorhexis rabbit models. Small molecule topical tyrosine kinase inhibitors, such as sunitinib and axitinib, FDA approved as chemotherapeutic agents to treat specific cancers, have also been found to be effective topical inhibitors of CNV in animal and human trials. Rho-kinase inhibitors, such as ripasudil and netarsudil, that are currently approved agents for the treatment of glaucoma in some countries, have been shown to stimulate corneal endothelial proliferation in animal studies and human trials, and may accelerate the regeneration of Descemet's membrane. These agents, as well as other drugs in development, will be used in targeted combinations to treat corneal pathophysiology associated with epithelial healing disorders, stromal scarring fibrosis, CNV, and corneal endothelial injury during the next decade.

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.

Defective perlecan-associated basement membrane regeneration and altered modulation of transforming growth factor beta in corneal fibrosis.

In the cornea, the epithelial basement membrane (EBM) and corneal endothelial Descemet's basement membrane (DBM) critically regulate the localization, availability and, therefore, the functions of transforming growth factor (TGF)ß1, TGFß2, and platelet-derived growth factors (PDGF) that modulate myofibroblast development. Defective regeneration of the EBM, and notably diminished perlecan incorporation, occurs via several mechanisms and results in excessive and prolonged penetration of pro-fibrotic growth factors into the stroma. These growth factors drive mature myofibroblast development from both corneal fibroblasts and bone marrow-derived fibrocytes, and then the persistence of these myofibroblasts and the disordered collagens and other matrix materials they produce to generate stromal scarring fibrosis. Corneal stromal fibrosis often resolves completely if the inciting factor is removed and the BM regenerates. Similar defects in BM regeneration are likely associated with the development of fibrosis in other organs where perlecan has a critical role in the modulation of signaling by TGFß1 and TGFß2. Other BM components, such as collagen type IV and collagen type XIII, are also critical regulators of TGF beta (and other growth factors) in the cornea and other organs. After injury, BM components are dynamically secreted and assembled through the cooperation of neighboring cells-for example, the epithelial cells and keratocytes for the corneal EBM and corneal endothelial cells and keratocytes for the corneal DBM. One of the most critical functions of these reassembled BMs in all organs is to modulate the pro-fibrotic effects of TGFßs, PDGFs and other growth factors between tissues that comprise the organ.