Tissue-targeted and localized AAV5-DCN and AAV5-PEDF combination gene therapy abrogates corneal fibrosis and concurrent neovascularization in rabbit eyes in vivo.

PURPOSE: Corneal fibrosis and neovascularization (CNV) after ocular trauma impairs vision. This study tested therapeutic potential of tissue-targeted adeno-associated virus5 (AAV5) mediated decorin (DCN) and pigment epithelium-derived factor (PEDF) combination genes in vivo. METHODS: Corneal fibrosis and CNV were induced in New Zealand White rabbits via chemical trauma. Gene therapy in stroma was delivered 30-min after chemical-trauma via topical AAV5-DCN and AAV5-PEDF application using a cloning cylinder. Clinical eye examinations and multimodal imaging in live rabbits were performed periodically and corneal tissues were collected 9-day and 15-day post euthanasia. Histological, cellular, and molecular and apoptosis assays were used for efficacy, tolerability, and mechanistic studies. RESULTS: The AAV5-DCN and AAV5-PEDF combination gene therapy significantly reduced corneal fibrosis (p < 0.01 or p < 0.001) and CNV (p < 0.001) in therapy-given (chemical-trauma and AAV5-DCN + AAV5-PEDF) rabbit eyes compared to the no-therapy given eyes (chemical-trauma and AAV5-naked vector). Histopathological analyses demonstrated significantly reduced fibrotic α-smooth muscle actin and endothelial lectin expression in therapy-given corneas compared to no-therapy corneas on day-9 (p < 0.001) and day-15 (p < 0.001). Further, therapy-given corneas showed significantly increased Fas-ligand mRNA levels (p < 0.001) and apoptotic cell death in neovessels (p < 0.001) compared to no-therapy corneas. AAV5 delivered 2.69 × 107 copies of DCN and 2.31 × 107 copies of PEDF genes per µg of DNA. AAV5 vector and delivered DCN and PEDF genes found tolerable to the rabbit eyes and caused no significant toxicity to the cornea. CONCLUSION: The combination AAV5-DCN and AAV5-PEDF topical gene therapy effectively reduces corneal fibrosis and CNV with high tolerability in vivo in rabbits. Additional studies are warranted.

Toxicological effects of ocular acrolein exposure to eyelids in rabbits in vivo.

Acrolein is a highly reactive volatile toxic chemical that injures the eyes and many organs. It has been used in wars and terrorism for wounding masses on multiple occasions and is readily accessible commercially. Our earlier studies revealed acrolein's toxicity to the cornea and witnessed damage to other ocular tissues. Eyelids play a vital role in keeping eyes mobile, moist, lubricated, and functional utilizing a range of diverse lipids produced by the Meibomian glands located in the upper and lower eyelids. This study sought to investigate acrolein's toxicity to eyelid tissues by studying the expression of inflammatory and lipid markers in rabbit eyes in vivo utilizing our reported vapor-cap model. The study was approved by the institutional animal care and use committees and followed ARVO guidelines. Twelve New Zealand White Rabbits were divided into 3 groups: Naïve (group 1), 1-min acrolein exposure (group 2), or 3-min acrolein exposure (group 3). The toxicological effects of acrolein on ocular health in live animals were monitored with regular clinical eye exams and intraocular pressure measurements and eyelid tissues post-euthanasia were subjected to H&E and Masson's trichrome histology and qRT-PCR analysis. Clinical eye examinations witnessed severely swollen eyelids, abnormal ocular discharge, chemosis, and elevated intraocular pressure (p < 0.001) in acrolein-exposed eyes. Histological studies supported clinical findings and exhibited noticeable changes in eyelid tissue morphology. Gene expression studies exhibited significantly increased expression of inflammatory and lipid mediators (LOX, PAF, Cox-2, and LTB4; p < 0.001) in acrolein-exposed eyelid tissues compared to naïve eyelid tissues. The results suggest that acrolein exposure to the eyes causes acute damage to eyelids by altering inflammatory and lipid mediators in vivo.

Spherical Aberration of Cataractous Eyes and Its Relationship With Age, Ocular Biometry, and Various IOL Platforms.

PURPOSE: To determine the median spherical aberration (SA) of the cataractous population, how it relates to biometry, and the theoretical effect of different intraocular lens (IOL) platforms. METHODS: A retrospective chart review of patients undergoing cataract surgery evaluation with a high quality Pentacam (Oculus Optikgeräte GmbH) were included. Age, gender, Q-value, mean total SA, higher order aberration root mean square wavefront error, and equivalent keratometry were collected from the Holladay report and axial length and anterior chamber depth (ACD) from the IOLMaster 700 (Carl Zeiss Meditec AG). RESULTS: Data from 1,725 eyes of 999 patients were collected. SA had a median of 0.37 µm (95% confidence interval: 0.36 to 0.38. Age (r = .136, P < .001), Q-factor (r = .743, P < .001), and higher order aberration root mean square wavefront error (r = .307, P < .001) were positively correlated with SA. Average equivalent keratometry (r = -.310, P < .001) was negatively correlated with SA. Axial length (r = -0.037, P = .120) and ACD (r = .004, P = .856) had no association with SA. Up to 1,499 (86.9%) theoretically had SA moved closer to zero with IOLs that had negative SA. Up to 102 (5.9%) had SA theoretically worsened. CONCLUSIONS: SA is not normally distributed, suggesting that there may be no "average" SA that IOLs should aim to correct. Patients might benefit from tailoring IOL choice to individual SA. Without access to SA data, eyes with steeper average keratometry or younger patients may have less SA, which could influence IOL choice. [J Refract Surg. 2023;39(2):89-94.].

Corneal fibrosis abrogation by a localized AAV-mediated inhibitor of differentiation 3 (Id3) gene therapy in rabbit eyes in vivo.

Previously we found that inhibitor of differentiation 3 (Id3) gene, a transcriptional repressor, efficiently inhibits corneal keratocyte differentiation to myofibroblasts in vitro. This study evaluated the potential of adeno-associated virus 5 (AAV5)-mediated Id3 gene therapy to treat corneal scarring using an established rabbit in vivo disease model. Corneal scarring/fibrosis in rabbit eyes was induced by alkali trauma, and 24 h thereafter corneas were administered with either balanced salt solution AAV5-naked vector, or AAV5-Id3 vector (n = 6/group) via an optimized reported method. Therapeutic effects of AAV5-Id3 gene therapy on corneal pathology and ocular health were evaluated with clinical, histological, and molecular techniques. Localized AAV5-Id3 gene therapy significantly inhibited corneal fibrosis/haze clinically from 2.7 to 0.7 on the Fantes scale in live animals (AAV5-naked versus AAV5-Id3; p < 0.001). Furthermore, AAV5-Id3 treatment significantly reduced profibrotic gene mRNA levels: α-smooth muscle actin (α-SMA) (2.8-fold; p < 0.001), fibronectin (3.2-fold; p < 0.001), collagen I (0.8-fold; p < 0.001), and collagen III (1.4-fold; p < 0.001), as well as protein levels of α-SMA (23.8%; p < 0.001) and collagens (1.8-fold; p < 0.001). The anti-fibrotic activity of AAV5-Id3 is attributed to reduced myofibroblast formation by disrupting the binding of E-box proteins to the promoter of α-SMA, a transforming growth factor-ß signaling downstream target gene. In conclusion, these results indicate that localized AAV5-Id3 delivery in stroma caused no clinically relevant ocular symptoms or corneal cellular toxicity in the rabbit eyes.

Effects of Regular/Dilute Proparacaine Anesthetic Eye Drops in Combination with Ophthalmic Antibiotics on Corneal Wound Healing.

Purpose: Topical, local anesthetic eye drops in conjunction with antibiotics are commonly used to reduce ocular pain and treat patients in emergency clinics; however, their effects on corneal healing are poorly understood. This study examined whether regular or diluted proparacaine eye drops given in combination with common ophthalmic antibiotics affect corneal wound healing parameters using in vitro and in vivo models. Methods: Primary human corneal fibroblasts generated from donor corneas and New Zealand white rabbits were used. Regular (0.5%) and diluted (0.05%) proparacaine eye drops, twice daily for 3 days, were applied to cultures and rabbit eyes, with or without ophthalmic antibiotics (polymyxin B sulfate and trimethoprim). Trypan blue, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), and scratch wound assays measured cellular viability, proliferation, and migration, respectively, in vitro. Slit lamp biomicroscopy, tonometry, fluorescein eye test, hematoxylin and eosin (H&E) staining, and 4',6-diamidino-2-phenylindole (DAPI) immunofluorescence were used for in vivo studies. Results: Both regular and diluted proparacaine affected wound healing response in the cornea in vitro and in vivo in a time-dependent manner. Adjunct antibiotic treatments had additive effects characterized by reduced corneal fibroblast viability, proliferation, and migration in vitro and corneal epithelial recovery in vivo. Regular proparacaine with antibiotics showed most pronounced effects on corneal wound healing parameters, and diluted proparacaine without antibiotics had minimal negative effects in vitro and in vivo. Conclusion: Both methods of regular (0.5%) and diluted (0.05%) proparacaine topical application to the cornea are safe, but impede corneal wound healing in vitro and in vivo. Adjunct antibiotic treatments had additive negative effects on corneal wound repair.

Long-Term Safety and Tolerability of BMP7 and HGF Gene Overexpression in Rabbit Cornea.

Purpose: Tissue-targeted localized BMP7+HGF genes delivered into the stroma via nanoparticle effectively treats corneal fibrosis and rehabilitates transparency in vivo without acute toxicity. This study evaluated the long-term safety and tolerability of BMP7+HGF nanomedicine for the eye in vivo. Methods: One eye each of 36 rabbits received balanced salt solution (group 1, naïve; n = 12), naked vector with polyethylenimine-conjugated gold nanoparticles (PEI2-GNP; group 2, naked-vector; n = 12), or BMP7+HGF genes with PEI2-GNP (group 3, BMP7+HGF; n = 12) via a topical delivery technique. Safety and tolerability measurements were performed by clinical biomicroscopy in live rabbits at predetermined time intervals up to 7 months. Corneal tissues were collected at 2 months and 7 months after treatment and subjected to histology, immunofluorescence, and quantitative real-time PCR analyses. Results: Clinical ophthalmic examinations and modified MacDonald-Shadduck scores showed no significant changes in corneal thickness (P = 0.3389), tear flow (P = 0.2121), intraocular pressure (P = 0.9958), epithelial abrasion, or ocular abnormality. Slit-lamp, stereo, confocal, and specular biomicroscopy showed no signs of blepharospasm chemosis, erythema, epiphora, abnormal ocular discharge, or changes in epithelium, stroma, and endothelium after BMP7+HGF therapy for up to 7 months, as compared with control groups. Throughout the 7-month period, no significant changes were recorded in endothelial density (P = 0.9581). Histological and molecular data were well corroborated with the subjective clinical analyses and showed no differences in the naïve, naked-vector, and BMP7+HGF groups. Conclusions: Localized BMP7+HGF therapy is a safe, tolerable, and innovative modality for the treatment of corneal fibrosis. Translational Relevance: Nanoparticle-mediated BMP7+HGF combination gene therapy has the potential to treat corneal fibrosis in vivo without short- or long-term toxicity.

Six-Month In Vivo Safety Profiling of Topical Ocular AAV5-Decorin Gene Transfer.

Purpose: A significant remission of corneal fibrosis and neovascularization in rabbit eye in vivo was observed from a tissue-selective localized adeno-associated virus (AAV)5-Decorin (Dcn) gene therapy. This study sought to investigate 6-month toxicity profiling of this gene therapy for the eye in vivo using a rabbit model. Methods: A small epithelial scrape followed by corneal drying was performed unilaterally in 12 rabbit eyes and either AAV5-Dcn (n = 6) or naked vector (n = 6) was delivered topically using a cloning cylinder technique. Contralateral eyes served as naïve control (n = 6). Safety and tolerability measurements in live rabbits were performed periodically until month 6 using multimodel clinical ophthalmic imaging tools-a slit lamp, stereomicroscope, and HRT3-RCM in vivo confocal microscope. Thereafter, corneas were excised and subjected to hematoxylin and eosin staining, Mason trichome staining, propidium iodide nuclear staining, and quantitative real-time polymerase chain reaction analyses. Results: Clinical eye examinations based on the modified Hackett-McDonald ocular scoring system, and in vivo confocal imaging of the cornea showed no signs of ocular toxicity in rabbit eyes given AAV5-Dcn gene transfer vs control eyes (P > 0.05) through 6 months after treatment. The histologic and molecular analyses showed no significant differences in AAV5-Dcn vs AAV naked or naïve control groups (P > 0.05) and were in accordance with the masked clinical ophthalmic observations showing no abnormalities. Conclusions: Topical tissue-targeted localized AAV5-Dcn gene therapy seems to be safe and nontoxic to the rabbit eye in vivo. Translational Relevance: AAV5-Dcn gene therapy has the potential to treat corneal fibrosis and neovascularization in vivo safely without significant ocular toxicity.

Review on the Myopia Pandemic: Epidemiology, Risk Factors, and Prevention.

Nearsightedness, or myopia, is becoming more prevalent worldwide. The eye experiences dynamic growth throughout adolescence, but the etiopathogenesis of myopia progression is not fully understood. Myopia is associated with several pathologic eye conditions, leading to irreversible vision loss. Treatment for preventing myopia progression is reliant on effective screening and initiating treatment early in life. This article will review risk factors for myopia progression and discuss treatment strategies that are most effective in halting its spread.

Role of inhibitor of differentiation 3 gene in cellular differentiation of human corneal stromal fibroblasts.

Purpose: Inhibitor of differentiation (Id) proteins are helix-loop-helix (HLH) transcriptional repressors that modulate a range of developmental and cellular processes, including cell differentiation and cell cycle mobilization. The inhibitor of differentiation 3 (Id3) gene, a member of the Id gene family, governs the expression and progression of transforming growth factor beta (TGFß)-mediated cell differentiation. In the face of mechanical, chemical, or surgical corneal insults, corneal keratocytes differentiate into myofibroblasts for wound repair. Excessive development or persistence or both of myofibroblasts after wound repair results in corneal haze that compromises corneal clarity and visual function. The objective of this study was to investigate whether Id3 overexpression in human corneal stromal fibroblasts governs TGFß-driven cellular differentiation and inhibits keratocyte to myofibroblast transformation. Methods: Primary human corneal stromal fibroblast (h-CSF) cultures were generated from donor human corneas. Human corneal myofibroblasts (h-CMFs) were produced by growing h-CSF in the presence of TGFß1 under serum-free conditions. The Id3 gene was cloned into a mammalian expression vector (pcDNA3 mCherry LIC cloning vector), and the nucleotide sequence of the vector constructs was confirmed with sequencing as well as through restriction enzyme analysis. The Id3 mammalian overexpression vector was introduced into h-CSFs using a lipofectamine transfection kit. The expression of Id3 in selected clones was characterized with quantitative real-time PCR (qRT-PCR), immunocytochemistry, and western blotting. Phase contrast microscopy and trypan blue exclusion assays were used to evaluate the effects of the transfer of the Id3 gene on the hCSF phenotype and viability, respectively. To analyze the inhibitory effects of the Id3 gene transfer on TGFß-induced formation of h-CMFs, expression of the mRNA and protein of the myofibroblast marker alpha smooth muscle actin (α-SMA) was examined with qRT-PCR, western blotting, and immunocytochemistry. Student t test, analysis of variance (ANOVA), and Bonferroni adjustment for repeated measures were used for statistical analysis. Results: The results indicate that Id3 overexpression does not alter the cellular phenotype or viability of h-CSFs. Overexpression of the Id3 gene in h-CSF cells grown in the presence of TGFß1 under serum-free conditions showed a statistically significant decrease (76.3±4.3%) in α-SMA expression (p<0.01) compared to the naked-vector transfected or non-transfected h-CSF cells. Id3-transfected, naked-vector transfected, and non-transfected h-CSF cells grown in the absence of TGFß1 showed the expected low expression of α-SMA (0-5%). Furthermore, Id3 overexpression statistically significantly decreased TGFß-induced mRNA levels of profibrogenic genes such as fibronectin, collagen type I, and collagen type IV (1.80±0.26-, 1.70±0.35- and 1.70±0.36-fold, respectively; p<0.05) that a play role in stromal matrix modulation and corneal wound healing. Results of the protein analysis with western blotting indicated that Id3 overexpression in h-CSF cells effectively slows TGFß-driven differentiation and formation of h-CMFs. Results for subsequent overexpression studies showed that this process occurs through the regulation of E2A, a TATA box protein. Conclusions: Id3 regulates TGFß-driven differentiation of h-CSFs and formation of h-CMFs in vitro. Targeted Id3 gene delivery has potential to treat corneal fibrosis and reestablish corneal clarity in vivo.

A Novel Topical Ophthalmic Formulation to Mitigate Acute Mustard Gas Keratopathy In Vivo: A Pilot Study.

Purpose: This pilot study investigated the in vivo therapeutic potential and tolerability of a multimodal ophthalmic formulation, topical eye drops (TED), for acute mustard gas keratopathy (MGK) using a rabbit model. Methods: Twenty New Zealand White rabbits were used. Only right eyes of 18 rabbits (oculus dexter [OD]) received single sulfur mustard gas (SM) vapor injury, whereas contralateral eyes were left untreated or received TED for tolerabilty evaluation. Two rabbit eyes received no treatment and served as age-matched naive control. The four groups were: Naive (oculus sinister [OS] untreated eyes; n = 9); TED (OS treated only with TED BID for 3 days; n = 9); SM (OD exposed to SM vapor; n = 9); and SM+TED (OD exposed to SM+TED BID for 3 days; n = 9). Ocular examination in live rabbits were performed utilizing slit-lamp biomicroscopy, Fantes grading system, fluorescein staining, Schirmer's tests, pachymetry, and applanation tonometry. Cellular and molecular changes in rabbit corneas were assessed after humane euthanasia on day-3 and day-7 with histopathological and real-time polymerase chain reaction PCR techniques. Results: TED to rabbit eyes was found tolerable in vivo. SM-exposed eyes showed significant increase in Fantes scores, central corneal thickness (CCT), Schirmer's test, epithelium-stroma separation, and corneal edema. TED mitigated clinical symptoms by reducing corneal edema, Fantes scores, CCT, and Schirmer's test. Further, TED decreased SM-induced corneal haze, inflammatory and profibrotic markers, transforming growth factor-TGF-ß1 and cyclooxygenase-2COX-2, and damage to corneal structure, including epithelial-stromal integrity. Conclusions: The developed multimodal eyedrop formulation, TED, has potential to mitigate acute MGK effectively in vivo. Translational Relevance: TED is effective against MGK.

A rabbit model for evaluating ocular damage from acrolein toxicity in vivo.

Acrolein is a highly reactive and volatile unsaturated aldehyde commonly used for producing scores of commercial products. It has been recognized as a chemical weapon since its use during World War I, and more recently, in Syria. Acrolein exposure causes severe eye, skin, and lung damage in addition to many casualties. In the eye, it causes severe pain, eyelid swelling, corneal burns, and vision impairment. Very little information is available about how acrolein damages the cornea and causes vision loss. At present, the lack of clinically relevant animal models limits evaluation of acrolein toxicity and mechanisms specific to the eye. We aim to standardize the mode of delivery and exposure duration of acrolein, damaging the rabbit eye in vivo as an ocular injury model for studying the toxicity of acrolein and developing medical countermeasures. Rabbit eyes were exposed to two modes of delivery (topical and vapor) for different durations (1-5 minutes). Clinical ophthalmic examinations with a slit lamp, stereomicroscope, fluorescein dye, pachymeter, tonometer, and tearing examinations in live rabbits were performed at various times up to 4 weeks. Corneas were histologically diagnosed for transparency, fibrosis, collagens, and neovascularization. Our study successfully established an in vivo rabbit model for evaluating acrolein toxicity to the eye, accounting for different modes and durations of exposure.

Is sex a biological variable in corneal wound healing?

Wound healing differs significantly between men and women in a tissue-dependent manner. Dermal wounds heal faster in women whereas mucosal wounds heal faster in men. However, the effect of sex as a variable in corneal wound healing is largely unknown. The primary objective of this study was to test whether sex is a biological variable in corneal wound healing activated by the trauma or injury using an established in vivo rabbit model with male and female New Zealand White rabbits. Corneal wounds in rabbits were produced by a single topical alkali (0.5N Sodium hydroxide) application. Serial slit-lamp, stereo biomicroscopy, and applanation tonometry evaluated corneal opacity, anterior segment ocular health, and intraocular pressure (IOP), respectively, at various times during the study. Fourteen days after alkali-wound, corneal tissues were collected after humane euthanasia to examine cellular and molecular wound healing parameters. Quantitative PCR (qPCR) and immunofluorescence were used to quantify changes in the extracellular modeling protein levels of alpha-smooth muscle actin (α-SMA), Fibronectin (FN), Collagen-I (Col-I), and Transforming growth factor beta 1 (TGFß1) involved in corneal healing. Hematoxylin and Eosin (H&E) staining was used to study histopathological changes in morphology and TUNEL assay to evaluate levels of apoptotic cell death. Male and female rabbits showed no significant differences in corneal opacity (Fantes score) or intraocular pressure (IOP) values (9.5 ±â€¯0.5 mm Hg) in live animals. Likewise, no statistically significant sex-based differences in the mRNA levels of α-SMA (male = 5.95 ±â€¯0.21 fold vs. female = 5.32 ±â€¯0.043), FN (male = 3.02 ±â€¯0.24 fold vs. female = 3.23 ±â€¯0.27), Col-I (male = 3.12 ±â€¯0.37 fold vs. female = 3.31 ±â€¯0.24), TGFß1 (male = 1.65 ±â€¯0.06 fold vs. female = 1.59 ±â€¯0.053); and protein levels of α-SMA (male = 74.16 ±â€¯4.6 vs. female = 71.58 ±â€¯7.1), FN (male = 60.11 ±â€¯4.6 vs. female = 57.41 ±â€¯8.3), Col-I (male = 84.11 ±â€¯2.8 vs. female = 84.55 ±â€¯3.6), TGFß1 (male = 11.61 ±â€¯2.8 vs. female = 9.5 ±â€¯3.04) were observed. Furthermore, H&E and TUNEL analyses found no statistically significant differences in cellular structures and apoptosis, respectively, in male vs. female corneas. Consistent with earlier reports, wounded corneas showed significantly increased levels of these parameters compared to the unwounded corneas. Our data suggest that sex is not a major biological variable during active early stages of corneal wound healing in rabbits in vivo.

Decorin antagonizes corneal fibroblast migration via caveolae-mediated endocytosis of epidermal growth factor receptor.

Decorin (Dcn), a small leucine-rich proteoglycan, is involved in the regulation of corneal wound healing. Epidermal growth factor receptor (EGFR) plays a critical role in corneal fibroblasts proliferation, migration and extracellular matrix (ECM) modulation upon injury or infection. The present study aimed to investigate the mechanistic role of Dcn in EGFR internalization to the regulation of corneal stromal fibroblasts (CSFs) migration, a key step in the corneal wound healing. Human corneal stromal fibroblasts (hCSF) cultures were generated from donor corneas. At 70% confluence, cells were switched to serum-free conditions for 48 h and then treated with decorin (250 nM) in the presence or absence of EGF (100 ng/ml) for various time points (10-60 min). Cell lysates were subjected to proteome array analysis screening for 42 different phosphorylated human receptor tyrosine kinases (RTKs), immunocytochemistry, and western blots to analyze EGFR phosphorylation. The scratch-wound assay was performed to evaluate the effects of decorin on EGF-mediated hCSF migration. Dcn caused a rapid EGFR phosphorylation within 10 min of exposure in RTK blot defining its role as a biological ligand for EGFR in hCSFs. Prolonged exposure to Dcn caused complete disappearance of EGFR and inhibition of the hCSF migration in the scratch wound assay suggesting Dcn binding to EGFR causes EGFR down-regulation. Immunostaining studies indicated that Dcn-treatment to hCSFs internalizes Dcn-EGFR complex, which does not require tyrosine kinase activity when treated with the AG1478 inhibitor and co-localizes the complex to the perinuclear region. Next, we found that Dcn-EGFR complex does not follow canonical early endosome internalization as revealed by the EEA1 antibody instead binds to the CD63 antibody directed for degradation by the late endosome. We also found that Dcn regulates the EGFR recycling by preventing its binding to Rab11, a specific antibody for recycling endosome. Further, hCSFs-pretreated with pharmacological inhibitors, methyl-ß-cyclodextrin and chlorpromazine and supplemented with Dcn suggested EGFR trafficking via the caveolae-mediated pathway. These results suggest that Dcn acts as a biological ligand for EGFR and modulates hCSF migration via EGFR down-regulation, thus playing a vital role in corneal wound healing.

Novel Combination BMP7 and HGF Gene Therapy Instigates Selective Myofibroblast Apoptosis and Reduces Corneal Haze In Vivo.

Purpose: We tested the potential of bone morphogenic protein 7 (BMP7) and hepatocyte growth factor (HGF) combination gene therapy to treat preformed corneal fibrosis using established rabbit in vivo and human in vitro models. Methods: Eighteen New Zealand White rabbits were used. Corneal fibrosis was produced by alkali injury. Twenty-four hours after scar formation, cornea received topically either balanced salt solution (BSS; n = 6), polyethylenimine-conjugated gold nanoparticle (PEI2-GNP)-naked plasmid (n = 6) or PEI2-GNP plasmids expressing BMP7 and HGF genes (n = 6). Donor human corneas were used to obtain primary human corneal fibroblasts and myofibroblasts for mechanistic studies. Gene therapy effects on corneal fibrosis and ocular safety were evaluated by slit-lamp microscope, stereo microscopes, quantitative real-time PCR, immunofluorescence, TUNEL, modified MacDonald-Shadduck scoring system, and Draize tests. Results: PEI2-GNP-mediated BMP7+HGF gene therapy significantly decreased corneal fibrosis in live rabbits in vivo (Fantes scale was 0.6 in BMP7+HGF-treated eyes compared to 3.3 in -therapy group; P < 0.001). Corneas that received BMP7+HGF demonstrated significantly reduced mRNA levels of profibrotic genes: α-SMA (3.2-fold; P < 0.01), fibronectin (2.3-fold, P < 0.01), collagen I (2.1-fold, P < 0.01), collagen III (1.6-fold, P < 0.01), and collagen IV (1.9-fold, P < 0.01) compared to the -therapy corneas. Furthermore, BMP7+HGF-treated corneas showed significantly fewer myofibroblasts compared to the -therapy controls (83%; P < 0.001). The PEI2-GNP introduced >104 gene copies per microgram DNA of BMP7 and HGF genes. The recombinant HGF rendered apoptosis in corneal myofibroblasts but not in fibroblasts. Localized topical BMP7+HGF therapy showed no ocular toxicity. Conclusions: Localized topical BMP7+HGF gene therapy treats corneal fibrosis and restores transparency in vivo mitigating excessive healing and rendering selective apoptosis in myofibroblasts.

KCa3.1 ion channel: A novel therapeutic target for corneal fibrosis.

Vision impairment from corneal fibrosis is a common consequence of irregular corneal wound healing after injury. Intermediate-conductance calmodulin/calcium-activated K+ channels 3.1 (KCa3.1) play an important role in cell cycle progression and cellular proliferation. Proliferation and differentiation of corneal fibroblasts to myofibroblasts can lead to corneal fibrosis after injury. KCa3.1 has been shown in many non-ocular tissues to promote fibrosis, but its role in corneal fibrosis is still unknown. In this study, we characterized the expression KCa3.1 in the human cornea and its role in corneal wound healing in vivo using a KCa3.1 knockout (KCa3.1-/-) mouse model. Additionally, we tested the hypothesis that blockade of KCa3.1 by a selective KCa3.1 inhibitor, TRAM-34, could augment a novel interventional approach for controlling corneal fibrosis in our established in vitro model of corneal fibrosis. The expression of KCa3.1 gene and protein was analyzed in human and murine corneas. Primary human corneal fibroblast (HCF) cultures were used to examine the potential of TRAM-34 in treating corneal fibrosis by measuring levels of pro-fibrotic genes, proteins, and cellular migration using real-time quantitative qPCR, Western blotting, and scratch assay, respectively. Cytotoxicity of TRAM-34 was tested with trypan blue assay, and pro-fibrotic marker expression was tested in KCa3.1-/-. Expression of KCa3.1 mRNA and protein was detected in all three layers of the human cornea. The KCa3.1-/- mice demonstrated significantly reduced corneal fibrosis and expression of pro-fibrotic marker genes such as collagen I and α-smooth muscle actin (α-SMA), suggesting that KCa3.1 plays an important role corneal wound healing in vivo. Pharmacological treatment with TRAM-34 significantly attenuated corneal fibrosis in vitro, as demonstrated in HCFs by the inhibition TGFß-mediated transcription of pro-fibrotic collagen I mRNA and α-SMA mRNA and protein expression (p<0.001). No evidence of cytotoxicity was observed. Our study suggests that KCa3.1 regulates corneal wound healing and that blockade of KCa3.1 by TRAM-34 offers a potential therapeutic strategy for developing therapies to cure corneal fibrosis in vivo.

Blockade of KCa3.1: A novel target to treat TGF-ß1 induced conjunctival fibrosis.

Postoperative conjunctival fibrosis is common in patients after glaucoma filtration surgery. The calcium activated potassium (KCa3.1) channel has been shown to inhibit fibrosis in many non-ocular tissues. However, its potential in treating ocular fibrosis remains unknown. We tested the anti-fibrotic potential of TRAM34, a selective blocker of KCa3.1 channel, in treating conjunctival fibrosis. Primary human conjunctival fibroblast (HCF) cultures derived from donor tissues. Myofibroblasts causing conjunctival fibrosis were generated by growing HCFs in the presence of TGFß1 for 72 h. KCa3.1 mRNA and protein expression in HCF was examined with PCR and western blot. The anti-fibrotic potential of TRAM34 was examined by measuring fibrotic gene expression with quantitative PCR (qPCR), immunofluorescence, and western blotting in HCFs in ±â€¯TGFß1 (5 ng/ml) and TRAM34 (0-25 µM). The cytotoxicity of Tram34 was analyzed with trypan blue assay and its role in Smad signaling was studied with immunofluorescence. Expression of KCa3.1 mRNA and protein was detected in HCFs and TGFß1 treatment to HCFs significantly increased expression of KCa3.1. TRAM34 treatment attenuated transcription of fibrotic markers, αSMA (p < .001), fibronectin (p < .05), collagen I (p < .001) and collagen IV (p < .001) in TGFß1-induced HCFs. Further, TRAM34 significantly inhibited TGFß1-stimulated αSMA protein expression (p < .01) and nuclear translocation of fibrotic Smad2/3 in HCFs and showed no significant cytotoxicity (p < .05). The KCa3.1 potassium channel plays a significant role in the prevention of conjunctival fibrosis and TRAM34 has potential to control post surgical bleb fibrosis in patients. In vivo studies are warranted.

Efficacy and Safety Comparison Between Suberoylanilide Hydroxamic Acid and Mitomycin C in Reducing the Risk of Corneal Haze After PRK Treatment In Vivo.

PURPOSE: This study compared the efficacy and safety of suberoylanilide hydroxamic acid (SAHA) and mitomycin C (MMC) up to 4 months in the prevention of corneal haze induced by photorefractive keratectomy (PRK) in rabbits in vivo. METHODS: Corneal haze in rabbits was produced with -9.00 diopter PRK. A single application of SAHA (25 µM) or MMC (0.02%) was applied topically immediately after PRK. Effects of the two drugs were analyzed by slit-lamp microscope, specular microscope, TUNEL assay, and immunofluorescence. RESULTS: Single topical adjunct use of SAHA (25 µM) or MMC (0.02%) after PRK attenuated more than 95% corneal haze and myofibroblast formation (P < .001). SAHA did not reduce keratocyte density, cause keratocyte apoptosis, or increase immune cell infiltration compared to MMC (P < .01 or .001). Furthermore, SAHA dosing did not compromise corneal endothelial phenotype, density, or function in rabbit eyes, whereas MMC application did (P < .01 or .001). CONCLUSIONS: SAHA and MMC significantly decreased corneal haze after PRK in rabbits in vivo. SAHA exhibited significantly reduced short- and long-term damage to the corneal endothelium compared to MMC in rabbits. SAHA is an effective and potentially safer alternative to MMC for the prevention of corneal haze after PRK. Clinical trials are warranted. [J Refract Surg. 2017;33(12):834-839.].

Targeted AAV5-Smad7 gene therapy inhibits corneal scarring in vivo.

Corneal scarring is due to aberrant activity of the transforming growth factor ß (TGFß) signaling pathway following traumatic, mechanical, infectious, or surgical injury. Altered TGFß signaling cascade leads to downstream Smad (Suppressor of mothers against decapentaplegic) protein-mediated signaling events that regulate expression of extracellular matrix and myogenic proteins. These events lead to transdifferentiation of keratocytes into myofibroblasts through fibroblasts and often results in permanent corneal scarring. Hence, therapeutic targets that reduce transdifferentiation of fibroblasts into myofibroblasts may provide a clinically relevant approach to treat corneal fibrosis and improve long-term visual outcomes. Smad7 protein regulates the functional effects of TGFß signaling during corneal wound healing. We tested that targeted delivery of Smad7 using recombinant adeno-associated virus serotype 5 (AAV5-Smad7) delivered to the corneal stroma can inhibit corneal haze post photorefractive keratectomy (PRK) in vivo in a rabbit corneal injury model. We demonstrate that a single topical application of AAV5-Smad7 in rabbit cornea post-PRK led to a significant decrease in corneal haze and corneal fibrosis. Further, histopathology revealed lack of immune cell infiltration following AAV5-Smad7 gene transfer into the corneal stroma. Our data demonstrates that AAV5-Smad7 gene therapy is relatively safe with significant potential for the treatment of corneal disease currently resulting in fibrosis and impaired vision.