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-PEI nanoconstruct attenuates equine corneal fibroblast differentiation.

OBJECTIVE: To explore (i) the potential of polyethylenimine (PEI) nanoparticles as a vector for delivering genes into equine corneal fibroblasts (ECFs) using green fluorescent protein (GFP) marker gene, (ii) whether PEI nanoparticle-mediated decorin (DCN) gene therapy could be used to inhibit fibrosis in the equine cornea using an in vitro model. PROCEDURE: Polyethylenimine-DNA nanoparticles were prepared at nitrogen-to-phosphate (N-P) ratio of 15 by mixing 22 kDa linear PEI and a plasmid encoding either GFP or DCN. ECFs were generated from donor corneas as previously described. Initially, GFP was introduced into ECFs using PEI nanoparticles to confirm gene delivery, then DCN was introduced to evaluate for antifibrotic effects. GFP gene delivery was confirmed with real-time qPCR and ELISA. Changes in fibrosis after DCN therapy were quantified by measuring α-smooth muscle actin (αSMA) mRNA and protein levels with qPCR, immunostaining, and immunoblotting. Cytotoxicity was determined by evaluating cell morphology, cellular viability, and TUNEL assay. RESULTS: Polyethylenimine-green fluorescent protein-treated cultures showed 2.2 × 10(4) GFP plasmid copies/µg of cellular DNA and 2.1 pg of GFP/100 µL of lysate. PEI-DCN delivery significantly attenuated TGFß-induced transdifferentiation of fibroblasts to myofibroblasts (2-fold decrease of αSMA mRNA; P = 0.05) and significant inhibition of αSMA (49 ± 14.2%; P < 0.001) in immunocytochemical staining and immunoblotting were found. Furthermore, PEI-DNA nanoparticle delivery did not alter cellular phenotype at 24 h and cellular viability was maintained. CONCLUSIONS: Twenty-two kilo dalton Polyethylenimine nanoparticles are safe and effective for equine corneal gene therapy in vitro. PEI-mediated DCN gene delivery is effective at inhibiting TGFß-mediated fibrosis in this model.

Corneal gene therapy: basic science and translational perspective.

Corneal blindness is the third leading cause of blindness worldwide. Gene therapy is an emerging technology for corneal blindness due to the accessibility and immune-privileged nature of the cornea, ease of vector administration and visual monitoring, and ability to perform frequent noninvasive corneal assessment. Vision restoration by gene therapy is contingent upon vector and mode of therapeutic gene introduction into targeted cells/tissues. Numerous efficacious vectors, delivery techniques, and approaches have evolved in the last decade for developing gene-based interventions for corneal diseases. Maximizing the potential benefits of gene therapy requires efficient and sustained therapeutic gene expression in target cells, low toxicity, and a high safety profile. This review describes the basic science associated with many gene therapy vectors and the present progress of gene therapy carried out for various ocular surface disorders and diseases.

Attenuation of corneal myofibroblast development through nanoparticle-mediated soluble transforming growth factor-ß type II receptor (sTGFßRII) gene transfer.

PURPOSE: To explore (i) the potential of polyethylenimine (PEI)-DNA nanoparticles as a vector for delivering genes into human corneal fibroblasts, and (ii) whether the nanoparticle-mediated soluble extracellular domain of the transforming growth factor-ß type II receptor (sTGFßRII) gene therapy could be used to reduce myofibroblasts and fibrosis in the cornea using an in vitro model. METHODS: PEI-DNA nanoparticles were prepared at a nitrogen-to-phosphate ratio of 30 by mixing linear PEI and a plasmid encoding sTGFßRII conjugated to the fragment crystallizable (Fc) portion of human immunoglobulin. The PEI-DNA polyplex formation was confirmed through gel retardation assay. Human corneal fibroblasts (HCFs) were generated from donor corneas; myofibroblasts and fibrosis were induced with TGFß1 (1 ng/ml) stimulation employing serum-free conditions. The sTGFßRII conjugated to the Fc portion of human immunoglobulin gene was introduced into HCF using either PEI-DNA nanoparticles or Lipofectamine. Suitable negative and positive controls to compare selected nanoparticle and therapeutic gene efficiency were included. Delivered gene copies and mRNA (mRNA) expression were quantified with real-time quantitative PCR (qPCR) and protein with enzyme-linked immunosorbent assay (ELISA). The changes in fibrosis parameters were quantified by measuring fibrosis marker α-smooth muscle actin (SMA) mRNA and protein levels with qPCR, immunostaining, and immunoblotting. Cytotoxicity was determined using cellular viability, proliferation, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. RESULTS: PEI readily bound to plasmids to form nanoparticular polyplexes and exhibited much greater transfection efficiency (p<0.01) than the commercial reagent Lipofectamine. The PEI-DNA-treated cultures showed 4.5×10(4) plasmid copies/µg DNA in real-time qPCR and 7,030±87 pg/ml sTGFßRII protein in ELISA analyses, whereas Lipofectamine-transfected cultures demonstrated 1.9×10(3) gene copies/µg DNA and 1,640±100 pg/ml sTGFßRII protein during these assays. The PEI-mediated sTGFßRII delivery remarkably attenuated TGFß1-induced transdifferentiation of corneal fibroblasts to myofibroblasts in cultures, as indicated by threefold lower levels of SMA mRNA (p<0.01) and significant inhibition of SMA protein (up to 96±3%; p<0.001 compared to no-gene-delivered cultures) in immunocytochemical staining and immunoblotting. The nanoparticle-mediated delivery of sTGFßRII showed significantly better antifibrotic effects than the Lipofectamine under similar experimental conditions. However, the inhibition of myofibroblast in HCF cultures by sTGFßRII overexpression by either method was significantly higher than the naked vector transfection. Furthermore, PEI- or Lipofectamine-mediated sTGFßRII delivery into HCF did not alter cellular proliferation or phenotype at 12 and 24 h post-treatment. Nanoparticles treated with HCF showed more than 90% cellular viability and very low cell death (2-6 TUNEL+ cells), suggesting that the tested doses of PEI-nanoparticles do not induce significant cell death. CONCLUSIONS: This study demonstrated that PEI-DNA nanoparticles are an attractive vector for the development of nonviral corneal gene therapy approaches and that the sTGFßRII gene delivery into keratocytes could be used to control corneal fibrosis in vivo.

Glutathione is a potential therapeutic target for acrolein toxicity in the cornea.

Toxic and volatile chemicals are widely used in household products and previously used as warfare agents, causing a public health threat worldwide. This study aimed to evaluate the extent of injury and mechanisms of acrolein toxicity in the cornea. Primary human corneal stromal fibroblasts cultures (hCSFs) from human donor cornea were cultured and exposed to acrolein toxicity with -/+ N-acetylcysteine (NAC) to study the mode of action in the presence of Buthionine sulphoximine (BSO). PrestoBlue and MTT assays were used to optimize acrolein, NAC, and BSO doses for hCSFs. Cell-based assays and qRT-PCR analyses were performed to understand the acrolein toxicity and mechanisms. Acrolein exposure leads to an increased reactive oxygen species (ROS), compromised glutathione (GSH) levels, and mitochondrial dysfunction. The TUNEL and caspase assays showed that acrolein caused cell death in hCSFs. These deleterious effects can be mitigated using NAC in hCSFs, suggesting that GSH can be a potential target for acrolein toxicity in the cornea.

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.

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.

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.

Linear Polyethylenimine-DNA Nanoconstruct for Corneal Gene Delivery.

PURPOSE: This study investigated the efficiency and potential toxicity of a linear 22-kDa polyethylenimine (PEI)-DNA nanoconstruct for delivering genes to corneal cells and the effects of PEI nitrogen-to-DNA phosphate (N:P) ratio on gene transfer efficiency in vitro and in vivo. METHODS: A gel retardation assay, zeta potential measurement, bright-field microscopy, transfection with green fluorescent protein (GFP), immunofluorescence, and enzyme-linked immunosorbent assay (ELISA) were used to characterize the physicochemical and biological properties and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH), and reactive oxygen species (ROS) assay for cytotoxicity of the linear PEI-DNA nanoconstruct using in vitro cultured primary human corneal fibroblast and in vivo mouse models. RESULTS: Of the several evaluated N:P ratios, the highest gene transfection efficiency achieved without any notable cytotoxicity was observed at an N:P ratio of 30:1 (N:P 30). In vivo gene transfer studies revealed substantial GFP gene delivery into the corneas of mice 3 days after a single 5-min topical application without any significant adverse ocular effects. Slit-lamp biomicroscope ophthalmic examination of the mouse exposed to the linear PEI-DNA nanoconstruct showed no evidence of hyperemia (redness), corneal edema, ocular inflammation, or epiphora (excessive tearing). CONCLUSIONS: The 22-kDa linear PEI-DNA nanoconstruct is an efficient and well-tolerated vector for corneal gene therapy in vitro and in vivo and could be used as a platform for developing novel gene-based nanomedicine approaches for corneal diseases.

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.

BMP7 gene transfer via gold nanoparticles into stroma inhibits corneal fibrosis in vivo.

This study examined the effects of BMP7 gene transfer on corneal wound healing and fibrosis inhibition in vivo using a rabbit model. Corneal haze in rabbits was produced with the excimer laser performing -9 diopters photorefractive keratectomy. BMP7 gene was introduced into rabbit keratocytes by polyethylimine-conjugated gold nanoparticles (PEI2-GNPs) transfection solution single 5-minute topical application on the eye. Corneal haze and ocular health in live animals was gauged with stereo- and slit-lamp biomicroscopy. The levels of fibrosis [α-smooth muscle actin (αSMA), F-actin and fibronectin], immune reaction (CD11b and F4/80), keratocyte apoptosis (TUNEL), calcification (alizarin red, vonKossa and osteocalcin), and delivered-BMP7 gene expression in corneal tissues were quantified with immunofluorescence, western blotting and/or real-time PCR. Human corneal fibroblasts (HCF) and in vitro experiments were used to characterize the molecular mechanism mediating BMP7's anti-fibrosis effects. PEI2-GNPs showed substantial BMP7 gene delivery into rabbit keratocytes in vivo (2×10(4) gene copies/ug DNA). Localized BMP7 gene therapy showed a significant corneal haze decrease (1.68±0.31 compared to 3.2±0.43 in control corneas; p<0.05) in Fantes grading scale. Immunostaining and immunoblot analyses detected significantly reduced levels of αSMA (46±5% p<0.001) and fibronectin proteins (48±5% p<0.01). TUNEL, CD11b, and F4/80 assays revealed that BMP7 gene therapy is nonimmunogenic and nontoxic for the cornea. Furthermore, alizarin red, vonKossa and osteocalcin analyses revealed that localized PEI2-GNP-mediated BMP7 gene transfer in rabbit cornea does not cause calcification or osteoblast recruitment. Immunofluorescence of BMP7-transefected HCFs showed significantly increased pSmad-1/5/8 nuclear localization (>88%; p<0.0001), and immunoblotting of BMP7-transefected HCFs grown in the presence of TGFß demonstrated significantly enhanced pSmad-1/5/8 (95%; p<0.001) and Smad6 (53%, p<0.001), and decreased αSMA (78%; p<0.001) protein levels. These results suggest that localized BMP7 gene delivery in rabbit cornea modulates wound healing and inhibits fibrosis in vivo by counter balancing TGFß1-mediated profibrotic Smad signaling.