Rayne R. Lim

Elevated expression of matrix metalloproteinase-9 and inflammatory cytokines in keratoconus patients is inhibited by cyclosporine A.

PURPOSE The present study was designed to understand the role of inflammatory cytokines secreted by corneal epithelial cells in keratoconus (KC) and the response to treatment with cyclosporine A (CyA). METHODS The study involved 129 Indian KC patients clinically graded according to Amsler-Krumeich classification and 20 healthy, nonectatic subjects as controls. Tear levels of matrix metalloproteinase-9 (MMP9), interleukin-6 (IL6), and tumor necrosis factor-α (TNFα) were measured using ELISA kits. Gene expression was measured by qPCR in corneal epithelial cells obtained by debridement from subjects undergoing ocular surface surgeries. In addition, epithelial cells were stimulated with TNFα and treated with CyA to study its role on MMP9 expression. Finally, 20 KC patients (27 eyes) with inflammatory symptoms were treated with topical CyA application. RESULTS We observed that MMP9, TNFα, and IL6 levels were strongly upregulated at the mRNA level in KC patient epithelia. Similarly, tears collected from KC patients exhibited high levels of MMP9 and IL6 protein. Cyclosporine A treatment significantly reduced the mRNA expression levels of IL6 and TNFα in both short- and long-term treatments; however, it reduced MMP9 levels only in long-term treatment in cultured corneal epithelial cells. Subsequent treatment of KC patients with CyA for approximately 6 months reduced tear MMP9 levels and led to local reduction in corneal curvatures as determined by corneal topography maps. CONCLUSIONS The data indicate that corneal epithelium contributes to elevated MMP9 and inflammatory cytokine expression in tears of KC patients. Cyclosporine A treatment reduced MMP9 and inflammatory cytokine levels in an in vitro inflammation model system. In KC patients, CyA treatment reduced MMP9 levels measured in tears with concomitant arrest of disease progression. Therefore, CyA might be a novel treatment strategy in KC patients but requires additional evaluation in larger cohorts. (ClinicalTrials.gov number, NCT01746823.).

Nanomedicine Approaches for Corneal Diseases

Corneal diseases are the third leading cause of blindness globally. Topical nonsteroidal anti-inflammatory drugs (NSAIDs), steroids, antibiotics and tissue transplantation are currently used to treat corneal pathological conditions. However, barrier properties of the ocular surface necessitate high concentration of the drugs applied in the eye repeatedly. This often results in poor efficacy and several side-effects. Nanoparticle-based molecular medicine seeks to overcome these limitations by enhancing the permeability and pharmacological properties of the drugs. The promise of nanomedicine approaches for treating corneal defects and restoring vision without side effects in preclinical animal studies has been demonstrated. Numerous polymeric, metallic and hybrid nanoparticles capable of transporting genes into desired corneal cells to intercept pathologic pathways and processes leading to blindness have been identified. This review provides an overview of corneal diseases, nanovector properties and their applications in drug-delivery and corneal disease management.

Insight into membrane selectivity of linear and branched polyethylenimines and their potential as biocides for advanced wound dressings.

UNLABELLED We report here structure-property relationship between linear and branched polyethylene imines by examining their antimicrobial activities against wide range of pathogens. Both the polymers target the cytoplasmic membrane of bacteria and yeasts, eliciting rapid microbicidal properties. Using multiscale molecular dynamic simulations, we showed that, in both fully or partially protonated forms LPEI discriminates between mammalian and bacterial model membranes whereas BPEI lacks selectivity for both the model membranes. Simulation results suggest that LPEI forms weak complex with the zwitterionic lipids whereas the side chain amino groups of BPEI sequester the zwitterionic lipids by forming tight complex. Consistent with these observations, label-free cell impedance measurements, cell viability assays and high content analysis indicate that BPEI is cytotoxic to human epithelial and fibroblasts cells. Crosslinking of BPEI onto electrospun gelatin mats attenuate the cytotoxicity for fibroblasts while retaining the antimicrobial activity against Gram-positive and yeasts strains. PEI crosslinked gelatin mats elicit bactericidal activity by contact-mediated killing and durable to leaching for 7days. The potent antimicrobial activity combined with enhanced selectivity of the crosslinked ES gelatin mats would expand the arsenel of biocides in the management of superficial skin infections. The contact-mediated microbicidal properties may avert antimicrobial resistance and expand the diversity of applications to prevent microbial contamination. STATEMENT OF SIGNIFICANCE Current commercially available advanced wound dressings are either impregnated with metallic silver or silver salts which have side effects or may not avert antimicrobial resistance. In this article, we have used multidisciplinary approach comprising of computational, chemical and biological methods to understand the antimicrobial properties and biocompatibility of linear (LPEI) and branched (BPEI) polyethylenimines. We then applied this knowledge to develop dual purpose wound dressings containing these polymers, which encourages healing while maintain antimicrobial activity. In addition, the approach can be expanded to rationalize the antimicrobial vs. cytotoxicity of other cationic polymers and the method of crosslinking would enhance their potentials as biocides for advanced materials.

ITF2357 transactivates Id3 and regulate TGFβ/BMP7 signaling pathways to attenuate corneal fibrosis.

Corneal fibrosis is often seen in patients with ocular trauma and infection that compromises corneal transparency resulting in vision loss. Treatment strategies including NSAIDs, steroids, MMC and corneal transplants have shown tremendous success but with several side effects and cellular toxicity. Histone deacetylase inhibitors (HDACi) have been shown to inhibit corneal fibrosis via TGFβ signaling pathway. In this study, we investigated safety, efficacy and mechanism of action of a HDACi, ITF2357 in TGFβ-stimulated in vitro primary human cornea stromal fibroblasts (pHCSFs) and in vivo in a photorefractive keratectomy-treated rabbit model of corneal fibrosis. We found that in vivo ITF2357 decreased collagen I, collagen IV, fibronectin, integrin αVβ3 expression with a reduction in corneal haze. In addition, ITF2357 reduced myofibroblast formation, suppressed phosphorylation of Smad proteins in TGFβ pathway and inhibited key responsive protein, P4HA1 involved in pro-collagen synthesis. Treatment of pHCSFs with ITF2357 activated BMP7 levels and expressed all the members of inhibitor of differentiation proteins (Id1-Id4), however, it failed to rescue TGFβ-driven transdifferentiation of fibroblasts to myofibroblasts in the presence of siRNA specific to Id3. We conclude that ITF2357 is a potential anti-fibrotic drug that exerts its action via activation of Id3, a downstream target of TGFβ/BMP7 signaling pathways.

Hevin Plays a Pivotal Role in Corneal Wound Healing

Background Hevin is a matricellular protein involved in tissue repair and remodeling via interaction with the surrounding extracellular matrix (ECM) proteins. In this study, we examined the functional role of hevin using a corneal stromal wound healing model achieved by an excimer laser-induced irregular phototherapeutic keratectomy (IrrPTK) in hevin-null (hevin-/-) mice. We also investigated the effects of exogenous supplementation of recombinant human hevin (rhHevin) to rescue the stromal cellular components damaged by the excimer laser. Methodology/Principal Findings Wild type (WT) and hevin -/- mice were divided into three groups at 4 time points- 1, 2, 3 and 4 weeks. Group I served as naïve without any treatment. Group II received epithelial debridement and underwent IrrPTK using excimer laser. Group III received topical application of rhHevin after IrrPTK surgery for 3 days. Eyes were analyzed for corneal haze and matrix remodeling components using slit lamp biomicroscopy, in vivo confocal microscopy, light microscopy (LM), transmission electron microscopy (TEM), immunohistochemistry (IHC) and western blotting (WB). IHC showed upregulation of hevin in IrrPTK-injured WT mice. Hevin -/- mice developed corneal haze as early as 1-2 weeks post IrrPTK-treatment compared to the WT group, which peaked at 3-4 weeks. They also exhibited accumulation of inflammatory cells, fibrotic components of ECM proteins and vascularized corneas as seen by IHC and WB. LM and TEM showed activated keratocytes (myofibroblasts), inflammatory debris and vascular tissues in the stroma. Exogenous application of rhHevin for 3 days reinstated inflammatory index of the corneal stroma similar to WT mice. Conclusions/Significance Hevin is transiently expressed in the IrrPTK-injured corneas and loss of hevin predisposes them to aberrant wound healing. Hevin -/- mice develop early corneal haze characterized by severe chronic inflammation and stromal fibrosis that can be rescued with exogenous administration of rhHevin. Thus, hevin plays a pivotal role in the corneal wound healing.

pH Induced Conformational Transitions in the Transforming Growth Factor β-Induced Protein (TGFβIp) Associated Corneal Dystrophy Mutants

Most stromal corneal dystrophies are associated with aggregation and deposition of the mutated transforming growth factor-β induced protein (TGFβIp). The 4th_FAS1 domain of TGFβIp harbors ~80% of the mutations that forms amyloidogenic and non-amyloidogenic aggregates. To understand the mechanism of aggregation and the differences between the amyloidogenic and non-amyloidogenic phenotypes, we expressed the 4th_FAS1 domains of TGFβIp carrying the mutations R555W (non-amyloidogenic) and H572R (amyloidogenic) along with the wild-type (WT). R555W was more susceptible to acidic pH compared to H572R and displayed varying chemical stabilities with decreasing pH. Thermal denaturation studies at acidic pH showed that while WT did not undergo any conformational transition, the mutants exhibited a clear pH-dependent irreversible conversion from αβ conformation to β-sheet oligomers. The β-oligomers of both mutants were stable at physiological temperature and pH. Electron microscopy and dynamic light scattering studies showed that β-oligomers of H572R were larger compared to R555W. The β-oligomers of both mutants were cytotoxic to primary human corneal stromal fibroblast (pHCSF) cells. The β-oligomers of both mutants exhibit variations in their morphologies, sizes, thermal and chemical stabilities, aggregation patterns and cytotoxicities.

Conventional Versus Inverted Side-cut Flaps for Femtosecond Laser-Assisted LASIK: Laboratory and Clinical Evaluation.

PURPOSE To study the differences in early corneal cellular events and biomechanical properties after femtosecond laser-assisted LASIK performed using conventional or inverted side-cut angles. METHODS In the laboratory study, left eyes of 24 rabbits underwent LASIK flap creation with a 70° or 115° side-cut. The contralateral eyes served as controls. The corneas were harvested 24 hours postoperatively. In the clinical study, 2 eyes of each patient (n = 29) were randomized to corneal flap creation with 70° or 115° side-cut angles during LASIK. The Ocular Response Analyzer (ORA; Reichert Ophthalmic Instruments, Depew, NY) was used to assess biomechanical properties of the cornea. RESULTS In rabbit eyes, epithelial ingrowth was observed more frequently in flaps with 70° side cuts compared to flaps with 115° side-cuts. Corneas with 70° side-cuts showed significantly increased apoptotic cells compared to 115° side-cuts in the central (P = .001) and peripheral (P = .004) regions. Fifty-eight eyes of 29 patients were included in the clinical study. An overall reduction in Goldmann-correlated intraocular pressure, corneal-compensated intraocular pressure, corneal resistance factor, corneal hysteresis measurements, p1 area, p2 area, and p1 area 1 and p2 area 1 was noted 37 ± 2 months after surgery (P < .001). No significant difference was observed in the change of any of these parameters between both groups (P ≥ .146). CONCLUSIONS Significant differences in wound healing were observed in rabbit corneas that underwent LASIK with conventional or inverted side-cuts. Variation in flap side-cut angles did not alter the long-term biomechanical properties measured with the ORA in patients after LASIK. [J Refract Surg. 2017;33(2):96-103.].

Branched Peptide, B2088, Disrupts the Supramolecular Organization of Lipopolysaccharides and Sensitizes the Gram-negative Bacteria

Dissecting the complexities of branched peptide-lipopolysaccharides (LPS) interactions provide rationale for the development of non-cytotoxic antibiotic adjuvants. Using various biophysical methods, we show that the branched peptide, B2088, binds to lipid A and disrupts the supramolecular organization of LPS. The disruption of outer membrane in an intact bacterium was demonstrated by fluorescence spectroscopy and checkerboard assays, the latter confirming strong to moderate synergism between B2088 and various classes of antibiotics. The potency of synergistic combinations of B2088 and antibiotics was further established by time-kill kinetics, mammalian cell culture infections model and in vivo model of bacterial keratitis. Importantly, B2088 did not show any cytotoxicity to corneal epithelial cells for at least 96 h continuous exposure or hemolytic activity even at 20 mg/ml. Peptide congeners containing norvaline, phenylalanine and tyrosine (instead of valine in B2088) displayed better synergism compared to other substitutions. We propose that high affinity and subsequent disruption of the supramolecular assembly of LPS by the branched peptides are vital for the development of non-cytotoxic antibiotic adjuvants that can enhance the accessibility of conventional antibiotics to the intracellular targets, decrease the antibiotic consumption and holds promise in averting antibiotic resistance.

The NLRP3 Inflammasome May Contribute to Pathologic Neovascularization in the Advanced Stages of Diabetic Retinopathy

Diabetic retinopathy (DR) is a retinal microvascular disease characterized by inflammatory and angiogenic pathways. In this study, we evaluated NLRP3 inflammasome in a double transgenic mouse model, Akimba (Ins2AkitaxVEGF+/−), which demonstrates hyperglycemia, vascular hyperpermeability and neovascularization seen in the proliferative DR. Retinal structural integrity, vascular leakage and function were examined by fundus photography, fluorescein angiography, optical coherence tomography, retinal flat mounts, laser speckle flowgraphy (LSFG), and electroretinography in Akimba and its parental strains, Akita (Ins2Akita) and Kimba (trVEGF029) mice. Inflammatory mechanisms involving NLRP3 inflammasome were investigated using real time-PCR, immunohistochemistry, ELISA and western blots. We observed an increased vascular leakage, reduced retinal thickness, and function in Akimba retina. Also, Akimba retina depicts decreased relative flow volume measured by LSFG. Most importantly, high levels of IL-1β along with increased NLRP3, ASC, and Caspase-1 at mRNA and protein levels were observed in Akimba retina. However, the in vivo functional role remains undefined. In conclusion, increased activation of macroglia (GFAP), microglia (Iba-1 and OX-42) and perivascular macrophages (F4/80 and CD14) together with pro-inflammatory (IL-1β and IL-6) and pro-angiogenic markers (PECAM-1, ICAM-1, VEGF, Flt-1, and Flk-1), suggested a critical role for NLRP3 inflammasome in the Akimba mouse model depicting advanced stages of DR pathogenesis.

Young Ossabaw Pigs Fed a Western Diet Exhibit Early Signs of Diabetic Retinopathy

Purpose Recent clinical data suggest an increasing prevalence of obesity and type 2 diabetes in adolescents, placing them at high risk of developing diabetic retinopathy during adult working years. The present study was designed to characterize the early retinal and microvascular alterations in young Ossabaw pigs fed a Western diet, described as a model of metabolic syndrome genetically predisposed to type 2 diabetes. Methods Four-month-old Ossabaw miniature pigs were divided into two groups, lean and diet-induced obesity. Obese pigs were fed a Western diet with high-fat/high-fructose corn syrup/high-choleric content for 10 weeks. Blood and retina were collected for biochemical profiling, trypsin digest, flatmounts, Fluoro-Jade C staining, electron microscopy, quantitative PCR, immunohistochemistry, and Western blots. Results Young Ossabaw pigs had elevated fasting blood glucose after feeding on a Western diet for 10 weeks. Their retina showed disrupted cellular architecture across neural layers, with numerous large vacuoles seen in cell bodies of the inner nuclear layer. Microvessels in the obese animals exhibited thickened basement membrane, along with pericyte ghosts and acellular capillaries. The pericyte to endothelial ratio decreased significantly. Retina flatmounts from obese pigs displayed reduced capillary density, numerous terminal capillary loops, and string vessels, which stained collagen IV but not isolectin IB4. Quantitative PCR and Western blots showed significantly high levels of basement membrane proteins collagen IV and fibronectin in obese pigs. Conclusions This is the first study to describe the ultrastructural neuronal and vascular changes in the retina of young Ossabaw pigs fed a Western diet, simulating early signs of diabetic retinopathy pathogenesis.