CFH Haploinsufficiency and Complement Alterations in Early-Onset Macular Degeneration.

Purpose: Complement dysregulation is a key component in the pathogenesis of age-related macular degeneration (AMD) and related diseases such as early-onset macular drusen (EOMD). Although genetic variants of complement factor H (CFH) are associated with AMD risk, the impact of CFH and factor H-like protein 1 (FHL-1) expression on local complement activity in human retinal pigment epithelium (RPE) remains unclear. Methods: We identified a novel CFH variant in a family with EOMD and generated patient induced pluripotent stem cell (iPSC)-derived RPE cells. We assessed CFH and FHL-1 co-factor activity through C3b breakdown assays and measured complement activation by immunostaining for membrane attack complex (MAC) formation. Expression of CFH, FHL-1, local alternative pathway (AP) components, and regulators of complement activation (RCA) in EOMD RPE cells was determined by quantitative PCR, western blot, and immunostaining. Isogenic EOMD (cEOMD) RPE was generated using CRISPR/Cas9 gene editing. Results: The CFH variant (c.351-2A>G) resulted in loss of CFH and FHL-1 expression and significantly reduced CFH and FHL-1 protein expression (∼50%) in EOMD iPSC RPE cells. These cells exhibited increased MAC deposition upon exposure to normal human serum. Under inflammatory or oxidative stress conditions, CFH and FHL-1 expression in EOMD RPE cells paralleled that of controls, whereas RCA expression, including MAC formation inhibitors, was elevated. CRISPR/Cas9 correction restored CFH/FHL-1 expression and mitigated alternative pathway complement activity in cEOMD RPE cells. Conclusions: Identification of a novel CFH variant in patients with EOMD resulting in reduced CFH and FHL-1 and increased local complement activity in EOMD iPSC RPE supports the involvement of CFH haploinsufficiency in EOMD pathogenesis.

Metabolic phenotyping of healthy and diseased human RPE cells.

Purpose: Metabolic defects in retinal pigment epithelium (RPE) are underlying many retinal degenerative diseases. This study aims to identify the nutrient requirements of healthy and diseased human RPE cells. Methods: We profiled the utilization of 183 nutrients in human RPE cells: 1) differentiated and dedifferentiated fetal RPE (fRPE), 2) induced pluripotent stem cell derived-RPE (iPSC RPE), 3) Sorsby fundus dystrophy (SFD) patient-derived iPSC RPE and its CRISPR-corrected isogenic SFD (cSFD) iPSC RPE, and 5) ARPE-19 cell lines cultured under different conditions. Results: Differentiated fRPE cells and healthy iPSC RPE cells can utilize 51 and 48 nutrients respectively, including sugars, intermediates from glycolysis and tricarboxylic acid (TCA) cycle, fatty acids, ketone bodies, amino acids, and dipeptides. However, when fRPE cells lose epithelial phenotype through dedifferentiated, they can only utilize 17 nutrients, primarily sugar and glutamine-related amino acids. SFD RPE cells can utilize 37 nutrients; however, Compared to cSFD RPE and healthy iPSC RPE, they are unable to utilize lactate, some TCA cycle intermediates, and short-chain fatty acids. Nonetheless, they show increased utilization of branch-chain amino acids (BCAAs) and BCAA-containing dipeptides. The dedifferentiated ARPE-19 cells in traditional culture media cannot utilize lactate and ketone bodies. In contrast, nicotinamide supplementation promotes differentiation into epithelial phenotype, restoring the ability to use these nutrients. Conclusions: Epithelial phenotype confers metabolic flexibility to the RPE for utilizing various nutrients. SFD RPE cells have reduced metabolic flexibility, relying on the oxidation of BCAAs. Our findings highlight the importance of nutrient availability and utilization in RPE differentiation and diseases.

ITF2357 regulates NF-κB signaling pathway to protect barrier integrity in retinal pigment epithelial cells.

The robust integrity of the retinal pigment epithelium (RPE), which contributes to the outer brain retina barrier (oBRB), is compromised in several retinal degenerative and vascular disorders, including diabetic macular edema (DME). This study evaluates the role of a new generation of histone deacetylase inhibitor (HDACi), ITF2357, in regulating outer blood-retinal barrier function and investigates the underlying mechanism of action in inhibiting TNFα-induced damage to RPE integrity. Using the immortalized RPE cell line (ARPE-19), ITF2357 was found to be non-toxic between 50 nM and 5 µM concentrations. When applied as a pre-treatment in conjunction with an inflammatory cytokine, TNFα, the HDACi was safe and effective in preventing epithelial permeability by fortifying tight junction (ZO-1, -2, -3, occludin, claudin-1, -2, -3, -5, -19) and adherens junction (E-cadherin, Nectin-1) protein expression post-TNFα stress. Mechanistically, ITF2357 depicted a late action at 24 h via attenuating IKK, IκBα, and p65 phosphorylation and ameliorated the expression of IL-1ß, IL-6, and MCP-1. Also, ITF2357 delayed IκBα synthesis and turnover. The use of Bay 11-7082 and MG132 further uncovered a possible role for ITF2357 in non-canonical NF-κB activation. Overall, this study revealed the protection effects of ITF2357 by regulating the turnover of tight and adherens junction proteins and modulating NF-κB signaling pathway in the presence of an inflammatory stressor, making it a potential therapeutic application for retinal vascular diseases such as DME with compromised outer blood-retinal barrier.

Acetyl-CoA carboxylase Inhibition increases RPE cell fatty acid oxidation and limits apolipoprotein efflux.

Purpose: In age-related macular degeneration (AMD) and Sorsby's fundus dystrophy (SFD), lipid-rich deposits known as drusen accumulate under the retinal pigment epithelium (RPE). Drusen may contribute to photoreceptor and RPE degeneration in AMD and SFD. We hypothesize that stimulating ß-oxidation in RPE will reduce drusen accumulation. Inhibitors of acetyl-CoA carboxylase (ACC) stimulate ß-oxidation and diminish lipid accumulation in fatty liver disease. In this report we test the hypothesis that an ACC inhibitor, Firsocostat, limits the accumulation of lipid deposits in cultured RPE cells. Methods: We probed metabolism and cellular function in mouse RPE-choroid, human fetal- derived RPE cells, and induced pluripotent stem cell-derived RPE cells. We used 13 C6-glucose and 13 C16-palmitate to determine the effects of Firsocostat on glycolytic, Krebs cycle, and fatty acid metabolism. 13 C labeling of metabolites in these pathways were analyzed using gas chromatography-linked mass spectrometry. We quantified ApoE and VEGF release using enzyme-linked immunosorbent assays. Immunostaining of sectioned RPE was used to visualize ApoE deposits. RPE function was assessed by measuring the trans-epithelial electrical resistance (TEER). Results: ACC inhibition with Firsocostat increases fatty acid oxidation and remodels lipid composition, glycolytic metabolism, lipoprotein release, and enhances TEER. When human serum is used to induce sub-RPE lipoprotein accumulation, fewer lipoproteins accumulate with Firsocostat. In a culture model of Sorsby's fundus dystrophy, Firsocostat also stimulates fatty acid oxidation, improves morphology, and increases TEER. Conclusions: Firsocostat remodels intracellular metabolism and improves RPE resilience to serum-induced lipid deposition. This effect of ACC inhibition suggests that it could be an effective strategy for diminishing drusen accumulation in the eyes of patients with AMD.

New Insights into RPE-Photoreceptor Complex Ultrastructure using Focused Ion Beam-Scanning Election Microscopy (FIB-SEM).

Photoreceptors in the retina are specialized neuronal cells that perceive light and play a central role in the visual system. Damage to photoreceptors is a clinical feature often associated with various retinal degenerative disorders. The photoreceptor bed comprises a unique extracellular matrix (ECM) scaffold often described as the interphotoreceptor matrix (IPM) in the subretinal space, vital during retinal development and homeostasis. In this study, we used focused ion beam scanning electron microscopy (FIB-SEM) and transmission electron microscopy (TEM) to analyze the ultrastructural architecture of the retinal pigmented epithelium (RPE)-photoreceptor complex in mice. Additionally, we describe methods for retinal preparation in EM imaging. TEM images display ultrastructural retina layers, including Bruch's membrane and the interdigitation zone (IZ). The 3-dimensional reconstruction of the outer retina revealed individual photoreceptors, the connection between their inner and outer segment via the photoreceptor cilia, and photoreceptor interaction with the RPE ciliary processes. Our findings highlight the importance of FIB-SEM in deciphering the ultrastructural details of RPE-photoreceptor interactions in the IPM complex which are essential for the maintenance of retinal architecture.

Maintaining and Assessing Various Tissue and Cell Types of the Eye Using a Novel Pumpless Fluidics System.

Many in vitro models used to investigate tissue function and cell biology require a flow of media to provide adequate oxygenation and optimal cell conditions required for the maintenance of function and viability. Toward this end, we have developed a multi-channel flow culture system to maintain tissue and cells in culture and continuously assess function and viability by either in-line sensors and/or collection of outflow fractions. The system combines 8-channel, continuous optical sensing of oxygen consumption rate with a built-in fraction collector to simultaneously measure production rates of metabolites and hormone secretion. Although it is able to maintain and assess a wide range of tissue and cell models, including islets, muscle, and hypothalamus, here we describe its operating principles and the experimental preparations/protocols that we have used to investigate bioenergetic regulation of isolated mouse retina, mouse retinal pigment epithelium (RPE)-choroid-sclera, and cultured human RPE cells. Innovations in the design of the system, such as pumpless fluid flow, have produced a greatly simplified operation of a multi-channel flow system. Videos and images are shown that illustrate how to assemble, prepare the instrument for an experiment, and load the different tissue/cell models into the perifusion chambers. In addition, guidelines for selecting conditions for protocol- and tissue-specific experiments are delineated and discussed, including setting the correct flow rate to tissue ratio to obtain consistent and stable culture conditions and accurate determinations of consumption and production rates. The combination of optimal tissue maintenance and real-time assessment of multiple parameters yields highly informative data sets that will have great utility for research in the physiology of the eye and drug discovery for the treatment of impaired vision.

Extracellular matrix dysfunction in Sorsby patient-derived retinal pigment epithelium.

Sorsby Fundus Dystrophy (SFD) is a rare form of macular degeneration that is clinically similar to age-related macular degeneration (AMD), and a histologic hallmark of SFD is a thick layer of extracellular deposits beneath the retinal pigment epithelium (RPE). Previous studies of SFD patient-induced pluripotent stem cell (iPSC) derived RPE differ as to whether these cultures recapitulate this key clinical feature by forming increased drusenoid deposits. The primary purpose of this study is to examine whether SFD patient-derived iPSC-RPE form basal deposits similar to what is found in affected family member SFD globes and to determine whether SFD iPSC RPE may be more oxidatively stressed. We performed a careful comparison of iPSC RPE from three control individuals, multiple iPSC clones from two SFD patients' iPSC RPE, and post-mortem eyes of affected SFD family members. We also examined the effect of CRISPR-Cas9 gene correction of the S204C TIMP3 mutation on RPE phenotype. Finally, targeted metabolomics with liquid chromatography and mass spectrometry analysis and stable isotope-labeled metabolite analysis were performed to determine whether SFD RPE are more oxidatively stressed. We found that SFD iPSC-RPE formed significantly more sub-RPE deposits (∼6-90 µm in height) compared to control RPE at 8 weeks. These deposits were similar in composition to the thick layer of sub-RPE deposits found in SFD family member globes by immunofluorescence staining and TEM imaging. S204C TIMP3 correction by CRISPR-Cas9 gene editing in SFD iPSC RPE cells resulted in significantly reduced basal laminar and sub-RPE calcium deposits. We detected a ∼18-fold increase in TIMP3 accumulation in the extracellular matrix (ECM) of SFD RPE, and targeted metabolomics showed that intracellular 4-hydroxyproline, a major breakdown product of collagen, is significantly elevated in SFD RPE, suggesting increased ECM turnover. Finally, SFD RPE cells have decreased intracellular reduced glutathione and were found to be more vulnerable to oxidative stress. Our findings suggest that elements of SFD pathology can be demonstrated in culture which may lead to insights into disease mechanisms.

Proline metabolism and transport in retinal health and disease.

The retina is one of the most energy-demanding tissues in the human body. Photoreceptors in the outer retina rely on nutrient support from the neighboring retinal pigment epithelium (RPE), a monolayer of epithelial cells that separate the retina and choroidal blood supply. RPE dysfunction or cell death can result in photoreceptor degeneration, leading to blindness in retinal degenerative diseases including some inherited retinal degenerations and age-related macular degeneration (AMD). In addition to having ready access to rich nutrients from blood, the RPE is also supplied with lactate from adjacent photoreceptors. Moreover, RPE can phagocytose lipid-rich outer segments for degradation and recycling on a daily basis. Recent studies show RPE cells prefer proline as a major metabolic substrate, and they are highly enriched for the proline transporter, SLC6A20. In contrast, dysfunctional or poorly differentiated RPE fails to utilize proline. RPE uses proline to fuel mitochondrial metabolism, synthesize amino acids, build the extracellular matrix, fight against oxidative stress, and sustain differentiation. Remarkably, the neural retina rarely imports proline directly, but it uptakes and utilizes intermediates and amino acids derived from proline catabolism in the RPE. Mutations of genes in proline metabolism are associated with retinal degenerative diseases, and proline supplementation is reported to improve RPE-initiated vision loss. This review will cover proline metabolism in RPE and highlight the importance of proline transport and utilization in maintaining retinal metabolism and health.

Collagen matrix perturbations in corneal stroma of Ossabaw mini pigs with type 2 diabetes.

Purpose: Diabetes mellitus (DM) is a metabolic disorder that affects over 450 million people worldwide. DM is characterized by hyperglycemia, causing severe systemic damage to the heart, kidneys, skin, vasculature, nerves, and eye. Type 2 diabetes (T2DM) constitutes 90% of clinical cases and is the most common cause of blindness in working adults. Also, about 70% of T2DM patients show corneal complications including delayed wound healing, often described as diabetic keratopathy (DK). Despite the increasing severity of DM, the research on DK is bleak. This study investigated cellular morphology and collagen matrix alterations of the diabetic and non-diabetic corneas collected from Ossabaw mini pigs, a T2DM animal model with a "thrifty genotype." Methods: Pig corneas were collected from six-month-old Ossabaw miniature pigs fed on a western diet (WD) for ten weeks. The tissues were processed for immunohistochemistry and analyzed using hematoxylin and eosin staining, Mason Trichrome staining, Picrosirus Red staining, Collage I staining, and TUNEL assay. mRNA was prepared to quantify fibrotic gene expression using quantitative reverse-transcriptase PCR (qRT-PCR). Transmission electron microscopy (TEM) was performed to evaluate stromal fibril arrangements to compare collagen dynamics in WD vs. standard diet (SD) fed Ossabaw pig corneas. Results: Ossabaw mini pigs fed on a WD for 10 weeks exhibit classic symptoms of metabolic syndrome and hyperglycemia seen in T2DM patients. We observed significant disarray in cornea stromal collagen matrix in Ossabaw mini pigs fed on WD compared to the age-matched mini pigs fed on a standard chow diet using Masson Trichome and Picrosirius Red staining. Furthermore, ultrastructure evaluation using TEM showed alterations in stromal collagen fibril size and organization in diabetic corneas compared to healthy age-matched corneas. These changes were accompanied by significantly decreased levels of Collagen IV and increased expression of matrix metallopeptidase 9 in WD-fed pigs. Conclusions: This pilot study indicates that Ossabaw mini pigs fed on WD showed collagen disarray and altered gene expression involved in wound healing, suggesting that corneal stromal collagens are vulnerable to diabetic conditions.

NOD-like Receptors in the Eye: Uncovering Its Role in Diabetic Retinopathy.

Diabetic retinopathy (DR) is an ocular complication of diabetes mellitus (DM). International Diabetic Federations (IDF) estimates up to 629 million people with DM by the year 2045 worldwide. Nearly 50% of DM patients will show evidence of diabetic-related eye problems. Therapeutic interventions for DR are limited and mostly involve surgical intervention at the late-stages of the disease. The lack of early-stage diagnostic tools and therapies, especially in DR, demands a better understanding of the biological processes involved in the etiology of disease progression. The recent surge in literature associated with NOD-like receptors (NLRs) has gained massive attraction due to their involvement in mediating the innate immune response and perpetuating inflammatory pathways, a central phenomenon found in the pathogenesis of ocular diseases including DR. The NLR family of receptors are expressed in different eye tissues during pathological conditions suggesting their potential roles in dry eye, ocular infection, retinal ischemia, cataract, glaucoma, age-related macular degeneration (AMD), diabetic macular edema (DME) and DR. Our group is interested in studying the critical early components involved in the immune cell infiltration and inflammatory pathways involved in the progression of DR. Recently, we reported that NLRP3 inflammasome might play a pivotal role in the pathogenesis of DR. This comprehensive review summarizes the findings of NLRs expression in the ocular tissues with special emphasis on its presence in the retinal microglia and DR pathogenesis.

Correlation between systemic S100A8 and S100A9 levels and severity of diabetic retinopathy in patients with type 2 diabetes mellitus.

AIMS: S100A8 and S100A9 are myeloid-related damage-associated molecular patterns (DAMPs) primarily involved in the modulation of innate immune response to cellular injury. This study evaluated the correlation between circulating concentrations of S100A8 and S100A9 proteins with the severity of diabetic retinopathy (DR) in patients with type 2 diabetes (T2DM). METHODS: T2DM patients with HbA1c levels >7%, fasting blood glucose >126 mg/dl and history of diabetes were included in this study. DR severity was graded based on ETDRS and Gloucestershire classifications. Plasma samples were evaluated for S100A8 and S100A9 levels using ELISA. RESULTS: In this comparative study, DR patients (n = 89) had increased plasma S100A8 and S100A9 proteins compared to age-matched T2DM controls (n = 28), which was directly related to the severity of DR. Female DR subjects had increased S100A8 expression compared to their male counterparts. Substantial retention of S100A8 and S100A9 production was seen in DR patients above 50 years of age. Duration of T2DM was not found to affect protein levels, however T2DM onset at >50 years old significantly increased S100A8 and S100A9 concentrations. CONCLUSIONS: Our findings suggest that systemic circulation levels of S100A8 and S100A9 are correlated with the progression of DR in T2DM patients, indicating their potential role in DR pathogenesis.

Characterization of a functionally active primary microglial cell culture from the pig retina.

Retinal inflammation is an integral component of many retinal diseases including diabetic retinopathy (DR), age-related macular degeneration (AMD) and retinopathy of prematurity (ROP). Inflammation is commonly initiated and perpetuated by myeloid-derived immune cells. In the retina, microglial cells are resident macrophages with myeloid origins, which acts as the first responders involved in the innate immune system. To understand the disease pathogenesis, the use of isolated retinal cell culture model is vital for the examination of multiple cellular responses to injury or trauma. The pig retina resembles human retina in terms of tissue architecture, vasculature, and topography. Additionally, it is a better model than the rodent retina because of the presence of the pseudomacula. In the present study, we sought to establish and characterize pig retinal primary microglial cell (pMicroglia) culture. We used pig eyes from the local abattoir and optimized pMicroglia cultures using multiple cell culture conditions and methods. The best results were obtained by seeding cells in DMEM-high glucose media for 18 days followed by shaking of the culture plate. The resulting pMicroglia were characterized by cellular morphology, phenotype, and immunostaining with Iba-1, CD68, P2Y12, CD163, CD14, and Isolectin GS-IB4. Generated pMicroglia were found functionally active in phagocytosis assay and responsive to lipopolysaccharides (LPS) in dose-dependent production of IL-1ß. Furthermore, they showed increased secretion of pro-inflammatory cytokines with LPS treatment. Thus, we report a novel and reproducible method for the isolation of primary microglial cells from pig eyes, which may be useful for studying retinal diseases.

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.

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 (Ins2 Akita xVEGF+/-), 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 (Ins2 Akita ) 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-1ß 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-1ß 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.

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.

pH Induced Conformational Transitions in the Transforming Growth Factor ß-Induced Protein (TGFßIp) Associated Corneal Dystrophy Mutants.

Most stromal corneal dystrophies are associated with aggregation and deposition of the mutated transforming growth factor-ß induced protein (TGFßIp). The 4(th)_FAS1 domain of TGFßIp 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 4(th)_FAS1 domains of TGFßIp 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 αß conformation to ß-sheet oligomers. The ß-oligomers of both mutants were stable at physiological temperature and pH. Electron microscopy and dynamic light scattering studies showed that ß-oligomers of H572R were larger compared to R555W. The ß-oligomers of both mutants were cytotoxic to primary human corneal stromal fibroblast (pHCSF) cells. The ß-oligomers of both mutants exhibit variations in their morphologies, sizes, thermal and chemical stabilities, aggregation patterns and cytotoxicities.

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 TGFß/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 TGFß signaling pathway. In this study, we investigated safety, efficacy and mechanism of action of a HDACi, ITF2357 in TGFß-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 αVß3 expression with a reduction in corneal haze. In addition, ITF2357 reduced myofibroblast formation, suppressed phosphorylation of Smad proteins in TGFß 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 TGFß-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 TGFß/BMP7 signaling pathways.

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.