Publications by authors named "Tina T Wong"

105 Publications

Altered Iris Aquaporin Expression and Aqueous Humor Osmolality in Glaucoma.

Invest Ophthalmol Vis Sci 2021 Feb;62(2):34

Singapore National Eye Centre, Singapore.

Purpose: Aquaporins (AQPs) facilitate transmembrane osmotic water transport and may play a role in iris fluid conductivity, which is implicated in the pathophysiology of glaucoma. In this study, we compared the iris expression of AQPs and aqueous osmolality between primary angle closure glaucoma (PACG), primary open-angle glaucoma (POAG), and nonglaucoma eyes.

Methods: AQP1-5 transcripts from a cohort of 36 PACG, 34 POAG and 26 nonglaucoma irises were measured by quantitative real-time PCR. Osmolality of aqueous humor from another cohort of 49 PACG, 50 POAG, and 50 nonglaucoma eyes were measured using an osmometer. The localization of AQP1 in both glaucoma and nonglaucoma irises was determined by immunofluorescent analysis.

Results: Of the five AQP genes evaluated, AQP1 and AQP2 transcripts were significantly upregulated in both PACG (3.48- and 8.07-fold, respectively) and POAG (3.12- and 11.58-fold, respectively) irises relative to nonglaucoma counterparts. The aqueous osmolalities of PACG (303.68 mmol/kg) and POAG (300.79 mmol/kg) eyes were significantly lower compared to nonglaucoma eyes (312.6 mmol/kg). There was no significant difference in expression of AQP transcripts or aqueous osmolality between PACG and POAG eyes.

Conclusions: PACG and POAG eyes featured significant increase in AQP1 and AQP2 expression in the iris and reduced aqueous osmolality compared to nonglaucoma eyes. These findings suggest that the iris may be involved in altered aqueous humor dynamics in glaucoma pathophysiology. Because PACG did not differ from POAG in both properties studied, it is likely that they are common to glaucoma disease in general.
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http://dx.doi.org/10.1167/iovs.62.2.34DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7910645PMC
February 2021

Liposomal drug delivery system for anti-inflammatory treatment after cataract surgery: a phase I/II clinical trial.

Drug Deliv Transl Res 2021 Feb 10. Epub 2021 Feb 10.

Singapore National Eye Centre (SNEC), 11 Third Hospital Avenue, Singapore City, 168751, Singapore.

Liposomes as a drug delivery system may overcome the problems associated with non-compliance to eyedrops and inadequate control of inflammation after cataract surgery. We evaluated the safety and efficacy of a single subconjunctival injection of liposomal prednisolone phosphate (LPP) for the treatment of post-cataract surgery inflammation. This is a phase I/II, open-label non-comparative interventional trial of patients undergoing cataract surgery. All patients received a single injection of subconjunctival LPP intraoperatively. The primary outcome measure was the proportion of eyes with an anterior chamber cell count of 0 at postoperative month 1. Ocular and non-ocular adverse events, including elevated intraocular pressure, rebound iritis and pseudophakic macular edema were monitored. Five patients were enrolled in this study. The mean age was 66.6 ± 6.2 and 4 (80%) were male. The proportion of patients with AC cell grading of 0 was 0%, 80%, 80%, and 100% at day 1, week 1, month 1, and month 2 after cataract surgery, respectively. Mean laser flare photometry readings were significantly elevated at week 1 after cataract surgery (48.8 ± 18.9, p = 0.03) compared with baseline, decreasing to 25.8 ± 9.2 (p = 0.04) at month 1 and returned to baseline by month 2 (10.9 ± 5.1, p = 1.0). No ocular or non-ocular adverse events were observed. Liposomal prednisolone phosphate, administered as a single subconjunctival injection intraoperatively, can be a safe and effective treatment for post-cataract surgery inflammation. The delivery of steroids with a liposomal drug delivery system could potentially replace eyedrops as anti-inflammatory therapy following cataract surgery.
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http://dx.doi.org/10.1007/s13346-021-00912-xDOI Listing
February 2021

Positive-charge tuned gelatin hydrogel-siSPARC injectable for siRNA anti-scarring therapy in post glaucoma filtration surgery.

Sci Rep 2021 Jan 14;11(1):1470. Epub 2021 Jan 14.

School of Chemical and Biomedical Engineering, Nanyang Technological University, 62 Nanyang Dr, Singapore, 637459, Singapore.

Small interfering RNA (siRNA) therapy is a promising epigenetic silencing strategy. However, its widespread adoption has been severely impeded by its ineffective delivery into the cellular environment. Here, a biocompatible injectable gelatin-based hydrogel with positive-charge tuned surface charge is presented as an effective platform for siRNA protection and delivery. We demonstrate a two-step synthesis of a gelatin-tyramine (Gtn-Tyr) hydrogel with simultaneous charge tunability and crosslinking ability. We discuss how different physiochemical properties of the hydrogel interact with siSPARC (siRNA for secreted protein, acidic and rich in cysteine), and study the positive-charge tuned gelatin hydrogel as an effective delivery platform for siSPARC in anti-fibrotic treatment. Through in vitro studies using mouse tenon fibroblasts, the positive-charge tuned Gtn-Tyr hydrogel shows sustained siSPARC cellular internalization and effective SPARC silencing with excellent biocompatibility. Similarly, the same hydrogel platform delivering siSPARC in an in vivo assessment employing a rabbit model shows an effective reduction in subconjunctival scarring in post glaucoma filtration surgery, and is non-cytotoxic compared to a commonly used anti-scarring agent, mitomycin-C. Overall, the current siRNA delivery strategy involving the positive-charge tuned gelatin hydrogel shows effective delivery of gene silencing siSPARC for anti-fibrotic treatment. The current charge tunable hydrogel delivery system is simple to fabricate and highly scalable. We believe this delivery platform has strong translational potential for effective siRNA delivery and epigenetic silencing therapy.
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http://dx.doi.org/10.1038/s41598-020-80542-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7809290PMC
January 2021

Assessment of progressive alterations in collagen organization in the postoperative conjunctiva by multiphoton microscopy.

Biomed Opt Express 2020 Nov 19;11(11):6495-6515. Epub 2020 Oct 19.

Ocular Therapeutics and Drug Delivery, Singapore Eye Research Institute, Singapore.

Glaucoma filtration surgery (GFS) commonly fails due to excessive fibrosis. As collagen structure aberrations is implicated in adverse fibrotic progression, this study aims to uncover collagen organization alterations during postoperative scarring. Via quantitative second harmonic generation/two photon excitation multiphoton imaging, we reveal the scar development and phenotype in the mouse model of conjunctival scarring. We also show that multiphoton imaging corroborated the collagen ultrastructure anomaly characteristic of the SPARC-/- mouse postoperative conjunctiva. These data improve our understanding of postoperative conjunctival scarring and further enhance the utility of this model for the development of anti-fibrotic therapeutics for GFS.
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http://dx.doi.org/10.1364/BOE.403555DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7687938PMC
November 2020

Nanotechnology for the Treatment of Allergic Conjunctival Diseases.

Pharmaceuticals (Basel) 2020 Oct 29;13(11). Epub 2020 Oct 29.

Tissue Engineering and Cell Therapy Group, Singapore Eye Research Institute, Singapore 169856, Singapore.

Allergic conjunctivitis is one of the most common external eye diseases and the prevalence has been increasing. The mainstay of treatment is topical eye drops. However, low bioavailability, low ocular drug penetration, transient resident time on the ocular surface due to tear turnover, frequent topical applications and dependence on patient compliance, are the main drawbacks associated with topical administration. Nanotechnology-based medicine has emerged to circumvent these limitations, by encapsulating the drugs and preventing them from degradation and therefore providing sustained and controlled release. Using a nanotechnology-based approach to load the drug is particularly useful for the delivery of hydrophobic drugs such as immunomodulatory agents, which are commonly used in allergic conjunctival diseases. In this review, different nanotechnology-based drug delivery systems, including nanoemulsions, liposomes, nanomicelles, nanosuspension, polymeric and lipid nanoparticles, and their potential ophthalmic applications, as well as advantages and disadvantages, are discussed. We also summarize the results of present studies on the loading of immunomodulators or nonsteroidal anti-inflammatory drugs to nano-scaled drug delivery systems. For future potential clinical use, research should focus on the optimization of drug delivery designs that provide adequate and effective doses with safe and satisfactory pharmacokinetic and pharmaco-toxic profiles.
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http://dx.doi.org/10.3390/ph13110351DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7694068PMC
October 2020

A review of the clinical applications of drug delivery systems for the treatment of ocular anterior segment inflammation.

Br J Ophthalmol 2020 Oct 30. Epub 2020 Oct 30.

Singapore National Eye Centre, Singapore

Ocular anterior segment inflammation is a medical problem that is seen in cases of cataract surgery and non-infectious anterior uveitis. Inadequately treated anterior segment inflammation can lead to sight-threatening conditions such as corneal oedema, glaucoma and cystoid macular oedema. The mainstay of treatment for anterior segment inflammation is topical steroid eye-drops. However, several drawbacks limit the critical value of this treatment, including low bioavailability, poor patient compliance, relatively difficult administration manner and risk of blurring of vision and ocular irritation. A drug delivery system (DDS) that can provide increased bioavailability and sustained delivery while being specifically targeted towards inflamed ocular tissue can potentially replace daily eye-drops as the gold standard for management of anterior segment inflammation. The various DDS for anti-inflammatory drugs for the treatment of anterior segment inflammation are listed and summarised in this review, with a focus on commercially available products and those in clinical trials. Dextenza, INVELTYS, Dexycu and Bromsite are examples of DDS that have enjoyed success in clinical trials leading to FDA approval. Nanoparticles and ocular iontophoresis form the next wave of DDS that have the potential to replace topical steroids eye-drops as the treatment of choice for anterior segment inflammation. With the current relentless pace of ophthalmic drug delivery research, the pursuit of a new standard of treatment that eliminates the problems of low bioavailability and patient compliance may soon be realised.
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http://dx.doi.org/10.1136/bjophthalmol-2020-315911DOI Listing
October 2020

Endogenous or Exogenous Retinal Pigment Epithelial Cells: A Comparison of Two Experimental Animal Models of Proliferative Vitreoretinopathy.

Transl Vis Sci Technol 2020 08 31;9(9):46. Epub 2020 Aug 31.

Singapore National Eye Centre, Singapore.

Purpose: Proliferative vitreoretinopathy (PVR) is a blinding condition that can occur following ocular penetrating injury and retinal detachment. To develop effective therapeutics for PVR, it is imperative to establish an animal model that is reproducible, closest in anatomy to the human eye, and most representative of the human disease. We compared two in vivo models of PVR in minipig eyes to assess reproducibility and consistency.

Methods: Six minipigs underwent PVR induction with procedure A and six underwent procedure B. In both procedures, PVR was induced with vitrectomy, bleb retinal detachment, retinotomy, and injection of platelet-rich plasma. In procedure A, retinal pigment epithelial (RPE) cells were harvested from cadaveric pig eyes and injected at the end of surgery. In procedure B, native RPE cells were released into the vitreous cavity by creating a RPE detachment and scraping the RPE layer. PVR severity was graded on fundoscopic examination with a modified Silicone Study Classification System for PVR. Severe PVR was defined as stages 2 to 5.

Results: Three eyes (50%) and five eyes (83.3%) developed re-detachment of the retina from severe PVR in procedures A and B, respectively ( = 0.55). Median PVR stage was higher in eyes that underwent procedure B compared to eyes that underwent procedure A, although the difference was not statistically significant (2.5 vs. 1.5, = 0.26).

Conclusions: This new model utilizing native RPE cells achieved a high consistency in inducing severe PVR in the minipig.

Translational Relevance: Our model closely follows pathogenic events in human PVR, making it ideal for preclinical testing of novel therapeutics for PVR.
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http://dx.doi.org/10.1167/tvst.9.9.46DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7463202PMC
August 2020

Hydroxyapatite Particles Induced Modulation of Collagen Expression and Secretion in Primary Human Dermal Fibroblasts.

Int J Nanomedicine 2020 13;15:4943-4956. Epub 2020 Jul 13.

School of Materials Science and Engineering, Nanyang Technological University, Singapore 639798, Singapore.

Background: Hydroxyapatite (HA) [Ca(PO4)(OH)] is a naturally occurring calcium phosphate which makes up 60-70% of the dry weight of human bones. Nano-scale HA particles are increasingly being used as carriers for controlled and targeted delivery of bioactive agents like drugs, proteins, and nucleic acids due to their high porosity, negative charge, and biodegradability.

Purpose: Although much effort has been devoted to understanding the delivery kinetics and effects of the payloads in such carriers, a thorough understanding of the influence of the carriers themselves is lacking.

Methods: HA particles (300 µg/mL) were administered to primary human dermal fibroblasts (HDFs). The uptake and intracellular localization of the particles were determined by flow cytometry, confocal imaging, and transmission electron microscopy (TEM). Immunological assays and PCR were performed to determine the levels of pro-inflammatory cytokines and collagens in cell lysates and media supernatant.

Results: The current study explores the effects of poly-dispersed HA particles on primary HDFs as a model system. The majority of the particles were determined to range between 150 and 200 nm in diameter. Upon exposure to HA suspensions, primary HDFs internalized the particles by endocytosis within 6 hours of exposure, showing maximum uptake at 72 hours following which the particles were exocytosed by 168 hours. This correlated to reduced secretion of various pro-inflammatory and pro-collagenic cytokines. Biochemical analysis further revealed a reduction in Type I collagen expression and secretion.

Conclusion: HA particles have an immune-modulatory effect on dermal fibroblasts and reduce collagen production, which may impact the integrity of the extracellular matrix (ECM). This study demonstrates the need to consider the secondary effects of particulate carriers like HA, beyond basic cytotoxicity, in the specific tissue environment where the intended function is to be realized.
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http://dx.doi.org/10.2147/IJN.S245500DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7367744PMC
September 2020

Plateau Iris and Severity of Primary Angle Closure Glaucoma.

Am J Ophthalmol 2020 12 29;220:1-8. Epub 2020 Jul 29.

Singapore Eye Research Institute, Singapore; Singapore National Eye Centre, Singapore; Duke-National University of Singapore Medical School, Singapore; Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore. Electronic address:

Purpose: To compare the distribution of plateau iris in eyes across varying severity of primary angle closure glaucoma (PACG) using standardized ultrasound biomicroscopy (UBM) criteria.

Design: Cross-sectional study.

Methods: UBM was performed on 210 patients with PACG who had previously undergone laser peripheral iridotomy. Plateau iris was defined as the presence of all the following UBM criteria in ≥2 quadrants of the angle: anteriorly directed ciliary body, absent ciliary sulcus, iris angulation, flat iris plane, and iridotrabecular contact. Disease severity was based on the visual field mean deviation (MD) and classified as early-to-moderate (MD ≥ -12 dB), advanced (-12.01 dB to -20 dB), and severe (MD < -20 dB).

Results: Of 210 subjects recruited, 23 were excluded because of poor quality UBM images. The remaining 187 patients were categorized as having early-to-moderate (n = 103), advanced (n = 38), and severe PACG (n = 46). Of these subjects, 48.1% were male, and 90.9% were of Chinese ethnicity. The overall proportion of plateau iris was 36.9%, with 32.0% (33/103) in early-to-moderate, 34.2% (13/38) in advanced, and 50% (23/46) in severe PACG (P = .03, comparing severe PACG with early-to-moderate groups). Among the severe PACG group, those with plateau iris configuration had significantly smaller anterior chamber area (P = .03) and volume (P = .01) compared with those without plateau iris.

Conclusion: The higher proportion of plateau iris configuration in eyes with severe PACG compared with early-to-moderate PACG suggest that this may be a contributory factor for disease severity.
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http://dx.doi.org/10.1016/j.ajo.2020.07.033DOI Listing
December 2020

Asian-specific vertical cup-to-disc ratio cut-off for glaucoma screening: An evidence-based recommendation from a multi-ethnic Asian population.

Clin Exp Ophthalmol 2020 Dec 20;48(9):1210-1218. Epub 2020 Aug 20.

Singapore Eye Research Institute, Singapore National Eye Centre, Singapore, Singapore.

Importance: Evidence-based guidelines are essential for glaucoma screening to work effectively.

Background: To derive a vertical cup-to-disc ratio (VCDR) cut-off for glaucoma screening in a multi-ethnic Asian population.

Design: The Singapore Epidemiology of Eye Diseases (SEED) study is a population-based study conducted from 2004 to 2011 in a single tertiary care research institute.

Participants: SEED comprised of 10 033 Chinese, Malay and Indian adults aged ≥40 (response rate 75.6%). After excluding participants with a history of glaucoma medication or surgery, 9673 participants were included for analysis.

Methods: A systematic eye examination, which included applanation tonometry, visual field testing, gonioscopy and dilated fundus examination was conducted.

Main Outcome Measure: Diagnosis of glaucoma.

Results: The distribution of VCDR and VCDR asymmetry were relatively homogenous in this multi-ethnic Asian population, with a 97.5th percentile value of 0.67 and 0.17, respectively. In the absence of more definite signs of glaucoma, VCDR ≥0.60 and VCDR asymmetry ≥0.20 provided the best balance between sensitivity (95.1%) and specificity (90.9%) in detecting glaucoma. For larger optic disc (≥2.0 mm), VCDR ≥0.65 with VCDR asymmetry ≥0.20 provided the best balance between sensitivity (84.8%) and specificity (93.2%).

Conclusion And Relevance: Overall, VCDR ≥0.60 with VCDR ≥0.20 asymmetry provides a good balance between sensitivity and specificity in detecting glaucoma. For larger optic disc, VCDR ≥0.65 should be considered instead to mitigate against false-referrals due to larger physiological disc cupping. Our findings may act as a reference to populations with similar VCDR distribution.
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http://dx.doi.org/10.1111/ceo.13836DOI Listing
December 2020

Experimental models of glaucoma filtration surgery.

Acta Ophthalmol 2021 Feb 26;99(1):9-15. Epub 2020 Jul 26.

Singapore National Eye Centre, Singapore, Singapore.

Glaucoma filtration surgery plays an important role in achieving intraocular pressure (IOP) reduction in patients who have high IOP despite maximum medical therapy. Preclinical experimental models of glaucoma filtration surgery contribute a great deal to our knowledge of the wound healing processes that predispose to scarring and may lead to poor outcomes. However, this research needs to be interpreted in the light of the specific study design, animal model and methods used. We review the existing literature addressing various models of experimental glaucoma filtration surgery, discuss the considerations in assessing these models and describe future steps in evaluating potential therapeutics and bleb characteristics that could impact translational research in this field.
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http://dx.doi.org/10.1111/aos.14485DOI Listing
February 2021

Evaluation of Primary Angle-Closure Glaucoma Susceptibility Loci for Estimating Angle Closure Disease Severity.

Ophthalmology 2021 Mar 16;128(3):403-409. Epub 2020 Jul 16.

Singapore Eye Research Institute, Singapore National Eye Centre, Singapore, Republic of Singapore; Ophthalmology and Visual Sciences Academic Clinical Program (Eye ACP), DukeNUS Medical School, Singapore, Republic of Singapore; Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.

Purpose: To investigate whether recently identified genetic loci for primary angle-closure glaucoma (PACG) are associated with disease severity.

Design: Case-control study.

Participants: Eight hundred four PACG patients and 943 control participants of Chinese ethnicity from Singapore.

Methods: The 8 PACG-associated single nucleotide polymorphisms (SNPs; rs11024102 at PLEKHA7, rs3753841 at COL11A1, rs1015213 located between PCMTD1 and ST18 on chromosome 8q, rs3816415 at EPDR1, rs1258267 at CHAT, rs736893 at GLIS3, rs7494379 at FERMT2, and rs3739821 mapping in between DPM2 and FAM102A) identified from genome-wide association studies were tested for association with disease severity using logistic regression adjusted for age and gender. A P value of 0.006 was set as significant after Bonferroni correction for testing of 8 loci. We also calculated the weighted genetic risk score (GRS) weighted by the estimated individual SNP effect size on PACG calculated as logarithm of the odds ratio (OR). Disease severity was based on the visual field mean deviation (MD) and classified as early to moderate (MD, >-12 dB) and severe (MD, <-20 dB).

Main Outcome Measures: Association of PACG loci with severe disease.

Results: Of the 804 PACG patients, genotyping data were available for 768 individuals and included 436 with mild-to-moderate PACG and 206 with severe PACG. The PACG patients were significantly older (mean age, 64.3 ± 9.1 years vs. 56.4 ± 8.9 years; P < 0.001) and there were proportionately more women compared with control participants (58.4% vs. 49.0%; P < 0.001). Of the 8 loci investigated, we observed significant evidence of association with severe PACG at 1 SNP, namely rs3816415 in EPDR1 (OR, 2.03; 95% confidence interval [CI], 1.49-2.78; P = 1 × 10). A higher-weighted GRS was associated significantly with severe PACG, with an OR of 3.11 (95% CI, 1.95-4.96) comparing the lowest quartile with the highest quartile.

Conclusions: Our results show that EPDR1 is associated significantly with severe PACG, suggesting that it may predispose patients to more aggressive disease development. Individuals with PACG with a higher GRS were associated with a higher risk of severe PACG.
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http://dx.doi.org/10.1016/j.ophtha.2020.07.027DOI Listing
March 2021

Organogenesis and distribution of the ocular lymphatic vessels in the anterior eye.

JCI Insight 2020 07 9;5(13). Epub 2020 Jul 9.

Department of Surgery and.

Glaucoma surgeries, such as trabeculectomy, are performed to lower intraocular pressure to reduce risk of vision loss. These surgeries create a new passage in the eye that reroutes the aqueous humor outflow to the subconjunctival space, where the fluid is presumably absorbed by the conjunctival lymphatics. Here, we characterized the development and function of the ocular lymphatics using transgenic lymphatic reporter mice and rats. We found that the limbal and conjunctival lymphatic networks are progressively formed from a primary lymphatic vessel that grows from the nasal-side medial canthus region at birth. This primary lymphatic vessel immediately branches out, invades the limbus and conjunctiva, and bidirectionally encircles the cornea. As a result, the distribution of the ocular lymphatics is significantly polarized toward the nasal side, and the limbal lymphatics are directly connected to the conjunctival lymphatics. New lymphatic sprouts are produced mainly from the nasal-side limbal lymphatics, posing the nasal side of the eye as more responsive to fluid drainage and inflammatory stimuli. Consistent with this polarized distribution of the ocular lymphatics, a higher drainage efficiency was observed in the nasal side than the temporal side of the eye when injected with a fluorescent tracer. In contrast, blood vessels are evenly distributed at the anterior surface of the eyes. Also, we found that these distinct vascular distribution patterns were conserved in human eyes. Together, our study demonstrated that the ocular surface lymphatics are more densely present in the nasal side and uncovered the potential clinical benefits in selecting the nasal side as a glaucoma surgery site to improve fluid drainage.
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http://dx.doi.org/10.1172/jci.insight.135121DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7406257PMC
July 2020

Functional, structural, and molecular identification of lymphatic outflow from subconjunctival blebs.

Exp Eye Res 2020 07 6;196:108049. Epub 2020 May 6.

Doheny Eye Institute and Department of Ophthalmology, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA. Electronic address:

The purpose of this study is to evaluate outflow pathways from subconjunctival blebs and to identify their identity. Post-mortem porcine (n = 20), human (n = 1), and bovine (n = 1) eyes were acquired, and tracers (fluorescein, indocyanine green, or fixable/fluorescent dextrans) were injected into the subconjunctival space to create raised blebs where outflow pathways were visualized qualitatively and quantitatively. Rodents with fluorescent reporter transgenes were imaged for structural comparison. Concurrent optical coherence tomography (OCT) was obtained to study the structural nature of these pathways. Using fixable/fluorescent dextrans, tracers were trapped to the bleb outflow pathway lumen walls for histological visualization and molecular identification using immunofluorescence against lymphatic and blood vessel markers. Bleb outflow pathways could be observed using all tracers in all species. Quantitative analysis showed that the nasal quadrant had more bleb-related outflow pathways compared to the temporal quadrant (nasal: 1.9±0.3 pathways vs. temporal: 0.7±0.2 pathways; p = 0.003). However, not all blebs resulted in an outflow pathway (0-pathways = 18.2%; 1-pathway = 36.4%; 2-pathways = 38.6%; and 3-pathways = 6.8%). Outflow signal was validated as true luminal pathways using optical coherence tomography and histology. Bicuspid valves were identified in the direction of flow in porcine eyes. Immunofluorescence of labeled pathways demonstrated a lymphatic (Prox-1 and podoplanin) but not a blood vessel (CD31) identity. Therefore, subconjunctival bleb outflow occurs in discrete luminal pathways. They are lymphatic as assessed by structural identification of valves and molecular identification of lymphatic markers. Better understanding of lymphatic outflow may lead to improved eye care for glaucoma surgery and ocular drug delivery.
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http://dx.doi.org/10.1016/j.exer.2020.108049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7328765PMC
July 2020

Characterisation of the inflammatory cytokine and growth factor profile in a rabbit model of proliferative vitreoretinopathy.

Sci Rep 2019 10 28;9(1):15419. Epub 2019 Oct 28.

Singapore National Eye Centre (SNEC), 11 Third Hospital Avenue, Singapore, 168751, Singapore.

To clarify the mechanisms and their temporal relationship in the development of proliferative vitreoretinopathy (PVR), we measured vitreous levels of pro-inflammatory cytokines and growth factors in a rabbit model of PVR. PVR was surgically induced in 11 rabbit eyes by vitrectomy, retinotomy, cryotherapy and injection of platelet-rich plasma at baseline. Severity of PVR was assessed on dilated fundal examination with indirect binocular ophthalmoscopy and graded based on the revised experimental PVR classification. Severe PVR was defined as stage 5 or worse. Vitreous concentrations of interleukin 6 (IL-6), interleukin 8 (IL-8), interleukin 1 beta (IL-1 β), tumor necrosis factor beta (TNF-β), granulocyte macrophage colony stimulating factor (GM-CSF), interferon gamma (IFN-γ), C reactive protein; (CRP), placental growth factor (PlGF), platelet derived growth factor BB (PDGF-BB), vascular endothelial growth factor (VEGF) and angiopoietin 2 (Ang-2) at weeks 2, 3 and 4 were compared to baseline and correlations between the cytokines with PVR severity were assessed. Four weeks after PVR induction, 5 eyes (45.5%) had developed severe PVR. IL-8 was raised at 2 weeks post PVR induction (1.46 ± 0.48 pg/ml vs 0.53 ± 0.25 pg/ml, p = 0.04) and remained significantly elevated at week 4 (2.6 ± 3.1 pg/ml, p = 0.03). CRP was significantly raised at week 4 (34.8 ± 12.0 pg/ml vs 13.0 ± 13.1 pg/ml, p < 0.001). Among the growth factors, PDGF-BB was the earliest to show significantly elevated levels, at 3 weeks (50.4 ± 19.0 pg/ml vs 6.2 ± 10.1 pg/ml) and remained elevated at week 4 (p = 0.002), while PlGF (11.2 ± 7.7 pg/ml vs 5.3 ± 3.8 pg/ml, p = 0.002) and Ang2 (13617.0 ± 8170.2 pg/ml vs 38593.8 ± 8313.4, p = 0.02) were significantly raised at week 4. IFN-γ (p = 0.03), PDGF-BB (p = 0.02) and VEGF (p = 0.02) were significantly associated with PVR severity. We demonstrated that inflammatory cytokines IL-6, -8, elevation post PVR induction is followed by elevated levels of fibroproliferative growth factors, Ang2, PlGF, VEGF and PDGF-BB in the development of PVR. These findings will guide future studies targeting appropriate therapeutic strategies for the treatment of PVR.
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http://dx.doi.org/10.1038/s41598-019-51633-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6817814PMC
October 2019

RelB regulates basal and proinflammatory induction of conjunctival CCL2.

Ocul Immunol Inflamm 2021 Jan 16;29(1):29-42. Epub 2019 Oct 16.

Ocular Therapeutics and Drug Delivery, Singapore Eye Research Institute, Singapore.

: This study investigated the involvement of NF-kB in regulating postoperative conjunctival inflammation.: Experimental surgery was performed as described for the mouse model of conjunctival scarring. Expression of NF-κB in postoperative conjunctival tissues or conjunctival fibroblasts were assessed by real-time PCR, immunoblotting and immunofluorescence analyses. Downregulation of RelB was achieved using small interfering RNA. Cellular cytokine secretion was determined using multiplex cytokine assay.: RelB was the most highly induced member of the NF-kB family on day 2 post-surgery. Elevated RelB may be found associated with vimentin-positive cells and fibroblasts in vivo and in vitro. In conjunctival fibroblasts, RelB may be induced by TNF-α but not TGF-β2 while its silencing caused selective induction of CCL2 secretion by both basal and TNF-α-stimulated fibroblasts.: High RelB induction in the inflammatory phase and the selective modulation of CCL2 suggest a specific anti-inflammatory role for RelB in the postoperative conjunctiva.
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http://dx.doi.org/10.1080/09273948.2019.1662060DOI Listing
January 2021

Integration of Genetic and Biometric Risk Factors for Detection of Primary Angle Closure Glaucoma.

Am J Ophthalmol 2019 12 1;208:160-165. Epub 2019 Aug 1.

Singapore Eye Research Institute, Singapore National Eye Centre, Singapore; Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore. Electronic address:

Purpose: The purpose of this study was to investigate whether the addition of primary angle closure glaucoma (PACG)-associated genetic loci allows improved detection of PACG, compared to anterior segment parameters measured by imaging.

Design: Case-control study.

Methods: Genotype data of the 8 PACG single-nucleotide polymorphisms (SNPs) (rs11024102 at PLEKHA7, rs3753841 at COL11A1, rs1015213 located between PCMTD1 and ST18 on Chromosome 8q, rs3816415 at EPDR1, rs1258267 at CHAT, rs736893 at GLIS3, rs7494379 at FERMT2, and rs3739821 mapping in between DPM2 and FAM102A) were available. Customized software was used to measure anterior segment optical coherence tomography (ASOCT) parameters, namely, anterior chamber depth, width, and area (ACD, ACW, and ACA) and lens vault (LV). Statistical analysis for positive predictive values was modeled using the receiver operating characteristic curve (AUC). Statistical significance comparing predictive power of the different parameters was calculated using permutation.

Results: A total of 388 PACG subjects and 751 controls with both ASOCT and genetic data were available for analysis. Anterior segment parameters including ACD, ACA, and LV had excellent predictive value (AUCs >0.94), except ACW (AUC=0.65), for identifying PACG. The inclusion of genetic risk alleles (either singly or as a composite genetic risk score for 8 genomewide association study SNPs) to ACD only provided a +0.50% improvement in reclassifying PACG cases and controls over and above the discriminatory value of ACD. This +0.50% improvement was not statistically significant (P > .05).

Conclusions: Although significant on their own, the incorporation of genetic data in the context of anterior segment imaging parameters like ACD provided only a marginal improvement of PACG detection.
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http://dx.doi.org/10.1016/j.ajo.2019.07.022DOI Listing
December 2019

Using Adherence-Contingent Rebates on Chronic Disease Treatment Costs to Promote Medication Adherence: Results from a Randomized Controlled Trial.

Appl Health Econ Health Policy 2019 12;17(6):841-855

Health Services & Systems Research Program, Duke-NUS Medical School, Singapore, Singapore.

Background: Poor adherence to medications is a global public health concern with substantial health and cost implications, especially for chronic conditions. In the USA, poor adherence is estimated to cause 125,000 deaths and cost $US100 billion annually. The most successful adherence-promoting strategies that have been identified so far have moderate effect, are relatively costly, and raise availability, feasibility, and/or scalability issues.

Objective: The main objective of SIGMA (Study on Incentives for Glaucoma Medication Adherence) was to measure the effectiveness on medication adherence of a novel incentive strategy based on behavioral economics that we refer to as adherence-contingent rebates. These rebates offered patients a near-term benefit while leveraging loss aversion and regret and increasing the salience of adherence.

Methods: SIGMA is a 6-month randomized, controlled, open-label, single-center superiority trial with two parallel arms. A total of 100 non-adherent glaucoma patients from the Singapore National Eye Centre were randomized into intervention (adherence-contingent rebates) and usual care (no rebates) arms in a 1:1 ratio. The primary outcome was the mean change from baseline in percentage of adherent days at Month 6. The trial registration number is NCT02271269 and a detailed study protocol has been published elsewhere.

Findings: We found that participants who were offered adherence-contingent rebates were adherent to all their medications on 73.1% of the days after 6 months, which is 12.2 percentage points (p = 0.027) higher than in those not receiving the rebates after controlling for baseline differences. This better behavioral outcome was achieved by rebates averaging 8.07 Singapore dollars ($US5.94 as of 2 November 2017) per month during the intervention period.

Conclusion: This study shows that simultaneously leveraging several insights from behavioral economics can significantly improve medication adherence rates. The relatively low cost of the rebates and significant health and cost implications of medication non-adherence suggest that this strategy has the potential to cost-effectively improve health outcomes for many conditions.
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http://dx.doi.org/10.1007/s40258-019-00497-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6885505PMC
December 2019

Young's Modulus Determination of Normal and Glaucomatous Human Iris.

Invest Ophthalmol Vis Sci 2019 06;60(7):2690-2695

Singapore Eye Research Institute and Singapore National Eye Center, Singapore.

Purpose: To evaluate the biomechanical properties (Young's modulus) of normal (control) and glaucomatous human iris using atomic force microscopy (AFM).

Methods: Iris tissue obtained from eighteen glaucomatous subjects (equal number of eyes with primary angle closure glaucoma (PACG) and primary open angle glaucoma (POAG) and five normal subjects who underwent elective eye surgery were subjected to the estimation of Young's modulus by AFM. Force measurements were done at room temperature using Nanowizard II BioAFM. The iris samples were immersed in the liquid media (PBS with 0.1% BSA) during force measurements. Young's modulus values were calculated for each recorded curve using JPK Data Processing Software, which uses a Hertz's contact model for spherical indenters fitted to the extend curves.

Results: The iris from the normal controls had the least Young's modulus (0.85 ± 0.31 kPa) while those from PACG patients had the highest Young's modulus (2.40 ± 0.82 kPa). The Young's modulus of PACG iris was significantly higher compared to that of the normal controls (P = 0.005) and POAG iris (P = 0.001). However, there was no significant difference in the Young's modulus of POAG iris (1.13 ± 0.36 kPa) compared to that of the normal controls (P = 0.511).

Conclusions: Variations in biomechanical properties of iris tissue may have a significant role in the pathogenesis of angle closure glaucoma. This study suggests the existence of fundamental biomechanical differences in eyes with angle closure versus open angle glaucoma. An understanding of this basis creates a new platform to understand disease pathology better and work on therapeutic strategies that will address the same.
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http://dx.doi.org/10.1167/iovs.18-26455DOI Listing
June 2019

Posterior segment drug delivery for the treatment of exudative age-related macular degeneration and diabetic macular oedema.

Br J Ophthalmol 2019 10 30;103(10):1356-1360. Epub 2019 Apr 30.

Duke-NUS Graduate Medical School, Singapore, Singapore

Inhibitors of vascular endothelial growth factors are used to treat a myriad of retinal conditions, including exudative age-related macular degeneration (AMD), diabetic macular oedema (DME) and diabetic retinopathy. Although effective, long-term efficacy is limited by the need for frequent and invasive intravitreal injections. The quest for sustained action therapeutics that can be delivered to target tissue in the least invasive manner is an arduous endeavour that has ended in premature failure for several technologies in Phase II or III trials. Nevertheless, there have been promising preclinical studies, and more are on the horizon: port delivery systems for the treatment of exudative AMD have entered Phase III trials and a wide array of preclinical studies have demonstrated the potential for nanoparticles, such as liposomes, dendrimers and cell penetrating peptides to deliver therapeutics into the posterior segment via minimally invasive routes. In this review, we discuss the challenges posed by ocular barriers for drug penetration and present the recent advancements of the most pertinent drug delivery platforms with a focus on the treatment of exudative AMD and DME.
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http://dx.doi.org/10.1136/bjophthalmol-2018-313462DOI Listing
October 2019

Valproic acid exerts specific cellular and molecular anti-inflammatory effects in post-operative conjunctiva.

J Mol Med (Berl) 2019 01 19;97(1):63-75. Epub 2018 Nov 19.

Ocular Therapeutics and Drug Delivery, Singapore Eye Research Institute, The Academia, 20 College Road, Singapore, 169856, Singapore.

Valproic acid (VPA) is a histone deacetylase inhibitor used clinically for neurological disorders. It is also potentially useful as anti-fibrotic therapy as it reduced collagen deposition in the post-operative conjunctiva. In this study, we further evaluated the effects of VPA on post-operative inflammation using the mouse model of conjunctival scarring. VPA, injected into the subconjunctiva immediately after surgery, did not cause any adverse tissue response when examined by live microscopy and produced an apparent reduction of proinflammatory and proangiogenic markers in immunohistological examinations. In-depth analyses of the treated operated tissues revealed that VPA selectively inhibited the CD45F4/80 macrophage subset as well as the production of specific proinflammatory cytokines/ chemokines, including CXCL1, IL-5, IL-6, and IL-10 which were reduced by ≥ 2.0-fold. VPA also specifically reduced tissue NF-кB2 p100 protein by mean 3.87-fold. On conjunctival fibroblasts, VPA treatment resulted in decreased secretion of specific cytokines, including CCL2, VEGF-A, and IL-15. In the presence of TNF-α, VPA inhibited the induction of specific cytokines/chemokines, notably CCL5 and VEGF-A, as well as NF-кB2 p100. In corroboration, VPA suppressed TNF-α stimulation of NF-кB reporter transcription by 1.51-fold. These data indicate that VPA can modulate both proinflammatory cellular and molecular targets in a selective manner and may therefore attenuate surgery-induced conjunctival inflammation. These and previous findings suggest that, by suppressing key mediators of both inflammation and fibrosis, VPA is a useful therapeutic for improving surgical outcome involving the conjunctiva. KEY MESSAGES: VPA inhibited recruitment of a CD45F4/80 macrophage subset. VPA reduced chemokine and cytokine levels in treated tissues. VPA selectively suppressed tissue NF-кB2 p100 levels. VPA suppressed TNF-α induction of chemokines, cytokines and NF-кB2 p100 expression. VPA suppressed TNF-α stimulation of NF-кB reporter.
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http://dx.doi.org/10.1007/s00109-018-1722-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6326969PMC
January 2019

Characteristics of the Corneal Endothelium Across the Primary Angle Closure Disease Spectrum.

Invest Ophthalmol Vis Sci 2018 09;59(11):4525-4530

Singapore Eye Research Institute, Singapore National Eye Centre, Singapore.

Purpose: To evaluate the corneal endothelial characteristics across the primary angle closure (PAC) disease spectrum amongst patients diagnosed as PAC suspects (PACS), PAC, PAC glaucoma (PACG), and previous acute PAC (APAC).

Methods: We analyzed a total of 529 subjects (51 PACS, 170 PAC, 234 PACG, and 74 with previous APAC). All subjects had undergone laser peripheral iridotomy prior to study recruitment. Corneal endothelial parameters were measured using a noncontact specular microscope and the following parameters were obtained: mean central endothelial cell density (ECD; cells/mm2), coefficient of variation (CV) in cell area, and percentage of hexagonal cells.

Results: The mean age of the subjects was 65.1 ± 8.2 years, and 55.2% were females. The mean central ECD was 2582.0 ± 472.8 cells/mm2 in PACS, 2566.0 ± 408.3 cells/mm2 in PAC, 2523.8 ± 406.8 cells/mm2 in PACG, and 2504.0 ± 558.1 cells/mm2 in APAC, with no significant differences in ECD across the subgroups (P = 0.61). The CV was lowest in PACS (34.38 ± 6.05 μm2/cell), and highest in APAC (37.61 ± 7.98 μm2/cell), but the differences were not significant (P = 0.07). Likewise, the percentage of hexagonality was not significantly different between the groups. A subgroup analysis on the eyes with previous APAC with their fellow eye also showed no significant differences in the corneal endothelial characteristics.

Conclusions: The corneal ECD and morphological characteristics such as CV and hexagonality are not significantly different across the PAC disease spectrum. This may reflect the lack of a sustained and/or dramatic IOP insult and/or an insignificant deleterious effect from medications, age, and chronicity on corneal endothelial parameters.
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http://dx.doi.org/10.1167/iovs.18-24939DOI Listing
September 2018

Design and in vitro release study of siRNA loaded Layer by Layer nanoparticles with sustained gene silencing effect.

Expert Opin Drug Deliv 2018 10 10;15(10):937-949. Epub 2018 Sep 10.

a School of Materials Science and Engineering , Nanyang Technological University , Singapore.

Objectives: Clinical translation of siRNA therapeutics has been severely limited due to the lack of stable and sustained siRNA delivery systems. Furthermore, when nanocarrier systems with siRNA are administered systemically to treat diseases, insufficient doses reach the target tissue. Here we report the successful development of a new nanocarrier system for the management of fibrosis.

Methods: The new carrier has a hydroxyapatite core, with alternating layers of siRNA and a cationic peptide. The siRNA used here targets secreted protein acidic and rich in cysteine (SPARC), a key matricellular protein involved in the regulation of collagen fibrillogenesis and assembly. We have also used FRET studies to elucidate the fate of the particles inside cells, including the mechanistic details of layer-by-layer detachment.

Results: In vitro studies using murine conjunctiva fibroblasts show sustained release over 2 weeks, and that such released siRNA sustained SPARC knockdown without affecting cell growth, and maintained siRNA presence in the cells for at least two weeks with a single-dose treatment. Release studies of siRNA from particles in vitro gave insight on how the particles delivered prolonged gene-silencing effects.

Conclusion: A single treatment of the layer-by-layer nanoparticle designed can achieve sustained gene silencing over 2 weeks. Localized delivery of stabilized siRNA with sustained-release capabilities opens the door for many other applications of siRNA-based gene regulation.
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http://dx.doi.org/10.1080/17425247.2018.1518426DOI Listing
October 2018

Targeted therapy for the post-operative conjunctiva: SPARC silencing reduces collagen deposition.

Br J Ophthalmol 2018 10 18;102(10):1460-1470. Epub 2018 Jul 18.

Ocular Therapeutics and Drug Delivery, Singapore Eye Research Institute, Singapore, Singapore

Background: To develop targeted antifibrotic therapy for glaucoma filtration surgery; this study determines the effectiveness of small interfering RNA (siRNA) to reduce in vivo secreted protein acidic and rich in cysteine (SPARC) expression using the mouse model of conjunctival scarring.

Methods: Experimental surgery was performed as described for the mouse model of conjunctival scarring. Scrambled (siScram) or Sparc (siSparc) siRNAs, loaded on layer-by-layer (LbL) nanoparticles, were injected into the conjunctiva immediately after surgery. Expression of , , and was measured by real-time PCR and immunoblotting on days 7 and 14 postsurgery. Live imaging of the operated eyes was performed using slit lamp, anterior segment-optical coherence tomography and confocal microscopy. Tissue pathology was evaluated by histochemical and immunofluorescent analyses of operated conjunctival cryosections. Tissue apoptosis was quantitated by annexin V assay. RESULTS : siSparc, delivered via expanded LbL nanoparticles, significantly inhibited transcription in both day 7 (2.04-fold) and day 14 (1.39-fold) treated tissues. suppression on day 7 was associated with a significant reduction of (2.52-fold), (2.89-fold) and (2.23-fold) mRNAs. At the protein level, both SPARC and collagen 1A1 (COL1A1) were significantly reduced at both time points with siSparc treatment. Nanoparticles were visualised within cell-like structures by confocal microscopy, while overt tissue response or apoptosis was not observed. CONCLUSIONS : SPARC targeted therapy effectively reduced both SPARC and collagen production in the operated mouse conjunctiva. This proof-of-concept study suggests that targeted treatment of fibrosis in glaucoma surgery is safe and feasible, with the potential to extend to a range of potential genes associated with fibrosis.
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http://dx.doi.org/10.1136/bjophthalmol-2018-311937DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6173823PMC
October 2018

Ocular surface status in glaucoma and ocular hypertension patients with existing corneal disorders switched from latanoprost 0.005% to tafluprost 0.0015%: comparison of two prostaglandin analogues with different concentrations of benzalkonium chloride.

Clin Exp Ophthalmol 2018 12 26;46(9):1028-1034. Epub 2018 Jun 26.

Singapore National Eye Centre (SNEC), Singapore, Singapore.

Importance: Glaucoma treatment has often been associated with adverse side-effects from preservatives that are included in the used eye drops.

Background: To evaluate changes in the ocular surface and the presence of prostaglandin-induced corneal disorders after being switched from latanoprost 0.005% to low preservative tafluprost 0.0015% ophthalmic solution.

Design: Single centre, prospective study.

Participants: Patients with primary open-angle glaucoma or ocular hypertension that had received treatment with once daily latanoprost 0.005% ophthalmic solution for control of intraocular pressure (IOP) for 3 months, with a score of above 1 on the National Eye Institute (NEI) ocular surface staining scale.

Methods: Following the ≥3 month latanoprost treatment period, patients were switched to once daily low preservative tafluprost 0.0015% ophthalmic solution. Patients were followed for a minimum of 3 months.

Main Outcome Measures: Ocular surface changes were assessed by fluorescein staining score (NEI scale). Additional evaluations included tear break-up time, hyperaemia score, subjective symptoms, changes in intraocular pressure and presence of adverse reactions.

Results: Out of 59 patients enrolled, 51 were included in the final analysis. Fluorescein staining scores at baseline, prior to treatment switch, were 6.9 ± 3.1 and 3.3 ± 2.7 at the end of the study period (change in scores was -3.6 ± 2.2 [P < 0.001]). At last follow-up, significant improvements were observed in tear break-up time, hyperaemia score and subjective symptoms (all P < 0.05).

Conclusions And Relevance: The clinical signs of ocular surface disease and subjective symptoms of dry eyes improved following the switch to low preservative tafluprost and demonstrated comparable IOP lowering effectiveness.
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http://dx.doi.org/10.1111/ceo.13329DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6585858PMC
December 2018

Evaluation of subconjunctival liposomal steroids for the treatment of experimental uveitis.

Sci Rep 2018 04 26;8(1):6604. Epub 2018 Apr 26.

Singapore National Eye Centre (SNEC), 11 Third Hospital Avenue, Singapore, 168751, Singapore.

Non-infectious anterior uveitis (AU) is a potentially sight threatening inflammatory condition. The current gold standard for treatment is topical steroids, but low ocular bioavailability and compliance issues with the intensive dosing regimen limit the efficacy of this treatment. Liposomes as a drug delivery system may help to overcome these problems. We studied the efficacy of a PEG-liposomal formulation of liposomal steroids, administered as a single subconjunctival dose, in the treatment of experimental uveitis in rabbit eyes. Rabbits that received subconjunctival liposomal triamcinolone acetonide phosphate (LTAP) or liposomal prednisolone phosphate (LPP) had significantly lower mean inflammatory scores than untreated controls on Day 4 after induction of uveitis (LPP vs controls, p = 0.049) and 8 (LPP vs controls, p = 0.007; LTAP vs controls, p = 0.019), and lower scores than rabbits given topical PredForte1% 4 times a day on Day 8 (p = 0.03). After antigen rechallenge, the subconjunctival liposomal steroid groups continued to have greater suppression of inflammation than untreated controls on Day 11 (p = 0.02). Localization of liposomes in inflamed ocular tissue was confirmed by histology and immunostaining, and persisted in the eye for at least one month. Our study demonstrates that a single subconjunctival injection of liposomal steroids induces effective and sustained anti-inflammatory action.
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http://dx.doi.org/10.1038/s41598-018-24545-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5919899PMC
April 2018

Bevacizumab Promotes T-Cell-Mediated Collagen Deposition in the Mouse Model of Conjunctival Scarring.

Invest Ophthalmol Vis Sci 2018 03;59(3):1682-1692

Singapore Eye Research Institute, Singapore.

Purpose: We determine the effects of bevacizumab on collagen production in a mouse model of conjunctival scarring.

Methods: Experimental surgery was performed as described for the mouse model of conjunctival scarring, and bevacizumab was introduced by conjunctival injection. The capacity of bevacizumab to recognize conjunctival VEGF-A was determined by ELISA. Col1a1 was measured by real-time PCR and immunoblotting. T cells and collagen were visualized by immunofluorescence and picrosirius red staining of bleb cryosections. Conjunctival CD4+ or CD8a+ T cells were counted by flow cytometry. Mouse splenic T cells were cultured with bevacizumab/IgG and their numbers, cell cycle, and collagen production were measured using a cell counter, flow cytometry, and sircol soluble collagen assay, respectively. Reconstitution experiments in severe combined immunodeficiency (SCID) mice were performed by injection of freshly isolated T cells on day 2 postoperatively.

Results: Bevacizumab recognized approximately 20% of endogenous murine VEGF-A. Injection of bevacizumab raised Col1a1 expression in the blebs at mRNA and protein levels. Bevacizumab did not induce collagen in conjunctival fibroblasts, but increased CD4+ and CD8a+ cell numbers as well as collagen production by these cells. Collagen appeared to accumulate in the vicinity of T cells in the bevacizumab-treated blebs. While SCID blebs did not show elevated collagen levels, reconstitution with CD4+ or CD8a+ cells resulted in increased Col1a1 expression at mRNA and protein levels.

Conclusions: Bevacizumab increased collagen production in the mouse model of conjunctival scarring. This collagen induction was mediated by T cells that were also stimulated by bevacizumab to increase in numbers.
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http://dx.doi.org/10.1167/iovs.17-22694DOI Listing
March 2018

A Biodegradable, Sustained-Released, Tacrolimus Microfilm Drug Delivery System for the Management of Allergic Conjunctivitis in a Mouse Model.

Invest Ophthalmol Vis Sci 2018 02;59(2):675-684

Singapore Eye Research Institute, Singapore.

Purpose: To investigate the drug release profiles of a tacrolimus-loaded poly(D,L-lactide-co-ε-caprolactone) (PLC) microfilm, and to evaluate its efficacy on the treatment of allergic conjunctivitis using a mouse model.

Methods: The in vitro and in vivo drug release profiles were first characterized. Balb/c mice were immunized with short ragweed (SRW) injection followed by re-challenges with topical SRW solution. The mice were divided into six groups (n = 12 in each): negative control (NC); positive control (PC); tacrolimus eye drops (Te); subconjunctival tacrolimus microfilm (Tm); dexamethasone eye drops (De); and tacrolimus + dexamethasone eye drops (Te+De). The mice were evaluated for 28 days by a scoring system for allergic conjunctivitis. Histopathologic and immunohistochemical staining with CD11c, CD4, and IL-4 were performed.

Results: The microfilms were biocompatible and delivered clinically sufficient dose in a sustained manner, with a steady rate of 0.212 to 0.243 μg/day in vivo. Compared to the PC groups, the Te, Tm, De, and Te+De groups significantly reduced the allergic clinical scores throughout the study period (all P < 0.01; 0.0 ± 0.0, 5.6 ± 0.9, 3.3 ± 0.9, 3.2 ± 0.9, 1.9 ± 0.4 and 1.7 ± 0.8 for the NC, PC, Tm, Te, De, and Te+De groups, respectively, at 4 weeks after treatment). The suppressed eosinophils, CD11c, CD4, and IL-4 expression were also observed in all treatment groups, with more reduction in the Te+De group.

Conclusions: Tacrolimus-loaded microfilms display good biocompatibility and desirable sustained drug release. It was as effective as conventional tacrolimus eye drops on the treatment of allergic conjunctivitis, providing a promising clinically applicable alternative for controlling allergic disease activity, or other immune-mediated ocular diseases.
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http://dx.doi.org/10.1167/iovs.17-23066DOI Listing
February 2018

Evaluation of Primary Angle-Closure Glaucoma Susceptibility Loci in Patients with Early Stages of Angle-Closure Disease.

Ophthalmology 2018 05 6;125(5):664-670. Epub 2018 Jan 6.

Singapore Eye Research Institute, Singapore National Eye Centre, Singapore, Republic of Singapore; Duke-National University of Singapore Medical School, Singapore, Republic of Singapore. Electronic address:

Purpose: To investigate whether newly identified genetic loci for primary angle-closure glaucoma (PACG) are associated with early stage angle-closure disease defined as primary angle closure suspect (PACS).

Design: Case-control study.

Participants: A total of 1397 PACS patients and 943 controls of Chinese ethnicity from Singapore and 604 PACS patients and 287 controls of Indian ethnicity.

Methods: The 8 PACG single nucleotide polymorphisms (SNPs; rs11024102 at PLEKHA7, rs3753841 at COL11A1, rs1015213 located between PCMTD1 and ST18 son chromosome 8q, rs3816415 at EPDR1, rs1258267 at CHAT, rs736893 at GLIS3, rs7494379 at FERMT2, and rs3739821 mapping in between DPM2 and FAM102A) were genotyped by Taqman assays. The association between SNP genotypes and PACS status was measured using logistic regression. A P value of 0.006 was set to account for the testing of 8 genetic loci using a Bonferroni correction. A meta-analysis was conducted to calculate the overall P value and accompanying per-allele odds ratios for each SNP analyzed.

Main Outcome Measures: Association of PACG loci with PACS status.

Results: The PACS patients were significantly older in both cohorts (Chinese, P < 0.001; Indian, P = 0.002), and there were also more women (P < 0.001, both Chinese and Indian cohorts). In the Chinese cohort, significant evidence of association was noted at 3 SNPs: rs1015213 [A] in PCMTD1-ST18 (odds ratio [OR], 2.36; 95% confidence interval [CI], 1.36-4.11; P = 0.002), rs3816415 [A] in EPDR1 (OR, 1.49; 95% CI, 1.19-1.85; P < 0.001), and rs3739821 [G] in DPM2-FAM102A (OR, 1.40; 95% CI, 1.18-1.65; P < 0.001). Only PCMTD1-ST-18 was replicated modestly in the Indian population (P = 0.056). Meta-analysis showed significant evidence of association for PCMTD1-ST-18 (OR, 1.55; 95% CI, 1.18-2.04; P = 0.002) and DPM2-FAM102A (OR, 1.27; 95% CI, 1.12-1.45; P = 0.0002).

Conclusions: In this study, 2 of 8 PACG-associated loci were associated significantly with PACS status, the earliest stage in the angle-closure glaucoma disease course. The association of these PACG loci with PACS status suggests that these loci may confer susceptibility to a narrow angle configuration.
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http://dx.doi.org/10.1016/j.ophtha.2017.11.016DOI Listing
May 2018

Characterization of liposomal carriers for the trans-scleral transport of Ranibizumab.

Sci Rep 2017 12 1;7(1):16803. Epub 2017 Dec 1.

School of Materials Science and Engineering, Nanyang Technological University, Singapore, Singapore.

Age-related macular degeneration (AMD) is a leading cause of blindness in the modern world. The standard treatment regimen for neovascular AMD is the monthly/bimonthly intravitreal injection of anti-VEGF agents such as ranibizumab or aflibercept. However, these repeated invasive injections can lead to sight-threatening complications. Sustained delivery by encapsulation of the drug in carriers is a way to reduce the frequency of these injections. Liposomes are biocompatible, non-toxic vesicular nanocarriers, which can be used to encapsulate therapeutic agents to provide sustained release. The protein encapsulation was performed by a modified dehydration-rehydration (DRV) method. The liposomes formed were characterized for size, zeta potential, encapsulation efficiency, stability, in vitro release, and ex vivo release profiles. In addition, the localization of the liposomes themselves was studied ex vivo. Entrapment-efficiency of ranibizumab into 100-nm liposomes varied from 14.7 to 57.0%. Negatively-charged liposomes prepared from DPPC-DPPG were found to have the slowest release with a low initial burst release compared to the rest of liposomal formulations. The ex vivo protein release was found to slower than the in vitro protein release for all samples. In conclusion, the DPPC-DPPG liposomes significantly improved the encapsulation and release profile of ranibizumab.
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http://dx.doi.org/10.1038/s41598-017-16791-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5711922PMC
December 2017