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Purpose This study aimed to compare the effectiveness of combination therapy consisting of low-dose mitomycin C (MMC) and valproic acid (VPA) against high-dose MMC for improving the scar phenotype in minimally invasive glaucoma surgery (MIGS). Methods A rabbit model of MIGS incorporating the PreserFlo MicroShunt was treated with high (0.4 mg/mL) or low (0.1 mg/mL) doses of MMC or with combination therapy consisting of low-dose (0.1 mg/mL) MMC and VPA. Operated eyes were examined by live ocular imaging, histochemical evaluation, multiphoton quantitation of collagen characteristics, and molecular analyses. Results Although high-dose MMC obliterated the vasculature, combination therapy vastly improved the postoperative tissue morphology by maintaining the vasculature without increased vascularization. Combination therapy also altered collagen morphology and reduced encapsulation of the MicroShunt distal end, which remained at risk with MMC treatment alone. Multiphoton quantitation indicated that the combination therapy significantly reduced collagen density and fiber dimensions compared with monotherapy. At the molecular level, combination therapy significantly reduced Vegfa, Vegfc, and Vegfd expression and inhibited Col1a1 upregulation from baseline levels, all of which low-dose MMC alone was unable to achieve. Notably, COL1A1 protein levels appeared more consistently suppressed by combination therapy compared with high-dose MMC alone. Conclusions Compared with high-dose MMC, combination therapy was less toxic by sparing the vasculature and potentially more effective in reducing scarring via the regulation of collagen content and organization. Translational Relevance VPA may be combined with low-dose MMC to replace high-dose MMC to deliver safe and effective anti-scarring outcomes.

Valproic acid (VPA), widely used for the treatment of neurological disorders, has anti-fibrotic activity by reducing collagen production in the postoperative conjunctiva. In this study, we investigated the capacity of VPA to modulate the postoperative collagen architecture. Histochemical examination revealed that VPA treatment was associated with the formation of thinner collagen fibers in the postoperative days 7 and 14 scars. At the micrometer scale, measurements by quantitative multiphoton microscopy indicated that VPA reduced mean collagen fiber thickness by 1.25-fold. At the nanometer scale, collagen fibril thickness and diameter measured by transmission electron microscopy were decreased by 1.08- and 1.20-fold, respectively. Moreover, delicate filamentous structures in random aggregates or closely associated with collagen fibrils were frequently observed in VPA-treated tissue. At the molecular level, VPA reduced Col1a1 but induced Matn2, Matn3, and Matn4 in the postoperative day 7 conjunctival tissue. Elevation of matrilin protein expression induced by VPA was sustained till at least postoperative day 14. In primary conjunctival fibroblasts, Matn2 expression was resistant to both VPA and TGF-β2, Matn3 was sensitive to both VPA and TGF-β2 individually and synergistically, while Matn4 was modulable by VPA but not TGF-β2. MATN2, MATN3, and MATN4 localized in close association with COL1A1 in the postoperative conjunctiva. These data indicate that VPA has the capacity to reduce collagen fiber thickness and potentially collagen assembly, in association with matrilin upregulation. These properties suggest potential VPA application for the prevention of fibrotic progression in the postoperative conjunctiva. KEY MESSAGES: VPA reduces collagen fiber and fibril thickness in the postoperative scar. VPA disrupts collagen fiber assembly in conjunctival wound healing. VPA induces MATN2, MATN3, and MATN4 in the postoperative scar.

Purpose: This study investigated the involvement of NF-kB in regulating postoperative conjunctival inflammation.Methods: 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.Results: 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.Conclusions: 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.

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 CD45highF4/80low 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 CD45highF4/80low 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.

PurposeTo determine the effect of valproic acid (VPA) on bleb morphology and scar characteristics in a rabbit model of minimally invasive glaucoma surgery (MIGS).MethodsNine New Zealand white rabbits were subjected to MIGS with intraoperative implantation of the PreserFlo MicroShunt. Rabbits were then administered with subconjunctival injections of phosphate buffered saline (PBS) (n=4) or with VPA (n=5). Bleb morphology was examined by slit-lamp biomicroscopy and in vivo confocal microscopy. Postoperative day 28 tissues were examined by immunohistochemical evaluation and label-free multiphoton microscopy to visualise the collagen matrix, by terminal deoxynucleotidyl transferase dUTP nick-end labelling assay and immunofluorescent labelling for Ki67 expression to detect apoptosis and cell growth, and by real-time quantitative PCR to measure Col1a1, Fn, and Smad6 transcript expression.ResultsVPA-treated blebs were detectable on day 28, while the PBS-treated blebs were not detectable by day 14. VPA-treated blebs were diffuse, extended posteriorly with near normal conjunctival vascularity and featured a combination of reticular/blurred stromal pattern with evidence of relatively large stromal cysts. Instead of the deposition of thick, disorganised collagen fibres characteristic of the PBS bleb, the VPA bleb contained conspicuously thinner collagen fibres which were associated with similarly thinner fibronectin fibres. In corroboration, Col1a1 and Fn mRNA expression was reduced in the VPA blebs, while increased Smad6 expression implicated the disruption of the transforming growth factor beta pathway. Apoptosis and cell growth profiles appeared similar with both treatments.ConclusionsThe results support the application of VPA to enhance bleb morphology associated with good bleb function in MIGS with no apparent cytotoxicity.

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. Ministry of Education (MOE) Nanyang Technological University National Medical Research Council (NMRC) National Research Foundation (NRF) Published version This research was supported by an NTU internal grant (M4081851), Singapore Ministry of Education NTUitive Gap Fund (NGF-2019-07-004) and the Singapore National Research Foundation under its Translational and Clinical Research (TCR) Programme (NMRC/TCR/008-SERI/2013). Animal studies were partially funded by the SERI core grant (NMRC/CG/015/2013). We would also like to acknowledge Nyein Chan Lwin from SingHealth Experimental Medicine Centre (SEMC), Singapore for assisting the in vivo studies.

BackgroundTo 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.MethodsExperimental 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 ofSparc,Col1a1,Fn1andMmp14was 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 inhibitedSparctranscription in both day 7 (2.04-fold) and day 14 (1.39-fold) treated tissues.Sparcsuppression on day 7 was associated with a significant reduction ofCol1a1(2.52-fold),Fn1(2.89-fold) andMmp14(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.

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.

Background This study aimed to evaluate differences in iris gene expression profiles between primary angle closure glaucoma (PACG) and primary open angle glaucoma (POAG) and their interaction with biometric characteristics. Design Prospective study. Participants Thirty-five subjects with PACG and thirty-three subjects with POAG who required trabeculectomy were enrolled at the Singapore National Eye Centre, Singapore. Methods Iris specimens, obtained by iridectomy, were analysed by real-time polymerase chain reaction for expression of type I collagen, vascular endothelial growth factor (VEGF)-A, -B and -C, as well as VEGF receptors (VEGFRs) 1 and 2. Anterior segment optical coherence tomography (ASOCT) imaging for biometric parameters, including anterior chamber depth (ACD), anterior chamber volume (ACV) and lens vault (LV), was also performed pre-operatively. Main Outcome Measures Relative mRNA levels between PACG and POAG irises, biometric measurements, discriminant analyses using genes and biometric parameters. Results COL1A1, VEGFB, VEGFC and VEGFR2 mRNA expression was higher in PACG compared to POAG irises. LV, ACD and ACV were significantly different between the two subgroups. Discriminant analyses based on gene expression, biometric parameters or a combination of both gene expression and biometrics (LV and ACV), correctly classified 94.1%, 85.3% and 94.1% of the original PACG and POAG cases, respectively. The discriminant function combining genes and biometrics demonstrated the highest accuracy in cross-validated classification of the two glaucoma subtypes. Conclusions Distinct iris gene expression supports the pathophysiological differences that exist between PACG and POAG. Biometric parameters can combine with iris gene expression to more accurately define PACG from POAG.

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.

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. NRF (Natl Research Foundation, S’pore) NMRC (Natl Medical Research Council, S’pore) MOH (Min. of Health, S’pore)

Overproduction of type I collagen is associated with a wide range of fibrotic diseases as well as surgical failure such as in glaucoma filtration surgery (GFS). Its modulation is therefore of clinical importance. Valproic acid (VPA) is known to reduce collagen in a variety of tissues with unclear mechanism of action. In this report, we demonstrate that VPA inhibited collagen production in both conjunctival fibroblasts and the mouse model of GFS. In fibroblasts, VPA decreased type I collagen expression which intensified with longer drug exposure and suppressed steady-state type I collagen promoter activity. Moreover, VPA decreased Smad2, Smad3 and Smad4 but increased Smad6 expression with a similar intensity-exposure profile. Reduction of Smad3 using small hairpin RNA and/or overexpression of Smad6 resulted in decreased collagen expression which was exacerbated when VPA was simultaneously present. Furthermore, fibrogenic TGF-β2 failed to induce collagen when VPA was present, as opposed to the myofibroblast markers, beta-actin, alpha-smooth muscle actin and tenascin-C, which were elevated by TGF-β2. VPA suppressed p3TP-Lux luciferase activity and selectively rescued Smad6 expression from suppression by TGF-β2. Notably, SMAD6 overexpression reduced the effectiveness of TGF-β2 in inducing collagen expression. In corroboration, VPA inhibited type I collagen but increased Smad6 expression in the late phase of wound healing in the mouse model of GFS. Taken together, our data indicate that VPA has the capacity to effectively suppress both steady-state and fibrogenic activation of type I collagen expression by modulating Smad expression. Hence, VPA is potentially applicable as an anti-fibrotic therapeutic by targeting collagen. Key message • VPA modulates type I collagen expression via members of the Smad family. •VPA suppresses Smad2, Smad3 and Smad4 but upregulates Smad6. •Smad3 and Smad6 are involved in VPA regulation of steady-state collagen expression. •Smad6 is involved in VPA modulation of TGF-β-stimulated collagen expression. •VPA reduces collagen and upregulates Smad6 in the mouse model of glaucoma filtration surgery.

Purpose: Secreted protein acidic and rich in cysteine (SPARC) and Hevin are structurally related matricellular proteins involved in extracellular matrix assembly. In this study, we compared the anterior chamber biometric parameters and iris collagen properties in SPARC-, Hevin- and SPARC-/Hevin-null with wild-type (WT) mice. Methods: The right eyes of 53 WT, 35 SPARC-, 56 Hevin-, and 63 SPARC-/Hevin-null mice were imaged using the RTVue-100 Fourier-domain optical coherence tomography system. The parameters measured were anterior chamber depth (ACD), trabecular-iris space area (TISA), angle opening distance (AOD), and pupil diameter. Biometric data were analyzed using analysis of covariance and adjusted for age, sex, and pupil diameter. Expression of Col1a1, Col8a1, and Col8a2 transcripts in the irises was measured by quantitative polymerase chain reaction. Collagen fibril thickness was evaluated by transmission electron microscopy. Results: Mice that were SPARC- and SPARC-/Hevin-null had 1.28- and 1.25-fold deeper ACD, 1.45- and 1.53-fold larger TISA, as well as 1.42- and 1.51-fold wider AOD than WT, respectively. These measurements were not significantly different between SPARC- and SPARC-/Hevin-null mice. The SPARC-null iris expressed lower Col1a1, but higher Col8a1 and Col8a2 transcripts compared with WT. Collagen fibrils in the SPARC- and SPARC-/Hevin-null irises were 1.5- and 1.7-fold thinner than WT, respectively. The Hevin-null iris did not differ from WT in these collagen properties. Conclusions: SPARC-null mice have deeper anterior chamber as well as wider drainage angles compared with WT. Therefore, SPARC plays a key role in influencing the spatial organization of the anterior segment, potentially via modulation of collagen properties, while Hevin is not likely to be involved. NRF (Natl Research Foundation, S’pore) NMRC (Natl Medical Research Council, S’pore) Published version

Excessive accumulation of collagen is often used to assess the development of fibrosis. This study aims to identify collagen genes that define fibrosis in the conjunctiva following glaucoma filtration surgery (GFS). Using the mouse model of GFS, we have identified collagen transcripts that were upregulated in the fibrotic phase of wound healing via RNA-seq. The collagen transcripts that were increased the most were encoded by Col8a1, Col11a1 and Col8a2. Further analysis of the Col8a1, Col11a1 and Col8a2 transcripts revealed their increase by 67-, 54- and 18-fold, respectively, in the fibrotic phase, compared with 12-fold for Col1a1, the most commonly evaluated collagen gene for fibrosis. However, only type I collagen was significantly upregulated at the protein level in the fibrotic phase. Type VIII and type I collagens colocalized in fibrous structures and in ACTA2-positive pericytes, and appeared to compensate for each other in expression levels. Type XI collagen showed low colocalization with both type VIII and type I collagens but can be found in association with macrophages. Furthermore, we show that both mouse and human conjunctival fibroblasts expressed elevated levels of the most highly expressed collagen genes in response to TGFβ2 treatment. Importantly, conjunctival tissues from individuals whose GF surgeries have failed due to scarring showed 3.60- and 2.78-fold increases in type VIII and I collagen transcripts, respectively, compared with those from individuals with no prior surgeries. These data demonstrate that distinct collagen transcripts are expressed at high levels in the conjunctiva after surgery and their unique expression profiles may imply differential influences on the fibrotic outcome., Summary: As well as providing an objective quantitative measure, distinct collagen genes may further aid in the characterization and definition of the development of fibrosis.

Failure after glaucoma filtration surgery is attributed to fibrosis at the operated site. To understand the wound healing process after glaucoma filtration surgery, we have developed a mouse model for glaucoma filtration surgery which closely mimics the clinical response. In this study, we describe a systematic analysis of the wound healing response in vivo. Our data revealed that the post-surgical tissue response was separable into two distinguishable phases. The early "acute inflammatory" phase was characterized by significantly increased transcript expression of Vegfa, Cxcl1, Cxcl5, Ccl2, Ccl3, Ccl4, Gmcsf and specific Mmps as well as greater infiltration of monocytes/macrophages and T cells. The late "fibrotic" phase was characterized by an increased expression of Tgfb2 and extracellular matrix genes as well as a notable reduction of infiltrating inflammatory cells. Significantly, more mitotic cells were observed at both time points post-surgery. Subconjunctival fibroblasts may be involved in both phases since they have the capacity to reiterate the in vivo gene expression profiles upon either pro-inflammatory or pro-fibrotic cytokine stimulation. Given that the cellular and molecular targets that govern the early and late phases of wound healing are distinct and time sensitive, a multi-targeted therapeutic approach to sequentially inhibit inflammation and fibrogenesis at the critical time point may lead to improved surgical outcomes in glaucoma filtration surgery.

Summary Myopia is a huge public health problem worldwide, reaching the highest incidence in Asia. Identification of susceptible genes is crucial for understanding the biological basis of myopia. In this paper, we have identified and characterized a functional myopia-associated gene using a specific mouse-knockout model. Mice lacking the muscarinic cholinergic receptor gene (M2; also known as Chrm2) were less susceptible to lens-induced myopia compared with wild-type mice, which showed significantly increased axial length and vitreous chamber depth when undergoing experimental induction of myopia. The key findings of this present study are that the sclera of M2 mutant mice has higher expression of collagen type I and lower expression of collagen type V than do wild-type mice and mice that are mutant for other muscarinic subtypes, and, therefore, M2 mutant mice were resistant to the development of experimental myopia. Pharmacological blockade of M2 muscarinic receptor proteins retarded myopia progression in the mouse. These results suggest for the first time a role of M2 in growth-related changes in extracellular matrix genes during myopia development in a mammalian model. M2 receptor antagonists might thus provide a targeted therapeutic approach to the management of this refractive error.

The maintenance of cellular homeostasis and execution of regulatory mechanisms to dynamically govern various cellular processes require the correct delivery of proteins to their target subcellular compartments. It is estimated that over 30% of the proteins encoded by the human genome, projected to encode about 25 000 proteins and other macromolecules, are delivered to the secretory and endocytic pathways where movement of proteins between various compartments is primarily mediated by vesicles/carriers budding from one compartment for delivery to another. Sorting of cargo proteins into budding vesicles/carriers is mediated by adaptors that link the cargo proteins to the coat proteins. The adaptor function of VHS domain proteins, GGA proteins, STAM proteins and Hrs is well-established and is evolutionarily conserved from yeast to humans. Recent studies suggest that Tom1, Tom1L1 and Tom1L2 subfamily of VHS domain proteins, which do not exist in yeast, are emerging as novel regulators for post-Golgi trafficking and signaling.

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. NRF (Natl Research Foundation, S’pore) NMRC (Natl Medical Research Council, S’pore) Published version

Valproic acid (VPA) is a histone deacetylase inhibitor previously shown to promote the proliferation and self-renewal of CD34(+) hematopoietic cells. We tested the effect of VPA in conjunction with the selective amplification technology developed by Viacell Inc. Stem cells enriched from frozen cord blood were cultured for 7 d, subjected to reselection and grown in fresh medium for a further 7 d. Treatment with VPA resulted in an average two-fold higher expansion of CD45(+)34(+) cells compared with control. Furthermore, VPA-treatment induced higher numbers of CD45(+)34(+) cells to reside in the S phase than control cultured cells and resulted in a 2.5-fold upregulation in HOXB4 expression. Importantly, VPA-treated cells reconstituted hematopoiesis in non-obese diabetic/severe combined immunodeficient mice with a six-fold higher efficiency than control cells. Collectively, our results indicate that VPA, already used clinically for neurologic disorder treatment, is a useful additive for the ex vivo culture of hematopoietic stem/progenitor cells to enhance engraftment efficiency.

Phosphoinositides (PIs) are phosphorylated derivatives of phosphatidylinositol (PtdIns) that regulate many cellular and physiological processes. Most PIs act by serving as membrane docking sites for proteins harboring specific PI-binding domains so that the location and function of these proteins could be dynamically governed. The Phox (PX) domain represents a novel PI-binding module capable of regulating membrane targeting of about 47 mammalian proteins, 30 of which are tentatively referred to as sorting nexins (SNXs). Some SNXs have been implicated in regulating membrane trafficking in the endocytic pathway. We discuss here recent development and progress in the study of the PX domain-containing proteins.

TOM1 and its related proteins, TOM1-like1 (TOM1-L1) and TOM1-like2 (TOM1-L2), constitute a subfamily of the VHS domain protein family. We have recently shown that endofin, a FYVE domain protein associated with the early endosome, is able to recruit cytosolic TOM1 onto endosomal membranes. To reveal the biological consequence of endofin-mediated endosomal recruitment of TOM1, we have identified the clathrin heavy chain as a major interacting protein for TOM1. Optimal clathrin binding by TOM1 involves three sites: residues 300-321, 321-326 and a putative clathrin-binding box at residues 362-366 (362LEDEF366). Although residues 321-326 could function independently as a weak clathrin-binding motif, deletion of amino acids 300-321 or mutation of 362Leu and 364Asp to Ala residues reduced the binding of clathrin to TOM1. A fragment lacking amino acids 300-322 and containing 362Leu and 364Asp to Ala mutations lost the ability to interact with clathrin. Remarkably, overexpression of endofin led to a massive and specific recruitment of clathrin [but not dynamin, or the adaptor protein (AP) complexes, AP1, AP2 or AP3] onto endofin-positive endosomes. Although SARA is homologous to endofin, it did not interact with the C-terminal region of TOM1. Examination of chimeric proteins of endofin and SARA suggests that the C-terminal half of endofin is responsible for interaction with the C-terminal region of TOM1 and for recruitment of TOM1 and clathrin to endosomes. The correlation between the ability of endofin to interact with the C-terminal domain of TOM1 and clathrin recruitment suggests that endofin may recruit clathrin via TOM1. Indeed, a chimeric protein consisting of TOM1 fused to two FYVE domains derived from endofin has the ability to recruit clathrin onto endosomal structures. Moreover, we show that affinity-purified TOM1 antibody can abolish binding of clathrin to the C-terminal region of TOM1. Upon microinjection into cells, this antibody reduced the membrane association of clathrin. These results, taken together, suggest that TOM1 is an important molecule for membrane recruitment of clathrin, and that endofin is able to exploit this recruitment at the endosome.

Endofin is an endosomal protein implicated in regulating membrane trafficking. It is characterized by the presence of a phosphatidylinositol 3-phosphate-binding FYVE domain positioned in the middle of the molecule. To determine its potential effectors or binding partners, we used the carboxyl-terminal half of endofin as bait to screen a human brain cDNA library in a yeast two-hybrid system. Three clones that encode TOM1 were recovered. TOM1 is a protein closely related to the VHS (VPS-27, Hrs, and STAM) domain-containing GGA family. Although the function of the GGAs in mediating Golgi to lysosomal trafficking is well established, the subcellular localization and function of TOM1 remain unknown. Glutathione S-transferase pull-down assays as well as co-immunoprecipitation experiments confirmed that the carboxyl-terminal half of endofin binds specifically to the carboxyl-terminal region of TOM1. Neither SARA nor Hrs, two other FYVE domain proteins, interact with this region of TOM1. Moreover, endofin does not interact with the analogous region of two other members of the TOM1 protein family, namely, TOM1-like 1 (TOM1-L1) or TOM1-like 2 (TOM1-L2). The carboxyl-terminal region of TOM1 was used as immunogen to generate TOM1-specific antibody. This antibody can distinguish TOM1 from the other family members as well as coimmunoprecipitate endogenous endofin. It also revealed the primarily cytosolic distribution of TOM1 in a variety of cell types by immunofluorescence analyses. In addition, sucrose density gradient analysis showed that both TOM1 and endofin can be detected in cellular compartments marked by the early endosomal marker EEA1. A marked recruitment of TOM1 to endosomes was observed in cells overexpressing endofin or its carboxyl-terminal fragment, indicating TOM1 to be an effector for endofin and suggesting a possible role for TOM1 in endosomal trafficking.

Synaptojanin 2 is an inositol polyphosphate 5'-phosphatase that appears to be regulated by alternative splicing. By screening mouse cDNA libraries derived from either mouse day 16 embryo or adult liver, we have identified additional synaptojanin 2 cDNAs that represent six new isoforms of the protein. This finding, together with other reports, indicates the presence of eight isoforms of synaptojanin 2. Sequence analysis of our cDNA clones suggests that there are at least two putative initiation sites and at least six different sequences coding for the carboxyl-terminus of the molecule. In addition, we have mapped synaptojanin 2 to mouse chromosome 17 band A2-3.1 by fluorescence in situ hybridization.

To investigate the effects of SLC4A11 gene depletion in human corneal endothelial cells.To achieve stable downregulation of SLC4A11 gene expression in immortalized human corneal endothelial cells (HCECs), short-hairpin RNA (shRNA) targeted against SLC4A11 was used. Cell growth and viability were determined using the real-time cell analyzer and trypan blue staining respectively. Apoptosis was investigated by Annexin V and TUNEL assays. Alterations in apoptotic gene expression following SLC4A11 silencing were determined using the RT(2)Profiler PCR array for human apoptosis while activation of the apoptotic pathway was ascertained by western analysis.SLC4A11 silencing in HCECs could be achieved by stable expression of shRNA targeted against SLC4A11. SLC4A11 knockdown suppressed HCEC growth and reduced HCEC viability compared to the control. This reduction in cell growth is associated with increased apoptosis in SLC4A11-silenced cells.Our data suggest that the reduction of cell number with time in SLC4A11-depleted HCECs is due to an increase in cell death by apoptosis. This suggests that SLC4A11 is necessary for cell survival and may explain the pathologic corneal endothelial cell loss in endotheliopathies due to SLC4A11 mutations.

Failure of glaucoma filtration surgery (GFS) is commonly attributed to scarring at the surgical site. The human Tenon’s fibroblasts (HTFs) are considered the major cell type contributing to the fibrotic response. We previously showed that SPARC (secreted protein, acidic, rich in cysteine) knockout mice had improved surgical success in a murine model of GFS. To understand the mechanisms of SPARC deficiency in delaying subconjunctival fibrosis, we used the gene silencing approach to reduce SPARC expression in HTFs and examined parameters important for wound repair and fibrosis. Mitomycin C-treated HTFs were used for comparison. We demonstrate that SPARC-silenced HTFs showed normal proliferation and negligible cellular necrosis but were impaired in motility and collagen gel contraction. The expression of pro-fibrotic genes including collagen I, MMP-2, MMP-9, MMP-14, IL-8, MCP-1 and TGF-β2 were also reduced. Importantly, TGF-β2 failed to induce significant collagen I and fibronectin expressions in the SPARC-silenced HTFs. Together, these data demonstrate that SPARC knockdown in HTFs modulates fibroblast functions important for wound fibrosis and is therefore a promising strategy in the development of anti-scarring therapeutics.

Glaucoma Is a progressive optic neuropathy, which, If left untreated, leads to blindness. The most common and most modifiable risk factor in glaucoma is elevated intraocular pressure (IOP), which can be managed surgically by filtration surgery. The postoperative subconjunctival scarring response, however, remains the major obstacle to achieving long-term surgical success. Antiproliferatives such as mitomycin C are commonly used to prevent postoperative scarring. Efficacy of these agents has been tested extensively on monkey and rabbit models of glaucoma filtration surgery. As these models have inherent limitations, we have developed a model of glaucoma filtration surgery in the mouse. We show, for the first time, that the mouse model typically scarred within 14 d, but when augmented with mitomycin C, more animals maintained lower intraocular pressures for a longer period of time concomitant with prolonged bleb survival to beyond 28 d. The morphology of the blebs following mitomycin C treatment also resembled well-documented clinical observations, thus confirming the validity and clinical relevance of this model. We demonstrate that the antiscarring response to mitomycin C is likely to be due to its effects on conjunctival fibroblast proliferation, apoptosis and collagen deposition and the suppression of inflammation. Indeed, we verified some of these properties on mouse conjunctival fibroblasts cultured in vitro. These data support the suitability of this mouse model for studying the wound healing response in glaucoma filtration surgery, and as a potentially useful tool for the in vivo evaluation of antifibrotic therapeutics in the eye.

Homozygous mutations in the Borate Cotransporter SLC4A11 cause two early-onset corneal dystrophies: congenital hereditary endothelial dystrophy (CHED) and Harboyan syndrome. More recently, four sporadic patients with late-onset Fuchs corneal dystrophy (FCD), a common age-related disorder, were also reported to harbor heterozygous mutations at this locus. We therefore tested the hypothesis that SLC4A11 contributes to FCD and asked whether mutations in SLC4A11 are responsible for familial cases of late-onset FCD. We sequenced SLC4A11 in 192 sporadic and small nuclear late-onset FCD families and found seven heterozygous missense novel variations that were absent from ethnically matched controls. Familial data available for one of these mutations showed segregation under a dominant model in a three-generational family. In silico analyses suggested that most of these substitutions are intolerant, whereas biochemical studies of the mutant protein indicated that these alleles impact the localization and/or posttranslational modification of the protein. These results suggest that heterozygous mutations in SLC4A11 are modest contributors to the pathogenesis of adult FCD, suggesting a causality continuum between FCD and CHED. Taken together with a recent model between FCD and yet another early onset corneal dystrophy, PPCD, our data suggest a shared pathomechanism and genetic overlap across several corneal dystrophies.

Glaucoma is a disease frequently associated with elevated intraocular pressure that can be alleviated by filtration surgery. However, the post-operative subconjunctival scarring response which blocks filtration efficiency is a major hurdle to the achievement of long-term surgical success. Current application of anti-proliferatives to modulate the scarring response is not ideal as these often give rise to sight-threatening complications. SPARC (secreted protein, acidic and rich in cysteine) is a matricellular protein involved in extracellular matrix (ECM) production and organization. In this study, we investigated post-operative surgical wound survival in an experimental glaucoma filtration model in SPARC-null mice. Loss of SPARC resulted in a marked (87.5%) surgical wound survival rate compared to 0% in wild-type (WT) counterparts. The larger SPARC-null wounds implied that aqueous filtration through the subconjunctival space was more efficient in comparison to WT wounds. The pronounced increase in both surgical survival and filtration efficiency was associated with a less collagenous ECM, smaller collagen fibril diameter, and a loosely-organized subconjunctival matrix in the SPARC-null wounds. In contrast, WT wounds exhibited a densely packed collagenous ECM with no evidence of filtration capacity. Immunolocalization assays confirmed the accumulation of ECM proteins in the WT but not in the SPARC-null wounds. The observations in vivo were corroborated by complementary data performed on WT and SPARC-null conjunctival fibroblasts in vitro. These findings indicate that depletion of SPARC bestows an inherent change in post-operative ECM remodeling to favor wound maintenance. The evidence presented in this report is strongly supportive for the targeting of SPARC to increase the success of glaucoma filtration surgery.

Although many proteins have been shown to participate in ligand-stimulated endocytosis of EGF receptor (EGFR), the adaptor protein responsible for interaction of activated EGFR with endocytic machinery remains elusive. We show here that EGF stimulates transient tyrosine phosphorylation of Tom1L1 by the Src family kinases, resulting in transient interaction of Tom1L1 with the activated EGFR bridged by Grb2 and Shc. Cytosolic Tom1L1 is recruited onto the plasma membrane and subsequently redistributes into the early endosome. Mutant forms of Tom1L1 defective in Tyr-phosphorylation or interaction with Grb2 are incapable of interaction with EGFR. These mutants behave as dominant-negative mutants to inhibit endocytosis of EGFR. RNAi-mediated knockdown of Tom1L1 inhibits endocytosis of EGFR. The C-terminal tail of Tom1L1 contains a novel clathrin-interacting motif responsible for interaction with the C-terminal region of clathrin heavy chain, which is important for exogenous Tom1L1 to rescue endocytosis of EGFR in Tom1L1 knocked-down cells. These results suggest that EGF triggers a transient Grb2/Shc-mediated association of EGFR with Tyr-phosphorylated Tom1L1 to engage the endocytic machinery for endocytosis of the ligand–receptor complex.

Phosphoinositides are key regulators of diverse cellular processes. The pleckstrin homology (PH) domain mediates the action of PtdIns(3,4)P2, PtdIns(4,5)P2 and PtdIns(3,4,5)P3, while the FYVE domain relays the pulse of PtdIns3P. The recent establishment that the Phox homology (PX) domain interacts with PtdIns3P and other phosphoinositides suggests another mechanism by which phosphoinositides can regulate/integrate multiple cellular events via a spectrum of PX domain-containing proteins. Together with the recent discovery that the epsin N-terminal homologue (ENTH) domain interacts with PtdIns(4,5)P2, it is becoming clear that phosphoinositides regulate diverse cellular events through interactions with several distinct structural motifs present in many different proteins.

KIAA0305 is an uncharacterized member of the FYVE domain protein family. It is closely related to SARA, with about 50% identity in the carboxyl-terminal 800-amino acid region. Indirect immunofluorescence microscopy using polyclonal antibodies raised against KIAA0305 revealed that it is enriched in early endosomes. The Myc-tagged version is also faithfully targeted to the early endosome. We have tentatively called KIAA0305 endofin (forendosome-associated FYVE-domain protein). The association of endofin with endosomes is mediated by its FYVE domain because deletion mutants lacking the central FYVE finger motif are distributed in the cytoplasm. In addition, a single point mutation in the FYVE finger motif at cysteine residue 753 (C753S) is sufficient to abolish its endosomal association. Its endosomal localization is also sensitive to the phosphatidylinositol 3-kinase inhibitor, wortmannin. Usingin vitro liposome binding assays, we demonstrate that Myc-tagged endofin associates preferentially with phosphatidylinositol 3-phosphate, whereas the C753S point mutant was unable to do so. We also show that endofin co-localizes with SARA but that they are not associated in a common complex because they failed to co-immunoprecipitate in co-expressing cells. Endofin also does not associate with Smad2 nor behave like SARA in affecting transforming growth factor-β signaling. At high levels of expression, both endofin and SARA can cause an endosome aggregation/fusion effect. In COS7 cells, which can support high levels of exogenous protein expression, both proteins can also cause other structural anomalies in the endocytic pathway, as represented by enlarged vesicular structures. These endosomal aggregates/fusions accumulated endocytosed epidermal growth factor. Taken together, this report provides evidence to suggest that endofin and the highly related SARA are endosomal proteins with potential roles in regulating membrane traffic.

The sorting nexin (SNX) protein family is implicated in regulating membrane traffic, but the mechanism is still unknown. We show that SNX3 is associated with the early endosome through a novel motif (PX domain) capable of interaction with phosphatidylinositol-3-phosphate (PtdIns(3)P). Overexpression of SNX3 alters endosomal morphology and delays transport to the lysosome. Transport from the early to the recycling endosome is affected upon microinjection of SNX3 antibodies. Our results highlight a novel mechanism by which SNX proteins regulate traffic and uncover a novel class of effectors for PtdIns(3)P.

PURPOSE. To investigate the expression of SPARC and matrix metalloproteinases (MMPs) in normal conjunctiva and pterygium tissues. METHODS. This study involved paired control or uninvolved conjunctiva and pterygium tissue from 21 patients. Quantitative real-time PCR was performed to assess SPARC and MMP mRNA expression, whereas Western blot analysis was performed to assess SPARC protein levels in normal conjunctiva and pterygium tissue. Tissue localization of SPARC, extracellular matrix proteins, and MMPs were determined by immunofluorescence analyses. RESULTS. SPARC transcript and protein levels were upregulated in pterygium compared with normal conjunctiva. Immunofluorescence analyses showed localization of SPARC to the epithelial basement membrane and stroma of normal conjunctiva tissue. Increased SPARC in the pterygium stroma colocalized partially with elevated collagen I, fibronectin, -SMA, and MMP-3. SPARC and MMP-3 also colocalized in the pterygium epithelium. CONCLUSIONS. SPARC was upregulated in pterygium and may collaborate with increased MMP-3 in some patients to account for many of the phenotypic properties characteristic of pterygium. (Invest Ophthalmol Vis Sci. 2012;53:587‐595)

Purpose Pterygium is a prevalent ocular surface disease with unknown pathogenesis. The authors investigated the role of nuclear factor kappa B (NF-κB) transcription factors in pterygium. Methods Surgically excised primary pterygia were studied compared with uninvolved conjunctiva tissues. NF-κB activation was evaluated using Western blot analysis, ELISA, and DNA-binding assays. Primary pterygium fibroblasts were treated with TNF-α (20 ng/mL), and NF-κB activation was evaluated using immunocytochemistry, Western blot analysis, phospho-IκBα ELISA, and DNA-binding assays. TNF-α stimulation of NF-κB target genes RelB, NFKB2, RANTES, MCP-1, ENA-78, MMP-1, MMP-2, and MMP-3 in pterygium fibroblasts was compared with that in primary tenon fibroblasts by real-time PCR. Results Phosphorylation of IκBα (Ser32) was increased in pterygia tissues compared with uninvolved conjunctiva tissues, as determined by Western blot analysis and ELISA. IκBα expression was decreased, whereas nuclear RelA and p50 DNA-binding capacities were increased. Within 30 minutes of treatment with TNF-α, pterygium fibroblasts showed increased IκBα phosphorylation and nuclear translocation of RelA and p50. Treatment with TNF-α beyond 12 hours resulted in increased nuclear expression of RelB, p100, and p52. Furthermore, the upregulation of RANTES, MCP-1, ENA-78, MMP-1, MMP-2, and MMP-3 expression was more pronounced in TNF-α-treated pterygium fibroblasts than in tenon fibroblasts. Conclusions The NF-κB pathway is shown for the first time to be activated in pterygia tissues compared with normal conjunctiva tissues. Stimulation by the inflammatory cytokine TNF-α can activate both canonical and noncanonical NF-κB pathways in pterygium fibroblasts with concomitant upregulation of NF-κB target genes.

Despite our general understanding that members of the SNARE superfamily participate in diverse intracellular docking/fusion events, the physiological role of the majority of SNAREs in the intact organism remains elusive. In this study, through targeted gene knockout in mice, we establish that VAMP8/endobrevin is a major player in regulated exocytosis of the exocrine pancreas. VAMP8 is enriched on the membrane of zymogen granules and exists in a complex with syntaxin 4 and SNAP-23. VAMP8 −/− mice developed normally but showed severe defects in the pancreas. VAMP8 null acinar cells contained three times more zymogen granules than control acinar cells. Furthermore, secretagogue-stimulated secretion was abolished in pancreatic fragments derived from VAMP8 −/− mice. In addition, VAMP8 −/− mice were partially resistant to supramaximal caerulein-induced pancreatitis. These results suggest a major physiological role of VAMP8 in regulated exocytosis of pancreatic acinar cells by serving as a v-SNARE of zymogen granules.