Effect of peripheral iridectomy on VEGF-A and TGF-ß levels in rabbit aqueous humour.

BACKGROUND: Peripheral iridectomy (PI), routinely performed during glaucoma filtration surgery, may contribute to scarring. This study aims to determine whether PI alters the concentrations of VEGF-A and TGF-ß isoforms in the rabbit aqueous humour. METHODS: Anterior chamber paracentesis (ACP) was performed in both eyes of six New Zealand white rabbits, with additional surgical PI performed in the right eyes. Eyes were examined on postoperative days (PODs) 1, 7, 30 and 60 by means of the tonopen, slit-lamp biomicroscopy, and bead-based cytokine assays for TGF-ß and VEGF-A concentrations in the aqueous humor. RESULTS: ACP caused a significant reduction in intraocular pressure (IOP) from mean preoperative 11.47 ± 1.01 mmHg to 5.67 ± 1.63 mmHg on POD 1 while PI did not cause further IOP reduction. Limbal conjunctival vasculature appeared slightly increased on POD 1 in both ACP and PI eyes with PI also causing mild bleeding from damaged iris vessels. Two PI eyes developed fibrinous anterior chamber reaction and/ or peripheral anterior synechiae. Aqueous VEGF-A levels were not significantly different between eyes treated with ACP and PI. Aqueous TGF-ß concentrations distributed in the ratio of 4:800:1 for TGF-ß1:TGF-ß2:TGF-ß3 respectively. While aqueous TGF-ß2 was not significantly induced by either procedure at any time point, TGF-ß1 and TGF-ß3 were significantly induced above baseline levels by PI on POD 1. CONCLUSION: PI increases the risk of inflammation. The combined induction of aqueous TGF-ß1 and TGF-ß3 by PI in glaucoma surgery may impact surgery success in glaucoma subtypes sensitive to these isoforms.

Distinct iris gene expression profiles of primary angle closure glaucoma and primary open angle glaucoma and their interaction with ocular biometric parameters.

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.

Participation of Tom1L1 in EGF-stimulated endocytosis of EGF receptor.

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.

The emerging role of VHS domain-containing Tom1, Tom1L1 and Tom1L2 in membrane trafficking.

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.

In vitro analyses of the anti-fibrotic effect of SPARC silencing in human Tenon's fibroblasts: comparisons with mitomycin C.

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-ß(2) were also reduced. Importantly, TGF-ß(2) 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.

Valproic acid modulates collagen architecture in the postoperative conjunctival scar.

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-ß2, Matn3 was sensitive to both VPA and TGF-ß2 individually and synergistically, while Matn4 was modulable by VPA but not TGF-ß2. 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.

Effects of Valproic Acid and Mitomycin C Combination Therapy in a Rabbit Model of Minimally Invasive Glaucoma Surgery.

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.

Effect of valproic acid on functional bleb morphology in a rabbit model of minimally invasive surgery.

PURPOSE: To determine the effect of valproic acid (VPA) on bleb morphology and scar characteristics in a rabbit model of minimally invasive glaucoma surgery (MIGS). METHODS: Nine 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. RESULTS: VPA-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. CONCLUSIONS: The results support the application of VPA to enhance bleb morphology associated with good bleb function in MIGS with no apparent cytotoxicity.

Validation of the glaucoma filtration surgical mouse model for antifibrotic drug evaluation.

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.

RelB regulates basal and proinflammatory induction of conjunctival CCL2.

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-ß2 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.

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

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.

Altered Iris Aquaporin Expression and Aqueous Humor Osmolality in Glaucoma.

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.

Missense mutations in the sodium borate cotransporter SLC4A11 cause late-onset Fuchs corneal dystrophy.

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.

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

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.

A role of VAMP8/endobrevin in regulated exocytosis of pancreatic acinar cells.

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.

Valproic acid enhances the engraftability of human umbilical cord blood hematopoietic stem cells expanded under serum-free conditions.

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.

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

Increased SPARC expression in primary angle closure glaucoma iris.

PURPOSE: SPARC (secreted protein, acidic, and rich in cysteine) is involved in extracellular matrix (ECM) organization. The purpose of this study was to evaluate the expression of SPARC in iris tissue from primary angle closure glaucoma (PACG) eyes. METHODS: Iris tissue was obtained from peripheral iridectomies performed during trabeculectomy surgery in nine PACG and 16 primary open-angle glaucoma (POAG) eyes at the Singapore National Eye Centre. Three non-glaucoma control iris specimens were obtained from patients who underwent Descemet's stripping automated endothelial keratoplasty (DSAEK) procedure. SPARC and collagen I expression were quantified by real-time polymerase chain reaction (PCR). The histological distribution of collagen I and III in the iris stroma was determined using picrosirius red polarization. Density of the iris stromal vasculature was also calculated. RESULTS: The mean age was 68.9+/-10.9 years and 65.7+/-12.2 years in POAG and PACG groups, respectively. The PACG iris expressed SPARC 13.6-fold more and collagen I 5.2 fold more compared to non-glaucoma control iris. The PACG iris also demonstrated 3.3 fold higher SPARC and 2.0 fold higher collagen I expression relative to the POAG iris. The density of collagen I was greater in PACG eyes than in POAG and control eyes (p<0.001). The mean density of iris stromal blood vessels per micron square area was similar in all three groups. CONCLUSIONS: SPARC was significantly increased in the PACG iris. The data suggest that SPARC could play a role in the development of PACG by influencing the biomechanical properties of the iris through a change in ECM organization.

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

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.

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

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.