Li-Fong Seet

SPARC Deficiency Results in Improved Surgical Survival in a Novel Mouse Model of Glaucoma Filtration Surgery

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.

Depletion of SLC4A11 Causes Cell Death by Apoptosis in an Immortalized Human Corneal Endothelial Cell Line

PURPOSE To investigate the effects of SLC4A11 gene depletion in human corneal endothelial cells. METHODS 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. RESULTS 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. CONCLUSIONS 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.

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‐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.

The Nuclear-Factor κB Pathway Is Activated in Pterygium

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.

Involvement of SPARC and MMP-3 in the Pathogenesis of Human Pterygium

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, α-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.

Valproic acid suppresses collagen by selective regulation of Smads in conjunctival fibrosis

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.

Altered Anterior Segment Biometric Parameters in Mice Deficient in SPARC

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.

Upregulation of distinct collagen transcripts in post-surgery scar tissue : a study of conjunctival fibrosis

ABSTRACT 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.

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

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.

Distinct iris gene expression profiles of PACG and POAG and their interaction with ocular biometric parameters

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.