Decursin inhibits retinal neovascularization via suppression of VEGFR-2 activation

Kim, Jeong Hun,Kim, Jin Hyoung,Lee, You Mie,Ahn, Eun-Mi,Kim, Kyu-Won,Yu, Young Suk Molecular Vision 2009 Molecular vision Vol.15 No.-

<P><B>Purpose</B></P><P>Pathologic angiogenesis in the retina leads to the catastrophic loss of vision. Retinopathy of prematurity (ROP), a vasoproliferative retinopathy, is a leading cause of blindness in children. We evaluated the inhibitory effect of decursin on retinal neovascularization.</P><P><B>Methods</B></P><P>Anti-angiogenic activity of decursin was evaluated by vascular endothelial growth factor (VEGF)-induced proliferation, migration, and in vitro tube formation assay of human retinal microvascular endothelial cells (HRMECs). We also used western blot analysis to assess inhibition of vascular endothelial growth factor receptor-2 (VEGFR-2) phosphorylation by decursin. After intravitreal injection of decursin in a mouse model of ROP, retinal neovascularization was examined by fluorescence angiography and vessel counting in cross-sections. The toxicity of decursin was evaluated through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in HRMECs as well as histologic and immunohistochemistry examination for glial fibrillary acidic protein in the retina.</P><P><B>Results</B></P><P>Decursin significantly inhibited VEGF-induced proliferation, migration, and the formation of capillary-like networks of retinal endothelial cells in a dose-dependent manner. Decursin inhibited VEGF-induced phosphorylation of VEGFR-2, blocking the VEGFR-2 signaling pathway. When intravitreously injected, decursin dramatically suppressed retinal neovascularization in a mouse model of ROP. Even in a high concentration, decursin never induced any structural or inflammatory changes to cells in retinal or vitreous layers. Moreover, the upregulation of glial fibrillary acidic protein expression was not detected in Mueller cells.</P><P><B>Conclusions</B></P><P>Our data suggest that decursin may be a potent anti-angiogenic agent targeting the VEGFR-2 signaling pathway, which significantly inhibits retinal neovascularization without retinal toxicity and may be applicable in various other vasoproliferative retinopathies as well.</P>

Cyclooxygenase-2-expressing macrophages in human pterygium co-express vascular endothelial growth factor

Park, Choul Yong,Choi, Jong Sun,Lee, Sung Jun,Hwang, Sang Won,Kim, Eo-Jin,Chuck, Roy S. Molecular Vision 2011 Molecular vision Vol.17 No.-

<P><B>Purpose</B></P><P>To evaluate cyclooxygenase-2 (COX-2) expression and to characterize COX-2-expressing stromal cells in human pterygium.</P><P><B>Methods</B></P><P>Primary pterygium tissue of Korean patients (eight males and nine females) was analyzed. The clinical characteristics were classified, and immunohistochemical staining using primary antibodies against cyclooxygenease-2, vascular endothelial growth factor-A, cluster of differentiation (CD)68, CD3, CD20, and leukocyte common antigen was performed.</P><P><B>Results</B></P><P>COX-2 expression was detected in all pterygium tissues evaluated (17 primary pterygia). Diffuse expression of COX-2 in the epithelial layer was observed in nine samples. Infiltration of strongly positive COX-2 cells into the epithelial layer was a more common observation than diffuse epithelial COX-2 expression. Scattered COX-2-expressing cells in the stromal layer were found in all samples. Some COX-2-positive cells were found within microvessels. In addition to stromal COX-2-expressing cells, a few vascular endothelial cells strongly expressed COX-2; however most of the vessels were negative for COX-2 expression. Stromal COX-2-expressing cells were positive for the macrophage marker CD68 and co-expressed vascular endothelial growth factor. COX-2 expression in normal conjunctiva was not observed in seven control samples.</P><P><B>Conclusions</B></P><P>These COX-2- and vascular endothelial growth factor-expressing macrophages may have relevance to the pathogenesis of pterygium.</P>

Effects of amniotic membrane suspension in human corneal wound healing in vitro

Choi, Jin A,Jin, Hyun-Jin,Jung, Samhyun,Yang, Eunkyung,Choi, Jun-Sub,Chung, So-Hyang,Joo, Choun-Ki Molecular Vision 2009 Molecular vision Vol.15 No.-

<P><B>Purpose</B></P><P>To investigate the biochemical mechanism of amniotic membrane (AM) suspension on corneal wound healing, particularly on epithelial proliferation and migration.</P><P><B>Methods</B></P><P>Human corneal epithelial cells (HCECs) were cultured in media with different concentrations of AM suspension (5% and 30%), Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (negative control), and serum containing Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (positive control). In an effort to evaluate the migratory potential of AM, migration assays were conducted via the manual scraping of HCECs and immunocytochemical staining of cell adhesion molecules (E-cadherin). The relative expression of matrix metallopeptidase 9 (MMP9) and adhesion molecules (E-cadherin, fibronectin) was determined via reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis. The proliferative potential of AM was evaluated via a proliferation assay using 5-Bromo-2-deoxyuridine (BrdU) incorporation and western blot analysis for proliferating cell nuclear antigen (PCNA). In addition, enzyme-linked immunosorbent assay<B> (</B>ELISA) was used to measure the protein concentrations of mitogenic growth factors (e<I>pidermal growth factor [</I>EGF],<I/>k<I>eratinocyte growth factor [</I>KGF], h<I>epatocyte growth factor [</I>HGF], and b<I>asic fibroblast growth factor [</I>bFGF]) in AM suspensions.</P><P><B>Results</B></P><P>Migration assay rates were enhanced as AM concentrations increased, with statistically significant changes seen in 30% AM-treated and positive control cells, compared to negative control cells (p<0.05). RT-PCRs revealed that the expression of the MMP9 gene was upregulated by AM, and the expressions of E-cadherin and fibronectin genes were downregulated by AM. Western blot analysis demonstrated significantly higher MMP9 expression in AM-treated groups, versus significantly lower levels of E-cadherin and fibronectin expression in AM-treated groups. Immunocytochemistry showed large quantities of E-cadherin near the wound edges after 24 h of injury in the AM-treated groups. The proliferation assay showed that the BrdU positive cell counts/total cell counts (labeling index) were augmented by AM to a statistically significant degree (p<0.05 in the 30% AM and positive control groups). Western blot analysis showed that the expression cell cycle-associated protein, PCNA, increased gradually as a result of AM treatment. ELISA showed that our AM suspension contained 4 growth factors (HGF, EGF, KGF, and FGF). The amount of HGF was especially large, followed by that of EGF.</P><P><B>Conclusions</B></P><P>These results demonstrate that the suspension form of AM maintains its beneficial effect on corneal epithelial wound healing in vitro, and that AM suspension leads to significant increases in corneal epithelial migration and proliferation with increasing AM concentrations.</P>

Antiangiogenic effect of dasatinib in murine models of oxygen-induced retinopathy and laser-induced choroidal neovascularization

Seo, Songyi,Suh, Wonhee Molecular Vision 2017 Molecular vision Vol.23 No.-

<P><B>Purpose</B></P><P>Vascular endothelial growth factor (VEGF) is a principal mediator of pathological ocular neovascularization, which is the leading cause of blindness in various ocular diseases. As Src, a non-receptor tyrosine kinase, has been implicated as one of the major signaling molecules in VEGF-mediated neovascularization, the present study aimed to investigate whether dasatinib, a potent Src kinase inhibitor, could suppress pathological ocular neovascularization in murine models of oxygen-induced retinopathy (OIR) and choroidal neovascularization (CNV).</P><P><B>Methods</B></P><P>Tube formation, scratch wounding migration, and cell proliferation assays were performed to measure the inhibitory effect of dasatinib on VEGF-induced angiogenesis in human retinal microvascular endothelial cells. Murine models of OIR and laser-induced CNV were used to assess the preventive effect of an intravitreal injection of dasatinib on pathological neovascularization in the retina and choroid. Neovascularization and Src phosphorylation were evaluated with immunofluorescence staining.</P><P><B>Results</B></P><P>Dasatinib efficiently inhibited VEGF-induced endothelial proliferation, wounding migration, and tube formation. In mice with OIR and laser injury-induced CNV, eyes treated with a single intravitreal injection of dasatinib exhibited significant decreases in pathological neovascularization compared with that of controls injected with vehicle. The dasatinib-treated OIR mice also showed a decrease in Src phosphorylation in the periretinal tufts. The intravitreal injection of dasatinib did not cause ocular toxicity at the treatment dose administered.</P><P><B>Conclusions</B></P><P>These results demonstrated that dasatinib suppressed pathological neovascularization in the mouse retina and choroid. Therefore, dasatinib may be indicated for the treatment of ischemia-induced proliferative retinopathy and neovascular age-related macular degeneration.</P>

Association of −31T>C and −511 C>T polymorphisms in the interleukin 1 beta ( <i>IL1B</i> ) promoter in Korean keratoconus patients

Kim, So-Hee,Mok, Jee-Won,Kim, Hyun-Seok,Joo, C.K. Molecular Vision 2008 Molecular vision Vol.14 No.-

<P><B>Purpose</B></P><P>To investigate the genetic association between unrelated Korean keratoconus patients and interleukin 1 alpha (<I>IL1A</I>), interleukin 1 beta (<I>IL1B</I>), and IL1 receptor antagonist (<I>IL1RN)</I> gene polymorphisms.</P><P><B>Methods</B></P><P>We investigated the association between <I>IL1A</I> (rs1800587, rs2071376, and rs17561), <I>IL1B</I> (rs1143627, rs16944, rs1143634, and rs1143633), and <I>IL1RN</I> (rs419598, rs423904, rs424078, and rs315952, variable number tandem repeat [VNTR]) polymorphisms in 100 unrelated Korean keratoconus patients. One hundred control individuals without any corneal disease were selected from the general population. Polymerase chain reaction (PCR) – restriction fragment length polymorphism (RFLP) analysis and direct sequencing were used to screen for genetic variations in the <I>IL1</I> gene cluster. Haplotypes for the <I>IL1</I> gene cluster were constructed using Haploview version 4.0.</P><P><B>Results</B></P><P>We analyzed a total of 12 polymorphic sites in the <I>IL1</I> gene cluster. Among them, the −511 (rs16944) and −31 (rs1143627) positions in the promoter region of <I>IL1B</I> were significantly different between patient and control groups. The C allele of rs16944 (−511C>T, p=0.022, odds ratio of risk [OR]=1.46, 95% confidence intervals [CI] 0.94<2.27) and the T allele of rs1143627 (−31T>C, p=0.025, OR=1.43, 95% CI 0.92<2.22) were associated with a significantly increased risk of keratoconus in Korean patients. Linkage of the two alleles, −31*C and −511*T, was associated with an increased risk for keratoconus with OR=2.38 (p=0.012, 95% CI=1.116–5.046). The *C/*A genotype of rs2071376 in <I>IL1A</I> intron 6 was significantly different between the keratoconus patients and control subjects (p=0.034, OR=0.59, 95% CI 0.32<1.11). Other polymorphisms did not show an association with keratoconus risk.</P><P><B>Conclusions</B></P><P>This is the first report of <I>IL1</I> gene cluster mutation screening in Korean keratoconus patients. Significant differences in allelic frequency of <I>IL1B</I> between keratoconus patients and the control group suggest that <I>IL1B</I> polymorphisms may play a role in the susceptibility of unrelated Koreans to develop keratoconus.</P>

Mitomycin C induces apoptosis in cultured corneal fibroblasts derived from type II granular corneal dystrophy corneas

Kim, Tae-im,Choi, Seung-il,Lee, Hyung Keun,Cho, Young Jae,Kim, Eung Kweon Molecular Vision 2008 Molecular vision Vol.14 No.-

<P><B>Purpose</B></P><P>The present study investigated the effect of mitomycin C (MMC) on cell viability, apoptosis, and transforming growth factor beta-induced protein (TGFBIp) expression in cultured normal corneal fibroblasts and heterozygote or homozygote granular corneal dystrophy type II (GCD II) corneal fibroblasts.</P><P><B>Methods</B></P><P>Keratocytes were obtained from normal cornea or from heterozygote or homozygote GCD II patients after lamellar or penetrating keratoplasty. To measure cell viability, corneal fibroblasts were incubated with 0.02% MMC for 3 h, 6 h, and 24 h or with 0%, 0.01%, 0.02%, and 0.04% MMC for 24 h and then tested using lactate dehydrogenase (LDH) and 3-[4,5-demethylthiazol-2,5-diphenyl-2H-tetrazolium bromide] (MTT) assays. To measure apoptosis, cells were analyzed by FACS analysis and annexin V staining. <I>Bcl-xL</I>, <I>Bax</I>, and <I>TGFBI</I> mRNA expression was measured using reverse transcription polymerase chain reaction (RT–PCR) assays. Cellular and media levels of TGFBIp protein were measured by immunoblotting.</P><P><B>Results</B></P><P>MTT and LDH assays showed that MMC reduced cell viability in all three cell types in a dose-dependent and time-dependent manner (p<0.05). FACS analysis and annexin V staining showed that MMC caused apoptosis with GCD II homozygote cells being most affected. RT–PCR analysis showed that MMC decreased <I>Bcl-xL</I> mRNA expression and increased <I>Bax</I> mRNA expression in all cell types. RT–PCR and immunoblotting analysis showed that MMC reduced <I>TGFBI</I> mRNA levels and cellular and media TGFBIp protein levels in all cell types.</P><P><B>Conclusions</B></P><P>MMC induced apoptosis, and the effects of MMC were greatest in GCD II homozygote cells. MMC also reduced the production of TGFBIp in all three types of corneal fibroblasts. These findings may explain the additional therapeutic effect of MMC in GCD II patients.</P>

Molecular genetic characteristics of X-linked retinoschisis in Koreans

Kim, So Yeon,Ko, Hyun Soo,Yu, Young Suk,Hwang, Jeong-Min,Lee, Jong Joo,Kim, Sung Yeun,Kim, Ji Yeon,Seong, Moon-Woo,Park, Kyu Hyung,Park, Sung Sup Molecular Vision 2009 Molecular vision Vol.15 No.-

<P><B>Purpose</B></P><P>X-linked retinoschisis (XLRS) is a recessively inherited disorder that causes macular degeneration and resultant visual defect in young males. Many genetic studies had focused on the patients in Western countries. We characterized the mutational spectrum of the <I>RS1</I> gene in Korean patients with XLRS, and aimed to provide genetic information of XLRS in an Asian population.</P><P><B>Methods</B></P><P>This study enrolled 17 unrelated probands and their mothers for molecular genetic evaluation. All exons and the flanking intronic regions of <I>RS1</I> were analyzed by direct sequencing. We performed gene dosage analysis by semiquantitative multiplex PCR to rule out the possibility of duplication in a patient without a sequence variation. We also tried RT–PCR analysis in a case with a putative splicing mutation.</P><P><B>Results</B></P><P>Genetic tests revealed 16 Korean patients (94.1%) had <I>RS1</I> mutations. In one patient, neither sequence variation nor deletion or duplication in <I>RS1</I> was detected. One case with de novo mutation was confirmed by familial analysis. Identified were 14 causative mutations, three of which were novel: one missense mutation (c.227T>G, p.V76G) and two splice-site mutations (c.78+1G>T and c.78+5G>A). No obvious genotype-phenotype relationship was observed.</P><P><B>Conclusions</B></P><P>A missense mutation was the predominant type, and common or founder mutations were not observed in the Korean patients in this study who had XLRS. This study provides molecular genetic characteristics about an Asian population previously unexplored. The genetic characteristics of Korean XLRS will be helpful for understanding the worldwide spectrum of <I>RS1</I> mutation.</P>

The role of macrophage migration inhibitory factor in ocular surface disease pathogenesis after chemical burn in the murine eye

Oh, Sei Yeul,Choi, Jong-Sun,Kim, Eo-Jin,Chuck, Roy S.,Park, Choul Yong Molecular Vision 2010 Molecular vision Vol.16 No.-

<P><B>Purpose</B></P><P>To evaluate the role of macrophage migration inhibitory factor (MIF) in the wound healing process following severe chemical burns to the ocular surface.</P><P><B>Methods</B></P><P>Chemical burning of the ocular surface was induced in mice (C57BL/6) via the application of 0.1 M NaOH. Macrophage migration inhibitory factor (<I>MIF</I>), tumor necrosis factor-α (<I>TNF-α</I>), and interleukin-1β (<I>IL-1β</I>) mRNA expression in the ocular surface and lacrimal gland was evaluated via real-time reverse transcription PCR on days 2, 7, and 30 after induction of the chemical burn. The expression of MIF protein in the ocular surface and lacrimal gland was evaluated via western blot analysis. Immunohistochemical staining was conducted to detect MIF and vasculoendothelial growth factor in the cornea during the wound healing process. The angiogenic role of MIF was further evaluated using an 8–0 polyglactin suture technique to induce corneal neovascularization.</P><P><B>Results</B></P><P><I>MIF</I>, <I>TNF-α</I>, and <I>IL-1β</I> mRNA expression were elevated significantly in the ocular surface up to day 30 after chemical burn induction. <I>TNF-α</I> alone was elevated in the lacrimal gland. MIF protein elevation was confirmed via western blot analysis, and the spatial similarity of MIF and VEGF expression in the cornea was noted during the wound healing process. 8–0 polyglactin sutures soaked in MIF induced significantly higher numbers of new vessels on the mouse cornea after 7 days (p=0.003, Mann–Whitney test).</P><P><B>Conclusions</B></P><P>These findings indicate that MIF performs a crucial role in wound healing on the ocular surface after the induction of chemical burns.</P>

Dual localization of wild-type myocilin in the endoplasmic reticulum and extracellular compartment likely occurs due to its incomplete secretion

Sohn, Seongsoo,Joe, Myung Kuk,Kim, Tae Eun,Im, Ji-eun,Choi, Young Ran,Park, Hwayong,Kee, Changwon Molecular Vision 2009 Molecular vision Vol.15 No.-

<P><B>Purpose</B></P><P>Wild-type myocilin is known to be secreted extracellularly, but a significant amount of the protein is also present in the endoplasmic reticulum (ER). The present study was undertaken to address whether intracellular myocilin is a true ER resident protein.</P><P><B>Methods</B></P><P>Human wild-type myocilin was adenovirally expressed in human trabecular meshwork cells, and general characteristics of both intracellular and extracellular myocilins including molecular weight, pI, glycosylation state, and cleavage site of the signal peptide were examined by biochemical analyses. Topology and decay kinetics of myocilin were also examined by protease protection assay and pulse chase analysis, respectively. The expression pattern and cytopathic effect of myocilin were analyzed in individual cells by immunocytochemistry.</P><P><B>Results</B></P><P>Intracellular myocilin were very similar to secreted myocilin in characteristics such as molecular weight, pI, glycosylation state, and cleavage site of the signal peptide. The intracellular protein was found to be present in the lumen of the ER where it appeared to be retained without further export to the Golgi apparatus. The kinetics of myocilin turnover clearly showed that it was intrinsically a very stable but incompletely secreted protein. The expression of myocilin was confined to a subset of cells and accompanied by the upregulation of a 78 kDa glucose-regulated protein, suggesting that it was not properly folded or processed in the ER.</P><P><B>Conclusions</B></P><P>Based on these findings and the fact that myocilin has no known ER retention signals, the ER localization of wild-type myocilin is likely a consequence of its incomplete secretion due to its misfolding.</P>

Pirfenidone inhibits transforming growth factor-β1-induced fibrogenesis by blocking nuclear translocation of Smads in human retinal pigment epithelial cell line ARPE-19

Choi, Kyungsun,Lee, Kihwang,Ryu, Seung-Wook,Im, Minju,Kook, Koung Hoon,Choi, Chulhee Molecular Vision 2012 Molecular vision Vol.18 No.-

<P><B>Purpose</B></P><P>Transforming growth factor-β (TGF-β) plays a key role in transforming retinal pigment epithelial (RPE) cells into mesenchymal fibroblastic cells, which are implicated in proliferative vitreoretinopathy. Herein, we tested the effect of pirfenidone, a novel antifibrotic agent, on TGF-β1-mediated fibrogenesis in the human RPE cell line ARPE-19.</P><P><B>Methods</B></P><P>The effect of pirfenidone on the TGF-β1-induced phenotype in ARPE-19 cells was measured with immunocytochemistry as the change in F-actin. Fibronectin and collagen production was measured with enzyme-linked immunosorbent assay, and cell migration activity was investigated using a scratch assay. Immunoblot analyses of cofilin, sma and mad protein (smad) 2/3, p38 mitogen-activated protein kinase, c-Jun N-terminal kinase, and extracellular signal-related kinase expression were conducted to elucidate the cell signaling networks that contribute to the antifibrotic effect of pirfenidone.</P><P><B>Results</B></P><P>Treatment with TGF-β1 induced typical phenotypic changes such as formation of stress fiber running parallel to the long axis of cells and enhanced migration and production of extracellular matrix components such as collagen type I and fibronectin. This fibroblast-like phenotype induced by TGF-β1 was significantly inhibited by pretreatment with pirfenidone in a dose-dependent manner. We also elucidated the TGF-β signaling pathways as the target of the inhibitory effect of pirfenidone. Pirfenidone inhibited TGF-β signaling by preventing nuclear accumulation of active Smad2/3 complexes rather than phosphorylation of Smad2/3.</P><P><B>Conclusions</B></P><P>These results collectively provide a rational background for future evaluation of pirfenidone as a potential antifibrotic agent for treating proliferative vitreoretinopathy and other fibrotic retinal disorders.</P>