Publications by authors named "Chang Sik Cho"

20 Publications

  • Page 1 of 1

Anti-tumor Activity of Novel STAT3 Inhibitors on Retinoblastoma.

Mol Pharmacol 2021 May 20. Epub 2021 May 20.

Seoul National University, Korea, Republic of

Signal transducer and activator of transcription 3 (STAT3) is a therapeutic target in the treatment of retinoblastoma, the most common intraocular malignant tumor in children. STAT3, a transcription factor for several genes related to tumorigenesis, is activated in retinoblastoma tumors as well as other cancers. In this study, we investigated the structure-activity relationship of a library of STAT3 inhibitors including a novel series of derivatives of the previously reported compound with a Michael acceptor (compound ). We chose two novel STAT3 inhibitors, compounds and , from the library based on their inhibitory effects on the phosphorylation and transcription activity of STAT3. These STAT3 inhibitors effectively suppressed the phosphorylation of STAT3 and inhibited the expression of STAT3-related genes, , , , , and Intraocularly administered STAT3 inhibitors decreased the degree of tumor formation in the vitreous cavity of nude mice of an orthotopic transplantation model. It is noteworthy that compounds 11 and 15 did not induce and toxicity on retinal constituent cells and retinal tissues, respectively, despite their potent anti-tumor effects. We suggest that these novel STAT3 inhibitors be utilized in the treatment of retinoblastoma. In the current study, we suggest the novel STAT3 inhibitors with Michael acceptors possess anti-tumor activity on retinoblastoma, the most common intraocular cancer in children. Based on a detailed structure-activity relationship studies, we found a 4-fluoro and 3-trifluoro analog (compound ) and a mono-chloro analog (compound ) of the parental compound (compound ) inhibited STAT3 phosphorylation, leading to suppress retinoblastoma and .
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http://dx.doi.org/10.1124/molpharm.120.000231DOI Listing
May 2021

Histamine causes an imbalance between pro-angiogenic and anti-angiogenic factors in the retinal pigment epithelium of diabetic retina via H4 receptor/p38 MAPK axis.

BMJ Open Diabetes Res Care 2020 12;8(2)

Department of Ophthalmology, Ajou University School of Medicine and Graduate School of Medicine, Suwon, Gyeonggi-do, Republic of Korea

Introduction: Systemic histaminergic activity is elevated in patients with diabetes mellitus. There are a few studies suggesting that histamine is implicated in the pathogenesis of diabetes, but the exact role of histamine in the development of diabetic retinopathy is unclear. The aim of this study was to investigate the role of histamine receptor H4 (HRH4) in the regulation of retinal pigment epithelium (RPE)-derived pro-angiogenic and anti-angiogenic factors under diabetic conditions.

Research Design And Methods: The levels of vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), histamine and histidine decarboxylase (HDC) in the serum and vitreous samples of patients with diabetes were compared with those of patients without diabetes. The effect of hyperglycemia on expression levels of HRH4, VEGF, IL-6 and pigment epithelium-derived factor (PEDF) in the RPE was determined. The role of HRH4 in high glucose-induced regulation of VEGF, IL-6 and PEDF in ARPE-19 cells and the underlying regulatory mechanism were verified using an RNA interference-mediated knockdown study.

Results: The serum and vitreous levels of VEGF, IL-6, histamine and HDC were more increased in patients with diabetic retinopathy than in patients without diabetes. HRH4 was overexpressed in RPE both in vitro and in vivo. Histamine treatment upregulated VEGF and IL-6 and downregulated PEDF expression in ARPE-19 cells cultivated under hyperglycemic conditions. Hyperglycemia-induced phosphorylation of p38 and subsequent upregulation of VEGF and IL-6 and downregulation of PEDF were dampened by small interfering RNA-mediated knockdown of HRH4 in ARPE-19 cells.

Conclusions: Taken together, HRH4 was a critical regulator of VEGF, IL-6 and PEDF in the RPE under hyperglycemic conditions and the p38 mitogen-activated protein kinase pathway mediated this regulatory mechanism.
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http://dx.doi.org/10.1136/bmjdrc-2020-001710DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7745681PMC
December 2020

Intracellular amyloid-β disrupts tight junctions of the retinal pigment epithelium via NF-κB activation.

Neurobiol Aging 2020 11 21;95:115-122. Epub 2020 Jul 21.

Fight Against Angiogenesis-Related Blindness (FARB) Laboratory, Biomedical Research Institute, Seoul National University Hospital, Seoul, Republic of Korea; Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, Republic of Korea; Department of Ophthalmology, Seoul National University College of Medicine, Seoul, Republic of Korea. Electronic address:

Drusen are focal deposits between the retinal pigment epithelium (RPE) and Bruch's membrane in the retina of patients with age-related macular degeneration. Amyloid-β is one of the important components of drusen, which leads to local inflammation. Furthermore, intracellular amyloid-β disrupts tight junctions of the RPE. However, the intracellular mechanisms linking intracellular amyloid-β and tight-junction disruption are not clear. In this study, intracellular amyloid-β oligomers activated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65, leading to the disorganization of tight junctions of the RPE in mice after subretinal injection of amyloid-β. Amyloid-β also triggered NF-κB activation in the RPE cells in confluent culture, which was inhibited by the suppression of the advanced glycosylation end product-specific receptor. NF-κB inhibition by an IκB kinase inhibitor prevented the suppression of expression of tight-junction proteins, zonula occuludens-1 and occludin in RPE cells. In addition, tight-junction complexes remained intact in the RPE of mice with NF-κB inhibition, although there were intracellular amyloid-β oligomers. These data suggested that NF-κB inhibition might be a therapeutic approach to prevent amyloid-β-mediated tight-junction disruption.
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http://dx.doi.org/10.1016/j.neurobiolaging.2020.07.013DOI Listing
November 2020

CRISPR-Cas9-mediated therapeutic editing of ameliorates the disease phenotypes in a mouse model of Leber congenital amaurosis.

Sci Adv 2019 10 30;5(10):eaax1210. Epub 2019 Oct 30.

ToolGen Inc., Seoul, Republic of Korea.

Leber congenital amaurosis (LCA), one of the leading causes of childhood-onset blindness, is caused by autosomal recessive mutations in several genes including . In this study, we performed CRISPR-Cas9-mediated therapeutic correction of a disease-associated nonsense mutation in in mice, a model of human LCA. Subretinal injection of adeno-associated virus carrying CRISPR-Cas9 and donor DNA resulted in >1% homology-directed repair and ~1.6% deletion of the pathogenic stop codon in in retinal pigment epithelial tissues of mice. The a- and b-waves of electroretinograms were recovered to levels up to 21.2 ± 4.1% and 39.8 ± 3.2% of their wild-type mice counterparts upon bright stimuli after dark adaptation 7 months after injection. There was no definite evidence of histologic perturbation or tumorigenesis during 7 months of observation. Collectively, we present the first therapeutic correction of an nonsense mutation using CRISPR-Cas9, providing new insight for developing therapeutics for LCA.
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http://dx.doi.org/10.1126/sciadv.aax1210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6821465PMC
October 2019

Development of a patient-derived xenograft model of glioblastoma via intravitreal injection in mice.

Exp Mol Med 2019 04 16;51(4):1-9. Epub 2019 Apr 16.

Department of Neurosurgery, Seoul National University Hospital, Seoul, 03080, Republic of Korea.

Currently, the two primary patient-derived xenograft (PDX) models of glioblastoma are established through intracranial or subcutaneous injection. In this study, a novel PDX model of glioblastoma was developed via intravitreal injection to facilitate tumor formation in a brain-mimicking microenvironment with improved visibility and fast development. Glioblastoma cells were prepared from the primary and recurrent tumor tissues of a 39-year-old female patient. To demonstrate the feasibility of intracranial tumor formation, U-87 MG and patient-derived glioblastoma cells were injected into the brain parenchyma of Balb/c nude mice. Unlike the U-87 MG cells, the patient-derived glioblastoma cells failed to form intracranial tumors until 6 weeks after tumor cell injection. In contrast, the patient-derived cells effectively formed intraocular tumors, progressing from plaques at 2 weeks to masses at 4 weeks after intravitreal injection. The in vivo tumors exhibited the same immunopositivity for human mitochondria, GFAP, vimentin, and nestin as the original tumors in the patient. Furthermore, cells isolated from the in vivo tumors also demonstrated morphology similar to that of their parental cells and immunopositivity for the same markers. Overall, a novel PDX model of glioblastoma was established via the intravitreal injection of tumor cells. This model will be an essential tool to investigate and develop novel therapeutic alternatives for the treatment of glioblastoma.
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http://dx.doi.org/10.1038/s12276-019-0241-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6467997PMC
April 2019

Long-Term Effects of In Vivo Genome Editing in the Mouse Retina Using Campylobacter jejuni Cas9 Expressed via Adeno-Associated Virus.

Mol Ther 2019 01 17;27(1):130-136. Epub 2018 Oct 17.

Fight against Angiogenesis-Related Blindness (FARB) Laboratory, Clinical Research Institute, Seoul National University Hospital, Seoul, Republic of Korea; Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, Republic of Korea; Department of Ophthalmology, Seoul National University College of Medicine, Seoul, Republic of Korea. Electronic address:

Genome editing with CRISPR systems provides an unprecedented opportunity to modulate cellular responses in pathological conditions by inactivating undruggable targets, such as transcription factors. Previously, we demonstrated that the smallest Cas9 ortholog characterized to date, from Campylobacter jejuni (CjCas9) targeted to Hif1a and delivered in an adeno-associated virus (AAV) vector, effectively suppressed pathological choroidal neovascularization in the mouse retina. Before implementation of CjCas9 as an in vivo therapeutic modality, it is essential to investigate the long-term effects of target gene disruption via AAV-mediated delivery of CjCas9 in vivo. In this study, histologic and electroretinographic analyses demonstrated that CjCas9 targeted to Hif1a did not induce any definite toxicity in the retina, although the target gene was mutated with a frequency ranging from 45% to 79% in retinal or retinal pigment epithelial cells. Importantly, at 14 months after injection, no indels were detected at potential off-target sites identified using Digenome-seq and Cas-OFFinder, suggesting that long-term expression of CjCas9 does not aggravate off-target effects. Taken together, our results show that intravitreal injection of AAV encoding CjCas9 targeted to Hif1a effectively induced and maintained mutations in retinal tissues for more than 1 year and did not affect retinal histologic integrity or functions.
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http://dx.doi.org/10.1016/j.ymthe.2018.10.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6318782PMC
January 2019

Interaction between microglia and retinal pigment epithelial cells determines the integrity of outer blood-retinal barrier in diabetic retinopathy.

Glia 2019 02 16;67(2):321-331. Epub 2018 Nov 16.

Vascular Microenvironment Laboratory, Department of Pharmacology and Ischemic/Hypoxic Disease Institute, Seoul National University College of Medicine, Seoul, Republic of Korea.

Inner and outer blood-retinal barriers (BRBs), mainly composed of retinal endothelial cells and retinal pigment epithelial (RPE) cells, respectively, maintain the integrity of the retinal tissues. In this study, we aimed to investigate the mechanisms of the outer BRB disruption regarding the interaction between RPE and microglia. In mice with high-fat diet-induced obesity and streptozotocin-induced hyperglycemia, microglia accumulated on the RPE layer, as in those after intravitreal injection of interleukin (IL)-6, which is elevated in ocular fluids of patients with diabetic retinopathy. Although IL-6 did not directly affect the levels of zonula occludens (ZO)-1 and occludin in RPE cells, IL-6 increased VEGFA mRNA in RPE cells to recruit microglial cells. In microglial cells, IL-6 upregulated the mRNA levels of MCP1, MIP1A, and MIP1B, to amplify the recruitment of microglial cells. In this manner, IL-6 modulated RPE and microglial cells to attract microglial cells on RPE cells. Furthermore, IL-6-treated microglial cells produced and secreted tumor necrosis factor (TNF)-α, which activated NF-κB and decreased the levels of ZO-1 in RPE cells. As STAT3 inhibition reversed the effects of IL-6-treated microglial cells on the RPE monolayer in vitro, it reduced the recruitment of microglial cells and the production of TNF-α in RPE tissues in streptozotocin-treated mice. Taken together, IL-6-treated RPE and microglial cells amplified the recruitment of microglial cells and IL-6-treated microglial cells produced TNF-α to disrupt the outer BRB in diabetic retinopathy.
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http://dx.doi.org/10.1002/glia.23542DOI Listing
February 2019

Depthwise-controlled scleral insertion of microneedles for drug delivery to the back of the eye.

Eur J Pharm Biopharm 2018 Dec 26;133:31-41. Epub 2018 Sep 26.

Department of Mechanical Engineering, Yonsei University, Seoul 03722, Republic of Korea. Electronic address:

To treat retinal diseases, intravitreal injection is commonly performed to deliver therapeutic agents to the eye. However, intravitreal injection poses potential risks of ocular complications such as endophthalmitis, retinal detachment, and ocular hemorrhage. Thus, it is desired to develop a minimally invasive and therapeutically effective ocular drug delivery system without full penetration into the sclera. Here, we studied the possibility of precisely-controlled insertion of microneedles (MNs) into the sclera to different levels of depths and how different insertion depths could affect drug delivery into the sclera and to the back of the eye. A microneedle pen (MNP) was developed for depth-controlled scleral delivery by controlling insertion speeds, and it was confirmed that the insertion depths of MNs could be finely controlled by insertion speeds in ex vivo studies. Finite element modeling analyses were also conducted to understand how the depth-controlled insertion of MNs could significantly influence the diffusion distances of drug molecules. Finally, in vivo experiments demonstrated that this MNP system could be applied to the beagle eyes comparable to human ones for the scleral administration of therapeutic agents through the scleral tissues.
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http://dx.doi.org/10.1016/j.ejpb.2018.09.021DOI Listing
December 2018

Novel Hypoxia-Inducible Factor 1α (HIF-1α) Inhibitors for Angiogenesis-Related Ocular Diseases: Discovery of a Novel Scaffold via Ring-Truncation Strategy.

J Med Chem 2018 10 9;61(20):9266-9286. Epub 2018 Oct 9.

College of Pharmacy , Seoul National University , Seoul 08826 , Republic of Korea.

Ocular diseases featuring pathologic neovascularization are the leading cause of blindness, and anti-VEGF agents have been conventionally used to treat these diseases. Recently, regulating factors upstream of VEGF, such as HIF-1α, have emerged as a desirable therapeutic approach because the use of anti-VEGF agents is currently being reconsidered due to the VEGF action as a trophic factor. Here, we report a novel scaffold discovered through the complete structure-activity relationship of ring-truncated deguelin analogs in HIF-1α inhibition. Interestingly, analog 6i possessing a 2-fluorobenzene moiety instead of a dimethoxybenzene moiety exhibited excellent HIF-1α inhibitory activity, with an IC value of 100 nM. In particular, the further ring-truncated analog 34f, which showed enhanced HIF-1α inhibitory activity compared to analog 2 previously reported by us, inhibited in vitro angiogenesis and effectively suppressed hypoxia-mediated retinal neovascularization. Importantly, the heteroatom-substituted benzene ring as a key structural feature of analog 34f was identified as a novel scaffold for HIF-1α inhibitors that can be used in lieu of a chromene ring.
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http://dx.doi.org/10.1021/acs.jmedchem.8b00971DOI Listing
October 2018

Lactic Acid Upregulates VEGF Expression in Macrophages and Facilitates Choroidal Neovascularization.

Invest Ophthalmol Vis Sci 2018 07;59(8):3747-3754

Department of Microbiology and Immunology, Institute of Endemic Disease, Seoul National University College of Medicine, Chongno-gu, Seoul, South Korea.

Purpose: Lactic acid, the end product of glycolysis, has emerged as an immune-modulating metabolite in various diseases. In this study, we aimed to examine whether lactic acid contributes to the disease pathogenesis of choroidal neovascularization (CNV) and to investigate the role of macrophages in CNV pathogenesis.

Methods: CNV was induced by laser photocoagulation in C57BL/6J mice. Lactic acid concentration was measured in the RPE-choroid region. Macrophage infiltration and VEGF were quantified by flow cytometry. VEGF-positive areas and CNV lesions were measured by flat-mount immunofluorescence staining. To inhibit lactic acid uptake in vivo, alpha-cyano-4-hydroxycinnamic acid (α-CHC), a monocarboxylate transporter (MCT) blocker, was injected intravitreally 1 day after laser. VEGF productions were measured in ARPE-19, THP-1 cells, and human umbilical vein endothelial cells (HUVECs) by quantitative PCR and ELISA. Angiogenic activity of lactic acid-treated macrophages was assessed by HUVEC tube formation assay.

Results: Lactic acid was significantly increased in the RPE-choroid region of CNV-induced mice. Lactic acid upregulated VEGFA mRNA and VEGF protein expressions in THP-1 macrophages, but did not in ARPE-19 or HUVECs. THP-1 macrophages treated with lactic acid increased the angiogenesis of endothelial cells independent of MCT activity. Intravitreal injection of α-CHC substantially reduced the VEGF-positive area that colocalized with F4/80-positive macrophages. CNV lesions were also significantly reduced following α-CHC injection compared with vehicle-injected controls.

Conclusions: To our knowledge, these results show for the first time the role of lactic acid in facilitating neovascularization through macrophage-induced angiogenesis. We suggest that targeting macrophage metabolism can be a promising strategy for CNV treatment.
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http://dx.doi.org/10.1167/iovs.18-23892DOI Listing
July 2018

Chronological Changes in Tip Cells during Sprouting Angiogenesis of Development of the Retinal Vasculature in Newborn Mice.

Curr Eye Res 2017 11 19;42(11):1511-1517. Epub 2017 Sep 19.

a Fight against Angiogenesis-Related Blindness (FARB) Laboratory, Clinical Research Institute , Seoul National University Hospital , Seoul , South Korea.

Purpose: To investigate a sequential chronological change in tip cells during the development of the retinal vasculature in newborn mice.

Materials And Methods: Newborn C57BL/6 mice were used for this study. To elucidate the patterns in the developing retinal vasculature, histology, and immunohistochemistry-antiplatelet endothelial cell adhesion molecule-1, anticollagen type IV, isolectin IB-were performed on sections of mouse retina on postnatal days (P)-4, -8, and -12. Staining patterns of isolectin IB-stained arterial and venous tip cells were compared in retinal wholemounts, in which the numbers and characteristics of tip cells were compared between arteries and veins on P-4, -6, and -8. In addition, vascular densities and branching patterns were compared between arterial and venous vascular forefront areas.

Results: Tip cells in the superficial vascular plexus were observed until P-8. The number of tip cells was highest on P-6, decreasing dramatically from P-6 to P-8 (P-4, 165.2 ± 10.1, n = 17; P-6, 183.8 ± 19.4, n = 15; P8, 21.4 ± 6.4, n = 15) (p < 0.05, respectively, t-test). There was a greater number of tip cells in veins versus arteries on P-4 and P-6 (P-4, 91.0 ± 9.2 veins versus 74.2 ± 10.4 arteries; P-6, 104.0 ± 10.2 veins versus 79.8 ± 11.3 arteries) (p < 0.05, respectively). Arterial tip cells had thinner and longer sprouts compared with venous tip cells (basal thickness: 15.7 ± 8.7 veins versus 9.9 ± 3.5 μm arteries) (length, 20.3 ± 9.1 veins versus 37.1 ± 13.2 μm arteries on P-4) (p < 0.05, respectively). Vessel areas and densities of vascular branch points were significantly higher around veins compared to arteries (vessel areas: 58.9 ± 1.2% veins versus 40.8 ± 1.9% arteries; vascular branch points, 1371.9 ± 136.7/mm veins versus 1046.7 ± 175.5/mm arteries) (p < 0.05, respectively).

Conclusion: The number of tip cells increased to a greater extent in the superficial vascular plexus of veins versus arteries until P-6. Consequently, there are more vessel areas and vascular branch points near retinal veins versus arteries. Arterial tip cells are longer and thinner than the shorter and thicker venous tip cells.
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http://dx.doi.org/10.1080/02713683.2017.1347691DOI Listing
November 2017

Intravitreally Injected Anti-VEGF Antibody Reduces Brown Fat in Neonatal Mice.

PLoS One 2015 30;10(7):e0134308. Epub 2015 Jul 30.

Fight against Angiogenesis-Related Blindness (FARB) Laboratory, Clinical Research Institute, Seoul National University Hospital, Seoul, Republic of Korea; Department of Biomedical Sciences, College of Medicine, Seoul National University, Seoul, Republic of Korea; Department of Ophthalmology, College of Medicine, Seoul National University, Seoul, Republic of Korea.

Anti-vascular endothelial growth factor (VEGF) agents are the mainstay treatment for various angiogenesis-related retinal diseases. Currently, bevacizumab, a recombinant humanized anti-VEGF antibody, is trailed in retinopathy of prematurity, a vasoproliferative retinal disorder in premature infants. However, the risks of systemic complications after intravitreal injection of anti-VEGF antibody in infants are not well understood. In this study, we show that intravitreally injected anti-VEGF antibody is transported into the systemic circulation into the periphery where it reduces brown fat in neonatal C57BL/6 mice. A considerable amount of anti-VEGF antibody was detected in serum after intravitreal injection. Furthermore, in interscapular brown adipose tissue, we found lipid droplet accumulation, decreased VEGF levels, loss of vascular network, and decreased expression of mitochondria-related genes, Ppargc1a and Ucp1, all of which are characteristics of "whitening" of brown fat. With increasing age and body weight, brown fat restored its morphology and vascularity. Our results show that there is a transient, but significant impact of intravitreally administered anti-VEGF antibody on brown adipose tissue in neonatal mice. We suggest that more attention should be focused on the metabolic and developmental significance of brown adipose tissue in bevacizumab treated retinopathy of prematurity infants.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0134308PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4520452PMC
May 2016

STAT3 inhibition suppresses proliferation of retinoblastoma through down-regulation of positive feedback loop of STAT3/miR-17-92 clusters.

Oncotarget 2014 Nov;5(22):11513-25

Fight against Angiogenesis-Related Blindness (FARB) Laboratory, Clinical Research Institute, Seoul National University Hospital, Seoul, Republic of Korea. Tumor Microenvironment Research Center, Global Core Research Center, Seoul National University, Seoul, Republic of Korea. Department of Biomedical Sciences, College of Medicine, Seoul National University, Seoul, Republic of Korea. Department of Ophthalmology, College of Medicine, Seoul National University, Seoul, Republic of Korea.

Retinoblastoma, the most common intraocular malignant tumor in children, is characterized by the loss of both functional alleles of RB1 gene, which however alone cannot maintain malignant characteristics of retinoblastoma cells. Nevertheless, the investigation of other molecular aberrations such as matrix metalloproteinases (MMPs) and miRNAs is still lacking. In this study, we demonstrate that STAT3 is activated in retinoblastoma cells, Ki67-positive areas of in vivo orthotopic tumors in BALB/c nude mice, and human retinoblastoma tissues of the advanced stage. Furthermore, target genes of STAT3 including BCL2, BCL2L1, BIRC5, and MMP9 are up-regulated in retinoblastoma cells compared to other retinal constituent cells. Interestingly, STAT3 inhibition by targeted siRNA suppresses the proliferation of retinoblastoma cells and the formation of in vivo orthotopic tumors. In line with these results, STAT3 siRNA effectively induces down-regulation of target genes of STAT3. In addition, miRNA microarray analysis and further real-time PCR experiments with STAT3 siRNA treatment show that STAT3 activation is related to the up-regulation of miR-17-92 clusters in retinoblastoma cells via positive feedback loop between them. In conclusion, we suggest that STAT3 inhibition could be a potential therapeutic approach in retinoblastoma through the suppression of tumor proliferation.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4294389PMC
http://dx.doi.org/10.18632/oncotarget.2546DOI Listing
November 2014

Hypoxia-mediated retinal neovascularization and vascular leakage in diabetic retina is suppressed by HIF-1α destabilization by SH-1242 and SH-1280, novel hsp90 inhibitors.

J Mol Med (Berl) 2014 Oct 31;92(10):1083-92. Epub 2014 May 31.

Fight against Angiogenesis-Related Blindness (FARB) Laboratory, Clinical Research Institute, Seoul National University Hospital, Seoul, 110-744, Republic of Korea.

In diabetic retinopathy (DR), visual deterioration is related with retinal neovascularization and vascular hyperpermeability. Anti-vascular endothelial growth factor (VEGF) agents are currently utilized to suppress retinal neovascularization and macular edema (ME); however, there are still concerns on the widespread use of them because VEGF is a trophic factor for neuronal and endothelial cells in the retina. As an alternative treatment strategy for DR, it is logical to address hypoxia-related molecules to treat DR because the retina is in relative hypoxia as DR progresses. In this study, we demonstrate that destabilization of hypoxia-inducible factor-1α (HIF-1α) by SH-1242 and SH-1280, novel heat shock protein 90 (hsp90) inhibitors, leads to suppression of hypoxia-mediated retinal neovascularization and vascular leakage in diabetic retina. In vitro experiments showed that these inhibitors inhibited hypoxia-induced upregulation of target genes of HIF-1α and further secretion of VEGF. Furthermore, these inhibitors effectively suppressed expression of target genes of HIF-1α including vegfa in the retina of oxygen-induced retinopathy (OIR) mice. Interestingly, despite hsp90 inhibition, these inhibitors do not induce definite toxicity at the level of gene expression, cellular viability, and histologic integrity. We suggest that SH-1242 and SH-1280 can be utilized in the treatment of DR, as an alternative treatment of direct VEGF inhibition. Key message: SH-1242 and SH-1280 are novel hsp90 inhibitors similar to deguelin. HIF-1α destabilization by hsp90 inhibition leads to anti-angiogenic effects. Despite hsp90 inhibition, both inhibitors do not induce definite toxicity. HIF-1α modulation can be a safer therapeutic option than direct VEGF inhibition.
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http://dx.doi.org/10.1007/s00109-014-1168-8DOI Listing
October 2014

Animal models of diabetic retinopathy: doors to investigate pathogenesis and potential therapeutics.

J Biomed Sci 2013 Jun 20;20:38. Epub 2013 Jun 20.

Fight against Angiogenesis-Related Blindness (FARB) Laboratory, Clinical Research Institute, Seoul National University, Seoul 110-744, Republic of Korea.

Effective and validated animal models are valuable to investigate the pathogenesis and potential therapeutics for human diseases. There is much concern for diabetic retinopathy (DR) in that it affects substantial number of working population all around the world, resulting in visual deterioration and social deprivation. In this review, we discuss animal models of DR based on different species of animals from zebrafish to monkeys and prerequisites for animal models. Despite criticisms on imprudent use of laboratory animals, we hope that animal models of DR will be appropriately utilized to deepen our understanding on the pathogenesis of DR and to support our struggle to find novel therapeutics against catastrophic visual loss from DR.
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http://dx.doi.org/10.1186/1423-0127-20-38DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3694455PMC
June 2013

Anti-angiogenic effect of luteolin on retinal neovascularization via blockade of reactive oxygen species production.

Invest Ophthalmol Vis Sci 2012 Nov 19;53(12):7718-26. Epub 2012 Nov 19.

Fight against Angiogenesis-Related Blindness Laboratory, Clinical Research Institute, Seoul National University Hospital, Seoul, Korea.

Purpose: Oxidative stress-induced vascular endothelial growth factor (VEGF) is thought to play a critical role in the pathogenesis of retinopathy of prematurity (ROP). This study was performed to investigate the anti-angiogenic effect of luteolin against reactive oxygen species (ROS)-induced retinal neovascularization.

Methods: The toxicity of luteolin was evaluated through modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay in human retinal microvascular endothelial cells (HRMECs) as well as TUNEL staining in the retina of C57BL/6J mice. After intravitreal injection of luteolin in the mouse model of ROP, retinal neovascularization was examined by fluorescence angiography and vessel counting. Anti-angiogenic activity of luteolin was evaluated by VEGF-induced migration and tube formation assay. The effect of luteolin on tertiary-butylhydroperoxide (t-BH)-induced ROS production was measured with 2'7'-dichlorofluorescein diacetate. The effect of luteolin on t-BH-induced and hypoxia-induced VEGF transcription and expression were evaluated by RT-PCR and Western blot, respectively.

Results: Luteolin never affected the viability of HRMECs up to 10 μM, where luteolin never induced any structural change in all retinal layers. Luteolin inhibited retinal neovascularization in the mouse model of ROP. Moreover, VEGF-induced migration and tube formation were significantly decreased by cotreatment of luteolin. Luteolin attenuated VEGF transcription via blockade of t-BH-induced ROS production. Luteolin suppressed hypoxia-induced VEGF expression via attenuating hypoxia inducible factor 1 α expression.

Conclusions: Our results suggest that luteolin could be a potent anti-angiogenic agent for retinal neovascularization, which is related to anti-oxidative activity to block ROS production and to subsequently suppress VEGF expression and the pro-angiogenic effect of VEGF.
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http://dx.doi.org/10.1167/iovs.11-8790DOI Listing
November 2012

Inhibitory activity of bevacizumab to differentiation of retinoblastoma cells.

PLoS One 2012 22;7(3):e33456. Epub 2012 Mar 22.

Department of Ophthalmology, Seoul National University College of Medicine, Seoul National University, Seoul, Korea.

Vascular endothelial growth factor (VEGF) is a major regulator in retinal and choroidal angiogenesis, which are common causes of blindness in all age groups. Recently anti-VEGF treatment using anti-VEGF antibody has revolutionarily improved the visual outcome in patients with vaso-proliferative retinopathies. Herein, we demonstrated that bevacizumab as an anti-VEGF antibody could inhibit differentiation of retinoblastoma cells without affection to cellular viability, which would be mediated via blockade of extracellular signal-regulated kinase (ERK) 1/2 activation. The retinoblastoma cells expressed VEGFR-2 as well as TrkA which is a neurotrophin receptor associated with differentiation of retinoblastoma cells. TrkA in retinoblastoma cells was activated with VEGF treatment. Interestingly even in the concentration of no cellular death, bevascizumab significantly attenuated the neurite formation of differentiated retinoblastoma cells, which was accompanied by inhibition of neurofilament and shank2 expression. Furthermore, bevacizumab inhibited differentiation of retinoblastoma cells by blockade of ERK 1/2 activation. Therefore, based on that the differentiated retinoblastoma cells are mostly photoreceptors, our results suggest that anti-VEGF therapies would affect to the maintenance or function of photoreceptors in mature retina.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0033456PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3310877PMC
August 2012

Differential roles of matrix metalloproteinase-9 and -2, depending on proliferation or differentiation of retinoblastoma cells.

Invest Ophthalmol Vis Sci 2010 Mar 20;51(3):1783-8. Epub 2009 Nov 20.

Fight Against Angiogenesis-Related Blindness Laboratory, Department of Ophthalmology, College of Medicine, Seoul National University, Seoul, Korea.

Purpose: To investigate the differential roles of matrix metalloproteinase (MMP)-9 and MMP-2 in the proliferation or differentiation of retinoblastoma cells.

Methods: Cell proliferation assay with an MMP-9 inhibitor and cell viability assay with an MMP-2 inhibitor were performed in retinoblastoma cells with 5 ng/mL fibroblast growth factor 2 for proliferation, 0.1% bovine serum albumin for differentiation, or reverse transcriptase-polymerase chain reaction (RT-PCR) for MMP-9, MMP-2, and their tissue inhibitors TIMP-1 and TIMP-2. Immunohistochemistry for MMP-2 and nm23 was performed using an experimental model of retinoblastoma. With the use of an MMP-2 inhibitor, Western blot analysis was performed for neurofilament, extracellular signal-regulated kinases 1 and 2 (ERK 1/2), and phospho-ERK 1/2, and neurite length was measured in differentiated retinoblastoma cells.

Results: With the proliferation of retinoblastoma cells, MMP-9 expression was upregulated without alteration of MMP-2, TIMP-1, or TIMP-2. However, proliferation was not affected by the inhibition of MMP-9 activity. Interestingly, only MMP-2 expression, colocalized with differentiated cells in retinoblastoma tissue, was significantly increased in the differentiation of retinoblastoma cells. Inhibition of MMP-2 activity did not affect cellular viability but attenuated neurite outgrowth and neurofilament expression of differentiated retinoblastoma cells, which was mediated through the suppression of ERK 1/2 activation.

Conclusions: The authors suggest that differential expression of MMP-9 and -2 could reflect biological features, such as proliferation and differentiation, of retinoblastoma cells. In particular, MMP-2 could be directly involved in the regulation of differentiation of retinoblastoma cells. Therefore, therapeutic targeting to MMP-2 may prove useful for reducing malignancy through the differentiation of retinoblastoma cells.
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http://dx.doi.org/10.1167/iovs.09-3990DOI Listing
March 2010

Neurotrophin receptors TrkA and TrkB in retinoblastoma are differentially expressed depending on cellular differentiation.

Tumour Biol 2009 7;30(5-6):233-41. Epub 2009 Oct 7.

Department of Ophthalmology, College of Medicine, Seoul National University, Seoul, Korea.

Retinoblastoma is the most common primary intraocular malignancy in children. With the progression of retinoblastoma, retinoblastoma cells lose their ability to differentiate. Regardless of many attempts to identify prognostic factors in retinoblastoma, further investigation for prognostic factors of retinoblastoma progression is still required because of the lack of sensitivity and specificity of these prognostic factors in predicting disease progression. We demonstrated that the differential expression of the neurotrophin receptors TrkA and TrkB is closely related to the differentiation of retinoblastoma cells. While retinoblastoma cells expressed TrkA as well as TrkB, their growth rates were not influenced by the addition of nerve growth factor to the culture medium. In experimental animal models of retinoblastoma, TrkA expression was primarily detected in more differentiated areas with high nm23 immunoreactivity whereas TrkB expression was apparent in more proliferative areas with high Ki67 immunoreactivity. With retinoic-acid-induced differentiation of retinoblastoma cells, TrkA expression significantly increased whereas TrkB significantly decreased. The differential expression of TrkA and TrkB with differentiation of retinoblastoma cells was mediated by extracellular-signal-regulated kinase 1/2 activation, which was confirmed by immunocytochemistry of TrkA. Therefore, our results suggest that the differential expression of TrkA and TrkB could be valuable as a therapeutic target, for instance using specific inhibitors.
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http://dx.doi.org/10.1159/000243766DOI Listing
February 2010

Blockade of angiotensin II attenuates VEGF-mediated blood-retinal barrier breakdown in diabetic retinopathy.

J Cereb Blood Flow Metab 2009 Mar 24;29(3):621-8. Epub 2008 Dec 24.

Fight against Angiogenesis-Related Blindness (FARB) Laboratory, Department of Ophthalmology, Seoul National University College of Medicine & Seoul Artificial Eye Center, Clinical Research Institute, Seoul National University Hospital, Seoul, Korea.

Diabetic retinopathy (DR) is the leading cause of vision loss as a major complication of diabetes mellitus. The blood-retinal barrier (BRB) breakdown is a critical early event in the pathogenesis of DR. It has been known that the rennin-angiotensin system (RAS) is important in the progression of the DR via angiotensin II (Ang II), the effector of RAS. In this study, we showed that blockade of Ang II attenuates vascular endothelial growth factor (VEGF)-mediated BRB breakdown in DR. In streptozotocin-induced diabetes, retinal vascular permeability increased with upregulation of VEGF, where Ang II and its receptors were upregulated. Ang II induced VEGF expression in retinal endothelial cells accompanied by loss of tight junction proteins. However, the blockade of Ang II by perindopril, an angiotensin converting enzyme (ACE) inhibitor, inhibited upregulation of VEGF, and prevented the loss of tight junction proteins. Moreover, inhibition of Ang II by perindopril attenuated increased vascular permeability of diabetic retina accompanied by recovery of tight junction proteins in retinal vessels. Therefore, we suggest that the RAS involves in increased vascular permeability during early stage of DR, which is mediated by VEGF. Furthermore, the ACE inhibitor may have a therapeutic potential in the treatment of diabetic BRB breakdown.
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http://dx.doi.org/10.1038/jcbfm.2008.154DOI Listing
March 2009