Kun-Che (Gary) Chang was born in Kaohsiung, Taiwan. He obtained his B.S. in Life Science from National Dong Hwa University (Taiwan) in 2006 and his M.S. in Biotechnology from National Tsing Hua University (Taiwan) in 2008. After worked at National Taiwan University for 1 year, he transited to University of Minnesota, Dept. of Pharmacology as a research assistant from 2010 to 2011. He joined the Toxicology PhD program at University of Colorado and was mentored by Dr. J. Mark Petrash at Dept. of Ophthalmology. He obtained his PhD degree in 2015. His PhD thesis focuses on prevention of ocular inflammation. He joined Dr. Jeffrey Goldberg's lab's for his post-doctoral training at Dept. of Ophthalmology, Stanford University in 2016.
Honors & Awards
Ruth L. Kirschstein National Research Service Award Individual Postdoctoral Fellowship (F32), National Eye Institute (2018-present)
JNS Travel Award, The 41th Annual Meeting of the Japan Neuroscience Society (2018)
NEI-T32 awardee, Stanford Vision Research Training Program (2018-2019)
ARVO Travel Grant, The 2018 annual meeting of the Association for Research in Vision and Ophthalmology (2018)
Harold C. Heim Awards for Excellence in Graduate Research, University of Colorado Denver, Skaggs School of Pharmacy and Pharmaceutical Sciences (2015)
Award for best poster of vision sciences class, University of Colorado Denver Anschutz Medical Campus 29th Annual Student Research Forum (2014)
Trainee Award, The 17th International Workshop on the Enzymology and Molecular Biology of Carbonyl Metabolism (2014)
Award for School of Medicine Class of 2017 Poster Choice, University of Colorado Denver Anschutz Medical Campus 28th Annual Student Research Forum (2013)
C. Werner and Kitty Hirs Research Award, Graduate School at Anschutz Medical Campus, University of Colorado (2013, 2014, 2015)
MWSOT Student Travel Award, The 30th Annual Regional Meeting of the Mountain West Society of Toxicology (2012)
Book Aroma Award, National Dong Hwa University (2003, 2004)
Doctor of Philosophy, University of Colorado Denver (2015)
Master of Science, National Tsinghua University (2008)
Bachelor of Science, National Dong Hwa University (2006)
EMT signaling: potential contribution of CRISPR/Cas gene editing.
Cellular and molecular life sciences : CMLS
Epithelial to mesenchymal transition (EMT) is a complex plastic and reversible cellular process that has critical roles in diverse physiological and pathological phenomena. EMT is involved in embryonic development, organogenesis and tissue repair, as well as in fibrosis, cancer metastasis and drug resistance. In recent years, the ability to edit the genome using the clustered regularly interspaced palindromic repeats (CRISPR) and associated protein (Cas) system has greatly contributed to identify or validate critical genes in pathway signaling. This review delineates the complex EMT networks and discusses recent studies that have used CRISPR/Cas technology to further advance our understanding of the EMT process.
View details for DOI 10.1007/s00018-020-03449-3
View details for PubMedID 32008085
Mouse gamma-Synuclein Promoter-Mediated Gene Expression and Editing in Mammalian Retinal Ganglion Cells.
The Journal of neuroscience : the official journal of the Society for Neuroscience
Optic neuropathies are a group of optic nerve (ON) diseases caused by various insults including glaucoma, inflammation, ischemia, trauma and genetic deficits, which are characterized by retinal ganglion cell (RGC) death and ON degeneration. An increasing number of genes involved in RGC intrinsic signaling have been found to be promising neural repair targets that can potentially be modulated directly by gene therapy, if we can achieve RGC specific gene targeting. To address this challenge, we first used adeno associated virus (AAV)-mediated gene transfer to perform a low throughput in vivo screening in both male and female mouse eyes and identified the mouse γ-synuclein (mSncg) promoter, which specifically and potently sustained transgene expression in mouse RGCs and also works in human RGCs. We further demonstrated that gene therapy that combines AAV-mSncg promoter with CRISPR/Cas9 gene editing can knockdown pro-degenerative genes in RGCs and provide effective neuroprotection in optic neuropathies.Significance Statement:Here we present an RGC-specific promoter, mouse γ-synuclein (mSncg) promoter, and perform extensive characterization and proof-of-concept studies of mSncg promoter-mediated gene expression and CRISPR/Cas9 gene editing in RGCs in vivo To our knowledge, this is the first report demonstrating in vivo neuroprotection of injured RGCs and optic nerve by AAV-mediated CRISPR/Cas9 inhibition of genes that are critical for neurodegeneration. It represents a powerful tool to achieve RGC-specific gene modulation, and also opens up a promising gene therapy strategy for optic neuropathies, the most common form of eye diseases that cause irreversible blindness.
View details for DOI 10.1523/JNEUROSCI.0102-20.2020
View details for PubMedID 32300046
Role of aldose reductase in diabetes-induced retinal microglia activation.
Diabetes-induced hyperglycemia plays a key pathogenic role in degenerative retinal diseases. In diabetic hyperglycemia, aldose reductase (AR) is elevated and linked to the pathogenesis of diabetic retinopathy (DR) and cataract. Retinal microglia (RMG), the resident immune cells in the retina, are thought to contribute to the proinflammatory phenotype in the diabetic eye. However, we have a limited understanding of the potential role of AR expressed in RMG as a mediator of inflammation in the diabetic retina. Glycated proteins accumulate in diabetes, including Amadori-glycated albumin (AGA) which has been shown to induce a proinflammatory phenotype in various tissues. In this study, we investigated the ability of AGA to stimulate inflammatory changes to RMG and macrophages, and whether AR plays a role in this process. In macrophages, treatment with an AR inhibitor (Sorbinil) or genetic knockdown of AR lowered AGA-induced TNF-α secretion (56% and 40%, respectively) as well as cell migration. In a mouse RMG model, AR inhibition attenuated AGA-induced TNF-α secretion and cell migration (67% and 40%, respectively). To further mimic the diabetic milieu in retina, we cultured RMG under conditions of hypoxia and observed the induction of TNF-α and VEGF protein expression. Downregulation of AR in either a pharmacological or genetic manner prevented hypoxia-induced TNF-α and VEGF expression. In our animal study, increased numbers of RMG observed in streptozotocin (STZ)-induced diabetic retina was substantially lower when diabetes was induced in AR knockout mice. Thus, in vitro and in vivo studies demonstrated that AR is involved in diabetes-induced RMG activation, providing a rationale for targeting AR as a therapeutic strategy for DR.
View details for DOI 10.1016/j.cbi.2019.01.020
View details for PubMedID 30682331
Cell transplantation of retinal ganglion cells derived from hESCs.
Restorative neurology and neuroscience
Glaucoma, the number one cause of irreversible blindness, is characterized by the loss of retinal ganglion cells (RGCs), which do not regenerate in humans or mammals after cell death. Cell transplantation provides an opportunity to restore vision in glaucoma, or other optic neuropathies. Since transplanting primary RGCs from deceased donor tissues may not be feasible, stem cell-derived RGCs could provide a plausible alternative source of donor cells for transplant.We define a robust chemically defined protocol to differentiate human embryonic stem cells (hESCs) into RGC-like neurons.Human embryonic stem cell lines (H7-A81 and H9) and induced pluripotent stem cell (iPSC) were used for RGC differentiation. RGC immaturity was measured by calcium imaging against muscimol. Cell markers were detected by immunofluorescence staining and qRT-PCR. RGC-like cells were intravitreally injected to rat eye, and co-stained with RBPMS and human nuclei markers. All experiments were conducted at least three times independently. Data were analyzed by ANOVA with Tukey's test with P value of <0.05 considered statistically significant.We detected retinal progenitor markers Rx and Pax6 after 15 days of differentiation, and the expression of markers for RGC-specific differentiation (Brn3a and Brn3b), maturation (synaptophysin) and neurite growth (β-III-tubulin) after an additional 15 days. We further examined the physiologic differentiation of these hESC-derived RGC-like progeny to those differentiated in vitro from primary rodent retinal progenitor cells (RPCs) with calcium imaging, and found that both populations demonstrate the immature RGC-like response to muscimol, a GABAA receptor agonist. By one week after transplant to the adult rat eye by intravitreal injection, the human RGC-like cells successfully migrated into the ganglion cell layer.Our protocol provides a novel, short, and cost-effective approach for RGC differentiation from hESCs, and may broaden the scope for cell replacement therapy in RGC-related optic neuropathies such as glaucoma.
View details for DOI 10.3233/RNN-190941
View details for PubMedID 31815704
MTP18 is a Novel Regulator of Mitochondrial Fission in CNS Neuron Development, Axonal Growth, and Injury Responses.
2019; 9 (1): 10669
The process of mitochondrial fission-fusion has been implicated in diverse neuronal roles including neuronal survival, axon degeneration, and axon regeneration. However, whether increased fission or fusion is beneficial for neuronal health and/or axonal growth is not entirely clear, and is likely situational and cell type-dependent. In searching for mitochondrial fission-fusion regulating proteins for improving axonal growth within the visual system, we uncover that mitochondrial fission process 1,18 kDa (MTP18/MTFP1), a pro-fission protein within the CNS, is critical to maintaining mitochondrial size and volume under normal and injury conditions, in retinal ganglion cells (RGCs). We demonstrate that MTP18's expression is regulated by transcription factors involved in axonal growth, Kruppel-like factor (KLF) transcription factors-7 and -9, and that knockdown of MTP18 promotes axon growth. This investigation exposes MTP18's previously unexplored role in regulating mitochondrial fission, implicates MTP18 as a downstream component of axon regenerative signaling, and ultimately lays the groundwork for investigations on the therapeutic efficacy of MTP18 expression suppression during CNS axon degenerative events.
View details for DOI 10.1038/s41598-019-46956-5
View details for PubMedID 31337818
Magnetic Human Corneal Endothelial Cell Transplant: Delivery, Retention, and Short-Term Efficacy.
Investigative ophthalmology & visual science
2019; 60 (7): 2438–48
Corneal endothelial dysfunction leads to corneal edema, pain, and vision loss. Adequate animal models are needed to study the safety and efficacy of novel cell therapies as an alternative to corneal transplantation.Primary human corneal endothelial cells (HCECs) were isolated from cadaveric donor corneas, expanded in vitro, transduced to express green fluorescent protein (GFP), loaded with superparamagnetic nanoparticles, and injected into the anterior chamber of adult rabbits immediately after endothelial cell or Descemet's membrane stripping. The same volume of balanced salt solution plus (BSS+) was injected in control eyes. We compared different models for inducing corneal edema in rabbits, and examined the ability of transplanted HCECs to reduce corneal edema over time by measuring central corneal thickness and tracking corneal clarity. GFP-positive donor cells were tracked in vivo using optical coherence tomography (OCT) fluorescence angiography module, and the transplanted cells were confirmed by human nuclei immunostaining.Magnetic HCECs integrated onto the recipient corneas with intact Descemet's membrane, and donor identity was confirmed by GFP expression and immunostaining for human nuclei marker. Donor HCECs formed a monolayer on the posterior corneal surface and expressed HCEC functional markers of tight junction formation. No GFP-positive cells were observed in the trabecular meshwork or on the iris, and intraocular pressure remained stable through the length of the study.Our results demonstrate magnetic cell-based therapy efficiently delivers HCECs to restore corneal transparency without detectable toxicity or adverse effect on intraocular pressure. Magnetic delivery of HCECs may enhance corneal function and should be explored further for human therapies.
View details for DOI 10.1167/iovs.18-26001
View details for PubMedID 31158276
Opposing Effects of Growth and Differentiation Factors in Cell-Fate Specification.
Current biology : CB
Following ocular trauma or in diseases such as glaucoma, irreversible vision loss is due to the death of retinal ganglion cell (RGC) neurons. Although strategies to replace these lost cells include stem cell replacement therapy, few differentiated stem cells turn into RGC-like neurons. Understanding the regulatory mechanisms of RGC differentiation in vivo may improve outcomes of cell transplantation by directing the fate of undifferentiated cells toward mature RGCs. Here, we report a new mechanism by which growth and differentiation factor-15 (GDF-15), a ligand in the transforming growth factor-beta (TGF-β) superfamily, strongly promotes RGC differentiation in the developing retina in vivo in rodent retinal progenitor cells (RPCs) and in human embryonic stem cells (hESCs). This effect is in direct contrast to the closely related ligand GDF-11, which suppresses RGC-fate specification. We find these opposing effects are due in part to GDF-15's ability to specifically suppress Smad-2, but not Smad-1, signaling induced by GDF-11, which can be recapitulated by pharmacologic or genetic blockade of Smad-2 in vivo to increase RGC specification. No other retinal cell types were affected by GDF-11 knockout, but a slight reduction in photoreceptor cells was observed by GDF-15 knockout in the developing retina in vivo. These data define a novel regulatory mechanism of GDFs' opposing effects and their relevance in RGC differentiation and suggest a potential approach for advancing ESC-to-RGC cell-based replacement therapies.
View details for DOI 10.1016/j.cub.2019.05.011
View details for PubMedID 31155355
Retinal Cell Fate Specification.
Trends in neurosciences
2018; 41 (4): 165–67
How are different neural cell types generated from progenitor cells? In 1990, Turner et al. used new lineage tracing techniques to show that different cells in the mammalian retina share their progenitor origin. The findings established a key step toward our understanding of how multipotent progenitor cells give rise to complex circuitry in the retina.
View details for PubMedID 29602333
Aldo-Keto Reductases: Multifunctional Proteins as Therapeutic Targets in Diabetes and Inflammatory Disease.
Advances in experimental medicine and biology
2018; 1032: 173–202
Aldose reductase (AR) is an NADPH-dependent aldo-keto reductase that has been shown to be involved in the pathogenesis of several blinding diseases such as uveitis, diabetic retinopathy (DR) and cataract. However, possible mechanisms linking the action of AR to these diseases are not well understood. As DR and cataract are among the leading causes of blindness in the world, there is an urgent need to explore therapeutic strategies to prevent or delay their onset. Studies with AR inhibitors and gene-targeted mice have demonstrated that the action of AR is also linked to cancer onset and progression. In this review we examine possible mechanisms that relate AR to molecular signaling cascades and thus explain why AR inhibition is an effective strategy against colon cancer as well as diseases of the eye such as uveitis, cataract, and retinopathy.
View details for DOI 10.1007/978-3-319-98788-0_13
View details for PubMedID 30362099
Induced Pluripotent Stem Cells Promote Retinal Ganglion Cell Survival After Transplant.
Investigative ophthalmology & visual science
2018; 59 (3): 1571–76
The purpose of this study was to characterize whether induced pluripotent stem cells (iPSCs) affect survival of grafted retinal ganglion cells (RGCs) after transplantation.For in vitro studies, human iPSCs were either directly cocultured with mouse RGCs or plated in hanging inserts in RGC cultures for 1 week. For ex vivo studies, RGCs and iPSCs were seeded onto the inner surface of an adult rat retina explant and cultured for 1 week. For in vivo studies, RGCs and iPSCs were intravitreally coinjected into an adult rat eye 1 week before examining retinas by explant and immunostaining.A dose-dependent increase in RGC survival was observed in RGC-iPSC direct cocultures, and RGC-iPSC indirect cocultures showed a similar RGC protective effect, but to a lesser extent than in direct coculture. Enhanced RGC survival was also identified in RGC-iPSC cotransplantations to adult retinas ex vivo and in vivo. In addition, RGCs with iPSC cotransplantation extended significantly longer neurites than RGC-only transplants.Human iPSCs promote transplanted RGC survival and neurite extension. This effect may be mediated at least partially through secretion of diffusible neuroprotective factors.
View details for PubMedID 29625481
Novel Regulatory Mechanisms for the SoxC Transcriptional Network Required for Visual Pathway Development
JOURNAL OF NEUROSCIENCE
2017; 37 (19): 4967-4981
What pathways specify retinal ganglion cell (RGC) fate in the developing retina? Here we report on mechanisms by which a molecular pathway involving Sox4/Sox11 is required for RGC differentiation and for optic nerve formation in mice in vivo, and is sufficient to differentiate human induced pluripotent stem cells into electrophysiologically active RGCs. These data place Sox4 downstream of RE1 silencing transcription factor (REST) in regulating RGC fate, and further describe a newly identified, Sox4-regulated SUMOylation site in Sox11, which suppresses Sox11's nuclear localization and its ability to promote RGC differentiation, providing a mechanism for the SoxC familial compensation observed here and elsewhere in the nervous system. These data define novel regulatory mechanisms for this SoxC molecular network, and suggest pro-RGC molecular approaches for cell replacement-based therapies for glaucoma and other optic neuropathies.SIGNIFICANCE STATEMENTGlaucoma is the most common cause of blindness worldwide and along with other optic neuropathies is characterized by loss of retinal ganglion cells (RGCs). Unfortunately, vision and RGC loss are irreversible, and lead to bilateral blindness in around 14% of all diagnosed patients. Differentiating and transplanting RGC-like cells derived from stem cells have the potential to replace neurons that have already been lost and thereby restore visual function. These data uncover new mechanisms of retinal progenitor cell (RPC)- and human stem cell-to-RGC fate specification, and take a significant step towards understanding neuronal and retinal development and ultimately cell transplant therapy.
View details for DOI 10.1523/JNEUROSCI.3430-13.2017
View details for PubMedID 28411269
Influence of aldose reductase on epithelial-to-mesenchymal transition signaling in lens epithelial cells.
Cataract is the most frequent cause of blindness worldwide and is treated by surgical removal of the opaque lens to restore the light path to the retina. While cataract surgery is a safe procedure, some patients develop a complication of the surgery involving opacification and wrinkling of the posterior lens capsule. This process, called posterior capsule opacification (PCO), requires a second clinical treatment that can in turn lead to additional complications. Prevention of PCO is a current unmet need in the vision care enterprise. The pathogenesis of PCO involves the transition of lens epithelial cells to a mesenchymal phenotype, designated epithelial-to-mesenchymal transition (EMT). Our previous studies showed that transgenic mice designed for overexpression of human aldose reductase developed lens defects reminiscent of PCO. In the current study, we evaluated the impact of aldose reductase (AR) on expression of expression of EMT markers in the lens. Primary lens epithelial cells from AR-transgenic mice showed downregulated expression of Foxe3 and Pax6 and increased expression of α-SMA, fibronectin and snail, a pattern of gene expression typical of cells undergoing EMT. A role for AR in these changes was further confirmed when we observed that they could be normalized by treatment of cells with Sorbinil, an AR inhibitor. Smad-dependent and Smad-independent pathways are known to contribute to EMT. Interestingly, AR overexpression induced ERK but not Smad-2 activation. These results suggest that elevation of AR may lead to activation of ERK signaling and thus play a role in TGF-β/Smad independent induction of EMT in lens epithelial cells.
View details for DOI 10.1016/j.cbi.2017.01.017
View details for PubMedID 28137510
SoxC transcription factors in retinal development and regeneration.
Neural regeneration research
2017; 12 (7): 1048–51
Glaucoma and other optic neuropathies result in optic nerve degeneration and the loss of retinal ganglion cells (RGCs) through complex signaling pathways. Although the mechanisms that regulate RGC development remain unclear, uncovering novel developmental pathways may support new strategies to regenerate the optic nerve or replace RGCs. Here we review recent studies that provide strong evidence that the Sry-related high-mobility-group C (SoxC) subfamily of transcription factors (TFs) are necessary and sufficient for axon guidance and RGC fate specification. These findings also uncover novel SoxC-dependent mechanisms that serve as master regulators during important steps of RGC development. For example, we review work showing that SoxC TFs regulate RGC axon guidance and direction through the optic chiasm towards their appropriate targets in the brain. We also review work demonstrating that Sox11 subcellular localization is, in part, controlled through small ubiquitin-like post-translational modifier (SUMO) and suggest compensatory cross-talk between Sox4 and Sox11. Furthermore, Sox4 overexpression is shown to positively drive RGC differentiation in human induced pluripotent stem cells (hiPSCs). Finally, we discuss how these findings may contribute to the advancement of regenerative and cell-based therapies to treat glaucoma and other optic nerve neuropathies.
View details for PubMedID 28852381
View details for PubMedCentralID PMC5558478
Characterization of Emodin as a Therapeutic Agent for Diabetic Cataract
JOURNAL OF NATURAL PRODUCTS
2016; 79 (5): 1439-1444
Aldose reductase (AR) in the lens plays an important role in the pathogenesis of diabetic cataract (DC) by contributing to osmotic and oxidative stress associated with accelerated glucose metabolism through the polyol pathway. Therefore, inhibition of AR in the lens may hold the key to prevent DC formation. Emodin, a bioactive compound isolated from plants, has been implicated as a therapy for diabetes. However, its inhibitory activity against AR remains unclear. Our results showed that emodin has good selectively inhibitory activity against AR (IC50 = 2.69 ± 0.90 μM) but not other aldo-keto reductases and is stable at 37 °C for at least 7 days. Enzyme kinetic studies demonstrated an uncompetitive inhibition against AR with a corresponding inhibition constant of 2.113 ± 0.095 μM. In in vivo studies, oral administration of emodin reduced the incidence and severity of morphological markers of cataract in lenses of AR transgenic mice. Computational modeling of the AR-NADP(+)-emodin ternary complex indicated that the 3-hydroxy group of emodin plays an essential role by interacting with Ser302 through hydrogen bonding in the specificity pocket of AR. All the findings above provide encouraging evidence for emodin as a potential therapeutic agent to prevent cataract in diabetic patients.
View details for DOI 10.1021/acs.jnatprod.6b00185
View details for Web of Science ID 000377150900027
View details for PubMedID 27140653
Aldose reductase mediates retinal microglia activation
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
2016; 473 (2): 565-571
Retinal microglia (RMG) are one of the major immune cells in charge of surveillance of inflammatory responses in the eye. In the absence of an inflammatory stimulus, RMG reside predominately in the ganglion layer and inner or outer plexiform layers. However, under stress RMG become activated and migrate into the inner nuclear layer (INL) or outer nuclear layer (ONL). Activated RMG in cell culture secrete pro-inflammatory cytokines in a manner sensitive to downregulation by aldose reductase inhibitors. In this study, we utilized CX3CR1(GFP) mice carrying AR mutant alleles to evaluate the role of AR on RMG activation and migration in vivo. When tested on an AR(WT) background, IP injection of LPS induced RMG activation and migration into the INL and ONL. However, this phenomenon was largely prevented by AR inhibitors or in AR null mice, or was exacerbated in transgenic mice that over-express AR. LPS-induced increases in ocular levels of TNF-α and CX3CL-1 in WT mice were substantially lower in AR null mice or were reduced by AR inhibitor treatment. These studies demonstrate that AR expression in RMG may contribute to the proinflammatory phenotypes common to various eye diseases such as uveitis and diabetic retinopathy.
View details for DOI 10.1016/j.bbrc.2016.03.122
View details for Web of Science ID 000374809700031
View details for PubMedID 27033597
Aldose Reductase Mediates Transforming Growth Factor beta 2 (TGF-beta 2)-InducedMigration and Epithelial-To-Mesenchymal Transition of Lens-Derived Epithelial Cells
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE
2015; 56 (8): 4198-4210
Cataract surgery involves removal of lens tissue, but is associated with a high complication rate due to regrowth of residual lens epithelial cells to produce posterior capsule opacification (PCO) and diminished visual acuity. As inhibitors of aldose reductase (AR) have been shown to suppress markers of PCO, our studies were designed to identify a role for AR in the pathogenesis of PCO.Sorbinil-mediated AR inhibition was determined by measuring sorbitol accumulation. Cell migration was measured using both transwell and scratch assays. Proteins in the SMAD signaling pathway were measured by Western blotting. The interactions of AR and SMADs were demonstrated by co-immunoprecipitation (Co-IP) and proximity ligation assay (PLA). Epithelial-to-mesenchymal transition (EMT) expression was measured by Western blot and quantitative PCR (q-PCR). Matrix metalloproteinase (MMP)-2 and MMP-9 activities were measured in conditioned medium by zymography.We observed that either Sorbinil-mediated AR inhibition or siRNA-mediated AR gene knockdown prevented migration of lens epithelial cells following exposure to TGF-β2. AR inhibition or AR knockdown reduced SMAD and MMP activation triggered by TGF-β2. In addition, we demonstrated AR inhibition or AR knockdown decreased TGF-β2-induced expression of EMT markers. Co-IP studies and PLA were used to demonstrate that AR and SMAD2 interact either directly or in close concert with additional factor(s) in a nonenzymatic manner.This study demonstrates that AR participates in the response of lens epithelial cells to TGF-β2. Our studies raise the possibility that AR inhibition may be effective in preventing development of PCO by disrupting the TGF-β2/SMAD pathway.
View details for DOI 10.1167/iovs.15-16557
View details for Web of Science ID 000362882700004
View details for PubMedID 26132779
View details for PubMedCentralID PMC4495811
Aldose reductase inhibition alleviates hyperglycemic effects on human retinal pigment epithelial cells
2015; 234: 254-260
Chronic hyperglycemia is an important risk factor involved in the onset and progression of diabetic retinopathy (DR). Among other effectors, aldose reductase (AR) has been linked to the pathogenesis of this degenerative disease. The purpose of this study was to investigate whether the novel AR inhibitor, beta-glucogallin (BGG), can offer protection against various hyperglycemia-induced abnormalities in human adult retinal pigment epithelial (ARPE-19) cells. AR is an enzyme that contributes to cellular stress by production of reactive oxygen species (ROS) under high glucose conditions. A marked decrease in cell viability (from 100% to 78%) following long-term exposure (4 days) of RPE cells to high glucose (HG) was largely prevented by siRNA-mediated knockdown of AR gene expression (from 79% to 97%) or inhibition using sorbinil (from 66% to 86%). In HG, BGG decreased sorbitol accumulation (44%), ROS production (27%) as well as ER stress (22%). Additionally, we demonstrated that BGG prevented loss of mitochondrial membrane potential (MMP) under HG exposure. We also showed that AR inhibitor pretreatment reduced retinal microglia-induced apoptosis in APRE-19 cells. These results suggest that BGG may be useful as a therapeutic agent against retinal degeneration in the diabetic eye by preventing RPE cell death.
View details for DOI 10.1016/j.cbi.2014.10.007
View details for Web of Science ID 000355044000030
View details for PubMedID 25451566
View details for PubMedCentralID PMC4402120
Aldose reductase expression as a risk factor for cataract
2015; 234: 247-253
Aldose reductase (AR) is thought to play a role in the pathogenesis of diabetic eye diseases, including cataract and retinopathy. However, not all diabetics develop ocular complications. Paradoxically, some diabetics with poor metabolic control appear to be protected against retinopathy, while others with a history of excellent metabolic control develop severe complications. These observations indicate that one or more risk factors may influence the likelihood that an individual with diabetes will develop cataracts and/or retinopathy. We hypothesize that an elevated level of AR gene expression could confer higher risk for development of diabetic eye disease. To investigate this hypothesis, we examined the onset and severity of diabetes-induced cataract in transgenic mice, designated AR-TG, that were either heterozygous or homozygous for the human AR (AKR1B1) transgene construct. AR-TG mice homozygous for the transgene demonstrated a conditional cataract phenotype, whereby they developed lens vacuoles and cataract-associated structural changes only after induction of experimental diabetes; no such changes were observed in AR-TG heterozygotes or nontransgenic mice with or without experimental diabetes induction. We observed that nondiabetic AR-TG mice did not show lens structural changes even though they had lenticular sorbitol levels almost as high as the diabetic AR-TG lenses that showed early signs of cataract. Over-expression of AR led to increases in the ratio of activated to total levels of extracellular signal-regulated kinase (ERK1/2) and c-Jun N-terminal (JNK1/2), which are known to be involved in cell growth and apoptosis, respectively. After diabetes induction, AR-TG but not WT controls had decreased levels of phosphorylated as well as total ERK1/2 and JNK1/2 compared to their nondiabetic counterparts. These results indicate that high AR expression in the context of hyperglycemia and insulin deficiency may constitute a risk factor that could predispose the lens to disturbances in signaling through the ERK and JNK pathways and thereby alter the balance of cell growth and apoptosis that is critical to lens transparency and homeostasis.
View details for DOI 10.1016/j.cbi.2014.12.017
View details for Web of Science ID 000355044000029
View details for PubMedID 25541468
Aldose Reductase Inhibition Prevents Endotoxin-Induced Inflammatory Responses in Retinal Microglia
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE
2014; 55 (5): 2853-2861
Retinal microglia become activated in diabetes and produce pro-inflammatory molecules associated with changes in retinal vasculature and increased apoptosis of retinal neurons and glial cells. We sought to determine if the action of aldose reductase (AR), an enzyme linked to the pathogenesis of diabetic retinopathy, contributes to activation of microglial cells.Involvement of AR in the activation process was studied using primary cultures of retinal microglia (RMG) isolated from wild-type and AR-null mice, or in mouse macrophage cultures treated with either AR inhibitors or small interfering RNA (siRNA) directed to AR. Inflammatory cytokines were measured by ELISA. Cell migration was measured using a transwell assay. Gelatin zymography was used to detect active matrix metalloproteinase (MMP)-9, while RMG-induced apoptosis of adult retinal pigment epithelium (ARPE-19) cells was studied in a cell coculture system.Aldose reductase inhibition or genetic deficiency substantially reduced lipopolysacharide (LPS)-induced cytokine secretion from macrophages and RMG. Aldose reductase inhibition or deficiency also reduced the activation of MMP-9 and attenuated LPS-induced cell migration. Additionally, blockade of AR by sorbinil or through genetic means caused a reduction in the ability of activated RMG to induce apoptosis of ARPE-19 cells.These results demonstrate that the action of AR contributes to the activation of RMG. Inhibition of AR may be a therapeutic strategy to reduce inflammation associated with activation of RMG in disease.
View details for DOI 10.1167/iovs.13-13487
View details for Web of Science ID 000339484800006
View details for PubMedID 24677107
View details for PubMedCentralID PMC4010364
Design of an Amide N-Glycoside Derivative of beta-Glucogallin: A Stable, Potent, and Specific Inhibitor of Aldose Reductase
JOURNAL OF MEDICINAL CHEMISTRY
2014; 57 (1): 71-77
β-Glucogallin (BGG), a major component of the Emblica officinalis medicinal plant, is a potent and selective inhibitor of aldose reductase (AKR1B1). New linkages (ether/triazole/amide) were introduced via high yielding, efficient syntheses to replace the labile ester, and an original two-step (90%) preparation of BGG was developed. Inhibition of AKR1B1was assessed in vitro and using transgenic lens organ cultures, which identified the amide linked glucoside (BGA) as a stable, potent, and selective therapeutic lead toward the treatment of diabetic eye disease.
View details for DOI 10.1021/jm401311d
View details for Web of Science ID 000329677800006
View details for PubMedID 24341381
Beta-glucogallin reduces the expression of lipopolysaccharide-induced inflammatory markers by inhibition of aldose reductase in murine macrophages and ocular tissues
2013; 202 (1-3): 283-287
Aldose reductase (AR) catalyzes the reduction of toxic lipid aldehydes to their alcohol products and mediates inflammatory signals triggered by lipopolysaccharide (LPS). Beta-glucogallin (BGG), a recently described AR inhibitor, was purified from extracts of the Indian gooseberry (Emblica officinalis). In this study, we found that BGG showed low cytotoxicity in Raw264.7 murine macrophages and effectively inhibited AR activity as measured by a decrease in sorbitol accumulation. In addition, BGG-mediated inhibition of AR prevented LPS-induced activation of JNK and p38 and lowered ROS levels, which could inhibit LPS-induced apoptosis. Uveitis is a disease of the eye associated with chronic inflammation. In this study, we also demonstrated that treatment with BGG decreased the number of inflammatory cells that infiltrate the ocular media of mice with experimental uveitis. Accordingly, these results suggest BGG is a potential therapy for inflammatory diseases.
View details for DOI 10.1016/j.cbi.2012.12.001
View details for Web of Science ID 000317539500035
View details for PubMedID 23247009
View details for PubMedCentralID PMC3656825
Eicosapentaenoic acid and docosahexaenoic acid inhibit macrophage-induced gastric cancer cell migration by attenuating the expression of matrix metalloproteinase 10
JOURNAL OF NUTRITIONAL BIOCHEMISTRY
2012; 23 (11): 1434-1439
Uptake of docosahexaenoic acid (DHA)/eicosapentaenoic acid (EPA) improves the treatment of cancer and reduces tumor-associated macrophage count. However, the mechanism of this relationship is still unclear. In this study, macrophages enhanced gastric cancer cell migration ability and induced the differentially expressed matrix metalloproteinase genes (MMP1, MMP3 and MMP10) of N87 as identified by polymerase chain reaction array. Furthermore, DHA and EPA inhibited macrophage-enhanced cancer cell migration and attenuated MMP10 at both the RNA and protein level. The suppression of MMP10 expression was further verified by zymography and antibody blocking experiments. Additionally, DHA and EPA attenuated expression of macrophage-activated extracellular-signal-regulated kinase (ERK) and signal transducers and activators of transcription 3 (STAT3) in cancer cells. Attenuation was verified by demonstrating blockade with specific inhibitors and thereby increased MMP10 expression. Accordingly, we hypothesized that macrophage enhances cancer cell migration through ERK and STAT3 phosphorylation and subsequent increased MMP10 expression and that DHA and EPA could attenuate these signals. These findings not only explain the beneficial effects of DHA/EPA, but also point to ERK/STAT3/MMP10 as the potential targets for gastric cancer treatment.
View details for DOI 10.1016/j.jnutbio.2011.09.004
View details for Web of Science ID 000310490000009
View details for PubMedID 22285823
Cholesterol regulation of receptor-interacting protein 140 via microRNA-33 in inflammatory cytokine production
2011; 25 (5): 1758-1766
Receptor interacting protein 140 (RIP140) is a nuclear receptor coregulator that affects a wide spectrum of biological processes. It is unclear whether and how the expression level of RIP140 can be modulated and whether RIP140 is involved in inflammatory diseases. Here, we examine how intracellular cholesterol regulates RIP140 expression, and we evaluate the effect of RIP140 expression on macrophage proinflammatory potential. Macrophages treated with modified low-density lipoprotein express higher RIP140 mRNA and protein levels. Consistently, simvastatin reduces RIP140 levels, which can be reversed by mevalonate. Moreover, a high-fat diet elevates RIP140 but lowers miR-33 levels in peritoneal macrophages, and increases the production of IL-1β and TNF-α in macrophages. Mechanistically, miR-33 targets RIP140 mRNA by recognizing its target located in a highly conserved sequence of the 3'-untranslated region (3'-UTR) of RIP140 mRNA. Consequentially, miR-33 reduces RIP140 coactivator activity for NF-κB, which is supported by the reduction in NF-κB reporter activity and the inflammatory potential in macrophages. This study uncovers a cholesterol-miR-33-RIP140 regulatory pathway that modulates the proinflammatory potential in macrophages in response to an alteration in the intracellular cholesterol status, and identifies RIP140 as a direct target of miR-33 that mediates simvastatin-triggered anti-inflammation.
View details for DOI 10.1096/fj.10-179267
View details for Web of Science ID 000290023800031
View details for PubMedID 21285396
Serum vascular endothelial growth factor-D levels correlate with cervical lymph node metastases in papillary thyroid carcinoma
2011; 29 (2-3): 57-62
The aim of this multicenter study was to evaluate the clinical relevance of serum vascular endothelial growth factor-D (VEGF-D) in papillary thyroid carcinoma (PTC). This prospective study consisted of 74 patients with primary PTC and 15 patients with benign thyroid nodules treated from 2008 to 2009. VEGF-D concentration was compared with patient clinicopathologic features and lymph node metastases. There was no significant difference in mean serum VEGF-D levels between the PTC and benign thyroid nodule groups. Within the PTC group, serum VEGF-D levels were significantly higher in patients with lymph node metastases than in patients without metastases (241.92 vs. 213.89 pg/ml, respectively; P = 0.035). Receiver operating characteristic curve analysis revealed that preoperative serum VEGF-D levels were predictive of lymph node metastases in the patients >45 years. Serum VEGF-D level that was correlated with the presence of cervical lymph node metastases in PTC patients might be a useful prognostic indicator.
View details for DOI 10.3109/08977194.2011.557373
View details for Web of Science ID 000290407300002
View details for PubMedID 21319951
Gefitinib-induced epidermal growth factor receptor-independent keratinocyte apoptosis is mediated by the JNK activation pathway
BRITISH JOURNAL OF DERMATOLOGY
2011; 164 (1): 38-46
Gefitinib (ZD1839) is a selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor with a significant antitumour effect on various cancers. Skin toxicity induced by gefitinib is common, and has been shown to be related to the inhibition of EGFR signalling pathways. However, other mechanisms may be involved in gefitinib-induced skin toxicity.To study the possible EGFR-independent mechanisms of gefitinib-induced skin toxicity.The human immortalized keratinocyte cell line HaCaT and human lung adenocarcinoma cell lines (A549 and PC9) were treated with different concentrations of gefitinib for 24, 48 and 72 h. Cell viability was measured by MTT assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] after EGFR gene silencing. The signalling pathways were investigated by immunoblot analysis. Keratinocyte apoptosis was evaluated by nuclear condensation and flow cytometric analysis.Gefitinib maintained its cytotoxicity to HaCaT cells after EGFR gene silencing, indicating that an EGFR-independent mechanism exists. Increased phosphorylation of p38 mitogen-activated protein kinase and JNK by gefitinib was observed in a dose-dependent manner in HaCaT cells. The JNK inhibitor, SP600125, attenuated the gefitinib-induced cytotoxicity and apoptosis of HaCaT cells. Immunohistochemical examination of patient specimens showed an increased expression of phosphorylated JNK in lesional epidermis compared with nonlesional epidermis.Gefitinib can induce keratinocyte apoptosis through an EGFR-independent JNK activation pathway.
View details for DOI 10.1111/j.1365-2133.2010.10038.x
View details for Web of Science ID 000285752000008
View details for PubMedID 20846305
TNF-alpha Mediates Eosinophil Cationic Protein-induced Apoptosis in BEAS-2B Cells
BMC CELL BIOLOGY
Eosinophilic granulocytes are important for the human immune system. Many cationic proteins with cytotoxic activities, such as eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN), are released from activated eosinophils. ECP, with low RNase activity, is widely used as a biomarker for asthma. ECP inhibits cell viability and induces apoptosis to cells. However, the specific pathway underlying the mechanisms of ECP-induced cytotoxicity remains unclear. This study investigated ECP-induced apoptosis in bronchial epithelial BEAS-2B cells and elucidated the specific pathway during apoptosis.To address the mechanisms involved in ECP-induced apoptosis in human BEAS-2B cells, investigation was carried out using chromatin condensation, cleavage of poly (ADP-ribose) polymerase (PARP), sub-G1 distribution in cell cycle, annexin V labeling, and general or specific caspase inhibitors. Caspase-8-dependent apoptosis was demonstrated by cleavage of caspase-8 after recombinant ECP treatment, accompanied with elevated level of tumor necrosis factor alpha (TNF-alpha). Moreover, ECP-induced apoptosis was effectively inhibited in the presence of neutralizing anti-TNF-alpha antibody.In conclusion, our results have demonstrated that ECP increased TNF-alpha production in BEAS-2B cells and triggered apoptosis by caspase-8 activation through mitochondria-independent pathway.
View details for Web of Science ID 000274599300002
View details for PubMedID 20089176
Control Mechanisms of Differential Translation of Hsp90 Isoforms in 9L Rat Gliosarcoma Cells
JOURNAL OF CELLULAR BIOCHEMISTRY
2009; 107 (3): 418-427
Although the differential expression of heat shcok proteins, Hsp90alpha and Hsp90beta was extensively studied in many kinds of cells, the post-transcriptional regulation of Hsp90 isoforms remains unclear. In control and GA-treated rat gliosarcoma cells, it has been reported that the translational efficiency of hsp90alpha is higher than hsp90beta. In this study, we present evidences identifying the roles for leaky scanning and 5'-UTR sequence in translational regulation of Hsp90beta. The result of in vitro transcription and translation (IVTT) experiment showed that hsp90alpha exhibited higher translation efficiency than hsp90beta. Sequence analysis revealed that there is an out-of-frame downstream AUG codon in hsp90beta gene. However, elimination of the downstream AUG by site-directly mutagenesis or introducing Kozak context sequence around the initiator AUG of hsp90beta open reading frame increased its translational efficiency, which indicated that leaky scanning might be a possible mechanism regulating hsp90beta. Furthermore, we also constructed a firefly luciferase reporter system to verify the effect of subsequent translation at the downstream out-of-frame AUG codon in 9L and A549 cells. Furthermore, it is believed that 5'-untranslated region (5'-UTR) also plays a significant role in translational control. We showed hsp90beta 5'-UTR gives rise to the reduction of the translation efficiency in IVTT experiment. Additionally, the reductive effect of hsp90beta 5'-UTR was further confirmed by luciferase reporter assay using truncated deletion analyses of 5'-UTR of hsp90beta. Our results support the hypothesis that ribosome leaky scanning mechanism and 5'-UTR sequence acts as negative regulators in hsp90beta mRNA.
View details for DOI 10.1002/jcb.22138
View details for Web of Science ID 000266872300006
View details for PubMedID 19308988
Concerted actions of multiple transcription elements confer differential Transactivation of HSP90 isoforms in geldanamycin-treated 9L rat gliosarcoma cells
JOURNAL OF CELLULAR BIOCHEMISTRY
2008; 104 (4): 1286-1296
HSP90 chaperones are transducer proteins of many signaling pathways in cells. Using a highly specific inhibitor, geldanamycin (GA), an increasing number of the HSP90 client proteins have been identified. Nevertheless, there is little information on the differential transactivation of the two isoforms of the hsp90 genes, hsp90alpha and beta, in cells under stress conditions. Here, we demonstrate the differential expression of the HSP90 isoforms, HSP90alpha and beta, in rat gliosarcoma 9L cells using a modified SDS-PAGE system that allowed us to distinguish the isoforms. We subsequently assessed the transcriptional controls involving the transcription elements located in the promoter regions of the hsp90 genes. At the protein level, HSP90alpha is more responsive to GA in terms of rate of de novo synthesis and amount of accumulation, as shown by metabolic-labeling and Western-blotting analyses. Upregulation of the hsp90 genes was demonstrated by real-time qPCR. The promoter elements hsp90alpha-HSE2 and hsp90beta-HSE1 were also identified to be the major transcription elements involved in GA-activated gene expression, as shown by EMSA, whereas the results of supershift showed that the transcription factor HSF1 is also involved. Moreover, EMSA results of analysis of the GC box showed differences in both the initial amounts and inductive response of hsp90s transcripts, whereas analysis of the TATA box showed GA responsiveness in hsp90alpha only. Collectively, these results indicate that GA exerts its regulatory effects through transcription elements including heat-shock elements (HSEs), GC boxes and TATA boxes, resulting in differential transactivation of hsp90alpha and hsp90beta in rat gliosarcoma 9L cells.
View details for DOI 10.1002/jcb.21705
View details for Web of Science ID 000257567300014
View details for PubMedID 18320580