Browsing by Author "Qi, Xiaoping"
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Item BACE1 Inhibition Increases Susceptibility to Oxidative Stress by Promoting Mitochondrial Damage(MDPI, 2021-09-28) Francelin, Carolina; Mitter, Sayak K.; Qian, Qingwen; Barodia, Sandeep Kumar; Ip, Colin; Qi, Xiaoping; Gu, Hongmei; Quigley, Judith; Goldberg, Matthew S.; Grant, Maria B.; Boulton, Michael E.; Ophthalmology, School of MedicineBACE1 is a key enzyme facilitating the generation of neurotoxic β-amyloid (Aβ) peptide. However, given that BACE1 has multiple substrates we explored the importance of BACE1 in the maintenance of retinal pigment epithelial (RPE) cell homeostasis under oxidative stress. Inhibition of BACE1 reduced mitochondrial membrane potential, increased mitochondrial fragmentation, and increased cleaved caspase-3 expression in cells under oxidative stress. BACE1 inhibition also resulted in significantly lower levels of mitochondrial fusion proteins OPA1 and MFN1 suggesting a higher rate of mitochondrial fission while increasing the levels of mitophagic proteins Parkin and PINK1 and autophagosome numbers. In contrast, BACE2 had minimal effect on cellular response to oxidative stress. In summary, our results emphasize the importance of BACE1 in augmenting cellular defense against oxidative stress by protecting mitochondrial dynamics.Item Chemical Proteomics Reveals Soluble Epoxide Hydrolase as a Therapeutic Target for Ocular Neovascularization(ACS, 2018) Sulaiman, Rania S.; Park, Bomina; Sardar Pasha, Sheik Pran Babu; Si, Yubing; Kharwadkar, Rakshin; Mitter, Sayak K.; Lee, Bit; Sun, Wei; Qi, Xiaoping; Boulton, Michael E.; Meroueh, Samy; Fei, Xiang; Seo, Seung-Yong; Corson, Timothy W.; Ophthalmology, School of MedicineThe standard-of-care therapeutics for the treatment of ocular neovascular diseases like wet age-related macular degeneration (AMD) are biologics targeting vascular endothelial growth factor signaling. There are currently no FDA approved small molecules for treating these blinding eye diseases. Therefore, therapeutic agents with novel mechanisms are critical to complement or combine with existing approaches. Here, we identified soluble epoxide hydrolase (sEH), a key enzyme for epoxy fatty acid metabolism, as a target of an antiangiogenic homoisoflavonoid, SH-11037. SH-11037 inhibits sEH in vitro and in vivo and docks to the substrate binding cleft in the sEH hydrolase domain. sEH levels and activity are up-regulated in the eyes of a choroidal neovascularization (CNV) mouse model. sEH is overexpressed in human wet AMD eyes, suggesting that sEH is relevant to neovascularization. Known sEH inhibitors delivered intraocularly suppressed CNV. Thus, by dissecting a bioactive compound’s mechanism, we identified a new chemotype for sEH inhibition and characterized sEH as a target for blocking the CNV that underlies wet AMD.Item Decreased Expression of Soluble Epoxide Hydrolase Suppresses Murine Choroidal Neovascularization(MDPI, 2022-12) Park, Bomina; Sardar Pasha, Sheik Pran Babu; Sishtla, Kamakshi L.; Hartman, Gabriella D.; Qi, Xiaoping; Boulton, Michael E.; Corson, Timothy W.; Ophthalmology, School of MedicineNeovascular or “wet” age-related macular degeneration (nAMD) is a leading cause of blindness among older adults. Choroidal neovascularization (CNV) is a major pathological feature of nAMD, in which abnormal new blood vessel growth from the choroid leads to irreversible vision loss. There is a critical need to develop novel therapeutic strategies to address limitations of the current anti-vascular endothelial growth factor biologics. Previously, we identified soluble epoxide hydrolase (sEH) as a possible therapeutic target for CNV through a forward chemical genetic approach. The purpose of this study was to validate sEH as a target by examining retinal expression of sEH protein and mRNA by immunohistochemistry and RNAscope in situ hybridization, respectively, and to assess the efficacy of an adeno-associated virus (AAV) vector designed to knock down the sEH gene, Ephx2, in the murine laser-induced (L-) CNV model. nAMD patient postmortem eye tissue and murine L-CNV showed overexpression of sEH in photoreceptors and retinal pigment epithelial cells. Ephx2 knockdown significantly reduced CNV and normalized mRNA expression levels of CNV-related inflammatory markers. Thus, this study further establishes sEH as a promising therapeutic target against CNV associated with nAMD.Item Dysregulated autophagy in the RPE is associated with increased susceptibility to oxidative stress and AMD(Landes Bioscience, 2014) Mitter, Sayak K.; Song, Chunjuan; Qi, Xiaoping; Mao, Haoyu; Rao, Haripriya; Akin, Debra; Lewin, Alfred; Grant, Maria; Dunn, William; Ding, Jindong; Bowes Rickman, Catherine; Boulton, Michael; Department of Ophthalmology, IU School of MedicineAutophagic dysregulation has been suggested in a broad range of neurodegenerative diseases including age-related macular degeneration (AMD). To test whether the autophagy pathway plays a critical role to protect retinal pigmented epithelial (RPE) cells against oxidative stress, we exposed ARPE-19 and primary cultured human RPE cells to both acute (3 and 24 h) and chronic (14 d) oxidative stress and monitored autophagy by western blot, PCR, and autophagosome counts in the presence or absence of autophagy modulators. Acute oxidative stress led to a marked increase in autophagy in the RPE, whereas autophagy was reduced under chronic oxidative stress. Upregulation of autophagy by rapamycin decreased oxidative stress-induced generation of reactive oxygen species (ROS), whereas inhibition of autophagy by 3-methyladenine (3-MA) or by knockdown of ATG7 or BECN1 increased ROS generation, exacerbated oxidative stress-induced reduction of mitochondrial activity, reduced cell viability, and increased lipofuscin. Examination of control human donor specimens and mice demonstrated an age-related increase in autophagosome numbers and expression of autophagy proteins. However, autophagy proteins, autophagosomes, and autophagy flux were significantly reduced in tissue from human donor AMD eyes and 2 animal models of AMD. In conclusion, our data confirm that autophagy plays an important role in protection of the RPE against oxidative stress and lipofuscin accumulation and that impairment of autophagy is likely to exacerbate oxidative stress and contribute to the pathogenesis of AMD.Item Ferrochelatase is a therapeutic target for ocular neovascularization(Wiley, 2017) Basavarajappa, Halesha D.; Sulaiman, Rania S.; Qi, Xiaoping; Shetty, Trupti; Babu, Sardar Sheik Pran; Sishtla, Kamakshi L.; Lee, Bit; Quigley, Judith; Alkhairy, Sameerah; Briggs, Christian M.; Gupta, Kamna; Tang, Buyun; Shadmand, Mehdi; Grant, Maria B.; Boulton, Michael E.; Seo, Seung-Yong; Corson, Timothy W.; Department of Ophthalmology, IU School of MedicineOcular neovascularization underlies major blinding eye diseases such as “wet” age-related macular degeneration (AMD). Despite the successes of treatments targeting the vascular endothelial growth factor (VEGF) pathway, resistant and refractory patient populations necessitate discovery of new therapeutic targets. Using a forward chemical genetic approach, we identified the heme synthesis enzyme ferrochelatase (FECH) as necessary for angiogenesis in vitro and in vivo. FECH is overexpressed in wet AMD eyes and murine choroidal neovascularization; siRNA knockdown of Fech or partial loss of enzymatic function in the Fechm1Pas mouse model reduces choroidal neovascularization. FECH depletion modulates endothelial nitric oxide synthase function and VEGF receptor 2 levels. FECH is inhibited by the oral antifungal drug griseofulvin, and this compound ameliorates choroidal neovascularization in mice when delivered intravitreally or orally. Thus, FECH inhibition could be used therapeutically to block ocular neovascularization.Item Impaired Autophagy Diurnal Rhythmicity in Rodent Diabetic Retinopathy(Office of the Vice Chancellor for Research, 2015-04-17) Qi, Xiaoping; Mitter, Sayak; Yan, Yuanqing; Dunn, William; Busik, Juliet; Grant, Maria; Boulton, MichelePurpose: Retinal homeostasis is under both diurnal and circadian regulation. However, diurnal changes in retinal autophagy have not been hitherto explored. We sought to investigate the diurnal expression of autophagy proteins/genes in normal rodent retina to determine if this is impaired in diabetic retinopathy. Methods: Eyes from C57BL/6 mice and BBZ rats maintained under a 12h/12h; 6am/6pm light/dark cycle were enucleated every 2 or 3 hours over a 24 hour period. Eyes were also collected from C57BL/6 induced STZ for 2 or 9 month as type 1 and BBZDR/wor type 2 diabetic rats for 4 months. Immunohistochemistry, Western-blot and real-time PCR were performed for Atg7, Atg9, LC3 and Beclin. Retina vessel pathology and superoxide were assessed by enzyme digestion and a spectrofluorometer. Results: Autophagy proteins (Atgs) were abundantly expressed in neural retina and endothelia cells in both mice and rats with differential staining pattern across the retinas and demonstrated a distinctive diurnal rhythmicity. All Atgs showed localization to retinal blood vessels with Atg7 being the most highly expressed. Analysis of the immunostaining demonstrated distinctive diurnal rhythmicity of which Atg9 and LC3 shared a biphasic expression cycle with the highest level at 8:15 am and 8:15 pm. By contrast, Beclin revealed a 24-hour cycle with the highest level observed at midnight. Atg7 was also on a 24-hour cycle with peak expression at 8:15am, coinciding with the first peak expression of Atg9 and LC3. In diabetic animals, immunohistochemistry showed dramatic reduction in all four Atgs and this was further confirmed by Western Blot, especially a decrease in LC3II/LC3I ratio (a measure of autophagy flux). Furthermore, the distinctive diurnal rhythmicity of these autophagy proteins was significantly impaired and phase shifted in diabetic animals. Conclusions: Autophagy proteins show both spatial and diurnal-dependent expression in normal rodent retinas and this is severely impaired and phase shifted in both type 1 and type 2 diabetic animals. Decreased autophagy in diabetic animals may in part explain the increased generation of reactive oxygen species in diabetic retinopathy. Therefore, restoration of diurnal rhythmicity and facilitating autophagy pathway expression may provide new treatment strategies for diabetic retinopathy.Item Improving the Transduction of Bone Marrow-Derived Cells with an Integrase-Defective Lentiviral Vector(Mary Ann Liebert, Inc., 2018-02) Pay, S. Louise; Qi, Xiaoping; Willard, Jeffrey F.; Godoy, Juliana; Sankhavaram, Kavya; Horton, Ranier; Mitter, Sayak K.; Quigley, Judith L.; Chang, Lung-Ji; Grant, Maria B.; Boulton, Michael E.; Medical and Molecular Genetics, School of MedicineIn lentiviral vector (LV) applications where transient transgene expression is sufficient, integrase-defective lentiviral vectors (IDLVs) are beneficial for reducing the potential for off-target effects associated with insertional mutagenesis. It was previously demonstrated that human RPE65 mRNA expression from an integrating lentiviral vector (ILV) induces endogenous Rpe65 and Cralbp mRNA expression in murine bone marrow-derived cells (BMDCs), initiating programming of the cells to retinal pigment epithelium (RPE)-like cells. These cells regenerate RPE in retinal degeneration models when injected systemically. As transient expression of RPE65 is sufficient to activate endogenous RPE-associated genes for programming BMDCs, use of an ILV is an unnecessary risk. In this study, an IDLV expressing RPE65 (IDLV3-RPE65) was generated. Transduction with IDLV3-RPE65 is less efficient than the integrating vector (ILV3-RPE65). Therefore, IDLV3-RPE65 transduction was enhanced with a combination of preloading 20 × -concentrated viral supernatant on RetroNectin at a multiplicity of infection of 50 and transduction of BMDCs by low-speed centrifugation. RPE65 mRNA levels increased from ∼12-fold to ∼25-fold (p < 0.05) after modification of the IDLV3-RPE65 transduction protocol, achieving expression similar to the ∼27-fold (p < 0.05) increase observed with ILV3-RPE65. Additionally, the study shows that the same preparation of RetroNectin can be used to coat up to three wells with no reduction in transduction. Critically, IDLV3-RPE65 transduction initiates endogenous Rpe65 mRNA expression in murine BMDCs and Cralbp/CRALBP mRNA in both murine and human BMDCs, similar to expression observed in ILV3-RPE65-transduced cells. Systemic administration of ILV3-RPE65 or IDLV3-RPE65 programmed BMDCs in a mouse model of retinal degeneration is sufficient to retain visual function and reduce retinal degeneration compared to mice receiving no treatment or naïve BMDC. It is concluded that IDLV3-RPE65 is appropriate for programming BMDCs to RPE-like cells.Item A novel small molecule ameliorates ocular neovascularisation and synergises with anti-VEGF therapy(Nature, 2016-05-05) Sulaiman, Rania S.; Merrigan, Stephanie; Quigley, Judith; Qi, Xiaoping; Lee, Bit; Boulton, Michael E.; Kennedy, Breandán; Seo, Seung-Yong; Corson, Timothy W.; Department of Ophthalmology, IU School of MedicineOcular neovascularisation underlies blinding eye diseases such as retinopathy of prematurity, proliferative diabetic retinopathy, and wet age-related macular degeneration. These diseases cause irreversible vision loss, and provide a significant health and economic burden. Biologics targeting vascular endothelial growth factor (VEGF) are the major approach for treatment. However, up to 30% of patients are non-responsive to these drugs and they are associated with ocular and systemic side effects. Therefore, there is a need for small molecule ocular angiogenesis inhibitors to complement existing therapies. We examined the safety and therapeutic potential of SH-11037, a synthetic derivative of the antiangiogenic homoisoflavonoid cremastranone, in models of ocular neovascularisation. SH-11037 dose-dependently suppressed angiogenesis in the choroidal sprouting assay ex vivo and inhibited ocular developmental angiogenesis in zebrafish larvae. Additionally, intravitreal SH-11037 (1 μM) significantly reduced choroidal neovascularisation (CNV) lesion volume in the laser-induced CNV mouse model, comparable to an anti-VEGF antibody. Moreover, SH-11037 synergised with anti-VEGF treatments in vitro and in vivo. Up to 100 μM SH-11037 was not associated with signs of ocular toxicity and did not interfere with retinal function or pre-existing retinal vasculature. SH-11037 is thus a safe and effective treatment for murine ocular neovascularisation, worthy of further mechanistic and pharmacokinetic evaluation.Item Oxidative stress-mediated NFκB phosphorylation upregulates p62/SQSTM1 and promotes retinal pigmented epithelial cell survival through increased autophagy(Plos, 2017-02-21) Song, Chunjuan; Mitter, Sayak K.; Qi, Xiaoping; Beli, Eleni; Rao, Haripriya V.; Ding, Jindong; Ip, Colin S.; Gu, Hongmei; Akin, Debra; Dunn, William A. Jr.; Bowes Rickman, Catherine; Lewin, Alfred S.; Grant, Maria B.; Boulton, Michael E.; Department of Ophthalmology, IU School of Medicinep62 is a scaffolding adaptor implicated in the clearance of protein aggregates by autophagy. Reactive oxygen species (ROS) can either stimulate or inhibit NFκB-mediated gene expression influencing cellular fate. We studied the effect of hydrogen peroxide (H2O2)-mediated oxidative stress and NFκB signaling on p62 expression in the retinal pigment epithelium (RPE) and investigated its role in regulation of autophagy and RPE survival against oxidative damage. Cultured human RPE cell line ARPE-19 and primary human adult and fetal RPE cells were exposed to H2O2-induced oxidative stress. The human apolipoprotein E4 targeted-replacement (APOE4) mouse model of AMD was used to study expression of p62 and other autophagy proteins in the retina. p62, NFκB p65 (total, phosphorylated, nuclear and cytoplasmic) and ATG10 expression was assessed by mRNA and protein analyses. Cellular ROS and mitochondrial superoxide were measured by CM-H2DCFDA and MitoSOX staining respectively. Mitochondrial viability was determined using MTT activity. qPCR-array system was used to investigate autophagic genes affected by p62. Nuclear and cytoplasmic levels of NFκB p65 were evaluated after cellular fractionation by Western blotting. We report that p62 is up-regulated in RPE cells under H2O2-induced oxidative stress and promotes autophagic activity. Depletion of endogenous p62 reduces autophagy by downregulation of ATG10 rendering RPE more susceptible to oxidative damage. NFκB p65 phosphorylation at Ser-536 was found to be critical for p62 upregulation in response to oxidative stress. Proteasome inhibition by H2O2 causes p62-NFκB signaling as antioxidant pre-treatment reversed p62 expression and p65 phosphorylation when RPE was challenged by H2O2 but not when by Lactacystin. p62 protein but not RNA levels are elevated in APOE4-HFC AMD mouse model, suggesting reduction of autophagic flux in disease conditions. Our findings suggest that p62 is necessary for RPE cytoprotection under oxidative stress and functions, in part, by modulating ATG10 expression. NFκB p65 activity may be a critical upstream initiator of p62 expression in RPE cells under oxidative stress.Item PPARβ/δ selectively regulates phenotypic features of age-related macular degeneration.(Impact Journals, 2016-09) Choudhary, Mayur; Ding, Jin-dong; Qi, Xiaoping; Boulton, Michael E.; Yao, Pei-Li; Peters, Jeffrey M.; Malek, Goldis; Department of Ophthalmology, IU School of MedicinePeroxisome proliferator-activated receptor-β/δ (PPARβ/δ) is a nuclear receptor that regulates differentiation, inflammation, lipid metabolism, extracellular matrix remodeling, and angiogenesis in multiple tissues. These pathways are also central to the pathogenesis of age-related macular degeneration (AMD), the leading cause of vision loss globally. With the goal of identifying signaling pathways that may be important in the development of AMD, we investigated the impact of PPARβ/δ activation on ocular tissues affected in the disease. PPARβ/δ is expressed and can be activated in AMD vulnerable cells, including retinal pigment epithelial (RPE) and choroidal endothelial cells. Further, PPARβ/δ knockdown modulates AMD-related pathways selectively. Specifically, genetic ablation of Pparβ/δ in aged mice resulted in exacerbation of several phenotypic features of early dry AMD, but attenuation of experimentally induced choroidal neovascular (CNV) lesions. Antagonizing PPARβ/δ in both in vitro angiogenesis assays and in the in vivo experimentally induced CNV model, inhibited angiogenesis and angiogenic pathways, while ligand activation of PPARβ/δ, in vitro, decreased RPE lipid accumulation, characteristic of dry AMD. This study demonstrates for the first time, selective regulation of a nuclear receptor in the eye and establishes that selective targeting of PPARβ/δ may be a suitable strategy for treatment of different clinical sub-types of AMD.