Browsing by Author "Luo, Qianyi"
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Item Advanced glycation end (AGE) product modification of laminin downregulates Kir4.1 in retinal Müller cells(PLOS, 2018-02-23) Thompson, Kayla; Chen, Jonathan; Luo, Qianyi; Xiao, Yucheng; Cummins, Theodore R.; Bhatwadekar, Ashay D.; Ophthalmology, School of MedicineDiabetic retinopathy (DR) is a major cause of adult blindness. Retinal Müller cells maintain water homeostasis and potassium concentration via inwardly rectifying Kir4.1 channels. Accumulation of advanced glycation end products (AGEs) is a major pathologic event in DR. While diabetes leads to a decrease in the Kir4.1 channels, it remains unknown whether AGEs-linked to the basement membrane (BM) affect normal Kir4.1 channels. For this study, we hypothesized that AGE-modification of laminin is detrimental to Kir4.1 channels, therefore, disrupting Müller cell function. The AGE-modified laminin-coated substrates were prepared by incubating Petri-dishes with laminin and methylglyoxal for seven days. The rat Müller cells (rMC-1) were propagated on AGE-modified laminin, and Kir4.1 expression and function were evaluated. Quantification of AGEs using ELISA revealed a dose-dependent increase in methylglyoxal-hydro-imidazolone adducts. The rMC-1 propagated on AGE-modified laminin demonstrated a decrease in Kir4.1 levels in immunofluorescence and western blot studies and a decrease in the Kir4.1 channel function. Kir4.1 decrease on AGE-modified laminin resulted in a disorganization of an actin cytoskeleton and disruption of α-dystroglycan-syntrophin-dystrophin complexes. Our studies suggest that AGE-modification of laminin is detrimental to Kir4.1 channels. By studying the role of AGEs in Kir4.1 channels we have identified a novel mechanism of Müller cell dysfunction and its subsequent involvement in DR.Item Anti-integrin therapy for retinovascular diseases(Taylor & Francis, 2020) Bhatwadekar, Ashay D.; Kansara, Viral; Luo, Qianyi; Ciulla, Thomas; Ophthalmology, School of MedicineIntegrins are a family of multi-functional cell-adhesion molecules, heterodimeric receptors that connect extracellular matrix (ECM) to actin cytoskeleton in the cell cortex, thus regulating cellular adhesion, migration, proliferation, invasion, survival, and apoptosis. Consequently, integrins play a role in inflammation, angiogenesis and fibrosis.Item BMAL1 Overexpression in Suprachiasmatic Nucleus Protects from Retinal Neurovascular Deficits in Diabetes(bioRxiv, 2025-02-06) Mahajan, Neha; Luo, Qianyi; Lukkes, Jodi; Abhyankar, Surabhi D.; Bhatwadekar, Ashay D.; Ophthalmology, School of MedicineThe suprachiasmatic nucleus (SCN) regulates circadian rhythms and influences physiological and behavioral processes. Disruptions in circadian rhythms (CRD) are observed in type 2 diabetes (T2D), and importantly, CRD acts as an independent risk factor for T2D and its associated complications. BMAL1, a circadian clock gene, is vital for sustaining an optimal circadian rhythm and physiological function. However, the therapeutic potential of BMAL1 overexpression in the SCN to rectify the neurovascular deficits of T2D has yet to be investigated. In this study, db/db mice, a well-established model of T2D exhibiting arrhythmic behavior and the complications of diabetes, were injected stereotaxically with AAV8-Bmal1 or a control virus in the SCN to evaluate the protective effects of correcting the central clock on neurovascular deficits. Given the complex neurovascular network and the eye's unique accessibility as a transparent system, ocular complications were selected as a model to examine the neuronal functional, behavioral, and vascular benefits of correcting the central clock. BMAL1 overexpression normalized the circadian rhythms, as demonstrated by improvements in the free-running period. The retinal neuronal function improved on electroretinogram, along with optomotor behavior and visual acuity enhancements. Retinal vascular deficits were also significantly reduced. Notably, our approach helped decrease fat content in genetically predisposed obese animals. Since the SCN is known to regulate hepatic glucose production via sympathetic mechanisms, glycemic control, and pyruvate tolerance tests were conducted. Systemically, we observed improved glucose homeostasis in BMAL1-overexpressing mice alongside a substantial reduction in hepatic gluconeogenesis. BMAL1 overexpression lowered plasma norepinephrine and liver TH levels, indicating a protective regulation of adrenergic signaling. Thus, this study underscores the therapeutic potential of targeting circadian clock genes like BMAL1 in the SCN to alleviate metabolic and neurovascular deficits associated with T2D. Our research offers a compelling framework for integrating circadian rhythms into managing diabetes and its complications.Item Characterization of the Ocular Phenotype in a Col4a3 Knockout Mouse Model of Alport Syndrome(Association for Research in Vision and Ophthalmology, 2024) Belamkar, Ameya; Luo, Qianyi; Mahajan, Neha; Abhyankar, Surabhi; Jones, Bryce A.; Sodhi, Rupinder Kaur; Pattabiraman, Padmanabhan P.; Levi, Moshe; Bhatwadekar, Ashay D.; Ophthalmology, School of MedicinePurpose: Alport syndrome (AS) is a genetic condition caused by a dysfunctional collagen (IV) α3α4α5 heterotrimer, leading to basement membrane instability and, ultimately, abnormalities in the kidney, inner ear, and eyes. This study aimed to characterize ocular pathology of AS by focusing on inflammatory and fibrotic markers. Methods: Col4a3tm1Dec knockout (KO) mice eyes were evaluated for the localization of collagen (IV) α3 and collagen (IV) α4, then stained for transforming growth factor-β1 (TGF-β1), TGF-β2, connective tissue growth factor (CTGF), and β-catenin. mRNA levels of the profibrotic genes S100a4, Acta2, Col1a1, Snai1, Snai2, and Twist1 were assessed using real-time reverse transcription quantitative PCR (RT-qPCR). Results: Collagen (IV) α3 and collagen (IV) α4 were co-expressed in Descemet's and Bruch's membrane but not in the retina, lens, or other corneal substructures. Immunofluorescence quantitation revealed upregulation of TGF-β1 in the anterior lens and TGF-β2 in the retina of KO eyes. Conversely, CTGF and β-catenin were shown to be elevated in the corneal epithelium but not the retina or lens. RT-qPCR showed an increase in the transcription of Acta2, Col1a1, and Snai2 in the retinas and Snai2 in anterior segments of KO mice. Conclusions: Col4a3 KO mice exhibited a differential inflammatory and profibrotic response in the cornea, retina, and lens, which may play a role in the ocular pathology of AS.Item Circadian Rhythm Disruption Results in Visual Dysfunction(Wiley, 2022-02-07) Mathew, Deepa; Luo, Qianyi; Bhatwadekar, Ashay D.; Ophthalmology, School of MedicineArtificial light has been increasingly in use for the past 70 years. The aberrant light exposure and round‐the‐clock nature of work lead to the disruption of biological clock. Circadian rhythm disruption (CRD) contributes to multiple metabolic and neurodegenerative diseases. However, its effect on vision is not understood. Moreover, the mammalian retina possesses an autonomous clock that could be reset with light exposure. We evaluated the impact of CRD on retinal morphology, physiology, and vision after housing mice in a disruption inducing shorter light/dark cycle (L10:D10). Interestingly, the mice under L10:D10 exhibited three different entrainment behaviors; “entrained,” “free‐running,” and “zigzagging.” These behavior groups under CRD exhibited reduced visual acuity, retinal thinning, and a decrease in the number of photoreceptors. Intriguingly, the electroretinogram response was decreased only in the mice exhibiting “entrained” behavior. The retinal proteome showed distinct changes with each entrainment behavior, and there was a dysfunctional oxidative stress‐antioxidant mechanism. These results demonstrate that CRD alters entrainment behavior and leads to visual dysfunction in mice. Our studies uniquely show the effect of entrainment behavior on retinal physiology. Our data have broader implications in understanding and mitigating the impact of CRD on vision and its potential role in the etiology of retinal diseases.Item Conditional Deletion of Bmal1 Accentuates Microvascular and Macrovascular Injury(Elsevier, 2017-06) Bhatwadekar, Ashay D.; Beli, Eleni; Diao, Yanpeng; Chen, Jonathan; Luo, Qianyi; Alex, Alpha; Caballero, Sergio; Dominguez, James M., II; Salazar, Tatiana E.; Busik, Julia V.; Segal, Mark S.; Grant, Maria B.; Ophthalmology, School of MedicineThe brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein (BMAL)-1 constitutes a major transcriptional regulator of the circadian clock. Here, we explored the impact of conditional deletion of Bmal1 in endothelium and hematopoietic cells in murine models of microvascular and macrovascular injury. We used two models of Bmal1fx/fx;Tek-Cre mice, a retinal ischemia/reperfusion model and a neointimal hyperplasia model of the femoral artery. Eyes were enumerated for acellular capillaries and were stained for oxidative damage markers using nitrotyrosine immunohistochemistry. LSK (lineage-negative, stem cell antigen-1-positive, c-Kit-positive) cells were quantified and proliferation assessed. Hematopoiesis is influenced by innervation to the bone marrow, which we assessed using IHC analysis. The number of acellular capillaries increased threefold, and nitrotyrosine staining increased 1.5-fold, in the retinas of Bmal1fx/fx;Tek-Cre mice. The number of LSK cells from the Bmal1fx/fx;Tek-Cre mice decreased by 1.5-fold and was accompanied by a profound decrease in proliferative potential. Bmal1fx/fx;Tek-Cre mice also exhibited evidence of bone marrow denervation, demonstrating a loss of neurofilament-200 staining. Injured femoral arteries showed a 20% increase in neointimal hyperplasia compared with similarly injured wild-type controls. Our study highlights the importance of the circadian clock in maintaining vascular homeostasis and demonstrates that specific deletion of BMAL1 in endothelial and hematopoietic cells results in phenotypic features similar to those of diabetes.Item Dapagliflozin protects neural and vascular dysfunction of the retina in diabetes(BMJ, 2022) Luo, Qianyi; Leley, Sameer P.; Bello, Erika; Dhami, Hurshdeep; Mathew, Deepa; Bhatwadekar, Ashay Dilip; Ophthalmology, School of MedicineIntroduction: Dapagliflozin, a sodium-glucose transporter inhibitor, effectively reduces blood glucose and is indicated for individuals with kidney diseases and cardiovascular disorders. In this study, we further expand the therapeutic benefit of dapagliflozin in the neural and vascular retina, with the potential to effectively manage diabetic retinopathy (DR), the most common complication of diabetes. Research design and methods: Db/db mice, an animal model of type 2 diabetes, were treated with dapagliflozin orally, and the electroretinogram (ERG) response and acellular capillary numbers were assessed. Messenger RNA levels of inflammatory cytokines were studied using real-time quantitative (q)PCR. We assessed endothelial cell migration in a scratch wound assay and retinal glucose uptake using human retinal endothelial cells. Results: The dapagliflozin treatment improved the ERG b-wave amplitude and decreased acellular capillary numbers. The scratch wound assay demonstrated a reduction in wound closure after dapagliflozin treatment. Retinal glucose uptake reduced after dapagliflozin treatment compared with the respective controls. Conclusions: Our studies suggest that dapagliflozin treatment effectively corrects neural and vascular dysfunction of the retina in diabetes. This effect is mediated by a decrease in inflammation and improved glycemic control. In addition, dapagliflozin exhibits decreased wound healing and glucose uptake, which could benefit the retina. Thus, dapagliflozin could be helpful in the management of DR, with multimodal therapeutic effects.Item Diabetes Alters Diurnal Rhythm of Electroretinogram in db/db Mice(Yale School of Medicine, 2019-06-27) Di, Rong; Luo, Qianyi; Mathew, Deepa; Bhatwadekara, Ashay D.; Ophthalmology, School of MedicineDiabetic retinopathy (DR) is the most common complications of diabetes and a leading cause of blindness in the United States. The retinal neuronal changes precede the vascular dysfunction observed in DR. The electroretinogram (ERG) determines the electrical activity of retinal neural and non-neuronal cells. The retinal ERG amplitude is reduced gradually on the progression of DR to a more severe form. Circadian rhythms play an important role in the physiological function of the body. While ERG is known to exhibit a diurnal rhythm, it is not known whether a progressive increase in the duration of diabetes affects the physiological rhythm of retinal ERG. To study this, we determined the ERG rhythm of db/db mice, an animal model of type 2 diabetes at 2, 4, and 6 months of diabetes under a regular light-dark cycle and constant dark. Our studies demonstrate that the diurnal rhythm of ERG amplitude for retinal a-wave and b-wave was altered in diabetes. The implicit time was increased in db/db mice while the oscillatory potential was reduced. Moreover, there was a progressive decline in an intrinsic rhythm of ERG upon an increase in the duration of diabetes. In conclusion, our studies provide novel insights into the pathogenic mechanism of DR by showing an altered circadian rhythm of the ERG.Item The Diurnal Rhythm of Insulin Receptor Substrate-1 (IRS-1) and Kir4.1 in Diabetes: Implications for a Clock Gene Bmal1(ARVO, 2019-05) Luo, Qianyi; Xiao, Yucheng; Alex, Alpha; Cummins, Theodore R.; Bhatwadekar, Ashay D.; Ophthalmology, School of MedicinePurpose: Diabetes leads to the downregulation of the retinal Kir4.1 channels and Müller cell dysfunction. The insulin receptor substrate-1 (IRS-1) is a critical regulator of insulin signaling in Müller cells. Circadian rhythms play an integral role in normal physiology; however, diabetes leads to a circadian dysrhythmia. We hypothesize that diabetes will result in a circadian dysrhythmia of IRS-1 and Kir4.1 and disturbed clock gene function will have a critical role in regulating Kir4.1 channels. Methods: We assessed a diurnal rhythm of retinal IRS-1 and Kir4.1 in db/db mice. The Kir4.1 function was evaluated using a whole-cell recording of Müller cells. The rat Müller cells (rMC-1) were used to undertake in vitro studies using a siRNA. Results: The IRS-1 exhibited a diurnal rhythm in control mice; however, with diabetes, this natural rhythm was lost. The Kir4.1 levels peaked and troughed at times similar to the IRS-1 rhythm. The IRS-1 silencing in the rMC-1 led to a decrease in Kir4.1 and BMAL1. The insulin treatment of retinal explants upregulated Kir4.1 possibly via upregulation of BMAL1 and phosphorylation of IRS-1 and Akt-1. Conclusions: Our studies highlight that IRS-1, by regulating BMAL1, is an important regulator of Kir4.1 in Müller cells and the dysfunctional signaling mediated by IRS-1 may be detrimental to Kir4.1.Item Further Characterization of Multi-Organ DEARE and Protection by 16,16 Dimethyl Prostaglandin E2 in a Mouse Model of the Hematopoietic Acute Radiation Syndrome(BioOne, 2023) Wu, Tong; Pelus, Louis M.; Plett, P. Artur; Sampson, Carol H.; Chua, Hui Lin; Fisher, Alexa; Feng, Hailin; Liu, Liqiong; Li, Hongge; Ortiz, Miguel; Chittajallu, Supriya; Luo, Qianyi; Bhatwadekar, Ashay D.; Meyer, Timothy B.; Zhang, Xin; Zhou, Daohong; Fischer, Kathryn D.; McKinzie, David L.; Miller, Steven J.; Orschell, Christie M.; Medicine, School of MedicineSurvivors of acute radiation exposure suffer from the delayed effects of acute radiation exposure (DEARE), a chronic condition affecting multiple organs, including lung, kidney, heart, gastrointestinal tract, eyes, and brain, and often causing cancer. While effective medical countermeasures (MCM) for the hematopoietic-acute radiation syndrome (H-ARS) have been identified and approved by the FDA, development of MCM for DEARE has not yet been successful. We previously documented residual bone marrow damage (RBMD) and progressive renal and cardiovascular DEARE in murine survivors of H-ARS, and significant survival efficacy of 16,16-dimethyl prostaglandin E2 (dmPGE2) given as a radioprotectant or radiomitigator for H-ARS. We now describe additional DEARE (physiological and neural function, progressive fur graying, ocular inflammation, and malignancy) developing after sub-threshold doses in our H-ARS model, and detailed analysis of the effects of dmPGE2 administered before (PGE-pre) or after (PGE-post) lethal total-body irradiation (TBI) on these DEARE. Administration of PGE-pre normalized the twofold reduction of white blood cells (WBC) and lymphocytes seen in vehicle-treated survivors (Veh), and increased the number of bone marrow (BM) cells, splenocytes, thymocytes, and phenotypically defined hematopoietic progenitor cells (HPC) and hematopoietic stem cells (HSC) to levels equivalent to those in non-irradiated age-matched controls. PGE-pre significantly protected HPC colony formation ex vivo by >twofold, long term-HSC in vivo engraftment potential up to ninefold, and significantly blunted TBI-induced myeloid skewing. Secondary transplantation documented continued production of LT-HSC with normal lineage differentiation. PGE-pre reduced development of DEARE cardiovascular pathologies and renal damage; prevented coronary artery rarefication, blunted progressive loss of coronary artery endothelia, reduced inflammation and coronary early senescence, and blunted radiation-induced increase in blood urea nitrogen (BUN). Ocular monocytes were significantly lower in PGE-pre mice, as was TBI-induced fur graying. Increased body weight and decreased frailty in male mice, and reduced incidence of thymic lymphoma were documented in PGE-pre mice. In assays measuring behavioral and cognitive functions, PGE-pre reduced anxiety in females, significantly blunted shock flinch response, and increased exploratory behavior in males. No effect of TBI was observed on memory in any group. PGE-post, despite significantly increasing 30-day survival in H-ARS and WBC and hematopoietic recovery, was not effective in reducing TBI-induced RBMD or any other DEARE. In summary, dmPGE2 administered as an H-ARS MCM before lethal TBI significantly increased 30-day survival and ameliorated RBMD and multi-organ and cognitive/behavioral DEARE to at least 12 months after TBI, whereas given after TBI, dmPGE2 enhances survival from H-ARS but has little impact on RBMD or other DEARE.