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Browsing by Author "Bhatwadekar, Ashay D."
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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 Ataxia Telangiectasia Mutated Dysregulation Results in Diabetic Retinopathy(Wiley Blackwell (John Wiley & Sons), 2016-02) Bhatwadekar, Ashay D.; Duan, Yaqian; Chakravarthy, Harshini; Korah, Maria; Caballero, Sergio; Busik, Julia V.; Grant, Maria B.; Department of Ophthalmology, IU School of MedicineAtaxia telangiectasia mutated (ATM) acts as a defense against a variety of bone marrow (BM) stressors. We hypothesized that ATM loss in BM-hematopoietic stem cells (HSCs) would be detrimental to both HSC function and microvascular repair while sustained ATM would be beneficial in disease models of diabetes. Chronic diabetes represents a condition associated with HSC depletion and inadequate vascular repair. Gender mismatched chimeras of ATM(-/-) on wild type background were generated and a cohort were made diabetic using streptozotocin (STZ). HSCs from the STZ-ATM(-/-) chimeras showed (a) reduced self-renewal; (b) decreased long-term repopulation; (c) depletion from the primitive endosteal niche; (d) myeloid bias; and (e) accelerated diabetic retinopathy (DR). To further test the significance of ATM in hematopoiesis and diabetes, we performed microarrays on circulating angiogenic cells, CD34(+) cells, obtained from a unique cohort of human subjects with long-standing (>40 years duration) poorly controlled diabetes that were free of DR. Pathway analysis of microarrays in these individuals revealed DNA repair and cell-cycle regulation as the top networks with marked upregulation of ATM mRNA compared with CD34(+) cells from diabetics with DR. In conclusion, our study highlights using rodent models and human subjects, the critical role of ATM in microvascular repair in DR.Item Automated Computer-Based Enumeration of Acellular Capillaries for Assessment of Diabetic Retinopathy(SPIE, 2020-02) Tuceryan, Mihran; Hemmady, Anish N.; Schebler, Craig; Alex, Alpha; Bhatwadekar, Ashay D.; Computer and Information Science, School of ScienceDiabetic retinopathy (DR) is the most common complications of diabetes; if untreated the DR can lead to a vision loss. The treatment options for DR are limited and the development of newer therapies are of considerable interest. Drug screening for the retinopathy treatment is undertaken using animal models in which the quantification of acellular capillaries (capillary without any cells) is used as a marker to assess the severity of retinopathy and the treatment response. The traditional approach to quantitate acellular capillaries is through manual counting. The purpose of this investigation was to develop an automated technique for the quantitation of acellular capillaries using computer-based image processing algorithms. We developed a custom procedure using the Python, the medial axis transform (MAT) and the connected component algorithm. The program was tested on the retinas of wild-type and diabetic mice and the results were compared to single blind manual counts by two independent investigators. The program successfully identified and enumerated acellular capillaries. The acellular capillary counts were comparable to the traditional manual counting. In conclusion, we developed an automated computer-based program, which can be effectively used for future pharmacological development of treatments for DR. This algorithm will enhance consistency in retinopathy assessment and reduce the time for analysis, thus, contributing substantially towards the development of future pharmacological agents for the treatment of DR.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(Cold Spring Harbor Laboratory Press, 2020) Mathew, Deepa; Bhatwadekar, Ashay D.; Ophthalmology, School of MedicineCircadian rhythm disruption (CRD) contributes to the development of multiple metabolic and neurodegenerative diseases. However, its effect on vision is not understood. 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’, ‘freerunning’, and ‘zigzagging.’ These behavior groups under CRD exhibited reduced visual acuity, retinal thinning, and a decrease in the number of rod photoreceptors. Intriguingly, the electroretinogram response was decreased only in the mice exhibiting ‘entrained’ behavior. The retinal proteome showed distinct changes with each entrainment behavior. These results demonstrate that CRD leads to photoreceptor degeneration and visual dysfunction. We uniquely show the effect of entrainment behavior on retinal protein composition and physiology. Our data has broader implications in understanding and mitigating the effect of CRD on vision health.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 Circadian rhythms in diabetic retinopathy: an overview of pathogenesis and investigational drugs(Taylor & Francis, 2020) Bhatwadekar, Ashay D.; Rameswara, Varun; Ophthalmology, School of MedicineIntroduction: Circadian rhythm is a natural endogenous process occurring roughly every 24 hours. Circadian rhythm dysfunction is involved in diabetic retinopathy (DR) pathogenesis. Interestingly, there are investigational drugs that exhibit potential in the treatment of DR by targeting circadian rhythm dysfunction. Areas covered: We performed a literature search in June 2020 using PubMed's Medical Subject Heading (MeSH) terms 'circadian clock,' 'circadian rhythms,' and 'diabetic retinopathy.' This article offers an overview of the physiology of the biological clock and clock regulatory genes and presents an examination of the retinal clock. It discusses the pathogenic mechanisms of DR and emphasizes how circadian rhythm dysfunction at structural, physiological, metabolic and cellular levels, plays a critical role in the development of DR. The latter part of the paper sheds light on those investigational drugs (such as melatonin, tasimelteon and metformin) which exhibit potential in the treatment of DR by the targeting of circadian rhythm dysfunction. Expert opinion: An enhanced understanding of circadian rhythm and its role in DR could offer therapeutic potential by targeting of circadian rhythm dysfunction.Item Combining transfer learning with retinal lesion features for accurate detection of diabetic retinopathy(Frontiers Media, 2022-11-08) Hassan, Doaa; Gill, Hunter Mathias; Happe, Michael; Bhatwadekar, Ashay D.; Hajrasouliha, Amir R.; Janga, Sarath Chandra; BioHealth Informatics, School of Informatics and ComputingDiabetic retinopathy (DR) is a late microvascular complication of Diabetes Mellitus (DM) that could lead to permanent blindness in patients, without early detection. Although adequate management of DM via regular eye examination can preserve vision in in 98% of the DR cases, DR screening and diagnoses based on clinical lesion features devised by expert clinicians; are costly, time-consuming and not sufficiently accurate. This raises the requirements for Artificial Intelligent (AI) systems which can accurately detect DR automatically and thus preventing DR before affecting vision. Hence, such systems can help clinician experts in certain cases and aid ophthalmologists in rapid diagnoses. To address such requirements, several approaches have been proposed in the literature that use Machine Learning (ML) and Deep Learning (DL) techniques to develop such systems. However, these approaches ignore the highly valuable clinical lesion features that could contribute significantly to the accurate detection of DR. Therefore, in this study we introduce a framework called DR-detector that employs the Extreme Gradient Boosting (XGBoost) ML model trained via the combination of the features extracted by the pretrained convolutional neural networks commonly known as transfer learning (TL) models and the clinical retinal lesion features for accurate detection of DR. The retinal lesion features are extracted via image segmentation technique using the UNET DL model and captures exudates (EXs), microaneurysms (MAs), and hemorrhages (HEMs) that are relevant lesions for DR detection. The feature combination approach implemented in DR-detector has been applied to two common TL models in the literature namely VGG-16 and ResNet-50. We trained the DR-detector model using a training dataset comprising of 1,840 color fundus images collected from e-ophtha, retinal lesions and APTOS 2019 Kaggle datasets of which 920 images are healthy. To validate the DR-detector model, we test the model on external dataset that consists of 81 healthy images collected from High-Resolution Fundus (HRF) dataset and MESSIDOR-2 datasets and 81 images with DR signs collected from Indian Diabetic Retinopathy Image Dataset (IDRID) dataset annotated for DR by expert. The experimental results show that the DR-detector model achieves a testing accuracy of 100% in detecting DR after training it with the combination of ResNet-50 and lesion features and 99.38% accuracy after training it with the combination of VGG-16 and lesion features. More importantly, the results also show a higher contribution of specific lesion features toward the performance of the DR-detector model. For instance, using only the hemorrhages feature to train the model, our model achieves an accuracy of 99.38 in detecting DR, which is higher than the accuracy when training the model with the combination of all lesion features (89%) and equal to the accuracy when training the model with the combination of all lesions and VGG-16 features together. This highlights the possibility of using only the clinical features, such as lesions that are clinically interpretable, to build the next generation of robust artificial intelligence (AI) systems with great clinical interpretability for DR detection. The code of the DR-detector framework is available on GitHub at https://github.com/Janga-Lab/DR-detector and can be readily employed for detecting DR from retinal image datasets.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.
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