Browsing by Author "Abhyankar, Surabhi"

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    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 Medicine
    Purpose: 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.
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    Global mapping of BMAL1 protein-DNA interactions in human retinal Müller cells
    (Molecular Vision, 2024-11-10) Luo, Qianyi; Sangani, Neel; Abhyankar, Surabhi; Somalraju, Sahiti; Janga, Sarath Chandra; Bhatwadekar, Ashay D.; Ophthalmology, School of Medicine
    The circadian clock, a conserved biologic timekeeping mechanism, is pivotal in orchestrating rhythmic physiologic processes. While extensively studied in the central clock, the involvement of BMAL1 in peripheral clocks, particularly in human Müller cells, remains underexplored. Müller cells, critical for retinal homeostasis, may unveil novel insights into circadian regulation. Employing ChIP-sequencing, we comprehensively mapped BMAL1 binding sites in human Müller cells. The analysis identified 275 reproducible peaks, with predominant distribution across promoters (26.6%), intronic (26.3%), and intergenic (22.1%) regions, with 80% of these confident peaks linked to protein-coding genes. Differential peak analysis revealed 89 unique genes significantly enriched with BMAL1 sites in their promoters, while functional enrichment of the associated genes indicated key biologic processes such as circadian regulation of gene expression, photoperiodism, and glucocorticoid receptor signaling pathway regulation. Motif analysis revealed a highly conserved 6-nucleotide motif, CACGTG, appearing in 89.09% of the peaks. Analysis of the binding sites across genomic regions highlighted the robust BMAL1 binding, further confirmed by qPCR validation of circadian targets such as G6PC3, CIART, PER1, and TXNIP, which are critical for Müller cell health, along with SHMT2 and MALAT1, which have emerged as novel genes that may have implications for Müller cell health. Our findings unveil the regulatory landscape of BMAL1 in Müller cells, contributing to a broader understanding of the clock-mediated mechanism in ocular tissues. These insights hold therapeutic potential for circadian-related retinal diseases, presenting avenues for chronotherapeutic interventions.
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    Transcriptomic Profile of Lin-Sca1+c-kit (LSK) cells in db/db mice with long-standing diabetes
    (Springer Nature, 2024-08-12) Mahajan, Neha; Luo, Qianyi; Abhyankar, Surabhi; Bhatwadekar, Ashay D.; Ophthalmology, School of Medicine
    Background: The Lin-Sca1+c-Kit+ (LSK) fraction of the bone marrow (BM) comprises multipotent hematopoietic stem cells (HSCs), which are vital to tissue homeostasis and vascular repair. While diabetes affects HSC homeostasis overall, the molecular signature of mRNA and miRNA transcriptomic under the conditions of long-standing type 2 diabetes (T2D;>6 months) remains unexplored. Methods: In this study, we assessed the transcriptomic signature of HSCs in db/db mice, a well-known and widely used model for T2D. LSK cells of db/db mice enriched using a cell sorter were subjected to paired-end mRNA and single-end miRNA seq library and sequenced on Illumina NovaSeq 6000. The mRNA sequence reads were mapped using STAR (Spliced Transcripts Alignment to a Reference), and the miRNA sequence reads were mapped to the designated reference genome using the Qiagen GeneGlobe RNA-seq Analysis Portal with default parameters for miRNA. Results: We uncovered 2076 out of 13,708 mRNAs and 35 out of 191 miRNAs that were expressed significantly in db/db animals; strikingly, previously unreported miRNAs (miR-3968 and miR-1971) were found to be downregulated in db/db mice. Furthermore, we observed a molecular shift in the transcriptome of HSCs of diabetes with an increase in pro-inflammatory cytokines (Il4, Tlr4, and Tnf11α) and a decrease in anti-inflammatory cytokine IL10. Pathway mapping demonstrated inflammation mediated by chemokine, cytokine, and angiogenesis as one of the top pathways with a significantly higher number of transcripts in db/db mice. These molecular changes were reflected in an overt defect in LSK mobility in the bone marrow. miRNA downstream target analysis unveils several mRNAs targeting leukocyte migration, microglia activation, phagosome formation, and macrophage activation signaling as their primary pathways, suggesting a shift to an inflammatory phenotype. Conclusion: Our findings highlight that chronic diabetes adversely alters HSCs' homeostasis at the transcriptional level, thus potentially contributing to the inflammatory phenotype of HSCs under long-term diabetes. We also believe that identifying HSCs-based biomarkers in miRNAs or mRNAs could serve as diagnostic markers and potential therapeutic targets for diabetes and associated vascular complications.