👤 Padmanabhan P Pattabiraman

Glaucoma is a leading cause of irreversible blindness, often associated with elevated intraocular pressure (IOP) due to trabecular meshwork (TM) dysfunction. Diabetes mellitus (DM) is recognized as a significant risk factor for glaucoma; however, the molecular mechanisms through which hyperglycemia affects TM function remain unclear. This study investigated the impact of high glucose on gene expression in human TM (HTM) cells to uncover pathways that contribute to TM dysfunction and glaucoma pathogenesis under diabetic conditions. Primary HTM cells were cultured under normoglycemic (5.5 mM) and hyperglycemic (30 mM) conditions for seven days, followed by mRNA sequencing (mRNA-seq) to identify differentially expressed genes, with quantitative PCR (qPCR) used for confirmatory analysis. STRING network analysis was performed to predict potential interactions among upregulated and downregulated genes. mRNA-seq analysis revealed 25 significantly differentially expressed genes in high glucose conditions, including upregulated genes associated with oxidative stress, apoptosis, autophagy, immune response, and fibrosis. Notably, TXNIP gene was significantly upregulated, indicating increased oxidative stress and apoptosis in TM cells, while downregulation of autophagy-related genes, such as HSPA6 and LAMP3, suggests compromised protein quality control. Immune response genes, including CCL7 and CHI3L1, were upregulated, suggesting an inflammatory response to oxidative stress. Increased expression of fibrosis-related genes, such as SNAI1, FGF7, and KRT19, and an increase in ECM proteins like Collagen 1 and FN accumulation and fibril formation suggest increased fibrosis of TM in diabetic conditions, potentially elevating IOP. Metabolic changes in diabetic patients could therefore lead to TM dysfunction, impair aqueous humor outflow, and elevate IOP, thereby increasing glaucoma risk. Targeting oxidative stress and fibrosis pathways offers therapeutic strategies to mitigate glaucoma progression in diabetic populations. Show less

Glaucoma is a leading cause of irreversible blindness, often associated with elevated intraocular pressure (IOP) due to trabecular meshwork (TM) dysfunction. Diabetes mellitus (DM) is recognized as a significant risk factor for glaucoma; however, the molecular mechanisms through which hyperglycemia affects TM function remain unclear. This study investigated the impact of high glucose on gene expression in human TM (HTM) cells to uncover pathways that contribute to TM dysfunction and glaucoma pathogenesis under diabetic conditions. Primary HTM cells were cultured under normoglycemic (5.5 mM) and hyperglycemic (30 mM) conditions for seven days, followed by mRNA sequencing (mRNA-seq) to identify differentially expressed genes, with quantitative PCR (qPCR) used for confirmatory analysis. STRING network analysis was performed to predict interactions among upregulated and downregulated proteins. mRNA-seq analysis revealed 25 significantly differentially expressed genes in high glucose conditions, including upregulated genes associated with oxidative stress, apoptosis, autophagy, immune response, and fibrosis. Notably, TXNIP was significantly upregulated, indicating increased oxidative stress and apoptosis in TM cells, while downregulation of autophagy-related genes, such as HSPA6 and LAMP3, suggests compromised protein quality control. Immune response genes, including CCL7 and CHI3L1, were upregulated, suggesting an inflammatory response to oxidative stress. Increased expression of fibrosis-related genes, such as SNAI1, FGF7, and KRT19, supports the hypothesis of ECM accumulation in diabetic conditions, potentially elevating IOP. Chronic hyperglycemia in diabetic patients could therefore lead to TM dysfunction, impair aqueous humor outflow, and elevate IOP, thereby increasing glaucoma risk. Targeting oxidative stress and fibrosis pathways offers therapeutic strategies to mitigate glaucoma progression in diabetic populations. Show less

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. 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). 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. 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. Show less

Postmeiotic gene expression is essential for development and maturation of sperm and eggs. We report that the dual bromodomain-containing protein BRWD1, which is essential for both male and female fertility, promotes haploid spermatid-specific transcription but has distinct roles in oocyte meiotic progression. Brwd1 deficiency caused down-regulation of ∼300 mostly spermatid-specific transcripts in testis, including nearly complete elimination of those encoding the protamines and transition proteins, but was not associated with global epigenetic changes in chromatin, which suggests that BRWD1 acts selectively. In females, Brwd1 ablation caused severe chromosome condensation and structural defects associated with abnormal telomere structure but only minor changes in gene expression at the germinal vesicle stage, including more than twofold overexpression of the histone methyltransferase MLL5 and LINE-1 elements transposons. Thus, loss of BRWD1 function interferes with the completion of oogenesis and spermatogenesis through sexually dimorphic mechanisms: it is essential in females for epigenetic control of meiotic chromosome stability and in males for haploid gene transcription during postmeiotic sperm differentiation. Show less

A novel mutation, repro5, was isolated in a forward genetic screen for infertility mutations induced by ENU mutagenesis. Homozygous mutant mice were phenotypically normal but were infertile. Oocytes from mutant females appeared normal, but were severely maturation-defective in that they had reduced ability to progress to metaphase II (MII), and those reaching MII were unable to progress beyond the two pronuclei stage following in vitro fertilization (IVF). Mutant males exhibited defective spermiogenesis, resulting in oligoasthenoteratospermia. Genetic mapping, positional cloning, and complementation studies with a disruption allele led to the identification of a mutation in Brwd1 (Bromodomain and WD repeat domain containing 1) as the causative lesion. Bromodomain-containing proteins typically interact with regions of chromatin containing histones hyperacetylated at lysine residues, a characteristic of chromatin in early spermiogenesis before eventual replacement of histones by the protamines. Previous data indicated that Brwd1 is broadly expressed, encoding a putative transcriptional regulator that is believed to act on chromatin through interactions with the Brg1-dependent SWI/SNF chromatin-remodeling pathway. Brwd1 represents one of a small number of genes whose elimination disrupts gametogenesis in both sexes after the major events of meiotic prophase I have been completed. Show less