👤 Angad S Johar

Proliferative retinopathies are associated with formation of fibrous epiretinal membranes. At present, there is no pharmacological intervention for the treatment of retinopathies. Cytokines such as TGFβ are elevated in the vitreous humor of the patients with proliferative vitro-retinopathy, diabetic retinopathy and age-related macular degeneration. TGFβ isoforms lead to epithelial-mesenchymal transition (EMT) or trans-differentiation of the retinal pigment epithelial (RPE) cells. PI3K/Akt and MAPK/Erk pathways play important roles in the EMT of RPE cells. Therefore, inhibition of EMT by pharmacological agents is an important therapeutic strategy in retinopathy. Dichloroacetate (DCA) is shown to prevent proliferation and EMT of cancer cell lines but its effects are not explored on the prevention of EMT of RPE cells. In the present study, we have investigated the role of DCA in preventing TGFβ2 induced EMT of RPE cell line, ARPE-19. A wound-healing assay was utilized to detect the anti-EMT effect of DCA. The expressions of EMT and cell adhesion markers were carried out by immunofluorescence, western blotting, and quantitative real-time PCR. The expression of MAPK/Erk and PI3K/Akt pathway members was carried out using western blotting. We found that TGFβ2 exposure leads to an increase in the wound healing response, expression of EMT markers (Fibronectin, Collagen I, N-cadherin, MMP9, S100A4, α-SMA, Snai1, Slug) and a decrease in the expression of cell adhesion/epithelial markers (ZO-1, Connexin 43, E-cadherin). These changes were accompanied by the activation of PI3K/Akt and MAPK/Erk pathways. Simultaneous exposure of DCA along with TGFβ2 significantly inhibited wound healing response, expression of EMT markers and cell adhesion/epithelial markers. Furthermore, DCA and TGFβ2 effectively attenuated the activation of MAPK/Erk/JNK and PI3K/Akt/GSK3β pathways. Our results demonstrate that DCA has a strong anti-EMT effect on the ARPE-19 cells and hence can be utilized as a therapeutic agent in the prevention of proliferative retinopathies. Show less

Multiple autoimmune syndrome (MAS), an extreme phenotype of autoimmune disorders, is a very well suited trait to tackle genomic variants of these conditions. Whole exome sequencing (WES) is a widely used strategy for detection of protein coding and splicing variants associated with inherited diseases. The DNA of eight patients affected by MAS [all of whom presenting with Sjögren's syndrome (SS)], four patients affected by SS alone and 38 unaffected individuals, were subject to WES. Filters to identify novel and rare functional (pathogenic-deleterious) homozygous and/or compound heterozygous variants in these patients and controls were applied. Bioinformatics tools such as the Human gene connectome as well as pathway and network analysis were applied to test overrepresentation of genes harbouring these variants in critical pathways and networks involved in autoimmunity. Eleven novel and rare functional variants were identified in cases but not in controls, harboured in: MACF1, KIAA0754, DUSP12, ICA1, CELA1, LRP1/STAT6, GRIN3B, ANKLE1, TMEM161A, and FKRP. These were subsequently subject to network analysis and their functional relatedness to genes already associated with autoimmunity was evaluated. Notably, the LRP1/STAT6 novel mutation was homozygous in one MAS affected patient and heterozygous in another. LRP1/STAT6 disclosed the strongest plausibility for autoimmunity. LRP1/STAT6 are involved in extracellular and intracellular anti-inflammatory pathways that play key roles in maintaining the homeostasis of the immune system. Further; networks, pathways, and interaction analyses showed that LRP1 is functionally related to the HLA-B and IL10 genes and it has a substantial impact within immunological pathways and/or reaction to bacterial and other foreign proteins (phagocytosis, regulation of phospholipase A2 activity, negative regulation of apoptosis and response to lipopolysaccharides). Further, ICA1 and STAT6 were also closely related to AIRE and IRF5, two very well known autoimmunity genes. Novel and rare exonic mutations that may account for autoimmunity were identified. Among those, the LRP1/STAT6 novel mutation has the strongest case for being categorised as potentially causative of MAS given the presence of intriguing patterns of functional interaction with other major genes shaping autoimmunity. Show less