Date of Award

30-4-2024

Document Type

Thesis

School

School of Chemical & Biotechnology

Programme

Ph.D.-Doctoral of Philosophy

First Advisor

Dr.S.R.Bharathi Devi

Second Advisor

Dr.Shanker Jha

Keywords

Paraoxonase 2, Endothelial Dysfunction, ER Stress, Inflammation, Site Directed Mutagenesis

Abstract

Diabetes mellitus (DM) is a metabolic disorder associated with various vascular complications and affects the function of the endothelial cells. Elevated blood glucose levels accelerate the formation of advanced glycation end products (AGEs) by Amadori rearrangement and bind to their Receptor for the advanced glycation end product (RAGE) and evoke release of key pro-inflammatory cytokine proteins leading to endothelial dysfunction. Paraoxonase 2 (PON2) is an endogenous intracellular enzyme, localised in mitochondria, endoplasmic reticulum (ER), and nuclear membrane with numerous properties, like anti-inflammatory, anti-apoptotic and anti-oxidant.

Our present study aims to ascertain the effect of AGEs on PON2 activity and expression, and its consequences on endothelial dysfunction (ED) in both macrovascular and microvascular endothelial cells. In Human umbilical vein endothelial cells (HUVECs), AGEs (Glycated Albumin (GA) or CML) exposure significantly increased the expression of various receptors, which ultimately decreased the activity and expression of PON2 with elevated level of Reactive oxygen species (ROS), ER stress proteins and pro-inflammatory cytokines levels. Overexpression of PON2 significantly reduced ROS and ER stress through reduced Nuclear factor kappa B (NFκB), and extracellular signal-regulated protein kinase (ERK1/2) phosphorylation.

Mitochondrial dysfunction is one of the key pathological changes seen in diabetes patients and also induces dysfunctional endothelial cells thereby contributing to the pathology of DR. We found that PON2 protein was reduced in human retinal microvascular endothelial cells (HRECs) upon CML treatment and also in the diabetic retina (p=0.035). Silencing of PON2 augments Fission-1 (Fis1), resulting in fragmentation of mitochondria, and facilitates mitochondrial permeability transition pore (mPTP) opening, which induces the release of cytochrome-C (Cyt-c), and activates pro-apoptotic pathway.

While PON2 overexpression was similar to SP600125 (a specific JNK (c-Jun Nterminal kinases) inhibitor) in decreasing Fis1 (p=0.036) and reduces the phosphorylation of JNK1/2 signaling pathway confirming the anti-apoptotic role of PON2 in CML-mediated mitochondrial dysfunction. Since, PON2 a multifaceted enzyme, decreased in diabetes and glycation decreases its activity in diabetes, therefore we aimed at constructing a mutated PON2 that can defy glycation.

To deduce the glycation-prone residues in PON2, we used an in silico approach and developed a mutated mPON2 by SDM assay, and compared the efficiency of both wPON2 (wildtype PON2) and mPON2 (mutant PON2-PON2-K70A) in HRECs upon CML treatment. We deciphered CML glycates wPON2 and reduces its activity and mPON2 designed using in silico studies found to interact much better with its substrates than wPON2. Further overexpression of mPON2 show enhanced inhibition of CML-induced oxidative, ER stress, pro-inflammation, and mitochondrial fission than wPON2.

Additionally, mPON2 inhibited the CML-induced phosphorylation of NFĸB, similar to Pyrrolidine Dithiocarbamate (PTDC-an inhibitor of NFĸB) treatment. Hence we report inferring the functional implications of mPON2 in mitigating ER stress and inflammation against CML induced vascular dysfunction. Our Overall, analysis of all these results showed a PON2 as a highly promising potential therapeutic target against diabetic micro and macro vascular disease reducing the AGEs induced ROS, ER stress, inflammation and mitochondrial dysfunction.

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