Author: Negara Tajbakhsh
Tajbakhsh, Negara, 2022 The role of DDAH1 in angiogenesis, Flinders University, College of Medicine and Public Health
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Angiogenesis is the formation of new blood vessels from pre-existing ones. Although angiogenesis provides organs and tissues with oxygen and nutrients, excessive angiogenesis could potentially contribute to a variety of diseases including Alzheimer’s, AIDS, cancer, ocular diseases, arthritis, and psoriasis.
The vascular endothelial growth factor-A (VEGF-A) in endothelial cells plays a crucial role in angiogenesis. VEGF-mediated angiogenesis requires nitric oxide (NO˙), a key signalling molecule produced by the nitric oxide synthase (NOS) family of enzymes. However, overproduction of this molecule can lead to various disorders.
Dimethylarginine dimethylaminohydrolase-1 (DDAH1) is a key enzyme in the NO˙ regulation, metabolising asymmetric dimethylarginine (ADMA) and monomethyl arginine (L-NMMA). These methylarginines are endogenous inhibitors of NOS. The resulting decrease in NO˙ production leads to angiogenesis downregulation. Inhibition of DDAH1 reduces excessive angiogenesis via increasing ADMA and L-NMMA accumulation, representing a promising strategy in the treatment of NO˙ overproduction.
In this work, the anti-angiogenic effect of novel small molecule DDAH1 inhibitors was tested. The toxic effect of three of the most potent DDAH1 inhibitor molecules (ZST316, ZST152, and ZST086) were investigated using cell viability assays (Sulforhodamine B and crystal violet) and real-time analysis of cell proliferation (IncuCyte and xCELLigence). The results indicated no toxic effects in neuronal or endothelial cell models (PC-12, SH-SY5Y, and VeraVec cells).
Pharmacological DDAH1 inhibition significantly reduced endothelial cell tube formation in-vitro. Furthermore, we investigated the potential positive correlation between DDAH1 and VEGF-A (as an essential mediator of angiogenesis), by testing the effect of VEGF-A up- (hypoxia) and down-regulation (siRNA) on DDAH1 expression. While hypoxia-mediated VEGF-A overexpression suppressed DDAH1 protein expression, siRNA-mediated knockdown of VEGF-A, led to DDAH1 protein rise. Mass spectrometry (MS) analysis, was planned to investigate the affected angiogenic signalling pathways by the inhibitors. However, due to limited endothelial cell availability, an alternative set of experiments was conducted in non-endothelial cells that contain proteins involved in angiogenesis, HEK-293T cells, aiming to investigate the potential off-target effects of the putative drug candidates in non-endothelial cells. ZST316 showed some off-target effects based on protein abundance measurements. However, the level of changes was only 1-2% of all analysed proteins. Importantly, while ZST316 treatment caused no change in DDAH1 expression, whereas DDAH2 protein was upregulated by two-folds, which might represent a compensatory effect of the isoforms on ADMA metabolism. Pathway analysis (InnateDB) was performed to identify the main biochemical pathways enriched by the differentially expressed proteins. Limited changes were observed in the signalling pathway profile of HEK-293T cells, which might be due to a DDAH2-mediated compensatory effect following DDAH1 inhibition. Overall, the minimal effect of ZST316 treatment on the biological pathways of HEK-293T cells, including apoptosis and angiogenesis, rules out the presence of tangible off-target effects at least in this cell line.
The mass spectrometry analysis requires further investigations on the effect of DDAH1 inhibitors on endothelial and non-endothelial cells, followed by in vivo studies to confirm the presence of tangible anti-angiogenic effects without overt systemic toxicity.
Keywords: Dimethylarginine dimethylaminohydrolase-1 (DDAH1), angiogenesis, ZST
Subject: Clinical Pharmacology thesis
Thesis type: Doctor of Philosophy
Completed: 2022
School: College of Medicine and Public Health
Supervisor: Prof. Arduino Mangoni