Smith, Renee Melissa (2018) The Role Of Putative Nox Inhibitors In Homocysteine-Induced Vascular Dysfunction. PhD thesis, Victoria University.

Abstract

Excess plasma homocysteine (Hcy; hyperhomocysteinemia, HHcy) remains an independent risk factor for cardiovascular disease (CVD) and treatments remain elusive. The source of Hcy, methionine, is an essential amino acid acquired by ingestion of animal foods. Normal methionine metabolism effectively removes Hcy via recycling back into methionine or excretion via the kidney. However, in aberrant methionine metabolism, Hcy accumulates and causes damage to the vascular system by increasing oxidative stress; the exact mechanism of how this occurs is unknown. Importantly, the B vitamins B6, B9 and B12 are essential to proper methionine/Hcy metabolism and are often found in low levels in patients presenting with HHcy; this has provided a potentially viable treatment strategy in the clinical setting. Disappointingly, clinical trials administering B vitamins to reduce HHcy have been unsuccessful in reducing CVD and treatments continue to be sought. The NADPH oxidase (Nox) family of enzymes are expressed in a broad range of cell types throughout the body and are the primary source of superoxide (Nox1, Nox2) and hydrogen peroxide (Nox4) within the vasculature under both physiological and pathological conditions. Nox1, 2 and 4 are of primary interest in vascular disease, as there is evidence that Hcy can interfere with the proper function of Nox1, 2 and 4 signalling, potentially leading to an over-expression of pro-oxidants. Nox1, 2 and 4 have been implicated in vascular disease (endothelial dysfunction), hypertension, vascular inflammation, stroke, diabetes, and atherosclerosis, and putative inhibitors of these enzymes are now available. Additionally, nitric oxide (NO) is also measured as a marker of proper vascular function; indeed, the current gold standard of assessing NO availability is by indirectly measuring vascular responses to acetylcholine. Accordingly, a loss of NO bioavailability is linked to the development of many of the same vascular pathologies caused by HHcy and also potentially increased Nox1, 2, and 4 activity. Thus, this thesis examined if current putative Nox inhibitors could prevent vascular dysfunction caused by homocysteine (as indirectly measured by acetylcholine-mediated vasorelaxation). Using New Zealand white rabbits, C57BL/6 mice and a Nox2-/- (C57BL/6 background) mouse models, we observed that pharmacological intervention with single Nox1, 2 and 4 inhibitors reduced the effect of acetylcholine on vasorelaxation. In 1% methionine-fed Nox2-/- mice, we observed an improvement in function. We also assessed combinations of Nox1, 2 and 4 inhibitors and found that, although function was not restored to control levels, it was improved compared with single Nox inhibition. Due to these results, we performed a gp91ds-tat dose response in rabbit aorta. We found that in our models of vascular dysfunction, lower doses of gp91ds-tat significantly improved acetylcholine-mediated vasorelaxation. These results showed for the first time that in both pharmacological and diet-induced HHcy, high dose putative Nox inhibitors might not be effective. In conclusion, the observations made in this thesis highlight the important role that Hcy plays in the redox balance in the context of vascular function. Future work in this area should focus on low dose Nox inhibition in Hcy induced disease in in vivo models, in order to better determine which drug can be used to HHcy induced vascular damage.

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