Semicarbazide-sensitive amine oxidase (SSAO) and its interaction with lysyl oxidase (LOX) in rat aortic vascular smooth muscle cells

Introduction: Semicarbazide-sensitive amine oxidase (SSAO) is both a soluble and membrane bound transmembrane protein expressed in the vasculature. In endothelial cells, SSAO is involved in leukocyte rolling, adhesion, and transmigration into inflammatory sites which may imply a pathological role in the development of atherosclerosis. In vascular smooth muscle cells (VSMCs - the main stromal cells located in the middle layer of the arteries), SSAO has been highlighted as important factor in the oxidation of low-density cholesterol (LDL) and inducer of cell death. Lysyl oxidase (LOX) is another vascular enzyme which contributes to VSMCs extra cellular matrix (ECM) formation and stability. In these cells LOX has been associated with chemotaxis, which may imply a pathological role in the development of atherosclerosis. There is insufficient evidence regarding the interaction of these enzymes in VSMCs, their toxicological consequence, and the mechanism through which they contribute to LDL oxidation and the induction of chemotaxis. This thesis aims to investigate SSAO and LOX catalytic activity in rat aortic VSMCs, and their contributory role in oxidative stress and chemotaxis (important hallmarks in atherosclerosis), through ROS formation and vascular remodelling. Methods: To address this, an in vitro assay was developed and optimised for assessing SSAO and LOX activity in rat cultured aortic VSMCs through H2O2 generation. Toxicity of SSAO substrates and inhibitors was assessed colourimetrically. Cells at passage 3, 5 & 8 and confluency of ~80-90% were treated with the substrates benzylamine and cadaverine, and with LOX and SSAO irreversible suicide inhibitors βAPN and MDL72527. Western blot analysis was utilised to assess the membrane bound forms of the enzymes, vascular adhesion protein 1 (VAP-1) and Pro-LOX, the contractile markers smooth muscle 22α (SM22α) and alpha actin 2 (ACTA2) which indicate VSMCs physiological - contractile phenotype, and the expression of platelet derived growth factor receptor β (PDGFRβ), a receptor implicated in the mechanism of VSMCs chemotaxis. A quantitative polymerase chain reaction (qPCR) was applied to study mRNA levels of the gene that encodes for SSAO, amine oxidase copper containing 3 (AOC3) and LOX (LOX) in the presence of their respective irreversible inhibitors, and after silencing LOX gene. ROS and total glutathione (GSH) content were evaluated with ROS and the recycling GSH assay. Results: SSAO activity was maximal at 6h from addition of reaction mixture. There was a passage dependent increase in SSAO affinity for its substrate benzylamine (**p < 0.01) as shown by a 3-fold reduction in the Km as passage increased from 3 (Km = 0.2084) to 8 (Km = 0.07267). Methylhydrazine was identified as the most potent reversible, and MDL72527 as the most potent irreversible inhibitor. Aminoacetone and methylamine induced VSMCs death at concentrations of 50 & 1000µM, and higher SSAO affinity was detected for aminoacetone comparing to methylamine (Km = 12µM vs 65µM). The cytotoxicity was reversed with MDL72527 which completely abolished cell death. Enhanced cytotoxicity was detected after simultaneous addition of aldehydes and H2O2. VAP-1, Pro-LOX and PDGFRβ were expressed in cultured VSMCs under control conditions with levels maintained between passages. At basal level, LOX activity decreased with passage and its affinity and protein expression were maintained between passages. βAPN abolished LOX activity (**p < 0.01 for 3 vs 8 and *p < 0.05 for 5 vs 8) and had no effect on Pro-LOX expression and LOX mRNA levels; and MDL72527 had no effect on LOX activity, protein, and mRNA expression. At basal level, SSAO activity also decreased with passage and its protein expression was maintained between passages. MDL72527 abolished SSAO activity (****p < 0.0001 for 3 vs 8 and *p < 0.05 for 5 vs 8), VAP-1 expression at passage 5 (**p < 0.01) and passage 8 (****p < 0.0001), and AOC3 mRNA levels at passage 8 (*p < 0.05). βAPN inhibited SSAO activity (****p < 0.0001 for 3 vs 5 and 3 vs 8 and *p < 0.05 for 5 vs 8), VAP-1 expression at passage 3 (*p < 0.05), and AOC3 mRNA levels at passage 3 (*p < 0.05). Benzylamine did not alter VAP-1 and LOX expression and cadaverine reduced VAP-1 at passage 3 (**p < 0.01), passage 5 (*p < 0.05), and passage 8 (**p < 0.01). Loss of contractile markers was observed in non-treated cells and after treatment with MDL72527, cadaverine and benzylamine. PDGFRβ expression was reduced at passage 8 after LOX (*p < 0.05) and passage 3 (*p < 0.05), 5 (*p < 0.05), and 8 (**p < 0.01) after SSAO inhibition. PDGFRβ expression was also reduced at passage 3 (*p < 0.05) after benzylamine and cadaverine treatment. Silencing LOX gene at passage 3 reduced AOC3 mRNA (####p < 0.0001 for SiRNA6 and ###p < 0.001 for SiRNA8) and VAP-1 protein (#p < 0.05 for SiRNA8). Highest ROS production was detected with aminoacetone and benzylamine treatment. MDL72527 abolished ROS in benzylamine (****p < 0.0001), methylamine (****p < 0.0001), and aminoacetone (****p < 0.0001), and βAPN in benzylamine (*p < 0.05) treated cells. Total GSH levels were reduced after benzylamine, methylamine and aminoacetone treatment (****p < 0.0001); however, failed to be restored after addition of MDL72527 and βAPN. Conclusion: A robust method was optimised and validated to effectively characterise SSAO and LOX activity in vitro. A cytotoxic consequence of SSAO catalytic activity was observed in cultured VSMCs and a contributory role of SSAO and LOX was detected in VSMCs chemotaxis, through changes in PDGFRβ expression. A synergistic relationship between SSAO and LOX was identified in early passage VSMCs. In late passage VSMCs, SSAO expressed predominant activity over LOX, and was demonstrated as the leading enzyme in ROS formation. Overall, these novel findings suggest a passage dependent interplay between SSAO and LOX in rat aortic primary VSMCs and associate their activity with VSMCs phenotyping modulation and ROS formation.