Effects of oxidized lipids on PCSK6 modulation in human vascular cells

Atherosclerosis is a multifactorial disease characterized by oxidized LDL accumulation in the intima that promotes oxidative stress and inflammation, triggering atherosclerotic plaque development. After oxidation of the LDL lipid moiety different toxic compounds are formed. Nowadays, there is no more doubt that cholesterol oxidation products (oxysterols) and the aldehyde 4-hydroxy-2-nonenal (HNE) play a key role in all steps of atheroma formation. The clinical manifestations of atherosclerosis are the consequence of an unstable atherosclerotic plaque that is prone to rupture. The thinning of the cap is due to ECM component degradation by MMPs, which are released by inflammatory cells within the plaque. Among them, MMP-9 is highly involved in matrix degradation, showing a crucial contribution in plaque instability. Recently, there is a great effort to discover new biomarkers in order to estimate the patients with high risk to develop atherosclerosis. A recent work underlines the PCSK6, a pro-protein convertase, as a novel biomarker of unstable atherosclerotic plaques and it has been shown that PCSK6 and MMP-9 co-localize in the fibrous cap. On this basis, the aim of the project was to investigate whether an oxysterol mixture, of a composition similar to that found in human carotid plaques, and HNE, the more reactive aldehyde, might modulate the levels and the enzymatic activity of PCSK6. Moreover, we investigated whether the activity of PCSK6 might induce the activity of MMP-9 contributing to atherosclerotic plaque instability. In addition, to clarify if the inflammation could be the driving force of PCSK6 up-regulation, we incubated the cells with some anti-inflammatory compounds: NS-398 or acetylsalicylic acid, both inhibitors of COX enzymes, or epigallocatechin gallate, a polyphenol with anti-inflammatory properties. The expression, synthesis, and enzymatic activity of PCSK6 were significantly induced by the oxysterol mixture (20 M) or HNE (5 M) in human promonocytic U937 cells and in human umbilical vein endothelial cells (HUVECs). The effect of PCSK6 activity on MMP-9 activation was proved using a PCSK6 siRNA: the silencing of PCSK6 significantly decreased MMP-9 activity induced by the oxidized lipids in both cell types. Using anti-inflammatory molecules, the enzymatic activity of PCSK6 was decreased, pointing out that inflammation plays a key role in stimulating the activity of the pro-protein convertase PCSK6. These data demonstrate that oxysterols and HNE are able to induce PCSK6 levels and its enzymatic activity and that PCSK6 is an inflammation-dependent protein; in addition, PCSK6, induced by the oxidized lipids, triggers MMP-9 activation, a key enzyme in matrix remodeling and plaque destabilization, thus contributing to the fibrous cap degradation and plaque rupture. Although further investigations are needed, PCSK6 could be a promising new biomarker for atherosclerosis prevention and a target for developing novel therapeutic strategies.