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Proprotein convertases (PCSKs) process matrix metalloproteases and cytokines, but their function in the vasculature is largely unknown. Previously, we demonstrated upregulation of PCSK6 in atherosclerotic plaques from symptomatic patients, localization to smooth muscle cells (SMCs) in the fibrous cap and positive correlations with inflammation, extracellular matrix remodeling and cytokines. Here, we hypothesize that PCSK6 could be involved in flow-mediated vascular remodeling and aim to evaluate its role in the physiology of this process using knockout mice. Pcsk6−/− and wild type mice were randomized into control and increased blood flow groups and induced in the right common carotid artery (CCA) by ligation of the left CCA. The animals underwent repeated ultrasound biomicroscopy (UBM) examinations followed by euthanization with subsequent evaluation using wire myography, transmission electron microscopy or histology. The Pcsk6−/− mice displayed a flow-mediated increase in lumen circumference over time, assessed with UBM. Wire myography revealed differences in the flow-mediated remodeling response detected as an increase in lumen circumference at optimal stretch with concomitant reduction in active tension. Furthermore, a flow-mediated reduction in expression of SMC contractile markers SMA, MYH11 and LMOD1 was seen in the Pcsk6−/− media. Absence of PCSK6 increases outward remodeling and reduces medial contractility in response to increased blood flow.

Cardiovascular diseases and their complications account for one-third of deaths worldwide, an estimated 18.6 million per year. One underlying cause of cardiovascular diseases is atherosclerotic plaque formation and subsequent rupture or erosion. Atherosclerotic plaques are formed in response to high plasma lipid levels, endothelial dysfunction allowing for entrapment of cholesterol within the vessel wall and a subsequent inflammatory response. Currently, the gold standard treatment for atherosclerosis is lowering plasma lipids with statins, as well as surgical intervention to remove the plaque.The role of the proprotein convertase subtilisin/kexin (PCSK) family in vascular disease is largely unknown, with the exception of PCSK9. The PCSK family constitutes of 9 different enzymes. The only PCSK that has received broad attention in the cardiovascular field so far was PCSK9, because of its involvement in lipid metabolism through regulation of the surface expression of the low-density lipoprotein receptor on hepatocytes. We have previously shown upregulation of an enzyme called PCSK6 in atherosclerotic plaques, particularly those from symptomatic patients, localized to smooth muscle cells (SMC) in the fibrous cap and with positive correlations to inflammation, extracellular matrix remodeling and cytokines. In this Ph.D. thesis we aimed to further expand upon this finding and to investigate the expression and localization of the entire PCSK family in vascular disease, as well as their mechanisms and translational potential.In Study I we hypothesized that PCSK6 could be involved in flow-mediated vascular remodeling. Using several in vivo and ex vivo models, we evaluated the role of PCSK6 in the physiology of this process. Pcsk6-/- (mice without expression of Pcsk6) and control mice were subjected to carotid ligation of the left common carotid artery, resulting in increased blood flow in the right common carotid artery. We detected a progressive increase in vessel circumference and decreased vessel strength in the knockout mice, but not in control mice. On a cellular level, this could be attributed to decreased expression of the typical contractile SMC markers, SMA, MYH11 and LMOD1.In Study II we aimed to elucidate the mechanistic role of PCSK6 in SMCs in the context of vascular injury. Using various human biobanks, we investigated the genetic locus surrounding the PCSK6 gene and identified one SNP in particular (rs1531817) to be associated with vessel intima-media thickness progression, a surrogate marker for atherosclerosis. Neointima formation in response to carotid artery ligation was found to be decreased in Pcsk6-/- mice compared to control, concomitant with decreased activity of another downstream enzyme MMP14. Taken together, our results implicate PCSK6 as a key protease in modulating SMC control in vascular injury. Mechanistically, this likely happens through activation of the PCSK6- MMP14-MMP2 axis.In Study III we hypothesized that PCSK6 may be involved in modulating immune responses, particularly in vascular diseases. Splenocytes isolated from Pcks6-/- mice secreted higher levels of several cytokines upon stimulation with a-CD3 and a-CD28 antibodies, and were more proliferative. Bone marrow-derived macrophages from Pcks6-/- mice were more prone to lipid uptake. Transplantation of Pcks6-/- bone marrow to atherosclerotic Ldlr-/- mice led to increased plaque burden compared to controls. However, these plaques were more stable, attributed to increased collagen deposition, SMC presence and IL-17 content.