CLINICAL AND EXPERIMENTAL EVIDENCES OF DIRECT VASCULAR EFFECT OF PROPROTEIN CONVERTASE SUBTILISIN/KEXIN TYPE 9

Proprotein Convertase Subtilisin Kexin type 9 (PCSK9), together with LDLR and APOB genes, had been identified as the third gene associated with Familial Hypercholesterolemia (FH). Secreted PCSK9 in fact, targets the hepatic LDL receptor (LDLR) for degradation thus preventing its recycling on the cell surface. The decreased expression of LDLR determines an increase in the circulating LDL particles, leading to increased cholesterol levels. Secreted PCSK9 is mainly derived from the liver, but it is also expressed in other tissues such as the brain, the kidney, the pancreas as well as the cells that composed the arterial wall. It is therefore possible that it could exert multiple paracrine effects. Our research group found that PCSK9 is expressed and secreted by smooth muscles cells (SMCs) which constitute the atherosclerotic plaque. The aim of my project was to determine the paracrine role of PCSK9 on the neointima formation through preclinical and clinical approaches. The immunohistochemical analysis of Pcsk9-/- and Pcsk9+/+ mice revealed that, after vascular manipulation, the PCSK9-null mice were protected from the formation of neointima with lower intima area (28100 ± 4901 µm2 and 14350 ± 2990 µm2 for Pcsk9+/+ and Pcsk9-/- mice respectively, p<0.05), associated with decreased intima/media ratio of 1.48 ± 0.34 and 0.60 ± 0.18 for Pcsk9+/+ and Pcsk9-/- mice respectively, (p<0.05). The in vitro studies on isolated SMCs from Pcsk9-/- and Pcsk9+/+ mice showed that the absence of PCSK9 induced a more contractile phenotype, associated with a reduced proliferation rate (doubling time were 57.3 ± 2.1h and 106.3 ± 4.5h, respectively [p<0.001]). The response to the chemotactic agent PDGF-BB (Platelet-derived growth factor), measured with Boyden’s chamber assay, was also impaired in the absence of PCSK9. These were rescued after the reconstitution of PCSK9 in the Pcsk9-/- cell line, which led to a more synthetic phenotype associated with a doubling time of 32.2± 3.1h and 41.2 ± 1.9h [p< 0.001], for Pcsk9-/- and Pcsk9-/-REC SMCs, respectively. The difference in proliferation between Pcsk9-/- and Pcsk9-/-REC SMCs was maintained also after the incubation with 40µM of simvastatin, suggesting that PCSK9 could improve SMCs proliferation through mechanism independently from cholesterol levels. The cell cycle analyses of the Pcsk9-/- and Pcsk9-/-REC SMCs showed a decreased activation of p21 and p27, associated with an increased expression of cyclin E and cyclin D1; presumably due to a different activation of the PDGF receptor pathway mediated by LRP1. Finally, the observational study, carried out in collaboration with the Brisighella Heart Study research group, demonstrated that serum levels of PCSK9, together with aging, is positively correlated to the pulse wave velocity. This is an indirect parameter used to evaluate the arterial stiffness and hence the presence of atherosclerotic plaques. Taken together, these results demonstrated that aside from its function on in regulating cholesterol homeostasis, PCSK9 plays a direct pro-atherogenic role in the arterial wall by sustaining SMC synthetic phenotype, proliferation, and migration.