MACROPHAGE EXPRESSION OF PCSK9 INFLUENCES ATHEROSCLEROSIS DEVELOPMENT

Proprotein Convertase Subtilisin/Kexin 9 (PCSK9) regulates low\u2010density lipoprotein (LDL) cholesterol levels by binding and degrading hepatic LDL receptor (LDLR), thus promoting atherosclerosis. Little is known of PCSK9 effect in macrophages and whether this contributes to the development of the atheroma. To test the effect of human (h) PCSK9 expression on atherosclerosis we developed transgenic mice expressing hPCSK9 on wild type (WT), LDLR\u2010/\u2010 or apoE\u2010/\u2010 background. We first demonstrated that both mPCSK9 and hPCSk9 are expressed at the mRNA level and secreted in the culture medium by MPM. As in hepatocytes, hPCSK9 reduced LDLR levels in the murine macrophage cell line J774A.1 and in inflammatory MPM. On the contrary, hPCSK9 did not reduce LRP1 expression, another member of the LDL\u2010R gene family involved in the development of atherosclerosis through its effects on macrophage inflammatory responses and promotion of cell survival. To test the effects of PCSK9 in the atheroma, we fed our transgenic mice a high fat diet for 8 weeks. As expected, serum cholesterol levels were increased by 2 fold in hPCSK9tg compared to WT mice (325\ub164 vs. 158\ub144 mg/dl, respectively, p<0.05) and hPCSK9 was detected in atherosclerotic plaques of hPCSK9 tg. In contrast, there was no effect of PCSK9 expression in apoE\u2010/\u2010 mice on serum cholesterol levels compared with apoE\u2010/\u2010 controls (1066\ub1161 vs. 964\ub1188 mg/dl, respectively, NS). Surprisingly, hPCSK9 expressing apoE\u2010/\u2010 mice showed increased proximal aorta lesion area. Lesion composition analysis revealed that lesions of PCSK9/apoE\u2010/\u2010 mice have a higher content of Ly6Chigh positive cells (6.7\ub10.2%) compared to apoE\u2010/\u2010 controls (5.7\ub10.4%). Moreover, analysis of spleen lysates revealed an increase in the percentage of Ly6Chigh positive cells in hPCSK9tg compared to control apoE\u2010/\u2010, suggesting a direct effect of PCSK9 in macrophage inflammation and plaque development. On the contrary, despite an increase in both cholesterol levels and lesion size in hPCSK9 tg/LDLR\u2010/\u2010 compared to LDLR\u2010/\u2010, no differences in Ly6Chigh positive cells were found between the two groups. To study the effect of macrophage PCSK9 in the atheroma, bone marrow cells from PCSK9/apoE\u2010/\u2010 or apoE\u2010/\u2010 mice were transplanted into apoE\u2010/\u2010 recipients. hPCSK9 was detected in serum and lesions from PCSK9/apoE\u2010/\u2010 mice but there was no effect of PCSK9 expression on serum cholesterol levels compared with apoE\u2010/\u2010 controls. Interestingly, lesion composition analysis showed significantly higher levels of Ly6Chigh positive cells in recipients of hPCSK9/apoE\u2010/\u2010 bone marrow cells compared to controls (7.4\ub11.5% vs. 5.6\ub11.1%, respectively, p<0.05). To test whether the effects of hPCSK9 on inflammation were dependent on binding and degradation of LDLR in macrophages, we transplanted bone marrow cells from PCSK9/LDLR\u2010/\u2010 or LDLR\u2010/\u2010 mice into LDLR\u2010/\u2010 recipients. We observed that, despite a large amount of PCSK9 accumulated in the serum of transgenic mice, nearly undetectable levels were found in the plaque. No differences were found between the two groups in terms of cholesterol levels, lesion size and Ly6Chigh positive cells between the two groups. Our results show for first time that human PCSK9 expression in macrophages directly influences atherosclerotic plaque composition in the absence of changes in serum cholesterol levels, suggesting a direct effect of PCSK9 in macrophage inflammation and plaque development. The effect on inflammation is dependent on LDLR since no effects in lesion composition were found in its absence

New developments in atherosclerosis: clinical potential of PCSK9 inhibition

Ilaria Giunzioni, Hagai Tavori Knight Cardiovascular Institute, Center for Preventive Cardiology, Oregon Health and Science University, Portland, OR, USA Abstract: Pro-protein convertase subtilisin/kexin type 9 (PCSK9) is a secreted 692-amino acid protein that binds surface low-density lipoprotein (LDL) receptor (LDLR) and targets it toward lysosomal degradation. As a consequence, the number of LDLRs at the cell surface is decreased, and LDL-cholesterol (LDL-C) clearance is reduced, a phenomenon that is magnified by gain-of-function mutations of PCSK9. In contrast, loss-of-function mutations of PCSK9 result in increased surface LDLR and improved LDL-C clearance. This provides the rationale for targeting PCSK9 in hypercholesterolemic subjects as a means to lower LDL-C levels. Monoclonal antibodies (mAbs) against PCSK9 that block its interaction with the LDLR have been developed in the past decade. Two companies have recently received the approval for their anti-PCSK9 mAbs by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) Regeneron/Sanofi, with alirocumab (commercial name &ndash; PRALUENT&reg;) and, Amgen with evolocumab (commercial name &ndash; Repatha&trade;). The introduction of anti-PCSK9 mAbs will provide an alternative therapeutic strategy to address many of the unmet needs of current lipid-lowering therapies, such as inability to achieve goal LDL-C level, or intolerance and aversion to statins. This review will focus on the kinetics of PCSK9, pharmacokinetics and pharmacodynamics of anti-PCSK9 mAbs, and recent data linking PCSK9 and anti-PCSK9 mAbs to cardiovascular events. Moreover, it will highlight the unanswered questions that still need to be addressed in order to understand the physiologic function, kinetics, and dynamics of PCSK9. Keywords: PCSK9, LDLR, monoclonal antibodies, pharmacokinetics, cardiovascular ris

Cigarette Smoke Condensate Affects Monocyte Interaction With Endothelium

Objective Circulating monocytes adhere to the endothelium and migrate into the intima contributing to atherosclerotic plaque growth. Cigarette smoke is a risk factor for atherosclerosis, but it is not completely known how it affects monocyte behavior in atherogenesis. Methods We studied the effects of cigarette smoke condensate (CSC) on human monocytes (HM) chemotaxis and transmigration through an endothelial cell (EC) monolayer. Results Pre-treatment with CSC caused a decrease in HM chemotaxis and transmigration (-55% and -18% vs control, p<0.05, respectively), paralleled by a reduced expression of Rac 1 GTPase. On the contrary, direct exposure of both HM and EC to CSC increased (+23% vs control, p<0.05) HM transmigration, paralleled by a strong stimulation of VCAM1 and ICAM1 expression by ECs, and by a slight increase in monocyte integrin expression. An enhancement of monocyte transmigration was obtained after the exposure of both HM and EC to medium conditioned by HM previously incubated with CSC (+265% vs control, p<0.001). CSC showed a stimulatory effect on the expression by HM of TLR4, MCP1, IL8, IL1beta, and TNFalfa, which was ablated by pretreatment with PDTC. Incubation with neutralizing antibodies against both MCP1 or IL8 completely abolished the CSC-conditioned medium induced HM transmigration. Conclusions CSC induces HM to release chemotactic factor(s), which amplify the recruitment and transmigration of inflammatory cells through EC, but CSC may also reduce HM migratory capacity. Therefore, exposure to CSC affects monocyte behavior and interaction with the endothelium, thus potentially facilitating and/or further aggravating the atherogenic process

Pleiotropic Anti-atherosclerotic Effects of PCSK9 Inhibitors From Molecular Biology to Clinical Translation

PURPOSE OF REVIEW: Clinical trials with PCSK9 inhibitors have shown a robust decrease in plasma LDL levels and a significant reduction in the incidence of cardiovascular atherosclerotic events. However, the role of PCSK9 in atherosclerosis is not well investigated and it remains unclear whether PCSK9 inhibition has direct, LDL-independent, anti-atherosclerotic effects. This review outlines the molecular pathways and targets of PCSK9 in atherosclerosis and summarizes the experimental and clinical data supporting the anti-atherosclerotic (pleiotropic) actions of PCSK9 inhibitors. RECENT FINDINGS: PCSK9 is expressed by various cell types that are involved in atherosclerosis (e.g., endothelial cell, smooth muscle cell, and macrophage) and is detected inside human atherosclerotic plaque. Preclinical studies have shown that inhibition of PCSK9 can attenuate atherogenesis and plaque inflammation. Besides increasing plasma LDL, PCSK9 appears to promote the initiation and progression of atherosclerosis. Inhibition of PCSK9 may confer atheroprotection that extends beyond its lipid-lowering effects