MACROPHAGE EXPRESSION OF PCSK9 INFLUENCES ATHEROSCLEROSIS DEVELOPMENT
Creators: name: GIUNZIONI, ILARIA
Publisher: name: Universit� degli Studi di Milano
Year: 2014

description: Proprotein Convertase Subtilisin/Kexin 9 (PCSK9) regulates low?density 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?/? or apoE?/? 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?R 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�64 vs. 158�44 mg/dl, respectively, p&lt;0.05) and hPCSK9 was detected in atherosclerotic plaques of hPCSK9 tg. In contrast, there was no effect of PCSK9 expression in apoE?/? mice on serum cholesterol levels compared with apoE?/? controls (1066�161 vs. 964�188 mg/dl, respectively, NS). Surprisingly, hPCSK9 expressing apoE?/? mice showed increased proximal aorta lesion area. Lesion composition analysis revealed that lesions of PCSK9/apoE?/? mice have a higher content of Ly6Chigh positive cells (6.7�0.2%) compared to apoE?/? controls (5.7�0.4%). Moreover, analysis of spleen lysates revealed an increase in the percentage of Ly6Chigh positive cells in hPCSK9tg compared to control apoE?/?, 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?/? compared to LDLR?/?, 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?/? or apoE?/? mice were transplanted into apoE?/? recipients. hPCSK9 was detected in serum and lesions from PCSK9/apoE?/? mice but there was no effect of PCSK9 expression on serum cholesterol levels compared with apoE?/? controls. Interestingly, lesion composition analysis showed significantly higher levels of Ly6Chigh positive cells in recipients of hPCSK9/apoE?/? bone marrow cells compared to controls (7.4�1.5% vs. 5.6�1.1%, respectively, p&lt;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?/? or LDLR?/? mice into LDLR?/? 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.