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Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that binds and escorts the low density lipoprotein receptor (LDLR) into the lysosomal degradation pathway. Prescribed monoclonal antibodies (mAbs) against PCSK9 prevent its binding to the LDLR, and result in ~60% lower LDL cholesterol (LDLc) levels. Although efficient, mAbs are expensive. Hence other PCSK9 inhibitors are needed. For screening purpose, we developed C57BL/6J mice expressing the human PCSK9 gene under the control of its own promoter, but lacking endogenous mouse PCSK9. All lines recapitulate the endogenous PCSK9 expression pattern. The Tg2 line that expresses physiological levels of human PCSK9 (hPCSK9) was selected to characterize the inhibitory properties of a previously reported single domain antibody (sdAb), PKF8-mFc, which binds the C-terminal domain of PCSK9. Upon intraveinous injection of 10 mg/kg, PKF8-mFc and the mAb evolocumab neutralized ~50% and 100% of the hPCSK9 impact on total cholesterol (TC) levels, respectively, but PKF8-mFc had a more sustained effect. PKF8-mFc barely affected hPCSK9 levels, whereas evolocumab promoted a 4-fold increase 3 days post-injection, suggesting very different inhibitory mechanisms. The present study also shows that the new transgenic mice are well suited to screen a variety of hPCSK9 inhibitors. [ABSTRACT FROM AUTHOR]

Background: Given the link between cholesterol and activation of inflammation via interleukin 1β (IL-1β), we tested the effects of IL-1β inhibition on atherosclerotic calcification in mice. Patients with familial hypercholesterolemia develop extensive aortic calcification and calcific aortic stenosis. Although statins delay this process, low-density lipoprotein (LDL) cholesterol lowering alone is not enough to avert it. Data suggest that vascular inflammation initiated by hypercholesterolemia is followed by unchecked mineralization at sites of atherosclerotic plaques. The LDL-receptor (LDLR)-deficient (Ldlr−/−) and LDLR-attenuated Pcsk9(Tg) mice are available animal models for pharmacological testing. Methods: A mouse monoclonal antibody (mAb) against IL-1β or placebo was administered subcutaneously in Ldlr−/− and Pcsk9(Tg) models fed a Western diet. Drug level, anthropometric, lipid, and glucose profiles were determined. Expressions of proprotein convertase subtilisin/kexin type 9 (PCSK9), serum amyloid A1, and cytokine were measured by enzyme-linked immunosorbent assay. Aortic calcification was determined by microcomputerized tomography (micro-CT) and X-ray densitometry, and aortic flow velocity was assessed by ultrasound. Results: Circulating levels of IL-1β in Ldlr−/− mice were significantly greater (2-fold) than observed in Pcsk9(Tg) mice. Placebo- and mAb-treated mice did not differ in their growth, lipid, glucose profiles, and other cytokines. Calcifications were significantly diminished in mAb-treatment Ldlr−/− mice (a reduction of ∼75% by X-ray and ∼90% by micro-CT) and reduced insignificantly in mAb-treatment Pcsk9(Tg) mice, whereas aortic flow velocity was unchanged in both models. Conclusions: Herein, we demonstrate that aortic calcifications can be inhibited by an IL-1β mAb in LDLR-deficient mice. These results have a translational component to prevent vascular calcification in human and represent new evidence to rationalize targeting inflammation in cardiovascular disease. [ABSTRACT FROM AUTHOR]

Background--The proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes independently of its enzymatic activity the degradation of the low-density lipoprotein (LDL) receptor. PCSK9 gain of function in humans leads to autosomal dominant hypercholesterolemia, whereas the absence of functional PCSK9 results in 7-fold lower levels of LDL cholesterol. This suggests that lowering PCSK9 may protect against atherosclerosis. Methods and Results--We investigated the role of PCSK9 in atherosclerosis in C57BL/6 wild-type (WT), apolipoprotein E-deficient, and LDL receptor-deficient mouse models. Circulating cholesterol levels, fast protein liquid chromatog-raphy profiles, aortic cholesteryl esters (CE), and plaque sizes were determined. Intima-media thicknesses were measured by ultrasound biomicroscopy. First, mice expressing null (knockout [KO]), normal (WT), or high (transgenic ITg]) levels of PCSK9 were fed a 12-month Western diet. KO mice accumulated 4-fold less aortic CE than WT mice, whereas Tg mice exhibited high CE and severe aortic lesions. Next we generated apolipoprotein E-deficient mice, known to spontaneously develop lesions, that expressed null (KO/e), normal (WT/e), or high (Tg/e) levels of PCSK9. After a 6-month regular diet, KO/e mice showed a 39% reduction compared with WT/e mice in aortic CE accumulation, whereas Tg/e mice showed a 137% increase. Finally, LDL receptor-deficient mice expressing no (KO/L), normal (WT/L), or high (Tg/L) levels of PCSK9 were fed a Western diet for 3 months. KO/L and Tg/L mice exhibited levels of plasma cholesterol and CE accumulation similar to those of WT/L mice, suggesting that PCSK9 modulates atherosclerosis mainly via the LDL receptor. Conclusions--Altogether, our results show a direct relationship between PCSK9 and atherosclerosis. PCSK9 overexpres-sion is proatherogenic, whereas its absence is protective. [ABSTRACT FROM AUTHOR]