PCSK9 Function Research Articles

Lipid-lowering drugs and risk of rapid renal function decline: a mendelian randomization study.

Chronic kidney disease (CKD) patients face the risk of rapid kidney function decline leading to adverse outcomes like dialysis and mortality. Lipid metabolism might contribute to acute kidney function decline in CKD patients. Here, we utilized the Mendelian Randomization approach to investigate potential causal relationships between drug target-mediated lipid phenotypes and rapid renal function decline. In this study, we utilized two methodologies: summarized data-based Mendelian randomization (SMR) and inverse variance-weighted Mendelian randomization (IVW-MR), to approximate exposure to lipid-lowering drugs. This entailed leveraging expression quantitative trait loci (eQTL) for drug target genes and genetic variants proximal to drug target gene regions, which encode proteins associated with low-density lipoprotein (LDL) cholesterol, as identified in genome-wide association studies. The objective was to investigate causal associations with the progression of rapid kidney function decline. The SMR analysis revealed a potential association between high expression of PCSK9 and rapid kidney function decline (OR = 1.11, 95% CI= [1.001-1.23]; p = 0.044). Similarly, IVW-MR analysis demonstrated a negative association between LDL cholesterol mediated by HMGCR and kidney function decline (OR = 0.74, 95% CI = 0.60-0.90; p = 0.003). Genetically predicted inhibition of HMGCR is linked with the progression of kidney function decline, while genetically predicted PCSK9 inhibition is negatively associated with kidney function decline. Future research should incorporate clinical trials to validate the relevance of PCSK9 in preventing kidney function decline.

Abstract Tu105: Inhibition of Queuine tRNA-Ribosyltransferase 1 Ameliorates Heaptic Lipogenesis and Atherosclerosis

Background: Atherosclerosis threatens cardiovascular health, which is featured by abnormal lipid metabolism and chronic inflammation in arterial wall. Queuine tRNA-Ribosyltransferase 1 (QTRT1) , is an enzyme responsible for tRNA modification at the wobble base. Research Questions: It is unclear whether the function of QTRT1 is involved in metabolism of non-cancer cells and tissues, especially lipid metabolism mainly orchestrated by liver. Aims: We aimed to explore the role of QTRT1 deficiency in hyperlipidemia, liver steatosis and atherosclerosis. Methods: Transgenic mice such as Qtrt1fl/flAlb-iCre+/- (Qtrt1LKO), Qtrt1fl/fl(WT), ApoE-/-, and gain of function of PCSK9 by adeno-associated virus gene transfer were used to determine the functional significance of QTRT1 in atherosclerosis. Synthesis, transportation and oxidation of lipids in livers and hepatocytes were examined by quantitative real-time polymerase chain reaction. Moreover, primary hepatocytes with overexpression and downregulation of QTRT1 were followed by RNA sequencing to identify downstream targets. Results: Deficiency of hepatic QTRT1 significantly attenuated hyperlipidemia, liver steatosis and atherosclerotic burden, but increased plaque stability in the aorta of mice. Depletion of QTRT1 markedly downregulated de novo lipogenesis (DNL) without influence on lipoprotein transportation and fatty acid oxidation. Qtrt1LKO mice on a high-carbohydrate diet displayed relatively reduced hyperlipidemia and alleviation of DNL. RNA-seq indicated that odorant binding protein 2A (OBP2A) was upregulated in Qtrt1LKO hepatocytes, which reversed the promotion of DNL by QTRT1 in hepatocytes. Conclusion: Inhibition of QTRT1 in hepatocytes ameliorates hepatic lipogenesis and atherosclerosis in mice. Targeting of QTRT1 is a promising strategy to improve therapeutic effectiveness in both hyperlipidemia and atherosclerosis.

Insights into PCSK9-LDLR Regulation and Trafficking via the Differential Functions of MHC-I Proteins HFE and HLA-C.

PCSK9 is implicated in familial hypercholesterolemia via targeting the cell surface PCSK9-LDLR complex toward lysosomal degradation. The M2 repeat in the PCSK9's C-terminal domain is essential for its extracellular function, potentially through its interaction with an unidentified "protein X". The M2 repeat was recently shown to bind an R-x-E motif in MHC-class-I proteins (implicated in the immune system), like HLA-C, and causing their lysosomal degradation. These findings suggested a new role of PCSK9 in the immune system and that HLA-like proteins could be "protein X" candidates. However, the participation of each member of the MHC-I protein family in this process and their regulation of PCSK9's function have yet to be determined. Herein, we compared the implication of MHC-I-like proteins such as HFE (involved in iron homeostasis) and HLA-C on the extracellular function of PCSK9. Our data revealed that the M2 domain regulates the intracellular sorting of the PCSK9-LDLR complex to lysosomes, and that HFE is a new target of PCSK9 that inhibits its activity on the LDLR, whereas HLA-C enhances its function. This work suggests the potential modulation of PCSK9's functions through interactions of HFE and HLA-C.

Abstract 1311: PCSK9 expression in liver sinusoidal endothelial cells during liver metastasis

Abstract PCSK9 is a serin protein kinase of the family of convertases. It has been extensively studied in hypercholesterolaemia, but has recently been shown to be involved in cancer. It is overexpressed in several types of cancer, especially during the process of metastasis. One of the most common is colon liver metastasis. In this process of dissemination, the tumor microenvironment plays a key role. Our research focuses on the function of PCSK9 in liver sinusoidal endothelial cells (LSECs) involved in angiogenesis. We have shown in a study in vitro by qPCR and western blotting (WB), that PCSK9 protein is expressed in LSECs under basal conditions. Furthermore, we activated the cells with media from SW620 tumor cells as well as from SW620-derived cancer stem cells (CSCs) during 24 hours. Thus, we have shown that activation of LSECs with CSC conditioned media significantly increases PCSK9 expression at the mRNA level. This is the first time this convertase is detected in LSECs. In addition, we performed an immunofluorescence (IF) staining of PCSK9 to determine the cellular localization of the protein in this cell type. We found PCSK9 in the nucleus of LSECs in cell culture. Furthermore, we have confirmed the nuclear location by a WB analysis of the cytoplasmatic and nuclear fraction of the cells. As far as we know, this localization of PCSK9 has never been observed which open the possibility of new function for PCSK9 in LSECs. In addition, we performed IF staining in human tissues, both adjacent and metastatic livers. Thus, LSECs were labelled with the specific marker CD31 and PCSK9 demonstrating that PSCK9 is also expressed in patient’s LSECs. Regarding cellular localization, the protein appears to be localized in the nucleus according to the observations made in vitro. In order to determine PCSK9 function, we overexpressed PCSK9 in HEK cells and we co-immunoprecipitated it to study the related proteins. Some protein of interest appeared as far as EpCAM and CDK6 which are being studied. Furthermore, we inhibited PCSK9 with siRNA and chemical inhibitor (PF-06446846). We performed a proliferation test in both conditions and we showed that LSEC proliferation significantly decrease when we inhibit PCSK9. Finally, we did a RNAseq study in LSEC cells inhibiting PCSK9 after their activation with CSC media. This result showed us a significant decrease expression of ERN1 or FAP at mRNA level when PCSK9 is inhibited. We can conclude that PCSK9 plays a key role in LSEC during Liver Colorectal Metastasis affecting significantly on proliferation and showing a particular nuclear location. Although the metabolic pathway remains undetermined, the proteomic and RNAseq studies have provided us with some molecules that seem to interact with PCSK9 and that could explain the particular function of PCSK9 in LSECs during the metastatic process Citation Format: Ander Martin-San Sebastian, Helena García-García, Géraldine Siegfried, Abdel-Majid Khatib, Iker Badiola. PCSK9 expression in liver sinusoidal endothelial cells during liver metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1311.

PCSK9-D374Y Suppresses Hepatocyte Migration through Downregulating Free Cholesterol Efflux Rate and Activity of Extracellular Signal-Regulated Kinase.

Proprotein convertase subtilisin/kexin type 9 can mediate the intracellular lysosomal degradation of the low-density lipoprotein receptor protein in hepatocytes and decrease the liver's ability to scavenge low-density lipoprotein cholesterol from circulation, resulting in high levels of cholesterol in the circulatory system. Current studies have primarily focused on the relationship between PCSK9 and blood lipid metabolism; however, the biological function of PCSK9 in hepatocytes is rarely addressed. In this study, we evaluate its effects in the human hepatoma carcinoma cell line HepG2, including proliferation, migration, and free cholesterol transport. PCSK9-D374Y is a gain-of-function mutation that does not affect proliferation but significantly suppresses the migration and cholesterol efflux capacity of these cells. The suppression of the transmembrane outflow of intracellular-free cholesterol regulates small G proteins and the suppression of extracellular signal-regulated kinase. In summary, PCSK9-D374Y affects hepatocyte features, including their migration and free cholesterol transport capabilities.

Molecular interactions of PCSK9 with an inhibitory nanobody, CAP1 and HLA-C: Functional regulation of LDLR levels

ObjectiveThe liver-derived circulating PCSK9 enhances the degradation of the LDL receptor (LDLR) in endosomes/lysosomes. PCSK9 inhibition or silencing is presently used in clinics worldwide to reduce LDL-cholesterol, resulting in lower incidence of cardiovascular disease and possibly cancer/metastasis. The mechanism by which the PCSK9-LDLR complex is sorted to degradation compartments is not fully understood. We previously suggested that out of the three M1, M2 and M3 subdomains of the C-terminal Cys/His-rich-domain (CHRD) of PCSK9, only M2 is critical for the activity of extracellular of PCSK9 on cell surface LDLR. This likely implicates the binding of M2 to an unknown membrane-associated “protein X” that would escort the complex to endosomes/lysosomes for degradation. We reported that a nanobody P1.40 binds the M1 and M3 domains of the CHRD and inhibits the function of PCSK9. It was also reported that the cytosolic adenylyl cyclase-associated protein 1 (CAP1) could bind M1 and M3 subdomains and enhance the activity of PCSK9. In this study, we determined the 3-dimensional structure of the CHRD-P1.40 complex to understand the intricate interplay between P1.40, CAP1 and PCSK9 and how they regulate LDLR degradation. MethodsX-ray diffraction of the CHRD-P1.40 complex was analyzed with a 2.2 Å resolution. The affinity and interaction of PCSK9 or CHRD with P1.40 or CAP1 was analyzed by atomic modeling, site-directed mutagenesis, bio-layer interferometry, expression in hepatic cell lines and immunocytochemistry to monitor LDLR degradation. The CHRD-P1.40 interaction was further analyzed by deep mutational scanning and binding assays to validate the role of predicted critical residues. Conformational changes and atomic models were obtained by small angle X-ray scattering (SAXS). ResultsWe demonstrate that PCSK9 exists in a closed or open conformation and that P1.40 favors the latter by binding key residues in the M1 and M3 subdomains of the CHRD. Our data show that CAP1 is well secreted by hepatic cells and binds extracellular PCSK9 at distinct residues in the M1 and M3 modules and in the acidic prodomain. CAP1 stabilizes the closed conformation of PCSK9 and prevents P1.40 binding. However, CAP1 siRNA only partially inhibited PCSK9 activity on the LDLR. By modeling the previously reported interaction between M2 and an R-X-E motif in HLA-C, we identified Glu567 and Arg549 as critical M2 residues binding HLA-C. Amazingly, these two residues are also required for the PCSK9-induced LDLR degradation. ConclusionsThe present study reveals that CAP1 enhances the function of PCSK9, likely by twisting the protein into a closed configuration that exposes the M2 subdomain needed for targeting the PCSK9-LDLR complex to degradation compartments. We hypothesize that “protein X”, which is expected to guide the LDLR-PCSK9-CAP1 complex to these compartments after endocytosis into clathrin-coated vesicles, is HLA-C or a similar MHC-I family member. This conclusion is supported by the PCSK9 natural loss-of-function Q554E and gain-of-function H553R M2 variants, whose consequences are anticipated by our modeling.

Expanding Biology of PCSK9: Roles in Atherosclerosis and Beyond.

Purpose of ReviewSince the discovery of PCSK9 in 2003, this proprotein convertase was shown to target specific receptors for degradation in endosomes/lysosomes, including LDLR and other family members and hence to enhance the levels of circulating LDL-cholesterol (LDLc). Accordingly, inhibitors of PCSK9, including monoclonal antibodies blocking its circulating activity and siRNA silencers of its hepatic expression, are now used in clinics worldwide to treat hypercholesterolemia patients effectively and safely in combination with statins and/or ezetimibe. These powerful treatments reduce the incidence of atherosclerosis by at least 20%. Since 2008, novel targets of PCSK9 began to be defined, thereby expanding its roles beyond LDLc regulation into the realm of inflammation, pathogen infections and cellular proliferation in various cancers and associated metastases.Recent FindingsSome pathogens such as dengue virus exploit the ability of PCSK9 to target the LDLR for degradation to enhance their ability to infect cells. Aside from increasing the degradation of the LDLR and its family members VLDLR, ApoER2 and LRP1, circulating PCSK9 also reduces the levels of other receptors such as CD36 (implicated in fatty acid uptake), oxidized LDLR receptor (that clears oxidized LDLc) as well as major histocompatibility class-I (MHC-I) receptors (implicated in the immune response to antigens). Thus, these novel targets provided links between PCSK9 and inflammation/atherosclerosis, viral infections and cancer/metastasis. The functional activities of PCSK9, accelerated the development of novel therapies to inhibit PCSK9 functions, including small molecular inhibitors, long-term vaccines, and possibly CRISPR-based silencing of hepatic expression of PCSK9.SummaryThe future of inhibitors/silencers of PCSK9 function or expression looks bright, as these are expected to provide a modern armamentarium to treat various pathologies beyond hypercholesterolemia and its effects on atherosclerosis.

Relationship between PCSK9 and endothelial function in patients with acute myocardial infarction

Background and aimsWhile the role of PCSK9 in lipid metabolism is well established, its link with endothelial function is less clear. The aim of the present study is to evaluate the relationship between PCSK9 and endothelial dysfunction in the setting of acute myocardial infarction. Methods and resultsTo this purpose, we analyzed the serum of 74 patients with ST-elevation myocardial infarction (STEMI) at the time of admission and after 5 days. Endothelial dysfunction was evaluated as rate of apoptosis (AR) of human umbilical vein endothelial cells incubated with patients’ serum. There was a good correlation between PCSK9 and the apoptosis rate values, both at baseline (r = 0.649) and 5-day (r = 0.648). In the 5 days after STEMI, PCSK9 increased significantly (242–327 ng/ml, p < 0.001), while AR did not (p = 0.491). Overall, 21 (28%) patients showed a reduction of PCSK9, and they had a significantly higher decrease of AR as compared to others (−13.87 vs 5.8%, p = 0.002). At the univariable analysis, the 5-day change of PCSK9 resulted to be the only variable associated with the 5-day change of the apoptosis rate (beta 0.217, 95%CI 0.091–0.344, p = 0.001). ConclusionThe variation of endothelial function and PCKS9 in the first days after an acute myocardial infarction are related. Further validation and research are necessary to confirm our findings. Clinical trialNCT02438085.

Proprotein Convertase Subtilisin/Kexin Type 9 Inhibitors Use for Atherogenic Dyslipidemia in Solid Organ Transplant Patients.

Dyslipidemia is a widespread risk factor in solid organ transplant patients, due to many reasons, such as the use of immunosuppressive drugs, with a consequent increase in cardiovascular diseases in this population. PCSK9 is an enzyme mainly known for its role in altering LDL levels, consequently increasing cardiovascular risk. Monoclonal antibody PCSK9 inhibitors demonstrated remarkable efficacy in the general population in reducing LDL cholesterol levels and preventing cardiovascular disease. In transplant patients, these drugs are still poorly used, despite having comparable efficacy to the general population and giving fewer drug interactions with immunosuppressants. Furthermore, there is enough evidence that PCSK9 also plays a role in other pathways, such as inflammation, which is particularly dangerous for graft survival. In this review, the current evidence on the function of PCSK9 and the use of its inhibitors will be discussed, particularly in transplant patients, in which they may provide additional benefits.

The Multifaceted Biology of PCSK9.

This article reviews the discovery of PCSK9, its structure–function characteristics, and its presently known and proposed novel biological functions. The major critical function of PCSK9 deduced from human and mouse studies, as well as cellular and structural analyses, is its role in increasing the levels of circulating low-density lipoprotein (LDL)-cholesterol (LDLc), via its ability to enhance the sorting and escort of the cell surface LDL receptor (LDLR) to lysosomes. This implicates the binding of the catalytic domain of PCSK9 to the EGF-A domain of the LDLR. This also requires the presence of the C-terminal Cys/His-rich domain, its binding to the secreted cytosolic cyclase associated protein 1, and possibly another membrane-bound “protein X”. Curiously, in PCSK9-deficient mice, an alternative to the downregulation of the surface levels of the LDLR by PCSK9 is taking place in the liver of female mice in a 17β-estradiol-dependent manner by still an unknown mechanism. Recent studies have extended our understanding of the biological functions of PCSK9, namely its implication in septic shock, vascular inflammation, viral infections (Dengue; SARS-CoV-2) or immune checkpoint modulation in cancer via the regulation of the cell surface levels of the T-cell receptor and MHC-I, which govern the antitumoral activity of CD8+ T cells. Because PCSK9 inhibition may be advantageous in these processes, the availability of injectable safe PCSK9 inhibitors that reduces by 50% to 60% LDLc above the effect of statins is highly valuable. Indeed, injectable PCSK9 monoclonal antibody or small interfering RNA could be added to current immunotherapies in cancer/metastasis.

Proprotein convertase subtilisin/kexin Type 9 is required for Ahnak-mediated metastasis of melanoma into lung epithelial cells.

Previously we demonstrated that Ahnak mediates transforming growth factor-β (TGFβ)-induced epithelial-mesenchymal transition (EMT) during tumor metastasis. It is well-known that circulating tumor cells (CTCs) invade the vasculature of adjacent target tissues before working to adapt to the host environments. Currently, the molecular mechanism by which infiltrated tumor cells interact with host cells to survive within target tissue environments is far from clear. Here, we show that Ahnak regulates tumor metastasis through PCSK9 expression. To validate the molecular function of Ahnak in metastasis, B16F10 melanoma cells were injected into WT and Ahnak knockout (Ahnak-/-) mice. Ahnak-/- mice were more resistant to the pulmonary metastasis of B16F10 cells compared to wild-type (WT) mice. To investigate the host function of Ahnak in recipient organs against metastasis of melanoma cells, transcriptomic analyses of primary pulmonary endothelial cells from WT or Ahnak-/- mice in the absence or presence of TGFβ stimulation were performed. We found PCSK9, along with several other candidate genes, was involved in the invasion of melanoma cells into lung tissues. PCSK9 expression in the pulmonary artery was higher in WT mice than Ahnak-/- mice. To evaluate the host function of PCSK9 in lung tissues during the metastasis of melanoma cells, we established lung epithelial cell-specific tamoxifen-induced PCSK9 conditional KO mice (Scgb1a1-Cre/PCSK9fl/fl). The pulmonary metastasis of B16F10 cells in Scgb1a1-Cre/PCSK9fl/fl mice was significantly suppressed, indicating that PCSK9 plays an important role in the metastasis of melanoma cells. Taken together, our data demonstrate that Ahnak regulates metastatic colonization through the regulation of PCSK9 expression.

Evolocumab and alirocumab increases total PCSK9 plasma levels in hypocholesterolemic patients: Investigation of the basic molecular mechanisms

Background and Aims: Evolocumab and alirocumab (mAbs anti-PCSK9) block the function of PCSK9 but determine a significant increase of its plasma levels, an effect that can be determined by 1) an increased synthesis or 2) reduced clearance by the liver.

Effects of proprotein convertase subtilisin kexin type 9 modulation in human pancreatic beta cells function

Background and aimsProprotein Convertase Subtilisin Kexin Type 9 (PCSK9) is an endogenous inhibitor of the LDL receptor (LDLR). Mendelian randomization studies suggest that PCSK9 deficiency increases diabetes risk, but the underlying mechanisms remain unknown. The aim of our study was to investigate whether PCSK9 or its inhibition may modulate beta cell function. MethodsWe assessed PCSK9 and insulin colocalization in human pancreatic sections by epifluorescent and confocal microscopy. We also investigated the expression and the function of PCSK9 in the human EndoC−βH1 beta cell line, by ELISA and flow cytometry, respectively. PCSK9 was inhibited with Alirocumab or siRNA. LDLR expression and LDL uptake were assessed by flow cytometry. ResultsPCSK9 was expressed and secreted from beta cells isolated from human pancreas as well as from EndoC−βH1 cells. PCSK9 secretion was enhanced by statin treatment. Recombinant PCSK9 decreased LDLR abundance at the surface of these cells, an effect abrogated by Alirocumab. Alirocumab as well as PCSK9 silencing increased LDLR expression at the surface of EndoC−βH1 cells. Neither exogenous PCSK9, nor Alirocumab, nor PCSK9 silencing significantly altered glucose-stimulated insulin secretion (GSIS) from these cells. High-low density lipoproteins (LDL) concentrations decreased GSIS, but the addition of PCSK9 or its inhibition did not modulate this phenomenon. ConclusionsWhile PCSK9 regulates LDLR abundance in beta cells, inhibition of exogenous or endogenous PCSK9 does not appear to significantly impact insulin secretion. This is reassuring for the safety of PCSK9 inhibitors in terms of beta cell function.

Construction of point mutation rabbits using CRISPR/Cas9.

To establish a rabbit model of proprotein convertase subtilisin/kexin type9 () point mutation with CRISPR/Cas9 gene editing technique. According to the PubMed gene protein data, the PCSK9 protein functional regions of human and rabbit were analyzed by Blast. The 386S (Ser) amino acid functional region of human gene was homologous to the 485S of rabbit gene. Three small guide RNAs and one single-stranded donor oligonucleotide were designed according to the 485S base substitution position and sequence analysis of rabbit gene. The synthetic small guide RNAs, Cas9 mRNA and single-stranded donor oligonucleotide were co-injected into the cytoplasm of rabbit fertilized eggs and the embryos were transferred into the pregnant rabbits. PCR, TA cloning and off-target analysis were performed on the F0 rabbits to identify whether the PCSK9 mutation was successful. Fifteen F0 rabbits were obtained. The sequencing results showed that one of them was PCSK9 point mutation homozygote and two of them were PCSK9 point mutation heterozygotes, and the mutation could be stably inherited. The rabbit model of PCSK9 point mutation was successfully constructed by CRISPR/Cas9 technique, which provides an animal model for exploring the molecular mechanism of impaired PCSK9 function and developing reliable and effective diagnosis and treatment measures.

PCSK9 loss of function is protective against extra-coronary atherosclerotic cardiovascular disease in a large multi-ethnic cohort.

BackgroundTherapeutic inhibition of PCSK9 protects against coronary artery disease (CAD) and ischemic stroke (IS). The impact on other diseases remains less well characterized.MethodsWe created a genetic risk score (GRS) for PCSK9 using four single nucleotide polymorphisms (SNPs) at or near the PCSK9 locus known to impact lower LDL-Cholesterol (LDL-C): rs11583680, rs11591147, rs2479409, and rs11206510. We then used our GRS to calculate weighted odds ratios reflecting the impact of a genetically determined 10 mg/dL decrease in LDL-C on several pre-specified phenotypes including CAD, IS, peripheral artery disease (PAD), abdominal aortic aneurysm (AAA), type 2 diabetes, dementia, chronic obstructive pulmonary disease, and cancer. Finally, we used our weighted GRS to perform a phenome-wide association study.ResultsGenetic and electronic health record data that passed quality control was available in 312,097 individuals, (227,490 White participants, 58,907 Black participants, and 25,700 Hispanic participants). PCSK9 mediated reduction in LDL-C was associated with a reduced risk of CAD and AAA in trans-ethnic meta-analysis (CAD OR 0.83 [95% CI 0.80–0.87], p = 6.0 x 10−21; AAA OR 0.76 [95% CI 0.68–0.86], p = 2.9 x 10−06). Significant protective effects were noted for PAD in White individuals (OR 0.83 [95% CI 0.71–0.97], p = 2.3 x 10−04) but not in other genetic ancestries. Genetically reduced PCSK9 function associated with a reduced risk of dementia in trans-ethnic meta-analysis (OR 0.86 [95% CI 0.78–0.93], p = 5.0 x 10−04).ConclusionsGenetically reduced PCSK9 function results in a reduction in risk of several important extra-coronary atherosclerotic phenotypes in addition to known effects on CAD and IS, including PAD and AAA. We also highlight a novel reduction in risk of dementia, supporting a well-recognized vascular component to cognitive impairment and an opportunity for therapeutic repositioning.

Genetic Inhibition of PCSK9 and Liver Function

This study investigates associations of the lifelong genetic inhibition of PCSK9 with plasma liver enzymes and nonalcoholic fatty liver disease by using the loss-of-function variant PCSK9-p.Arg46Leu as a genetic instrument.

P704Nanoliposomal anti-PCSK9 vaccine ameliorates glucose intolerance and insulin resistance in diabetic rats

Abstract Background PCSK9 inhibitors have emerged as an effective lipid-lowering approach. Although the results of available studies suggest a positive association of plasma PCSK9 levels with glycemic parameters and risk of DM, the effects of PCSK9 inhibitors on glucose intolerance and insulin resistance as the key features of DM remain unclear. Purpose The present study was directed to determine effect of vaccine-mediated PCSK9 inhibition on glucose intolerance and insulin resistance in experimental diabetic rats. Methods Nanoliposomal vaccine composing from PCSK9-linked nanoliposome particles mixed in Alum adjuvant was subcutaneously injected four times with bi-weekly intervals in Wistar-Albino rats. Two weeks after the last immunization, vaccinated and non-vaccinated rats were subjected to diabetes experiment induced by single intraperitoneal injection of streptozotocin (STZ). One week after STZ injection, glucose tolerance ability of each animal was evaluated by using oral glucose tolerance test (OGTT) on the overnight fasted rats with glucose dose at 2 g/kg. Two weeks after STZ injection, insulin tolerance test (ITT) viaintraperitoneal injection of insulin (0.8 U/kg) was performed to determine the measure of peripheral utilization of glucose. The plasma concentrations of total cholesterol (TC), LDL-C, HDL-C, and TG were measured. Results Nanoliposomal vaccine exposing PCSK9 peptide was found to provoke high-titers IgG antibody response against PCSK9 in rats, which was associated with the decrease plasma levels and function of plasma PCSK9. During the first week after STZ injection, it was indicated that the fasting blood glucose (FBG) level was 49% (−171.7±35 mg/dL, p&lt;0.0001) lower in the vaccinated diabetic (VD) rats, when compared to the diabetic control (DC) rats. OGTT assessment revealed that the VS rats had significantly improved glucose tolerance ability and recorded significant reduction in the level of blood glucose over the period of 180 min, when compared with the DC rats. Measurement of integrated areas under the glucose curve values demonstrated that blood glucose levels were significantly (p&lt;0.0001) decreased by 34.5% in the VS rats than the DC rats. In addition, ITT analysis showed that after insulin administration blood glucose level was decreased by 49.3% in the VS group compared with the DC group. The VS rats showed significantly lower (−26.65%, p=0.02) plasma LDL-C levels than the DC rats (Figure). Histopathology examination indicated that the pancreatic islet of the VS rats had a slightly decreased population of β-cells and few α-cells. Histopathology examination of the liver tissues of both VS and DC rats exhibited the normal histology with the normal hepatic architecture composed of hepatic lobules with normal central vein. Conclusions PCSK9 inhibition using liposomal vaccine can protect from glucose and insulin tolerance impairments in diabetic rats through an unknown and pancreatic-independent mechanism.

457-P: One-Year Administration of Anti-PCSK9 Antibody Is Enough to Stabilize Vulnerable Coronary Plaques in Diabetic Patients, which Are Resistant to Intensive Statin Therapy

Purpose: Coronary plaque progression despite very low levels of LDL-C has been reported in the patients with coronary artery disease (CAD) who received intensive statin therapy (IST). Coronary CT Angiography (CCTA) has been applied to evaluate vulnerable coronary plaques having low attenuation of CT value. The major function of PCSK9 is the binding to hepatic LDL receptors, leading to their degradation. PCSK9 is produced also in vascular smooth muscle cells constituting the atherosclerotic lesion in addition to hepatocytes. PCSK9 promotes foam cell formation by suppressing the excretion of cholesterol from macrophages. Statin is demonstrated to promote PCSK9 production. PCSK9 has been proposed to play a crucial role in the pathogenesis of IST resistance. The present study was performed to establish a novel therapeutic approach to IST resistant vulnerable plaque of coronary artery in diabetic patients. Methods: The present study included 142 T2DM patients with non-FHC asymptomatic CAD who developed vulnerable coronary plaques despite IST over 2 years. CCTA by use of 320-slice CT was applied to evaluate vulnerable coronary plaques. During the administration of anti-PCSK9 antibody, changes in serum lipids and CT value of vulnerable coronary plaques were determined. Results: During 1 year administration of anti-PCSK9 antibody, 74% decrease in serum LDL-C was observed. Also, improvement of vulnerable coronary plaque, e.g., rise in the CT value of plaque (from 43.2 ±12.0 HU to 128.5 ± 52.3 HU, p&amp;lt;0.0001) was observed. Of the 142 patients, 95 patients finished the administration of the drug. Conclusion: One year administration of anti-PCSK9 antibody produced a significant stabilization of vulnerable coronary plaques in the patients with asymptomatic CAD who are resistant to IST. An analysis of the plaque quality by CCTA is useful method for the evaluation of the effect of the drug on vulnerable coronary plaques. Disclosure A. Hirai: None. K. Fujimura: None. K. Hirai: None. S. Kondo: None. A. Shirakami: None. T. Sakai: None. K. Murata: None. H. Taniai: None. K. Okuzaki: None. T. Kageyama: None.

A Novel Therapeutic Approach to Intensive Statin Therapy–Resistant Vulnerable Plaque of Coronary Artery in Diabetic Patients by Use of Anti-PCSK9 Antibody and Coronary CT Angiography (CCTA)

Purpose: Coronary plaque progression despite very low levels of LDL-C has been reported in the patients with coronary artery disease (CAD) who received intensive statin therapy (IST). CCTA has been applied to detect vulnerable coronary plaques having low attenuation of CT value. The best characterized function of PCSK9 is the binding to hepatic LDL receptors, leading to their degradation. PCSK9 is produced also in vascular smooth muscle cells constituting the atheriosclerotic lesion in addition to hepatocytes. PCSK9 promotes foam cell formation by suppressing the excretion of cholesterol from macrophages. Recently, statin is demonstrated to promote PCSK9 production. PCSK9 has been proposed to play a crucial role in the pathogenesis of IST resistance. The present study was performed to establish a novel therapeutic approach to IST resistant vulnerable plaque of coronary artery in diabetic patients. Methods: The present study included 1T2DM patients with non-FHC asymptomatic CAD who developed vulnerable plaques in coronary artery despite IST over 1 year. CCTA by use of 320-slice CT was applied to evaluate vulnerable plaques in coronary artery. During the administration of anti-PCSK9 antibody, changes in serum lipids and CT value of vulnerable coronary plaques were determined. Results: After the administration of anti-PCSK9 antibody, 72% decrease in serum LDL-C was observed. Also, improvement of vulnerable coronary plaque e.g., rise in the CT value of plaque (from 45.2 ± 12.0 HU to 107.5 ± 42.3 HU, p&amp;lt;0.0001) was observed in 6 months after administration of the drug. Conclusion: A significant improvement in vulnerable coronary plaques was observed in the patients with asymptomatic CAD who are resistant to IST after 6 months administration of anti-PCSK9 antibody. An analysis of the plaque quality by CCTA is useful method for the evaluation of the effect of the drug on vulnerable coronary plaques. Disclosure A. Hirai: None. K. Fujimura: None. S. Kondo: None. T. Sakai: None. S. Ikejima: None. H. Taniai: None. K. Hirai: None. K. Murata: None. A. Shirakami: None. K. Okuzaki: None. T. Kageyama: None.

Abstract 451: Radiation Induced Atherosclerosis on Mice

Rationale: The high prevalence of cardiovascular disease (CVD) in cancer survivors after ionizing radiation (IR) therapy has long been recognized, but the mechanism for this is unknown. Previous studies reported that the plaques in IR therapy survivors exhibit characteristics of increased instability without changes in the plaque size. Objective: To study the mechanism of IR induced CVD in humans, we decided to develop an IR mouse model that mimics human CV events. Methods and Results: We fed LDLR-/- mice or C57 mice with a single injection of adeno -associated virus vector(AAV) encoding a gain-of function of PCSK9 with a high fat diet, then irradiated the mice with different doses of IR (2 Gy, 5 Gy, 10 Gy). Whole body radiation was given 2 Gy, while localized IR to the neck and thoracic area was given at 5 Gy (1 time) or 10Gy (weekly 5 Gy, twice).About 2~3 weeks after the IR treatment, we performed left partial carotid artery ligation (PCL), and 3 weeks later examined the extent of plaque formation and cardiac abnormality by comparing those detected in non-irradiated control mice. The lesion area ratio between the partially ligated left carotid artery (LCA) and the right carotid artery (RCA) was significantly increased in the IR-treated group compared to the non-IR-treated group (3.1 ± 0.3 vs.1.8 ± 0.4, n = 7-8, p = 0.03). In addition, the lesion in the IR-treated of mice had significantly larger necrotic cores with a thinner fibrous cap when compared to those in non-irradiate mice. Furthermore, IR-treated mice showed cardiac hypertrophy, in which we found a significant increase of the wall thickness of mid-to small sized arteries as well as perivascular fibrosis, which were not noticed in the non-IR-treated mice. Conclusions: Our results demonstrate that IR not only increases atherosclerotic and vulnerable plaque formation but also cardiac hypertrophy with mid- and micro-vascular wall swelling. The increase of mid- and micro-vascular wall thickness may explain the heart failure preserved heart failure, which is observed in the majority of heart failure patients after IR treatment.