Regulation of PCSK9 Expression and Function: Mechanisms and Therapeutic Implications.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes degradation of low-density lipoprotein receptor (LDLR) and plays a central role in regulating plasma levels of LDL cholesterol levels, lipoprotein(a) and triglyceride-rich lipoproteins, increasing the risk of cardiovascular disease. Additionally, PCSK9 promotes degradation of major histocompatibility protein class I and reduces intratumoral infiltration of cytotoxic T cells. Inhibition of PCSK9 increases expression of LDLR, thereby reducing plasma levels of lipoproteins and the risk of cardiovascular disease. PCSK9 inhibition also increases cell surface levels of major histocompatibility protein class I in cancer cells and suppresses tumor growth. Therefore, PCSK9 plays a vital role in the pathogenesis of cardiovascular disease and cancer, the top two causes of morbidity and mortality worldwide. Monoclonal anti-PCSK9 antibody-based therapy is currently the only available treatment that can effectively reduce plasma LDL-C levels and suppress tumor growth. However, high expenses limit their widespread use. PCSK9 promotes lysosomal degradation of its substrates, but the detailed molecular mechanism by which PCSK9 promotes degradation of its substrates is not completely understood, impeding the development of more cost-effective alternative strategies to inhibit PCSK9. Here, we review our current understanding of PCSK9 and focus on the regulation of its expression and functions.

Membrane-type I matrix metalloproteinase (MT1-MMP), lipid metabolism, and therapeutic implications.

Lipids exert many essential physiological functions, such as serving as a structural component of biological membranes, storing energy, and regulating cell signal transduction. Dysregulation of lipid metabolism can lead to dyslipidemia related to various human diseases, such as obesity, diabetes, and cardiovascular disease. Therefore, lipid metabolism is strictly regulated through multiple mechanisms at different levels, including the extracellular matrix. Membrane-type I matrix metalloproteinase (MT1-MMP), a zinc-dependent endopeptidase, proteolytically cleaves extracellular matrix components, and non-matrix proteins, thereby regulating many physiological and pathophysiological processes. Emerging evidence supports the vital role of MT1-MMP in lipid metabolism. For example, MT1-MMP mediates ectodomain shedding of low-density lipoprotein receptor and increases plasma low-density lipoprotein cholesterol levels and the development of atherosclerosis. It also increases the vulnerability of atherosclerotic plaque by promoting collagen cleavage. Furthermore, it can cleave the extracellular matrix of adipocytes, affecting adipogenesis and the development of obesity. Therefore, the activity of MT1-MMP is strictly regulated by multiple mechanisms, such as autocatalytic cleavage, endocytosis and exocytosis, and post-translational modifications. Here, we summarize the latest advances in MT1-MMP, mainly focusing on its role in lipid metabolism, the molecular mechanisms regulating the function and expression of MT1-MMP, and their pharmacotherapeutic implications.

Membrane type 1 matrix metalloproteinase promotes LDL receptor shedding and accelerates the development of atherosclerosis.

Plasma low-density lipoprotein (LDL) is primarily cleared by LDL receptor (LDLR). LDLR can be proteolytically cleaved to release its soluble ectodomain (sLDLR) into extracellular milieu. However, the proteinase responsible for LDLR cleavage is unknown. Here we report that membrane type 1-matrix metalloproteinase (MT1-MMP) co-immunoprecipitates and co-localizes with LDLR and promotes LDLR cleavage. Plasma sLDLR and cholesterol levels are reduced while hepatic LDLR is increased in mice lacking hepatic MT1-MMP. Opposite effects are observed when MT1-MMP is overexpressed. MT1-MMP overexpression significantly increases atherosclerotic lesions, while MT1-MMP knockdown significantly reduces cholesteryl ester accumulation in the aortas of apolipoprotein E (apoE) knockout mice. Furthermore, sLDLR is associated with apoB and apoE-containing lipoproteins in mouse and human plasma. Plasma levels of sLDLR are significantly increased in subjects with high plasma LDL cholesterol levels. Thus, we demonstrate that MT1-MMP promotes ectodomain shedding of hepatic LDLR, thereby regulating plasma cholesterol levels and the development of atherosclerosis.

Tanshinone IIA Promotes Macrophage Cholesterol Efflux and Attenuates Atherosclerosis of apoE-/- Mice by Omentin-1/ABCA1 Pathway.

BACKGROUND: Tanshinone IIA (Tan IIA) and Omentin-1 have a protective role in the cardiovascular system. However, if and how Tan IIA and Omentin-1 regulate cholesterol metabolism in macrophages has not been fully elucidated. OBJECTIVE: To investigate the possible mechanisms of Tan IIA and Omentin-1 on preventing macrophage cholesterol accumulation and atherosclerosis development. METHODS: The effect of Tan IIA on the protein and mRNA levels of Omentin-1 and ATP-binding cassette transporter A1 (ABCA1) in macrophages was examined by Western blot and qRT-PCR assay, respectively. Cholesterol efflux was assessed by liquid scintillation counting (LSC). Cellular lipid droplet was measured by Oil Red O staining, and intracellular lipid content was detected by high performance liquid chromatography (HPLC). In addition, the serum lipid profile of apoE-/- mice was measured by enzymatic method. The size of atherosclerotic lesion areas and content of lipids and collagen in the aortic of apoE-/- mice were examined by Sudan IV, Oil-red O, and Masson staining, respectively. RESULTS: Tan IIA up-regulated expression of Omentin-1 and ABCA1 in THP-1 macrophages, promoting ABCA1-mediated cholesterol efflux and consequently decreasing cellular lipid content. Consistently, Tan IIA increased reverse cholesterol transport in apoE-/- mice. Plasma levels of high-density lipoprotein cholesterol (HDL-C), ABCA1 expression and atherosclerotic plaque collagen content were increased while plasma levels of low-density lipoprotein cholesterol (LDL-C) and atherosclerotic plaque sizes were reduced in Tan IIA-treated apoE-/- mice. These beneficial effects were, however, essentially blocked by knockdown of Omentin-1. CONCLUSION: Our results revealed that Tan IIA promotes cholesterol efflux and ameliorates lipid accumulation in macrophages most likely via the Omentin-1/ABCA1 pathway, reducing the development of aortic atherosclerosis.

Heat shock protein 70 accelerates atherosclerosis by downregulating the expression of ABCA1 and ABCG1 through the JNK/Elk-1 pathway.

BACKGROUND AND AIMS: Recent studies have suggested that heat shock protein 70 (HSP70) may play critical roles in cardiovascular disease. However, the effects of HSP70 on the development of atherosclerosis in apoE-/- mice remain largely unknown. This study was to investigate the role and potential mechanism of HSP70 in atherosclerosis. METHODS: HSP70 was overexpressed in apoE-/- mice and THP-1-derived macrophages with lentiviral vectors. Oil Red O, hematoxylin-eosin, and Masson staining were performed to evaluate atherosclerotic plaque in apoE-/- mice fed the Western type diet. Moreover, immunostaining was employed to detect the expression of relative proteins in aortic sinus. Reporter gene and chromatin immunoprecipitation were performed to analyze the effect of Elk-1 on the promoter activity of ABCA1 and ABCG1; [3H] labeled cholesterol was used to assess the capacity of cholesterol efflux and reverse cholesterol transport (RCT). RESULTS: Our results showed that HSP70 increased lipid accumulation in arteries and promoted the formation of atherosclerotic lesion. The capacity of cholesterol efflux was reduced in peritoneal macrophages isolated from HSP70-overexpressed apoE-/- mice. The levels of ABCA1 and ABCG1 expression were also reduced in the peritoneal macrophages and the aorta from apoE-/- mice in response to HSP70. The c-Jun N-terminal kinase (JNK) and ETS transcription factor (Elk-1) played a critical role in HSP70-induced downregulation ABCA1 and ABCG1. Further, HSP70 reduced RCT from macrophages to plasma, liver, and feces in apoE-/- mice. CONCLUSIONS: HSP70 promotes the progression of atherosclerosis in apoE-/- mice by suppressing the expression of ABCA1 and ABCG1 through the JNK/Elk-1 pathway.

MicroRNA-134 Promotes the Development of Atherosclerosis Via the ANGPTL4/LPL Pathway in Apolipoprotein E Knockout Mice.

AIMS: Atherosclerosis is the most common cause of cardiovascular disease, such as myocardial infarction and stroke. Previous study revealed that microRNA (miR)-134 promotes lipid accumulation and proinflammatory cytokine secretion through angiopoietin-like 4 (ANGPTL4)/lipid lipoprotein (LPL) signaling in THP-1 macrophages. METHODS: ApoE KO male mice on a C57BL/6 background were fed a high-fat/high-cholesterol Western diet, from 8 to 16 weeks of age. Mice were divided into four groups, and received a tail vein injection of miR-134 agomir, miR-134 antagomir, or one of the corresponding controls, respectively, once every 2 weeks after starting the Western diet. After 8 weeks we measured aortic atherosclerosis, LPL Activity, mRNA and protein levels of ANGPTL4 and LPL, LPL/ low-density lipoprotein receptor related protein 1 Complex Formation, proinflammatory cytokine secretion and lipid levels. RESULTS: Despite this finding, the influence of miR-134 on atherosclerosis in vivo remains to be determined. Using the well-characterized mouse atherosclerosis model of apolipoprotein E knockout, we found that systemic delivery of miR-134 agomir markedly enhanced the atherosclerotic lesion size, together with a significant increase in proinflammatory cytokine secretion and peritoneal macrophages lipid contents. Moreover, overexpression of miR-134 decreased ANGPTL4 expression but increased LPL expression and activity in both aortic tissues and peritoneal macrophages, which was accompanied by increased formation of LPL/low-density lipoprotein receptor-related protein 1 complexes in peritoneal macrophages. However, an opposite effect was observed in response to miR-134 antagomir. CONCLUSIONS: These findings suggest that miR-134 accelerates atherogenesis by promoting lipid accumulation and proinflammatory cytokine secretion via the ANGPTL4/LPL pathway. Therefore, targeting miR-134 may offer a promising strategy for the prevention and treatment of atherosclerotic cardiovascular disease.

MicroRNA-182 Promotes Lipoprotein Lipase Expression and Atherogenesisby Targeting Histone Deacetylase 9 in Apolipoprotein E-Knockout Mice.

BACKGROUND: Lipoprotein lipase (LPL) expressed in macrophages plays an important role in promoting the development of atherosclerosis or atherogenesis. MicroRNA-182 (miR-182) is involved in the regulation of lipid metabolism and inflammation. However, it remains unclear how miR-182 regulates LPL and atherogenesis.Methods and Results:Using bioinformatics analyses and a dual-luciferase reporter assay, we identified histone deacetylase 9 (HDAC9) as a target gene of miR-182. Moreover, miR-182 upregulated LPL expression by directly targetingHDAC9in THP-1 macrophages. Hematoxylin-eosin (H&E), Oil Red O and Masson's trichrome staining showed that apolipoprotein E (ApoE)-knockout (KO) mice treated with miR-182 exhibited more severe atherosclerotic plaques. Treatment with miR-182 increased CD68 and LPL expression in atherosclerotic lesions in ApoE-KO mice, as indicated by double immunofluorescence staining in the aortic sinus. Increased miR-182-induced increases in LPL expression in ApoE-KO mice was confirmed by real-time quantitative polymerase chain reaction and western blotting analyses. Treatment with miR-182 also increased plasma concentrations of proinflammatory cytokines and lipids in ApoE-KO mice. CONCLUSIONS: The results of the present study suggest that miR-182 upregulates LPL expression, promotes lipid accumulation in atherosclerotic lesions, and increases proinflammatory cytokine secretion, likely through targetingHDAC9, leading to an acceleration of atherogenesis in ApoE-KO mice.

Puerarin promotes ABCA1-mediated cholesterol efflux and decreases cellular lipid accumulation in THP-1 macrophages.

It was reported that puerarin decreases the total cholesterol, low-density lipoprotein cholesterol (LDL-C), triglyceride (TG) and increases high-density lipoprotein cholesterol (HDL-C) level, but the underlying mechanism is unclear. This study was designed to determine whether puerarin decreased lipid accumulation via up-regulation of ABCA1-mediated cholesterol efflux in THP-1 macrophage-derived foam cells. Our results showed that puerarin significantly promoted the expression of ATP-binding cassette transporter A1 (ABCA1) mRNA and protein via the AMP-activated protein kinase (AMPK)-peroxisome proliferator-activated receptor gamma (PPARγ)-liver X receptor-alpha (LXR-α) pathway and decreased cellular lipid accumulation in human THP-1 macrophage-derived foam cells. The miR-7 directly targeted 3' untranslated region of STK11 (Serine/Threonine Kinase 11), which activated the AMPK pathway. Transfection with miR-7 mimic significantly reduced STK11 expression in puerarin-treated macrophages, decreased the phosphorylation of AMPK, down-regulated the expression of the PPARγ-LXR-α-ABCA1 expression. Additionally, treatment with miR-7 decreased cholesterol efflux and increased cholesterol levels in THP-1 macrophage-derived foam cells. Our study demonstrates that puerarin promotes ABCA1-mediated cholesterol efflux and decreases intracellular cholesterol levels through the pathway involving miR-7, STK11, and the AMPK-PPARγ-LXR-α-ABCA1 cascade.

Apolipoprotein A-1 binding protein promotes macrophage cholesterol efflux by facilitating apolipoprotein A-1 binding to ABCA1 and preventing ABCA1 degradation.

RATIONALE: Previous studies have shown that apolipoprotein-1 (apoA-1) binding protein (AIBP) is highly associated with the regulation of apoA-1 metabolism, suggesting its role in the treatment of atherosclerosis. However, how AIBP regulates foam cell formation remains largely unexplored. OBJECTIVE: To investigate the mechanisms underlying AIBP inhibition of foam cell formation from macrophages. METHODS AND RESULTS: THP-1-derived macrophages were incubated without or with apoA-1 and AIBP, followed by assessing the formation of foam cells and the potential mechanisms. Our results showed that AIBP and apoA-1 enhanced cholesterol efflux, altered the levels of cellular free cholesterol and cholesterol ester and prevented lipid accumulation so as to reduce the formation of foam cells. Meanwhile, lack of AIBP 115-123 amino acids resulted in the loss of AIBP binding to apoA-1. Moreover, our chemiluminescent analysis showed that AIBP promoted biotin-labeled apoA-1 binding to macrophages. Besides with AIBP, more apoA-1 bound to ABCA1, a key transporter responsible for cholesterol efflux to apoA-1, as indicated by our co-immunoprecipitation assay. Our results also showed that AIBP did not regulate ABCA1 mRNA expression, but stabilized its protein from CSN2-mediated degradation. CONCLUSIONS: AIBP promotes apoA-1 binding to ABCA1 on the cell membrane of macrophages and prevents ABCA1 protein from CSN2-mediated degradation so as to prevent foam cell formation. AIBP 115-123 amino acids is at least partially responsible for its binding to apoA-1.

Cystathionine γ-lyase(CSE)/hydrogen sulfide system is regulated by miR-216a and influences cholesterol efflux in macrophages via the PI3K/AKT/ABCA1 pathway.

This study was designed to evaluate whether CSE/H2S system, which is regulated by miR-216a, regulated ABCA1-mediated cholesterol efflux and cholesterol contents in THP-1 macrophages-derived foam cells. Our qPCR and western blotting results showed that CSE/H2S significantly up-regulated the expression of ATP-binding cassette transporter A1 (ABCA1) mRNA and protein via PI3K/AKT pathway in foam cells derived from human THP-1 macrophages. The miR-216a directly targeted 3' untranslated region of CSE. It significantly reduced CSE and ABCA1 expression, and also decreased the phosphorylation of PI3K and AKT. Additionally, cholesterol efflux decreased, and cholesterol levels increased in THP-1 macrophage-derived foam cells in response to treatment with miR-216a. Our study demonstrates that CSE/H2S system is regulated by miR-216a, and regulates ABCA1-mediated cholesterol efflux and cholesterol levels through the PI3K/AKT pathway.