Proprotein Convertase Subtilisin/kexin Type 9 Secretion Research Articles

Chemical constituents from the roots of Cynanchum wilfordii with PCSK9 secretion inhibitory activities

From the Cynanchum wilfordii roots, 32 compounds, including 5 previously undescribed (1, 4–6, 12) and 27 known (2, 3, 7–11, 13–32) compounds, were isolated, and their structures were elucidated using NMR spectroscopic data and MS data aided by ECD calculations or the modified Mosher's reaction. All isolates were tested for their inhibitory effects on proprotein convertase subtilisin/kexin type 9 (PCSK9) secretion. Among the isolates, compound 4, a methyl cholesterol analog, exhibited the most potent effect in reducing PCSK9 secretion, along with PCSK9 downregulation at the mRNA and protein levels via FOXO1/3 upregulation. Moreover, compound 4 attenuated statin-induced PCSK9 expression and enhanced the uptake of DiI-LDL low-density lipoprotein. Thus, compound 4 is suggested to be a potential candidate for controlling cholesterol levels.

Endogenous SIRT6 in platelets negatively regulates platelet activation and thrombosis.

Thromboembolism resulting from platelet dysfunction constitutes a significant contributor to the development of cardiovascular disease. Sirtuin 6 (SIRT6), an essential NAD+-dependent enzyme, has been linked to arterial thrombosis when absent in endothelial cells. In the present study, we have confirmed the presence of SIRT6 protein in anucleated platelets. However, the precise regulatory role of platelet endogenous SIRT6 in platelet activation and thrombotic processes has remained uncertain. Herein, we present compelling evidence demonstrating that platelets isolated from SIRT6-knockout mice (SIRT6-/-) exhibit a notable augmentation in thrombin-induced platelet activation, aggregation, and clot retraction. In contrast, activation of SIRT6 through specific agonist treatment (UBCS039) confers a pronounced protective effect on platelet activation and arterial thrombosis. Moreover, in platelet adoptive transfer experiments between wild-type (WT) and SIRT6-/- mice, the loss of SIRT6 in platelets significantly prolongs the mean thrombus occlusion time in a FeCl3-induced arterial thrombosis mouse model. Mechanistically, we have identified that SIRT6 deficiency in platelets leads to the enhanced expression and release of proprotein convertase subtilisin/kexin type 9 (PCSK9), subsequently activating the platelet activation-associated mitogen-activated protein kinase (MAPK) signaling pathway. These findings collectively unveil a novel protective role of platelet endogenous SIRT6 in platelet activation and thrombosis. This protective effect is, at least in part, attributed to the inhibition of platelet PCSK9 secretion and mitogen-activated protein kinase signaling transduction. Our study provides valuable insights into the intricate interplay between SIRT6 and platelet function, shedding light on potential therapeutic avenues for managing thrombotic disorders.

High-salt diet induces dyslipidemia through the SREBP2/PCSK9 pathway in dahl salt-sensitive rats

A high-salt diet is known to increase serum cholesterol levels; however, the underlying mechanism of salt-induced dyslipidemia in patients with salt-sensitivity remains poorly understood. We aimed to investigate whether high-salt diet (HSD) can induce dyslipidemia and elucidate the underlying mechanism of salt-induced dyslipidemia in Dahl salt-sensitive (SS) rats. Metabolomic and biochemical analyses revealed that the consumption of an HSD (8 % NaCl) significantly increased the serum levels of total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) in SS rats. The enzyme-linked immunosorbent assay demonstrated an increase in circulating proprotein convertase subtilisin/kexin type 9 (PCSK9) levels, accompanied by a decrease in hepatic low-density lipoprotein receptor (LDLR) levels due to HSD consumption. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis revealed that HSD consumption activated sterol regulatory element-binding protein-2 (SREBP2) expression in the liver and kidney, resulting in upregulation of PCSK9 at the transcriptional level in the liver and at the translational level in the kidney, ultimately increasing circulating PCSK9 levels. The combined effects of HSD on the liver and kidney contributed to the development of hypercholesterolemia. Furthermore, an in vitro assay confirmed that high-salt exposure led to an increase in the protein expression of SREBP2 and PCSK9 secretion, thereby reducing low-density lipoprotein (LDL) uptake. This study, for the first time, shows that an HSD induces dyslipidemia through activation of the SREBP2/PCSK9 pathway, providing new insights into the prevention and treatment of dyslipidemia in patients with salt sensitivity.

Acyclic Triterpenoids from Alpinia katsumadai Seeds with Proprotein Convertase Subtilisin/Kexin Type 9 Expression and Secretion Inhibitory Activity.

Cholesterol is one of the primary causes of cardiovascular disease. Investigating and developing potential drugs to effectively treat hypercholesterolemia are therefore of critical importance. Proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors have been developed to lower the levels of low-density lipoprotein cholesterol in patients with hypercholesterolemia. In this study, we aimed to identify compounds that inhibit the PCSK9 mRNA expression and secretion. The bioassay-guided investigation of Alpinia katsumadai seeds utilizing a PCSK9 mRNA expression monitoring assay yielded the isolation and identification of seven new compounds. Among these were three acyclic triterpenoids (1-3), an acyclic sesquiterpenoid (5), one arylpentanoid (6), and two diarylheptanoids (7 and 8), alongside 10 known compounds. The structures of these compounds were determined using nuclear magnetic resonance (NMR) spectroscopy, vibrational circular dichroism (VCD), and electronic circular dichroism (ECD). The absolute configurations of compounds 1 and 2 were identified by comparing the calculated and experimental VCD data as the ECD method was unable to distinguish the diastereomers. All the isolated compounds were evaluated for their regulatory effects on the low-density lipoprotein receptor (LDLR) and PCSK9 mRNA expression, as well as PCSK9 secretion. Of the tested compounds, two of the acyclic triterpenoids (1 and 2) demonstrated potent effects in downregulating PCSK9 at both the mRNA and protein levels, compared with the positive control (berberine chloride). Additionally, compound 1 inhibited PCSK9 secretion to a level comparable to that of berberine chloride. This study identifies compounds that inhibit PCSK9 mRNA expression and secretion, offering significant contributions to the development of novel drugs for the effective treatment of hypercholesterolemia..

New Loss-of-Function Mutations in PCSK9 Reduce Plasma LDL Cholesterol.

Lower plasma levels of LDL (low-density lipoprotein) cholesterol (LDL-C) can reduce the risk of atherosclerotic cardiovascular disease. The loss-of-function mutations in PCSK9 (proprotein convertase subtilisin/kexin type 9) have been known to associate with low LDL-C in many human populations. PCSK9 genetic variants in Chinese Uyghurs who are at high risk of atherosclerotic cardiovascular disease due to their dietary habits have not been reported. The study involved the whole-exome and target sequencing of college students from Uyghur and other ethnic groups in Xinjiang, China, for the association of PCSK9 loss-of-function mutations with low plasma levels of LDL-C. The mechanisms by which the identified mutations affect the function of PCSK9 were investigated in cultured cells using biochemical and cell assays. The causal effects of the identified PCSK9 mutations on LDL-C levels were verified in mice injected with adeno-associated virus expressing different forms of PCSK9 and fed a high-cholesterol diet. We identified 2 PCSK9 mutations-E144K and C378W-in Chinese Uyghurs with low plasma levels of LDL-C. The E144K and C378W mutations impaired the maturation and secretion of the PCSK9 protein, respectively. Adeno-associated virus-mediated expression of E144K and C378W mutants in Pcsk9 KO (knockout) mice fed a high-cholesterol diet also hampered PCSK9 secretion into the serum, resulting in elevated levels of LDL receptor in the liver and reduced levels of LDL-C in the serum. Our study shows that E144K and C378W are PCSK9 loss-of-function mutations causing low LDL-C levels in mice and probably in humans as well.

Selective inhibition of protein secretion by abrogating receptor–coat interactions during ER export

Protein secretion is an essential process that drives cell growth, movement, and communication. Protein traffic within the secretory pathway occurs via transport intermediates that bud from one compartment and fuse with a downstream compartment to deliver their contents. Here, we explore the possibility that protein secretion can be selectively inhibited by perturbing protein-protein interactions that drive capture into transport vesicles. Human proprotein convertase subtilisin/kexin type 9 (PCSK9) is a determinant of cholesterol metabolism whose secretion is mediated by a specific cargo adaptor protein, SEC24A. We map a series of protein-protein interactions between PCSK9, its endoplasmic reticulum (ER) export receptor SURF4, and SEC24A that mediate secretion of PCSK9. We show that the interaction between SURF4 and SEC24A can be inhibited by 4-phenylbutyrate (4-PBA), a small molecule that occludes a cargo-binding domain of SEC24. This inhibition reduces secretion of PCSK9 and additional SURF4 clients that we identify by mass spectrometry, leaving other secreted cargoes unaffected. We propose that selective small-molecule inhibition of cargo recognition by SEC24 is a potential therapeutic intervention for atherosclerosis and other diseases that are modulated by secreted proteins.

Metformin enhances LDL-cholesterol uptake by suppressing the expression of the pro-protein convertase subtilisin/kexin type 9 (PCSK9) in liver cells.

Metformin (MF) intake associates with reduced levels of circulating low-density lipoprotein-cholesterol (LDL-C). This has been attributed to the activation of AMPK, which differentially regulates the expression of multiple genes involved in cholesterol synthesis and trafficking. However, the exact mechanism underlying the LDL-C lowering effect of MF remains ambiguous. MF-treated Hep-G2 and HuH7 cells were evaluated for cell viability and the expression status of key lipid metabolism-related genes along with LDL-C uptake efficiency. MF treatment resulted in decreased expression and secretion of PCSK9, increased expression of LDLR and enhanced LDL-C uptake in hepatocytes. It also resulted in increased expression of activated AMPK (p-AMPK) and decreased expression of SREBP2 and HNF-1α proteins. Transcriptomic analysis of MF-treated Hep-G2 cells confirmed these findings and showed that other key lipid metabolism-related genes including those that encode apolipoproteins (APOB, APOC2, APOC3 and APOE), MTTP and LIPC are downregulated. Lastly, MF treatment associated with reduced HMG-CoA reductase expression and activity. These findings suggest that MF treatment reduces circulating LDL-C levels by suppressing PCSK9 expression and enhancing LDLR expression; hence the potential therapeutic utility of MF in hypercholesterolemia.

Berberine: Ins and outs of a nature-made PCSK9 inhibitor.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein and a critical regulator of low-density lipoprotein-cholesterol (LDL-C) through inducing degradation of the LDL receptor (LDLR) within the hepatocyte lysosome. PCSK9 deficiency significantly improves the survival rate of cardiovascular disease (CVDs) patients. Up to now, various PCSK9 inhibition approaches have been tested. However, the currently available PCSK9 inhibitors' widespread use is limited due to their inconvenient method of administration and high cost. On the other hand, inhibiting PCSK9 with nutraceuticals is safe and affordable. The plant-derived compound berberine has shown anti-PCSK9 activity in several studies. Berberine is an isoquinoline quaternary alkaloid of phyto origin. Berberine treatment boosts the hepatic expression of LDLRs, while decreasing the expression and secretion of the LDLR modulator PCSK9. The current review presents a collection of in vitro and in vivo studies investigating berberine's effects on PCSK9 mRNA expression, protein level, and function.

Impact of Soy β-Conglycinin Peptides on PCSK9 Protein Expression in HepG2 Cells.

Background: Dyslipidaemias, particularly elevated plasma low-density lipoprotein cholesterol (LDL-C) levels, are major risk factors for cardiovascular disease (CVD). Besides pharmacological approaches, a nutritional strategy for CVD prevention has gained increasing attention. Among functional foods, the hypocholesterolemic properties of soy are driven by a stimulation of LDL-receptor (LDL-R) activity. Aim: To characterize the effect of two soy peptides, namely, β-conglycinin-derived YVVNPDNDEN and YVVNPDNNEN on the expression of proprotein convertase subtilisin/kexin type 9 (PCSK9), one of the key-regulators of the LDL-R. Methods: PCSK9 promoter activity (luciferase assay), PCSK9 protein expression (WB) and secretion (ELISA), PCSK9 interaction with LDL-R (binding assay) and human HepG2 cells were the objects of this investigation. Results: Treatment with YVVNPDNNEN peptide has led to a rise in PCSK9 gene expression (90.8%) and transcriptional activity (86.4%), and to a decrement in PCSK9 intracellular and secreted protein (−42.9%) levels. YVVNPDNNEN peptide reduced the protein expression of transcriptional factor HNF1α. Most changes driven by YVVNPDNDEN peptide were not statistically significant. Neither peptide inhibited the PCSK9–LDLR interaction. Conclusions: Although sharing a common effect on LDL-R levels through the inhibition of 3-hydroxy-3-methylglutaryl CoA reductase activity, only the YVVNPDNNEN peptide has an additional mechanism via the downregulation of PCSK9 protein levels.

Turnover Rates of the Low-Density Lipoprotein Receptor and PCSK9: Added Dimension to the Cholesterol Homeostasis Model.

We measured the turnover rates of the LDLR (low-density lipoprotein receptor) and PCSK9 (proprotein convertase subtilisin/kexin type 9) in mice by metabolic labeling with heavy water and mass spectrometry. Approach and Results: In liver of mice fed high-cholesterol diets, LDLR mRNA levels and synthesis rates were markedly lower with complete suppression of cholesterol synthesis and higher cholesterol content, consistent with the Brown-Goldstein model of tissue cholesterol homeostasis. We observed markedly lower PCSK9 mRNA levels and synthesis rates in liver and lower concentrations and synthesis rates in plasma. Hepatic LDLR half-life (t½) was prolonged, consistent with an effect of reduced PCSK9, and resulted in no reduction in hepatic LDLR content despite reduced mRNA levels and LDLR synthesis rates. These changes in PCSK9 synthesis complement and expand the well-established model of tissue cholesterol homeostasis in mouse liver, in that reduced synthesis and levels of PCSK9 counterbalance lower LDLR synthesis by promoting less LDLR catabolism, thereby maintaining uptake of LDL cholesterol into liver despite high intracellular cholesterol concentrations. Lower hepatic synthesis and secretion of PCSK9, an SREBP2 (sterol response element binding protein) target gene, results in longer hepatic LDLR t½ in response to cholesterol feeding in mice in the face of high intracellular cholesterol content. PCSK9 modulation opposes the canonical lowering of LDLR mRNA and synthesis by cholesterol surplus and preserves LDLR levels. The physiological and therapeutic implications of these opposing control mechanisms over liver LDLR are of interest and may reflect subservience of hepatic cholesterol homeostasis to whole body cholesterol needs.

High-Throughput Screening Identifies MicroRNAs Regulating Human PCSK9 and Hepatic Low-Density Lipoprotein Receptor Expression.

Investigations into the regulatory mechanisms controlling cholesterol homeostasis have proven fruitful in identifying low-density lipoprotein (LDL)-lowering therapies to reduce the risk of atherosclerotic cardiovascular disease. A major advance was the discovery of proprotein convertase subtilisin/kexin type 9 (PCSK9), a secreted protein that binds the LDL receptor (LDLR) on the cell surface and internalizes it for degradation, thereby blunting its ability to take up circulating LDL. The discovery that loss-of-function mutations in PCSK9 lead to lower plasma levels of LDL cholesterol and protection from cardiovascular disease led to the therapeutic development of PCSK9 inhibitors at an unprecedented pace. However, there remain many gaps in our understanding of PCSK9 regulation and biology, including its posttranscriptional control by microRNAs. Using a high-throughput region(3′-UTR) of human microRNA library screen, we identified microRNAs targeting the 3′ untranslated region of human PCSK9. The top 35 hits were confirmed by large-format PCSK9 3′-UTR luciferase assays, and 10 microRNAs were then selected for further validation in hepatic cells, including effects on PCSK9 secretion and LDLR cell surface expression. These studies identified seven novel microRNAs that reduce PCSK9 expression, including miR-221-5p, miR-342-5p, miR-363-5p, miR-609, miR-765, and miR-3165. Interestingly, several of these microRNAs were also found to target other genes involved in LDLR regulation and potently upregulate LDLR cell surface expression in hepatic cells. Together, these data enhance our understanding of post-transcriptional regulators of PCSK9 and their potential for therapeutic manipulation of hepatic LDLR expression.

The Modulation of PCSK9 and LDLR by Supercritical CO2 Extracts of Mentha longifolia and Isolated Piperitone Oxide, an In Vitro Study.

In the present study the ability of supercritical carbon dioxide (SCO2) extracts of M. longifolia L. leaves to modulate low-density lipoprotein receptor (LDLR) and proprotein convertase subtilisin/kexin type 9 (PCSK9) expression was evaluated in cultured human hepatoma cell lines Huh7 and HepG2. Two SCO2 extracts, one oil (ML-SCO2) and a semisolid (MW-SCO2), were subjected to detailed chemical characterization by mono- and bidimensional nuclear magnetic resonance (1D, 2D-NMR), gas chromatography coupled with mass spectrometry (GC-MS) and liquid chromatography coupled with mass spectrometry (LC-MS). Chemical analysis revealed significant amounts of fatty acids, phytosterols and terpenoids. ML-SCO2 was able to induce LDLR expression at a dose of 60 µg/mL in HuH7 and HepG2 cell lines. Furthermore, ML-SCO2 reduced PCSK9 secretion in a concentration-dependent manner in both cell lines. Piperitone oxide, the most abundant compound of the volatile constituent of ML-SCO2 (27% w/w), was isolated and tested for the same targets, showing a very effective reduction of PCSK9 expression. The overall results revealed the opportunity to obtain a new nutraceutical ingredient with a high amount of phytosterols and terpenoids using the SCO2 extraction of M. longifolia L., a very well-known botanical species used as food. Furthermore, for the first time we report the high activity of piperitone oxide in the reduction of PCSK9 expression.

Surf4 Knockdown Protects Against Cardiovascular Disease

Plasma low-density lipoprotein cholesterol (LDL-C) levels are positively correlated with risk of cardiovascular disease. LDL is produced from catabolism of very low-density lipoprotein (VLDL) that is exclusively secreted from hepatocytes. Inhibition of VLDL secretion reduces plasma LDL-C levels. LDL is cleared from circulation via hepatic LDL receptor (LDLR). Proprotein convertase subtilisin/kexin type 9 (PCSK9) is mainly secreted from the liver and promotes LDLR degradation, increasing plasma LDL-C levels. Understanding mechanisms of VLDL and PCSK9 secretion are crucial for identifying novel therapeutic targets. Surfeit 4 (Surf4) is a cargo receptor that resides in the endoplasmic reticulum (ER). Here, we investigated its role in VLDL and PCSK9 secretion. We generated Surf4 liver specific knockout (Surf4LKO) mice and found that knockout of Surf4 did not affect PCSK9 secretion but significantly reduced plasma levels of cholesterol, triglyceride, and apolipoprotein B (apoB). In cultured human hepatocytes, Surf4 co-immunoprecipitated and co-localized with apoB100, and Surf4 silencing reduced secretion of apoB100. VLDL secretion was impaired in Surf4LKO mice, causing the retention of small lipid particles in the hepatic ER lumen. However, liver lipid levels and plasma alanine aminotransferase activity were comparable in Surf4LKO and Surf4FLOXmice. Furthermore, knockdown of Surf4 in LDLR knockout (Ldlr-/-) mice significantly reduced triglyceride secretion and plasma levels of apoB and non-HDL cholesterol but had no effect on plasma HDL cholesterol or apoA-I levels. Surf4 silencing markedly reduced the development of atherosclerosis without causing notable liver damage. Expression of stearoyl-CoA-1 (SCD1) was reduced in the liver of Surf4LKO mice and Surf4 knockdown Ldlr-/- mice. In summary, Surf4 is not required for PCSK9 secretion, instead it interacts with apoB100 and facilitates VLDL secretion. Hepatic deficiency of Surf4 reduces VLDL secretion and the development of atherosclerosis but does not cause hepatic lipid accumulation or liver damage. Thus, hepatic Surf4 can be a novel therapeutic target to reduce LDL production and the risk of cardiovascular disease. Translational perspective. Patients with homozygous familial hypercholesterolemia harboring LDLR null mutations or with autosomal recessive hypercholesterolemia cannot be effectively treated by currently lipid-lowering drugs, statins and PCSK9 inhibitors. Inhibition of VLDL secretion can markedly reduce plasma levels of cholesterol and the development of atherosclerosis. Our findings that Surf4 facilitated VLDL secretion, and inhibition of Surf4 drastically reduced plasma cholesterol levels and the development of atherosclerosis without causing notable liver damage provide critical information for the development of novel LDL production-based therapies through inhibiting hepatic Surf4.

Purification, structural characterization, and PCSK9 secretion inhibitory effect of the novel alkali-extracted polysaccharide from Cordyceps militaris

One novel alkali-extracted polysaccharide, CM3-SII, was obtained from the fruiting body of C. militaris via column chromatography. Its structural characteristics were investigated via chemical and spectroscopic methods. The backbone of CM3-SII was composed of →4)-β-D-Manp(1→, →6)-β-D-Manp(1→, and →6)-α-D-Manp(1→ glycosyls, and branching at the O-4 positions of →6)-β-D-Manp(1→ glycosyls with β-D-Galp, (1→2) linked-β-D-Galf, and →2,6)-α-D-Manp(1→ residues. Furthermore, O-6 and O-2 positions of the →2,6)-α-D-Manp(1→ residues were substituted with methyl and β-D-Galp, respectively. This polysaccharide significantly enhanced the intracellular protein expression of low-density lipoprotein receptor and proprotein convertase subtilisin/kexin type 9 (PCSK9) via regulating sterol regulatory element-binding protein 2 in hepatoma Huh7 cells. Of note, CM3-SII significantly decreased PCSK9 secretion at the concentration of 200 μg/mL. Collectively, CM3-SII is different from the previously reported alkali-extracted polysaccharides isolated from the fruiting body of C. militaris, and it may have potential application in hypolipidemia or as a pharmaceutical additive.

Dynamin-related protein 1 inhibition reduces hepatic PCSK9 secretion.

Proteostasis maintains protein homeostasis and participates in regulating critical cardiometabolic disease risk factors including proprotein convertase subtilisin/kexin type 9 (PCSK9). Endoplasmic reticulum (ER) remodeling through release and incorporation of trafficking vesicles mediates protein secretion and degradation. We hypothesized that ER remodeling that drives mitochondrial fission participates in cardiometabolic proteostasis. We used in vitro and in vivo hepatocyte inhibition of a protein involved in mitochondrial fission, dynamin-related protein 1 (DRP1). Here, we show that DRP1 promotes remodeling of select ER microdomains by tethering vesicles at ER. A DRP1 inhibitor, mitochondrial division inhibitor 1 (mdivi-1) reduced ER localization of a DRP1 receptor, mitochondrial fission factor, suppressing ER remodeling-driven mitochondrial fission, autophagy, and increased mitochondrial calcium buffering and PCSK9 proteasomal degradation. DRP1 inhibition by CRISPR/Cas9 deletion or mdivi-1 alone or in combination with statin incubation in human hepatocytes and hepatocyte-specific Drp1-deficiency in mice reduced PCSK9 secretion (-78.5%). In HepG2 cells, mdivi-1 increased low-density lipoprotein receptor via c-Jun transcription and reduced PCSK9 mRNA levels via suppressed sterol regulatory binding protein-1c. Additionally, mdivi-1 reduced macrophage burden, oxidative stress, and advanced calcified atherosclerotic plaque in aortic roots of diabetic Apoe-deficient mice and inflammatory cytokine production in human macrophages. We propose a novel tethering function of DRP1 beyond its established fission function, with DRP1-mediated ER remodeling likely contributing to ER constriction of mitochondria that drives mitochondrial fission. We report that DRP1-driven remodeling of select ER micro-domains may critically regulate hepatic proteostasis and identify mdivi-1 as a novel small molecule PCSK9 inhibitor.

PCSK9 is not secreted from mature differentiated intestinal cells

Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes lysosomal degradation of the LDL receptor and is a key regulator of cholesterol metabolism. After the liver, the small intestine is the second organ that highly expresses PCSK9. However, the small intestine's ability to secrete PCSK9 remains a matter of debate. While liver-specific PCSK9-deficient mice present no PCSK9 in systemic blood, human intestinal Caco-2 cells can actively secrete PCSK9. This raises the possibility for active intestinal secretion via the portal blood. Here, we aimed to determine whether enterocytes can secrete PCSK9 using in vitro, ex vivo, and in vivo approaches. We first observed that PCSK9 secretion from Caco-2 cells was biphasic and dependent on Caco-2 maturation status. Transcriptional analysis suggested that this transient reduction in PCSK9 secretion might be due to loss of SREBP2-mediated transcription of PCSK9. Consistently, PCSK9 secretion was not detected ex vivo in human or mouse intestinal biopsies mounted in Ussing chambers. Finally, direct comparison of systemic versus portal blood PCSK9 concentrations in WT or liver-specific PCSK9-deficient mice confirmed the inability of the small intestine to secrete PCSK9 into the portal compartment. Altogether, our data demonstrate that mature enterocytes do not secrete PCSK9 and reinforce the central role of the liver in the regulation of the concentration of circulating PCSK9 and consequently of cellular LDL receptors.

Atherosclerosis-associated hepatic secretion of VLDL but not PCSK9 is dependent on cargo receptor protein Surf4

Plasma LDL is produced from catabolism of VLDL and cleared from circulation mainly via the hepatic LDL receptor (LDLR). Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes LDLR degradation, increasing plasma LDL-C levels. Circulating PCSK9 is mainly secreted by the liver, whereas VLDL is exclusively secreted by hepatocytes. However, the mechanism regulating their secretion is not completely understood. Surfeit 4 (Surf4) is a cargo receptor localized in the ER membrane. It recruits cargos into coat protein complex II vesicles to facilitate their secretion. Here, we investigated the role of Surf4 in VLDL and PCSK9 secretion. We generated Surf4 liver-specific knockout mice and found that knockout of Surf4 did not affect PCSK9 secretion, whereas it significantly reduced plasma levels of cholesterol, triglyceride, and lipid-binding protein apolipoprotein B (apoB). In cultured human hepatocytes, Surf4 coimmunoprecipitated and colocalized with apolipoprotein B100, and Surf4 silencing reduced secretion of apolipoprotein B100. Furthermore, knockdown of Surf4 in LDLR knockout (Ldlr−/−) mice significantly reduced triglyceride secretion, plasma levels of apoB and non-HDL-C, and the development of atherosclerosis. However, Surf4 liver-specific knockout mice and Surf4 knockdown in Ldlr−/− mice displayed similar levels of liver lipids and plasma alanine aminotransferase activity as control mice, indicating that inhibition of Surf4 does not cause notable liver damage. Expression of stearoyl-CoA desaturase-1 was also reduced in the liver of these mice, suggesting a reduction in de novo lipogenesis. In summary, hepatic deficiency of Surf4 reduced VLDL secretion and the development of atherosclerosis but did not cause significant hepatic lipid accumulation or liver damage.

Autocrine effects of PCSK9 on cardiomyocytes

Proprotein convertase subtilisin kexin type 9 (PCSK9) is in the focus of cardiovascular research due to its role in hepatic low density lipoprotein (LDL) clearance. However, extrahepatic expression of PCSK9 such as in cardiomyocytes and its regulation by oxidized LDL (oxLDL) put notion on extrahepatic effects of PCSK9 as well. This study was aimed to reveal the role of PCSK9 in oxLDL-dependent regulation of cardiomyocyte function. Adult rat and mouse ventricular cardiomyocytes and isolated perfused hearts were used. OxLDL was applied to increase PCSK9 expression in cardiomyocytes. Cell function was analyzed by load-free cell shortening as well as left ventricular developed pressure of isolated hearts. OxLDL decreased shortening in wild-type-derived mouse cardiomyocytes but not in those isolated from PCSK9 knockout mice. Overexpression of human PCSK9 in rat cardiomyocytes reduced shortening in the absence of oxLDL. Addition of recombinant PCSK9 mimicked these effects. In cardiomyocytes, oxLDL induced PCSK9 release into the supernatant. Inhibition of PCSK9 by Pep 2–8 or alirocumab attenuated the oxLDL-induced loss of cardiomyocyte shortening. Cardiomyocytes express surfeit locus protein 4 (SURF-4), a protein required for PCSK9 secretion in human embryonic kidney cells (HEK 293 T), and silencing of SURF-4 reduced the oxLDL effects on cardiomyocytes. In isolated perfused rat hearts PCSK9 inhibition by alirocumab improved the function. In addition, left ventricular function of isolated hearts from PCSK9 knockout mice was increased under basal conditions as well as at 10 min and 120 min of reperfusion following 45 min of ischemia. Collectively, the data show that cardiomyocytes express and release PCSK9 that acts in an autocrine way on cardiomyocytes and impairs their function.

Activation of estrogen receptor α (ERα) is required for Alisol B23-acetate to prevent post-menopausal atherosclerosis and reduced lipid accumulation

The risk of atherosclerosis (AS) ascends among post-menopausal women, while current hormone replacement therapy exerts several adverse effects. Alisol B 23-acetate (AB23A), a tetracyclic triterpenoid isolated from the rhizome of Alisma orientale, was reported to show multiple physiological activities, including regulating lipid metabolism. According to molecular docking analysis, it was predicted to bind with estrogen receptor α (ERα). In this study, we aimed to observe the effect of AB23A on preventing post-menopausal AS and explore whether the mechanism was mediated by ERα. In vitro, free fatty acid (FFA) was applied to induce the abnormal lipid metabolism of L02 cells. In vivo, the ApoE−/− mice were ovariectomized to mimic the cessation of estrogen. The high-fat diet was also given to induce post-menopausal AS. We demonstrated AB23A attenuated the accumulation of total cholesterol and triglyceride induced by free fatty acids in hepatocytes. In high-fat diet-ovariectomy-treated ApoE−/− mice, AB23A eliminated lipids in blood and liver. AB23A not only reduced the synthesis of proprotein convertase subtilisin/kexin type 9 (PCSK9) through sterol-regulatory element binding proteins (SREBPs) but also suppressed the secretion of PCSK9 through silent information regulator 1 (SIRT1). Notably, AB23A promoted the expression of ERα in vivo and in vitro. The both ERα inhibitor and ERα siRNA were also applied in confirming whether the hepatic protective effect of AB23A was mediated by ERα. We found that AB23A significantly promoted the expression of ERα. AB23A could inhibit the synthesis and secretion of PCSK9 through ERα, lower the accumulation of triglyceride and cholesterol, and prevent post-menopausal AS.

Pseurotin A as a novel suppressor of hormone dependent breast cancer progression and recurrence by inhibiting PCSK9 secretion and interaction with LDL receptor

Hypercholesterolemia has been documented to drive hormone-dependent breast cancer (BC) progression and resistance to hormonal therapy. Proprotein convertase subtilisin/kexin type-9 (PCSK9) regulates cholesterol metabolism through binding to LDL receptor (LDLR) and targeting the receptor for lysosomal degradation. Inhibition of PCSK9 is an established strategy to treat hypercholesterolemia. Pseurotin A (PS) is a unique spiro-heterocyclic γ-lactam alkaloid isolated from the fungus Aspergillus fumigatus. Preliminary studies indicated that PS lowered PCSK9 secretion in cultured HepG2 hepatocellular carcinoma cells, with an IC50 value of 1.20 μM. Docking studies suggested the ability of PS to bind at the PCSK9 narrow interface pocket that accommodates LDLR. Surface plasmon resonance (SPR) showed PS ability to inhibit the PCSK9-LDLR interaction at a concentration range of 10−150 μM. PS showed in vitro dose-dependent reduction of PCSK9, along with increased LDLR levels in hormone-dependent BT-474 and T47D breast cancer (BC) cell lines. In vivo, daily oral 10 mg/kg PS suppressed the progression of the hormone-dependent BT-474 BC cells in orthotopic nude mouse xenograft model. Immunohistochemistry (IHC) investigation of BT-474 breast tumor tissue proved the PS ability to reduce PCSK9 expression. PS also effectively suppressed BT-474 BC cells locoregional recurrence after primary tumor surgical excision. Western blot analysis showed decreased PCSK9 expression in liver tissues of PS-treated mice compared to vehicle-treated control group. PS treatment significantly reduced PCSK9 expression and normalized LDLR levels in collected primary and recurrent breast tumors at the study end. PS-treated mice showed reduced plasma cholesterol and 17β-estradiol levels. Inhibition of tumor recurrence was associated with significant reductions in plasma level of the human BC recurrence marker CA 15−3 in treated mice at the study end. Histopathological examination of various PS-treated mice organs indicated lack of metastatic tumor cells and any pathological changes. The results of this study provide the first evidence for the suppression of the hormone-dependent breast tumor progression and recurrence by targeting the PCSK9-LDLR axis. PS is a novel first-in-class PCSK9-targeting lead appropriate for the use to control hormone-dependent BC progression and recurrence.