Lipoprotein Homeostasis Research Articles

Effect of PCSK9 Inhibitors on Regulators of Lipoprotein Homeostasis, Inflammation and Coagulation

Background: PCSK9 inhibitors (PCSK9i) represent a newer form of atherosclerosis treatment. Inflammation and haemostasis are key processes in the development of atherosclerosis. In this study, we investigated the influence of therapy with PCSK9i in patients with coronary artery disease (CAD) on regulators for lipoprotein homeostasis, inflammation and coagulation. Methods: Using quantitative polymerase chain reaction (qPCR), we measured the expression of the genes involved in lipoprotein homeostasis, namely for sterol regulatory element-binding protein 1 (SREBP1), SREBP2, low-density lipoprotein receptor (LDLR), hepatic lipase type C (LIPC), LDLR-related protein 8 (LRP8), and the genes associated with inflammation and coagulation, such as cluster of differentiation (CD) 36 (CD36), CD63, and CD14 in 96 patients with CAD and 25 healthy subjects. Results: Significant differences in the expression of the investigated genes between patients and healthy controls were found. Treatment with PCSK9i also resulted in significant changes in the expression of all studied genes. Conclusions: We established that PCSK9i may have a significant effect on the gene expression of lipid regulators, inflammatory markers, and coagulation parameters, independent of their lipolytic effect.

Abstract P139: Exploring the cardiometabolic effects of lipid 12-hydroxyeicosapentaenoic acid through thromboxane receptor partial agonism

Introduction: It is well known that lipid mediators secreted by brown adipose tissue (BAT) during cold exposure or beta-adrenergic stimulation can exert beneficial effects on glucose and lipoprotein homeostasis. Recently, Leiria et al. demonstrated that one of these mediators, 12-hydroxyeicosapentaenoic acid (12-HEPE), secreted by BAT, enhances glucose tolerance and insulin sensitivity through the stimulation of a G protein-coupled receptor (GPCR). However, the receptor that 12(S)-HEPE binds to exert its functions was unknown. Therefore, this study aimed to identify the GPCR that 12-HEPE binds to and to investigate whether 12-HEPE affects vascular reactivity. Methods: Screening of 70 non-olfactory GPCR candidates was conducted in HEK293 cells, either stimulated or not with 12-HEPE (5µM). Receptor activation was measured using the dynamic mass redistribution (DRM) assay. After identifying the potential receptor, we validated the result by monitoring intracellular Ca2+ levels through a dose-response curve for 12-HEPE. To study the biological effects resulting from this ligand-receptor interaction, thoracic aortic artery rings were isolated from 12-week-old C57BL6 male mice, and their contractile responses were monitored using a myograph in response to cumulative doses ranging from 100pM to 10µM of 12-HEPE. Results: Through DRM screening, we found that the thromboxane receptor (TP) exhibited the strongest interaction with 12(S)-HEPE (5µM). To validate the screening, we incubated HEK293 cells with 12-HEPE or U46619 and quantified intracellular calcium mobilization. Our data demonstrate that 12-HEPE is a partial TP agonist, with an EC50 of 314nM compared to 7.2nM for U46619. Furthermore, incubation of the thoracic aorta with 12-HEPE produced dose-dependent increases in arterial constriction (pEC50: 6.129 ± 0.174; Emax: 95.39 ± 15.72), and this effect was completely prevented by the thromboxane antagonist, TPA (1µM). Conclusions: Our results suggest that 12-HEPE is an endogenous partial agonist of the TP receptor, and such interaction is necessary for the biological effects of these lipids. Importantly, the vasoconstrictor effect mediated by 12-HEPE may be an important physiological adaptation to prevent body heat loss upon cold exposure, thus allowing for adequate temperature maintenance.

Pla2g12b drives expansion of triglyceride-rich lipoproteins

Vertebrates transport hydrophobic triglycerides through the circulatory system by packaging them within amphipathic particles called Triglyceride-Rich Lipoproteins. Yet, it remains largely unknown how triglycerides are loaded onto these particles. Mutations in Phospholipase A2 group 12B (PLA2G12B) are known to disrupt lipoprotein homeostasis, but its mechanistic role in this process remains unclear. Here we report that PLA2G12B channels lipids within the lumen of the endoplasmic reticulum into nascent lipoproteins. This activity promotes efficient lipid secretion while preventing excess accumulation of intracellular lipids. We characterize the functional domains, subcellular localization, and interacting partners of PLA2G12B, demonstrating that PLA2G12B is calcium-dependent and tightly associated with the membrane of the endoplasmic reticulum. We also detect profound resistance to atherosclerosis in PLA2G12B mutant mice, suggesting an evolutionary tradeoff between triglyceride transport and cardiovascular disease risk. Here we identify PLA2G12B as a key driver of triglyceride incorporation into vertebrate lipoproteins.

ANGPTL3 Downregulation Increases Intracellular Lipids by Reducing Energy Utilization.

ANGPTL3 (angiopoietin-like protein 3) is a circulating protein with a key role in maintaining lipoprotein homeostasis. A monoclonal antibody against ANGPTL3 is an approved and well-tolerated treatment to reduce lipoproteins in familial hypercholesterolemia homozygotes. However, the reduction of hepatic ANGPTL3 synthesis using an antisense oligonucleotide unexpectedly resulted in a dose-dependent increase in liver lipid content and circulating transaminases, resulting in the termination of the clinical trial. Meanwhile, the use of silencing RNAs remains an area of active investigation. Our study sought to investigate whether intracellular downregulation of ANGPTL3 may lead to a primary increase in neutral lipids within the hepatocyte. We downregulated ANGPTL3 by silencing RNA in primary human hepatocytes 3-dimensional spheroids, HepG2/LX-2 3-dimensional spheroids, and in HepG2, Hep3B2, and Huh7 cultured in 2 dimensions. ANGPTL3 downregulation increased neutral lipids in all models investigated. Interestingly, ANGPTL3 induced lower intracellular deiodinase type 1 protein levels resulting in a reduction in beta-oxidation and causing an increase in triglycerides stored in lipid droplets. In conclusion, intracellular ANGPTL3 downregulation by silencing RNA led to an increase in triglycerides content due to a reduction in energy substrate utilization resembling a primary intracellular hepatocyte hypothyroidism.

SGLT2i Dapagliflozin decreases NLRP3, IL-1 and PCSK9 expression in preclinical models of short-term doxorubicin cardiotoxicity

Abstract Background Anthracyclines are an effective and widely used chemotherapy agent in the treatment of multiple solid organ tumors and hematologic malignancies. The use of anthracyclines as a standard cancer therapy is limited by the potential for the development of cardiac dysfunction, arrhythmias, and clinical heart failure. In recent five years, it was demonstrated that proprotein convertase subtilisin/kexin type 9 (PCSK9), a lipid metabolism-related protein, is a key orchestrator of immune infiltration in myocardial and cancer tissues and could regulate cardiac fibrosis and inflammation. PCSK9 is a protein with key roles in hepatic low density lipoprotein (LDL) homeostasis. PCSK9 systemic levels are associated to HOMA score and high insulin levels. Dapagliflozin exerts systemic ant-inflammatory properties and cardioprotective effects in diabetic and non-diabetic patients. Purpose We hypothesized that Dapagliflozin, administered during doxorubicin, could reduce PCSK9 systemic levels in preclinical models. Methods Female C57Bl/6 mice were untreated (Sham, n=6) or treated for 10 days with doxorubicin i.p at 2.17 mg/kg (DOXO, n=6), DAPA at 12 mg/kg (DAPA, n=6) or doxorubicin combined to DAPA (DOXO-DAPA, n=6). After treatments, plasma levels of PCSK9, IL-1β and CRP were analyzed through selective anti-mouse ELISA methods. Myocardial and liver expression of NLRP3-inflammasome and IL-1β were analyzed through ELISA method in tissue lysates after treatments. Results DAPA associated to DOXO reduces significantly systemic levels of PCSK9 ( -37,5% vs DOXO group, p<0,001). IL-1β and CRP levels were also reduced ( -47,3 and – 28,5 %, respectively; p<0.05 for both). Myocardial and liver IL-1β and NLRP3 inflammasome expression were also reduced in DAPA/DOXO group vs DOXO and control, indicated beneficial metabolic and anti-inflammatory effects of SGLT2i. Conclusion DAPA has been shown to reduce systemic levels of PCSK9 in preclinical models of short-term DOXO cardiotoxicity. To the best of our knowledge, this is the first evidence of SGLT-2/PCSK9 crass-talk in cardioncology therefore the overall picture of the study open a new window on the beneficial properties of DAPA against anthracyclines side effects.

Abstract 15043: Dapagliflozin Reduces Systemic NLRP-3, IL-1 and PCSK9 Levels in Preclinical Models of Short-Term Doxorubicin Cardiotoxicity: New Evidences of SGLT2i Use in Cardioncology

Background: Anthracyclines are an effective and widely used chemotherapy agent in the treatment of multiple solid organ tumors and hematologic malignancies. The use of anthracyclines as a standard cancer therapy is limited by the potential for the development of cardiac dysfunction, arrhythmias, and clinical heart failure. In recent five years, it was demonstrated that proprotein convertase subtilisin/kexin type 9 (PCSK9), a lipid metabolism-related protein, is a key orchestrator of immune infiltration in myocardial and cancer tissues and could regulate cardiac fibrosis and inflammation. PCSK9 is a protein with key roles in hepatic low density lipoprotein (LDL) homeostasis. PCSK9 systemic levels are associated to HOMA score and high insulin levels. Dapagliflozin exerts systemic ant-inflammatory properties and cardioprotective effects in diabetic and non-diabetic patients. Purpose: We hypothesized that Dapagliflozin, administered during doxorubicin, could reduce PCSK9 systemic levels in preclinical models. Methods: Female C57Bl/6 mice were untreated (Sham, n=6) or treated for 10 days with doxorubicin i.p at 2.17 mg/kg (DOXO, n=6), DAPA at 12 mg/kg (DAPA, n=6) or doxorubicin combined to DAPA (DOXO-DAPA, n=6). After treatments, plasma levels of PCSK9, IL-1β and CRP were analyzed through selective anti-mouse ELISA methods. Myocardial and liver expression of NLRP3-inflammasome and IL-1β were analyzed through ELISA method in tissue lysates after treatments. Results: DAPA associated to DOXO reduces significantly systemic levels of PCSK9 ( -37,5% vs DOXO group, p<0,001). IL-1β and CRP levels were also reduced ( -47,3 and - 28,5 %, respectively; p<0.05 for both). Myocardial and liver IL-1β and NLRP3 inflammasome expression were also reduced in DAPA/DOXO group vs DOXO and control, indicated beneficial metabolic and anti-inflammatory effects of SGLT2i. Conclusion: DAPA has been shown to reduce systemic levels of PCSK9 in preclinical models of short-term DOXO cardiotoxicity. To the best of our knowledge, this is the first evidence of SGLT-2/PCSK9 crass-talk in cardioncology therefore the overall picture of the study open a new window on the beneficial properties of DAPA against anthracyclines side effects.

P383 DAPAGLIFLOZIN REDUCES SYSTEMIC PCSK9 LEVELS IN PRECLINICAL MODELS OF SHORT–TERM DOXORUBICIN CARDIOTOXICITY THROUGH NLRP3 INFLAMMASOME/IL–1Β: A FIRST EVIDENCE OF SGLT–2/PCSK9 CROSS–TALK IN CARDIONCOLOGY

Abstract Background Anthracyclines are an effective and widely used chemotherapy agent in the treatment of multiple solid organ tumors and hematologic malignancies. The use of anthracyclines as a standard cancer therapy is limited by the potential for the development of cardiac dysfunction, arrhythmias, and clinical heart failure. In recent five years, it was demonstrated that proprotein convertase subtilisin/kexin type 9 (PCSK9), a lipid metabolism–related protein, is a key orchestrator of immune infiltration in myocardial and cancer tissues and could regulate cardiac fibrosis and inflammation. PCSK9 is a protein with key roles in hepatic low density lipoprotein (LDL) homeostasis. PCSK9 systemic levels are associated to HOMA score and high insulin levels. Dapagliflozin exerts systemic ant–inflammatory properties and cardioprotective effects in diabetic and non–diabetic patients. Purpose: We hypothesized that Dapagliflozin, administered during doxorubicin, could reduce PCSK9 systemic levels in preclinical models. Methods Female C57Bl/6 mice were untreated (Sham, n=6) or treated for 10 days with doxorubicin i.p at 2.17 mg/kg (DOXO, n=6), DAPA at 12 mg/kg (DAPA, n=6) or doxorubicin combined to DAPA (DOXO–DAPA, n=6). After treatments, plasma levels of PCSK9, IL–1β and CRP were analyzed through selective anti–mouse ELISA methods. Myocardial and liver expression of NLRP3–inflammasome and IL–1β were analyzed through ELISA method in tissue lysates after treatments. Results DAPA associated to DOXO reduces significantly systemic levels of PCSK9 ( –37,5% vs DOXO group, p<0,001). IL–1β and CRP levels were also reduced ( –47,3 and – 28,5 %, respectively; p<0.05 for both). Myocardial and liver IL–1β and NLRP3 inflammasome expression were also reduced in DAPA/DOXO group vs DOXO and control, indicated beneficial metabolic and anti–inflammatory effects of SGLT2i. Conclusion DAPA has been shown to reduce systemic levels of PCSK9 in preclinical models of short–term DOXO cardiotoxicity. To the best of our knowledge, this is the first evidence of SGLT–2/PCSK9 crass–talk in cardioncology therefore the overall picture of the study open a new window on the beneficial properties of DAPA against anthracyclines side effects.

Association of serum PCSK9 levels with platelet function in patients with acute coronary syndromes.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates low-density lipoprotein (LDL) homeostasis and plays a key role in acute coronary syndrome (ACS). The cardioprotective effect of PCSK9 inhibition extends beyond LDL cholesterol reduction, involving regulation of platelet function by not yet unraveled mechanisms. Oxidized-LDL (ox-LDL) is increased during ACS and induces platelet activation via binding to platelet surface. We will evaluate serum PCSK9 and its correlation with platelet reactivity and platelet-ox-LDL binding in Chinese ACS patients. In this pilot cross-sectional study, we will enroll 115 Chinese participants aged 30 to 75 years with ACS. Blood sample will be obtained after the first maintenance dose of aspirin and clopidogrel during morning time. Serum PCSK9 will be measured by an enzyme-linked immunoadsorbent assay. Platelet reactivity will be assessed by; Platelet activation (P-selectin and GPIIbIIIa expression using flow cytometry) and; Platelet aggregation using light transmission aggregometry in response to various stimuli. On-treatment platelet reactivity is measured by adenosine diphosphate-induced platelet aggregation. Binding of ox-LDL to platelet will be evaluated by flow cytometry. Spearman correlations will be used to determine association of serum PCSK9 with platelet functional parameters and platelet-ox-LDL binding. Additionally, continuous PCSK9 levels will be categorized into tertiles of equal size to investigate its association with on-treatment platelet reactivity. This study will reveal possible relationship between serum PCSK9 and platelet reactivity in the setting of ACS which may shed light on therapeutic potential in platelet inhibition by targeting PCSK9. The study will also explore the association of serum PCSK9 and platelet-ox-LDL binding, an important mechanism for platelet-LDL interplay, to provide mechanistic insight into PCSK9-mediated regulation of platelet reactivity.

PCSK9 promotes the progression and metastasis of colon cancer cells through regulation of EMT and PI3K/AKT signaling in tumor cells and phenotypic polarization of macrophages

BackgroundProprotein convertase subtilisin/kexin type 9 (PCSK9) is the ninth member of the proprotein convertase family that regulates lipoprotein homeostasis and altered PCSK9 expression was reportedly associated with tumor development and progression. This study assessed PCSK9 expression and functions in human colon cancer and then explored the underlying molecular events.MethodsColon cancer tissues were utilized for analysis of PCSK9 expression for association with clinicopathological factors from patients by immunohistochemistry assay. Manipulation of PCSK9 expression was assessed in vitro and in vivo for colon cancer cell proliferation, migration, and invasion using cell viability CCK-8, Transwell tumor cell migration and invasion, and wound-healing assays. Next, proteomic analysis, Western blot, qRT-PCR and Flow cytometry were conducted to assess downstream targets and tumor cell-derived PCSK9 action on macrophage polarization.ResultsPCSK9 expression was upregulated in colon cancer tissues versus the normal tissues, and associated with advanced tumor pathological grade. Knockdown of PCSK9 expression reduced colon cancer cell proliferation, migration, and invasion and suppressed tumor metastasis in vivo. PCSK9 directly or indirectly upregulated Snail 1 and in turn to downregulate E-cadherin expression, but upregulate N-cadherin and MMP9 levels and thereafter, to induce colon cancer cell epithelial-mesenchymal transition (EMT) process and activated PI3K/AKT signaling. However, PCSK9 overexpression showed the inverse effects on colon cancer cells. Knockdown of PCSK9 expression inhibited M2 macrophage polarization, but also promoted M1 macrophage polarization by reduction of lactate, protein lactylation and macrophage migration inhibitory factor (MIF) levels.ConclusionPCSK9 played an important role in the progression and metastasis of colon cancer by regulation of tumor cell EMT and PI3K/AKT signaling and in the phenotypic polarization of macrophages by mediating MIF and lactate levels. Targeting PCSK9 expression or activity could be used to effectively control colon cancer.

MiR-125a-3p aggravates ox-LDL-induced HUVEC injury through BAMBI.

Atherosclerosis (AS) is a chronic inflammatory disease characterized by the formation of atherosclerotic plaque in the intima of arteries. Among the known regulators of atherosclerosis, microRNAs (miRNAs) have been reported to play critical roles in lipoprotein homeostasis and plaque formation. But the roles of microRNA-125a-3p (miR-125a-3p) in the pathogenesis of AS remain unknown. Human umbilical vein endothelial cells (HUVECs) were treated with oxidized low-density lipoprotein (ox-LDL) to construct the vascular injury model of AS pathogenesis in vitro. miR-125a-3p and BMP and activin membrane-bound inhibitor (BAMBI) expression levels in HUVECs were then measured by quantitative real-time polymerase chain reaction and western blot. The viability and apoptosis of HUVECs were analyzed by Cell Counting Kit-8 assay, TUNEL assay, and flow cytometry, respectively. The relationship between BAMBI 3'-untranslated region and miR-125a-3p was validated by dual luciferase reporter gene assay. miR-125a-3p expression was raised in HUVECs induced with ox-LDL. In HUVECs, miR-125a-3p enhanced the effects of ox-LDL treatment on repressing the viability and promoting the apoptosis of cells. Additionally, BAMBI was confirmed as a direct target of miR-125a-3p and BAMBI overexpression reversed the effects of miR-125a-3p on HUVECs. miR-125a-3p aggravates the dysfunction of HUVECs induced by ox-LDL via BAMBI, which implies that miR-125a-3p is involved in the pathogenesis of AS.

Lipocalin-2 and insulin as new biomarkers of alopecia areata.

Lipocalin-2 and visfatin are proinflammatory adipokines involved in the regulation of glucose homeostasis. Their role has been described in numerous inflammatory skin diseases such as atopic dermatitis and psoriasis. Recently, an increased prevalence of metabolic abnormalities has been reported in patients with alopecia areata. The aim of the study is to determine the serum levels of lipocalin-2 and visfatin in patients with alopecia areata in comparison with healthy controls. Moreover, the serum levels of total cholesterol, low-density lipoprotein cholesterol (LDL-cholesterol), high-density lipoprotein cholesterol (HDL-cholesterol), triglycerides, fasting glucose, insulin, c-peptide, and homeostasis model assessment for insulin resistance (HOMA-IR) were evaluated. Fifty-two patients with alopecia areata and 17 control subjects were enrolled in the study. The serum levels of lipocalin-2 [mean ± standard deviation, SD: 224.55 ± 53.58 ng/ml vs. 188.64 ± 44.75, p = 0.01], insulin [median (interquartile range, IQR): 6.85 (4.7–9.8) μIU/ml vs. 4.5 (3.5–6.6), p<0.05], c-peptide [median (IQR): 1.63 (1.23–2.36) ng/ml vs. 1.37 (1.1–1.58), p<0.05)], and HOMA-IR [median (IQR): 1.44 (0.98–2.15) vs. 0.92 (0.79–1.44), p<0.05) were significantly higher in patients with alopecia areata compared to the controls. The serum concentration of insulin and HOMA-IR correlated with the number of hair loss episodes (r = 0.300, p<0.05 and r = 0.322, p<0.05, respectively). Moreover, a positive correlation occurred between insulin, HOMA-IR, c-peptide and BMI (r = 0.436, p <0.05; r = 0.384, p<0.05 and r = 0.450, p<0.05, respectively). In conclusion, lipocalin-2 and insulin may serve as biomarkers for alopecia areata. Further studies are needed to evaluate the role of insulin as a prognostic factor in alopecia areata.

Antitumor activity and molecular mechanism of proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibition.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a member of the proprotein convertase family of proteins that activate other proteins in cells. Functionally, PCSK9 binds to the receptor of low-density lipoprotein particles (LDL) to regulate cholesterol metabolism and lipoprotein homeostasis in human body. PCSK9 inhibition is a novel pharmacological strategy to control hypercholesterolemia and cardiovascular diseases. Recently accumulating evidence realizes that PCSK9 possesses other roles in cells, such as regulation of tissue inflammatory response, intratumoral immune cell infiltration, and tumor progression. This review discussed the advancement of PCSK9 research on its role and underlying mechanisms in tumor development and progression. For example, PCSK9 inhibition could attenuate progression of breast cancer, glioma, colon tumor, hepatocellular cancer, prostate cancer, and lung adenocarcinoma and promote apoptosis of glioma, prostate cancer, and hepatocellular cancer cells. PCSK9 deficiency could reduce liver metastasis of B16F1 melanoma cells by lowering the circulating cholesterol levels. PCSK9 gene knockdown substantially attenuated mouse tumor growth in vivo by activation of cytotoxic T cells, although PCSK9 knockdown had no effect on morphology and growth rate of different mouse cancer cell lines in vitro. PCSK9 inhibition thus can be used to control human cancers. Future preclinical and clinical studies are warranted to define anti-tumor activity of PCSK9 inhibition.

Hypolipemiant besides Antileukemic Effect of Imatinib Mesylate (Case Report).

Introduction. The tyrosine kinase inhibitor imatinib mesylate (IM) specifically inhibits the kinase activity of ABL, platelet-derived growth factor receptor beta (PDGFR[]), and c-KIT tyrosine kinases and has impressive clinical efficacy in chronic myeloid leukemia (CML)[1]. IM prevented also the development of atherosclerotic lesions and diabetes-induced inflammatory cytokine overexpression [2,3].Case report. A 36 years old male patient with a history of acute myocardial infarction and double coronarian by-pass eight years ago was admitted for the first time on December 2005 complaining of wheigt loss, intermittent discomfort in upper left abdomenum, polydipsia.and polyuria. By the examination of the peripheral blood and of the bone marrow the diagnosis of chronic phase CML-Ph(+) has been established. Simultaneously, type II diabetes mellitus and dyslipidemia (Table 1) were discovered. After a short course of Hydrea, associated with diet and oral antidiabetic therapy, IM 400 mg/d has been started on April 2006. The hematologic remission and a concomitant reduction of the serum levels of cholesterol, and of low-density lipoproteins (LDL) has been obtained (Table 1).Table 1The dynamics of the peripheral blood counts and lipidic profile during IM therapyParametersAprilMayJuneJullyHemoglobin (g/dl)9,611,11212Leukocyte count (×109/l)5.24.67.57.8Platelet count (×109/l)168287210215Serum total cholesterol (mg/dl)310189157152Serum LD lipoproteins (mg/dl)2191288587Serum HD lipoproteins(mg/dL)49535250Comments and conclusions. The effects observed in this reported case and in other similar patients [4] indicates that the treatment with IM induces a favourable control not only on cell growth and replication but also on the homeostasis of lipoproteins and glucose. A possible mechanism of action could be the inhibition by IM of PDGFR-dependent phosphorylation of the LDL-receptor related protein. IM appears to be a novel therapeutic option to retard the development of atherosclerosis, specifically in the context of diabetes and the treatment of choice for patients suffering of CML and dyslipidemia. Further prolonged observation of this and other similar patients is necessary for more pertinent data.

Lipoprotein Glomerulopathy-Like Lesions in Atherosclerotic Mice Defected With HDL Receptor SR-B1.

High-density lipoprotein (HDL) homeostasis is important in maintaining both cardiovascular and renal health. Scavenger receptor class B type 1 (SR-B1), the major HDL receptor in mammals, plays a crucial role in reverse cholesterol transport and HDL metabolism. Evidence from mouse study has well demonstrated that HDL disorders caused by Srb1 inactivation accelerate atherosclerosis and even induce lethal cardiovascular diseases. However, the renal consequences of Srb1 dysfunction are still unknown. Here we explored this issue in both Srb1 knockout (Srb1-/-) mice and atherosclerotic low-density lipoprotein receptor knockout (Ldlr-/-) mice with Srb1 deletion. Our data showed that no apparent renal damage was observed in 5-month-old Srb1-/- mice fed on standard rodent chow diet as well as Srb1-/- mice fed on a high-fat diet (HFD) for 12 weeks. However, 5-month-old Srb1/Ldlr-/- mice fed on rodent chow had increased urinary albumin excretion and developed spontaneous intraglomerular Oil-red O (ORO)-positive lipoprotein deposition that is similar to lesions observed in human lipoprotein glomerulopathy (LPG). HFD feeding accelerated LPG-like lesions in Srb1/Ldlr-/- mice, inducing severe proteinuria and significantly promoting intraglomerular ORO-positive lipoprotein deposition. Interestingly, probucol reversed HFD-induced HDL disorders and almost fully abrogated LPG-like lesions in Srb1/Ldlr-/- mice. In conclusion, the present study demonstrates that SR-B1 dysfunction leads to LPG-like lesions in atherosclerotic mice, which could be rescued by probucol. SR-B1 loss-of-function mutant carriers therefore might be susceptible to developing metabolic nephropathy in addition to cardiovascular diseases, and probucol might be a potential therapeutics.

The Dual Role of Low-Density Lipoprotein Receptor-Related Protein 1 in Atherosclerosis.

Low-density lipoprotein receptor–related protein-1 (LRP1) is a large endocytic and signaling receptor belonging to the LDL receptor (LDLR) gene family and that is widely expressed in several tissues. LRP1 comprises a large extracellular domain (ECD; 515 kDa, α chain) and a small intracellular domain (ICD; 85 kDa, β chain). The deletion of LRP1 leads to embryonic lethality in mice, revealing a crucial but yet undefined role in embryogenesis and development. LRP1 has been postulated to participate in numerous diverse physiological and pathological processes ranging from plasma lipoprotein homeostasis, atherosclerosis, tumor evolution, and fibrinolysis to neuronal regeneration and survival. Many studies using cultured cells and in vivo animal models have revealed the important roles of LRP1 in vascular remodeling, foam cell biology, inflammation and atherosclerosis. However, its role in atherosclerosis remains controversial. LRP1 not only participates in the removal of atherogenic lipoproteins and proatherogenic ligands in the liver but also mediates the uptake of aggregated LDL to promote the formation of macrophage- and vascular smooth muscle cell (VSMC)-derived foam cells, which causes a prothrombotic transformation of the vascular wall. The dual and opposing roles of LRP1 may also represent an interesting target for atherosclerosis therapeutics. This review highlights the influence of LRP1 during atherosclerosis development, focusing on its dual role in vascular cells and immune cells.

Evidences for lipid involvement in SARS-CoV-2 cytopathogenesis

The pathogenesis of SARS-CoV-2 remains to be completely understood, and detailed SARS-CoV-2 cellular cytopathic effects requires definition. We performed a comparative ultrastructural study of SARS-CoV-1 and SARS-CoV-2 infection in Vero E6 cells and in lungs from deceased COVID-19 patients. SARS-CoV-2 induces rapid death associated with profound ultrastructural changes in Vero cells. Type II pneumocytes in lung tissue showed prominent altered features with numerous vacuoles and swollen mitochondria with presence of abundant lipid droplets. The accumulation of lipids was the most striking finding we observed in SARS-CoV-2 infected cells, both in vitro and in the lungs of patients, suggesting that lipids can be involved in SARS-CoV-2 pathogenesis. Considering that in most cases, COVID-19 patients show alteration of blood cholesterol and lipoprotein homeostasis, our findings highlight a peculiar important topic that can suggest new approaches for pharmacological treatment to contrast the pathogenicity of SARS-CoV-2.

Pro-protein convertase subtilisin/kexin type 9 promotes intestinal tumor development by activating Janus kinase 2/signal transducer and activator of transcription 3/SOCS3 signaling in ApcMin/+ mice.

IntroductionPro-protein convertase subtilisin/kexin type 9 (PCSK9) regulates lipoprotein homeostasis in humans. Evolocumab is a selective PCSK9 inhibitor that can reduce low-density lipoprotein cholesterol (LDLC) level and decrease hypercholesterolemia. The current study aimed to explore whether PCSK9 increases the risk of colorectal cancer.MethodsFirst, we utilized the classic intestinal tumor ApcMin/+ mouse model and PCSK9 knock-in (KI) mice to establish ApcMin/+PCSK9(KI) mice. Then, we investigated the effect of PCSK9 overexpression in ApcMin/+PCSK9(KI) mice and PCSK9 inhibition using evolocumab on the progression of intestinal tumors in vivo by hematoxylin and eosin (HE) staining, Western blot, and immunohistochemistry (IHC) assay.ResultsApcMin/+PCSK9(KI) mice had higher numbers and larger sizes of adenomas, with 83.3% of these mice developing adenocarcinoma (vs. 16.7% of ApcMin/+ mice). However, treatment with evolocumab reduced the number and size of adenomas and prevented the development of adenocarcinomas in ApcMin/+ mice. PCSK9 overexpression reduced tumor cell apoptosis, the Bax/bcl-2 ratio, and the levels of cytokine signaling 3 protein (SOCS3) suppressors, but activated Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling in intestinal tumors. In contrast, evolocumab treatment had the opposite effect on ApcMin/+mice.ConclusionPCSK9 might act as an oncogene or have an oncogenic role in the development and progression of colorectal cancer in vivo via activation of JAK2/STAT3/SOCS3 signaling.

Regulation of Lipoprotein Homeostasis by Self-Assembling Peptides.

High levels of serum low-density lipoprotein (LDL) cholesterol contribute to atherosclerosis, a key risk factor of cardiovascular diseases. PCSK9 is a circulatory enzyme that downregulates expression of hepatic LDL receptors, concomitantly increasing serum LDL-C. This work investigates a small, self-assembling peptide, EPep2-8, as a peptide inhibitor of PCSK9. EPep2-8 is a multidomain peptide comprising a self-assembling domain, E2, conjugated to a bioactive domain, Pep2-8, previously shown to inhibit PCSK9. The E2 domain facilitates self-assembly of EPep2-8 into long, nanofibrous polymers with an underlying supramolecular β-sheet secondary structure. Intermolecular interactions between nanofibers drive EPep2-8 to form a thixotropic and cytocompatible hydrogel in aqueous and charge-neutral solutions. These properties enable EPep2-8 to be delivered as an in situ depot for regulation of lipoprotein homeostasis. In surface plasmon resonance studies, EPep2-8 bound specifically to PCSK9 with an apparent, noncovalent, and irreversible dissociation, significantly improving the binding affinity of Pep2-8 alone (KD = 667 ± 48 nM). Increased binding affinity of EPep2-8 is primarily due to the superstoichiometric interaction of the peptide with PCSK9. Promisingly, EPep2-8 retains bioactivity in vitro, engendering dose-dependent uptake of LDL-C in hepatocytes. This mechanism of self-assembly on a target site may be a simple method to improve the affinity of peptide inhibitors.

Hsa-miRNA-23a-3p promotes atherogenesis in a novel mouse model of atherosclerosis

Of the known regulators of atherosclerosis, miRNAs have been demonstrated to play critical roles in lipoprotein homeostasis and plaque formation. Here, we generated a novel animal model of atherosclerosis by knocking in LDLRW483X in C57BL/6 mice, as the W483X mutation in LDLR is considered the most common newly identified pathogenic mutation in Chinese familial hypercholesterolemia (FH) individuals. Using the new in vivo mouse model combined with a well-established atherosclerotic in vitro human cell model, we identified a novel atherosclerosis-related miRNA, miR-23a-3p, by microarray analysis of mouse aortic tissue specimens and human aortic endothelial cells (HAECs). miR-23a-3p was consistently downregulated in both models, which was confirmed by qPCR. Bioinformatics analysis and further validation experiments revealed that the TNFα-induced protein 3 (TNFAIP3) gene was the key target of miR-23a-3p. The miR-23a-3p-related functional pathways were then analyzed in HAECs. Collectively, the present results suggest that miR-23a-3p regulates inflammatory and apoptotic pathways in atherogenesis by targeting TNFAIP3 through the NF-κB and p38/MAPK signaling pathways.

LDL Receptor Pathway Regulation by miR-224 and miR-520d.

MicroRNAs (miRNA) have emerged as important post-transcriptional regulators of metabolic pathways that contribute to cellular and systemic lipoprotein homeostasis. Here, we identify two conserved miRNAs, miR-224, and miR-520d, which target gene networks regulating hepatic expression of the low-density lipoprotein (LDL) receptor (LDLR) and LDL clearance. In silico prediction of miR-224 and miR-520d target gene networks showed that they each repress multiple genes impacting the expression of the LDLR, including the chaperone molecules PCSK9 and IDOL that limit LDLR expression at the cell surface and the rate-limiting enzyme for cholesterol synthesis HMGCR, which is the target of LDL-lowering statin drugs. Using gain- and loss-of-function studies, we tested the role of miR-224 and miR-520d in the regulation of those predicted targets and their impact on LDLR expression. We show that overexpression of miR-224 or miR-520d dose-dependently reduced the activity of PCSK9, IDOL, and HMGCR 3′-untranslated region (3′-UTR)-luciferase reporter constructs and that this repression was abrogated by mutation of the putative miR-224 or miR-520d response elements in the PCSK9, IDOL, and HMGCR 3′-UTRs. Compared to a control miRNA, overexpression of miR-224 or miR-520d in hepatocytes inhibited PCSK9, IDOL, and HMGCR mRNA and protein levels and decreased PCSK9 secretion. Furthermore, miR-224 and miR-520d repression of PCSK9, IDOL, and HMGCR was associated with an increase in LDLR protein levels and cell surface expression, as well as enhanced LDL binding. Notably, the effects of miR-224 and miR-520d were additive to the effects of statins in upregulating LDLR expression. Finally, we show that overexpression of miR-224 in the livers of Ldlr+/− mice using lipid nanoparticle-mediated delivery resulted in a 15% decrease in plasma levels of LDL cholesterol, compared to a control miRNA. Together, these findings identify roles for miR-224 and miR-520d in the posttranscriptional control of LDLR expression and function.