SREBP-1-mediated lipogenesis confers resistance to ferroptosis and improves endothelial injury.

Atherosclerosis refers to a disease characterized by the formation of lipid plaque deposits within arterial walls, leading to reduced blood flow or blockage of blood outflow. The process of endothelial injury induced by oxidized low-density lipoprotein (ox-LDL) is considered the initial stage of atherosclerosis. Ferroptosis is a form of iron-dependent, non-apoptotic cell death, and current research suggests its association with coronary artery disease (CAD). In this study, we observed a correlation between reduced expression of SREBP-1 and the occurrence of stable CAD. Additionally, during the process of endothelial injury induced by ox-LDL, we also noted decreased expression of the SREBP-1/SCD1/FADS2 and involvement in the ferroptosis process. Mechanistically, ox-LDL induced endothelial injury by inhibiting the lipid biosynthesis process mediated by the SREBP-1/SCD1/FADS2, thereby inducing lipid peroxidation and ferroptosis. On the contrary, overexpression of SREBP-1 or supplementation with monounsaturated fatty acids counteracted iron accumulation, mitochondrial damage, and lipid peroxidation-induced ferroptosis, thereby improving endothelial injury. Our study indicated that the decreased expression of peripheral blood SREBP-1 mRNA is an independent risk factor for stable CAD. Furthermore, in endothelial cells, the lipid biosynthesis process mediated by SREBP-1 could ameliorate endothelial injury by resisting ferroptosis. The study has been registered with the Chinese Clinical Trial Registry, which serves as a primary registry in the World Health Organization International Clinical Trials Registry Platform (ChiCTR2300074315, August 3rd, 2023).

Huayu Qutan Recipe promotes lipophagy and cholesterol efflux through the mTORC1/TFEB/ABCA1-SCARB1 signal axis.

This study aims to investigate the mechanism of the anti-atherosclerosis effect of Huayu Qutan Recipe (HYQT) on the inhibition of foam cell formation. In vivo, the mice were randomly divided into three groups: CTRL group, MOD group and HYQT group. The HYQT group received HYQT oral administration twice a day (20.54 g/kg/d), and the plaque formation in ApoE-/- mice was observed using haematoxylin-eosin (HE) staining and oil red O (ORO) staining. The co-localization of aortic macrophages and lipid droplets (LDs) was examined using fluorescent labelling of CD11b and BODIPY fluorescence probe. In vitro, RAW 264.7 cells were exposed to 50 µg/mL ox-LDL for 48 h and then treated with HYQT for 24 h. The accumulation of LDs was evaluated using ORO and BODIPY. Cell viability was assessed using the CCK-8 assay. The co-localization of LC3b and BODIPY was detected via immunofluorescence and fluorescence probe. LysoTracker Red and BODIPY 493/503 were used as markers for lysosomes and LDs, respectively. Autophagosome formation were observed via transmission electron microscopy. The levels of LC3A/B II/LC3A/B I, p-mTOR/mTOR, p-4EBP1/4EBP1, p-P70S6K/P70S6K and TFEB protein level were examined via western blotting, while SQSTM1/p62, Beclin1, ABCA1, ABCG1 and SCARB1 were examined via qRT-PCR and western blotting. The nuclear translocation of TFEB was detected using immunofluorescence. The components of HYQT medicated serum were determined using Q-Orbitrap high-resolution MS analysis. Molecular docking was employed to identify the components of HYQT medicated serum responsible for the mTOR signalling pathway. The mechanism of taurine was illustrated. HYQT has a remarkable effect on atherosclerotic plaque formation and blood lipid level in ApoE-/- mice. HYQT decreased the co-localization of CD11b and BODIPY. HYQT (10% medicated serum) reduced the LDs accumulation in RAW 264.7 cells. HYQT and RAPA (rapamycin, a mTOR inhibitor) could promote cholesterol efflux, while chloroquine (CQ, an autophagy inhibitor) weakened the effect of HYQT. Moreover, MHY1485 (a mTOR agonist) also mitigated the effects of HYQT by reduced cholesterol efflux. qRT-PCR and WB results suggested that HYQT improved the expression of the proteins ABCA1, ABCG1 and SCARB1.HYQT regulates ABCA1 and SCARB1 protein depending on the mTORC1/TFEB signalling pathway. However, the activation of ABCG1 does not depend on this pathway. Q-Orbitrap high-resolution MS analysis results demonstrated that seven core compounds have good binding ability to the mTOR protein. Taurine may play an important role in the mechanism regulation. HYQT may reduce cardiovascular risk by promoting cholesterol efflux and degrading macrophage-derived foam cell formation. It has been observed that HYQT and ox-LDL regulate lipophagy through the mTOR/TFEB signalling pathway, rather than the mTOR/4EBP1/P70S6K pathway. Additionally, HYQT is found to regulate cholesterol efflux through the mTORC1/TFEB/ABCA1-SCARB1 signal axis, while taurine plays a significant role in lipophagy.

CircZNF609 sponges miR-135b to up-regulate SEMA3A expression to alleviate ox-LDL-induced atherosclerosis.

The initiation and progression of atherosclerotic plaque caused by abnormal lipid metabolism is one of the main causes of atherosclerosis (AS). Lipid droplet accumulation has become a novel research pointcut for AS treatment in recent years. In AS patients, miR-135b level was up-regulated relative to the normal cases, which showed negative correlations with the levels of Semaphorin 3A (SEMA3A) and circZNF609, separately. The U937-derived macrophages were cultured with ox-LDL to establish AS models in vitro. After that, the lipid accumulation, inflammation, mitochondrial dysfunction and cell death were evaluated by ORO, ELISA, RT-qPCR, western blot, JC-1 and FCM assays respectively. Transfection of the circZNF609 expression vector notably declined lipid accumulation, attenuated inflammation, reduced mitochondrial dysfunction and inhibited cell death in ox-LDL-stimulated cells. The direct binding of miR-135b to circZNF609 in vitro was confirmed using RIP assay, and SEMA3A expression was up-regulated by circZNF609 overexpression. After manipulating the endogenous expressions of circZNF609, miR-135b and SEMA3A, the above damages in ox-LDL-stimulated cells were rescued by inhibition of miR-135b expression and overexpression of circZNF609 or SEMA3A. Besides, the AS mice model was built to demonstrate the excessive lipid accumulation, increasing inflammation and cell death in AS pathogenesis according to the results of HE staining, ELISA and IHC assays, while these damages were reversed after overexpression of circZNF609 or SEMA3A. In AS models, overexpressed circZNF609 prevents the AS progression through depleting miR-135b expression and subsequent up-regulation of SEMA3A expression to overwhelm lipid accumulation, mitochondrial dysfunction and cell death.

14-3-3-η interacts with BCL-2 to protect human endothelial progenitor cells from ox-LDL-triggered damage.

Oxidized low-density lipoprotein (ox-LDL) causes dysfunction of endothelial progenitor cells (EPCs), and we recently reported that 14-3-3-η can attenuate the damage triggered by ox-LDL in EPCs. However, the molecular mechanisms by which 14-3-3-η protects EPCs from the damage caused by ox-LDL are not fully understood. In this study, we observed that the expression of 14-3-3-η and BCL-2 were downregulated in ox-LDL-treated EPCs. Overexpression of 14-3-3-η in ox-LDL-treated EPC significantly increased BCL-2 level, while knockdown of BCL-2 reduced 14-3-3-η expression and mitigated the protective effect of 14-3-3-η on EPCs. In addition, we discovered that 14-3-3-η colocalizes and interacts with BCL-2 in EPCs. Taken together, these data suggest that 14-3-3-η protects EPCs from ox-LDL-induced damage by its interaction with BCL-2.

The association between oxidized low-density lipoprotein and cancer: An emerging targeted therapeutic approach?

Lipids play an important role in varying vital cellular processes including cell growth and division. Elevated levels of low-density lipoprotein (LDL) and oxidized-LDL (ox-LDL), and overexpression of the corresponding receptors including LDL receptor (LDLR), lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), and cluster of differentiation 36 (CD36), have shown strong correlations with different facets of carcinogenesis including proliferation, invasion, and angiogenesis. Furthermore, a high serum level of LOX-1 is considered as a poor prognostic factor in many types of cancer including colorectal cancer. Ox-LDL could contribute to cancer progression and metastasis through endothelial-to-mesenchymal transition (EMT) and autophagy. Thus, many studies have shed light on the significant role of ox-LDL as a potential therapeutic target for cancer therapy. In various repurposing approaches, anti-dyslipidemia agents, phytochemicals, autophagy modulators as well as recently developed ldl-like nanoparticles have been investigated as potential tumor therapeutic agents by targeting oxidized-LDL/LOX-1 pathways. Herein, we reviewed the role of oxidized-LDL and LOX-1 in cancer progression, invasion, metastasis, and also cancer-associated angiogenesis. Moreover, we addressed therapeutic utility of several compounds that proved to be capable of targeting the metabolic moieties in cancer. This review provides insights on the potential impact of targeting LDL and ox-LDL in cancer therapy and their future biomedical implementations.

Ox-LDL promotes M1-like polarization of macrophages through the miR-21-5p/SKP2/EP300 pathway.

Oxidized low-density lipoprotein (ox-LDL) mediated inflammatory damage, which possibly induces atherosclerosis (AS); however, the role of miRNA in this process has rarely been reported. In this paper, we study the ox-LDL-related endothelial cell damage and changes of macrophages. The bioinformatics method was used to analyze the expression changes of miRNA in AS patients, luciferase assay was used to study the interaction of protein and miRNA, and co-IP and ubiquitination experiments were used to analyze protein interaction. Flow cytometry was used to detect the polarization of macrophages. Database analysis showed that the expression of miR-21-5p was upregulated in AS patients. Luciferase assay showed that miR-21-5p can bind to SKP2 and subsequently influence ubiquitination of EP300. Overexpression of EP300 strengthens the HMGB1-induced acetylation and subsequently mediates the dissociation of HMGB1 from SIRT1, and thus HMGB1 could be secreted outside the cell. The HMGB1 released from endothelial cells can promote macrophage M1 polarization. This study shows that ox-LDL activates the SKP2/EP300 pathway through promoting upregulation of miR-21-5p, thereby acetylating and secreting HMGB1 outside the endothelium, subsequently enhancing macrophage polarization to further stabilize the inflammation situation.

Procyanidin B2 alleviates oxidized low-density lipoprotein-induced cell injury, inflammation, monocyte chemotaxis, and oxidative stress by inhibiting the nuclear factor kappa-B pathway in human umbilical vein endothelial cells.

BACKGROUND: Oxidized low-density lipoprotein (ox-LDL) can initiate and affect almost all atherosclerotic events including endothelial dysfunction. In this text, the role and underlying molecular basis of procyanidin B2 (PCB2) with potential anti-oxidant and anti-inflammatory activities in ox-LDL-induced HUVEC injury were examined. METHODS: HUVECs were treated with ox-LDL in the presence or absence of PCB2. Cell viability and apoptotic rate were examined by CCK-8 assay and flow cytometry, respectively. The mRNA and protein levels of genes were tested by RT-qPCR and western blot assays, respectively. Potential downstream targets and pathways of apple procyanidin oligomers were examined by bioinformatics analysis for the GSE9647 dataset. The effect of PCB2 on THP-1 cell migration was examined by recruitment assay. The effect of PCB2 on oxidative stress was assessed by reactive oxygen species (ROS) level, malondialdehyde (MDA) content, and mitochondrial membrane potential (MMP). RESULTS: ox-LDL reduced cell viability, induced cell apoptosis, and facilitated the expression of oxidized low-density lipoprotein receptor 1 (LOX-1), C-C motif chemokine ligand 2 (MCP-1), vascular cell adhesion protein 1 (VCAM-1) in HUVECs. PCB2 alleviated ox-LDL-induced cell injury in HUVECs. Apple procyanidin oligomers triggered the differential expression of 592 genes in HUVECs (|log2fold-change| > 0.58 and adjusted p-value < 0.05). These dysregulated genes might be implicated in apoptosis, endothelial cell proliferation, inflammation, and monocyte chemotaxis. PCB2 inhibited C-X-C motif chemokine ligand 1/8 (CXCL1/8) expression and THP-1 cell recruitment in ox-LDL-stimulated HUVECs. PCB2 inhibited ox-LDL-induced oxidative stress and nuclear factor kappa-B (NF-κB) activation in HUVECs. CONCLUSION: PCB2 weakened ox-LDL-induced cell injury, inflammation, monocyte recruitment, and oxidative stress by inhibiting the NF-κB pathway in HUVECs.

LY86 facilitates ox-LDL-induced lipid accumulation in macrophages by upregulating SREBP2/HMGCR expression.

LY86, also known as MD1, has been implicated in various pathophysiological processes including inflammation, obesity, insulin resistance, and immunoregulation. However, the role of LY86 in cholesterol metabolism remains incompletely understood. Several studies have reported significant up-regulation of LY86 mRNA in atherosclerosis; nevertheless, the regulatory mechanism by which LY86 is involved in this disease remains unclear. In this study, we aimed to investigate whether LY86 affects ox-LDL-induced lipid accumulation in macrophages. Firstly, we confirmed that LY86 is indeed involved in the process of atherosclerosis and found high expression levels of LY86 in human atherosclerotic plaque tissue. Furthermore, our findings suggest that LY86 may mediate intracellular lipid accumulation induced by ox-LDL through the SREBP2/HMGCR pathway. This mechanism could be associated with increased cholesterol synthesis resulting from enhanced endoplasmic reticulum stress response.

Oxidized-LDL Induces Metabolic Dysfunction in Retinal Pigment Epithelial Cells.

Recently, mitochondrial dysfunction has gained attention as a causative factor in the pathogenesis and progression of age-related macular degeneration (AMD). Mitochondrial damage plays a key role in metabolism and disrupts the balance of intracellular metabolic pathways, such as oxidative phosphorylation (OXPHOS) and glycolysis. In this study, we focused on oxidized low-density lipoprotein (ox-LDL), a major constituent of drusen that accumulates in the retina of patients with AMD, and investigated whether it could be a causative factor for metabolic alterations in retinal pigment epithelial (RPE) cells. We found that prolonged exposure to ox-LDL induced changes in fatty acid ß-oxidation (FAO), OXPHOS, and glycolytic activity and increased the mitochondrial reactive oxygen species production in RPE cells. Notably, the effects on metabolic alterations varied with the concentration and duration of ox-LDL treatment. In addition, we addressed the limitations of using ARPE-19 cells for retinal disease research by highlighting their lower barrier function and FAO activity compared to those of induced pluripotent stem cell-derived RPE cells. Our findings can aid in the elucidation of mechanisms underlying the metabolic alterations in AMD.

Dendrobine regulates STAT3 to attenuate mitochondrial dysfunction and senescence in vascular endothelial cells triggered by oxidized low-density lipoprotein.

Our previous studies have highlighted the potential therapeutic efficacy of dendrobine, an alkaloid, in atherosclerosis (AS), nevertheless, the underlying mechanism remains unclear. This study employs a combination of network pharmacology and in vitro experiments to explore the regulatory pathways involved. Through network pharmacology, the biological function for intersection targets between dendrobine and AS were identified. Molecular docking was conducted to investigate the interaction between the dominant target and dendrobine. Human umbilical vein endothelial cells (HUVECs) were treated with oxidized low-density lipoprotein (ox-LDL) to mimic AS, and the effects of dendrobine on cell viability, lipid deposition, mitochondrial function, and cellular senescence were evaluated. Subsequently, cells were treated with the mitophagy inhibitor Mdivi-1 and the STAT3 agonist colivelin to assess the role of mitophagy and STAT3 signaling in dendrobine regulation. Intersection targets were associated with biological processes, including reactive oxygen species production. Dendrobine attenuated the effects of ox-LDL treatment on HUVECs, mitigating changes in cell activity, lipid deposition, mitochondrial function, and cellular senescence. Both Mdivi-1 and colivelin treatments resulted in decreased cell viability and increased cellular senescence, with colivelin suppressing mitophagy. Cotreatment with Mdivi-1 and colivelin further aggravated cellular senescence and inhibited FoxO signaling. Together, this study indicated that dendrobine regulated the STAT3/FoxO signaling pathway, alleviating mitochondrial dysfunction and cellular senescence. This study contributes valuable insights to the potential clinical application of dendrobine.

LOX-1 in Cardiovascular Disease: A Comprehensive Molecular and Clinical Review.

LOX-1, ORL-1, or lectin-like oxidized low-density lipoprotein receptor 1 is a transmembrane glycoprotein that binds and internalizes ox-LDL in foam cells. LOX-1 is the main receptor for oxidized low-density lipoproteins (ox-LDL). The LDL comes from food intake and circulates through the bloodstream. LOX-1 belongs to scavenger receptors (SR), which are associated with various cardiovascular diseases. The most important and severe of these is the formation of atherosclerotic plaques in the intimal layer of the endothelium. These plaques can evolve into complicated thrombi with the participation of fibroblasts, activated platelets, apoptotic muscle cells, and macrophages transformed into foam cells. This process causes changes in vascular endothelial homeostasis, leading to partial or total obstruction in the lumen of blood vessels. This obstruction can result in oxygen deprivation to the heart. Recently, LOX-1 has been involved in other pathologies, such as obesity and diabetes mellitus. However, the development of atherosclerosis has been the most relevant due to its relationship with cerebrovascular accidents and heart attacks. In this review, we will summarize findings related to the physiologic and pathophysiological processes of LOX-1 to support the detection, diagnosis, and prevention of those diseases.

Knockdown of ADAMDEC1 ameliorates ox-LDL-induced endothelial cell injury and atherosclerosis progression.

This study was designed to investigate the role of a disintegrin and metalloproteinase domain-like protein decysin 1 (ADAMDEC-1) in atherosclerosis (AS). The Gene Expression Omnibus (GEO) database was utilized to identify differentially expressed genes (DEGs) between carotid atheroma plaque and carotid tissue adjacent atheroma plaque obtained from AS patients. Gene functional enrichment analysis was conducted on DEGs using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). QRT-PCR was employed to quantify mRNAs expression. AS animal model was established using ApoE-/- mice; serum triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) levels were detected. Aortic sinus atherosclerotic lesions were observed using H&E staining and Oil Red O staining. ADAMDEC-1 was silenced using small interfering RNAs (siRNAs) in human vascular smooth muscle cells (HVSMCs). Cell proliferation, migration, and cell cycle progression were detected by cell count kit-8 (CCK8), 5-ethynyl-2'-deoxyuridine (EDU), wound scratch healing assay, transwell assay, and flow cytometry, respectively. Western blot was used to evaluate various protein expression levels. Our results showed that ADAMDEC-1 was highly expressed in the serum of AS patients, consistent with the in silico results. The elevated TG, LDL-C, and HDL-C levels along with H&E and Oil Red O staining confirmed the successful establishment of the AS mouse model. ADAMDEC-1 expression was also elevated in AS mice. ADAMDEC-1 knockdown in HVSMCs suppressed cell proliferation, inhibited the expression of proliferating cell nuclear antigen (PCNA), and reduced the levels of matrix metalloproteinases (MMP2 and MMP9) proteins. Protein-protein interaction (PPI) analysis indicated that ADAMDEC-1 was associated with CXCL9, CCR5, TNF-α, TNFR1, and NF-κB-p50. The expression levels of CXCL9, CCR5, TNF-α, TNFR1, and NF-κB-p50 increased, while ADAMDEC-1 knockdown attenuated the expression of these proteins. Our study findings substantiate that ADAMDEC-1 may represent a novel target for AS.

Klotho inhibits renal ox-LDL deposition via IGF-1R/RAC1/OLR1 signaling to ameliorate podocyte injury in diabetic kidney disease.

OBJECTIVE: Diabetic kidney disease (DKD) is characterized by the abnormal deposition of oxidized low-density lipoprotein (ox-LDL), which contributes to podocyte damage. Klotho, an aging suppressor that plays a critical role in protecting podocytes in DKD, is mainly expressed in kidney tubular epithelium and secreted in the blood. However, it has not been established whether Klotho can alleviate podocyte injury by inhibiting renal ox-LDL deposition, and the potential molecular mechanisms require further investigation. METHODS: We conducted a comprehensive analysis of serum and kidney biopsy samples obtained from patients diagnosed with DKD. Additionally, to explore the underlying mechanism of Klotho in the deposition of ox-LDL in the kidneys, we employed a mouse model of DKD with the Klotho genotype induced by streptozotocin (STZ). Furthermore, we conducted meticulous in vitro experiments on podocytes to gain further insights into the specific role of Klotho in the deposition of ox-LDL within the kidney. RESULTS: Our groundbreaking study unveiled the remarkable ability of the soluble form of Klotho to effectively inhibit high glucose-induced ox-LDL deposition in podocytes affected by DKD. Subsequent investigations elucidated that Klotho achieved this inhibition by reducing the expression of the insulin/insulin-like growth factor 1 receptor (IGF-1R), consequently leading to a decrease in the expression of Ras-related C3 botulinum toxin substrate 1 (RAC1) and an enhancement of mitochondrial function. Ultimately, this series of events culminated in a significant reduction in the expression of the oxidized low-density lipoprotein receptor (OLR1), thereby resulting in a notable decrease in renal ox-LDL deposition in DKD. CONCLUSION: Our findings suggested that Klotho had the potential to mitigate podocyte injury and reduced high glucose-induced ox-LDL deposition in glomerulus by modulating the IGF-1R/RAC1/OLR1 signaling. These results provided valuable insights that could inform the development of novel strategies for diagnosing and treating DKD.

CircCHMP5 Contributes to Ox-LDL-induced Endothelial Cell Injury Through the Regulation of MiR-532-5p/ROCK2 axis.

BACKGROUND: Aberrant expression of circular RNA (circRNA) has been demonstrated to be related to atherosclerosis (AS) formation. However, the mechanism of circCHMP5 (also known as hsa_circ_0003575) in AS formation remains unclear. METHODS: Oxidized low-density lipoprotein (ox-LDL) was used to treat human umbilical vein endothelial cells (HUVECs) to construct a cell injury model. The expression level of circCHMP5, miR-532-5p, and Rho-associated protein kinase 2 (ROCK2) was measured using quantitative real-time PCR. Cell cycle, apoptosis, proliferation, and angiogenesis were determined by flow cytometry, 5-ethynyl-2'-deoxyuridine (EdU) assay, and tube formation assay. In addition, the protein expression of apoptosis markers, inflammation factors, and ROCK2 was detected by western blot analysis. The interaction between miR-532-5p and circCHMP5 or ROCK2 was assessed by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. RESULTS: Our results indicated that circCHMP5 was overexpressed in ox-LDL-induced HUVECs. CircCHMP5 knockdown promoted cell cycle, proliferation, and angiogenesis while inhibiting apoptosis and inflammation in ox-LDL-induced HUVECs. MiR-532-5p could be sponged by circCHMP5, and its inhibitor reversed the negative regulation of si-circCHMP5 on ox-LDL-induced HUVECs injury. ROCK2, a target of miR-532-5p, reversed the inhibition effect of miR-532-5p on ox-LDL-induced HUVECs injury. Furthermore, we confirmed that circCHMP5 upregulated ROCK2 by sponging miR-532-5p. CONCLUSION: To sum up, our data showed that circCHMP5 regulated the miR-532-5p/ROCK2 axis to accelerate ox-LDL-induced HUVECs injury, confirming that circCHMP5 might be a potential target for AS treatment.

Interleukin-35 Mitigates ox-LDL-Induced Proatherogenic Effects via Modulating miRNAs Associated with Coronary Artery Disease (CAD).

PURPOSE: Recent emergence of miRNAs as important regulators of processes involving lesion formation and regression has highlighted miRNAs as potent therapeutic targets for the treatment of atherosclerosis. Few studies have reported the atheroprotective role of IL-35, a novel immunosuppressive and anti-inflammatory cytokine; however, miRNA-dependent regulation underlying the anti-atherosclerotic potential of IL-35 remains elusive. METHODS: THP-1 macrophages were incubated with human recombinant IL-35 (rIL-35) either in the presence or absence of ox-LDL. qRT-PCR was conducted to validate the expression levels of previously identified miRNAs including miR-197-5p, miR-4442, miR-324-3p, miR-6879-5p, and miR-6069 that were differentially expressed in peripheral blood mononuclear cells of coronary artery disease (CAD) patients vs. controls. Additionally, bioinformatic analysis was performed to predict miRNA-associated targets and their corresponding functional significance in CAD. RESULTS: Exogenous IL-35 significantly decreased the average area of ox-LDL-stimulated macrophages, indicating the inhibitory effect of IL-35 on lipid-laden foam cell formation. Furthermore, rIL-35 treatment alleviated the ox-LDL-mediated atherogenic effects by modulating the expression levels of aforementioned CAD-associated miRNAs in the cultured macrophages. Moreover, functional enrichment analysis of these miRNA-related targets revealed their role in the molecular processes affecting different stages of atheroslerotic plaque development, such as macrophage polarization, T cell suppression, lipoprotein metabolism, foam cell formation, and iNOS-mediated inflammation. CONCLUSION: Our observations uncover the novel role of IL-35 as an epigenetic modifier as it influences the expression level of miRNAs implicated in the pathogenesis of atherosclerosis. Thus, IL-35 cytokine therapy-mediated miRNA targeting could be an effective therapeutic strategy against the development of early atheromas in asymptomatic high-risk CAD patients.

miR-216b-5p regulates proliferation and apoptosis of ox-LDL-stimulated VSMCs and HUVECs via IGF2.

Atherosclerosis (AS) is one of the principal causes of cardiovascular disorder. Reportedly, vascular smooth muscle cells (VSMCs) and human umbilical vein endothelial cells (HUVECs) play key roles in AS development, and microRNAs (miRNAs) regulate their functions. The function of miR-216b-5p in AS remains unknown. Human VSMCs and human HUVECs were treated with ox-LDL to establish the in vitro model of AS. MiR-216b-5p and IGF2 expressions in VSMCs and HUVECs were probed by qRT-PCR and western blot. The viability, cell cycle progression, and apoptosis of VSMCs and HUVECs were evaluated by Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine, and flow cytometry assays, respectively. The binding sites between IGF2 3'UTR and miR-216b-5p were validated by dual-luciferase reporter assay. miR-216b-5p expression was declined in ox-LDL-induced VSMCs and HUVECs. In VSMCs, miR-216b-5p overexpression inhibited excessive proliferation and induced apoptosis. MiR-216b-5p could markedly restrain the viabiblity of VSMCs induced by ox-LDL and enhanced the viability of HUVECs. Additionally, IGF2 was confirmed as the direct target of miR-216b-5p and transfection of IGF2 overexpression plasmids rescued the effects of miR-216b-5p on VSMCs and HUVECs. miR-216b-5p alleviates the dysfunction of VSMCs and HUVECs caused by ox-LDL via repressing IGF2, and exerts protective functions to block the development of AS.

Ox-LDL aggravates contrast-induced injury of renal tubular epithelial cells.

Hypercholesterolemia can aggravate contrast-induced acute kidney injury, and the exacerbation of renal tubular epithelial cell (RTEC) injury is a major cause. However, the exact mechanisms remain obscure. Mitophagy, a type of autophagy, selectively eliminates damaged mitochondria and reduces mitochondrial oxidative stress, which is strongly implicated in cell homeostasis and acute kidney injury. Oxidized low-density lipoprotein (Ox-LDL) is accumulated in hypercholesterolemia and has a cytotoxic effect. This study aimed to determine whether and how ox-LDL exacerbates contrast-induced injury in RTECs and to further explore whether PINK1/Parkin-dependent mitophagy is involved in this process. Iohexol and ox-LDL were used alone or in combination to treat HK-2 cells. Rapamycin pretreatment was utilized to enhance mitophagy. Cell viability, apoptosis, mitochondrial membrane potential (MMP) and mitochondrial reactive oxygen species (mtROS) were detected by cell counting kit-8, TUNEL staining, JC-1 kit and MitoSOX fluorescence, respectively. The expression of mitophagy-related proteins (including PINK1, Parkin, and so on) and cleaved caspase-3 was confirmed by western blot. Colocalization of MitoTracker-labeled mitochondria and LysoTracker-labeled lysosomes was observed by fluorescence microscopy to evaluate mitophagy. The results of our study showed that ox-LDL aggravated MMP decline, mtROS release and apoptosis in iohexol-treated HK-2 cells, accompanied by a further increased autophagy level. Enhancement of PINK1/Parkin-dependent mitophagy by rapamycin alleviated apoptosis and mitochondrial injury in HK-2 cells in response to iohexol under ox-LDL condition. Therefore, our findings indicate that ox-LDL aggravates contrast-induced injury of RTECs by increasing mitochondrial damage and mitochondrial oxidative stress, which may be associated with the relative insufficiency of PINK1/Parkin-dependent mitophagy.

Combination of hyperlipidemia and 17ß-Estradiol induces TMJOA-like pathological changes in rats.

OBJECTIVE: We explored whether hyperlipidemia or combination of hyperlipidemia and E2 could induce TMJOA. MATERIALS AND METHODS: Four groups of female rats were treated with normal diet, normal diet with E2, high-fat diet, and high-fat diet with E2 (HFD/E2), respectively, to induce TMJOA till 8 weeks. Another three groups were then used for COX2 inhibitor celecoxib to block the induction of TMJOA. Primary condylar chondrocytes were treated with combination of E2, ox-LDL, and corresponding inhibitors for evaluating expressions of related molecules. RESULTS: Condylar cartilage proliferation with plenty of chondrocyte apoptosis and increased staining for LOX1, nuclear NF-κB, IL-1ß, and COX2 at 4 weeks and decreased condylar cartilage and increased subchondral bone density at 8 weeks were observed only in the HFD/E2 group. Celecoxib significantly alleviated the cartilage proliferation and apoptosis in the HFD/E2 group. Serum ox-LDL increased in both high-fat diet groups, while serum IL-1ß increased only in the HFD/E2 group. Combination of E2 and ox-LDL synergistically induced expressions of LOX1, phosphorylated NF-κB, IL-1ß, and COX2, while LOX1 inhibitor blocked the induction of phosphorylated NF-κB, and NF-κB inhibitor the induction of IL-1ß, and IL-1ß inhibitor the induction of COX2. CONCLUSION: Combination of hyperlipidemia and E2-induced TMJOA-like pathological changes through LOX1/NF-κB/IL-1ß/COX2-signaling pathway.

CircRNA-PTPRA Knockdown Inhibits Atherosclerosis Progression by Repressing ox-LDL-Induced Endothelial Cell Injury via Sponging of miR-671-5p.

Atherosclerosis (AS) is a chronic inflammatory disease with high morbidity and mortality rates worldwide. This study aimed to investigate the role of circular RNA protein tyrosine phosphatase receptor type A (circRNA_PTPRA) in oxidized low-density lipoprotein (ox-LDL)-induced human umbilical vein endothelial cell (HUVECs) injury and its underlying molecular mechanism. The expression of circRNA-PTPRA and microRNA (miR)-671-5p was assessed by quantitative reverse transcription PCR (qRT-PCR). The interaction between circRNA-PTPRA and miR-671-5p was predicted using bioinformatic analysis. Cell viability and apoptosis were determined using the Cell Counting Kit-8 (CCK-8) assay and flow cytometry, respectively. Inflammation in HUVECs was analyzed by measuring the secretion of tumor necrosis factor alpha (TNF-α), interleukin-1beta (IL-1ß), and IL-6 using enzyme-linked immunosorbent assay (ELISA). Cleaved-caspase-3 expression was assessed using western blotting. The results indicated that circRNA-PTPRA expression was significantly increased and miR-671-5p expression was decreased in the serum of patients with AS and in ox-LDL-treated HUVECs. The interaction between circRNA-PTPRA and miR-671-5p was verified by dual luciferase reporter and RNA pull-down assays. In HUVECs, downregulation of circRNA-PTPRA reversed ox-LDL-induced reduction in cell viability, increase in apoptosis, and enhanced inflammation, whereas all these effects mediated by circRNA-PTPRA downregulation in ox-LDL-treated HUVECs were abolished by miR-671-5p downregulation. In conclusion, circRNA-PTPRA downregulation protects against ox-LDL-induced HUVECs injury by upregulating miR-671-5p, thereby providing potential therapeutic targets for AS.

Overexpression of MiR-181c-5p Attenuates Human Umbilical Vascular Endothelial Cell Injury in Deep Vein Thrombosis by Targeting FOS.

Deep venous thrombosis (DVT) is the third most common cardiovascular disease. Its clinical therapeutic effect is unsatisfactory due to the high rate of postthrombotic syndrome. Several studies have demonstrated the involvement of miRNAs in DVT. Therefore, we identified differentially expressed miRNAs in patients with DVT and explored their effects and underlying mechanism on endothelial cell (EC) injury.Differentially expressed miRNAs were identified via microRNA sequencing and verified using real-time quantitative PCR. The biological function of miR-181c-5p in human umbilical vein endothelial cell (HUVEC) injury stimulated by oxidized low-density lipoprotein (ox-LDL) was investigated. The target gene of miR-181c-5p was analyzed using bioinformatics and verified via dual-luciferase reporter assay.miRNA sequencing showed that miR-181c-5p was downregulated in the peripheral blood of patients with DVT. Furthermore, miR-181c-5p had a high clinical diagnostic value for DVT by receiver operating characteristic curve analysis. An in vitro cell model of EC injury, miR-181c-5p, was repressed in ox-LDL-treated HUVECs. Enhancing miR-181c-5p expression could alleviate the inhibition cell viability, cell apoptosis, raising ROS and MDA production, the reducing SOD level, and the elevated levels of thrombosis-related factor, ET-1 and vWF induced by ox-LDL. Further analysis revealed that FBJ osteosarcoma oncogene (FOS) is a target of miR-181c-5p and could antagonize the protective role of miR-181c-5p in ox-LDL-induced HUVEC injury.Our research demonstrated that miR-181c-5p could attenuate ox-LDL-induced EC injury and thrombosis-related factor expression by negatively regulating FOS. These findings suggest that the miR-181c-5p/FOS axis is a promising therapeutic target for DVT.