oxLDL Stimulation Research Articles

Abstract 198: Metabolic Stress Causes Mitochondrial Dysfunction and Vascular Senescence via Angiotensin II Type I Receptor Signal / Rho Kinase Induced Mitochondrial Fission

Introduction: Metabolic stress, such as oxidized low density lipoprotein (ox-LDL) and advanced glycation end products (AGE) cause mitochondrial dysfunction and evoke vascular senescence and atherosclerosis. Mitochondria are highly dynamic organelles that constantly change their morphology to fusion or fission. This study aims to clarify whether mitochondrial dynamics is involved in the etiology of vascular senescence. Methods: We used C57BL6 (WT), apolipoprotein E deficient (ApoE KO) and db/db mice. We also conducted in vitro experiments using VSMC and HUVEC. Results: The degree of arterial senescence, arterial protein level of Drp1 in mitochondrial fraction and mitochondrial oxidative stress were higher and the number of fused mitochondria and mitochondrial function were lower in either ApoE KO or db/db mice than those of WT mice. Treatment with Drp1-specific inhibitor, mdivi-1, to these mice reduced excessive mitochondrial fission, oxidative stress and attenuated vascular senescence. Administration of either ox-LDL or AGE to cells also induced excessive mitochondrial fission though phosphorylation of Drp1 at Ser616, mitochondrial dysfunction, reactive oxygen species production and cellular senescence. Treatment with mdivi-1 to these cells restored imbalance of mitochondrial dynamics and these detrimental alterations. These results suggest that metabolic stress-induced mitochondrial dysfunction and cellular senescence were derived from Drp1-dependent mitochondrial fission. Treatment with angiotensin II type1 receptor (AT1R) blocker (ARB) to either Apo E KO mice or db/db in in vivo experiments and administration of ARB to cells with ox-LDL or AGE stimulation in in vitro experiments inactivated Drp1 and improved imbalance of mitochondrial dynamics, mitochondrial dysfunction and cellular senescence, suggesting that AT1R signal is involved in regulating metabolic stress-induced mitochondrial fission. Finally, inhibition of Rho kinase ROCK1 successfully attenuated Drp1-mediated mitochondrial fission and cellular senescence derived from metabolic stress. Conclusion: Metabolic stress causes cellular and vascular senescence through AT1R signal/Rho kinase-mediated mitochondrial fission.

Foam Cell-Derived CXCL14 Muti-Functionally Promotes Atherogenesis and Is a Potent Therapeutic Target in Atherosclerosis.

CXC chemokine family has been related to atherogenesis for long. However, the relationship between CXCL14 and atherogenesis is still unclear. This study preliminarily detected CXCL14 expression at foam cells in atherosclerosis specimens by immunohistochemistry. In vitro foam cells were derived from THP-1 after phorbol-12-myristate-13-acetate (PMA) and oxidized low-density lipoprotein (ox-LDL) stimulation. Immunoblotting and qPCR convinced CXCL14 expression variation during foam cell formation. We further demonstrated that ox-LDL regulated CXCL14 expression by AP-1. AP-1 could bind to CXCL14 promoter and up-regulate CXCL14 mRNA expression. Besides, CXCL14 promoted THP-1 migration, macrophage lipid phagocytosis, and smooth muscle cell migration as well as proliferation mainly via the ERK1/2 pathway. Additionally, a CXCL14 peptide-induced immune therapy showed efficacy in ApoE-/- mouse model. In conclusion, our study demonstrated that CXCL14 is highly up-regulated during foam cell formation and promotes atherogenesis in various ways. CXCL14 may be a potent therapeutic target for atherosclerosis.

PTEN inhibition attenuates endothelial cell apoptosis in coronary heart disease via modulating the AMPK–CREB–Mfn2‐mitophagy signaling pathway

Atherosclerosis (AS) is a major pathogenic factor in patients with cardiovascular diseases, and endothelial dysfunction (ED) plays a primary role in the occurrence and development of AS. In our study, we attempted to evaluate the role of phosphatase and tensin homolog (PTEN) in endothelial cellapoptosis under oxidized low-density lipoprotein (ox-LDL) stimulation and identify the associated mechanisms. The results of our study demonstrated that ox-LDL induced human umbilical vein endothelial cell (HUVEC) death via mitochondrial apoptosis, as evidenced by reduced mitochondrial potential, increased mitochondria permeability transition pore opening, cellular calcium overload, and caspase-9/-3 activation. In addition, ox-LDL also suppressed cellular energy production via downregulating the mitochondrial respiratory complex. Moreover, ox-LDL impaired HUVECs migration. Western blot analysis showed that PTEN expression was upregulated after exposure to ox-LDL and knockdown of PTEN could attenuate ox-LDL-mediated endothelial cell damage. Furthermore, we found that ox-LDL impaired mitophagy activity, whereas PTEN deletion could improve mitophagic flux and this effect relied on the activity of the AMP-activated protein kinase (AMPK)-cAMP-response element-binding protein (CREB)-Mitofusin-2 (Mfn2) axis. When the AMPK-CREB-Mfn2 pathway was inhibited, PTEN deletion-associated HUVECs protection was significantly reduced, suggesting that the AMPK-CREB-Mfn2-mitophagy axis is required for PTEN deletion-mediated endothelial cell survival under ox-LDL. Taken together, our results indicate that ox-LDL-induced endothelial cell damage is associated with PTEN overexpression, and inhibition of PTEN could promote endothelial survival via activating the AMPK-CREB-Mfn2-mitophagy signaling pathway.

Regulation of oxidized LDL-induced inflammatory process through NLRP3 inflammasome activation by the deubiquitinating enzyme BRCC36.

Oxidized Low-Density Lipoprotein (oxLDL) is a well-established pro-inflammatory marker that activates the NLRP3 inflammasome. Ubiquitination plays an important role in modulating the stability and functions of various proteins. BRCC36 is a ubiquitin-modifying enzyme that plays a crucial role in protein stabilization and activation in the cytosol, but its role in OxLDL-induced NLRP3 inflammasome activation is not known. Here, we have investigated the role of deubiquitinating enzyme BRCC36 in regulating NLRP3 inflammasome during oxLDL stimulation. Raw 264.7 murine macrophages were stimulated with oxLDL and effect of BRCC36 deubiquitination activity was assessed by fluorometric assay, and protein expression was assessed by Western blotting. The level of IL-1β measured by ELISA and LDH activity as pyroptotic cell death marker was assessed by fluorometric assay. The results showed that oxLDL increased the level of NLRP3 in macrophages and also the level of active caspase-1 and IL-1β. It also modulated the expression of deubiquitinating enzymes and caused pyroptotic cell death as indicated by LDH release. Inhibiting the proteasomal activity by MG132 and siRNA-mediated silencing of BRCC36 in macrophages potentially suppressed oxLDL-induced NLRP3 inflammasome activation and IL-1β secretion. Furthermore, the inhibition of proteasomal deubiquitinating activity with specific BRCC36 inhibitor G5 also reduced the inflammatory cell death. Taken together, our study suggests that deubiquitinating enzyme BRCC36 inhibition could potentially suppress oxLDL-induced inflammatory process by inhibiting NLRP3 activation and resultant IL-1β secretion.

Oxidized low-density lipoproteins induce tissue factor expression in T-lymphocytes via activation of lectin-like oxidized low-density lipoprotein receptor-1.

T-lymphocytes plays an important role in the pathophysiology of acute coronary syndromes. T-cell activation in vitro by pro-inflammatory cytokines may lead to functional tissue factor (TF) expression, indicating a possible contribution of immunity to thrombosis. Oxidized low-density lipoproteins (oxLDLs) are found abundantly in atherosclerotic plaques. We aimed at evaluating the effects of oxLDLs on TF expression in T cells and the role of the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1). CD3+ cells were isolated from healthy volunteers. Gene, protein, and surface expression of TF, as well as of LOX-1, were assessed at different time-points after oxLDL stimulation. To determine whether oxLDL-induced TF was LOX-1 dependent, T cells were pre-incubated with an LOX-1 inhibiting peptide (L-RBP) or with an anti-LOX-1 blocking antibody. To exclude that TF expression was mediated by reactive oxygen species (ROS) generation, oxLDL-stimulated T cells were pre-incubated with superoxide dismutase + catalase or with 4-Hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol), an intracellular free radical scavenger. Finally, to determine if the observed findings in vitro may have a biological relevance, the presence of CD3+/TF+/LOX-1+ cells was evaluated by immunofluorescence in human carotid atherosclerotic lesions. oxLDLs induced functionally active TF expression in T cells in a dose- and time-dependent manner, independently on ROS generation. No effect was observed in native LDL-treated T cells. LOX-1 expression was also induced by oxLDLs in a time- and dose-dependent manner. Pre-incubation with L-RBP or anti-LOX-1 antibody almost completely inhibited oxLDL-mediated TF expression. Interestingly, human carotid plaques showed significant infiltration of CD3+ cells (mainly CD8+ cells), some of which were positive for both TF and LOX-1. oxLDLs induce functional TF expression in T-lymphocytes in vitro via interaction of oxLDLs with LOX-1. Human carotid atherosclerotic plaques contain CD3+/CD8+cells that express both TF and LOX-1, indicating that also in patients these mechanisms may play an important role.

MicroRNA-181a regulates the activation of the NLRP3 inflammatory pathway by targeting MEK1 in THP-1 macrophages stimulated by ox-LDL.

Atherosclerosis (AS) is a chronic inflammatory disease that is characterized by the deposition of lipids in the vascular wall and the formation of foam cells. Macrophages play a critical role in the development of this chronic inflammation. An increasing amount of research shows that microRNAs affect many steps of inflammation. The goal of our study was to investigate the regulatory effect of miR-181a on theNLRP3 inflammasome pathway and explore its possible mechanism. Compared with the control group, the expression of miR-181a was downregulated in the carotid tissue of AS group mice, while the expression of MEK1 and NLRP3-related proteins was upregulated significantly. In vitro, when THP-1 macrophages were stimulated with oxidized low-density lipoprotein (ox-LDL), the expression of miR-181a was decreased, the MEK/ERK/NF-κB inflammatory pathways were activated and the expression of NLRP3 inflammasome-related proteins was upregulated. Exogenous overexpression of miR-181a downregulated the activation of the MEK/ERK/NF-κB pathway and decreased the expression of NLRP3 inflammasome-related proteins (such as NLRP3, caspase-1, interleukin-18 [IL-18], IL-1β, etc). Exogenous miR-181a knockdown showed the opposite results to those of overexpression group. A luciferase reporter assay proved that miR-181a inhibited the expression of MEK1 by binding to its 3'-untranslated region. When we knocked down miR-181a and then treated cells with U0126 before ox-LDL stimulation, we found that U0126 reversed the increased activation of the MEK/ERK/NF-κB pathway and upregulation of NLRP3 inflammasome-related proteins (NLRP3, caspase-1, IL-18, IL-1β) that resulted from miR-181a knockdown. Our study suggests that miR-181a regulates the activation of the NLRP3 inflammatorypathway by altering the activity of the MEK/ERK/NF-κB pathway via targeting of MEK1.

CTRP3 Alleviates Ox-LDL-Induced Inflammatory Response and Endothelial Dysfunction in Mouse Aortic Endothelial Cells by Activating the PI3K/Akt/eNOS Pathway.

C1q/tumor necrosis factor-related protein-3 (CTRP3) is a novel, certified, adipokine that beneficially regulates metabolism and inflammation in the cardiovascular system. Atherosclerotic plaque rupturing and secondary thrombosis cause vascular disorders, such as myocardial infarction and unstable angina. However, the underlying role of CTRP3 in atherosclerosis remains unclear. In this study, we aimed to elucidate whether and how CTRP3 ameliorates inflammation and endothelial dysfunction caused by oxidized low-density lipoprotein (ox-LDL). We first confirmed that CTRP3 expression was inhibited in ApoE-/- mice, compared to normal mice. Then, pcDNA-CTRP3 and siCTRP3 were transfected into mouse aortic endothelial cells after ox-LDL stimulation, and we observed that enhanced CTRP3 remarkably downregulated CRP, TNF-α, IL-6, CD40, and CD40L. We also observed that overexpression of CTRP3 elevated cell activity and decreased lactated hydrogenase release, accompanied by a marked reduction in cell apoptosis induced by ox-LDL. Meanwhile, overexpressed CTRP3 caused a decrease in Ang II, ICAM-1, and VCAM-1 expression, and it restored the balance between ET-1 and NO. Mechanism analysis confirmed that incremental CTRP3 upregulated p-PI3K, p-Akt, and p-eNOS expression, indicating that CTRP3 facilitated activation of the PI3K/Akt/eNOS pathway. On the contrary, siCTRP3 exerted the opposite effect to this activation. Blocking these pathways using LY294002 or L-NAME attenuated the protective role of CTRP3. Overall, these results suggest that CTRP3 can efficiently inhibit the inflammatory response and endothelial dysfunction induced by ox-LDL in mouse aortic endothelial cells, perhaps by activating the PI3K/Akt/eNOS pathway, indicating a promising strategy against atherosclerosis.

Atherosclerotic Conditions Promote the Packaging of Functional MicroRNA-92a-3p Into Endothelial Microvesicles.

Microvesicle-incorporated microRNAs (miRs) are biomarkers and effectors of cardiovascular disease. Whether microvesicle-miR expression is regulated in coronary artery disease (CAD) or not is unknown. Here, we explore the expression of circulating microvesicle-miRs in patients with CAD and investigate the role of microvesicle-miR in endothelial cells. Circulating microvesicles were isolated from patients' plasma by using ultracentrifugation. Electron microscopy was used to determine the size of the microvesicles. A Taqman miR array revealed certain microvesicle-miRs are significantly regulated in patients with stable CAD compared with patients with ACS. To validate the miR array results, 180 patients with angiographically excluded CAD (n=41), stable CAD (n=77), and acute coronary syndrome (n=62) were prospectively studied. Nine miRs involved in regulation of vascular performance-miR-126-3p, miR-222-3p, miR-let-7d-5p, miR-21-5p, miR-26a-5p, miR-92a-3p, miR-139-5p, miR-30b-5p, and miR-199a-5p-were quantified in circulating microvesicles by real-time polymerase chain reaction (PCR). Among these, miR-92a-3p was significantly increased in patients with CAD compared with non-CAD patients. Microvesicle-sorting experiments showed endothelial cells (ECs) were the major cell source for microvesicles containing miR-92a-3p. In vitro oxLDL (oxidized low-density lipoprotein) and IL-6 (interleukin-6) stimulation increased miR-92a-3p expression in parent ECs and upregulated the expression level of endothelial microvesicle (EMV)-incorporated miR-92a-3p. Labeling of miR-92a-3p and EMVs demonstrated that functional miR-92a-3p was transported into recipient ECs, which accelerated cell migration and proliferation. Knockdown of miR-92a-3p in EMVs abrogated EMV-mediated effects on EC migration, proliferation, and blocked vascular network formation in a matrigel plug. Polymerase chain reaction-based gene profiling showed that the expression of THBS1 (thrombospondin 1) protein-a target of miR-92a-3p and an inhibitor of angiogenesis-was significantly reduced in ECs by EMVs. Knockdown of miR-92a-3p in EMVs abrogated EMV-mediated inhibition of the THBS1 gene and protein expression. Atherosclerotic conditions promote the packaging of endothelial miR-92a-3p into EMVs. EMV-mediated transfer of functional miR-92a-3p regulates angiogenesis in recipient ECs by a THBS1-dependent mechanism.

The clickable activity-based probe of anti-apoptotic calenduloside E

Context: Calenduloside E (CE), one of the primary natural products found in Aralia elata (Miq.) Seem. (Araliaceae), possesses prominent anti-apoptotic potential. A previous study found that one of the anti-apoptotic CE targets is heat shock protein 90 AB1 (Hsp90AB1) by probe CE-P, while the other targets of CE still need to be identified with more efficient probes.Objective: This study investigates CE analogue (CEA) as one clickable activity-based probe for use in exploring anti-apoptotic CE targets.Materials and methods: Pretreatment of HUVECs with CEA (1.25 μM) for 8 hr, followed by ox-LDL stimulation for 24 h. Flow cytometry analysis and JC-1 staining assays were performed The kinetic constant measurements were tested by the Biacore T200, CM5 Sensor Chip which was activated by using sulpho-NHS/EDC. Ligands were dissolved and injected with a concentration of 12.5, 6.25, 3.125, 1.56, 0.78 and 0 μM.Results: CEA was confirmed to possess an anti-apoptotic effect. The probable targets of CE/CEA were calculated, and as one of the higher scores proteins (Fit values: 0.88/0.86), Hsp90 properly got our attention. Molecular modelling study showed that both CE and CEA could bind to Hsp90 with the similar interaction, and the docking scores (S value) were −7.61 and −7.33. SPR assay provided more evidence to prove that CEA can interact with Hsp90 with the KD value 11.7 µM.Discussion and conclusions: Our results suggest that clickable probe CEA could alleviate ox-LDL induced apoptosis by a similar mechanism of anti-apoptotic CE, and afforded the possibility of identifying additional anti-apoptotic targets of CE.

Paeoniflorin attenuates oxidized low-density lipoprotein-induced apoptosis and adhesion molecule expression by autophagy enhancement in human umbilical vein endothelial cells.

Oxidized low-density lipoprotein (ox-LDL)-induced endothelial dysfunction is recognized as a driving force in the development of atherosclerosis (AS). Paeoniflorin (Pae), a typical traditional herbal medicine, possesses anti-inflammatory, antioxidative, antihyperglycaemic, and antiapoptotic properties. Our study aimed to investigate the effects of Pae on ox-LDL-induced injury of the human umbilical vein endothelial cells (HUVECs) and to explore its molecular mechanism. We found that ox-LDL stimulation inhibited cell viability, activated autophagy, and induced apoptosis and adhesion molecule expression in HUVECs. Pae rescued ox-LDL-induced viability reduction and enhanced the ox-LDL-induced autophagy activation in HUVECs. Pae inhibited ox-LDL-induced apoptosis and adhesion molecule expression by autophagy enhancement in HUVECs. In addition, inhibition of SIRT1 by EX-527 abolished the promoting effect of Pae on autophagy and restored the inhibitory effect of Pae on apoptosis and adhesion molecule expression in the presence of ox-LDL. In conclusion, Pae attenuated ox-LDL-induced apoptosis and adhesion molecule expression by autophagy enhancement via upregulation of SIRT1 in HUVECs, shedding light on the mechanism underlying the protective effect of Pae on ox-LDL-induced injury of HUVECs.

Gingival mesenchymal stem cells attenuate pro-inflammatory macrophages stimulated with oxidized low-density lipoprotein and modulate lipid metabolism

ObjectiveTo examine the effects of gingival mesenchymal stem cells (GMSCs) on inflammatory macrophages upon oxidized low-density lipoprotein (ox-LDL) stimulation and evaluate therapeutic potential of GMSCs on mouse model of periodontitis associated with hyperlipidemia. Methodsin vitro, GMSCs were co-cultured with macrophages for 48 h in the absence or presence of M1 polarizing conditions and oxidized low-density lipoprotein in the transwell system. The supernatants were collected for ELISA. M1 and M2 markers of macrophages were analyzed by flow cytometry and PCR, and lipid accumulation was assessed by oil red O staining. in vivo, eighteen mice were divided into three groups (n = 6): Group A (periodontally healthy mice as control), Group B (periodontitis mice with hyperlipidemia), Group C (periodontitis mice with hyperlipidemia with the transplantation of GMSCs). The serum levels of cholesterol and inflammatory factors were measured by automatic analyzer. Bone regeneration was evaluated by Masson staining. ResultsWhen co-cultured with GMSCs, the M1 markers of Tumor Necrosis Factor (TNF) -α, Interleukin (IL) -6, Interleukin (IL) -1β, CD86, and Human Leukocyte Antigen (HLA) -DR were significantly reduced. In contrast, M2 markers such as Interleukin(IL) -10 and CD206 were moderately increased. Similar results were obtained in the cell culture supernatants. In animal experiment, GMSCs suppressed the expression of sterol regulatory element binding transcription factor 1c (SREBP-1c) and elevated the levels of peroxisome proliferator-activated receptor alpha (PPARα) and peroxisome proliferator activator receptor- coactivator 1(PGC-1α) in the liver, attenuated cholesterol dysfunction via the downregulation of low-density lipoprotein (LDL) and total cholesterol (TC), and the upregulation of high-density lipoprotein (HDL), and decreased the levels of TNF-α and IL-6. Moreover, GMSC treatment improved bone regeneration. ConclusionGMSCs inhibit the activation of M1 macrophages, regulate lipid metabolism and reduce inflammatory response, and promote bone regeneration in mouse model of periodontitis associated with hyperlipidemia.

Corin is highly expressed in atherosclerosis models

Corin is a serine protease mainly expressed in the heart, where it regulates blood pressure and cardiac function through activating pro-atrial natriuretic peptide (pro-ANP) to ANP. Its expression has also been detected in non-cardiac tissues. However, there is no report so far about the distribution and function of corin in aorta and in related diseases such as atherosclerosis. This study was the first to explore corin expression in aorta both under normal conditions and in atherosclerosis models. In vivo, we found corin had a basal level of expression in aortas, mainly in intimal endothelial cells and was significantly elevated in mouse atherosclerosis model. Moreover, we observed pro-ANP, the specific substrate of corin, was also expressed in mice aortas and increased in mouse atherosclerosis model. In vitro, we further demonstrated corin expression in cultured vascular endothelial cells and its induced expression after ox-LDL stimulation. Our results suggested that corin may play important roles in aorta physiology and in the pathophysiological process of atherosclerosis in an autocrine manner and has potential clinical value for the treatment of atherosclerosis.

Hydrogen attenuated oxidized low-density lipoprotein-induced inflammation through the stimulation of autophagy via sirtuin 1.

Chronic inflammation is a central pathogenic mechanism underlying the induction and progression of atherosclerosis (AS). Hydrogen has been demonstrated to serve a protective role in diverse models of disease. However, the potential effects and mechanism of hydrogen with respect to ox-LDL-induced inflammation have not yet been completely elucidated. In the present study, various concentrations (0, 50 and 100 mg/l) of oxidized low-density lipoprotein (ox-LDL) were used to treat RAW264.7 cells. A Cell Counting kit-8 assay was used to determine cell viability and western blot analysis was performed to determine the expression of proteins that are involved in autophagy. The expression of inflammatory cytokines in ox-LDL-treated macrophages was detected using ELISA. Small interfering (si)RNA against sirtuin 1 (SIRT1) was employed to investigate the mechanism underlying hydrogen-activated autophagy. The results indicated that ox-LDL stimulation promoted inflammatory cytokine expression and impaired autophagic flux in RAW264.7 cells. Furthermore, hydrogen inhibited ox-LDL-induced inflammatory cytokine expression by upregulating autophagic flux. SIRT1 mediated the upregulation of autophagic flux via hydrogen in ox-LDL-treated macrophages. To conclude, the present study provided novel insights into the role of defective autophagy in the pathogenesis of AS and identified autophagy to be a promising therapeutic target for the treatment of AS. Notably, hydrogen may represent a potential agent for the treatment of AS.

TRIF Regulates BIC/miR-155 via the ERK Signaling Pathway to Control the ox-LDL-Induced Macrophage Inflammatory Response.

Toll/IL-1R-domain-containing adaptor-inducing IFN-β (TRIF) is an important adaptor for TLR3- and TLR4-mediated inflammatory signaling pathways. Recent studies have shown that TRIF plays a key role in vessel inflammation and atherosclerosis; however, the precise mechanisms are unclear. We investigated the mechanisms of the TRIF-regulated inflammatory response in RAW264.7 macrophages under oxidized low-density lipoprotein (ox-LDL) stimulation. Our data show that ox-LDL induces TRIF, miR-155, and BIC expression, activates the ERK1/2 and SOCS1-STAT3-NF-κB signaling pathways, and elevates the levels of IL-6 and TNF-α in RAW264.7 cells. Knockdown of TRIF using TRIF siRNA suppressed BIC, miR-155, IL-6, and TNF-α expression and inhibited the ERK1/2 and SOCS1-STAT3-NF-κB signaling pathways. Inhibition of ERK1/2 signaling also suppressed BIC and miR-155 expression. These findings suggest that TRIF plays an important role in regulating the ox-LDL-induced macrophage inflammatory response and that TRIF modulates the expression of BIC/miR-155 and the downstream SOCS1-STAT3-NF-κB signaling pathway via ERK1/2. Therefore, TRIF might be a novel therapeutic target for atherosclerosis.

Silica nanoparticles promote oxLDL-induced macrophage lipid accumulation and apoptosis via endoplasmic reticulum stress signaling

Oxidized low-density lipoprotein (oxLDL), a marker of hyperlipidemia, plays a pivotal role in the development of atherosclerosis through the induction of macrophage-derived foam cell formation and thereafter apoptosis. Previous studies have indicated that silica nanoparticle (SiNPs) may exert a proatherogenic role, which could induce endothelial dysfunction, and monocytes infiltration. However, little is known about SiNPs' effects on macrophage-derived foam cell formation and apoptosis in the pathogenesis of atherosclerosis. In this study, we investigated the effects of SiNPs and oxLDL coexposure on macrophage-derived lipid metabolism, foam cell and apoptosis by using Raw264.7 cells. As a result, SiNPs enhanced cytotoxicity, apoptosis, and lipid accumulation upon oxLDL stimulation. Furthermore, quantitative determination of the expression levels of genes involved in cholesterol influx or efflux showed significantly up-regulated expressions of CD36 and SRA, whereas down-regulated expressions of ATP-binding cassette A1 (ABCA1), ABCG1, and SRB1 in oxLDL-treated macrophages, especially upon the co-exposure with SiNPs. It indicated that SiNPs promoted lipid accumulation in macrophage cells through not only facilitating cholesterol influx but also inhibiting cholesterol efflux. Endoplasmic reticulum (ER) is specialized for the production, modification, even trafficking of lipids. Interestingly, ER response was triggered upon oxLDL treatment, while SiNPs coexposure augmented the ER stress. Taken together, our results revealed that SiNPs promoted oxLDL-induced macrophage foam cell formation and apoptosis, which may be mediated by ER stress signaling. Thus we propose future researches needed for a better understanding of NPs' toxicity and their interactions with various pathophysiological conditions.

NR4A1 contributes to high-fat associated endothelial dysfunction by promoting CaMKII-Parkin-mitophagy pathways.

Parkin-related mitophagy is vital for endothelial cell viability and the development of atherosclerosis, although the upstream regulatory factor underlying Parkin-mediated mitophagy in endothelial apoptosis and atherosclerosis progression remains unknown. In the present study, we demonstrated that nuclear receptor subfamily 4 group A member 1 (NR4A1) is actually expressed in aortic endothelial cells (AECs) under oxidized low-density lipoprotein (ox-LDL) treatment in vitro or isolated from high-fat treated mice in vivo. Higher NR4A1 levels were associated with AEC apoptosis, mitochondrial dysfunction, and energy disorder. At the molecular level, ox-LDL stimulation increased NR4A1 expression, which evoked Parkin-mediated mitophagy. Excessive mitophagy overtly consumed mitochondrial mass, leading to an energy shortage and mitochondrial dysfunction. However, loss of NR4A1 protected AECs against ox-LDL induced apoptosis by inhibiting excessive mitophagy. Furthermore, we also identified that NR4A1 regulated Parkin activation via post-transcriptional modification by Ca2+/calmodulin-dependent protein kinase II (CaMKII). Activated CaMKII via NR4A1 induced the phosphorylated activation of Parkin. In summary, our data support the role of NR4A1/CaMKII/Parkin/mitophagy in AEC apoptosis and atherosclerosis formation and provide new insights into treating atherosclerosis with respect to endothelial viability, mitophagy, and NR4A1.

Construction of CD36 gene silencing cell lines by lentivirus-mediated RNA interference and the effect on protein expression of caveolin-1

CD36, the major scavenger receptor, is intimately involved in the uptake of oxLDL in macrophages. To further study the function of CD36 in macrophages, we constructed CD36 gene silence cell lines (J774A.1) by lentivirus-mediated RNA interference technique, and analyzed the effect of CD36 in caveolin-1 protein expression. At first, 5 shRNA fragments were designed and synthesized according to the coding sequence (CDS) region of CD36 gene. Next, the CD36-shRNA was inserted into lentiviral vector to yield pLKO.1-CD36-shRNA plasmid. After DNA sequencing, the pLKO.1-CD36-shRNA plasmid and psiCHECK-II-CD36 were co-transfected into the 293T cells to screen the efficient CD36-shRNA. The efficient CD36-shRNA plasmid and the helper plasmid were co-transfected into the 293T cells to package the lentivirus, and then infected the J774A.1 cells. After screening by puromycin, CD36 gene silence cell lines (J774A.1) was established. Western blotting and confocal fluorescence microscopy results showed that the CD36 silencing efficiency in the gene silence cell line was 90%. Accompanied by a decrease in CD36 protein on cell surface, oxLDL binding to CD36 was significantly inhibited, indicating that the CD36 gene silence cell line is successfully established. Finally, the oxLDL stimulation and inhibitor experiments results showed that the CD36 knockdown significantly suppresses the phosphorylation of JNK and ERK, thereby inhibiting the oxLDL-induced caveolin-1 protein expression, demonstrating that CD36 modulates the caveolin-1 protein expression through the JNK/ERK-mediated signaling transduction.

Precise theranostic nanomedicines for inhibiting vulnerable atherosclerotic plaque progression through regulation of vascular smooth muscle cell phenotype switching.

Coronary heart disease is a prevalent and fatal killer caused by vulnerable atherosclerotic plaques (VASPs). However, the precise detection and treatment of VASPs remains a difficult challenge. Here, we present the development of noninvasive human serum albumin (HSA)-based theranostic nanomedicines (NMs) for the specific diagnosis and effective therapy of VASPs.Methods: The ICG/SRT@HSA-pept NMs were formulated to contain payloads of the near-infrared (NIR) fluorescent dye indocyanine green (ICG) and the sirtuin 1 (Sirt1) activator SRT1720, and modified with a peptide moiety targeting osteopontin (OPN). The in vivo atherosclerotic mouse model was established with the high-fat diet (HFD). The in vitro vascular smooth muscle cells (VSMCs) phenotypic switching was induced using the ox-LDL stimulation.Results: Due to the overexpression of OPN in activated VSMCs and VASPs, the targeted NMs specifically accumulated within the VASPs region after intravenous injection into the atherosclerotic mice, achieving the precise detection of VASPs. In addition, in the presence of SRT1720, the NMs could activate intracellular Sirt1 and activate an antiatherogenesis effect by inhibiting the phenotypic switching of VSMCs, which is an essential contributor to the vulnerability and progression of atherosclerotic plaques. After therapeutic administration of the ICG/SRT@HSA-pept NMs for two weeks, the physiological sizes and plaque compositions of VASPs were markedly improved. Furthermore, ICG/SRT@HSA-pept NMs-treated mice presented a more favorable plaque phenotype than that was observed in free SRT1720-treated mice, suggesting the enhanced delivery of pharmaceutical agents to the atherosclerotic lesions and improved therapeutic efficacy of NMs compared with free SRT1720.Conclusions: The theranostic ICG/SRT@HSA-pept NMs showed great potential for the precise identification and targeted treatment of atherosclerotic diseases.

The macrophage C-type lectin receptor CLEC5A (MDL-1) expression is associated with early plaque progression and promotes macrophage survival

BackgroundBiomarkers of early plaque progression are still elusive. Myeloid DAP12-associating lectin-1 (MDL-1), also called CLEC5A, is a C-type lectin receptor implicated in the progression of multiple acute and chronic inflammatory diseases. However, the relationship between its level and atherosclerosis is unknown. In this study, we aimed to investigate the correlation between macrophage MDL-1 expression and early atherosclerosis progression.MethodsImmunofluorescence staining, real-time PCR and western blot were performed to analyze MDL-1 expression in aorta or mice macrophages. The role of MDL-1 in macrophage survival was further investigated by adenovirus infection and TUNEL assay.ResultsSignificant MDL-1 expression was found in advanced human and apoE−/− mice atherosclerotic plaques, especially in lesional macrophages. In the model of atherosclerosis regression, we found MDL-1 expression was highly downregulated in lesional macrophages from ldlr−/− mouse regressive plaques, coincident with a reduction in lesional macrophage content and marker of M1 proinflammatory macrophages. Furthermore, we found MDL-1 was significantly expressed in inflammatory M1 subtype polarized bone marrow-derived macrophages. In vitro experiments, the level of MDL-1 was remarkably elevated in macrophages treated with pathophysiological drivers of plaque progression, such as oxidized low-density lipoprotein (ox-LDL) and hypoxia. Mechanistically, we demonstrated that MDL-1 overexpression notably promoted macrophage survival and decreased cleaved caspase-3 expression under ox-LDL stimulation, which suggested that it could maintain lesional macrophage survival and cause its accumulation.ConclusionsThis study firstly demonstrated that MDL-1 is mainly expressed in atherosclerotic lesional macrophages and increased macrophage MDL-1 expression is associated with early plaque progression and promotes macrophage survival.

Paeoniflorin inhibits VSMCs proliferation and migration by arresting cell cycle and activating HO-1 through MAPKs and NF-κB pathway

The proliferation, migration and inflammation of vascular smooth muscle cells (VSMCs) contributes to the pathogenesis and progression of atherosclerosis. Paeoniflorin (PF) as active compound in the Rhizoma Atractylodes macrocephala has been used for various diseases like cancer, splenic asthenia, anaphylaxis and anorexia. This study aimed to explore whether and how PF regulated the inflammation, proliferation and migration of VSMCs under ox-LDL stimulation. Here, we found that PF dose-dependently inhibited ox-LDL-induced VSMCs proliferation and migration, and decreased inflammatory cytokines and chemokine overexpression. Mechanistically, PF prevented p38, ERK1/2 and NF-κB phosphorylation, and arrested cell cycle in S phase. Meanwhile, PF regulated the HO-1 and PCNA expression. Furthermore, PF blocked the foam cell formation in macrophages induced by ox-LDL. These results indicate that PF antagonizes the ox-LDL-induced VSMCs proliferation, migration and inflammation through activation of HO-1, cell cycle arrest and then suppression of p38, ERK1/2/MAPK and NF-κB signaling pathways.