Atherosclerotic Model Research Articles

Abstract 4138639: Exploring advanced therapeutic approaches for atherosclerosis: Targeting efferocytosis and EndoMT with a focus on the CD47

Background: The anti-phagocytic molecule CD47 is upregulated in the atherosclerotic plaques of patients with cerebrovascular disease; however, the molecular mechanisms responsible for the development and progression of atherosclerotic lesions have not been fully established. In this study, we postulate how endothelial-specific CD47 promotes endothelial-to-mesenchymal transition (EndoMT) in potentiating atherosclerosis and assess the efficacy of the development of novel therapeutic interventions for cardiovascular disease. Methods: Using single-cell RNA sequencing (scRNA-seq), combined with molecular, cellular, and biochemical analyses, we investigated the role of endothelial CD47 in stimulating EndoMT using knock-out in ApoE-/- atherosclerotic mouse models. Results: ScRNA-seq revealed that CD47 promotes EndoMT, and the loss of endothelial CD47 inhibits EndoMT marker expression as well as transforming growth factor-beta signaling in vitro and atherosclerotic mice, which is associated with smaller lesions in Apoe-/- mouse model. In endothelial cells, CD47 hinders efferocytosis of senescent ECs and represses macrophage’s efferocytotic capacity to propel arterial inflammation and atherogenesis. Mechanistically, the interaction between CD47 and efferocytic receptor MerTK exacerbates inflammation by inhibiting the process of efferocytosis in endothelial cells and macrophages under atherogenic conditions. In response to atherogenic stimuli, endothelial CD47 upregulates the endocytic adaptor protein epsin, causing fibroblast growth factor receptor-1 (FGFR1) signal attenuation that upregulates TGF-β signaling and promoting EndoMT and potentiating atherosclerosis. Conclusion: We conclude that endothelial CD47 potentiates EndoMT during atherogenesis by increasing TGF-β signaling through FGFR1 internalization and degradation and targeted manipulating CD47 expression with precision nanomedicine offering a novel approach for mitigating cardiovascular disease.

Abstract 4129709: Aspirin-Nanoparticle for Dual Therapies: Targeted Anti-Inflammatory and Prolonged Anti-Platelet Effects for Atherosclerosis

Background: The current unmet needs for aspirin usage in atherosclerosis lie in its short half-life and narrow indication for anti-platelet effects. Daily aspirin intake is mandatory, and the anti-inflammatory effects of aspirin for atherosclerosis have not successfully translated to clinical practice. Nanoparticles remain in circulation for 2-3 days, with a large portion being cleared by splenic monocytes, which are known to inherently target inflamed sites. Hypothesis: By altering the pharmacokinetics of aspirin through loading into nanoparticles, aspirin-nanoparticles can exert prolonged anti-platelet effects and target atherosclerosis sites via monocyte carriers for anti-inflammatory effects, resulting in dual therapies. Methods: Splenic monocytes were loaded with aspirin-liposomes and co-cultured with endothelial cells or platelets to examine the interactions between them using high-resolution time-series microscopy. Prolonged anti-platelet effects and targeted anti-inflammatory effects of aspirin were validated in intact mice and hindlimb ischemia models, respectively. Furthermore, the dual therapies of aspirin-liposomes were validated in an atherosclerotic mouse model created by partial carotid ligation and a western diet in apoE gene knock-out mice. Results: When splenic monocytes were loaded with aspirin-liposomes, they emitted extracellular vesicles (EVs) loaded with aspirin towards endothelial cells or platelets. As inflamed cells upregulate caveolin expression, they uptake an increased amount of transferred EVs compared to non-inflamed cells. Additionally, aspirin-liposomes showed prolonged circulation time and increased splenic accumulation compared to aspirin itself, resulting in prolonged anti-platelet effects (>7 days) and targeted anti-inflammatory effects at inflamed sites. Compared to the daily oral aspirin group in the atherosclerosis model, the weekly intravenous aspirin-liposome group showed superior therapeutic effects, including attenuated systemic and local inflammation and patent lumen in atherosclerotic sites. Conclusion: Aspirin-nanoparticles can exert prolonged anti-platelet effects combined with targeted anti-inflammatory effects, resulting in superior therapeutic effects on atherosclerosis.

Gegen Qinlian Decoction Modulates Atherosclerosis and Lipid Metabolism Through Cellular Interplay and Signaling Pathways.

The objective of this study is to investigate Gegen Qinlian decoction (GQD) effects on lipid metabolism and explore its mechanism for preventing and treating atherosclerosis. An atherosclerotic rat model was established, and after an 8-week high-fat diet, atherosclerosis and non-alcoholic fatty liver disease were assessed. Subsequently, GQD was administered at low and high doses. Histopathological aortic wall changes, hepatic lipid deposition, and blood lipid changes were evaluated. ELISA indicated the influence of TNF-α and IL-13, and Western blotting revealed MerTK, ABCA1, and LXR-α expression. A foam macrophage model was established, and Cell activity was detected by the MTT method. ELISA indicated the influence of PPAR-γ. The expression of ABCA1, ABCA7, ABCG1, GAS6, MerTK, SCARB1, LXR- α and LXR-β mRNA were detected by qPCR. and Western blotting revealed MerTK and LXR-α expression. The impact of drug-containing serum of GQD on efferocytosis-related factors was studied. GQD improved atherosclerosis and non-alcoholic fatty liver disease and reduced serum low-density lipoprotein levels in the high-dose group. The high- and low-dose groups showed upregulated ABCA1, MerTK, and LXR-α expression in blood vessels and the liver, respectively. GQD decreased serum TNF-α and increased IL-13 levels. PPAR-γ expression was elevated in the high-, and low-dose groups. In the high-and low-dose groups, ABCA7, GAS6, SCARB1, and LXR-α, ABCA1 and MerTK, and ABCG1 gene expression were upregulated, respectively. Both low- and high-dose serum-containing drugs promoted LXR-β gene expression, and LXR-α protein expression was improved in the high-dose group. GQD improves rat atherosclerosis and hepatic lipid metabolism by regulating PPAR-γ, LXR-α, LXR-β, ABCA1, ABCA7, and ABCG1 expression and augmenting cellular intercalation through the GAS6/TAM pathway.

An In Vitro Study of 355‐nm Laser Ablation of Atherosclerotic Lesions Model

ABSTRACTA study of 355 nm laser with high pulse energy across various types of atherosclerotic lesion models is presented. The 355 nm laser pulses (10 ns) are delivered via a single fiber (600 μm diameter), and the ablation of calcified tissue, lipid tissue, and thrombus‐like tissue are studied under varied laser fluence (40–70 mJ/mm2) and repetition rate (5–30 Hz). The contact and noncontact ablation processes of chicken tibia samples (calcified tissue) are compared at 60 mJ/mm2 and 30 Hz, and the size of ablation particles is in the range of 0.1–1 μm. At the same repetition rate, the advancement rate of tricalcium phosphate samples reaches 150 μm/s at 70 mJ/mm2. Calcified and lipid models demonstrate predictable increases in ablation with higher laser fluence and repetition rate. The fresh porcine blood clot samples exhibit high‐quality ablation with good channel effect at 50 mJ/mm2 and 30 Hz.

Engineering Protein-Nanoparticle Hybrids as Targeted Contrast Agents.

Iron oxide nanoparticles (IONPs) have shown great promise in biomedical applications, particularly as MRI contrast agents due to their magnetic properties and biocompatibility. Although several IONPs have been approved by regulatory agencies as MRI contrast agents, their primary application as negative contrast agents limits their usage. Additionally, there is an emerging need for the development of molecular contrast agents that can specifically target biomarkers, enabling more accurate and sensitive diagnostics. To address these challenges, we exploited the engineerability of proteins to stabilize IONPs with tailored magnetic properties, creating protein-stabilized iron oxide nanoparticles (Prot-IONPs) and leveraged the chemical diversity of proteins to functionalize Prot-IONPs with targeting moieties. As a proof-of-concept, we used alendronate (Ald) to target atherosclerotic plaques in the aorta. Simple protein functionalization allowed targeting while maintaining the stability and relaxation properties of the Prot-IONPs. Prot-IONPs-Ald successfully enabled positive contrast imaging of atherosclerotic plaques in vivo in an atherosclerotic mouse model (ApoE-/- mice on a high-fat diet). This study demonstrates the potential of engineering protein-nanoparticle hybrids as versatile platforms for developing targeted in vivo MRI contrast agents.

METTL3-mediated m6A modification enhances lncRNA H19 stability to promote endothelial cell inflammation and pyroptosis to aggravate atherosclerosis.

This study explored the impact of N6-methyladenosine (m6A) modification on the regulation of long noncoding RNA (lncRNA) and atherosclerosis progression. An atherosclerosis cell model was established by treating human aortic endothelial cells (HAECs) with oxidized low-density lipoprotein. Additionally, an atherosclerotic animal model was developed using ApoE-/- C57BL/6 male mice fed a high-fat diet. Both models were employed to assess the expression changes of proteins associated with m6A modification. First, the effect of m6A modification writer protein methyltransferase-like 3 (METTL3) knockdown on changes in the level of pyroptosis in HAECs was investigated, and bioinformatic analysis confirmed that lncRNA H19 (H19) was the potential target of m6A modification. RNA-binding protein immunoprecipitation assays were subsequently performed to explore the interaction between H19 and the m6A writer protein METTL3, as well as the reader protein recombinant insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2). Finally, the effect of H19 expression on pyroptosis levels in HAECs was evaluated. In the aortas of atherosclerosis mice, overall m6A levels were significantly elevated compared with controls (p < .05), with METTL3 and METTL14 mRNA and protein levels notably increased (p < .05). Similarly, ox-LDL-treated HAECs showed a significant rise in m6A levels, along with increased METTL3 and METTL14 expression (p < .05). METTL3 knockdown in HAECs led to decreased pyroptosis, as evidenced by reduced lactate dehydrogenase release and lower levels of IL-1β, IL-18, and IL-6 (p < .05). Overexpression of H19 reversed these effects, indicating METTL3's role in promoting atherosclerosis by stabilizing H19 through m6A modification. H19 was the primary target lncRNA molecule of METTL3-mediated m6A modification in the pathogenesis of atherosclerosis. METTL3-mediated m6A modification regulated H19 expression, thereby aggravating atherosclerosis by activating pyroptosis.

CTRP9: An Anti-Atherosclerotic Factor in ApoE Knockout Mice through Oxidative Stress Inhibition

Background: C1q/tumor necrosis factor-related protein-9 (CTRP9) is critically involved in the pathophysiology of metabolic and cardiovascular disorders. This investigation aimed to clarify the mechanism underlying the role of CTRP9 in atherosclerosis in apolipoprotein E (ApoE) knockout (KO) mice. Methods: ApoE KO mice were fed a Western diet and injected with a virus which resulted in CTRP9 overexpression or knockdown for 12 weeks. The plasma lipid levels and atherosclerotic plaque areas were measured after the mice were euthanized. Aortas were isolated, and RNA sequencing was performed to identify the differentially expressed genes and related signaling pathways. Finally, plasma oxidative stress factors were measured to demonstrate the reliability of the RNA sequencing results. Results: The plasma lipid levels in the CTRP9 overexpression group did not significantly differ from those in the green fluorescence protein (GFP) group. Markablely, CTRP9 overexpression inhibited atherosclerotic plaque formation in ApoE KO mice, whereas CTRP9 knockdown promoted plaque formation. RNA sequencing analysis identified 3485 differentially expressed genes that were prominently enriched across 55 signaling pathways. Additionally, plasma oxidative stress factors were significantly reduced after CTRP9 overexpression, whereas these factors were increased after CTRP9 knockdown, which was consistent with the results of the RNA sequencing analysis. Conclusions: These findings demonstrated that CTRP9 alleviated inflammation and cholesterol metabolism, which reduced oxidative stress in an atherosclerotic animal model. These beneficial effects may mediate the suppression of lesion development in the aorta.

High expression levels of haem oxygenase-1 promote ferroptosis in macrophage-derived foam cells and exacerbate plaque instability

Plaque rupture with consequent thrombosis is the leading cause of acute cardiovascular events, during which macrophage death is a hallmark. Ferroptosis is a pivotal intermediate link between early and advanced atherosclerosis. Existing evidence indicates the involvement of macrophage ferroptosis in plaque vulnerability; however, the exact mechanism remains elusive. The aim of this study was to explore key ferroptosis-related genes (FRGs) involved in plaque progression and the underlying molecular mechanisms involved. The expression landscape of FRGs was obtained from atherosclerosis-related GEO datasets. Molecular mechanism studies of ferroptosis were performed using bone marrow-derived macrophages (BMDMs) and macrophage-derived foam cells (MDFCs). Bioinformatics analysis and immunohistochemistry revealed that macrophage haem oxygenase-1 (HMOX1) is the key FRG involved in plaque destabilization. Hypoxic conditions induced a significant increase in Hmox1 expression in MDFCs but not in macrophages. In addition, the beneficial or deleterious effects of Hmox1 were dependent on the degree of Hmox1 induction. Hmox1 overexpression drove inflammatory responses and ferroptotic oxidative stress in MDFCs and aggravated the plaque burden in atherosclerotic model mice. Further mechanistic investigations demonstrated that hypoxia-mediated degradation of egl-9 family hypoxia-inducible factor 3 (Egln3) stabilized Hif1a, which subsequently promoted Hmox1 transcription. Our findings suggest that high Hmox1 expression under hypoxia is deleterious to MDFC viability and plaque stability, providing a reference for the management of acute cardiovascular events.

Omega-3 polyunsaturated fatty acids, especially DHA and EPA, remold gut microbiota to suppress inflammation in rabbits with atherosclerosis

Atherosclerosis (AS) caused by a high-fat diet becomes a critical cause of cardiovascular diseases, which has been regarded as an urgent public health problem. Omega-3 polyunsaturated fatty acids (omega-3 PUFAs) have many beneficial effects on human health. However, in recent years, the critical role of gut microbiota in atherosclerosis has been gradually proposed, and the impact of omega-3 PUFAs on cardiovascular diseases via gut microbiota in rabbits remains unknown. Likewise, the role of omega-3 PUFAs in regulating endothelial lesions, visceral injury, and vascular inflammation caused by high-fat diet-induced trimethylamine-nitrogen oxide (TMAO) has not been reported. Here, we investigated the effects of omega-3 PUFAs administered orally on atherosclerosis via an atherosclerotic rabbit model induced by a high-fat diet. The results showed that omega-3 PUFAs obtained from deep-sea fish oil can alleviate high-fat diet-induced atherosclerosis by inhibiting the increase of plasma TMAO and the formation of liver foam cells, reducing inflammatory factors, fat deposition, and plasma lipid levels and suppressing organ damage. Furthermore, gut microbiota disrupted by a high-fat diet were remodeled in rabbits treated with omega-3 PUFAs. These results further confirm that omega-3 PUFAs are promising nutritional and medical health foods that can be supplemented daily to prevent cardiovascular diseases.

HCC-1 Accelerates Atherosclerosis by Inducing Endothelial Cell and Macrophage Pyroptosis and Serves as an Early Diagnostic Biomarker.

HCC-1 (hemofiltrate CC chemokine-1), a CC-type chemokine, exerts function to change intracellular calcium concentration, induce leukocyte, and manipulate enzyme release especially in monocytes. It has been reported that HCC-1 can predict the persistent acute kidney injury or suppress hepatocellular carcinoma by modulating cell cycle and promoting apoptosis; however, the effect of HCC-1 on atherosclerosis is poorly understood. Here, we aimed to clarify the function and mechanism of HCC-1 in atherosclerosis and whether it could serve as a novel biomarker for the diagnosis of atherosclerosis. HCC-1 expression in serum, atherosclerotic plaques, and normal arterial tissue from patients with atherosclerosis and control group was assessed by ELISA, immunohistochemistry and confocal microscope, and bioinformatic analysis. The atherosclerotic model of HCC-1 overexpressing and control mice was generated by tail vein injection of adeno-associated virus serotype 9-HCC-1 on an ApoE-/- background. Cell adhesion, polarization, and pyroptosis were evaluated in vitro. The relationship between HCC-1 concentration in serum and atherosclerosis was analyzed in patients with atherosclerosis. HCC-1 expression was positively correlated with the occurrence and stable-unstable switch of atherosclerosis under bioinformatic analysis, which is further supported by the results of increased HCC-1 expression in atherosclerosis patients both in serum and atherosclerotic plaque. adeno-associated virus serotype 9-HCC-1 mice had higher levels of inflammatory factors, increased macrophage accumulation and pyroptotic rate in plaque, and decreased atherosclerotic plaque stability. In vitro, HCC-1 promoted monocyte adhesion and M1 polarization and induced inflammation and pyroptosis both in endothelial cells and macrophages. HCC-1 expression was increased in patients with atherosclerosis, and HCC-1 overexpression accelerated atherosclerotic burden via an enhancement in monocyte recruitment, M1 polarization, and pyroptosis both in endothelial cells and macrophages. Our findings suggested that HCC-1 may serve as an early biomarker for the diagnosis of atherosclerosis, with the capacity to reflect the degree of stenosis.

Inhibition of the RXRA-PPARα-FABP4 signaling pathway alleviates vascular cellular aging by an SGLT2 inhibitor in an atherosclerotic mice model.

Atherosclerosis is the pathological cause of atherosclerotic cardiovascular disease (ASCVD), which rapidly progresses during the cellular senescence. Sodium-glucose cotransporter 2 inhibitors (SGLT2is) reduce major cardiovascular events in patients with ASCVD and have potential antisenescence effects. Here, we investigate the effects of the SGLT2 inhibitor dapagliflozin on cellular senescence in atherosclerotic mice. Compared with ApoE-/- control mice treated with normal saline, those in the ApoE-/- dapagliflozin group, receiving intragastric dapagliflozin (0.1 mg kg-1 d-1) for 14 weeks, exhibited the reduction in the total aortic plaque area (48.8%±6.6% vs. 74.6%±8.0%, P<0.05), the decrease in the lipid core area ((0.019±0.0037) mm2vs. (0.032±0.0062) mm2, P<0.05) and in the percentage of senescent cells within the plaques (16.4%±3.7% vs. 30.7%±2.0%, P<0.01), while the increase in the thickness of the fibrous cap ((21.6±2.1) µm vs. (14.6±1.5) µm, P<0.01). Transcriptome sequencing of the aortic arch in the mice revealed the involvement of the PPARα and the fatty acid metabolic signaling pathways in dapagliflozin's mechanism of ameliorating cellular aging and plaque progression. In vitro, dapagliflozin inhibited the expression of PPARα and its downstream signal FABP4, by which the accumulation of senescent cells in human aortic smooth muscle cells (HASMCs) was reduced under high-fat conditions. This effect was accompanied by a reduction in the intracellular lipid content and alleviation of oxidative stress. However, these beneficial effects of dapagliflozin could be reversed by the PPARα overexpression. Bioinformatics analysis and molecular docking simulations revealed that dapagliflozin might exert its effects by directly interacting with the RXRA protein, thereby influencing the expression of the PPARα signaling pathway. In conclusion, the cellular senescence of aortic smooth muscle cells is potentially altered by dapagliflozin through the suppression of the RXRA-PPARα-FABP4 signaling pathway, resulting in a deceleration of atherosclerotic progression.

Therapeutic biomaterials with liver X receptor agonists based on the horizon of material biology to regulate atherosclerotic plaque regression in situ for devices surface engineering.

Percutaneous coronary interventional is the main treatment for coronary atherosclerosis. At present, most studies focus on blood components and smooth muscle cells to achieve anticoagulation or anti-proliferation effects, while the mediated effects of materials on macrophages are also the focus of attention. Macrophage foam cells loaded with elevated cholesterol is a prominent feature of atherosclerotic plaque. Activation of liver X receptor (LXR) to regulate cholesterol efflux and efferocytosis and reduce the number of macrophage foam cells in plaque is feasible for the regression of atherosclerosis. However, cholesterol efflux promotion remains confined to targeted therapies. Herein, LXR agonists (GW3965) were introduced on the surface of the material and delivered in situ to atherogenic macrophages to improve drug utilization for anti-atherogenic therapy and plaque regression. LXR agonists act as plaque inhibition mediated by multichannel regulation macrophages, including lipid metabolism (ABCA1, ABCG1 and low-density lipoprotein receptor), macrophage migration (CCR7) and efferocytosis (MerTK). Material loaded with LXR agonists significantly reduced plaque burden in atherosclerotic model rats, most importantly, it did not cause hepatotoxicity and adverse reactions such as restenosis and thrombosis after material implantation. Both in vivo and in vitro evaluations confirmed its anti-atherosclerotic capability and safety. Overall, multi-functional LXR agonist-loaded materials with pathological microenvironment regulation effect are expected to be promising candidates for anti-atherosclerosis and have potential applications in cardiovascular devices surface engineering.

Effect of Parasteron on In Vivo Atherosclerotic model with Functional and Tissue Damage Mitigation

Background: Atherosclerosis, a leading cause of cardiovascular diseases, involves the buildup of plaques within arterial walls, leading to functional and tissue damage. Animal models, particularly rabbits, have been instrumental in studying this disease due to their lipoprotein metabolism and sensitivity to a cholesterol diet, similar to humans. The current study aims to evaluate the effects of the steroid hormone parasteron on the functional and tissue damage caused by atherosclerosis in adult female rabbits. Methods: The study was conducted over six weeks on four groups of adult female rabbits. The first group served as the control, while the second group was administered cholesterol to induce atherosclerosis. The third and fourth groups were treated with both cholesterol and the steroid hormone parasteron. Various parameters related to atherosclerosis, including functional metrics and tissue damage, were monitored and analyzed. Results: The control group showed no signs of atherosclerosis or tissue damage. The cholesterol-only group exhibited significant functional impairments and tissue damage characteristic of atherosclerosis. In the groups treated with parasteron, there was a notable reduction in both functional impairments and tissue damage compared to the cholesterol-only group. The extent of protection provided by parasteron was assessed through histological and biochemical analyses, which revealed a marked improvement in arterial health and function. Conclusion: The administration of parasteron significantly mitigates the functional and tissue damage associated with atherosclerosis in adult female rabbits. These findings suggest the potential of parasteron as a therapeutic agent in the management of atherosclerosis. Further research is warranted to explore the underlying mechanisms and to evaluate the long-term efficacy and safety of parasteron in larger clinical settings.

OGG1 prevents atherosclerosis-induced vascular endothelial cell injury through mediating DNA damage repair.

This study was designed to investigate the role of 8-oxoguanine DNA glycosylase 1 (OGG1) in preventing atherosclerosis-induced vascular EC injury, thereby providing a theoretical basis for the exploration of drug targets and treatment methods for atherosclerosis. Human umbilical vein cell line (EA.hy926) was treated with ox-LDL to construct an in vitro atherosclerotic cell model. pcDNA3.1-OGG1 was transfected into EA.hy926 cells to overexpress OGG1. qRT-PCR, CCK-8 assay, flow cytometry, oil red O staining, ELISA, comet assay and western blot were used to evaluate the OGG1 expression, viability, apoptosis level, lipid droplet content, 8-OHdG level and DNA damage of cells in each group. Compared with the Control group, ox-LDL stimulation of endothelial cells significantly decreased cell viability, promoted apoptosis and DNA damage, and increased intracellular levels of 8-OHdG and γH2AX, while decreasing protein levels of PPARγ, FASN, FABP4, RAD51 and POLB. However, overexpression of OGG1 can significantly inhibit ox-LDL damage to endothelial cells, promote lipid metabolism, decrease lipid droplet content, and improve DNA repair function. Over-expression of OGG1 improves DNA repair. Briefly, OGG1 over-expression enhances the DNA damage repair of ECs by regulating the expression levels of γH2AX, RAD51 and POLB, thereby enhancing cell viability and reducing apoptosis.

Jingzhi Guanxin Oral Liquids Attenuate Atherosclerotic Coronary Heart Disease via Modulating Lipid Metabolism and PPAR-Related Targets.

Jingzhi Guanxin Oral Liquids (JZGX), a traditional Chinese medicine formulation prepared from the decoction of five herbs, has been utilized to relieve chest pain with coronary artery disease (CAD). However, the chemical composition and therapeutic mechanisms of JZGX remain obscured. In this research, the potential targets and pathways of JZGX against CAD were anticipated through network pharmacology based on analyzing its chemical constituents using UPLC-Q-TOF-MS/MS. One hundred seven ingredients in JZGX were identified. The 39 active chemicals and 37 key targets were screened, and CAD-related signaling pathways were clustered, mainly associated with lipid metabolism. Subsequently, the atherosclerotic CAD animal model employing 24 weeks of high-fat diet (HFD) ApoE-/- mice was constructed to investigate the JZGX efficacy and underlying mechanisms validating network forecasts. The histological staining examination and cardiovascular biomarker tests confirmed that JZGX reduced plaque formation in the aorta and decreased blood lipids in vivo. It featured anti-inflammatory, anti-thrombotic, and myocardial protective effects. JZGX prevented excessive lipid deposits and inflammation within the liver and exhibited hepatoprotective properties. Serum untargeted metabolomics analysis indicated that JZGX ameliorated metabolic abnormalities in atherosclerotic CAD mice and prompted lipid metabolism, especially linoleic acid. The PPARs and attached critical targets (SREBP1, FASN, PTGS2, and CYP3A), filtered from the networks and connected with lipid metabolism, were dramatically modulated through JZGX administration, as revealed by western blotting. The molecular docking outcomes showed that all 39 active ingredients in JZGX had good binding activity with PPARα and PPARγ. These findings illustrate that JZGX alleviates atherosclerotic CAD progression by remodeling the lipid metabolism and regulating PPAR-related proteins.

Stability analysis of an atherosclerotic plaque formation model with time delay

Atherosclerosis is a chronic inflammatory disease that poses a serious threat to human health. It starts with the buildup of plaque in the artery wall, which results from the accumulation of pro‐inflammatory factors and other substances. In this paper, we propose a mathematical model of early atherosclerosis with a free boundary and time delay. The time delay represents the time required for macrophages to transit to foam cells through cholesterol accumulation. We obtain an explicit solution and analyze the stability of the model and the effect of the time delay on plaque size. We show that in the form of perturbation (where represents the mode of angle), when or 1, the steady‐state solution is linearly stable; when , there exists a critical parameter such that the steady‐state solution is linearly stable for and unstable for . Moreover, we find that smaller plaque are associated with the presence of time delay.

Untangling the inflammatory responses of liver x receptor (LXR) activation in human macrophages

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): Rembrandt Institute for Cardiovascular Science (grant 2022) The regulation of cholesterol and fatty acid homeostasis is essential and the Liver X Receptor (LXR) plays a crucial role in this process. When activated, LXR promotes cholesterol efflux and suppresses the transcription of pro-inflammatory genes in mouse macrophages, making it a promising therapy against atherosclerosis. Studies indeed have shown that LXR agonism is effective in pre-clinical atherosclerotic mouse models. However, the limited data on human macrophages suggests that LXR activation may have additional pro-inflammatory effects in humans, and the mechanisms contributing to this are not yet fully understood. To investigate the inflammatory properties of LXR activation in human macrophages, monocytes were isolated from buffy coats of healthy donors and differentiated into macrophages using M-CSF. The macrophages were exposed to synthetic and endogenous LXR ligands, such as GW3965, T0901317, and desmosterol, respectively, followed by stimulation with lipopolysaccharide (LPS) for six hours. We used qPCR and ELISA to analyze experiments and plan to supplement our data with RNA-Seq and multiplex assays. Preliminary results show that LXR activation reduces LPS-induced activation of interferon-mediated genes like CXCL9 and CXCL10, similar to mice. However, it promotes the transcription of NF-kB target genes like IL1B and IL12B upon LPS activation. These findings suggest that LXR activation in human macrophages not solely suppresses inflammatory responses in macrophages and that LXR activation has different effects on the regulation of genes mediated by interferon or NF-kB. Our research aims to further uncover the regulatory mechanisms behind LXR activation in human macrophages as central regulators in atherosclerosis.

Leukemia inhibitory factor receptor inhibition reduces stenosis grade and total serum cholesterol levels in a mouse model for atherosclerosis

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Dutch Heart Foundation Atherosclerosis is characterized by the accumulation of lipids and immune cells in the arterial wall and it is the main underlying pathology causing cardiovascular diseases (CVD). Cytokines are involved in all stages of atherosclerosis and most cytokines contribute to disease progression. For instance, IL-6 cytokine family members, such as IL-6, oncostatin-M and cardiotrophin-1, are closely related to CVDs. However, the role of leukemia inhibitory factor (LIF), a member of the IL-6 cytokine family, and its receptor (LIFR) in atherosclerosis remains unknown. Therefore, the aim of this study is to assess the contribution of LIFR signaling to atherosclerosis development by systemically inhibiting LIFR in an atherosclerotic mouse model. First, the expression of LIF and LIFR was examined using single-cell RNA sequencing data of human carotid artery plaques. Mast cells highly and exclusively express LIF, whereas LIFR was specifically expressed by activated endothelial cells. To validate LIFR signaling in vivo, female western-type diet fed Ldlr-/- mice were treated with LIFR inhibitor EC359 (5 mg/kg s.c., n=15) or control solvent (n=15) three times per week for eight weeks. During the experiment, weights of the mice did not differ between groups, whereas total serum cholesterol levels were significantly reduced in EC359 treated mice (p=0.03). In particular, EC359 treatment reduced VLDL levels. Additionally, EC359 treatment significantly reduced Sr1, Abca1 and Lrp1 expression in the liver, suggesting an altered lipid metabolism in EC359 treated mice. Furthermore, LIFR inhibition affected innate immunity, both systemically and locally in the plaque. Neutrophil numbers were significantly increased in the circulation (ctrl 18.7±1.7%; EC359 28.0±2.0%; p=0.002) and aortic plaques (ctrl 3.9±0.8%; EC359 9.9±1.9%; p=0.003) of mice treated with EC359 compared to control mice. Moreover, LIFR inhibition affected circulating monocytes resulting in less inflammatory monocytes. The adaptive immune cells, such as T and B cells, remained largely unaffected. Collectively, the reduction of serum total cholesterol and increase in neutrophil numbers in EC359 treated mice resulted in a trend towards reduced atherosclerotic stenosis grade compared to control mice (39.8±1.4% versus 34.9±1.9%; p=0.054), but it did not affect plaque collagen and monocyte/macrophage content in the aortic root. Conclusively, LIFR signaling inhibition reduces atherosclerotic stenosis grade, lowers plasma total cholesterol levels and affects innate immunity.

Mechanical Assessment in Atherosclerosis Based on Photoacoustic Viscoelasticity Imaging

Early identification of vulnerable plaques is a major challenge in diagnosis and assessment of atherosclerosis. In atherosclerotic plaque development, the proportion change in components caused plaque mechanical property change and induced plaque rupture. In this paper, a photoacoustic viscoelasticity imaging (PAVEI) technique was proposed to measure the viscosity–elasticity ratio of atherosclerotic plaque and evaluated for the potential in characterizing vulnerable plaques. Apolipoprotein E-knockout mice fed with a high-fat/high-cholesterol diet were chosen as the atherosclerotic model. Plaque component phantoms were examined to demonstrate the high efficiency of PAVEI in detecting the proportion change in components compared to single elasticity or viscosity detection. Finally, atherosclerotic plaques from mice aortas at different stages were imaged by PAVEI, which provided an insight into the compositional and functional characterization of vulnerability plaques and suggested its potential applications in the identification of high-risk plaques.

Sodium-glucose cotransporter 2 inhibitors attenuate vascular calcification by suppressing endoplasmic reticulum protein thioredoxin domain containing 5 dependent osteogenic reprogramming

AimsVascular calcification is strongly linked to the development of major adverse cardiovascular events, but effective treatments are lacking. Sodium-glucose cotransporter 2 (SGLT2) inhibitors are an emerging category of oral hypoglycemic drugs that have displayed marked effects on metabolic and cardiovascular diseases, including recently reported vascular medial calcification. However, the roles and underlying mechanisms of SGLT2 inhibitors in vascular calcification have not been fully elucidated. Thus, we aimed to further determine whether SGLT2 inhibitors protect against vascular calcification and to investigate the mechanisms involved. Methods and resultsA computed tomography angiography investigation of coronary arteries from 1554 patients with type 2 diabetes revealed that SGLT2 inhibitor use was correlated with a lower Agatston calcification score. In the vitamin D3 overdose, 5/6 nephrectomy chronic kidney disease-induced medial calcification and Western diet-induced atherosclerotic intimal calcification models, dapagliflozin (DAPA) substantially alleviated vascular calcification in the aorta. Furthermore, we showed that DAPA reduced vascular calcification via Runx2-dependent osteogenic transdifferentiation in vascular smooth muscle cells (VSMCs). Transcriptome profiling revealed that thioredoxin domain containing 5 (TXNDC5) was involved in the attenuation of vascular calcification by DAPA. Rescue experiments showed that DAPA-induced TXNDC5 downregulation in VSMCs blocked the protective effect on vascular calcification. Furthermore, TXNDC5 downregulation disrupted protein folding-dependent Runx2 stability and promoted subsequent proteasomal degradation. Moreover, DAPA downregulated TXNDC5 expression via amelioration of oxidative stress and ATF6-dependent endoplasmic reticulum stress. Consistently, the class effects of SGLT2 inhibitors on vascular calcification were validated with empagliflozin in intimal and medial calcification models. ConclusionsSGLT2 inhibitors ameliorate vascular calcification through blocking endoplasmic reticulum stress-dependent TXNDC5 upregulation and promoting subsequent Runx2 proteasomal degradation, suggesting that SGLT2 inhibitors are potentially beneficial for vascular calcification treatment and prevention.