Atherosclerotic Mice Research Articles

Colchicine delivered by a novel nanoparticle platform alleviates atherosclerosis by targeted inhibition of NF-κB/NLRP3 pathways in inflammatory endothelial cells

Atherosclerosis, a chronic inflammatory disease characterized by arterial plaque formation, is one of the most prominent causes of cardiovascular diseases. However, the current treatments often do not adequately compromise the chronic inflammation-mediated plaque accumulation and the disease progression. Therefore, a new and effective strategy that blocks atherosclerosis-associated inflammation is urgently needed to further reduce the risk. Colchicine, a potent anti-inflammatory medication, has shown great potential in the treatment of atherosclerosis, but its adverse effects have hampered its clinical application. Herein, we developed a novel delivery nanosystem encapsulated with colchicine (VHPK-PLGA@COL), which exhibited improved biosafety and sustained drug release along with the gradual degradation of PLGA and PEG as confirmed both in vitro and in vivo. Surface modification of the nanoparticles with the VHPK peptide ensured its capability to specifically target inflammatory endothelial cells and alleviate atherosclerotic plaque accumulation. In the ApoE − / − atherosclerotic mouse model, both colchicine and VHPK-PLGA@COL treatment significantly decreased the plaque area and enhanced plaque stability by blocking the NF-κB/NLRP3 pathways, while VHPK-PLGA@COL exhibited enhanced therapeutic effects due to its unique ability to target inflammatory endothelial cells without obvious long-term safety concerns. In summary, VHPK-PLGA@COL has the potential to overcome the key translational barriers of colchicine and open new avenues to repurpose this drug for anti-atherosclerotic therapy.

Assessing Early Atherosclerosis by Detecting and Imaging of Hypochlorous Acid and Phosphorylation Using Fluorescence Nanoprobe.

The assessment of early atherosclerosis (AS) is of great significance for the early diagnosis and mechanism research. Herein, a novel nanoprobe PCN@FL is developed to realize the simultaneous detection and imaging of phosphorylation and hypochlorous acid (HClO). The selective recognition of HClO is achieved through the specific interaction between DMTC and HClO, while the levels of phosphorylation are detected via the specific interaction between Zr (IV) and phosphates. The nanoprobe can be utilized to monitor the fluctuations in HClO and phosphate in early atherosclerosis. It is observed that the levels of HClO and phosphate in the serum of early AS mice are higher than those of the normal mice. Ultimately, the levels of hypochlorous acid and phosphorylation in the inner wall of aortic vessels are imaged by two-photon microscope. The results show that the levels of HClO and phosphorylation in the early atherosclerotic mice are significantly higher than those of in normal mice. The nanoprobe provides a suitable fluorescent tool for simultaneous detection and imaging of HClO and phosphorylation, which holds promise for early atherosclerotic disease assessment.

Exploring the adverse effects of 1,3,6,8-tetrabromo-9H-carbazole in atherosclerotic model mice by metabolomic profiling integrated with mechanism studies in vitro

Given its wide distribution in the environment and latent toxic effects, 1,3,6,8-tetrabromo-9H-carbazole (1368-BCZ) is an emerging concern that has gained increasing attention globally. 1368-BCZ exposure is reported to have potential cardiovascular toxicity. Although atherosclerosis is a cardiovascular disease and remains a primary cause of mortality worldwide, no evidence has been found regarding the impact of 1368-BCZ on atherosclerosis. Therefore, we aimed to explore the deleterious effects of 1368-BCZ on atherosclerosis and the underlying mechanisms. Serum samples from 1368-BCZ-treated atherosclerotic model mice were subjected to metabolomic profiling to investigate the adverse influence of the pollutant. Subsequently, the molecular mechanism associated with the metabolic pathway of atherosclerotic mice that was identified following 1368-BCZ exposure was validated in vitro. Serum metabolomics analysis revealed that 1368-BCZ significantly altered the tricarboxylic acid cycle, causing a disturbance in energy metabolism. In vitro, we further validated general markers of energy metabolism based on metabolome data: 1368-BCZ dampened adenosine triphosphate (ATP) synthesis and increased reactive oxygen species (ROS) production. Furthermore, blocking the aryl hydrocarbon receptor (AhR) reversed the high production of ROS induced by 1368-BCZ. It is concluded that 1368-BCZ decreased the ATP synthesis by disturbing the energy metabolism, thereby stimulating the AhR-mediated ROS production and presumably causing aggravated atherosclerosis. This is the first comprehensive study on the cardiovascular toxicity and mechanism of 1368-BCZ based on rodent models of atherosclerosis and integrated with in vitro models.

2-(2-Phenylethyl)chromone-enriched extract of Chinese agarwood (Aquilaria sinensis) inhibits atherosclerosis progression through endoplasmic reticulum stress-mediated CD36 expression in macrophages

Ethnopharmacological relevanceChinese agarwood, derived from the Aquilaria sinensis (Lour.) Gilg (Thymelaeaceae), has a long history of use in Traditional Chinese Medicine for the management of cardiovascular disease. However, the specific active ingredients responsible for its impact on atherosclerosis are yet to be fully understood.Aim of the study: The aim of this study is to investigate the anti-atherosclerotic effectiveness of the 2-(2-phenylethyl)chromone-enriched extract derived from Chinese agarwood (CPE) through the ER stress-mediated CD36 pathway. Materials and methodsTo assess the effectiveness of CPE, an atherosclerotic mouse model was established using ApoE−/− mice with a high-fat diet. Then we assessed the impact of CPE on lipid accumulation in THP-1 macrophages that were exposed to oxLDL. Subsequently, the effect of CPE on the expression of CD36 and markers related to ER stress was characterized. ResultsOur in vivo research confirmed that CPE effectively reduces the formation of aortic plaques in atherosclerotic ApoE−/− mice. Additionally, our in vitro study observed that CPE inhibits the uptake of oxLDL and hinders the generation of foam cells. This effect is achieved by downregulating the level of CD36 in macrophages. Furthermore, our study revealed that the increase in CD36 expression, resulting from oxLDL exposure, is governed by the activation of JNK1/2/3 signaling pathways and the initiation of ER stress. ConclusionCPE demonstrated significant efficacy to inhibit the atherosclerosis. The ER stress/P-JNK/PPARγ/CD36 signaling pathway plays critical involvement in modulating the foam cell formation in vitro and in vivo. These findings underscore the efficacy of CPE as a viable therapeutic intervention for the treatment of atherosclerosis.

Sirtuin-1 directly binds and deacetylates hepatic PCSK9 to inhibit low-density lipoprotein receptor degradation

Abstract Background Low-density lipoprotein cholesterol (LDL-C) is causally involved in atherosclerotic cardiovascular disease (ASCVD). We have previously shown that pharmacological activation of Sirtuin-1 (SIRT1), a NAD+-dependent deacetylase, reduces plasma LDL-C levels by decreasing PCSK9 and increasing hepatic LDLR expression, thus exerts atheroprotective effects in mice; however, the mechanism of SIRT1 action remains elusive. Purpose We found diminished levels of circulating SIRT1 in atherosclerotic mice compared to wild-type healthy controls. We hypothesized that this decrease in SIRT1 levels may promote dysregulated lipid metabolism and ASCVD in humans. Herein, we aimed to restore the reduced systemic Sirtuin-1 plasma levels in ASCVD by injecting recombinant murine Sirtuin-1 (rmSIRT1) in atherosclerotic mice. Methods Twelve-week-old apolipoprotein E-deficient (ApoE-/-) male mice fed a high-cholesterol diet (1.25% w/w) were randomized to receive rmSIRT1 (n=6; 0.3mg/kg BW i.p.) or vehicle treatment (n=6) every third day over 4-weeks. Mice were euthanized and organs were harvested to study lipid metabolism and ASCVD progression. Human hepatocytes (Huh7) cells were used to perform radioactive assays to study cellular uptake of radioiodinated LDL, surface plasmon resonance to study binding and site-directed mutagenesis to dissect the underlying molecular mechanism exerted by circulating SIRT1. Results Boosting circulating SIRT1 levels increased hepatic LDLR expression, reduced plasma LDL-C levels and progression of plaques in ApoE-/- mice (Figure 1). rmSIRT1 treatment did not change hepatic expression of total PCSK9 but increased its deacetylated status. Mechanistically, rmSIRT1 directly bound to hepatic PCSK9 and deacetylated PCSK9 at 3 sites: Lys243, Lys421 and Lys506 as shown by Surface Plasmon Resonance and mass spectrometry, respectively. In vitro mutagenesis to triple deacetylation mimetic (3KR) in hepatocytes (Huh7 cells) reduced SIRT1-induced PCSK9 activity, as evidenced by increased cellular binding and association of 125I-LDL through LDLR in Huh7cells. Finally, plasma levels of SIRT1 and PCSK9 were assessed at baseline in patients with acute coronary syndromes (ACS) where plasma SIRT1 showed an inverse correlation with PCSK9 and conferred a reduced risk of major adverse cardiovascular events. Conclusion SIRT1 directly binds PCSK9 and decreases its activity by deacetylation, thereby enhancing LDL-C clearance by hepatic LDLR upregulation. Increased circulating SIRT1 exerts atheroprotective effects and is associated with improved prognosis in patients with ACS.Figure 1

Canagliflozin enhances endogenous eliminatiton of senescent cells through downregulation of PD-L1

Abstract Sodium-glucose co-transporter 2 (SGLT2) inhibitors have been developed as a new class of anti-diabetic drug, and shown cardio- and renal-protective effects in patients with diabetes, heart failure, and chronic kidney diseases. Also, there's evidence that canagliflozin, one of the SGLT2 inhibitors has expanded lifespan in aged mice, but their detailed protective machinery is still to be clarified. Here we demonstrate that canagliflozin has a effect to eliminate senescent cells from pathological tissues, so-called "senolysis" in some murine disease models and alleviates their pathology. We treated canagliflozin mixed in food at 0.03% (w/w) to high-fat-diet(HFD)-fed obese mice and found that canagliflozin reduced senescent cells in visceral adipose tissue within 1 week. Also, we found that senolytic effect of canagliflozin was provided through enhancing endogenous senolytic function by NK or T cells. This machinery was mediated by enhanced AMPK signaling, which leads to the downregulation of PD-L1 expression on senescent cells. Furthermore, we treated canagliflozin to ApoE-knockout atherosclerotic mice with western diet feeding, progeroid mice and chronological aged mice. Canagliflozin reduced their senescent cell burden, resulting in alleviation of atherosclerosis, extended lifespan of progeroid, and altered physical function due to chronological aging. Our results would be promising evidence that SGLT2 inhibitors can be used as not only cardio- or renal-protective drugs but also anti-aging drugs.

Abstract 18666: SR-BI and Tyrosine Kinase Inhibitors: A Novel Pathway for Atherosclerotic Cardiovascular Disease Therapeutic Development

Introduction: Cardiovascular disease (CVD) is the leading cause of death in the U.S., where coronary artery disease (CAD) accounts for 42.1% of all CVD deaths. Current therapeutics focus on lowering LDL-C, as previous attempts to raise HDL-C were not successful in altering CVD outcomes. However, the therapeutic potential of HDL has not been fully explored. Variants in SCARB1, the gene that encodes HDL receptor Scavenger Receptor B1 (SR-BI), are associated with dyslipidemia and atherosclerotic CVD (ASCVD), and we first to identified Mendelian inheritance of SCARB1 variants that cause severe early-onset CAD and dyslipidemia. Limited clinical evidence suggests imatinib reduces total cholesterol in chronic myeloid leukemia (CML). Additionally, TKI treatment in atherosclerotic mice reduced total cholesterol (imatinib) and atherosclerotic lesions (dasatinib). Yet, no data is available on the effects of TKIs on HDL and RCT. Hypothesis: Our findings suggest that HDL function (vs. HDL-C concentration) in reverse cholesterol transport (RCT) may be a promising target for cholesterol-based therapy and studying TKIs in RCT may provide a novel mechanism for therapeutic development. Methods: We performed a high throughput drug screen with 788 FDA-approved compounds, using HepG2 cells to measure endogenous HDL binding. We identified four compounds that significantly increased HDL binding, of which, imatinib was the only one to increase SR-BI. Wildtype C57Bl/6 mice on a high fat, high cholesterol diet, underwent imatinib treatment for 4 weeks and measured biweekly serum lipids. Results: We have found that imatinib and dasatinib significantly enhance HDL binding, where imatinib increased SR-BI, ABCA1, and ABCG1 in vitro. Furthermore, in vivo imatinib treatment decreased plasma total cholesterol, HDL-C and triglyceride levels, and elevated hepatic SR-BI. Knockdown of SR-BI in HepG2 cells ameliorated the imatinib-induced HDL binding increase suggesting an SR-BI-specific mechanism. Mass spectrometry revealed a novel SR-BI methionine oxidation site with imatinib treatment. Conclusions: Our data supports the exploration of TKI-mediated SR-B1 regulation, HDL metabolism, and RCT mechanism to identify new therapeutic targets for dyslipidemia and ASCVD.

Abstract 14285: Sodium Glucose Co-Transporter 2 Inhibition Eliminates Senescent Cells Through Downregulation of PD-L1 and Improves Pathological Aging

Introduction: Sodium-glucose co-transporter 2 (SGLT2) inhibitors have been developed as a new class of anti-diabetic drug, and shown cardio- and renal-protective effects in patients with diabetes, heart failure, and chronic kidney diseases. SGLT2 inhibitor also has effect to expand lifespan in aged mice, suggesting anti-aging effect. But the detailed machinery of these protective effects is still to be clarified. Here we demonstrate that canagliflozin has a effect to eliminate senescent cells from pathological tissues, so-called "senolysis" in murine aging or age-related disease models. Methods: Canagliflozin mixed in food at 0.03% (w/w) was administered for up to 4 weeks to high-fat-fed diabetic mice, for 2 weeks to ApoE-knockout atherosclerotic mice model, for 20 weeks for chronological aged mice, and for lifetime to Zmpste-knockout progeroid mice. Burden of senescent cell accumilation as well as pathological or aged phenotype were measured in each mice model. Results: Canagliflozin reduced senescent cells in visceral adipose tissue within 1 week. Also, we found that senolytic effect of canagliflozin was provided through enhancing endogenous senolytic function by immune cells such as CD8+T cells. This machinery was mediated by enhanced AMPK signaling through incrase of endogenous AICAR production, which leads to the downregulation of PD-L1 expression on senescent cells. Oppositely, inhibition of AMPK signaling with Compound C administration (10mg/kg ip daily) resulted in abolishing senolytic effect of canagliflozin. Furthermore, canagliflozin reduced their senescent cell burden and alleviated atherosclerosis in ApoE-knockout mice, extended lifespan in progeroid mice, and altered physical function in aged mice. Conclusions: Our results showed new aspect of protective efficacy of SGLT2 inhibitors and would be promising evidence that SGLT2 inhibitors can be used as not only cardio- or renal-protective drugs but also anti-aging drugs.

Abstract 17202: Cross-Species Evaluation of Cellular TNF Receptor-Associated Factor (TRAF) Expression, Regulation and Function in Murine and Human Atherosclerosis

Background: TNF receptor-associated factors (TRAFs) potently modulate immune cell function and activity in a variety of inflammatory diseases. However, due to the limited availability of viable animal models, the roles of TRAF3, TRAF4 and TRAF7 in atherosclerosis remain unknown to date. Here, we present a stepwise multiparametric approach to delineate cellular expression, regulation and implications on cardiovascular outcomes of TRAFs in murine and human atherosclerosis. Methods: To induce atherosclerotic lesion formation, LDLR-/- mice were fed a high cholesterol diet for 20 weeks and expression of TRAF1-7 was determined in whole tissue lysates of the murine aorta as well as on sorted leukocytes. We then performed scRNAseq and bioinformatical integration of leukocytes from human and mouse atherosclerotic plaques to analyze and compare single cell TRAF expression in both species. Finally, potential significance of cellular TRAF3, 4 and 7 expression in human atherosclerosis was assessed in three independent clinical study cohorts. Results: Aortic TRAF3 and TRAF4 expression was 2-fold higher in atherosclerotic mice compared to controls, whereas TRAF7 expression was significantly lower. Between species, humans exhibited higher cellular TRAF3 expression than mice. Further, TRAF7 blood expression was lower in patients with an unstable CAD angiogram than in patients with stable CAD while expression of TRAF3 in human atherosclerotic plaques was significantly higher in unstable compared to stable lesions. Finally, TRAF3 expression on circulating CD16+ monocytes determined by scRNAseq of PBMCs was substantially higher in atherosclerotic patients that died from a cardiovascular cause during 20 years of follow-up compared to survivors from the same study collective, indicating that cellular TRAF expression may predict cardiovascular long-term outcomes. Conclusion: Here, we present the first cross-species assessment of cellular TRAF3, 4 and 7 expression in murine and human atherosclerosis by utilizing a multiparametric approach to circumvent the lack of viable knockout models. Finally, we provide first evidence that cellular TRAF expression assessed by scRNAseq may add to the clinicians armamentarium for MACE prediction in the future.

QiShenYiQi pill inhibits atherosclerosis by promoting TTC39B-LXR mediated reverse cholesterol transport in liver

BackgroundTetranucleotide repeat domain protein 39B (TTC39B) was found to combine with ubiquitin ligase E3, and promote the ubiquitination modification of liver X receptor (LXR), which led to the inhibition of reverse cholesterol transport and development of atherosclerosis. QiShenYiQi pill (QSYQ) is a modern Chinese patent drug for treating ischemic cardiovascular diseases, the underlying mechanism is found to promote the expression of LXR-α/ ATP-binding cassette transporter G5 (ABCG5) in the liver of atherosclerotic mice. PurposeThe aim of this study is to investigate the effect of QSYQ on TTC39B-LXR mediated reverse cholesterol transport in atherosclerotic mice. Study design and methodsMale apolipoprotein E gene knockout mice (7 weeks old) were fed with high-fat diet and treated with low dose of QSYQ (QSYQ-l, 0.3 g/kg·d), high dose of QSYQ (QSYQ-H, 1.2 g/kg·d) and LXR-α agonist (LXR-A, GW3965 10 mg/kg·d) for 8 weeks. C57BL/6 J mice were fed with normal diet and used as negative control. Oil red O staining, HE staining, ELISA, RNA sequencing, western blot, immunohistochemistry, RT-PCR, cell culture and RNA interference were performed to analyze the effect of QSYQ on atherosclerosis. ResultsHE staining showed that QSYQ reduced the atherosclerotic lesion significantly when compared to the control group. ELISA measurement showed that QSYQ decreased serum VLDL and increased serum ApoA1. Oil Red O staining showed that QSYQ reduced the lipid content of liver and protect liver function. Comparative transcriptome RNA-sequence of liver showed that DEGs after QSYQ treatment enriched in high-density lipoprotein particle, ubiquitin ligase complex, bile secretion, etc. Immunohistochemical staining and western blot proved that QSYQ increased the protein expression of hepatic SR-B1, LXR-α, LXR-β, CYP7A1 and ABCG5. Targeted inhibiting Ttc39b gene in vitro further established that QSYQ inhibited the gene expression of Ttc39b, increased the protein expression of SR-B1, LXR-α/β, CYP7A1 and ABCG5 in rat hepatocyte. ConclusionOur results demonstrated the new anti-atherosclerotic mechanism of QSYQ by targeting TTC39B-LXR mediated reverse cholesterol transport in liver. QSYQ not only promoted reverse cholesterol transport, but also improved fatty liver and protected liver function.

Single-cell transcriptomics reveals subtype-specific molecular profiles in Nrf2-deficient macrophages from murine atherosclerotic aortas.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcriptional regulator of antioxidant and anti-inflammatory response in all cell types. It also activates the transcription of genes important for macrophage function. Nrf2 activity declines with age and has been closely linked to atherosclerosis, but its specific role in this vascular pathology is not clear. Atherosclerotic plaques contain several macrophage subsets with distinct, yet not completely understood, functions in the lesion development. The aim of this study was to analyze the transcriptome of diverse Nrf2-deficient macrophage subpopulations from murine atherosclerotic aortas. Mice with transcriptionally inactive Nrf2 in Cdh5-expressing cells (Nrf2 Cdh5tKO) were used in the experiments. These mice lack transcriptional Nrf2 activity in endothelial cells, but also in a proportion of leukocytes. We confirmed that the bone marrow-derived and tissue-resident macrophages isolated from Nrf2 Cdh5tKO mice exhibit a significant decline in Nrf2 activity. Atherosclerosis was induced in Nrf2 Cdh5tKO and appropriate control mice via adeno-associated viral vector (AAV)-mediated overexpression of murine proprotein convertase subtilisin/kexin type 9 (Pcsk9) in the liver and high-fat diet feeding. After 21 weeks, live aortic cells were sorted on FACS and single-cell RNA sequencing (scRNA-seq) was performed. Unsupervised clustering singled out 13 distinct aortic cell types. Among macrophages, 9 subclusters were identified. Differential gene expression analysis revealed cell subtype-specific expression patterns. A subset of inflammatory macrophages from atherosclerotic Nrf2 Cdh5tKO mice demonstrated downregulation of DNA replication genes (e.g. Mcm7, Lig1, Pola1) concomitant with upregulation of DNA damage sensor Atr gene. Atherosclerotic Nrf2 Cdh5tKO Lyve1+ resident macrophages showed strong upregulation of IFN-stimulated genes, as well as changes in the expression of death pathways-associated genes (Slc40a1, Bcl2a1). Furthermore, we observed subtype-specific expression of core ferroptosis genes (e.g. Cp, Hells, Slc40a1) in inflammatory versus tissue resident macrophages. This observation suggested a link between ferroptosis and inflammatory microenvironment appearing at a very early stage of atherogenesis. Our findings indicate that Nrf2 deficiency in aortic macrophages leads to subtype-specific transcriptomic changes associated with inflammation, iron homeostasis, cell injury or death pathways. This may help understanding the role of aging-associated decline of Nrf2 activity and the function of specific macrophage subtypes in atherosclerotic lesion development.

Metabolite acetyl-L-carnitine participates in Bifidobacterium animalis F1-7 to ameliorate atherosclerotic inflammation by downregulating theTLR4/NF-κB pathway

This study aimed to explore the effect of Bifidobacterium animalis F1-7 on the improvement of atherosclerotic inflammation. Arteriosclerosis model ApoE–/– mice were orally administered with B. animalis F1-7 for 12 weeks. The probiotic intervention reduced the plaque areas in aorta and the accumulation of macrophages, and downregulated the expression of toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) pathway to reduce the levels of inflammatory factors. The widely-targeted metabolomics analysis showed that acetyl-L-carnitine (ALC) in the intestine of atherosclerotic mice was significantly increased after B. animalis F1-7 intervention. Correlation analysis proved that ALC was associated with atherosclerotic inflammatory response. By using oxidized low density lipoprotein induced macrophage foam cells, we further verified that ALC could reduce lipid accumulation and inflammatory response in foam cells by downregulating the TLR4/NF-κB pathway. Finally, our results revealed that B. animalis F1-7 upregulated the metabolite ALC to downregulate the inflammatory responses, leading to the reduction of plaque accumulation of atherosclerosis.

Vascular Ultrasound for In Vivo Assessment of Arterial Pathologies in a Murine Model of Atherosclerosis and Aortic Aneurysm.

Vascular diseases like atherosclerosis and abdominal aortic aneurysm (AAA) are common pathologies in the western world, promoting various potentially fatal conditions. Here, we evaluate high-resolution (HR) ultrasound in mouse models of atherosclerosis and AAA as a useful tool for noninvasive monitoring of early vascular changes in vivo. We used Apolipoprotein E-deficient (ApoE-/-) mice as an atherosclerosis model and induced AAA development by the implementation of Angiotensin II-releasing osmotic minipumps. HR ultrasound of the carotid artery or the abdominal aorta was performed to monitor vascular remodeling in vivo. Images were analyzed by speckle tracking algorithms and correlated to histological analyses and subsequent automated collagen quantification. Consistent changes were observed via ultrasound in both models: Global radial strain (GRS) was notably reduced in the AAA model (23.8 ± 2.8% vs. 12.5 ± 2.5%, p = 0.01) and in the atherosclerotic mice (20.6 ± 1.3% vs. 15.8 ± 0.9%, p = 0.02). In mice with AAA, vessel distensibility was significantly reduced, whereas intima-media thickness was increased in atherosclerotic mice. The area and collagen content of the tunica media were increased in diseased arteries of both models as measured by automated image analysis of Picrosirius Red-stained aortic sections. Correlation analysis revealed a strong correlation of multiple parameters, predicting early vascular damage in HR ultrasound and histological examinations. In conclusion, our findings underscore the potential of HR ultrasound in effectively tracing early alterations in arterial wall properties in murine models of atherosclerosis and AAA.

Oxidative stress biomarker triggered multiplexed tool for auxiliary diagnosis of atherosclerosis.

Oxidative stress is integral in the development of atherosclerosis, but knowledge of how oxidative stress affects atherosclerosis remains insufficient. Here, we design a multiplexed diagnostic tool that includes two functions (photoacoustic imaging and urinalysis), for assessing intraplaque and urinary malondialdehyde (MDA), a well-recognized end-product of oxidative stress. Molecular design is conducted to develop the first near-infrared MDA-responsive molecule (MRM). Acid-unlocked ratiometric photoacoustic nanoprobe is designed to report intraplaque MDA, enabling it to reflect plaque burden. Furthermore, MRM is tailored for urinary MDA detection with excellent specificity in a blind study. Moreover, we found a significant difference in urinary MDA between healthy adults and atherosclerotic patients (more than 600 participants). Combining these two functions, such a multiplexed diagnostic tool can dynamically report intraplaque and systemic oxidative stress levels during atherosclerosis progression, pneumonia infection, and drug treatment in atherosclerotic mice, which is promising for the auxiliary diagnosis of atherosclerosis.

Immune-Related Genes in the Pathogenesis of Atherosclerosis: Based on Sex Differences.

Atherosclerosis is still a global public problem with increasing incidence rate and mortality. It has been found that gender factors play an important role in the progression of atherosclerosis. However, few people explore gender related atherosclerosis at the level of genes and immune cells. The purpose of this study was to determine genetic and immune cell differences between male and female samples. This study aims to identify differential genes between male and female samples in the GSE43292 dataset. The focus will be on identifying immune-related genes (IRGs) among these differentially expressed genes. Subsequently, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis will be employed to explore the enrichment of IRGs in biological processes, molecular functions, cellular components, and pathways. Furthermore, a protein-protein interaction (PPI) network for the IRGs will be constructed using Cytoscape software. To estimate the degree of immune cell infiltration, single-sample gene set enrichment analysis (ssGSEA) will be conducted. Moreover, the identified IRGs will be validated using GSE28829 dataset. Finally, we validated in atherosclerotic mice. Seven IRGs (CCL13, IL1RN, FPR2, S100A8, CCL19, CXCL1, CXCL8) were identified as being overexpressed in male atherosclerosis. GO and KEGG analysis revealed that these IRGs are primarily enriched in inflammatory response pathways, cytokine signaling pathways, and cytokine- cytokine receptor interactions. Notably, when compared to females, there was a significant infiltration of immune cells in male specimens. Importantly, all seven IRGs demonstrated high diagnostic value in GSE28829 dataset. The use of animal samples supports our results. This study demonstrates the effectiveness of seven IRGs and reveal sex differences in atherosclerosis. Notably, there is a significant presence of immune cells within the atherosclerotic plaque of men compared to women. These findings have potential implications for the development of personalized treatment approaches targeting gender-related atherosclerosis.

Oxidized Low-Density Lipoprotein Accumulation Suppresses Glycolysis and Attenuates the Macrophage Inflammatory Response by Diverting Transcription from the HIF-1α to the Nrf2 Pathway.

Lipid accumulation in macrophages (Mφs) is a hallmark of atherosclerosis, yet how lipid accumulation affects inflammatory responses through rewiring of Mφ metabolism is poorly understood. We modeled lipid accumulation in cultured wild-type mouse thioglycolate-elicited peritoneal Mφs and bone marrow-derived Mφs with conditional (Lyz2-Cre) or complete genetic deficiency of Vhl, Hif1a, Nos2, and Nfe2l2. Transfection studies employed RAW264.7 cells. Mφs were cultured for 24 h with oxidized low-density lipoprotein (oxLDL) or cholesterol and then were stimulated with LPS. Transcriptomics revealed that oxLDL accumulation in Mφs downregulated inflammatory, hypoxia, and cholesterol metabolism pathways, whereas the antioxidant pathway, fatty acid oxidation, and ABC family proteins were upregulated. Metabolomics and extracellular metabolic flux assays showed that oxLDL accumulation suppressed LPS-induced glycolysis. Intracellular lipid accumulation in Mφs impaired LPS-induced inflammation by reducing both hypoxia-inducible factor 1-α (HIF-1α) stability and transactivation capacity; thus, the phenotype was not rescued in Vhl-/- Mφs. Intracellular lipid accumulation in Mφs also enhanced LPS-induced NF erythroid 2-related factor 2 (Nrf2)-mediated antioxidative defense that destabilizes HIF-1α, and Nrf2-deficient Mφs resisted the inhibitory effects of lipid accumulation on glycolysis and inflammatory gene expression. Furthermore, oxLDL shifted NADPH consumption from HIF-1α- to Nrf2-regulated apoenzymes. Thus, we postulate that repurposing NADPH consumption from HIF-1α to Nrf2 transcriptional pathways is critical in modulating inflammatory responses in Mφs with accumulated intracellular lipid. The relevance of our invitro models was established by comparative transcriptomic analyses, which revealed that Mφs cultured with oxLDL and stimulated with LPS shared similar inflammatory and metabolic profiles with foamy Mφs derived from the atherosclerotic mouse and human aorta.

Dandelion Polysaccharides Ameliorate High-Fat-Diet-Induced Atherosclerosis in Mice through Antioxidant and Anti-Inflammatory Capabilities.

Dandelion (Taraxacum officinale), a member of the Asteraceae (Compositae) family, is well known as the traditional medical plant. Dandelion polysaccharides, a natural active ingredient extracted from the dandelion, possess immune regulation, anti-inflammatory, antioxidant, and anti-aggregation properties. These properties suggest that dandelion polysaccharides might alleviate atherosclerosis. Using an ApoE-/- atherosclerotic mice model fed a high-fat diet, we investigated the impact and potential mechanism of dandelion polysaccharides on atherosclerosis. We observed that dandelion polysaccharides significantly reduced the levels of triglyceride, total cholesterol, and low-density lipoprotein-cholesterol in serum, while elevated the high-density lipoprotein-cholesterol level. Concomitantly, dandelion polysaccharides reduced the area of atherosclerotic lesions and necrotic core of the aortic sinus, and increased the collagen content. Mechanistic studies showed that dandelion polysaccharides were effective in reducing serum malondialdehyde levels while elevating the enzymatic activities of superoxide dismutase and glutathione peroxidase. Furthermore, dandelion polysaccharides reduced the expression of chemotactic factor Mcp-1 and pro-inflammatory cytokines (Tnf-α, Il-1β, and Il-6) in atherosclerotic lesions. Overall, these results indicated that dandelion polysaccharides may take an important part in the attenuation of atherosclerosis via its antioxidant and anti-inflammatory properties.

Sirt6 enhances macrophage lipophagy and improves lipid metabolism disorder by regulating the Wnt1/β-catenin pathway in atherosclerosis

Lipid metabolism disorders are considerably involved in the pathology of atherosclerosis; nevertheless, the fundamental mechanism is still largely unclear. This research sought to examine the function of lipophagy in lipid metabolism disorder-induced atherosclerosis and its fundamental mechanisms. Previously, Sirt6 has been reported to stimulate plaque stability by promoting macrophage autophagy. However, its role in macrophage lipophagy and its relationship with Wnt1 remains to be established. In this study, ApoE−/−: Sirt6−/− and ApoE−/−: Sirt6Tg mice were used and lipid droplets were analysed via transmission electron microscopy and Bodipy 493/503 staining in vitro. Atherosclerotic plaques in ApoE−/−: Sirt6−/− mice showed greater necrotic cores and lower stability score. Reconstitution of Sirt6 in atherosclerotic mice improved lipid metabolism disorder and prevented the progression of atherosclerosis. Furthermore, macrophages with Ac-LDL intervention showed more lipid droplets and increased expression of adipophilin and PLIN2. Reconstitution of Sirt6 recruited using SNF2H suppressed Wnt1 expression and improved lipid metabolism disorder by promoting lipophagy. In addition, downregulation of Sirt6 expression in Ac-LDL-treated macrophages inhibited lipid droplet degradation and stimulated foam cell formation. Innovative discoveries in the research revealed that atherosclerosis is caused by lipid metabolism disorders due to downregulated Sirt6 expression. Thus, modulating Sirt6’s function in lipid metabolism might be a useful therapeutic approach for treating atherosclerosis.

Adrenomedullin, transcriptionally regulated by vitamin D receptors, alleviates atherosclerosis in mice through suppressing AMPK-mediated endothelial ferroptosis.

Vitamin D receptors (VDR) play important roles in cardiovascular, immune, metabolic and other functions. Activation of VDR may help improve endothelial dysfunction, atherosclerosis, vascular calcification, and cardiac hypertrophy. However, the specific target genes and mechanisms of VDR in improving Human Umbilical Vein Endothelial Cell (HUVEC) functions remain unclear. This study aims to investigate the function and mechanism of VDR in HUVECs. Endothelial dysfunction cell model was constructed by oxidized low-density lipoprotein (ox-LDL). An animal model of atherosclerosis was established in male homozygous Apoe-/- mice (6 weeks) on a high fat diet for 6 weeks. The relationship between VDR and adrenomedullin (ADM) was studied by bioinformatics analysis, ChIP, and luciferase reporter gene analysis. Endothelial cell function was evaluated by Transwell migration and Tube Formation tests. Ferroptosis was detected by measuring intracellular iron content, levels of oxidative stress markers, and ferroptosis related proteins. Overexpression of VDR in HUVECs inhibits ox-LDL-induced endothelial dysfunction and ferroptosis. VDR binds to the ADM promoter sequence and regulates the transcription of ADM. Inhibition of ADM promotes ox-LDL-induced endothelial dysfunction and ferroptosis. ADM regulates ox-LDL-induced endothelial dysfunction and ferroptosis through the AMPK signaling pathway. Overexpression of VDR in Apoe-/- mice inhibited lipid deposition and plaque area in atherosclerotic mice. VDR inhibits ox-LDL-induced endothelial dysfunction and ferroptosis by regulating ADM transcription and acting on AMPK signaling pathway. Overexpression of VDR in Apoe-/- mice reduced lipid deposition and plaque area in the thoracic aorta of atherosclerotic mice.

Rational Design of a Double-Locked Photoacoustic Probe for Precise In Vivo Imaging of Cathepsin B in Atherosclerotic Plaques.

Atherosclerotic plaque rupture is a significant cause of acute cardiovascular events such as heart attack and stroke, triggered by the decomposition of fiber caps induced by cysteine cathepsin. However, the accurate measurement of cathepsin B (CTB) activity in plaques is challenging due to the low specificity and insufficient penetration depth of available atherosclerosis-associated cathepsin fluorescent probes, hampering reliable assessment of plaque vulnerability. To address these limitations, we added both lipophilic alkyl chain and hydrophilic CTB substrate to the hemicyanine scaffold to develop a lipid-unlocked CTB responsive probe (L-CRP) that uses lipids and CTB as two keys to unlock photoacoustic (PA) signals for measuring CTB activity in lipophilic environments. Such properties allow L-CRP for the reliable imaging of specific CTB activities in foam cells and atherosclerotic plaques while keeping in silence toward CTB in lipid-deficient environments, such as M1-type macrophages and LPS-induced inflammatory lesions. Moreover, the activatable PA signals of L-CRP exhibit a deeper tissue penetration ability (>1.0 cm) than current CTB probes based on near-infrared fluorescent imaging (∼0.3 cm), suitable for atherosclerosis imaging in living mice. In atherosclerotic mice, L-CRP dynamically reports intraplaque CTB levels, which is well-correlated with the plaque vulnerability characteristics such as fiber cap thickness, macrophage recruitment, and necrotic core size, thus enabling risk stratification of atherosclerotic mice complicated with pneumonia. Moreover, L-CRP successfully identifies atherosclerotic plaques in excised human artery tissues, promising for auxiliary diagnosis of plaque vulnerability in clinical application.