Plaque Area Research Articles

Bone marrow mesenchymal stem cell-derived extracellular vesicles alleviate diabetes-exacerbated atherosclerosis via AMPK/mTOR pathway-mediated autophagy-related macrophage polarization

IntroductionBone marrow-derived mesenchymal stem cell-derived extracellular vesicles (BMSC-EVs) are widely used for therapeutic purposes in preclinical studies. However, their utility in treating diabetes-associated atherosclerosis remains largely unexplored. Here, we aimed to characterize BMSC-EV-mediated regulation of autophagy and macrophage polarization.MethodsEVs were isolated from the supernatant of cultured BMSCs and characterized with transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting. A diabetes-related atherosclerotic ApoE−/− mouse model was established through feeding with a high-fat diet (HFD) and streptozotocin (STZ). Histopathological analyses were carried out using Oil Red O, H&E, and Masson staining of the aorta. TEM and immunohistochemistry (IHC) were applied to evaluate autophagy, and immunofluorescence (IF) was used to identify macrophage polarization. RAW264.7 macrophages were induced with oxidized low-density lipoprotein (ox-LDL) and high glucose (HG), co-cultured with BMSC-EVs, and analyzed for macrophage proliferation, migration, and foam cell formation. RAW264.7 cells were transduced with autophagy marker mRFP-GFP-LC3 lentivirus and analyzed with IF and western blotting.ResultsDiabetic mice (DA group) had larger aortic plaque areas and lower collagen content than the HFD mice. BMSC-EV treatment significantly reduced blood glucose, LDL levels, and aortic plaque areas while increasing collagen content. BMSC-EV-treated aortas contained a higher number of autophagosomes/autolysosomes, with increased expression of LC3BII correlating with decreased P62 levels and a lower proportion of M1 macrophages. In vitro, BMSC-EVs inhibited proliferation, migration, and foam cell formation in ox-LDL and HG-induced activated RAW264.7 cells. These effects were reversed by the autophagy blocker bafilomycin A1. Consistent with the in vivo findings, BMSC-EVs elevated levels of the autophagy-related protein LC3BII/I and decreased P62 in ox-LDL and HG-induced RAW264.7 cells. These cells also expressed the M1 macrophage markers CD86 and iNOS, but showed reduced expression of the M2 marker Arg-1. Further, BMSC-EVs decreased AMPKα and mTOR phosphorylation levels, which were blocked by the AMPK inhibitor compound C.ConclusionsBMSC-EVs attenuate diabetes-exacerbated atherosclerosis by inhibiting vascular macrophage proliferation, migration, and foam cell formation via AMPK/mTOR signaling-regulated autophagy and macrophage polarization. BMSC-EVs thus hold promise as therapeutic agents for atherosclerosis.Graphical Extracellular vesicles derived from bone marrow mesenchymal stem cells may mitigate diabetes-aggravated atherosclerosis by regulating AMPK/mTOR-mediated autophagy-related macrophage polarization and inhibiting macrophage proliferation, migration, foam cell formation, and cholesterol transport.

Platelet membrane-modified exosomes targeting plaques to activate autophagy in vascular smooth muscle cells for atherosclerotic therapy.

Atherosclerosis is one of the leading causes of ischemic cardiovascular disease worldwide. Recent studies indicated that vascular smooth muscle cells (VSMCs) play an indispensable role in the progression of atherosclerosis. Exosomes derived from mesenchymal stem cells (MSCs) have demonstrated promising clinical applications in the treatment of atherosclerosis. However, there are still challenges and limitations persist in targeted therapy. This study aims to develop a bionic nano-delivery system by fusing platelet membranes with exosomes (MSC-ExoP) and explore the anti-atherosclerosis effect of MSC-ExoP by improving the targeting efficiency and participating in regulating the pathophysiological processes associated with VSMCs. The morphology, particle size, stability, and fusion efficiency of MSC-ExoP were assessed using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), immunofluorescence staining, and Western blotting, respectively. MSC-ExoP was administered intravenously into ApoE-/- mice via the tail vein. In vivo, immunofluorescence staining was used to assess the targeting efficacy of MSC-ExoP. The ORO staining, H&E staining, Masson staining, aortic root immunofluorescence staining, and Western blot were utilized to evaluate the VSMC autophagy and anti-atherosclerosis effects of MSC-ExoP. In vitro, the autophagy activation of MSC-ExoP on VSMCs was further assessed by immunofluorescence staining and Western blotting. The effects of MSC-ExoP on VSMCs proliferation, migration, and foam cell formation were detected by EdU experiment, Transwell experiment, wound healing experiment, ORO staining, and BODIPY staining. The TEM revealed that MSC-ExoP retained a ring nanostructure, which was similar to MSC-Exo in morphology. NTA analysis indicated the MSC-ExoP exhibited a slight increase after cell membrane fusion. Besides, the stability analysis of exosomes and MSC-ExoP resulted in no significant changes in particle size. Western blot analysis confirmed that MSC-ExoP simultaneously expressed platelet-specific markers (GPVI, GPIbα, CD62P) and exosome-specific markers (CD81, TSG101, and Alix). In ApoE-/- mice, the immunofluorescence of aorta and its roots was significantly enhanced after injection of DiI-labeled MSC-ExoP, indicating enhanced targeting of MSC-Exo to atherosclerotic plaques by platelets. In vivo experiments demonstrated that MSC-ExoP could significantly suppress the progression of atherosclerosis and reduce the area of atherosclerotic plaques by reducing lipid deposition and necrotic nucleus area and increasing collagen content. In vitro experiments further revealed that the uptake of MSC-ExoP by foam cells significantly increased, and their proliferation, migration, and foam formation were inhibited by autophagy activation. This study demonstrated successful fusion of platelet membranes with exosomes derived from MSCs. MSC-ExoP could significantly improve the targeting efficiency of atherosclerosis and play an anti-atherosclerosis effect by activating VSMC autophagy.

Prophylactic and therapeutic effects of EsV3 on atherosclerotic lesions in ApoE−/− mice

BackgroundAtherosclerosis (AS) is a major contributor to vascular disorders and represents a significant risk to human health. Currently, first-line pharmacotherapies are associated with substantial side effects, and the development of atherosclerosis is closely linked to dietary factors. This study evaluated the effects of a dietary supplement, EsV3, on AS in apolipoprotein E (ApoE) −/− model mice.MethodsThe study utilized a high-fat diet-induced ApoE−/− hyperlipidemic mouse model. EsV3 was administered in prophylactic (P-EsV3) and therapeutic regimens for 16 and 12 weeks, respectively, with distinct high- and low-dose groups (0.36 and 1.8 g/kg/day). Serum lipid levels were measured and monitored for body weight and food consumption alterations in murine models. Aortic oil red O staining was conducted to assess plaque formation and calculate the plaque-to-vessel area ratio. Liver tissue changes were examined via HE staining. Moreover, serum oxidative stress markers (MDA, GSH, SOD) were measured to evaluate oxidative damage and lipid metabolism.ResultsBoth atorvastatin and P-EsV3 treatments significantly lowered TC, TG, and LDL-C levels, with P-EsV3-H enhancing HDL-C levels (P < 0.05). Prophylactic EsV3 administration was more effective than therapeutic administration in regulating TG and LDL-C levels and had comparable effects to atorvastatin on TC and HDL-C. All treatment groups exhibited reduced body weight compared to the model group, with no significant differences in food intake. Additionally, EsV3 administration significantly reduced the aortic plaque area and liver lipid droplets compared to the model group, while mitigating oxidative stress, as evidenced by decreased MDA levels and increased SOD and GSH levels, with outcomes comparable to those observed with atorvastatin.ConclusionsIn ApoE−/− hyperlipidemic mice, EsV3 improved lipid profiles and reduced aortic plaque formation. EsV3's effects, attributed partly to its antioxidant properties, were comparable to atorvastatin, suggesting its potential as a preventive and therapeutic agent for hyperlipidemia and atherosclerosis.

Regression of carotid atherosclerosis in high-risk individuals with proprotein convertase subtilisin/kexin type 9 inhibitors.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors represent a novel approach for reducing cholesterol and, accordingly, the burden of atherosclerosis. However, limited data are available regarding the possible effects of PCSK9 inhibitors on atherosclerotic plaque. To evaluate the efficacy of PCSK9 inhibitors in reducing carotid plaque progression in individuals with high-risk carotid atherosclerotic disease. We used carotid total plaque area (TPA) to assess the burden of atherosclerosis. Ultrasound imaging of the carotid was acquired before and after the initiation of PCSK9 inhibitor therapy. We selected high-risk cases with atherosclerosis with a minimum of three ultrasound examinations, 1 year before, one at the time of initiation of a PCSK9 inhibitor, and 1 year after initiating a PCSK9 inhibitor. Statistical analysis was conducted using the mixed-effects model with Restricted Maximum Likelihood (REML). We reviewed data from 131 patients with a mean follow-up of 6 (±4) years. Patients were high-risk, with the majority having diabetes or hypertension. There was a decrease in TPA, particularly during the first 3 years after initiating PCSK9 inhibitor therapy (p < 0.05). Furthermore, we observed that individuals with higher baseline serum low-density lipoprotein cholesterol (LDL-C) levels experienced a greater decline in TPA (p < 0.05). PCSK9 inhibitors are effective in achieving plaque regression in high-risk patients with atherosclerosis. This is important, as plaque regression is associated with a lower risk of stroke, myocardial infarction, or vascular death.

Astaxanthin-loaded polylactic acid-glycolic acid nanoparticles alleviates atherosclerosis by suppressing macrophage ferroptosis via the NRF2/SLC7A11/GPX4 pathway.

Astaxanthin (ASX), a fat-soluble carotenoid mainly sourced from Haematococcus pluvialis, shows promise for clinical applications in chronic inflammatory diseases. This study investigates whether ASX can mitigate atherosclerosis (AS) by modulating macrophage ferroptosis and provides astaxanthin-loaded polylactic acid-glycolic acid nanoparticles (ASX-PLGA NPs) as comparison. ApoE-/- mice were fed a high-fat diet with ASX or statin intervention. Plaque area, lipid aggregation, collagen content, and ferroptosis-related indicators were assessed. Moreover, ASX-PLGA NPs were synthesized and characterized and were used to pretreat macrophages induced with oxidized low-density lipoprotein (ox-LDL). Indicators linked to ferroptosis and oxidative stress were detected. Finally, the expression of nuclear factor erythroid -related factor 2 (NRF2) was evaluated. ASX intervention significantly delayed the progression of AS plaques, characterized by reductions in plaque area and increased collagen fibers. The observed improvements in AS were consistent with statins. ASX-PLGA NPs demonstrate good safety and stability and have better therapeutic effects than ASX alone. Indicators linked to ferroptosis and oxidative stress were significantly improved in groups containing ASX in vivo and vitro. Additionally, ASX facilitated the nuclear translocation of NRF2, which could be attenuated with ML385, a specific inhibitor of NRF2. ASX-PLGA NPs have better therapeutic effects than ASX alone. The regulation of NRF2/SLC7A11/GPX4 represents a novel mechanism by which ASX can counteract ferroptosis and impede AS progression.

Dehydrocorydaline maintains the vascular smooth muscle cell contractile phenotype by upregulating Spta1.

Vascular smooth muscle cell (VSMC) phenotypic switching plays a crucial role in the initiation and progression of atherosclerosis. Dehydrocorydaline (DHC), a major active component of the traditional Chinese herbal medicine Rhizoma Corydalis, exhibits diverse pharmacological effects. However, its impact on VSMCs remains largely unknown. This study aims to investigate the effects and underlying mechanisms of DHC in phenotypic switching of VSMCs. Our study revealed that DHC increased the mRNA and protein levels of rat VSMC contractile phenotype markers, such as calponin 1 (Cnn1), myosin heavy chain (Myh11, SM-MHC), smooth muscle 22α (Sm22α), and alpha-smooth muscle actin (Acta2, α-SMA) in a time- and dose-dependent manner. Additionally, DHC inhibited platelet-derived growth factor-BB-induced VSMC proliferation and migration. In Apoe-/- mice, DHC treatment resulted in reduced carotid plaque areas and macrophage infiltration, along with increased contractile phenotype marker expression. RNA sequencing analysis revealed a significant upregulation of spectrin alpha, erythrocytic 1 (Spta1) in DHC-treated rat VSMCs. Strikingly, Spta1 knockdown effectively negated the increase in contractile phenotype marker expression in VSMCs that was initially prompted by DHC. Therefore, DHC preserves the VSMC contractile phenotype through Spta1, thereby attenuating carotid artery atherosclerotic plaques in Apoe-/- mice. This study provides evidence supporting the potential use of Chinese herbal medicines, particularly those containing DHC such as Rhizoma Corydalis, in the treatment of atherosclerotic cardiovascular disease, thus expanding the clinical application of such herbal remedies.

Association of stent thrombectomy and conventional treatment with neuroprotection, complications, anxiety, and depression in acute ischemic stroke patients.

Acute ischemic stroke (AIS) is an abrupt blood flow cessation to a specific brain region within a vascular zone, causing a subsequent decline in neurological capabilities. Stent thrombectomy is a recently established technique for treating AIS. It provides the benefits of being a relatively simple and safe procedure, capable of partially enhancing a patient's condition. However, some patients may experience endothelial damage and recurrent thrombosis, with clinical outcomes that are not always satisfactory. Hence, the efficacy of this method remains unclear. To survey the association of stent thrombectomy vs standard treatment with neurological function protection, complications, and short-term prognosis in patients diagnosed with AIS. This study assigned 90 patients with AIS to the observation and control groups (n = 45 patients) from December 2020 to December 2022. Stent thrombectomy was conducted in the observation group, whereas routine treatment was provided to the control group. The study assessed the therapeutic outcomes of two groups, including a comparison of their neurological function, living ability, anxiety and depression status, plaque area, serum inflammatory factors, serum Smur100 β protein, neuron-specific enolase (NSE), homocysteine (Hcy), and vascular endothelial function. Additionally, the incidence of complications was calculated and analyzed for each group. The total effective rate of treatment was 77.78% and 95.56% in the control and observation groups, respectively. After 8 weeks of treatment, the scores on the National Institutes of Health Stroke Scale, Hamilton Anxiety Scale, and Hamilton Depression Scale decreased remarkably; the Barthel index increased remarkably, with better improvement effects of the scores in the observation group (P < 0.05); total cholesterol, triglyceride, C-reactive protein, and plaque area lessened remarkably, with fewer patients in the observation group (P < 0.05); S-100β protein, NSE, and Hcy levels lessened remarkably, with fewer patients in the observation group (P < 0.05); serum vascular endothelial growth factor and nitric oxide synthase levels increased remarkably, whereas the endothelin-1 level decreased, with better improvement effect in the observation group (P < 0.05). Complications occurred in 8.88% of patients in the observation group compared with 33.33% in the control group. Stent thrombectomy appeared to provide more remarkable neuroprotective effects in patients with AIS compared to the intravenous thrombolysis regimen. Additionally, it has effectively improved the neurological function, daily activities, and vascular endothelial function of patients, while reducing the incidence of complications and improving short-term prognosis.

Cerebrovascular risk in rheumatoid arthritis patients: insights from carotid artery atherosclerosis in the Paracelsus 10,000 study

Rheumatoid arthritis (RA) is a chronic autoimmune disease marked by systemic inflammation. While RA primarily affects the joints, its systemic effects may lead to an increased cerebro- and cardiovascular risk. Atherosclerosis of the carotid arteries is a significant risk factor for cerebrovascular events and serves as a surrogate marker for cardiovascular risk. This study explores the link between RA and carotid artery atherosclerosis with data from the Paracelsus 10,000 Study. Baseline assessments were conducted on individuals randomly selected from Salzburg and its surrounding regions. Participants diagnosed with RA based on ACR-EULAR classification criteria and who underwent carotid artery ultrasound were included. Data were gathered from a total of 9729 participants, among whom 299 were diagnosed with RA. Carotid arteries were examined using ultrasound imaging. The primary endpoint was the difference in the prevalence of plaque presence between the RA and non-RA groups. One univariate (Model I) and three multivariate analyses were conducted, with adjustments in Model II incorporating SCORE 2, while Model III accounted for metabolic syndrome, age and sex. Additionally, Model IV included further adjustments for high-sensitivity C-reactive protein (hs-CRP). Plaque presence was defined as the ultrasound detection of plaque formation larger than 0 mm2, regardless of whether it was unilateral or bilateral. Additional assessments included carotid stenosis, intima-media thickness (IMT) and total plaque area (TPA). RA patients had a higher prevalence of plaque (50%) compared to non-RA individuals (38%). The odds ratio (OR) for plaque presence in RA patients versus non-RA individuals was 1.64 (95% CI 1.30–2.06). This association persisted after adjusting for SCORE2, with an adjusted odds ratio (aOR) of 1.65 (95% CI 1.26–2.15). The association remained significant when adjusting for metabolic syndrome, age and sex (aOR = 1.32, 95% CI 1.02–1.72) and also in Model IV, which included further adjustment for hs-CRP (OR = 1.33, 95% CI 1.02–1.74). The findings underscore an increased risk of cerebrovascular disease associated with RA. This study highlights the importance of thorough cerebrovascular and cardiovascular risk assessments, along with proactive management, for RA patients to reduce this risk. Recognizing the substantial impact of RA on stroke and cerebrovascular disease is important for enhancing patient care strategies. Carotid ultrasound appears to be an effective method for atherosclerosis screening in RA patients.

Selenium-Doped Copper Formate Nanozymes with Antisenescence and Oxidative Stress Reduction for Atherosclerosis Treatment.

Atherosclerosis, resulting from chronic inflammation of the arterial wall, serves as the underlying cause of multiple major cardiovascular diseases. Current anti-inflammatory therapies often exhibit limited and unsatisfactory efficacy. To address this, we have designed a selenium-doped copper formate (Cuf-Se) nanozyme for the treatment of atherosclerosis, which possesses superoxide dismutase (SOD) and glutathione peroxidase (GPx)-like activities. The Cuf-Se can efficiently scavenge reactive oxygen species (ROS), inhibit cellular senescence, and prevent the formation of foam cells. It acts on macrophages to reduce cellular ROS levels and lipid oxidation, thereby significantly inhibiting inflammation-related processes. Notably, while inhibiting foam cell formation, Cuf-Se also alleviates endothelial cells senescence. After intravenous administration, Cuf-Se effectively inhibits the formation of atherosclerosis in mice through the synergistic effects of antisenescence and antioxidant properties, reducing plaque area by approximately 5-fold. This study provides an effective strategy for the treatment of atherosclerosis.

ADAMTS4-Specific MR Peptide Probe for the Assessment of Atherosclerotic Plaque Burden in a Mouse Model

Introduction Atherosclerosis is the underlying cause of multiple cardiovascular pathologies. The present-day clinical imaging modalities do not offer sufficient information on plaque composition or rupture risk. A disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4) is a strongly upregulated proteoglycan-cleaving enzyme that is specific to cardiovascular diseases, inter alia, atherosclerosis. Materials and Methods Male apolipoprotein E-deficient mice received a high-fat diet for 2 (n = 11) or 4 months (n = 11). Additionally, a group (n = 11) receiving pravastatin by drinking water for 4 months alongside the high-fat diet was examined. The control group (n = 10) consisted of C57BL/6J mice on standard chow. Molecular magnetic resonance imaging was performed prior to and after administration of the gadolinium (Gd)-based ADAMTS4-specific probe, followed by ex vivo analyses of the aortic arch, brachiocephalic arteries, and carotid arteries. A P value &lt;0.05 was considered to indicate a statistically significant difference. Results With advancing atherosclerosis, a significant increase in the contrast-to-noise ratio was measured after intravenous application of the probe (mean precontrast = 2.25; mean postcontrast = 11.47, P &lt; 0.001 in the 4-month group). The pravastatin group presented decreased ADAMTS4 expression. A strong correlation between ADAMTS4 content measured via immunofluorescence staining and an increase in the contrast-to-noise ratio was detected (R 2 = 0.69). Microdissection analysis revealed that ADAMTS4 gene expression in the plaque area was significantly greater than that in the arterial wall of a control mouse (P &lt; 0.001). Laser ablation–inductively coupled plasma–mass spectrometry confirmed strong colocalization of areas positive for ADAMTS4 and Gd. Conclusions Magnetic resonance imaging using an ADAMTS4-specific agent is a promising method for characterizing atherosclerotic plaques and could improve plaque assessment in the diagnosis and treatment of atherosclerosis.

Artificial intelligence-based cardiovascular/stroke risk stratification in women affected by autoimmune disorders: a narrative survey.

Women are disproportionately affected by chronic autoimmune diseases (AD) like systemic lupus erythematosus (SLE), scleroderma, rheumatoid arthritis (RA), and Sjögren's syndrome. Traditional evaluations often underestimate the associated cardiovascular disease (CVD) and stroke risk in women having AD. Vitamin D deficiency increases susceptibility to these conditions. CVD risk prediction in AD can benefit from surrogate biomarker for coronary artery disease (CAD), such as carotid ultrasound. Due to non-linearity in the CVD risk stratification, we use artificial intelligence-based system using AD biomarkers and carotid ultrasound. Investigate the relationship between AD and CVD/stroke markers including autoantibody-influenced plaque load. Second, to study the surrogate biomarkers for the CAD and gather radiomics-based features such as carotid intima-media thickness (cIMT), and plaque area (PA). Third and final, explore the automated CVD/stroke risk identification using advanced machine learning (ML) and deep learning (DL) paradigms. Analysed biomarker data from women with AD, including carotid ultrasonography imaging, clinical parameters, autoantibody profiles, and vitamin D levels. Proposed artificial intelligence (AI) models to predict CVD/stroke risk accurately in AD for women. There is a strong association between AD duration and elevated cIMT/PA, with increased CVD risk linked to higher rheumatoid factor (RF) and anti-citrullinated peptide antibodies (ACPAs) levels. AI models outperformed conventional methods by integrating imaging data and disorder-specific factors. Interdisciplinary collaboration is crucial for managing CVD/stroke in women with chronic autoimmune diseases. AI-based assisted risk stratification methods may improve treatment decision-making and cardiovascular outcomes.

Association between carotid ultrasound features and the detection of functionally significant coronary artery stenosis: a prospective study based on quantitative flow ratio.

Carotid ultrasound is a helpful approach for classifying cardiovascular risk. Quantitative flow ratio (QFR) is used to evaluate functionally significant coronary artery stenosis (CAS). The aim of this prospective study was to investigate the correlation between carotid artery features from carotid ultrasound and functionally significant CAS. Furthermore, this study aimed to evaluate the diagnostic performance of carotid ultrasound in diagnosing functional CAS. Carotid ultrasound was performed in 82 patients with suspicious coronary artery disease, measuring carotid intima-media thickness (IMT), internal artery diameter (IAD), and carotid plaques. QFR values were measured in all patients, and functionally significant CAS was defined as QFR ≤0.8. Forty patients (48.8%) had non-functionally significant CAS with QFR >0.8, while 42 patients (51.2%) had functionally significant CAS with QFR ≤0.8. Logistic regression analyses were performed to evaluate the association among functionally significant CAS, carotid ultrasound features and clinical parameters. A receiver operating characteristic (ROC) curve was developed to assess the capability of carotid ultrasound to diagnose functionally significant CAS. Patients with functionally significant CAS (QFR ≤0.8) had greater IMT, carotid bifurcation IAD and internal carotid artery-IAD, compared to patients with non-functionally significant CAS, with P values of <0.001, 0.015, and 0.011, respectively. The presence of carotid plaque was significantly higher in the functionally significant CAS group (95.2%) compared to the non-functionally significant CAS group (60%), with a P value of <0.001. In multivariable logistic regression analysis, maximum plaque height (MPH) (OR: 1.777, P=0.018) was associated with functionally significant CAS in patients with coronary artery disease. ROC curves showed plaque area to be superior to IMT, MPH and plaque length in identifying functionally significant CAS. The cutoff value of the plaque area was 9.07, and the sensitivity and specificity were 85.7% and 70.0%. Carotid artery properties measured by carotid ultrasound were associated with functionally significant CAS. Plaque area is the most clinically useful parameter for detecting functionally significant CAS compared to IMT, MPH, and plaque length.

Disturbed shear stress promotes atherosclerosis through TRIM21-regulated MAPK6 degradation and consequent endothelial inflammation.

Coronary artery plaques often develop in regions subjected to disturbed shear stress (DSS), yet the mechanisms underlying this phenomenon remain poorly understood. Our study aimed to elucidate the unknown role of MAPK6 in shear stress and plaque formation. In vitro and in vivo experiments, RNA-seq, CO-IP and proteomic analysis, combined with single-cell RNA-seq datasets were used to reveal the upstream and downstream mechanisms involved. AAV-MAPK6, ApoE-/-MAPK6flox/floxTEKCre mice and the CXCL12 neutraligand were used to confirm the beneficial effects of MAPK6 against atherosclerosis. Our study revealed a substantial decrease in MAPK6 protein levels in endothelial cells in response to DSS, both in vivo and in vitro, which was contingent on the binding of the ubiquitin ligase TRIM21 to MAPK6. Endothelium-specific MAPK6 overexpression exerts antiatherosclerotic effects in ApoE-/- mice, elucidating the unexplored role of MAPK6 in atherosclerosis. Comprehensive RNA-seq, integrated single-cell mapping and further experiments unveiled the involvement of MAPK6 in inflammation through the EGR1/CXCL12 axis. ApoE-/-MAPK6flox/floxTEKCre mice finally confirmed that conditional MAPK6 knockout resulted in endothelial inflammation and significant increases in plaque areas. Notably, these effects could be reversed through the neutralization of CXCL12. Our study illuminates the advantages of MAPK6 in decelerating plaque progression, highlighting the potential of safeguarding MAPK6 as a novel therapeutic strategy against atherosclerosis. Disturbed flow activates the ubiquitin‒proteasome degradation pathway of MAPK6 in endothelial cells, which is contingent on the binding of the ubiquitin ligase TRIM21 to MAPK6. Endothelial MAPK6 has an advantageous impact on decelerating plaque progression. MAPK6 regulates endothelial inflammation via the EGR1/CXCL12 axis.

ALKBH5 alleviates lower extremity arteriosclerosis by regulating ITGB1 demethylation and influencing macrophage polarization.

M6A methylation-regulated macrophages play an important role in the occurrence and development of arteriosclerosis. However, their role in lower extremity arteriosclerosis remains unclear. Therefore, this study aims to explore the key factors that regulate arteriosclerosis methylation in the lower extremities and the mechanism by which they affect arteriosclerosis by influencing macrophage polarization. The m6A methylation levels in peripheral blood mononuclear cells (PBMCs) of patients with lower extremity atherosclerosis was investigated using the Dot blot method. Additionally, the expression levels of RNA methyltransferases and demethylases were examined using ELISA and Western blotting. Inflammatory macrophages were established using RAW264.7cells stimulated with LPS (100ng/mL), and the expression of ALKBH5 and ITGB1 was evaluated using Western blotting. Immunofluorescence staining was conducted to assess the expression of M1 macrophage markers (F4/80+CD86) and M2 macrophage markers (F4/80+CD206) in renal tissue. ELISA was employed to measure the levels of cytokines (IL-6, IL-1β, TNF-a, IL-10, and TGF-β) and plasma lipid levels in mice. An atherosclerosis model was established in ApoE-/- mice through balloon pull surgery and high-fat feeding. Oil Red O staining and hematoxylin and eosin (HE) staining were performed to measure the area of atherosclerotic plaques and the size of the necrotic core in mouse femoral artery, respectively. Additionally, Starbase2.0 was used for downstream target gene prediction of ALKBH5. The half-life of ITGB1 was evaluated using RT-qPCR. The m6A methylation levels were significantly increased in PBMCs of patients with lower extremity atherosclerosis. Among them, the expression of the RNA demethylase ALKBH5 was the lowest in PBMCs of patients with lower extremity atherosclerosis. Further analysis revealed that ALKBH5 can alleviate the progression of lower extremity atherosclerosis and promote the polarization of M2 macrophages. ALKBH5 can reduce the stability of ITGB1 through demethylation. Mechanistically, ALKBH5 influences macrophage polarization and mitigates lower extremity atherosclerosis by affecting the demethylation of ITGB1. ALKBH5 promotes M2 macrophage polarization and alleviates lower extremity atherosclerosis by regulating the demethylation of ITGB1. A deeper understanding of this process not only helps elucidate the molecular mechanisms of atherosclerosis but also provides possibilities for the development of new therapeutic strategies.

Endothelial cell (EC)-specific CTGF/CCN2 Expression Increases EC Reprogramming and Atherosclerosis.

Arterial endothelial cells (ECs) reside in a complex biomechanical environment. ECs sense and respond to wall shear stress. Low and oscillatory wall shear stress is characteristic of disturbed flow and commonly found at arterial bifurcations and around atherosclerotic plaques. Disturbed flow is pro-inflammatory to ECs. Arteries also stiffen with aging and/or the onset of vascular disease. ECs sense and respond to stiffening in a pro-fibrotic manner. Thus, flow and stiffening disturbances elicit EC responses that promote pathologic arterial remodeling. However, the pathways elicited by ECs under pathologic stiffening and disturbed flow are not well understood. The objective of this work was to discover and test the modifiability of key pathways in ECs. To do this we used the partial carotid ligation model to impose disturbed flow onto the precociously stiffened fibulin-5 knockout (Fbln5-/-) mouse carotid arteries. Biomechanical testing demonstrated that Fbln5-/- arteries under disturbed flow approximate the stiffness ratio of diseased human arteries, and the ECs in these Fbln5-/- arteries underwent rapid reprogramming via endothelial to mesenchymal transition (EndMT). Under atherogenic conditions, disturbed flow Fbln5-/- arteries developed more vulnerable plaques than the wild type (WT) mouse arteries. Connective tissue growth factor/cellular communication network factor 2 (Ctgf/Ccn2) was upregulated in vivo in ECs with aging, with stiffening in the Fbln5-/- arteries, and increased again by disturbed flow under stiffened conditions, supporting CTGF as a key biomarker for flow and stiffening. This was validated by immunohistochemistry, which demonstrated increased CTGF deposition in areas of disturbed flow in patient carotid endarterectomy and peripheral artery disease (PAD) specimens. Finally, to test the role of CTGF in regulating and combining these processes, we created an EC-specific Ctgf knockout (Ctgfecko). We identified that carotid arteries under disturbed flow and atherogenic conditions in male Ctgfecko, but not female, mice had decreased plaque area compared to WT control mice. We then tested the Ctgf expression in the carotid endothelium exposed to disturbed or stable flow in WT and Fbln5-/- mice. Here we found that under disturbed flow male mice had greater Ctgf expression than female mice. This work demonstrates that stiffened + disturbed flow conditions drive EC reprogramming, that CTGF is increased by these conditions, and that this increase is more prominent in male carotid arteries. Future exploration of sex-based differences in these fibrotic pathways are warranted to develop targeted therapeutics to limit pathologic arterial remodeling under pathologically stiffened + disturbed flow environments.

Immunization with the NcMYR1 gene knockout strain effectively protected C57BL/6 mice and their pups against the Neospora caninum challenge

ABSTRACT Neospora caninum is an important protozoan parasite that causes abortion in cattle and nervous system dysfunction in dogs. No effective drugs and vaccines for neosporosis are available. Further elucidation of proteins related to N. caninum virulence will provide potential candidates for vaccine development against neosporosis. In the present study, N. caninum c-Myc regulatory protein (NcMYR1) gene knockout strains (ΔNcMYR1-1, ΔNcMYR1-2, and ΔNcMYR1-3) were generated using the CRISPR-Cas9 gene editing system to investigate phenotype changes and the potential of the ΔNcMYR1-1 strain as an attenuated vaccine, and this is the first time of using the N. caninum CRISPR-Cas9 gene knockout strain as an attenuated vaccine. NcMYR1 was determined to be a cytoplasmic protein in N. caninum tachyzoites. The deficiency of NcMYR1 decreased the plaque area and the rate of invasion, replication, and egression of the parasites. ΔNcMYR1-1 strain-infected C57BL/6 mice had 100% survival rate, reduced parasite burden, and alleviated pathological changes in tissues compared with those in Nc-1 strain-infected mice. Immunization with ΔNcMYR1-1 tachyzoites increased the productions of cytokines in mice, with a survival rate reaching 80%, and the parasite burdens in the liver and spleen were greatly reduced when challenged with the Nc-1 strain with a lethal dose after 40 days of ΔNcMYR1-1 tachyzoite immunization. ΔNcMYR1 immunization could decrease the abortion rate of female mice from 71.4% to 12.5% and increase the survival rate of pups from 12.5% to 83.3% against the N. caninum challenge. Above all, NcMYR1 is a virulence factor and the ΔNcMYR1-1 strain could be used as a candidate vaccine against N. caninum infection and vertical transmission.

Klotho enhances stability of chronic kidney disease atherosclerotic plaques by inhibiting GRK2/PLC-β-mediated endoplasmic reticulum stress in macrophages via modulation of the ROS/SHP1 pathway

Klotho has been importantly linked to atherosclerosis, but little is known about its specific role. This study investigates the mechanism by which Klotho enhances the stability of atherosclerotic plaques in chronic kidney disease. apoE-/- knockout mice and C57BL/6 mice underwent 5/6 nephrectomy and then klotho-NC and klotho-mimic groups were set up to be fed a high-fat chow diet and a dummy group was created to be fed a normal chow diet. qPCR detected relative mRNA expression of klotho. Oil Red O and HE staining assessed lipid proportion in the aorta. Masson staining evaluated renal failure pathology in mice. Immunohistochemistry measured MAC-2 and α-SMA expression in the aorta. ELISA quantified urea, cholesterol, calcium ions, and triglycerides in mouse plasma. Western blotting detected associated protein expression, followed by cell-based experiments for validation. Compared with the Klotho-NC group, the plaque area and aortic lipid and renal fibrosis area were reduced in the Klotho-mimic group. Klotho-mimic reduced macrophage area, plasma urea, cholesterol, calcium ions, and triglyceride levels, and decreased the expression of p-PERK, NOX2, NOX4, Caspase-3, Caspase-9, Bax, p-GRK2, p-PLCβ, p-Src, and p-IP3R. Without ox-LDL stimulation, Klotho expression increased in the Klotho-mimic group, with no significant differences in NOX2, p-SHP1, p-Src, p-PERK, p-GRK2, and p-PLCβ. With ox-LDL in high-calcium medium, Klotho and p-SHP1 increased, while NOX2, p-Src, p-PERK, p-GRK2, and p-PLCβ decreased in the Klotho-mimic group. After ox-LDL and TPI-1 treatment, Klotho increased, NOX2 decreased, and other proteins showed no significant changes. Adding shRNA-GRK2 reduced NOX2, p-Src, and p-PERK, increased p-SHP1, with no changes in p-GRK2 and p-PLCβ. Differences in NOX2, p-GRK2, p-PLCβ, and p-PERK between groups were reduced in high-calcium medium, while p-SHP1 differences increased. Klotho enhances chronic kidney disease atherosclerotic plaque stability by inhibiting GRK2/PLC-β-mediated endoplasmic reticulum stress in macrophages via the ROS/SHP1 pathway.

Validation of a Prediction Model From Quantitative Coronary Angiography to Detect Ischaemic Lesions as Evaluated by Invasive Fractional Flow Reserve

Physician visual assessment (PVA) in invasive coronary angiography (ICA) is clinically used to determine stenosis severity and guide coronary intervention. However, PVA provides limited information regarding the haemodynamic significance of stenosis. This prospective study aimed to develop a model combining visual diameter stenosis (DSPVA) and quantitative coronary angiography (QCA)-derived parameters to diagnose ischaemic lesions using invasive fractional flow reserve (FFR) with pharmacologically induced maximal hyperaemia as the gold standard. A total of 103 patients (148 lesions) who underwent ICA and FFR measurement were included in the study. Quantitative coronary angiography was used to evaluate various parameters, including anatomical parameters such as lesion length (LL), minimal lumen diameter (MLD), and minimal lumen area, along with haemodynamic parameters like LL/MLD4 and stenotic flow reserve (SFR). Plaque area, a characteristic parameter of plaque, was also assessed. Lesion-specific ischaemia was defined as invasive FFR ≤0.8. The LL/MLD4 (r= -0.66, p<0.001) and SFR (r=0.66, p<0.001) exhibited inverse and positive correlations, respectively, with invasive FFR. In the multivariable logistic regression analysis, LL/MLD4 (≥10.6 mm-3 vs <10.6 mm-3; Odds ratio [OR] 10.59, 95% confidence interval [CI] 3.94-28.50; p<0.001) and SFR (≤2.85 vs >2.85; OR 4.38, 95% CI 1.63-11.79; p=0.004) were identified as the optimal dichotomised predictors for discriminating ischaemia. The area under the curve (AUC) was 0.77 using DSPVA ≥70% as a single predictor. Adding LL/MLD4 ≥10.6 mm-3 and SFR ≤2.85 into the model significantly increased the AUC to 0.87 (p<0.001). Incorporating QCA-derived haemodynamic parameters provided significant incremental value in the model's discriminatory capability for ischaemic lesions compared with visual diameter assessment alone.

Efficacy of Cetylpyridinium Chloride Mouthwash on Denture Plaque Reduction and Microbiome Alteration in a Randomized Crossover Trial.

Purpose This study aimed to evaluate the effectiveness of cetylpyridinium chloride (CPC) mouthwash in reducing denture plaque and its impact on the microbial composition of denture plaque. Materials and methods A randomized, placebo-controlled crossover trial included 29 participants with maxillary complete dentures. Participants used either CPC or a placebo mouthwash for one week each in a crossover design. The denture plaque area was quantified using image analysis, and microbiome composition was analyzed via 16S rRNA gene sequencing. Results The use of CPC mouthwash significantly reduced the denture plaque area compared to placebo (p<0.025). Microbiome analysis revealed a significant decrease in the relative abundance of the genusActinomyces(p=0.025) and a significant increase in the genusHaemophilus(p<0.001) after CPC use. While alpha diversity showed no significant changes, beta diversity analysis indicated a significant shift in microbial composition (p=0.002). Conclusion CPC mouthwash effectively reduces denture plaque accumulation and modifies its microbial composition. These findings highlight the potential of CPC mouthwash as an effective tool in denture hygiene management, which may help lower the risk of denture-related oral and systemic infections.

Association between perforator stroke after middle cerebral artery elective stenting and arterial remodeling patterns: a high-resolution MRI-based retrospective cohort study.

Patterns of arterial remodeling may be associated with outcomes in patients with severe middle cerebral artery (MCA) stenosis after endovascular treatment (EVT). This study aims to investigate the potential correlation between arterial remodeling patterns in patients with severe MCA stenosis, and plaque characteristics and procedure-related perforator stroke (PS). Consecutive patients with MCA atherosclerotic disease who underwent EVT at the First Affiliated Hospital of Zhengzhou University from January 2018 to June 2023 were retrospectively enrolled in this study. The clinical and surgical data of the patients were evaluated. Remodeling index values >1.05, 0.95-1.05, and <0.95 indicated positive remodeling (PR), intermediate remodeling (IR), and negative remodeling (NR), respectively. Based on the remodeling index values calculated by high-resolution magnetic resonance imaging (HR-MRI), the arterial remodeling patterns were classified as NR or non-NR; non-NR was defined as both PR and IR. The primary endpoint was procedure-related PS. Plaque characteristics included the plaque area, plaque burden, contrast-enhancement ratio, and plaque distribution. Plaque features and the incidence of procedure-related PS were compared between the NR and non-NR groups. Differences in the categorical variables between the patients with NR and non-NR were assessed using the Chi-square test (χ2) or Fisher's exact test. Differences in the continuous variables between the two groups were assessed using the Student t-test or Mann-Whitney U-test. A total of 65 patients (mean age: 57.6 years) were enrolled in the study. Of the patients, 45 (69.2%) showed NR and 20 (30.8%) showed non-NR. The primary endpoint of procedure-related PS occurred in 3 patients (3/65, 4.6%). On HR-MRI, the entire cohort had a mean plaque area of 10.0 mm2, a mean plaque burden of 92.0 %, and a mean enhancement ratio of 1.3. Compared to the non-NR group, the NR group had a smaller plaque area (9.2 vs. 11.6 mm2, P=0.001). The probability of the primary end point was 15.0% in the non-NR group and 0.0% in the NR group (P=0.026). PS after MCA elective stenting may be related to non-NR based on HR-MRI. Further prospective and multicenter studies need to be conducted to confirm these findings.