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Mammalian and reptilian vascular tissues present basal release of 6-nitrodopamine, which is reduced when the tissues are pre-incubated with the NO synthase inhibitor L-N G -Nitro arginine methyl ester (L-NAME), or when the endothelium is mechanically removed. 6-Nitrodopamine induces vasorelaxation in pre-contracted vascular rings by antagonizing the dopaminergic D 2-like receptor. Here it was investigated whether male swine vessels (including carotid, left descendent coronary, renal, and femoral arteries) release 6-nitrodopamine, dopamine, noradrenaline, and adrenaline, as measured by liquid chromatography coupled to tandem mass spectrometry. The in vitro vasorelaxant action of 6-nitrodopamine was evaluated in carotid, coronary, renal, and femoral arteries precontracted by U-46619 (3 nM), and compared to that induced by the dopamine D 2 -receptor antagonist L-741,626. Expression of tyrosine hydroxylase and the neuromaker calretinin was investigated by immunohistochemistry. All vascular tissues presented basal release of endothelium-derived catecholamines. The relaxation induced by 6-nitrodopamine was not affected by preincubation of the tissues with either L-NAME (100 μM, 30-min preincubation) or the heme-site inhibitor of soluble guanylyl cyclase ODQ (100 μM, 30-min preincubation). Electrical field stimulation (EFS)-induced contractions were significantly potentiated by previous incubation with L-NAME, but unaffected by ODQ preincubation. The contractions induced by EFS were reduced by preincubation with either 6-nitrodopamine or L-741,626. Immunohistochemistry in all arteries revealed the presence of tyrosine hydroxylase in the endothelium, whereas immunoreactivity for calretinin was negative. Swine vessels present basal release of endothelium-derived catecholamines and expression of tyrosine hydroxylase in the endothelium. The vasodilation induced by 6-nitrodopamine is due to blockade of dopaminergic D 2 -like receptors., Competing Interests: Declaration of competing interest The authors declare no competing or financial interests., (Copyright © 2024. Published by Elsevier Inc.)

Background: To date, PCSK9 inhibitors are well known for eliminating cardiac and cerebral artery ischemia events by lowering the serum lipid level. However, the pathophysiological value of in-plaque PCSK9 expression is still unclear., Methods: Advanced plaques removed by carotid endarterectomy were sectioned and stained to identify the PCSK9 expression pattern and its co-expression with rupture-relevant markers. To investigate the correlation of PCSK9 expression with regional blood shear flow, hemodynamic characteristics were analyzed using computational fluid dynamics, and representative parameters were compared between PCSK9 positive and negative staining plaques. To explore this phenomenon in vitro, human aortic vascular smooth muscle cells were used to overexpress and knock down PCSK9. The impacts of PCSK9 modulations on mechanical sensor activity were testified by western blot and immunofluorescence. Real-time polymerase chain reaction was used to evaluate the transcription levels of downstream rupture-prone effectors., Results: PCSK9 distribution in plaque preferred cap and shoulder regions, residing predominantly in smooth muscle actin-positive cells. Cap PCSK9 expression correlated with fibrous cap thickness negatively and co-expressed with MMP-9, both pointing to the direction of plaque rupture. A hemodynamic profile indicated a rupture-prone feature of cap PCSK9 expression. In vitro, overexpression and knockdown of PCSK9 in human aortic vascular smooth muscle cells has positive modulation on mechanical sensor Yes-associated protein 1 (YAP) activity and transcription levels of its downstream rupture-prone effectors. Serial section staining verified in situ colocalization among PCSK9, YAP, and downstream effectors., Conclusions: Cap PCSK9 possesses a biomarker for rupture risk, and its modulation may lead to a novel biomechanical angle for plaque interventions., Competing Interests: Declaration of conflicting interestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Background and Aims: Lipids constitute one of the main components of atherosclerosis lesions and are the mediators of many mechanisms involved in plaque progression and stability. Here we tested the hypothesis that lipids known to be involved in plaque development exhibited associations with plaque vulnerability. We used spatial lipidomics to overcome plaque heterogeneity and to compare lipids from specific regions of symptomatic and asymptomatic human carotid atherosclerotic plaques., Methods: Carotid atherosclerotic plaques were collected from symptomatic and asymptomatic patients. Plaque lipids were analyzed with the spatial lipidomics technique matrix-assisted laser desorption/ionization mass spectrometry imaging, and histology and immunofluorescence were used to segment the plaques into histomolecularly distinct regions., Results: Macrophage-rich regions from symptomatic lesions were found to be enriched in phosphatidylcholines (synthesized to counteract excess free cholesterol), while the same region from asymptomatic plaques were enriched in polyunsaturated cholesteryl esters and triglycerides, characteristic of functional lipid droplets. Vascular smooth muscle cells (VSMCs) of the fibrous cap of asymptomatic plaques were enriched in lysophosphatidylcholines and cholesteryl esters, know to promote VSMC proliferation and migration, crucial for the buildup of the fibrous cap stabilizing the plaque., Conclusions: The investigation of the region-specific lipid composition of symptomatic and asymptomatic human atherosclerotic plaques revealed specific lipid markers of plaque outcome, which could be linked to known biological characteristics of stable plaques., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background and Aims: Sodium-glucose co-transporter 2 (SGLT2) inhibitors have been shown to reduce the risk of cardiovascular events independently of glycemic control. However, the possibility that SGLT2 inhibitors improve vascular restenosis is unknown. The aim of this study was to examine whether dapagliflozin could prevent neointima thickening following balloon injury and, if so, to determine the underlying mechanisms., Methods: Saline, dapagliflozin (1.5 mg/kg/day), or losartan (30 mg/kg/day) was administered orally for five weeks to male Wistar rats. Balloon injury of the left carotid artery was performed a week after starting the treatment and rats were sacrificed 4 weeks later. The extent of neointima was assessed by histomorphometric and immunofluorescence staining analyses. Vascular reactivity was assessed on injured and non-injured carotid artery rings, changes of target factors by immunofluorescence, RT-qPCR, and histochemistry., Results: Dapagliflozin and losartan treatments reduced neointima thickening by 32 % and 27 %, respectively. Blunted contractile responses to phenylephrine and relaxations to acetylcholine and down-regulation of eNOS were observed in the injured arteries. RT-qPCR investigations indicated an increased in gene expression of inflammatory (IL-1beta, VCAM-1), oxidative (p47phox, p22phox) and fibrotic (TGF-beta1) markers in the injured carotid. While these changes were not affected by dapagliflozin, increased levels of AT1R and NTPDase1 (CD39) and decreased levels of ENPP1 were observed in the restenotic carotid artery of the dapagliflozin group., Conclusions: Dapagliflozin effectively reduced neointimal thickening. The present data suggest that dapagliflozin prevents restenosis through interfering with angiotensin and/or extracellular nucleotides signaling. SGLT2 represents potential new target for limiting vascular restenosis., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Excessive neointimal hyperplasia (NIH) of coronary vessels in patients is the main cause of restenosis (RS) after percutaneous coronary intervention (PCI). This study aimed to identify the regulatory genes related to NIH in a rat carotid artery balloon injury model.We established a rat model and performed RNA sequencing to identify differentially expressed long non-coding RNAs (DElncRNAs) and differentially expressed message RNAs (DEmRNAs). Immune cells were analyzed using a murine Microenvironment Cell Population counter. The Pearson correlation between DEmRNAs, DElncRNAs, and immune cells was analyzed, followed by function enrichment analysis. Core DEmRNA was identified using Cytoscape. Next, a core lncRNAs-mRNAs-immune cell regulatory network was constructed. NIH-related gene sets from the Gene Expression Omnibus and GeneCards databases were used for validation.A total of 2,165 DEmRNAs and 705 DElncRNAs were identified in rat carotid artery tissue. Four key immune cells were screened out, including mast cells, vessels, endothelial cells, and fibroblasts. Based on the Pearson correlation between DEmRNAs, DElncRNAs and 4 key immune cells, 246 DEmRNAs and 93 DElncRNAs were obtained. DEmRNAs that interact with lncRNAs were mainly involved in the cell cycle, MAPK signaling pathway, and PI3K-Akt signaling pathway. A core lncRNA-mRNA-immune cell regulatory network was constructed, including 9 mRNAs, 4 lncRNAs, and fibroblasts. External datasets validation confirmed the significant correlation of both these mRNAs and lncRNAs with NIH.In this study, an lncRNA-mRNA-immune cell regulatory network related to NIH was constructed, which provided clues for exploring the potential mechanism of RS in cardiovascular diseases.

Background: Atherosclerosis, serving as the primary pathological mechanism at the core of cardiovascular disease, is now widely acknowledged to be associated with DNA damage and repair, contributing to atherosclerotic plaque formation. Therefore, molecules involved in the DNA repair process may play an important role in the progression of atherosclerosis. Our research endeavors to explore the contributions of specific and interrelated molecules involved in DNA repair (APE1, BRCA1, ERCC2, miR-221-3p, miR-145-5p, and miR-155-5p) to the development of atherosclerotic plaque and their interactions with each other., Methods & Results: Gene expression study was conducted using the real-time polymerase chain reaction (qRT-PCR) method on samples from carotid artery atherosclerotic plaques and nonatherosclerotic internal mammary arteries obtained from 50 patients diagnosed with coronary artery disease and carotid artery disease. Additionally, 50 healthy controls were included for the determination of 8-hydroxy-2'-deoxyguanosine (8-OHdG). Although no difference was observed in mRNA gene expressions, we noted a decrease in miR-155-5p gene expression (p = 0.003) and an increase in miR-221-3p gene expression (p = 0.015) in plaque samples, while miR-145-5p gene expression remained unchanged (p = 0.57). Regarding serum 8-OHdG levels, patients exhibited significantly higher levels (1111.82 ± 28.64) compared to controls (636.23 ± 24.23) (p < 0.0001)., Conclusions: In our study demonstrating the role of miR-155-5p and miR-221-3p in atherosclerosis, we propose that these molecules are potential biomarkers and therapeutic targets for coronary artery diseases and carotid artery disease., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: Metabolism is increasingly recognized as a key regulator of the function and phenotype of the primary cellular constituents of the atherosclerotic vascular wall, including endothelial cells, smooth muscle cells, and inflammatory cells. However, a comprehensive analysis of metabolic changes associated with the transition of plaque from a stable to a hemorrhaged phenotype is lacking., Methods: In this study, we integrated two large mRNA expression and protein abundance datasets (BIKE, n = 126; MaasHPS, n = 43) from human atherosclerotic carotid artery plaque to reconstruct a genome-scale metabolic network (GEM). Next, the GEM findings were linked to metabolomics data from MaasHPS, providing a comprehensive overview of metabolic changes in human plaque., Results: Our study identified significant changes in lipid, cholesterol, and inositol metabolism, along with altered lysosomal lytic activity and increased inflammatory activity, in unstable plaques with intraplaque hemorrhage (IPH+) compared to non-hemorrhaged (IPH-) plaques. Moreover, topological analysis of this network model revealed that the conversion of glutamine to glutamate and their flux between the cytoplasm and mitochondria were notably compromised in hemorrhaged plaques, with a significant reduction in overall glutamate levels in IPH+ plaques. Additionally, reduced glutamate availability was associated with an increased presence of macrophages and a pro-inflammatory phenotype in IPH+ plaques, suggesting an inflammation-prone microenvironment., Conclusions: This study is the first to establish a robust and comprehensive GEM for atherosclerotic plaque, providing a valuable resource for understanding plaque metabolism. The utility of this GEM was illustrated by its ability to reliably predict dysregulation in the cholesterol hydroxylation, inositol metabolism, and the glutamine/glutamate pathway in rupture-prone hemorrhaged plaques, a finding that may pave the way to new diagnostic or therapeutic measures., (© 2024. The Author(s).)

Clinical failure of arteriovenous neointimal hyperplasia (NIH) fistulae (AVF) is frequently due to juxta-anastomotic NIH (JANIH). Although the mouse AVF model recapitulates human AVF maturation, previous studies focused on the outflow vein distal to the anastomosis. We hypothesized that the juxta-anastomotic area (JAA) has increased NIH compared with the outflow vein. AVF was created in C57BL/6 mice without or with chronic kidney disease (CKD). Temporal and spatial changes of the JAA were examined using histology and immunofluorescence. Computational techniques were used to model the AVF. RNA-seq and bioinformatic analyses were performed to compare the JAA with the outflow vein. The jugular vein to carotid artery AVF model was created in Wistar rats. The neointima in the JAA shows increased volume compared with the outflow vein. Computational modeling shows an increased volume of disturbed flow at the JAA compared with the outflow vein. Endothelial cells are immediately lost from the wall contralateral to the fistula exit, followed by thrombus formation and JANIH. Gene Ontology (GO) enrichment analysis of the 1,862 differentially expressed genes (DEG) between the JANIH and the outflow vein identified 525 overexpressed genes. The rat jugular vein to carotid artery AVF showed changes similar to the mouse AVF. Disturbed flow through the JAA correlates with rapid endothelial cell loss, thrombus formation, and JANIH; late endothelialization of the JAA channel correlates with late AVF patency. Early thrombus formation in the JAA may influence the later development of JANIH. NEW & NOTEWORTHY Disturbed flow and focal endothelial cell loss in the juxta-anastomotic area of the mouse AVF colocalizes with acute thrombus formation followed by late neointimal hyperplasia. Differential flow patterns between the juxta-anastomotic area and the outflow vein correlate with differential expression of genes regulating coagulation, proliferation, collagen metabolism, and the immune response. The rat jugular vein to carotid artery AVF model shows changes similar to the mouse AVF model.

Background: Neointimal hyperplasia (NIH) is the pathological basis of vascular injury disease. Vascular cells are the dominant cells in the process of NIH, but the extent of heterogeneity amongst them is still unclear., Methods: A mouse model of NIH was constructed by inducing carotid artery ligation. Single-cell sequencing was then used to analyze the transcriptional profile of vascular cells. Cluster features were determined by functional enrichment analysis, gene set scoring, pseudo-time analysis, and cell-cell communication analysis. Additionally, immunofluorescence staining was conducted on vascular tissues from fibroblast lineage-traced (PdgfraDreER-tdTomato) mice to validate the presence of Pecam1+Pdgfra+tdTomato+ cells., Results: The left carotid arteries (ligation) were compared to right carotid arteries (sham) from ligation-induced NIH C57BL/6 mice. Integrative analyses revealed a high level of heterogeneity amongst vascular cells, including fourteen clusters and seven cell types. We focused on three dominant cell types: endothelial cells (ECs), vascular smooth muscle cells (vSMCs), and fibroblasts. The major findings were: (1) four subpopulations of ECs, including ECs4, mesenchymal-like ECs (ECs1 and ECs2), and fibro-like ECs (ECs3); (2) four subpopulations of fibroblasts, including pro-inflammatory Fibs-1, Sca1+ Fibs-2, collagen-producing Fibs-3, and mesenchymal-like Fibs-4; (3) four subpopulations of vSMCs, including vSMCs-1, vSMCs-2, vSMCs-3, and vSMCs-3-derived vSMCs; (4) ECs3 express genes related to extracellular matrix (ECM) remodeling and cell migration, and fibro-like vSMCs showed strong chemokine secretion and relatively high levels of proteases; (5) fibro-like vSMCs that secrete Vegfa interact with ECs mainly through vascular endothelial growth factor receptor 2 (Vegfr2)., Conclusions: This study presents the dynamic cellular landscape within NIH arteries and reveals potential relationships between several clusters, with a specific focus on ECs3 and fibro-like vSMCs. These two subpopulations may represent potential target cells for the treatment of NIH., Competing Interests: The authors declare no conflict of interest., (© 2024 The Author(s). Published by IMR Press.)

Endothelium-dependent contraction (EDC) exists in blood vessels of normotensive animals, but is exaggerated in hypertension. An early signal in EDC is cytosolic Ca 2+ rise in endothelial cells. In this study we investigated the functional role of Orai1, a major endothelial cell Ca 2+ entry channel, in EDC. Hypertension model was established in WT mice by intake of L-NNA in the drinking water (0.5 g/L) for 4 weeks or osmotic pump delivery of Ang II (1.5 mg·kg -1 ·d -1 ) for 2 weeks. In TRPC5 KO mice, the concentration of L-NNA and Ang II were increased to 1 g/L or 2 mg·kg -1 ·d -1 , respectively. Arterial segments were prepared from carotid arteries and aortas, and EDC was elicited by acetylcholine in the presence of N ω -nitro-L-arginine methyl ester. We showed that low concentration of acetylcholine (3-30 nM) initiated relaxation in phenylephrine-precontracted carotid arteries of both normotensive and hypertensive mice, while high concentration of acetylcholine (0.1-2 μM) induced contraction. Application of selective Orai1 inhibitors AnCoA4 (100 μM) or YM58483 (400 nM) had no effect on ACh-induced relaxation but markedly reduced acetylcholine-induced EDC. We found that EDC was increased in hypertensive mice compared with that of normotensive mice, which was associated with increased Orai1 expression in endothelial cells of hypertensive mice. Compared to TRPC5 and TRPV4, which were also involved in EDC, endothelial cell Orai1 had relatively greater contribution to EDC than either TRPC5 or TRPV4 alone. We identified COX-2, followed by PGF2α, PGD2 and PGE2 as the downstream signals of Orai1/TRPC5/TRPV4. In conclusion, Orai1 coordinates together with TRPC5 and TRPV4 in endothelial cells to regulate EDC responses. This study demonstrates a novel function of Orai1 in EDC in both normotensive and hypertensive mice, thus providing a general scheme about the control of EDC by Ca 2+ -permeable channels., (© 2024. The Author(s), under exclusive licence to Shanghai Institute of Materia Medica, Chinese Academy of Sciences and Chinese Pharmacological Society.)

Aims: Elucidating the impacts of long-term spaceflight on cardiovascular health is urgently needed in face of the rapid development of human space exploration. Recent reports including the NASA Twins Study on vascular deconditioning and aging of astronauts in spaceflight are controversial. The aims of this study were to elucidate whether long-term microgravity promotes vascular aging and the underlying mechanisms., Methods and Results: Hindlimb unloading (HU) by tail suspension was used to simulate microgravity in rats and mice. The dynamic changes of carotid stiffness in rats during 8 weeks of HU were determined. Simulated microgravity led to carotid artery aging-like changes as evidenced by increased stiffness, thickness, fibrosis, and elevated senescence biomarkers in the HU rats. Specific deletion of the mechanotransducer Piezo1 in vascular smooth muscles significantly blunted these aging-like changes in mice. Mechanistically, mechanical stretch-induced activation of Piezo1 elevated microRNA-582-5p in vascular smooth muscle cells, with resultant enhanced synthetic cell phenotype and increased collagen deposition via PTEN/PI3K/Akt signalling. Importantly, inhibition of miRNA-582-5p alleviated carotid fibrosis and stiffness not only in HU rats but also in aged rats., Conclusions: Long-term simulated microgravity induces carotid aging-like changes via the mechanotransducer Piezo1-initiated and miRNA-mediated mechanism., Competing Interests: Conflict of interest: None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Orosomucoid (ORM) has been reported as a biomarker of carotid atherosclerosis, but the role of ORM 2, a subtype of ORM, in carotid atherosclerotic plaque formation and the underlying mechanism have not been established., Methods: Plasma was collected from patients with carotid artery stenosis (CAS) and healthy participants and assessed using mass spectrometry coupled with isobaric tags for relative and absolute quantification (iTRAQ) technology to identify differentially expressed proteins. The key proteins and related pathways were identified via western blotting, immunohistochemistry, and polymerase chain reaction of carotid artery plaque tissues and in vitro experiments involving vascular smooth muscle cells (VSMCs)., Results: We screened 33 differentially expressed proteins out of 535 proteins in the plasma. Seventeen proteins showed increased expressions in the CAS groups relative to the healthy groups, while 16 proteins showed decreased expressions during iTRAQ and bioinformatic analysis. The reactive oxygen species metabolic process was the most common enrichment pathway identified by Gene Ontology analysis, while ORM2, PRDX2, GPX3, HP, HBB, ANXA5, PFN1, CFL1, and S100A11 were key proteins identified by STRING and MCODE analysis. ORM2 showed increased expression in patients with CAS plaques, and ORM2 was accumulated in smooth muscle cells. Oleic acid increased the lipid accumulation and ORM2 and PRDX6 expressions in the VSMCs. The recombinant-ORM2 also increased the lipid accumulation and reactive oxygen species (ROS) in the VSMCs. The expressions of ORM2 and PRDX-6 were correlated, and MJ33 (an inhibitor of PRDX6-PLA2) decreased ROS production and lipid accumulation in VSMCs., Conclusion: ORM2 may be a biomarker for CAS; it induced lipid accumulation and ROS production in VSMCs during atherosclerosis plaque formation. However, the relationships between ORM2 and PRDX-6 underlying lipid accumulation-induced plaque vulnerability require further research., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Actin linked regulatory mechanisms are known to contribute contraction/relaxation in smooth muscle. In order to clarify whether modulation of polymerization/depolymerization of actin filaments affects relaxation process, we examined the effects of cytochalasin D on relaxation process by Ca 2+ removal after Ca 2+ -induced contraction of β-escin skinned (cell membrane permeabilized) taenia cecum and carotid artery preparations from guinea pigs. Cytochalasin D, an inhibitor of actin polymerization, significantly suppressed the force during relaxation both in skinned taenia cecum and carotid artery. The data fitting analysis of the relaxation processes indicates that cytochalasin D accelerates slow (latch-like) bridge dissociation. Cytochalasin D seems to directly disrupts actin filament organization or its length, resulting in modulation of actin filament structure that prevents myosin binding., (© 2024. The Author(s).)

Hutchinson-Gilford progeria syndrome (HGPS) is an extremely rare genetic disease caused by expression of progerin, a lamin A variant that is also expressed at low levels in non-HGPS individuals. Although HGPS patients die predominantly from myocardial infarction and stroke, the mechanisms that provoke pathological alterations in the coronary and cerebral arteries in HGPS remain ill defined. Here, we assessed vascular function in the coronary arteries (CorAs) and carotid arteries (CarAs) of progerin-expressing Lmna G609G/G609G mice (G609G), both in resting conditions and after hypoxic stimulus. Wire myography, pharmacological screening, and gene expression studies demonstrated vascular atony and stenosis, as well as other functional alterations in progeroid CorAs and CarAs and aorta. These defects were associated with loss of vascular smooth muscle cells and overexpression of the K V 7 family of voltage-dependent potassium channels. Compared with wild-type controls, G609G mice showed reduced median survival upon chronic isoproterenol exposure, a baseline state of chronic cardiac hypoxia characterized by overexpression of hypoxia-inducible factor 1α and 3α genes, and increased cardiac vascularization. Our results shed light on the mechanisms underlying progerin-induced coronary and carotid artery disease and identify K V 7 channels as a candidate target for the treatment of HGPS., (© 2023. The Author(s).)

Atherosclerosis is a chronic inflammatory disease characterized with innate and adaptive immunity but also involves pyroptosis. Few studies have explored the role of pyroptosis in advanced atherosclerotic plaques from different vascular beds. Here we try to identify the different underlying function of pyroptosis in the progression of atherosclerosis between carotid arteries and femoral. arteries. We extracted gene expression levels from 55 advanced carotid or femoral atherosclerotic plaques. The pyroptosis score of each sample was calculated by single-sample-gene-set enrichment analysis (ssGSEA). We then divided the samples into two clusters: high pyroptosis scores cluster (PyroptosisScoreH cluster) and low pyroptosis scores cluster (PyroptosisScoreL cluster), and assessed functional enrichment and immune cell infiltration in the two clusters. Key pyroptosis related genes were identified by the intersection between results of Cytoscape and LASSO (Least Absolute Shrinkage and Selection Operator) regression analysis. Finally, all key pyroptosis related genes were validated in vitro. We found all but one of the 29 carotid plaque samples belonged to the PyroptosisScoreH cluster and the majority (19 out of 26) of femoral plaques were part of the PyroptosisScoreL cluster. Atheromatous plaque samples in the PyroptosisScoreL cluster had higher proportions of gamma delta T cells, M2 macrophages, myeloid dendritic cells (DCs), and cytotoxic lymphocytes (CTLs), but lower proportions of endothelial cells (ECs). Immune full-activation pathways (e.g., NOD-like receptor signaling pathway and NF-kappa B signaling pathway) were highly enriched in the PyroptosisScoreH cluster. The key pyroptosis related genes GSDMD, CASP1, NLRC4, AIM2, and IL18 were upregulated in advanced carotid atherosclerotic plaques. We concluded that compared to advanced femoral atheromatous plaques, advanced carotid atheromatous plaques were of higher grade of pyroptosis. GSDMD, CASP1, NLRC4, AIM2, and IL18 were the key pyroptosis related genes, which might provide a new sight in the prevention of fatal strokes in advanced carotid atherosclerosis., (© 2024. The Author(s).)

Introduction: It is well documented that high-salt (HS) diet increases systemic and vascular oxidative stress in various animal models and in humans, leading to impairment of vascular reactivity. The present study examined the interaction of genotype and HS diet intake and the potential effects of oxidative stress - antioxidative system balance on the flow-induced dilation (FID) in pressurized carotid arteries of normotensive Tff3-/-/C57BL/6N knockout mice and their wild-type (WT) controls., Methods: Male, ten-week-old transgenic Tff3-/-/C57BL/6N (Tff3-/-) knockout mice and WT/C57BL/6N (WT) (parental strain) healthy mice were divided in LS (0.4% NaCl in rodent chow) and HS (4% NaCl in rodent chow fed for 1 week) groups. Additionally, LS and HS groups were treated with 1 mmol/L 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPOL) dissolved in the drinking water. After anesthesia with ketamine chloride (100 mg/kg) and midazolam (5 mg/kg), blood pressure was measured, carotid arteries and aortas were isolated, and blood samples were collected., Results: FID was decreased in WT&#95;HS mice and restored by superoxide scavenger TEMPOL in vivo. On the other hand, attenuated FID of Tff3-/- mice was not further affected by HS diet or TEMPOL in vivo treatment. Vascular superoxide/reactive oxygen species levels were increased with HS diet in both strains and restored by TEMPOL. HS upregulated glutathione peroxidase 1 (GPx1) gene expression in WT&#95;HS and Tff3-/-&#95;HS mice, while GPx activity was significantly decreased only in WT&#95;HS group. Systemic (serum) markers of oxidative stress (oxLDL and AOPP) and arterial blood pressure were similar among groups., Conclusion: HS diet increases vascular oxidative stress and impairs vasodilation in WT mice. Tff3 gene deficiency attenuates vasodilation per se, without further effects of HS intake. This can be attributed to vascular upregulation of antioxidative enzyme GPx1 in Tff3-/-/C57BL/6N mice conferring protection from oxidative stress., (© 2024 S. Karger AG, Basel.)

The rupture of carotid artery vulnerable plaque plays a critical role in ischemic stroke, and the widely spread new coronavirus in recent years plays a certain role in the development of human carotid artery vulnerable plaque, we screened out 27 differential expression genes (DEGs) of stable plaque and vulnerable plaque associated with the new coronavirus. Through the construction of the protein-protein interaction (PPI) network, the Cathepsin B (CTSB) and Niemann-Pick Disease Type 2 (NPC2) were identified as crucial expression genes, and further, we confirmed the validity of core gene expression in two validation sets. Additionally, we discovered a significant connection between CTSB, NPC2 and 28 different kinds of immune cells in carotid plaque tissue. We screened out 65 target interacting drugs based on 10 differentially expressed genes through online tools and finally verified the high expression of 2 core genes in fragile plaques through clinical sample experiments. These findings imply that two core genes may be novel targets for molecular diagnostics and immunotherapy of vulnerable plaques., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The aim of this study was to investigate how the concentration of interleukin-13 (IL-13) affects the regulation of endothelial cell migration after injury. The incubation of recombinant human interleukin-13 (rhIL-13) strongly increased the content of reactive oxygen species (ROS) in HUVECs via the JAK-1/STAT-3/NOX-4 signaling pathway. Antagonizing the high intracellular ROS that was induced by rhIL-13 promoted the migration of HUVECs. Furthermore, IL-13 neutralization not only inhibited intimal hyperplasia, but also promoted the migration of endothelial cells (ECs) after injury. The results suggest that IL-13 inhibition is a potential means of stimulating endothelial cells recovery after injury. Therefore, the attenuation of IL-13 activation may have therapeutic value for vascular disease.

Background Plaque myofibroblasts are critical players in the initiation and advancement of atherosclerotic disease. They are involved in the production of extracellular matrix, the formation of the fibrous cap, and the underlying lipidic core via modulation processes in response to different environmental cues. Despite clear phenotypic differences between myofibroblast cells and healthy vascular smooth muscle cells, smooth muscle cells are still widely used as a cellular model in atherosclerotic research. Methods and Results Here, we present a conditioned outgrowth method to isolate and culture myofibroblast cells from plaques. We obtained these cells from 27 donors (24 carotid and 3 femoral endarterectomies). We show that they keep their proliferative capacity for 8 passages, are transcriptionally stable, retain donor-specific gene expression programs, and express extracellular matrix proteins ( FN1 , COL1A1 , and DCN ) and smooth muscle cell markers ( ACTA2 , MYH11 , and CNN1 ). Single-cell transcriptomics reveals that the cells in culture closely resemble the plaque myofibroblasts. Chromatin immunoprecipitation sequencing shows the presence of histone H3 lysine 4 dimethylation at the MYH11 promoter, pointing to their smooth muscle cell origin. Finally, we demonstrated that plaque myofibroblasts can be efficiently transduced (>97%) and are capable of taking up oxidized low-density lipoprotein and undergoing calcification. Conclusions In conclusion, we present a method to isolate and culture cells that retain plaque myofibroblast phenotypical and functional capabilities, making them a suitable in vitro model for studying selected mechanisms of atherosclerosis.

Aims: The article investigates whether chronic hyperglycaemia in Type 1 diabetes (T1D) is associated with a proinflammatory immune signature and with arterial wall inflammation, driving the development of atherosclerosis., Methods and Results: Patients with T1D (n = 41), and healthy age-, sex-, and body mass index-matched controls (n = 20) were recruited. Arterial wall inflammation and haematopoietic activity were measured with 2'-deoxy-2'-(18F)-fluoro-D-glucose (18F-FDG) positron emission tomography/computed tomography. In addition, flow cytometry of circulating leucocytes was performed as well as targeted proteomics to measure circulating inflammatory markers. 18F-FDG uptake in the wall of the abdominal aorta, carotid arteries, and iliac arteries was higher in T1D compared with that in the healthy controls. Also, 18F-FDG uptake in the bone marrow and spleen was higher in patients with T1D. CCR2 and CD36 expressions on circulating monocytes were higher in patients with T1D, as well as several circulating inflammatory proteins. In addition, several circulating inflammatory markers (osteoprotegerin, transforming growth factor-alpha, CX3CL1, and colony-stimulating factor-1) displayed a positive correlation with FDG uptake. Within T1D, no differences were found between people with a high and low HbA1c., Conclusion: These findings strengthen the concept that chronic hyperglycaemia in T1D induces inflammatory changes that fuel arterial wall inflammation leading to atherosclerosis. The degree of hyperglycaemia appears to play a minor role in driving this inflammatory response in patients with T1D., Competing Interests: Conflict of interest: None declared., (© The Author(s) 2023. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Reversible lysine-63 (K63) polyubiquitination regulates proinflammatory signaling in vascular smooth muscle cells (SMCs) and plays an integral role in atherosclerosis. Ubiquitin-specific peptidase 20 (USP20) reduces NFκB activation triggered by proinflammatory stimuli, and USP20 activity attenuates atherosclerosis in mice. The association of USP20 with its substrates triggers deubiquitinase activity; this association is regulated by phosphorylation of USP20 on Ser334 (mouse) or Ser333 (human). USP20 Ser333 phosphorylation was greater in SMCs of atherosclerotic segments of human arteries as compared with nonatherosclerotic segments. To determine whether USP20 Ser334 phosphorylation regulates proinflammatory signaling, we created USP20-S334A mice using CRISPR/Cas9-mediated gene editing. USP20-S334A mice developed ∼50% less neointimal hyperplasia than congenic WT mice after carotid endothelial denudation. WT carotid SMCs showed substantial phosphorylation of USP20 Ser334, and WT carotids demonstrated greater NFκB activation, VCAM-1 expression, and SMC proliferation than USP20-S334A carotids. Concordantly, USP20-S334A primary SMCs in vitro proliferated and migrated less than WT SMCs in response to IL-1β. An active site ubiquitin probe bound to USP20-S334A and USP20-WT equivalently, but USP20-S334A associated more avidly with TRAF6 than USP20-WT. IL-1β induced less K63-linked polyubiquitination of TRAF6 and less downstream NFκB activity in USP20-S334A than in WT SMCs. Using in vitro phosphorylation with purified IRAK1 and siRNA-mediated gene silencing of IRAK1 in SMCs, we identified IRAK1 as a novel kinase for IL-1β-induced USP20 Ser334 phosphorylation. Our findings reveal novel mechanisms regulating IL-1β-induced proinflammatory signaling: by phosphorylating USP20 Ser334, IRAK1 diminishes the association of USP20 with TRAF6 and thus augments NFκB activation, SMC inflammation, and neointimal hyperplasia., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Alterations in gut microbiota have been implicated in HIV infection and cardiovascular disease. However, how gut microbial alterations relate to host inflammation and metabolite profiles, and their relationships with atherosclerosis, have not been well-studied, especially in the context of HIV infection. Here, we examined associations of gut microbial species and functional components measured by shotgun metagenomics with carotid artery plaque assessed by B-mode carotid artery ultrasound in 320 women with or at high risk of HIV (65% HIV +) from the Women's Interagency HIV Study. We further integrated plaque-associated microbial features with serum proteomics (74 inflammatory markers measured by the proximity extension assay) and plasma metabolomics (378 metabolites measured by liquid chromatography tandem mass spectrometry) in relation to carotid artery plaque in up to 433 women., Results: Fusobacterium nucleatum, a potentially pathogenic bacteria, was positively associated with carotid artery plaque, while five microbial species (Roseburia hominis, Roseburia inulinivorans, Johnsonella ignava, Odoribacter splanchnicus, Clostridium saccharolyticum) were inversely associated with plaque. Results were consistent between women with and without HIV. Fusobacterium nucleatum was positively associated with several serum proteomic inflammatory markers (e.g., CXCL9), and the other plaque-related species were inversely associated with proteomic inflammatory markers (e.g., CX3CL1). These microbial-associated proteomic inflammatory markers were also positively associated with plaque. Associations between bacterial species (especially Fusobacterium nucleatum) and plaque were attenuated after further adjustment for proteomic inflammatory markers. Plaque-associated species were correlated with several plasma metabolites, including the microbial metabolite imidazole-propionate (ImP), which was positively associated with plaque and several pro-inflammatory markers. Further analysis identified additional bacterial species and bacterial hutH gene (encoding enzyme histidine ammonia-lyase in ImP production) associated with plasma ImP levels. A gut microbiota score based on these ImP-associated species was positively associated with plaque and several pro-inflammatory markers., Conclusion: Among women living with or at risk of HIV, we identified several gut bacterial species and a microbial metabolite ImP associated with carotid artery atherosclerosis, which might be related to host immune activation and inflammation. Video Abstract., (© 2023. The Author(s).)

Vascular smooth muscle cells (VSMC) play a critical role in the development and pathogenesis of intimal hyperplasia indicative of restenosis and other vascular diseases. Fragile-X related protein-1 (FXR1) is a muscle-enhanced RNA binding protein whose expression is increased in injured arteries. Previous studies suggest that FXR1 negatively regulates inflammation, but its causality in vascular disease is unknown. In the current study, RNA-sequencing of FXR1-depleted VSMC identified many transcripts with decreased abundance, most of which were associated with proliferation and cell division. mRNA abundance and stability of a number of these transcripts were decreased in FXR1-depleted hVSMC, as was proliferation (P < 0.05); however, increases in beta-galactosidase (P < 0.05) and γH2AX (P < 0.01), indicative of senescence, were noted. Further analysis showed increased abundance of senescence-associated genes with FXR1 depletion. A novel SMC-specific conditional knockout mouse (FXR1 SMC/SMC ) was developed for further analysis. In a carotid artery ligation model of intimal hyperplasia, FXR1 SMC/SMC mice had significantly reduced neointima formation (P < 0.001) after ligation, as well as increases in senescence drivers p16, p21, and p53 compared with several controls. These results suggest that in addition to destabilization of inflammatory transcripts, FXR1 stabilized cell cycle-related genes in VSMC, and absence of FXR1 led to induction of a senescent phenotype, supporting the hypothesis that FXR1 may mediate vascular disease by regulating stability of proliferative mRNA in VSMC., (Copyright © 2023 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

17α-ethinylestradiol (EE2), a derivative of 17β-estradiol (E2), is a potent estrogenic substance that is used as the estrogenic component of oral contraceptives (OCPs). However, women who take OCPs have an increased risk of cardiovascular events. Since few studies have examined EE2 endothelial effects, we explored the effects of EE2 on endothelial function in ovariectomized and isoflavone-free rats. After ovariectomy, 12-week-old female Sprague-Dawley rats were assigned to EE2, E2 or control groups. After 16 weeks, the EE2 and E2 groups were orally administered EE2 (8.3 μg/day) and E2 (12.6 μg/day) for 4 weeks, respectively. At 18 weeks, endothelial denudation of the left common carotid arteries was performed, and they were harvested at 20 weeks. The rats in the EE2 and E2 groups exhibited significantly decreased body weights and significantly increased uterine weights, respectively, but no differences were observed between the EE2 and E2 groups. The EE2 and E2 groups showed significantly enhanced acetylcholine-induced endothelium-dependent relaxation, with apamin plus charybdotoxin inhibiting only the EE2 group. Endothelial nitric oxide (NO) synthase expression was significantly higher in the EE2 group than in the control, but lower than in the E2 group. The intima-to-media ratio of denuded arteries was significantly lower in the E2 group than in the other groups, suggesting that NO decreased in the EE2 group compared to the E2 group. We conclude that EE2 has a weaker ability than E2 to produce NO and, for the first time, we demonstrate the ability of EE2 to enhance the activity of endothelial-derived hyperpolarizing factor., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Extracellular vesicles (EV) are a novel biomarker source for diagnosis and prognosis of cardiovascular disease. A protein comparison of plasma EVs in relation to blood plasma and atherosclerotic plaque has not been performed but would provide insight into the origin and content of biomarker sources and their association with atherosclerotic progression. Using samples of 88 carotid endarterectomy patients in the Athero-Express, 92 proteins (Olink Cardiovascular III panel) were measured in citrate plasma, plasma derived LDL-EVs and atherosclerotic plaque. Proteins were correlated between sources and were related to pre-operative stroke and 3-year major adverse cardiovascular events (MACE). Plasma and EV proteins correlated moderately on average, but with substantial variability. Both showed little correlation with plaque, suggesting that these circulating biomarkers may not originate from the latter. Plaque (n = 17) contained most differentially-expressed proteins in patients with stroke, opposed to EVs (n = 6) and plasma (n = 5). In contrast, EVs contained most differentially-expressed proteins for MACE (n = 21) compared to plasma (n = 9) and plaque (n = 1). EVs appear to provide additional information about severity and progression of systemic atherosclerosis than can be obtained from plasma or atherosclerotic plaque., (© 2023. The Author(s).)

The time course of smooth muscle contraction can be divided into two phases, the initial phase is associated with force development, whereas the sustained phase is associated with force maintenance. Cumulative evidence suggests that the two phases are regulated by different signaling pathways and that ρ-kinase (ROCK) and protein kinase C (PKC) play an important role in regulating isometric force in sustained contractions. Since the maintenance of sustained force is critical to the function of vascular smooth muscle, unraveling the complex mechanism of force maintenance is crucial for understanding the cell biology of the muscle. The present study examined the effects of ROCK and PKC on the level of phosphorylation of the 20-kD myosin light chain (MLC20) and isometric force during a sustained contraction. We used partial activation and inhibition of ROCK and PKC to reduce the isometric force by 50% of the maximal isometric force in fully activated muscle, F max . We then examined the level of MLC20 phosphorylation in each case. We found that in partially activated muscle the level of MLC20 phosphorylation required to maintain 50% F max was much lower than that required in muscles where 50% reduction in F max was achieved by partial inhibition of ROCK and PKC. The results can be explained by a model containing a contractile apparatus and a cytoskeletal scaffold where force generated by the contractile apparatus is transmitted to the extracellular domain through the cytoskeleton. The results indicate that ROCK and PKC play an important role in force transmission through the cytoskeleton. NEW & NOTEWORTHY The study supports a model that the maintenance of sustained force during a contraction of arterial smooth muscle is dependent on the intracellular transmission of force through the cytoskeleton and that ρ-kinase and protein kinase C plays an important role in the regulation of cytoskeletal integrity and its efficiency in force transmission.

Aims: Trimethylamine-N-oxide (TMAO) is a metabolite generated from dietary choline, betaine, and l-carnitine, after their oxidization in the liver. TMAO has been identified as a novel independent risk factor for atherosclerosis through the induction of vascular inflammation. However, the effect of TMAO on neointimal formation in response to vascular injury remains unclear. Results: This study was conducted using a murine model of acutely disturbed flow-induced atherosclerosis induced by partial carotid artery ligation. 3,3-Dimethyl-1-butanol (DMB) was used to reduce TMAO concentrations. Wild-type mice were divided into four groups [regular diet, high-TMAO diet, high-choline diet, and high-choline diet+DMB] to investigate the effects of TMAO elevation and its inhibition by DMB. Mice fed high-TMAO and high-choline diets had significantly enhanced neointimal hyperplasia and advanced plaques, elevated arterial elastin fragmentation, increased macrophage infiltration and inflammatory cytokine secretion, and enhanced activation of nuclear factor (NF)-κB, the NLRP3 inflammasome, and endoplasmic reticulum (ER) stress relative to the control group. Mice fed high-choline diets with DMB treatment exhibited attenuated flow-induced atherosclerosis, inflammasome expression, ER stress, and reactive oxygen species expression. Human aortic smooth muscle cells (HASMCs) were used to investigate the mechanism of TMAO-induced injury. The HASMCs were treated with TMAO with or without an ER stress inhibitor to determine whether inhibition of ER stress modulates the TMAO-induced inflammatory response. Innovation: This study demonstrates that TMAO regulates vascular remodeling via ER stress. Conclusion: Our findings demonstrate that TMAO elevation promotes disturbed flow-induced atherosclerosis and that DMB administration mitigates vascular remodeling, suggesting a rationale for a TMAO-targeted strategy for the treatment of atherosclerosis. Antioxid. Redox Signal. 38, 215-233.

Ischemic stroke (IS) related to atherosclerosis of large arteries is one of the leading causes of mortality and disability in developed countries. Atherosclerotic internal carotid artery stenosis (ICAS) contributes to 20% of all cerebral ischemia cases. Nowadays, atherosclerosis prevention and treatment measures aim at controlling the atherosclerosis risk factors, or at the interventional (surgical or endovascular) management of mature occlusive lesions. There is a definite lack of the established circulating biomarkers which, once modulated, could prevent development of atherosclerosis, and consequently prevent the carotid-artery-related IS. Recent studies emphasize that microRNA (miRNA) are the emerging particles that could potentially play a pivotal role in this approach. There are some research studies on the association between the expression of small non-coding microRNAs with a carotid plaque development and vulnerability. However, the data remain inconsistent. In addition, all major studies on carotid atherosclerotic plaque were conducted on cell culture or animal models; very few were conducted on humans, whereas the accumulating evidence demonstrates that it cannot be automatically extrapolated to processes in humans. Therefore, this paper aims to review the current knowledge on how miRNA participate in the process of carotid plaque formation and rupture, as well as stroke occurrence. We discuss potential target miRNA that could be used as a prognostic or therapeutic tool.

Cardiovascular abnormality-mediated retinal ischemia causes severe visual impairment. Retinal ischemia is involved in enormous pathological processes including oxidative stress, reactive gliosis, and retinal functional deficits. Thus, maintaining retinal function by modulating those pathological processes may prevent or protect against vision loss. Over the decades, nicotinamide mononucleotide (NMN), a crucial nicotinamide adenine dinucleotide (NAD + ) intermediate, has been nominated as a promising therapeutic target in retinal diseases. Nonetheless, a protective effect of NMN has not been examined in cardiovascular diseases-induced retinal ischemia. In our study, we aimed to investigate its promising effect of NMN in the ischemic retina of a murine model of carotid artery occlusion. After surgical unilateral common carotid artery occlusion (UCCAO) in adult male C57BL/6 mice, NMN (500 mg/kg/day) was intraperitoneally injected to mice every day until the end of experiments. Electroretinography and biomolecular assays were utilized to measure ocular functional and further molecular alterations in the retina. We found that UCCAO-induced retinal dysfunction was suppressed, pathological gliosis was reduced, retinal NAD + levels were preserved, and the expression of an antioxidant molecule (nuclear factor erythroid-2-related factor 2; Nrf2 ) was upregulated by consecutive administration of NMN. Our present outcomes first suggest a promising NMN therapy for the suppression of cardiovascular diseases-mediated retinal ischemic dysfunction.

Backgruound: Excessive proliferation and migration of vascular smooth muscle cells (VSMCs), which contributes to the development of occlusive vascular diseases, requires elevated mitochondrial oxidative phosphorylation to meet the increased requirements for energy and anabolic precursors. Therefore, therapeutic strategies based on blockade of mitochondrial oxidative phosphorylation are considered promising for treatment of occlusive vascular diseases. Here, we investigated whether DN200434, an orally available estrogen receptor-related gamma inverse agonist, inhibits proliferation and migration of VSMCs and neointima formation by suppressing mitochondrial oxidative phosphorylation., Methods: VSMCs were isolated from the thoracic aortas of 4-week-old Sprague-Dawley rats. Oxidative phosphorylation and the cell cycle were analyzed in fetal bovine serum (FBS)- or platelet-derived growth factor (PDGF)-stimulated VSMCs using a Seahorse XF-24 analyzer and flow cytometry, respectively. A model of neointimal hyperplasia was generated by ligating the left common carotid artery in male C57BL/6J mice., Results: DN200434 inhibited mitochondrial respiration and mammalian target of rapamycin complex 1 activity and consequently suppressed FBS- or PDGF-stimulated proliferation and migration of VSMCs and cell cycle progression. Furthermore, DN200434 reduced carotid artery ligation-induced neointima formation in mice., Conclusion: Our data suggest that DN200434 is a therapeutic option to prevent the progression of atherosclerosis.

Aims: Arterial stiffness is a hallmark of vascular ageing that precedes and strongly predicts the development of cardiovascular diseases. Age-dependent stiffening of large elastic arteries is primarily attributed to increased levels of matrix metalloproteinase-2 (MMP-2). However, the mechanistic link between age-dependent arterial stiffness and MMP-2 remains unclear. Thus, we aimed to investigate the efficacy of MMP-2 knockdown using small-interfering RNA (siRNA) on age-dependent arterial stiffness., Methods and Results: Pulse wave velocity (PWV) was assessed in right carotid artery of wild-type (WT) mice from different age groups. MMP-2 levels in the carotid artery and plasma of young (3 months) and old (20-25 months) WT mice were determined. Carotid PWV as well as vascular and circulating MMP-2 were elevated with increasing age in mice. Old WT mice (18- to 21-month old) were treated for 4 weeks with either MMP-2 or scrambled (Scr) siRNA via tail vein injection. Carotid PWV was assessed at baseline, 2 and 4 weeks after start of the treatment. MMP-2 knockdown reduced vascular MMP-2 levels and attenuated age-dependent carotid stiffness. siMMP-2-treated mice showed increased elastin-to-collagen ratio, lower plasma desmosine (DES), enhanced phosphorylation of endothelial nitric oxide synthase (eNOS), and higher levels of vascular cyclic guanosine monophosphate (cGMP). An age-dependent increase in direct protein-protein interaction between MMP-2 and eNOS was also observed. Lastly, DES, an elastin breakdown product, was measured in a patient cohort (n = 64, 23-86 years old), where carotid-femoral PWV was also assessed; here, plasma levels of DES directly correlated with age and arterial stiffness., Conclusion: MMP-2 knockdown attenuates age-dependent carotid stiffness by blunting elastin degradation and augmenting eNOS bioavailability. Given the increasing clinical use of siRNA technology, MMP2 knockdown should be investigated further as a possible strategy to mitigate age-dependent arterial stiffness and related CV diseases., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions, please email: journals.permissions@oup.com.)

HIV-infected individuals have increased risk for cardiovascular disease (CVD) despite suppressive antiretroviral therapy (ART). This is likely a result of persistent immune activation and systemic inflammation. Extracellular vesicles (EVs) have emerged as critical mediators of intercellular communication and may drive inflammation contributing to CVD. EVs were characterized in plasma from 74 HIV-infected individuals on combination antiretroviral therapy (cART) and 64 HIV-uninfected controls with paired carotid intima-media thickness (cIMT) assessment. EVs were profiled with markers reflecting lymphoid, myeloid, and endothelial origin. Seventeen plasma inflammatory biomarkers were also assessed. Human umbilical vein endothelial cell (HUVEC) apoptosis was quantified after EV exposure. A significant correlation was observed in HIV-infected participants between cIMT and EVs expressing CD16, and the monocyte-related markers CD4, CD14, and CX3CR1 showed a similar but nonsignificant association with cIMT. No significant correlation between cIMT measurements from HIV-uninfected individuals and EVs was observed. Levels of serum amyloid A, C-reactive protein, and myeloperoxidase significantly correlated with CD14 + , CD16 + , and CX3CR1 + EVs. No correlation was noted between cIMT and soluble inflammatory markers. HUVECs showed increased necrosis after exposure to the EV-containing fraction of plasma derived from HIV-infected individuals compared to uninfected controls. Our study reveals that EVs expressing monocyte markers correlated with cIMT in HIV-infected individuals on cART. Moreover, EV fractions derived from HIV-infected individuals lead to greater endothelial cell death via necrotic pathways. Collectively, EVs have potential as biomarkers of and therapeutic targets in the pathogenesis of CVD in the setting of treated HIV disease. IMPORTANCE HIV-infected individuals have a 2-fold-increased risk of cardiovascular disease compared with the general population, yet the mechanisms underlying this comorbidity are unclear. Extracellular vesicles have emerged as important mediators in cell-cell communication and, given what we know of their biology, may drive inflammation contributing to cardiovascular disease in this vulnerable population.

Aims: Inflammation is involved in various processes of atherosclerosis development. Serum C-reactive protein (CRP) levels, a predictor for cardiovascular risk, are reportedly reduced by statins. However, several studies have demonstrated that CRP is a bystander during atherogenesis. While S100A12 has been focused on as an inflammatory molecule, it remains unclear whether statins affect circulating S100A12 levels. Here, we investigated whether atorvastatin treatment affected S100A12 and which biomarkers were correlated with changes in arterial inflammation., Methods: We performed a prospective, randomized open-labeled trial on whether atorvastatin affected arterial (carotid and thoracic aorta) inflammation using 18 fluorodeoxyglucose positron emission tomography/computed tomography ( 18 F-FDG-PET/CT) and inflammatory markers. Thirty-one statin-naïve patients with carotid atherosclerotic plaques were randomized to either a group receiving dietary management (n=15) or one receiving atorvastatin (10mg/day, n=16) for 12weeks. 18 F-FDG-PET/CT and flow-mediated vasodilation (FMD) were performed, the latter to evaluate endothelial function., Results: Atorvastatin, but not the diet-only treatment, significantly reduced LDL-cholesterol (LDL-C, -43%), serum CRP (-37%) and S100A12 levels (-28%) and improved FMD (+38%). 18 F-FDG-PET/CT demonstrated that atorvastatin, but not the diet-only treatment, significantly reduced accumulation of 18 F-FDG in the carotid artery and thoracic aorta. A multivariate analysis revealed that reduction in CRP, S100A12, LDL-C, oxidized-LDL, and increase in FMD were significantly associated with reduced arterial inflammation in the thoracic aorta, but not in the carotid artery., Conclusions: Atorvastatin treatment reduced S100A12/CRP levels, and the changes in these circulating markers mirrored the improvement in arterial inflammation. Our observations suggest that S100A12 may be an emerging therapeutic target for atherosclerosis.

Noninvasive imaging atherosclerotic (AS) plaque is of great importance for early diagnosis. Recently, CD93 in MΦ was linked to atherosclerosis development. Herein, we have investigated whether CD93 in MΦ is a potential novel target for atherosclerotic plaque imaging. CD93 hi and CD93 lo MΦ were prepared with or without LPS stimulation, before biological activity was evaluated. A rat AS model was produced with left carotid artery clamped. Whole-body/ex vivo phosphor autoradiography of the artery and biodistribution were investigated after incorporation of 3 H-2-DG into CD93 hi and CD93 lo MΦ or after 125 I-α-CD93 ( 125 I-anti-CD93mAb) injection. The plaque tissue was subjected to CD93/CD68 immunofluorescence/immunohistochemistry staining. CD93 hi and CD93 lo MΦ cells were successfully prepared without significant effect on bioactivity after incorporative labelled with 3 H-2-DG. The AS model was successfully established. Biodistribution studies showed that adoptive transfer of 3 H-2-DG-CD93 hi MΦ or 125 I- α-CD93 injection resulted in accumulation of radioactivity within the atherosclerotic plaque in the clamped left carotid artery. T/NT (target/non-target, left/right carotid artery) ratio was higher in the 3 H-2-DG-CD93 hi MΦ adoptive transfer group than in the 3 H-2-DG-CD93 lo MΦ group (p < .05). Plaque radioactivity in the 125 I-α-CD93 injection group was significantly higher than in the 125 I-IgG control group (p < .01). The higher radioactivity accumulated in the clamped left carotid artery was confirmed by phosphor autoradiography. More importantly, CD93/CD68 double-positive MΦ accumulated at the atherosclerotic plaque in 3 H-2-DG-CD93 hi MΦ adoptive transfer group, which correlated with plaque radioactivity (r = .99, p < .01). In summary, both adoptive-transferred 3 H-2-DG-labelled CD93 hi MΦ and 125 I-α-CD93 injection specifically targeted CD93 in atherosclerotic plaque. CD93 is a potential target in atherosclerotic plaque imaging., (© 2022 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Vascular endothelial function declines with age on average, but there is high variability in the magnitude of this decline within populations. Measurements of frailty, known as frailty index (FI), can be used as surrogates for biological age, but it is unknown if frailty relates to the age-related decline in vascular function. To examine this relation, we studied young (4-9 months) and old (23-32 months) C57BL6 mice of both sexes. We found that FI was greater in old compared with young mice, but did not differ between old male and female mice. Middle cerebral artery (MCA) and mesenteric artery endothelium-dependent dilation (EDD) also did not differ between old male and female mice; however, there were sex differences in the relations between FI and EDD. For the MCA, FI was inversely related to EDD among old female mice, but not old male mice. In contrast, for the mesenteric artery, FI was inversely related to EDD among old male mice, but not old female mice. A higher FI was related to a greater improvement in EDD with the superoxide scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl in the MCAs for old female mice and in the mesenteric arteries for old male mice. FI related to mesenteric artery gene expression negatively for extracellular superoxide dismutase (Sod3) and positively for interleukin-1β (Il1b). In summary, we found that the relation between frailty and endothelial function is dependent on sex and the artery examined. Arterial oxidative stress and proinflammatory signaling are potential mediators of the relations of frailty and endothelial function., (© The Author(s) 2021. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Persistent inflammation following vascular injury drives neointimal hyperplasia (NIH). Specialized lipid mediators (SPM) mediate resolution which attenuates inflammation and downstream NIH. We investigated the effects of a synthetic analogue of resolvin D1 (RvD1) on vascular cells and in a model of rat carotid angioplasty., Methods: Human venous VSMC and endothelial cells (EC) were employed in migration, cell shape, toxicity, proliferation and p65 nuclear translocation assays. Murine RAW 264.7 cells were utilized to test the effect of pro-resolving compounds on phagocytic activity. A model of rat carotid angioplasty was used to evaluate the effects of 17R/S-benzo-RvD1 (benzo-RvD1) and 17R-RvD1 applied to the adventitia via 25% Pluronic gel. Immunostaining was utilized to examine Ki67 expression and leukocyte recruitment. Morphometric analysis was performed on arteries harvested 14 days after injury., Results: Exposure to benzo-RvD1 attenuated PDGF- stimulated VSMC migration across a range of concentrations (0.1-100 nM), similar to that observed with 17R-RvD1. Pre-treatment with either Benzo-RvD1 or 17R-RvD1 (10, 100nM) attenuated PDGF-BB-induced VSMC cytoskeletal changes to nearly baseline dimensions. Benzo-RvD1 demonstrated modest anti-proliferative activity on VSMC and EC at various concentrations, without significant cytotoxicity. Benzo-RvD1 (10nM) inhibited p65 nuclear translocation in cytokine-stimulated EC by 21% (p<0.05), similar to 17R-RvD1. Consistent with pro-resolving activities of other SPM, both 17R-RvD1 and benzo-RvD1 increased the phagocytic activity of RAW 264.7 cells against S. Aureus and Zymosan particles. There were no significant differences in Ki-67 or CD45 staining observed on day 3 after angioplasty. Periadventitial treatment with benzo-RvD1 reduced carotid neointimal area at 14 days compared to control (0.08 mm2 v. 0.18 mm2; p<0.05), with similar efficacy to 17R-RvD1., Conclusions: 17R/S-benzo-RvD1 and 17R-RvD1 exhibit similar pro-resolving and anti-migratory activity in cell-based assays, and both compounds attenuated NIH following acute arterial injury in rats. Further studies of the mechanisms of resolution following vascular injury, and the translational potential of SPM analogues, are indicated., Competing Interests: MSC - co-Founder of VasaRx and co-inventor of IP related to this work with the Regents of University of California and Brigham and Women’s Hospital. This does not alter our adherence to PLOS-One policies on sharing data and materials.

Purpose: To optimize a sequence combining the delay alternating with nutation for tailored excitation (DANTE) preparative module with the variable-flip-angle rapid acquisition with relaxation enhancement (VF-RARE) sequence (DANTE-VF-RARE) and to investigate its feasibility for vessel wall imaging in Apolipoprotein E-Deficient (ApoE -/- ) mouse at 7 Tesla (T)., Materials and Methods: Specific T1/T2 values were used for producing a sharper vessel wall in the variable-flip-angle optimization scheme. The DANTE RF pulse flip angle and pulse train length were optimized for maximizing the wall-lumen contrast. ApoE -/- (fed high fat diet for 20/40/ 60 weeks, n = 9/4/4) and wild-type mice (controls, n = 3) were imaged at 7 T using VF-RARE, DANTE-VF-RARE, time-of-flight (TOF) angiography, and multi-slice T1-weighted 2D RARE coupled with inflow outflow saturation bands (IOSB-RARE). Wall-lumen contrast-to-noise-ratio efficiency (CNR eff ), lumen area (LA), and wall area (WA) were compared between DANTE-VF-RARE and 2D IOSB-RARE sequences. Additionally, linear regression analysis was conducted between MR measurements and histomorphometric planimetry results., Results: Residual blood signal was observed in the four out of eighteen carotids on VF-RARE images, whereas it was significantly suppressed on DANTE-VF-RARE images. Compared with IOSB-RARE, DANTE-VF-RARE offered significantly improved CNR eff (P < 0.001). The LA and WA were both comparable (P = 0.085 and 0.112, respectively) and showed excellent agreement between DANTE-VF-RARE and IOSB-RARE (ICC = 0.96 and 0.95, respectively). The luminal stenosis identified by DANTE-VF-RARE was in consistent with the results of TOF. Strong correlations were found between MR measurements and histopathological analysis for both WA (DANTE-VF-RARE: r = 0.92, slope = 0.94, P < 0.001; IOSB-RARE: r = 0.93, slope = 0.94, P < 0.001) and LA (DANTE-VF-RARE: r = 0.82, slope = 0.54, P < 0.001; IOSB-RARE: r = 0.78, slope = 0.50, P < 0.001)., Conclusion: DANTE-VF-RARE achieves effective blood signal suppression and is a feasible approach for the 3D carotid arterial wall imaging of ApoE -/- mouse at 7 T., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: The endothelium regulates vascular tonus by releasing nitric oxide (endothelium-derived nitric oxide, EDNO) and hyperpolarizing factor (endothelium-derived hyperpolarizing factor, EDHF). In vein grafts with poor runoff, lack of function of these factors causes severe intimal hyperplasia. This study evaluated how the functions of EDNO and EDHF are altered in artery grafts under poor runoff conditions., Materials and Methods: The right common carotid arteries of rabbits were excised and implanted in their original positions as autogenous grafts under normal runoff conditions ("nonoccluded grafts") or poor runoff conditions ("poor runoff grafts"). Histochemical changes, acetylcholine (ACh)-induced effects on endothelium-dependent relaxation and smooth muscle cell (SMC) hyperpolarization were examined., Results: Both artery graft types displayed negligible intimal hyperplasia. In the absence and presence of an EDNO synthase inhibitor, ACh-induced relaxation was lower in grafts with poor runoff than in nonoccluded grafts. Furthermore, ACh-induced but not nonreceptor agonist A23187-induced SMC hyperpolarization was lower in the poor runoff graft group than in the nonoccluded graft group., Conclusions: Unlike in those in vein grafts, the functions of EDNO and EDHF in autogenous carotid artery grafts under poor runoff conditions were reduced but partly maintained. In such artery grafts, intimal hyperplasia caused by surgical operation was not present. These results may explain some of the mechanisms underlying the improved patency of artery grafts compared with vein grafts., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Aims: Chronic kidney disease (CKD) is an independent risk factor for the development of coronary artery disease (CAD). For both, CKD and CAD, the intercellular transfer of microRNAs (miRs) through extracellular vesicles (EVs) is an important factor of disease development. Whether the combination of CAD and CKD affects endothelial function through cellular crosstalk of EV-incorporated miRs is still unknown., Methods and Results: Out of 172 screened CAD patients, 31 patients with CAD + CKD were identified and matched with 31 CAD patients without CKD. Additionally, 13 controls without CAD and CKD were included. Large EVs from CAD + CKD patients contained significantly lower levels of the vasculo-protective miR-130a-3p and miR-126-3p compared to CAD patients and controls. Flow cytometric analysis of plasma-derived EVs revealed significantly higher numbers of endothelial cell-derived EVs in CAD and CAD + CKD patients compared to controls. EVs from CAD + CKD patients impaired target human coronary artery endothelial cell (HCAEC) proliferation upon incubation in vitro. Consistent with the clinical data, treatment with the uraemia toxin indoxyl sulfate (IS)-reduced miR-130a-3p levels in HCAEC-derived EVs. EVs from IS-treated donor HCAECs-reduced proliferation and re-endothelialization in EV-recipient cells and induced an anti-angiogenic gene expression profile. In a mouse-experiment, intravenous treatment with EVs from IS-treated endothelial cells significantly impaired endothelial regeneration. On the molecular level, we found that IS leads to an up-regulation of the heterogenous nuclear ribonucleoprotein U (hnRNPU), which retains miR-130a-3p in the cell leading to reduced vesicular miR-130a-3p export and impaired EV-recipient cell proliferation., Conclusion: Our findings suggest that EV-miR-mediated vascular intercellular communication is altered in patients with CAD and CKD, promoting CKD-induced endothelial dysfunction., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2020. For permissions, please email: journals.permissions@oup.com.)

Objective: The aim: To study changes of the expression of synaptophysin (Syn) and vascular endothelial growth factor (VEGF) in neurons of the sensorimotor cortex (SMC) to reveal after unilateral ligation of the carotid artery, sensitization with brain antigen and their combination., Patients and Methods: Materials and methods: Experimental animals - Wistar rats (260-290 g). Experimental models: mobilization of the left common carotid artery, ligation of the indicated artery, sensitization with cerebral antigen, combination of sensitization with cerebral antigen and ligation of the carotid artery. Methods: immunohistochemistry, quantitative densitometric assessment., Results: Results: Dyscirculatory disorders of cerebral blood supply during unilateral mobilization or ligation of the common carotid artery, sensitization with cerebral antigen lead in rats to a transient decrease in synaptophysin expression and phase changes in VEGF expression in the SMC from the lesion side. These changes occur in the absence of morphological changes in the cerebral cortex., Conclusion: Conclusions: The absence of morphological changes in the SMC in the short term (10-30 days) after minor trauma to the common carotid artery (separation from the bed and n.vagus) or its ligation is accompanied by a transient decrease in Syn expression and some increase in VEGF, which may reflect a violation of synaptic function and the general metabolic activity of neurons. Sensitization with a brain antigen, leading to an increase in the level of anti-brain antibodies and immune complexes in the blood of rats, can act as an independent damaging factor for the brain, and also potentiates and prolongs changes caused by impaired blood circulation.

Phospholipase D (PLD) generates the signaling lipid phosphatidic acid (PA) and has been known to mediate proliferation signal in vascular smooth muscle cells (VSMCs). However, it remains unclear how PLD contributes to vascular diseases. VSMC proliferation directly contributes to the development and progression of cardiovascular disease, such as atherosclerosis and restenosis after angioplasty. Using the mouse carotid artery ligation model, we find that deletion of Pld1 gene inhibits neointima formation of the injuried blood vessels. PLD1 deficiency reduces the proliferation of VSMCs in both injured artery and primary cultures through the inhibition of ERK1/2 and AKT signals. Immunohistochemical staining of injured artery and flow cytometry analysis of VSMCs shows a reduction of the levels of reactive oxygen species (ROS) in Pld1 -/- VSMCs. An increase of intracellular ROS by hydrogen peroxide stimulation restored the reduced activities of ERK and AKT in Pld1 -/- VSMCs, whereas a reduction of ROS by N-acetyl-l-cysteine (NAC) scavenger lowered their activity in wild-type VSMCs. These results indicate that PLD1 plays a critical role in neointima, and that PLD1 mediates VSMC proliferation signal through promoting the production of ROS. Therefore, inhibition of PLD1 may be used as a therapeutic approach to suppress neointimal formation in atherosclerosis and restenosis after angioplasty., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Nucleotide-binding and oligomerization domain (NOD) receptor is a member of inherent immunity recognition receptor family. We investigated the NOD1/Rip2 signalling pathway on carotid arterial remodelling in spontaneously hypertensive rats (SHRs). SHRs were treated with NOD1 agonist (iE-DAP), inhibitor (ML130), or normal saline. We determined the NOD1 and Rip2 expression in carotid artery tissues, serum tumour necrosis factor-α (TNF-α) and monocyte chemotactic protein-1 (MCP-1). The carotid artery remodelling in 16-week SHRs was higher than that of 8-week SHRs and 16-week Wistar-Kyoto (WKY) rats. Expression of NOD1, Rip2, MCP-1 and TNF-α in 16-week SHRs was higher than that of 8-week SHRs and 16-week WKY rats. Blood pressure in iE-DAP-treated SHRs was higher than SHR-C group (no treatment), together with MCP-1, TNF-α, NOD1 and Rip2 expression, as well as carotid artery remodelling. In ML130-treated group, these aspects were completely the opposite. Taken together, inhibition of NOD1/Rip2 signalling pathway could delay the vascular remodelling process.

This study aimed to prepare an anti-Vascular cell adhesion protein 1 (VCAM-1) nanoscale ultrasound microbubble contrast agent using the hyperbranched self-assembly method for the molecular imaging diagnosis of atherosclerotic vulnerable plaques in rabbits. Twenty-five rabbits with carotid atherosclerosis were randomly divided into 5 groups, and the ear vein was injected with agents as follows: Groups A and B: nanoscale ultrasound microbubble contrast agent with and without anti-VCAM-1 agent; Groups C and D: SonoVue ultrasonic microbubble contrast agent, with and without anti-VCAM-1 agent; Control group: saline. The molecular imaging diagnosis of the atherosclerotic plaque, involved the examination of its vulnerability in the rabbit carotid artery was performed using the contrast ultrasound mode. The arrival and peaking time of the anti-VCAM-1 nanoscale ultrasound microbubble contrast agent (Group A) for plaque occurred earlier than those of the other groups ( p < 0.05), and with it, the plaque showed the strongest enhancement ( p < 0.05), followed by the SonoVue ultrasound microbubble contrast agent with anti-VCAM-1 group (Group C) and the self-made nanoscale ultrasound microbubble contrast agent group (Group B). No development was observed in the plaques of the SonoVue ultrasound microbubble contrast agent group and the control group. The anti-VCAM-1 nanoscale ultrasonic microbubble contrast agent, prepared using the self-assembly method, can facilitate the development effect of the carotid atherosclerotic vulnerable plaque, providing a basis for the molecular imaging diagnosis of carotid atherosclerotic vulnerable plaques., Competing Interests: The authors declare no conflict of interest., (© 2021 The Author(s). Published by IMR Press.)

The purpose of this study was to characterize the alterations in microstructural organization of arterial tissue using higher-order diffusion magnetic resonance schemes. Three porcine carotid artery models namely; native, collagenase treated and decellularized, were used to estimate the contribution of collagen and smooth muscle cells (SMC) on diffusion signal attenuation using gaussian and non-gaussian schemes. The samples were imaged in a 7 T preclinical scanner. High spatial and angular resolution diffusion weighted images (DWIs) were acquired using two multi-shell (max b-value = 3000 s/mm 2 ) acquisition protocols. The processed DWIs were fitted using monoexponential, stretched-exponential, kurtosis and bi-exponential schemes. Directionally variant and invariant microstructural parametric maps of the three artery models were obtained from the diffusion schemes. The parametric maps were used to assess the sensitivity of each diffusion scheme to collagen and SMC composition in arterial microstructural environment. The inter-model comparison showed significant differences across the considered models. The bi-exponential scheme based slow diffusion compartment (Ds) was highest in the absence of collagen, compared to native and decellularized microenvironments. In intra-model comparison, kurtosis along the radial direction was the highest. Overall, the results of this study demonstrate the efficacy of higher order dMRI schemes in mapping constituent specific alterations in arterial microstructure., (© 2021. The Author(s).)

Background: Vascular injury induces the exposure of subendothelial extracellular matrix (ECM) important to serve as substrate for platelets to adhere to the injured vessel wall to avoid massive blood loss. Different ECM proteins are known to initiate platelet adhesion and activation. In atherosclerotic mice, the small, leucine-rich proteoglycan biglycan is important for the regulation of thrombin activity via heparin cofactor II. However, nothing is known about the role of biglycan for hemostasis and thrombosis under nonatherosclerotic conditions., Methods: The role of biglycan for platelet adhesion and thrombus formation was investigated using a recombinant protein and biglycan knockout mice., Results: The present study identified biglycan as important ECM protein for the adhesion and activation of platelets, and the formation of three-dimensional thrombi under flow conditions. Platelet adhesion to immobilized biglycan induces the reorganization of the platelet cytoskeleton. Mechanistically, biglycan binds and activates the major collagen receptor glycoprotein (GP)VI, because reduced platelet adhesion to recombinant biglycan was observed when GPVI was blocked and enhanced tyrosine phosphorylation in a GPVI-dependent manner was observed when platelets were stimulated with biglycan. In vivo, the deficiency of biglycan resulted in reduced platelet adhesion to the injured carotid artery and prolonged bleeding times., Conclusions: Loss of biglycan in the vessel wall of mice but not in platelets led to reduced platelet adhesion at the injured carotid artery and prolonged bleeding times, suggesting a crucial role for biglycan as ECM protein that binds and activates platelets via GPVI upon vessel injury.

Background and Aims: Several studies demonstrated a role of active chronic inflammatory infiltrate in carotid plaques progression suggesting a possible link between cardiovascular risk factors and inflammation-related plaque instability. The aim of this study is therefore to evaluate the possible effects of cardiovascular risk factors on in situ expression of proinflammatory markers associated with carotid plaque instability., Methods and Results: A tissue microarray containing carotid plaques from 36 symptomatic (major stroke or transient ischemic attack) and 37 asymptomatic patients was built. Serial sections were employed to evaluate the expression of some inflammatory markers by immunohistochemistry [CD3, CD4a, CD8, CD20, CD86, CD163, interleukin (IL)-2, IL-6, IL-17]. Immunohistochemical data were analyzed to study the possible associations between in situ expression of inflammatory biomarker and the main cardiovascular risk factors. Our data demonstrated that plaque instability is associated with the high in situ expression of some cytokines, such as IL-2, IL-6, IL-17. Besides the female sex, none of the risk factors analyzed showed a significant association between the in situ expression of these markers and unstable plaques. A significant increase of IL-6-positive and IL-17-positive cells was observed in unstable atheromatous plaques of female patients, as compared with unstable plaques of male patients., Conclusions: Plaque destabilization is certainly correlated with the presence of the major cardiovascular risk factors, however, our results showed that, with the exception of sex, their action in the evolutive process of plaque instability seems rather nonspecific, favoring a general release of proinflammatory cytokines., Competing Interests: The authors declare no conflict of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Vascular restenosis remains a major problem in patients with coronary artery disease (CAD) and peripheral artery disease (PAD). Neointimal hyperplasia, defined by post-procedure proliferation and migration of vascular smooth muscle cells (VSMCs) is a key underlying pathology. Here we investigated the role of Interleukin 11 (IL-11) in a mouse model of injury-related plaque development. Apoe -/- mice were fed a hyperlipidaemic diet and subjected to carotid wire injury of the right carotid. Mice were injected with an anti-IL11 antibody (X203), IgG control antibody or buffer. We performed ultrasound analysis to assess vessel wall thickness and blood velocity. Using histology and immunofluorescence approaches, we determined the effects of IL-11 inhibition on VSMC and macrophages phenotypes and fibrosis. Treatment of mice with carotid wire injury using X203 significantly reduced post-endothelial injury vessel wall thickness, and injury-related plaque, when compared to control. Immunofluorescence staining of the injury-related plaque showed that X203 treatment did not reduce macrophage numbers, but reduced the number of VSMCs and lowered matrix metalloproteinase 2 (MMP2) levels and collagen content in comparison to control. X203 treatment was associated with a significant increase in smooth muscle protein 22α (SM22α) positive cells in injury-related plaque compared to control, suggesting preservation of the contractile VSMC phenotype. Interestingly, X203 also reduced the collagen content of uninjured carotid arteries as compared to IgG, showing an additional effect on hyperlipidemia-induced arterial remodeling in the absence of mechanical injury. Therapeutic inhibition of IL-11 reduced vessel wall thickness, attenuated neointimal hyperplasia, and has favorable effects on vascular remodeling following wire-induced endothelial injury. This suggests IL-11 inhibition as a potential novel therapeutic approach to reduce arterial stenosis following revascularization in CAD and PAD patients., (© 2021. The Author(s).)

Stroke models are vital tools in neuropharmacology and rehabilitation research. However, a classic and widely used model-the suture occlusion model-is not suitable for all research approaches, especially regarding thrombolysis. For embolic stroke models in thrombolytic research, the surgical procedures of thrombin injection in the middle cerebral artery or clot injection in the carotid artery involved are too sophisticated. Here, we report a new stroke model in mice that uses magnetic nanoparticle (MNP) cross-linked with thrombin to embolize. Briefly, after the magnet was positioned in the common carotid artery, MNP@Thrombin was injected from the tail vein. Within several minutes postinjection, the MNP@Thrombin accumulated in the carotid artery and induced thrombus formation. These complex clots were flushed into and subsequently blocked the cerebral artery. Collectively, these results suggested that this new method was a quick and easy stroke model that blocked hemisphere blood flow and damaged neural function. Importantly, this model had an excellent response to thrombolytic drugs. After urokinase injection, cerebral blood flow was restored and symptom scores were enhanced by nearly one. This method, including a quick synthesis of MNP and thrombin, provided an easy and minimally invasive process for a new stroke model that is usable in both pharmacological and rehabilitative research.

Perfusion catheters have recently emerged as a novel approach to deliver liquid anti-proliferative agents into flow obstructed arterial segments. The purpose of this study was to determine the impact of luminal delivery pressure on liquid drug penetration into the vessel wall. An ex vivo model using harvested porcine carotid arteries and a two-dimensional computational model were utilized to determine the impact of delivery pressure of liquid therapy into the arterial wall. A pig peripheral injury model determined the impact of intra-luminal delivery pressure on drug retention. Ex vivo results demonstrated that depth of fluid penetration varies from 6.93 ± 1.90% at 0 atm to 27.75 ± 6.61% penetration of the medial layer at 0.4 atm. Computational results had similar outcomes, as penetration varied between 4.4% and 22.84%. The in vivo results demonstrated significant increase in drug delivery to the arterial tissue at 0.4 atm versus 0.1 atm at 1 h (23.43 ± 13.59 ng/mg vs. 2.49 ± 1.81 ng/mg, p = 0.026) and 7 days (0.50 ± 0.39 ng/mg vs. 0.018 ± 0.023 ng/mg, p = 0.0496). The result of this study provides an innovative strategic and technical approach to enable targeted liquid therapy., (© 2021. The Author(s).)

Severe burns result in cardiovascular dysfunction, but responses in the peripheral vasculature are unclear. We hypothesize that severe burns disturb arterial contractility through acute changes in adrenergic and cholinergic receptor function. To address this, we investigated the changes in carotid artery contractility and relaxation following a severe burn. Thirty-four adult Sprague-Dawley male rats received a 40% total body surface area (TBSA) scald burn and fluid resuscitation using the Parkland formula. Control animals received sham burn procedure. Animals were serially euthanized between 6 h and 14 days after burn and endothelium-intact common carotid arteries were used for ex vivo force/relaxation measurements. At 6 h after burn, carotid arteries from burned animals demonstrated a > 50% decrease in cumulative dose-responses to norepinephrine (p < 0.05) and to 10 -7  M angiotensin II (p < 0.05). Notably, pre-constricted carotid arteries also demonstrated reduced relaxation responses to acetylcholine (p < 0.05) 6 h after burn, but not to sodium nitroprusside. Histologic examination of cross-sectional planes revealed significant increases in carotid artery wall thickness in burned rats at 6 h versus 3 days, with increased collagen expression in tunica media at 3 days (p < 0.05). Carotid artery dysfunction occurs within 6 h after severe burn, demonstrating decreased sensitivity to adrenergic- and angiotensin II-induced vasoconstriction and acetylcholine-induced relaxation., (© 2021. The Author(s).)