Preparation of a Single-Cell Suspension from Mouse Carotid Arteries for Single-Cell Sequencing.

Carotid arteries are major blood vessels in the neck that supply blood and oxygen to the brain, but carotid stenosis occurs when carotid arteries are clogged by plaque. Revealing the cellular composition of the carotid artery at the single-cell level is essential for treating carotid atherosclerosis. However, there is no ready-to-use protocol for the preparation of single-cell suspensions from carotid arteries. To obtain a suitable protocol for the dissociation of normal carotid arteries at the single-cell level with less damage to cells, we designed a two-step digestion method by integrating the digestion process of collagenase/DNase and trypsin. Acridine orange/propidium iodide (AO/PI) dual-fluorescence counting was used to detect cell viability and concentration, and it was found that the single-cell suspension satisfied the requirements for single-cell sequencing, with the viability of cells over 85% and a high cell concentration. After single-cell data processing, a median of ~2500 transcripts per cell were detected in each carotid artery cell. Notably, a variety of cell types of the normal carotid artery, including vascular smooth muscle cells (VSMCs), fibroblasts, endothelial cells (ECs), and macrophages and dendritic cells (Mφ/DCs), were concurrently detectable. This protocol may be applied to prepare a single-cell suspension of blood vessels from other tissues with appropriate modifications.

Dexmedetomidine Stereotaxic Injection Alleviates Neuronal Loss Following Bilateral Common Carotid Artery Occlusion via Up-Regulation of BDNF Expression.

BACKGROUND/AIM: Neurogenesis is an important process in the recovery from neurological damage caused by ischemic lesions. Endogenous neurogenesis is insufficient to restore neuronal damage following cerebral ischemia. Dexmedetomidine (DEX) exerts neuroprotective effects against cerebral ischemia and ischemia/reperfusion injury. DEX promotes neurogenesis, including neuronal proliferation and maturation in the hippocampus. In a previous study, we showed that early neurogenesis increased 3 days after bilateral common carotid artery occlusion (BCCAO). In this study, we investigated the effect of DEX on neurogenesis 3 days after BCCAO. MATERIALS AND METHODS: Male Sprague-Dawley (SD) rats (7-8 weeks old) were used as a BCCAO model. Right and left common carotid arteries of the rats were occluded using 4-0 silk sutures. Two hours after surgery, an intracranial DEX injection was administered to rats that underwent surgery using a stereotaxic injector. Brains were obtained from control and BCCAO rats 3 days after surgery. Immunohistochemistry was performed on the cortex and dentate gyrus of the hippocampus using a NeuN antibody. Western blot was performed with HIF1α and brain-derived neurotrophic factor (BDNF) antibodies. RESULTS: The number of mature neurons decreased 3 days after BCCAO, but DEX treatment alleviated neural loss in the parietal cortex and hippocampus. Up-regulation of BDNF was also observed after dexmedetomidine treatment. CONCLUSION: Stereotaxic injection of dexmedetomidine alleviates neural loss following BCCAO by up-regulating BDNF expression.

Okanin from Coreopsis tinctoria Nutt. alleviates cognitive impairment in bilateral common carotid artery occlusion mice by regulating the miR-7/NLRP3 axis in microglia.

Cognitive impairment is the main clinical feature following stroke, and microglia-mediated inflammatory response is a major contributor to it. Coreopsis tinctoria Nutt., an edible chrysanthemum, is commonly used as a functional ingredient in healthcare beverages and food. Okanin, the main active ingredient of Coreopsis tinctoria Nutt. flower, inhibits microglial activation. However, the role of okanin in cognitive impairment following ischemic stroke is still unknown. In this study, we investigated the effect of okanin on ischemic stroke and its underlying mechanism both in vivo and in vitro. Okanin was found to attenuate cognitive impairment in bilateral common carotid artery occlusion (BCCAO) mice, inhibit neuronal loss and microglial activation, decrease NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activation, and increase miR-7 expression. Okanin suppressed NLRP3 inflammasome activation in oxygen-glucose deprivation (OGD) and lipopolysaccharide (LPS)-stimulated microglia by increasing miR-7 expression and inhibited microglia-induced neuronal injury. This study provides new insights into the role of okanin in ischemic stroke and shows that the miR-7/NLRP3 axis plays an important role in mediating the beneficial effects of okanin on cerebral ischemia. These findings suggest that okanin has great potential as a functional food for stroke recovery.

EXPRESSION OF SYNAPTOFYSIN AND VEGF IN THE SENSOMOTOR CORTEX DURING THE CAROTID ARTERY LIGATION, THE BRAIN ANTIGEN SENSITIZATION AND THEIR COMBINATIONS.

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.

DN200434 Inhibits Vascular Smooth Muscle Cell Proliferation and Prevents Neointima Formation in Mice after Carotid Artery Ligation.

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.

Retinal Oxygen Delivery and Metabolism Response to Hyperoxia During Bilateral Common Carotid Artery Occlusion in Rats.

Purpose: The purpose of the current study was to test the hypothesis that responses of total retinal blood flow (TRBF), inner retinal oxygen delivery (DO2), metabolism (MO2), and extraction fraction (OEF) to hyperoxia are higher after minutes of bilateral common carotid artery occlusion (BCCAO) as compared to days of BCCAO. Methods: Twenty-eight rats were subjected to BCCAO for 30 minutes (n = 12), 1 day (n = 8), or 3 days (n = 8). Eight of the 12 rats were also evaluated at baseline, prior to BCCAO. During room air breathing (RA) and 100% O2 inspiration (hyperoxia), blood flow and phosphorescence lifetime imaging were performed to measure TRBF and vascular O2 contents, respectively. DO2, MO2, and OEF were calculated from these measurements. Results: After 30 minutes or 3 days of BCCAO, TRBF did not differ between RA and hyperoxia conditions (P ≥ 0.14) but decreased under hyperoxia after 1 day (P = 0.01). Compared to RA, DO2 and MO2 were increased under hyperoxia after 30 minutes of BCCAO (P ≤ 0.02). Additionally, MO2 was decreased under hyperoxia after 1 day of BCCAO (P = 0.04). OEF was decreased under hyperoxia compared to RA (P < 0.001). Under hyperoxia, TRBF and DO2 were reduced after all BCCAO durations compared to baseline (P ≤ 0.04), whereas MO2 did not differ from baseline after 30 minutes of BCCAO (P = 1.00). Conclusions: The findings indicate that hyperoxia introduced minutes after ischemia can reduce DO2 impairments and potentially return MO2 to approximately normal values. This information contributes to the knowledge of the effect of supplemental oxygen intervention on TRBF, DO2, MO2, and OEF outcomes after variable durations of ischemia.

The OPG/RANKL/RANK system modulates calcification of common carotid artery in simulated microgravity rats by regulating NF-κB pathway.

Functional and structural adaptation of common carotid artery could be one of the important causes of postflight orthostatic intolerance after microgravity exposure, the mechanisms of which remain unclear. Recent evidence indicates that long-term spaceflight increases carotid artery stiffness, which might present a high risk to astronaut health and postflight working ability. Studies have suggested that vascular calcification is a common pathological change in cardiovascular diseases that is mainly manifested as an increase in vascular stiffness. Therefore, this study investigated whether simulated microgravity induces calcification of common carotid artery and to elucidate the underlying mechanisms. Four-week-old hindlimb-unweighted (HU) rats were used to simulate the deconditioning effects of microgravity on cardiovascular system. We found that simulated microgravity induced vascular smooth muscle cell (VSMC) osteogenic differentiation and medial calcification, increased receptor activator of nuclear factor κB (NF-κB) ligand (RANKL) and RANK expression, and enhanced NF-κB activation in rat common carotid artery. In vitro activation of the RANK pathway with exogenous RANKL, a RANK ligand, increased RANK and osteoprotegerin (OPG) expression in HU rats. Moreover, the expression of osteogenic markers and activation of NF-κB in HU rats were further enhanced by exogenous RANKL but suppressed by the RANK inhibitor osteoprotegerin fusion protein (OPG-Fc). These results indicated that the OPG/RANKL/RANK system modulates VSMC osteogenic differentiation and medial calcification of common carotid artery in simulated microgravity rats by regulating the NF-kB pathway.

Exploring the mechanism of Danggui Buxue Decoction in regulating atherosclerotic disease network based on integrated pharmacological methods.

OBJECTIVE: To explore the mechanism of Danggui Buxue Decoction (DGBXD) in regulating Atherosclerosis (AS) network based on integrated pharmacological methods. METHODS: The active ingredients and targets of DGBXD are obtained from TCMSP database and ETCM. AS-related targets were collected from the Genecards and OMIM databases. The drug-disease protein interaction (PPI) networks were constructed by Cytoscape. Meanwhile, it was used to screen out densely interacting regions, namely clusters. Finally, Gene Ontology (GO) annotations are performed on the targets and genes in the cluster to obtain biological processes, and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations are performed on the targets of the PPI network to obtain signaling pathways. RESULTS: A total of 212 known targets, 265 potential targets and 229 AS genes were obtained. The 'DGBXD known-AS PPI network' and 'DGBXD-AS PPI Network' were constructed and analyzed. DGBXD can regulate inflammation, platelet activation, endothelial cell apoptosis, oxidative stress, lipid metabolism, vascular smooth muscle proliferation, angiogenesis, TNF, HIF-1, FoxO signaling pathway, etc. The experimental data showed that compared with the model group, the expressions of ICAM-1, VCAM-1, and interleukin (IL)-1ß protein and mRNA in the DGBXD group decreased (P<0.05). However, plasma IL-1ß, TNF-α, and MCP-1 in the DGBXD group were not significantly different from the model group (P>0.05). CONCLUSION: The mechanism of DGBXD in the treatment of AS may be related to the improvement of extracellular matrix (ECM) deposition in the blood vessel wall and the anti-vascular local inflammatory response, which may provide a reference for the study of the mechanism of DGBXD.

Corilagin ameliorates atherosclerosis by regulating MMP-1, -2, and -9 expression in vitro and in vivo.

Corilagin is a polyphenol has been identified anti-inflammatory properties. However, the anti-atherosclerotic effects of corilagin are not well understood. Here, we evaluated the anti-atherosclerotic effects and the underlying mechanisms of corilagin. We also verified whether corilagin can reverse atherosclerosis by regulating matrix metalloproteinase (MMP)-1, -2, and -9 in vitro and in vivo. An atherosclerosis model was established by feeding minipigs a high-fat diet combined with balloon injury, and the effects of different concentrations of corilagin on common carotid artery atherosclerosis in minipigs were monitored. Murine RAW264.7 macrophages were cultured and induced with oxidized low-density lipoprotein; fluorescence microscopy revealed the nuclear translocation of NF-κB. Furthermore, MMP-1, -2, and -9 expression in common carotid artery plaques and cellular models was detected by immunohistochemistry, western blotting, and RT-PCR. The pathological results suggested that the vascular intima of the model control group was significantly thickened, a large amount of collagen fibers was deposited, endothelial cells were damaged and detached, and plaque and foam cell formation occurred to varying degrees on the arterial wall, with lipid deposition. Corilagin treatment significantly reduced the degree of injury in the common carotid artery and decreased the number of lipid plaques and foam cells. Additionally, corilagin downregulated MMP-1, -2, and -9 expression in the common carotid artery plaques and cellular model. Moreover, corilagin significantly inhibited NF-κB nuclear translocation in vitro. Overall, corilagin exerted substantial therapeutic effects on experimental atherosclerotic minipigs via the downregulation of MMP-1, -2, and -9 expression.

MFG-E8 Regulates Vascular Smooth Muscle Cell Migration Through Dose-Dependent Mediation of Actin Polymerization.

Background Migration of vascular smooth muscle cells (VSMCs) is the main contributor to neointimal formation. The Arp2/3 (actin-related proteins 2 and 3) complex activates actin polymerization and is involved in lamellipodia formation during VSMC migration. Milk fat globule-epidermal growth factor 8 (MFG-E8) is a glycoprotein expressed in VSMCs. We hypothesized that MFG-E8 regulates VSMC migration through modulation of Arp2/3-mediated actin polymerization. Methods and Results To determine whether MFG-E8 is essential for VSMC migration, a model of neointimal hyperplasia was induced in the common carotid artery of wild-type and MFG-E8 knockout mice, and the extent of neointimal formation was evaluated. Genetic deletion of MFG-E8 in mice attenuated injury-induced neointimal hyperplasia. Cultured VSMCs deficient in MFG-E8 exhibited decreased cell migration. Immunofluorescence and immunoblotting revealed decreased Arp2 but not Arp3 expression in the common carotid arteries and VSMCs deficient in MFG-E8. Exogenous administration of recombinant MFG-E8 biphasically and dose-dependently regulated the cultured VSMCs. At a low concentration, MFG-E8 upregulated Arp2 expression. By contrast, MFG-E8 at a high concentration reduced the Arp2 level and significantly attenuated actin assembly. Arp2 upregulation mediated by low-dose MFG-E8 was abolished by treating cultured VSMCs with ß1 integrin function-blocking antibody and Rac1 inhibitors. Moreover, treatment of the artery with a high dose of recombinant MFG-E8 diminished injury-induced neointimal hyperplasia and reduced VSMC migration. Conclusions MFG-E8 plays a critical role in VSMC migration through dose-dependent regulation of Arp2-mediated actin polymerization. These findings suggest that high doses of MFG-E8 may have therapeutic potential for treating vascular occlusive diseases.

Protective Functions of Liver X Receptor α in Established Vulnerable Plaques: Involvement of Regulating Endoplasmic Reticulum-Mediated Macrophage Apoptosis and Efferocytosis.

Background Liver X receptor (LXR) belongs to the metabolic nuclear receptor superfamily, which plays a critical regulatory role in vascular physiology/pathology. However, effects of systemic LXR activation on established vulnerable plaques and the potential isotype-specific role involved remain unclear. Methods and Results The 8-week-old male apolipoprotein E-/- mice went through carotid branch ligation and renal artery constriction, combined with a high-fat diet. Plaques in the left carotid artery acquired vulnerable features 4 weeks later, confirmed by magnetic resonance imaging scans and histological analysis. From that time on, mice were injected intraperitoneally daily with PBS or GW3965 (10 mg/kg per day) for an additional 4 weeks. Treatment with LXR agonists reduced the lesion volume by 52.61%, compared with the vehicle group. More important, a profile of less intraplaque hemorrhage detection and necrotic core formation was found. These actions collectively attenuated the incidence of plaque rupture. Mechanistically, reduced lesional apoptosis, enhanced efferocytosis, and alleviated endoplasmic reticulum stress are involved in the process. Furthermore, genetic ablation of LXRα, but not LXRß, blunted the protective effects of LXR on the endoplasmic reticulum stress-elicited C/EBP-homologous protein pathway in peritoneal macrophages. In concert with the LXRα-predominant role in vitro, activated LXR failed to stabilize vulnerable plaques and correct the acquired cellular anomalies in LXRα-/- apolipoprotein E-/- mice. Conclusions Our results revealed that LXRα mediates the capacity of LXR activation to stabilize vulnerable plaques and prevent plaque rupture via amelioration of macrophage endoplasmic reticulum stress, lesional apoptosis, and defective efferocytosis. These findings might expand the application scenarios of LXR therapeutics for atherosclerosis.

Knockout of the NONO Gene Inhibits Neointima Formation in a Mouse Model of Vascular Injury.

[Figure: see text].

Truncated YY1 interacts with BASP1 through a 339KLK341 motif in YY1 and suppresses vascular smooth muscle cell growth and intimal hyperplasia after vascular injury.

AIMS: In-stent restenosis and late stent thrombosis are complications associated with the use of metallic and drug-coated stents. Strategies that inhibit vascular smooth muscle cell (SMC) proliferation without affecting endothelial cell (EC) growth would be helpful in reducing complications arising from percutaneous interventions. SMC hyperplasia is also a pathologic feature of graft stenosis and fistula failure. Our group previously showed that forced expression of the injury-inducible zinc finger (ZNF) transcription factor, yin yang-1 (YY1), comprising 414 residues inhibits neointima formation in carotid arteries of rabbits and rats. YY1 inhibits SMC proliferation without affecting EC growth in vitro. Identifying a shorter version of YY1 retaining cell-selective inhibition would make it more amenable for potential use as a gene therapeutic agent. METHODS AND RESULTS: We dissected YY1 into a range of shorter fragments (YY1A-D, YY1Δ) and found that the first two ZNFs in YY1 (construct YY1B, spanning 52 residues) repressed SMC proliferation. Receptor binding domain analysis predicts a three-residue (339KLK341) interaction domain. Mutation of 339KLK341 to 339AAA341 in YY1B (called YY1Bm) abrogated YY1B's ability to inhibit SMC but not EC proliferation and migration. Incubation of recombinant GST-YY1B and GST-YY1Bm with SMC lysates followed by precipitation with glutathione-agarose beads and mass spectrometric analysis identified a novel interaction between YY1B and BASP1. Overexpression of BASP1, like YY1, inhibited SMC but not EC proliferation and migration. BASP1 siRNA partially rescued SMC from growth inhibition by YY1B. In the rat carotid balloon injury model, adenoviral overexpression of YY1B, like full-length YY1, reduced neointima formation, whereas YY1Bm had no such effect. CD31+ immunostaining suggested YY1B could increase re-endothelialization in a 339KLK341-dependent manner. CONCLUSION: These studies identify a truncated form of YY1 (YY1B) that can interact with BASP1 and inhibit SMC proliferation, migration, and intimal hyperplasia after balloon injury of rat carotid arteries as effectively as full length YY1. We demonstrate the therapeutic potential of YY1B in vascular proliferative disease.

Arterial wall injury and miRNA expression induced by stent retriever thrombectomy under stenotic conditions in a dog model.

BACKGROUND: Acute ischemic stroke can be caused by in situ stenotic vessel occlusion. In the present study, we compared the extent of arterial wall damage and miRNA expression following stent retriever use under normal and stenotic conditions. METHODS: The stent retriever procedure was simulated in three dogs by the creation of four stenoses on each side of the common carotid artery (CCA) to allow five stent passages. Device safety was also assessed in normal control models by five passages through both CCAs. Device manipulation-related damage to the arterial walls was evaluated and compared between groups by angiography and pathological analysis. Real-time PCR was used to evaluate the differences in the expression of miRNAs between the two groups. RESULTS: Twenty-four stenoses were created in three model dogs, and the mean stenosis rate was 65.58%±18.95%. Angiography revealed greater vasospasm in the stenotic group than in the non-stenotic group (1.17±0.17 vs 0.5±0.23; P=0.04). Pathological examination revealed that SR passage through the stenotic lumen caused higher injury scores (1.63±0.19 vs 0.25±0.09 for the non-stenotic lumen; P<0.001), more endothelial denudation (1.79±0.13 vs 0.58±0.13 for the non-stenotic lumen; P<0.001), and increased thrombus deposition (0.71±0.14 vs 0±0 for the non-stenotic lumen; P<0.001). miR21-3p, miR29-3p, and miR26a were upregulated in stenotic vessels compared with non-stenotic vessels after SR thrombectomy (P<0.001). CONCLUSION: In our model dogs, SR thrombectomy resulted in more severe tissue damage to the arterial wall under stenotic conditions than under non-stenotic conditions. The damage may have resulted from upregulation of miR21-3p, miR29-3p, and miR26a expression.

Leptin Receptor Deficiency Protects Mice against Chronic Cerebral Hypoperfusion-Induced Neuroinflammation and White Matter Lesions.

Leptin participates in the inflammatory responses in multiple cell types and animal models. Chronic cerebral hypoperfusion (CCH) induces inflammation in the central nervous system (CNS), which acts as one of the main reasons for CCH-induced white matter lesions (WMLs). But whether leptin participates in the pathogenesis of CCH-induced WMLs remains unknown. Therefore, we performed bilateral common carotid artery stenosis (BCAS) to induce CCH on the leptin receptor- (LepR-) deficient db/db mice, aiming to evaluate the possible involvement of leptin in CCH-induced cognitive impairment, WMLs, and neuroinflammation, and further explore the effect of leptin on chronic hypoxia-induced inflammation using the BV2 microglial cell line. After four weeks of BCAS, wild-type mice showed significant working memory deficits, WMLs, activation of microglia and astrocytes, decrease in the number of oligodendrocytes, downregulation of myelin basic protein expression, and increase in the expression of TNF-α and IL-1ß; however, four weeks of BCAS failed to induce significant changes in the LepR-deficient db/db mice but elevated the production of anti-inflammatory cytokines and activated the M2 microglia. We further confirmed that leptin would aggravate the hypoxia-induced proinflammatory cytokine expression in the BV2 microglia cell line. These results suggested that LepR deficiency would protect mice against the CCH-induced cognitive impairment and WMLs by inhibiting glial activation and suppressing proinflammatory responses as well as promoting anti-inflammatory cytokine expression and M2 microglia activation in the white matter.

Elastin haploinsufficiency in mice has divergent effects on arterial remodeling with aging depending on sex.

Elastin is a primary structural protein in the arterial wall that contributes to vascular mechanical properties and degrades with aging. Aging is associated with arterial stiffening and an increase in blood pressure. There is evidence that arterial aging follows different timelines with sex. Our objective was to investigate how elastin content affects arterial remodeling in male and female mice with aging. We used male and female wild-type (Eln+/+) and elastin heterozygous (Eln+/-) mice at 6, 12, and 24 mo of age and measured their blood pressure and arterial morphology, wall structure, protein content, circumferential stress, stretch ratio, and stiffness. Two arteries were used with varying contents of elastin: the left common carotid and ascending aorta. We show that Eln+/- arteries start at a different homeostatic set point for circumferential wall stress, stretch, and material stiffness but show similar increases with aging to Eln+/+ mice. With aging, structural stiffness is greatly increased, while material stiffness and circumferential stress are only slightly increased, highlighting the importance of maintaining these homeostatic values. Circumferential stretch shows the smallest change with age and may be important for controlling cellular phenotype. Independent sex differences are mostly associated with males being larger than females; however, many of the measured factors show age × sex and/or genotype × sex interactions, indicating that males and females follow different cardiovascular remodeling timelines with aging and are differentially affected by reduced elastin content.NEW & NOTEWORTHY A comprehensive study on arterial mechanical behavior as a function of elastin content, aging, and sex in mice. Elastin haploinsufficient arteries start at a different homeostatic set point for mechanical parameters such as circumferential stress, stretch, and material stiffness. Structural stiffness of the arterial wall greatly increases with aging, as expected, but there are interactions between sex and aging for most of the mechanical parameters that are important to consider in future work.

An Effective and Simple Way to Establish Elastase-Induced Middle Carotid Artery Fusiform Aneurysms in Rabbits.

OBJECTIVE: Elastase-induced aneurysms in rabbits have been proposed as a preclinical tool for device development, but there is still much deficiency in those aneurismal models. So we need to explore the efficient and convenient animal models for the investigation of intracranial aneurysms. Then, we compared and analyzed three methods of elastase-induced carotid artery aneurysms in rabbits and aimed to find a simple, effective, and reproducible method for creating elastase-induced aneurysms. METHODS: 42 standard feeding male adult Japanese white rabbits (3.05 ± 0.65 kg) were randomly divided into 3 groups and treated with elastase ablation to create common carotid artery (RCCA) aneurysm models: Group A (root-RCCA medication group, n = 12), Group B (mid-RCCA medication group, n = 18), and Group C (ligated RCCA+medication group, n = 12). For Group A, the origin of the RCCA was blocked by two temporary aneurysm clips, and the resulting 2 cm cavity was infused with elastase for 20 min, then the clip was removed and the RCCA was not ligated. For Group B, the middle part of RCCA was treated the same way as Group A and the RCCA was not ligated. For Group C, the middle part of RCCA was treated as Group B, but the distal RCCA was ligated. After the aneurysm models were created for 3 weeks, prior to sacrificing the animals, color Doppler ultrasound and angiography were performed for blood flow measurements inside the aneurysms. Histological analysis (such as SMA-α, CD31, CD34, CD68, collagen IV, and Ki67) and the other relevant indexes were compared between the ideal model's aneurysmal tissues and the human intracranial aneurysm's tissues to confirm whether we have successfully established elastase-induced aneurysm models. RESULTS: Compared with human intracranial aneurysm specimens by the color Doppler ultrasound, angiography, and changes in the inner diameter of arteries, all three methods have successfully established the elastase-induced aneurysm models. Histology showed that biological responses were similar to both human cerebral aneurysms and previously published elastase-induced rabbit aneurysm models. Group A and Group B had the same morphology, but Group A had a higher mortality rate than Group B. Group B and Group C had different morphology. The aneurysm of Group C was more similar to human cerebral aneurysms but had a higher mortality rate than Group B. Group B was confirmed not only as an alternative method but also as a more safe and effective method for creating elastase-induced aneurysm models. CONCLUSION: Through analysis and comparison, the Group B is proven to be the simplest, reproducible, and most effective modeling method. The aneurysm model established by Group B can be used for basic research related to aneurysm mechanism. We have provided a new and effective method for basic research on aneurysm.

Moderate Alcohol Consumption Targets S100ß+ Vascular Stem Cells and Attenuates Injury-Induced Neointimal Hyperplasia.

BACKGROUND: Stem cells present in the vessel wall may be triggered in response to injurious stimuli to undergo differentiation and contribute to vascular disease development. Our aim was to determine the effect of moderate alcohol (EtOH) exposure on the expansion and differentiation of S100 calcium-binding protein B positive (S100ß+ ) resident vascular stem cells and their contribution to pathologic vessel remodeling in a mouse model of arteriosclerosis. METHODS AND RESULTS: Lineage tracing analysis of S100ß+ cells was performed in male and female S100ß-eGFP/Cre/ERT2-dTomato transgenic mice treated daily with or without EtOH by oral gavage (peak BAC: 15 mM or 0.07%) following left common carotid artery ligation for 14 days. Carotid arteries (ligated or sham-operated) were harvested for morphological analysis and confocal assessment of fluorescent-tagged S100 ß + cells in FFPE carotid cross sections. Ligation-induced carotid remodeling was more robust in males than in females. EtOH-gavaged mice had less adventitial thickening and markedly reduced neointimal formation compared to controls, with a more pronounced inhibitory effect in males compared to females. There was significant expansion of S100ß+ -marked cells in vessels postligation, primarily in the neointimal compartment. EtOH treatment reduced the fraction of S100ß+ cells in carotid cross sections, concomitant with attenuated remodeling. In vitro, EtOH attenuated Sonic Hedgehog-stimulated myogenic differentiation (as evidenced by reduced calponin and myosin heavy chain expression) of isolated murine S100ß+ vascular stem cells. CONCLUSIONS: These data highlight resident vascular S100ß+ stem cells as a novel target population for alcohol and suggest that regulation of these progenitors in adult arteries, particularly in males, may be an important mechanism contributing to the antiatherogenic effects of moderate alcohol consumption.

Expression of the C677T Polymorphism of the 5, 10-Methylenetetrahydrofolate Reductase (MTHFR) Gene in Patients with Carotid Artery Atherosclerosis.

BACKGROUND The C677T polymorphism of the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene polymorphism has been associated with hypertension and coronary heart disease, but its relationship with carotid artery remains unknown. This study aimed to investigate the association between the C677T polymorphism of the MTHFR gene in patients with confirmed carotid artery atherosclerosis. MATERIAL AND METHODS This retrospective study included 210 patients with carotid artery atherosclerosis (the patient group) and 210 controls (the control group). Color Doppler ultrasound was used to identify carotid artery intimo-medial thickness and atherosclerotic plaques. Sanger sequencing using the polymerase chain reaction (PCR) was used to detect the MTHFR C677T gene polymorphism. Systolic blood pressure (SBP), diastolic blood pressure (DBP), fasting plasma glucose (FPG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), triglyceride (TG), glycosylated hemoglobin (HbA1c), and other laboratory indicators were measured. RESULTS SBP, DBP, FPG, TC, LDL-C, HbA1c, and intimo-medial thickness were significantly increased in the patient group compared with the control group, and HDL-C was significantly lower. The allele frequencies of the C667T locus of MTHFR gene were significantly different between the two groups (P<0.05), and the TT genotype and the T allele frequencies in the patient group were higher than in the control group. Logistic regression analysis showed that SBP, TC, LDL-C, and the C667T MTHFR gene polymorphism were risk factors for carotid artery atherosclerosis. CONCLUSIONS The C677T polymorphism of the MTHFR gene was expressed in patients with carotid artery atherosclerosis.

Ginsenoside Rg1 prevents vascular intimal hyperplasia involved by SDF-1α/CXCR4, SCF/c-kit and FKN/CX3CR1 axes in a rat balloon injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Panax ginseng C. A. Mey. is a traditional tonic that has been used for thousands of years, and has positive effects on vascular diseases. Ginsenoside Rg1 (GS-Rg1) is one of the active ingredients of Panax ginseng C. A. Mey. and has been shown to have beneficial effects against ischemia/reperfusion injury. Our previously study has found that GS-Rg1 can mobilize bone marrow stem cells and inhibit vascular smooth muscle proliferation and phenotype transformation. However, pharmacological effects and mechanism of GS-Rg1 in inhibiting intimal hyperplasia is still unknown. AIM OF THE STUDY: This study was aimed to investigate whether GS-Rg1 prevented vascular intimal hyperplasia, and the involvement of stromal cell-derived factor-1α (SDF-1α)/CXCR4, stem cell factor (SCF)/c-kit and fractalkine (FKN)/CX3CR1 axes. MATERIALS AND METHODS: Rats were operated with carotid artery balloon injury. The treatment groups were injected with 4, 8 and 16 mg/kg of GS-Rg1 for 14 days. The degree of intimal hyperplasia was evaluated by histopathological examination. The expression of α-SMA (α-smooth muscle actin) and CD133 were detected by double-label immunofluorescence. Serum levels of SDF-1α, SCF and soluble FKN (sFKN) were detected by enzyme linked immunosorbent assay (ELISA). The protein expressions of SCF, SDF-1α and FKN, as well as the receptors c-kit, CXC chemokine receptor type 4 (CXCR4) and CX3C chemokine receptor type 1 (CX3CR1) were detected by immunochemistry. RESULTS: GS-Rg1 reduced intimal hyperplasia by evidence of the values of NIA, the ratio of NIA/MA, and the ratio of NIA/IELA and the ratio of NIA/LA, especially in 16 mg/kg group. Furthermore, GS-Rg1 8 mg/kg group and 16 mg/kg group decreased the protein expressions of the SDF-1α/CXCR4, SCF/c-kit and FKN/CX3CR1 axes in neointima, meanwhile GS-Rg1 8 mg/kg group and 16 mg/kg group also attenuated the expressions of SDF-1α, SCF and sFKN in serum. In addition, the expression of α-SMA and CD133 marked smooth muscle progenitor cells (SMPCs) was decreased after GS-Rg1 treatment. CONCLUSIONS: GS-Rg1 has a positive effect on inhibiting vascular intimal hyperplasia, and the underlying mechanism is related to inhibitory expression of SDF-1α/CXCR4, SCF/c-kit and FKN/CX3CR1 axes.