Rabbit Model Of Atherosclerosis Research Articles

Mechanistic exploration of the shenlian formula in the suppression of atherosclerosis progression via network pharmacology and in vivo experimental validation

Ethnopharmacological relevanceThe Shenlian formula (SL) is a Chinese medicine formula used to curb the development of atherosclerosis (AS) and cardiovascular disease in clinical practice. However, owing to the complexity of compounds and their related multiple targets in traditional Chinese medicine (TCM), it remains difficult and urgent to elucidate the underlying mechanisms at a holistic level. AimTo investigate the intrinsic mechanisms by which SL suppresses AS progression and to gain new insight into its clinical use. MethodsWe proposed a network pharmacology-based workflow to evaluate the mechanism by which SL affects AS via data analysis, target prediction, PPI network construction, GO and KEGG analyses, and a “drug-core ingredient-potential target-key pathway” network. Then, non-targeted lipidomic analysis was performed to explore the differential lipid metabolites in AS rats, revealing the possible mechanism by which SL affects atherosclerotic progression. Moreover, an AS rabbit model was constructed and gavaged for SL intervention. Serum lipid profiles and inflammatory cytokine indices were tested as an indication of the mitigating effect of SL on AS. ResultsA total of 89 bioactive compounds and 298 targets related to SL and AS, which play essential roles in this process, were identified, and a component-target-disease network was constructed. GO and KEGG analyses revealed that SL regulated metabolic pathway, lipids and atherosclerosis, the PI3K-Akt pathway, the MAPK pathway and so on. In vivo experimental validation revealed that a total of 43 different lipid metabolites regulated by SL were identified by non-targeted lipidomics, and glycerophospholipid metabolism was found to be an important mechanism for SL to interfere with AS. SL reduced the plaque area and decreased the levels of inflammatory cytokines (TNF-α and IL-4) and blood lipids (TC, TG, LDL-C, and ApoB) in HFD-induced AS models. In addition, HDL and ApoA1 levels are increased. PLA2 and Lipin1 are highly expressed in AS model, indicating their role in destabilizing glycerophosphatidylcholine metabolism and contributing to the onset and progression of ankylosing spondylitis. Moreover, SL intervention significantly reduced the level of pro-inflammatory cytokines; significantly down-regulated NF-kB/p65 expression, exhibiting anti-inflammatory activity. ConclusionThe Shenlian formula (SL) plays a pivotal role in the suppression of AS progression by targeting multiple pathways and mechanisms. This study provides novel insights into the essential genes and pathways associated with the prognosis and pathogenesis of AS.

Molecular Imaging of Fibroblast Activation in Rabbit Atherosclerotic Plaques: a Preclinical PET/CT Study.

Atherosclerosis remains the pathological basis of myocardial infarction and ischemic stroke. Early and accurate identification of plauqes is crucial to improve clinical outcomes of atherosclerosis patients. Our study aims to evaluate the potential value of fibroblast activation protein inhibitor (FAPI)-04 PET/CT in identifying plaques via a preclinical rabbit model of atherosclerosis. New Zealand white rabbits were fed high-fat diet (HFD), and randomly divided into the model group injured by the balloon, and the sham group only with incisions. Ultrasound was performed to detect plaques, and FAPI-avid was determined through Al18F-NOTA-FAPI-04 PET/CT. Mean standardized uptake values (SUVmean) in lesions were compared, and biodistribution of Al18F-NOTA-FAPI-04 and target-to-background ratios (TBRs) were calculated. Histological staining was performed to display arterial plaques, and autoradiography (ARG) was employed to measure the in vitro intensity of Al18F-NOTA-FAPI-04. At last, the correlation among FAP levels, plaque area, SUVmean values and fibrous cap thickness was assessed. The rabbit carotid and abdominal atherosclerosis model was established. Al18F-NOTA-FAPI-04 showed a higher uptake in carotid plaques (SUVmean 1.32 ± 0.11) and abdominal plaques (SUVmean 0.73 ± 0.13) compared to corresponding controls (SUVmean 1.07 ± 0.06; 0.46 ± 0.03) (P < 0.05). Biodistribution analysis of Al18F-NOTA-FAPI-04 revealed that the bigger plaques were delineated with higher TBRs. Pathological staining showed the formation of arterial plaques, and ARG staining exhibited a higher intensity of Al18F-NOTA-FAPI-04 in the bigger plaques. Lastly, plaque area was found to be positively correlated to FAP expression and SUVmean, while FAP expression was negatively correlated to fibrous cap thickness of plaques. We successfully achieve molecular imaging of fibroblast activation in atherosclerotic lesions of rabbits, suggesting Al18F-NOTA-FAPI-04 PET/CT may be a potentially valuable tool to identify plaques.

Accumulation of Iron Oxide-Based Contrast Agents in Rabbit Atherosclerotic Plaques in Relation to Plaque Age and Vulnerability Features.

In this study, a detailed characterization of a rabbit model of atherosclerosis was performed to assess the optimal time frame for evaluating plaque vulnerability using superparamagnetic iron oxide nanoparticle (SPION)-enhanced magnetic resonance imaging (MRI). The progression of atherosclerosis induced by ballooning and a high-cholesterol diet was monitored using angiography, and the resulting plaques were characterized using immunohistochemistry and histology. Morphometric analyses were performed to evaluate plaque size and vulnerability features. The accumulation of SPIONs (novel dextran-coated SPIONDex and ferumoxytol) in atherosclerotic plaques was investigated by histology and MRI and correlated with plaque age and vulnerability. Toxicity of SPIONDex was evaluated in rats. Weak positive correlations were detected between plaque age and intima thickness, and total macrophage load. A strong negative correlation was observed between the minimum fibrous cap thickness and plaque age as well as the mean macrophage load. The accumulation of SPION in the atherosclerotic plaques was detected by MRI 24 h after administration and was subsequently confirmed by Prussian blue staining of histological specimens. Positive correlations between Prussian blue signal in atherosclerotic plaques, plaque age, and macrophage load were detected. Very little iron was observed in the histological sections of the heart and kidney, whereas strong staining of SPIONDex and ferumoxytol was detected in the spleen and liver. In contrast to ferumoxytol, SPIONDex administration in rabbits was well tolerated without inducing hypersensitivity. The maximum tolerated dose in rat model was higher than 100 mg Fe/kg. Older atherosclerotic plaques with vulnerable features in rabbits are a useful tool for investigating iron oxide-based contrast agents for MRI. Based on the experimental data, SPIONDex particles constitute a promising candidate for further clinical translation as a safe formulation that offers the possibility of repeated administration free from the risks associated with other types of magnetic contrast agents.

An anti-inflammatory Glyburide-loaded nanoghost for atherosclerosis therapy: A red blood cell based bio-mimetic strategy

Inflammation control is becoming a critical strategy for atherosclerosis management, because inflammation is involved in plaque progression. In this regard, a biomimetic strategy using cell membrane-coated nanoparticles has some promising advantages. In the study, a RBC membrane-based nanoghost containing Glyburide (Glibenclamide) was prepared using an extrusion method. The hydrodynamic size and zeta potential of the nanoghost were changed compared to PLGA nanoparticles. In addition, a nanoghost with a diameter and shell size of 125 nm and 8.3 nm was obtained based on the TEM measurement. The fabricated nanoghost was not only hemocompatible but also was biocompatible, showing no significant induction of apoptosis. According to RT-PCR assay, the expression levels of inflammatory genes including NLRP3, IL-1β, IL-18 caspase1, 8 and 9 were decreased. In accordance with in vitro anti-inflammation properties, total foam cells, total surface area in tunica intima and population of CD14+ cells were decreased in the rabbit model of atherosclerosis upon nanoghost treatment, compared to positive control. Furthermore, macrophages in aorta sections exhibited M1 to M2 polarization. In general, the development of Glyburide-loaded nanoghost can be considered as a potential therapeutic for controlling the progression and inflammation of atherosclerotic plaque.

Drug-loaded balloon with built-in NIR controlled tip-separable microneedles for long-effective arteriosclerosis treatment

Drug-eluting balloon (DEB) angioplasty has emerged as an effective treatment for cardiovascular and cerebrovascular diseases. However, distal embolism and late lumen restenosis could be caused by drug loss during DEB handling and rapid drug metabolization. Here, a drug-loaded balloon equipped with tip-separable microneedles on the balloon surface (MNDLB) was developed. Inbuilt near-infrared (NIR) ring laser inside the catheter inner shaft was introduced to activate the biodegradable microneedle tips for the first time. The drug-loaded tips thus could be embedded in the vasculature and then released antiproliferative drug - paclitaxel slowly via polymer degradation for more than half a year. A significant increase in drug delivery efficiency and superior therapeutic effectiveness compared with the standard DEB were demonstrated using an atherosclerosis rabbit model.

Diagnostic Performance of 60 MHz High-Definition Intravascular Ultrasound versus Fourier Domain Optical Coherence Tomography for Identifying Plaque Rupture, Plaque Erosion, and Thrombosis in a Rabbit Model

Background: Most acute coronary syndromes occur due to coronary thrombosis caused by plaque rupture (PR) and plaque erosion (PE). Precise in vivo differentiation between PR and PE is challenging for intravascular imaging. This study is the first to determine the diagnostic performance of the novel 60 MHz high-definition intravascular ultrasound (HD-IVUS) for differentiating atherosclerotic plaque morphology influenced by local hemodynamic flow in rabbits. This study evaluated the diagnostic performance of 60 MHz HD-IVUS in identifying thrombosis in rabbits. Methods: We established 60 rabbit models of atherosclerosis with left common carotid artery (LCCA) stenosis and 30 FeCl3-induced LCCA thrombosis. Intravascular imaging was assessed with 60 MHz HD-IVUS and fourier-domain optical coherence tomography (FD-OCT). The present study investigated the diagnostic accuracy of 60 MHz HD-IVUS for PR and PE, as well as thrombosis, using OCT-diagnosis as a standard reference. Results: 60 MHz HD-IVUS for identifying atherosclerotic plaque morphology using plaque cavity and minor intimal irregularities showed high sensitivity and specificity; 92.0 and 90.0% for identifying OCT-defined PR, and 80.0 and 70.0% for OCT-defined PE, respectively. In a rabbit thrombus model, 60 MHz HD-IVUS showed high sensitivity (88.0%) and specificity (80.0%) in identifying OCT-defined thrombosis. Conclusions: 60 MHz HD-IVUS can accurately identify PR and thrombosis. Further studies should confirm the clinical value of this novel technique in PE diagnosis.

Neutrophil membrane camouflaged nanoprobes for NIR-II fluorescence imaging of inflamed, high-risk atherosclerotic plaques in mouse and rabbit models

Atherosclerosis is the leading cause of vascular death worldwide, and inflammation plays a crucial role in atherosclerotic plaque progression and rupture. Fluorescent nanoprobes that can detect inflamed, high-risk atherosclerotic plaques and evaluate the therapeutic efficacy at the animal level in vivo are particularly meaningful. Herein, we developed a kind of neutrophil membrane-camouflaged nanoprobe in the second near-infrared spectral region (NIR-II) for the imaging of inflamed, high-risk atherosclerotic plaques in both mouse and rabbit models. Compared with free indocyanine green (ICG), the NIR-II fluorescence intensity of neutrophil biomimetic nanoparticles (Neu-NPs) was increased by 276% and had good stability in serum. Neu-NPs could specifically target inflamed, high-risk atherosclerotic plaques through the interaction of the chemokine receptor LFA-1with ICAM-1, which are highly expressed in inflamed sites. In the ApoE−/− mouse model, the signal-to background (S/B) ratio of the Neu-NP-treated mice was 1.67-fold higher than that of NP-treated mice. Even in large animals, such as the New Zealand white rabbit atherosclerosis model, Neu-NPs exhibited excellent targeting to high-risk atherosclerotic plaques. Moreover, the Neu-NPs could be used to reflect the reduction in plaque inflammation after the treatment and could thus be used for therapeutic evaluation and drug screening. Therefore, this highly biocompatible neutrophil membrane-camouflaged nanoprobe may be a new nanocarrier for the treatment of chronic inflammatory diseases such as atherosclerosis. Moreover, the use of NIR-II fluorescence imaging in mice and rabbits with atherosclerosis extends the scope of this emerging imaging technology.

Norbixin, a natural dye that improves serum lipid profile in rabbits and prevents LDL oxidation

We hypothesized that norbixin, which is a carotenoid used as an orange/red natural food coloring additive, has anti-atherogenic properties. An in vitro oxidation assay with human LDL and a rabbit model of atherosclerosis were used to test this hypothesis. Norbixin inhibited the oxidation of isolated human LDL in a concentration-dependent manner. In the in vivo assay, rabbits were fed with a regular chow (control) or an atherogenic diet (0.5% cholesterol) alone or supplemented with norbixin (10, 30 or 100 mg/kg b.w.) for 60 days. Norbixin supplementation (30 and 100 mg/kg b.w.) increased HDL levels and reduced triglyceride levels and the atherogenic index of rabbits. This effect was associated with the decrease of serum levels of oxidized LDL, oxidized LDL antibodies and aortic tissue levels of lipid and protein oxidation in the atherogenic rabbits supplemented with norbixin. Atherogenic diet increased enzymatic (superoxide dismutase, catalase, glutathione reductase, and thioredoxin reductase-1) and non-enzymatic (non-protein thiol groups content) antioxidant defense systems in the aortic tissue but reduced the activity of paraoxonase-1 in the serum. All these changes were prevented by norbixin supplementation (10, 30 and 100 mg/kg b.w.). These results suggest that norbixin has atheroprotective potential by improving serum lipid profile and preventing oxidative modifications of circulating LDL and aortic tissue. Norbixin may, therefore, be beneficial in the control of atherosclerosis risk factors and can be further investigated as a candidate to be used not only as a functional food ingredient but also for therapeutic applications and in the nutraceutical industry.

Lipid-lowering treatment in a rabbit model of atherosclerosis: a vessel wall magnetic resonance imaging study.

BackgroundVessel wall magnetic resonance imaging (VWMRI) is currently one of the best imaging techniques for the non-invasive evaluation of intracranial atherosclerotic lesions. However, it is still impossible to obtain pathological specimens of intracranial atherosclerotic plaques in vivo. The establishment of experimental animal models of atherosclerotic lesions similar to those of humans could solve this deficiency.MethodsA model of abdominal aortic atherosclerosis in New Zealand white rabbits was established using abdominal aortic balloon dilatation combined with high-fat fodder. The pathological results were used as the reference standard for the successful establishment of the animal model. The rabbits with abdominal aortic atherosclerosis were randomly divided into the lipid-lowering treatment and the normal-fodder control groups. VWMRI and blood biochemical examinations were performed at 4, 12, and 24 weeks, and the radiologic-pathologic correlations were established.ResultsA rabbit abdominal aortic atherosclerosis model was established using balloon dilatation followed by high-fat fodder for 8 weeks. The lipid-lowering treatment reduced the plaque lipid core volume and decreased the plaque burden. However, it did not change plaque distribution, shape, or reverse vascular remodeling. Our pathological findings suggest that the lipid-lowering treatment reduced intraplaque macrophages but did not alter microvascular density.ConclusionsVWMRI accurately assessed the morphological changes of the plaques before and after the lipid-lowering treatment, and the results support the pathology results. VWMRI could be useful in experimental studies on the pathogenesis, diagnosis, and treatment of atherosclerotic lesions.

Tongxinluo May Alleviate Inflammation and Improve the Stability of Atherosclerotic Plaques by Changing the Intestinal Flora.

Intestinal flora plays an important role in atherosclerosis. Tongxinluo, as a multi-target Chinese medicine to improve atherosclerosis, whether it can improve atherosclerosis by affecting the intestinal flora is worth exploring. We established a vulnerable plaque model of atherosclerosis in New Zealand white rabbits by high cholesterol diet and balloon injury (HCB), and performed Tongxinluo intervention. We detected the level of inflammation by immunohistochemistry, Western Blot, and ELISA, analyzed plaque characteristics by calculating the vulnerability index, and analyzed the changes of gut microbiota and metabolites by 16S rRNA gene sequencing and untargeted metabolomic sequencing. The results showed that Tongxinluo intervention improved plaque stability, reduced inflammatory response, inhibited NLRP3 inflammatory pathway, increased the relative abundance of beneficial bacteria such as Alistipes which reduced by HCB, and increased the content of beneficial metabolites such as trans-ferulic acid in feces. Through correlation analysis, we found that some metabolites were significantly correlated with some bacteria and some inflammatory factors. In particular, the metabolite trans-ferulic acid was also significantly positively correlated with plaque stability. Our further studies showed that trans-ferulic acid could also inhibit the NLRP3 inflammatory pathway. In conclusion, Tongxinluo can improve plaque stability and reduce inflammation in atherosclerotic rabbits, which may be achieved by modulating intestinal flora and intestinal metabolism. Our study provides new views for the role of Tongxinluo in improving atherosclerotic vulnerable plaque, which has important clinical significance.

Delphinidin-3-O-glucoside, an active compound of Hibiscus sabdariffa calyces, inhibits oxidative stress and inflammation in rabbits with atherosclerosis

Context Delphinidin-3-O-glucoside (DP) is a bioactive compound of Hibiscus sabdariffa L. (Malvaceae) (Roselle) calyces and exerts endothelial protection and lipid-lowering activities, which provided a basis for the prevention and treatment of cardiovascular diseases. Objectives To investigate the therapeutic effects of DP against atherosclerosis. Materials and methods A rabbit model of atherosclerosis (AS) was established by 12 weeks of a high-fat diet (HFD). The rabbits were divided into five groups: control, AS, simvastatin (4 mg/kg), and two DP groups (10 and 20 mg/kg). After treatment with DP or simvastatin by oral gavage for 12 weeks, the lipid profiles were measured. Histopathological assessment of the aorta was performed by H&E staining. Oxidative stress and inflammation-related markers were analyzed by ELISA kit and real-time RT-PCR. Results DP (20 mg/kg) decreased serum TG (2.36 ± 0.66 vs. 4.33 ± 0.27 mmol/L for the AS group), TC, LDL-C, and HDL-C (all p < 0.05). DP (20 mg/kg) also reduced lipid levels in the liver and aorta. DP (20 mg/kg) down-regulated the mRNA levels of IL-6, VCAM-1, and NF-κB and up-regulated the mRNA levels of GSH-PX and SOD1. Conclusions This study proved that DP alleviated the HFD-induced oxidative stress and inflammation in atherosclerosis rabbits. These results provided the scientific basis for developing novel therapies.

Antiatherosclerotic effects of corilagin via suppression of the LOX-1/MyD88/NF-κB signaling pathway in vivo and in vitro.

Corilagin, a natural polyphenol compound isolated from Phyllanthus urinaria L., exerts various pharmacological effects, such as antihyperglycemic, antitumor, and antioxidative stress properties, but the mechanisms underlying the antiatherosclerotic effects of corilagin have not been entirely elucidated. In the present study, we investigated the antiatherosclerotic effects of corilagin using a high-fat diet (HFD)-induced atherosclerotic rabbit model and ox-LDL-induced vascular smooth muscle cells (VSMCs) and explored the underlying molecular mechanisms. The serum lipid levels were measured through an enzymatic colorimetric assay. A histological analysis of rabbit aortas was performed after hematoxylin-eosin and oil red O staining. The proliferation of ox-LDL-induced VSMCs was detected using MTT assays, and the migration of cells was determined by wound scratch assays. In addition, the mRNA and protein expression levels of lectin-like ox-LDL receptor-1 (LOX-1), myeloid differentiation factor 88 (MyD88), nuclear factor-kappa B (NF-κB), monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor α (TNF-α) were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting assays. Our results indicate that corilagin significantly reduced the serum levels of TC, TG and LDL-C, increased the HDL-C levels, decreased the intimal thickening in the thoracic aorta, and reduced the formation of foam cells in an HFD-induced rabbit atherosclerosis model. Moreover, corilagin suppressed the proliferation and migration of ox-LDL-induced VSMCs and reduced LOX-1, MyD88, NF-κB, MCP-1, and TNF-α mRNA and protein expression in vivo and in vitro. These data demonstrate that corilagin exerts antiatherosclerotic effects in vivo and in vitro and that the mechanisms may be closely associated with downregulation of the LOX-1/MyD88/NF-κB pathway.

Association Between Stent Implantation and Progression of Nontarget Lesions in a Rabbit Model of Atherosclerosis.

Progression of nontarget lesions (NTLs) after percutaneous coronary intervention (PCI) has been reported. However, it remains unknown whether progression of NTLs was causally related to stenting. This study was undertaken to test the hypothesis that stent implantation triggers acute phase response and systemic inflammation which may be associated with progression of NTLs. Thirty New Zealand rabbits receiving endothelial denudation and atherogenic diet were randomly divided into stenting, sham, and control groups. Angiography and intravascular ultrasonography were performed in the stenting and sham groups, and stent implantation performed only in the stenting group. Histopathologic study was conducted and serum levels of APPs (acute phase proteins) measured in all rabbits. Proteomics analysis was performed to screen the potential proteins related to NTLs progression after stent implantation. The serum levels of APPs and inflammatory cytokines were measured in 147 patients undergoing coronary angiography or PCI. Plaque burden in the NTLs was significantly increased 12 weeks after stent implantation in the stenting group versus sham group. Serum levels of APPs and their protein expression in NTLs were significantly increased and responsible for stenting-triggered inflammation. In patients receiving PCI, serum levels of SAA-1 (serum amyloid A protein 1), CRP (C-reactive protein), TNF (tumor necrosis factor)-α, and IL (interleukin)-6 were substantially elevated up to 1 month post-PCI. In a rabbit model of atherosclerosis, stent implantation triggered acute phase response and systemic inflammation, which was associated with increased plaque burden and pathological features of unstable plaque in NTLs. The potential mechanism involved vessel injury-triggered acute phase response manifested as increased serum levels of SAA-1, CRP, and LBP (lipopolysaccharide-binding protein) and their protein expression in NTLs. These findings provided a new insight into the relation between stent implantation and progression of NTLs, and further studies are warranted to clarify the detailed mechanism and clinical significance of these preliminary results. Registration: URL: http://www.chictr.org.cn; Unique identifier: ChiCTR1900026393. Graphic Abstract: A graphic abstract is available for this article.

Translocator protein imaging with 18F-FEDAC-positron emission tomography in rabbit atherosclerosis and its presence in human coronary vulnerable plaques

Background and aimsThis study aimed to investigate whether N-benzyl-N-methyl-2-[7,8-dihydro-7-(2-[18F]fluoroethyl)-8-oxo-2-phenyl-9H-purin-9-yl]acetamide (18F-FEDAC), a probe for translocator protein (TSPO), can visualize atherosclerotic lesions in rabbits and whether TSPO is localized in human coronary plaques. Methods18F-FEDAC-PET of a rabbit model of atherosclerosis induced by a 0.5% cholesterol diet and balloon injury of the left carotid artery (n = 7) was performed eight weeks after the injury. The autoradiography intensity of 18F-FEDAC in carotid artery tissue sections was measured, and TSPO expression was evaluated immunohistochemically. TSPO expression was examined in human coronary arteries obtained from autopsy cases (n = 16), and in human coronary plaques (n = 12) aspirated from patients with acute myocardial infarction (AMI). Results18F-FEDAC-PET visualized the atherosclerotic lesions in rabbits as high-uptake areas, and the standard uptake value was higher in injured arteries (0.574 ± 0.24) than in uninjured arteries (0.277 ± 0.13, p < 0.05) or myocardium (0.189 ± 0.07, p < 0.05). Immunostaining showed more macrophages and more TSPO expression in atherosclerotic lesions than in uninjured arteries. TSPO was localized in macrophages, and arterial autoradiography intensity was positively correlated with macrophage concentration (r = 0.64) and TSPO (r = 0.67). TSPO expression in human coronary arteries was higher in AMI cases than in non-cardiac death, or in the vulnerable plaques than in early or stable lesions, respectively. TSPO was localized in macrophages in all aspirated coronary plaques with thrombi. Conclusions18F-FEDAC-PET can visualize atherosclerotic lesions, and TSPO-expression may be a marker of high-risk coronary plaques.

In Vivo Human Single-Chain Fragment Variable Phage Display-Assisted Identification of Galectin-3 as a New Biomarker of Atherosclerosis.

BackgroundAtherosclerosis is a complex pathology in which dysfunctional endothelium, activated leucocytes, macrophages, and lipid‐laden foam cells are implicated, and in which plaque disruption is driven by many putative actors. This study aimed to identify accurate targetable biomarkers using new in vivo approaches to propose tools for improved diagnosis and treatment.Methods and ResultsHuman scFv (single‐chain fragment variable) selected by in vivo phage display in a rabbit model of atherosclerosis was reformatted as scFv fused to the scFv‐Fc (single‐chain fragment variable fused to the crystallizable fragment of immunoglobulin G format) antibodies. Their reactivity was tested using flow cytometry and immunoassays, and aorta sections from animal models and human carotid and coronary artery specimens. A pool of atherosclerotic proteins from human endarterectomies was co‐immunoprecipitated with the selected scFv‐Fc followed by mass spectrometry for target identification. Near‐infrared fluorescence imaging was performed in Apoe −/− mice after injection of an Alexa Fluor 647–labeled scFv‐Fc‐2c antibody produced in a baculovirus system with 2 additional cysteine residues (ie, 2c) for future coupling to nano‐objects for theranostic applications. One scFv‐Fc clone (P3) displayed the highest cross‐reactivity against atherosclerotic lesion sections (rabbit, mouse, and human) and was chosen for translational development. Mass spectrometry identified galectin‐3, a β‐galactoside‐binding lectin, as the leader target. ELISA and immunofluorescence assays with a commercial anti‐galectin‐3 antibody confirmed this specificity. P3 scFv‐Fc‐2c specifically targeted atherosclerotic plaques in the Apoe −/− mouse model.ConclusionsThese results provide evidence that the P3 antibody holds great promise for molecular imaging of atherosclerosis and other inflammatory pathologies involving macrophages. Recently, galectin‐3 was proposed as a high‐value biomarker for the assessment of coronary and carotid atherosclerosis.

Effects of different enantiomers of amlodipine on lipid profiles and vasomotor factors in atherosclerotic rabbits.

This research aimed to describe the functions of vascular endothelial cells (VECs) in protecting target organs and the anti-atherosclerotic effects of different enantiomers of amlodipine on a rabbit model of atherosclerosis. Thirty male New Zealand white rabbits were randomly allocated to four groups (nA = 9, nB = 7, nC = 7, and nD = 7 rabbits): rabbits in group-A (control group) were fed a high-fat diet, group-B rabbits were fed a high-fat diet plus 2.5 mg/kg/day S-amlodipine, group-C rabbits were fed a high-fat diet plus 2.5 mg/kg/day R-amlodipine, and group-D rabbits were fed a high-fat diet plus 5 mg/kg/day racemic amlodipine. Different enantiomers of amlodipine did not influence lipid profiles and serum level of eNOS in the rabbit atherosclerosis model but decreased ET-1 expression to some extent. The serum NO and iNOS levels in the drug intervention groups were significantly reduced. No significant differences in the rabbits’ body weights were observed. At the 4th and 8th weeks, the serum lipid profiles significantly increased in high cholesterol diet groups. The serum ET-1 level was significantly increased in each group of rabbits at the 8th week. Both S-amlodipine and R-amlodipine may protect the endothelium by reducing the serum ET-1 level, downregulating iNOS expression.

Colchicine reduces atherosclerotic plaque vulnerability in rabbits

Background and aimsThe anti-inflammatory agent colchicine is gaining interest as a treatment for coronary artery disease. However, the effects of colchicine in atherosclerotic animal models are mostly unknown. This study aimed to evaluate colchicine in a rabbit model of atherosclerosis. MethodsTwenty-two rabbits were fed a 0.5% cholesterol-enriched diet for 10 weeks and then randomized to receive either oral saline (n=11) or colchicine (350 μg/kg/day; n=11) for 6 weeks, with 0.2% cholesterol-diet during the treatment period. We performed intravascular ultrasound imaging (at start and end of treatment) and histology analyses of the descending thoracic aorta. Leucocyte activation was assessed in vitro on blood samples obtained during treatment. ResultsColchicine prevented positive aortic vascular remodelling (p=0.029 vs placebo). This effect was even more marked at high plasma cholesterol level (third quartile of plasma cholesterol, p=0.020). At high cholesterol level, both atherosclerotic plaque and media areas on histomorphology were reduced by colchicine compared to placebo (p=0.031 and p=0.039, respectively). Plaque fibrosis and macrophage area were reduced by colchicine (Masson's trichrome stain: p=0.038; RAM-11: p=0.026). The plaque vulnerability index, assessed by histology, was reduced by colchicine (p=0.040). Elastin/type I collagen ratio in media was significantly higher with colchicine compared to placebo (p=0.013). At a high level of plasma cholesterol, in vitro LPS challenge revealed a decrease in monocyte activation following treatment with colchicine (p<0.001) and no change in the placebo group (p=0.353). ConclusionsColchicine decreases plaque vulnerability with reductions in plaque inflammation, medial fibrosis, outward vascular remodelling and ex vivo monocyte activation.

Nanomicellar preparation, HB-ATV-8, protects against endothelial dysfunction induced by a high-fat diet

Background and Aims: Hypercholesterolemia promotes endothelial dysfunction, which might trigger the establishment of cardiovascular diseases. HB-ATV-8 comprises nanomicelles of membrane lipids of the T. aquaticus and an amphipathic peptide that protects against atherosclerotic lesions. However, it is unknown if HB-ATV-8 also maintains the function of the endothelial cells of the thoracic arteries in the model of atherosclerosis in rabbits.

Elevation of homocysteine levels in the plasma leads to deregulation of lipid metabolism in a rabbit model of atherosclerosis

Cardiovascular disease, the leading cause of death worldwide, is in the majority of cases a result of atherosclerosis, which can be only to 50% explained by established risk factors including hypercholesterolemia (HCL). Hyperhomocysteinemia (HHcy) is an independent risk factor for atherosclerosis. It is linked to numerous human diseases, strongly correlates with cardiovascular and all-cause mortality, and is associated with deregulation of lipid metabolism. HHcy is found in 5-10% of the general population and up to 30% of the elderly, and is mainly due to genetic defects or vitamin deficiency resulting in blocking of homocysteine (Hcy) degradation. We induced HHcy by dietary partial blocking of Hcy degradation and intravenous injections of Hcy into a rabbit model of atherosclerosis in the presence or absence of HCL. Deficiency of vitamins and choline required for Hcy degradation in the absence of HCL leads to an accumulation of lipid droplets in cells of the aorta including endothelial cells, smooth muscle cells, and fibroblasts, an accumulation of dilated ER in fibroblasts of the aorta as well as decreased LPC18:1 and 18:2 levels in muscles. In addition, while dietary blocking of Hcy degradation results in increased triacylglycerol (TAG) concentrations in blood cells, further elevation of Hcy levels leads to their decrease, but also to ceramide, lyso-phosphatidylcholine (LPC), phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin accumulation in blood cells, compared to the diet blocking Hcy degradation alone. A decrease of TAG levels was also observed in the liver, in response to intravenous injections of Hcy and in rabbits receiving a combined diet that blocked Hcy degradation and induced HCL. In conclusion our data suggest that dietary blocking of Hcy degradation as well as Hcy itself may contribute to different human pathologies by deregulation of lipid metabolism in different tissues.

Treatment of atherosclerosis through transplantation of endothelial progenitor cells overexpressing dimethylarginine dimethylaminohydrolase (DDAH) in rabbits

BackgroundEndothelial dysfunction is a key event in the development of vascular diseases, including atherosclerosis. Endothelial progenitor cells (EPCs) play an important role in vascular repair. Decreased dimethylarginine dimethylaminohydrolase (DDAH) activity is observed in several pathological conditions, and it is associated with an increased risk of vascular disease. We hypothesized that bone marrow-derived EPCs and combination therapy with DDAH2-EPCs could reduce plaque size and ameliorate endothelial dysfunction in an atherosclerosis rabbit model. MethodFour groups of rabbits (n = 8 per group) were subjected to a hyperlipidemic diet for a month. After establishing the atherosclerosis model, rabbits received 4 × 106 EPC, EPCs expressing DDAH2, through femoral vein injection, or saline (the control group with basic food and the untreated group). One month after transplantation, plaque thickness, endothelial function, oxidative stress, and inflammatory mRNAs, DDAH, and eNOS function were assessed. ResultsDDAH2-EPCs transplantation (p < 0.05) and EPCs transplantation (p < 0.05) were both associated with a reduction in plaque size compared to the control saline injection. The antiproliferative and antiatherogenic effects of EPCs were further enhanced by the overexpression of DDAH2 (p < 0.05, DDAH2-EPCs vs. EPCs). Furthermore, DDAH2-EPCs transplantation significantly increased endothelium integrity compared to the EPCs transplantation. ConclusionTransplantation of EPCs overexpressing DDAH2 may enhance the repair of injuredendothelium by reducing inflammation and restoring endothelial function. Therefore, pCMV6-mediated DDAH2 gene-transfected EPCs are a potentially valuable tool for the treatment of atherosclerosis.