Atherosclerotic Rabbit Model Research Articles

Omega-3 polyunsaturated fatty acids, especially DHA and EPA, remold gut microbiota to suppress inflammation in rabbits with atherosclerosis

Atherosclerosis (AS) caused by a high-fat diet becomes a critical cause of cardiovascular diseases, which has been regarded as an urgent public health problem. Omega-3 polyunsaturated fatty acids (omega-3 PUFAs) have many beneficial effects on human health. However, in recent years, the critical role of gut microbiota in atherosclerosis has been gradually proposed, and the impact of omega-3 PUFAs on cardiovascular diseases via gut microbiota in rabbits remains unknown. Likewise, the role of omega-3 PUFAs in regulating endothelial lesions, visceral injury, and vascular inflammation caused by high-fat diet-induced trimethylamine-nitrogen oxide (TMAO) has not been reported. Here, we investigated the effects of omega-3 PUFAs administered orally on atherosclerosis via an atherosclerotic rabbit model induced by a high-fat diet. The results showed that omega-3 PUFAs obtained from deep-sea fish oil can alleviate high-fat diet-induced atherosclerosis by inhibiting the increase of plasma TMAO and the formation of liver foam cells, reducing inflammatory factors, fat deposition, and plasma lipid levels and suppressing organ damage. Furthermore, gut microbiota disrupted by a high-fat diet were remodeled in rabbits treated with omega-3 PUFAs. These results further confirm that omega-3 PUFAs are promising nutritional and medical health foods that can be supplemented daily to prevent cardiovascular diseases.

A novel glycyrrhizin acid-coated stent reduces neointimal formation in a rabbit iliac artery model.

Introduction: Most drug-eluting stents (DESs) inhibit intimal hyperplasia but impair re-endothelialization. This study aimed to evaluate in vivo strut coverage and neointimal growth in a new glycyrrhizin acid (GA)-eluting stent. Methods: New Zealand White rabbits (n = 20) with atherosclerotic plaques were randomly divided into three groups based on implanted iliac artery stents: bare-metal stents (BMSs), rapamycin-eluting stents, and GA-eluting stents. After the in vivo intravascular ultrasound (IVUS) assessment at 28 days, the vessels were harvested for scanning electron microscopy (SEM) and histology. After 4 weeks of follow-up, the stent and external elastic lamina (EEL) areas were compared among the groups. Results: The rapamycin- or GA-eluting stents significantly reduced the neointimal area compared with BMSs, though GA-eluting stents had the lowest reduction. There were more uncovered struts for rapamycin-eluting stents than those for GA-eluting stents and bare-metal stents. The endothelial nitric oxide synthase (eNOS) expression in GA-eluting stents was much higher than that in BMSs and rapamycin-eluting stents, even though the endothelial coverage between struts was equivalent between BMSs and GA-eluting stents. Moreover, GA-eluting stents markedly promoted re-endothelialization and improved arterial healing compared to rapamycin-eluting stents in a rabbit atherosclerotic model. Conclusion: In conclusion, the novel GA-coated stent used in this study inhibited intimal hyperplasia and promoted re-endothelialization.

Preclinical Evaluation of Coronary Artery Stents.

Coronary artery disease is a major contributor to morbidity and mortality worldwide. While lifestyle changes and medication are the cornerstones of treatment, coronary artery balloon angioplasty and stenting are routinely performed on patients with acute coronary syndromes and chronic coronary artery disease who remain symptomatic with optical medical treatment. Several generations of coronary stents have been developed over recent decades. Balloon angioplasty and stenting are supported by the use of pharmaceutical agents applied onto balloons and the stent surface, either to advance the healing properties of the artery post-intervention or to prevent the formation of restenosis. New devices need to be rigorously tested for safety and efficacy before acceptance into clinical practice; thus, there is a continuing need for reliable and reproducible preclinical methods of stent evaluation. We describe here a pig coronary artery model as well as an atherosclerotic rabbit model for coronary artery stent research and describe basic steps in intravascular imaging and stent histology.

Hepatic Alarmins and Mitochondrial Dysfunction under Residual Hyperlipidemic Stress Lead to Irreversible NAFLD.

Nonalcoholic fatty liver disease (NAFLD) includes a range of progressive disorders generated by excess lipid accumulation in the liver leading to hepatic steatosis and eventually fibrosis. We aimed to identify by high performance mass spectrometry-based proteomics the main signaling pathways and liver proteome changes induced by hypercholesterolemia in a rabbit atherosclerotic model that induced high accumulation of lipids in the liver. The effect of combined lipid-lowering drugs (statins and anti-PCSK9 monoclonal antibody) were used after the interruption of the hypercholesterolemic diet to identify also the potential mediators, such as alarmins, responsible for the irreversible NAFLD build up under the hyperlipidemic sustained stress. Proteomic analysis revealed a number of proteins whose abundance was altered. They were components of metabolic pathways including fatty-acid degradation, glycolysis/gluconeogenesis, and nonalcoholic fatty liver disease. Mitochondrial dysfunction indicated alteration at the mitochondrial respiratory chain level and down-regulation of NADH: ubiquinone oxidoreductase. The expression of a majority of cytochromes (P4502E1, b5, and c) were up-regulated by lipid-lowering treatment. Long-term hyperlipidemic stress, even with a low-fat diet and lipid-lowering treatment, was accompanied by alarmin release (annexins, galectins, HSPs, HMGB1, S100 proteins, calreticulin, and fibronectin) that generated local inflammation and induced liver steatosis and aggressive fibrosis (by high abundance of galectin 3, fibronectin, and calreticulin). The novel findings of this study were related to the residual effects of hyperlipidemic stress with consistent, combined lipid-lowering treatment with statin and inhibitor of PCSK9.

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.

Abstract 13468: Single-Cell Driven Drug Repositioning Approach Identifies a New Anti-Atherosclerotic Compound Targeting Myeloid Cells

Introduction: Inflammation is a key driver of atherosclerosis. The identification of new treatments and strategies targeting the specific inflammatory signaling that persists in optimally treated atherosclerotic patients remain an unmet clinical need. Hypothesis: The integration of time-of-flight mass cytometry (CyTOF), gene expression analysis, and drug repositioning computational approaches applied to pre-clinical validation in models of the disease using non-invasive imaging will help to identify drugs tailored to residual inflammatory signaling in patients with atherosclerosis. Methods: We developed a new systems biology pipeline to identify new therapeutic/indication pair that combines CyTOF and RNA sequencing (RNA-seq) data analysis of clinical samples from patients with carotid and coronary artery disease (CVD) ( Fig. 1A ). This approach allowed the identification of new uses for existing compounds. The predicted anti-atherosclerotic effect was validated in ApoE-/- mice and in a rabbit model of atherosclerosis using 18 F-Fluorodeoxyglucose (FDG) PET-MRI. Results: We found a specific inflammatory signature in CD14 + monocytes stimulated with CVD plasma that was used to identify 50 small molecules predicted to reverse this signature. CyTOF screening of the top eight candidate compounds showed that Saracatinib—a phase 2a-ready Src/Abl inhibitor—blunted the inflammatory response found in CD14 + monocytes ( Fig. 1B ). The anti-atherosclerotic efficacy of Saracatinib was confirmed in ApoE-/- mice fed a high fat diet ( Fig.1C ). In the atherosclerotic rabbit model, Saracatinib reduced plaque inflammation measured as reduction of 18 F-FDG uptake by PET-MRI ( Fig. 1D ). Conclusions: These data support the use of our integrated systems biology pipeline as a powerful approach to identify new immune signature specific to patients with atherosclerosis that can be leveraged to identify new indications for existing drugs to treat cardiovascular disease.

IVUS\\IVPA hybrid intravascular molecular imaging of angiogenesis in atherosclerotic plaques via RGDfk peptide-targeted nanoprobes

Current intravascular imaging modalities face hurdles in the molecular evaluation of progressed plaques. This study aims to construct a novel hybrid imaging system (intravascular ultrasound/intravascular photoacoustic, IVPA/IVUS) via RGDfk peptide-targeted nanoparticles for monitoring angiogenesis in progressed atherosclerotic plaques in a rabbit model. An atherosclerotic rabbit model was induced by abdominal aorta balloon de-endothelialization followed by a high-fat diet. A human serum albumin (HSA)-based nanoprobe modified with RGDfk peptide was constructed by encapsulating indocyanine green (ICG) via electrostatic force (ICG-HSA-RGDfk NPs, IHR-NPs). A hybrid intravascular imaging system that combined IVUS and IVPA was self-assembled for RGDfk visualization within atherosclerotic plaques in the rabbit abdominal aorta. Through IHR-NPs and the hybrid IVUS/IVPA imaging platform, multiple comprehensive pieces of information on progressed plaques, including anatomical information, composition information and molecular information, can be obtained simultaneously, which may improve the precise diagnosis of plaque characteristics and the evaluation of early interventions for atherosclerosis.

Cholesterol crystal induced inflammation and mechanical cardiac valve injury: Implications for transcatheter aortic-valve replacement

The process of cardiac valve sclerosis and stenosis in non-rheumatic disease is related to inflammation via the innate immune system similar to atherosclerosis. Cardiac valve composition shares many features with arterial tissue. Cholesterol infiltration in the valve matrix results in cholesterol crystal formation and deposition that causes mechanical injury and triggers inflammation. Sclerotic human valve specimens as well as valves from atherosclerotic rabbit models reveal presence of cholesterol crystals and macrophage infiltration as seen in atherosclerosis. Lipid lowering by combination of simvastatin and ezetimibe demonstrates cholesterol reduction in valves only if used in a preventive manner. However, once the valve is infiltrated by cholesterol and crystals form in the tissue matrix, these become very difficult to extract and lipid lowering is no longer very effective. Furthermore, cholesterol crystals serve as a nidus for calcium phosphate deposition that distorts and stiffens the valve tissue leading to valve dysfunction. During Transcatheter Aortic Valve Replacement (TAVR) procedures, crystals can complicate the procedure by release of crystalline particles causing ischemic cerebral events and rupturing balloons.

MRI tracing of ultrasmall superparamagnetic iron oxide nanoparticle‑labeled endothelial progenitor cells for repairing atherosclerotic vessels in rabbits.

Endothelial progenitor cells (EPCs) have been discovered to be relevant to the prognosis of cardiovascular diseases. Previous research has demonstrated that EPCs serve vital roles in the occurrence and development of atherosclerosis. Significant improvements have been made in MRI technology and in the experimental use of EPCs for therapeutic angiogenesis and vascular repair. Nevertheless, the migratory, adhesive, proliferative and angiogenic properties of EPCs remain unknown. The aims of the present study were to investigate the potential of using non-invasive monitoring with ultrasmall superparamagnetic iron oxide nanoparticle (USPION)-labeled endothelial progenitor cells (EPCs) after transplantation, and to assess the treatment outcomes in an atherosclerotic rabbit model. EPCs derived from rabbit peripheral blood samples were labeled with USPION-poly-l-lysine (USPION-PLL). The morphology, proliferation, adhesive ability and labeling efficiency of the EPCs were determined by optical and electron microscopy. Moreover, biological activity was assessed by flow cytometry. In addition, T2-weighted image fast spin-echo MRI was used to detect cell labeling. USPION content in the labeled EPCs was determined by Prussian blue staining and scanning electron microscopy. Rabbit atherosclerosis model was established using a high-fat diet. USPION-labeled EPCs were transplanted into rabbits, and in vivo MRI was performed 1 and 7 days after transplantation. It was found that EPCs cultured on Matrigel formed capillary-like structures, and expressed the surface markers CD133, CD31, CD34 and vascular endothelial growth factor receptor 2 (VEGFR2). The optimal USPION concentration was 32 µg/ml, as determined by adhesion and proliferation assays. It was identified that USPION-PLL nanoparticles were 10–20 nm in diameter. Histopathological analysis results indicated that 1 day after transplantation of the labeled EPCs, blue-stained granules were observed in the intima of vascular lesions in rabbit models after Prussian blue staining. Therefore, the present results suggest that USPION-labeled EPCs may play a role in repairing endothelial injury and preventing atherosclerosis in vivo.

Systematic analysis of lncRNA expression profiles and atherosclerosis-associated lncRNA-mRNA network revealing functional lncRNAs in carotid atherosclerotic rabbit models.

Atherosclerosis, a multifactorial and chronic immune inflammatory disorder, is the main cause of multiple cardiovascular diseases. Researchers recently reported that lncRNAs may exert important functions in the progression of atherosclerosis (AS). Some studies found that lncRNAs can act as ceRNAs to communicate with each other by the competition of common miRNA response elements. However, lncRNA-associated ceRNA network in terms of atherosclerosis is limited. In present study, we pioneered to construct and systematically analyze the lncRNA-mRNA network and reveal its potential roles in carotid atherosclerotic rabbit models. Atherosclerosis was induced in rabbits (n = 3) carotid arteries via a high-fat diet and balloon injury, while age-matched rabbits (n = 3) were treated with normal chow as controls. RNA-seq analysis was conducted on rabbits carotid arteries (n = 6) with or without plaque formation. Based on the ceRNA mechanism, a ternary interaction network including lncRNA, mRNA, and miRNA was generated and an AS-related lncRNA-mRNA network (ASLMN) was extracted. Furthermore, we analyzed the properties of ASLMN and discovered that six lncRNAs (MSTRG.10603.16, 5258.4, 12799.3, 5352.1, 12022.1, and 12250.4) were highly related to AS through topological analysis. GO and KEGG enrichment analysis indicated that lncRNA MSTRG.5258.4 may downregulate inducible co-stimulator to perform a downregulated role in AS through T cell receptor signaling pathway and downregulate THBS1 to conduct a upregulated function in AS through ECM-receptor interaction pathway. Finally, our results elucidated the important function of lncRNAs in the origination and progression of AS. We provided an ASLMN of atherosclerosis development in carotid arteries of rabbits and probable targets which may lay the foundation for future research of clinical applications.

Prevention of in-stent restenosis with endothelial progenitor cell (EPC) capture stent placement combined with regional EPC transplantation: An atherosclerotic rabbit model.

Even with drug-eluting stents, the risk of in-stent restenosis (ISR) remains high. The goal of this study was to investigate the use of an endothelial progenitor cell (EPC) capture stent plus regional EPC transplantation to reduce the ISR rate. Endothelial progenitor cell capture stents were fabricated using fibrin gel and anti-CD34 plus anti-VEGFR-2 dual antibodies. Twenty male New Zealand white rabbits established as an atherosclerotic model were randomly divided into two groups: group 1 (n = 10), in which EPC capture stents were deployed into the right iliac artery; and group 2 (n = 10), in which sirolimus-eluting stents were placed. In both groups, EPCs were transplanted into target vessels beyond the stents, with outflow blocked. Radiologic-pathologic correlation outcomes were reviewed after 2 months. The technical success rate of EPC capture stent placement plus EPC transplantation was 100%. The ISR rate in group 1 was lower than in group 2 (1/10 vs. 4/10; p > 0.05). Minimal luminal diameters were larger in group 1 than in group 2 (computed tomographic angiography, 1.85 ± 0.15 mm vs. 1.50 ± 0.20 mm; duplex ultrasound, 1.90 ± 0.10 mm vs. 1.70 ± 0.30 mm; p > 0.05). Transplanted EPCs were tracked positively only in group 1. Pathologic analysis demonstrated neointimal hyperplasia thickness of 0.21 ± 0.09 mm in group 1 vs. 0.11 ± 0.07 mm in group 2 (p < 0.05). Endothelial progenitor cell capture stent placement plus local EPC transplant decreases the ISR rate through thrombosis reduction rather than through neointimal hyperplasia inhibition.

The atherogenic actions of LPC on vascular smooth muscle cells and its LPA receptor mediated mechanism

Lysophosphatidylcholine (LPC) is a bioactive lipid constituent of oxidized low density lipoprotein (ox-LDL). It regulates various cellular functions, including migration of circulating monocytes, expression of endothelial adhesion molecules, proliferation and migration of vascular smooth muscle cells (VSMCs). LPC can also be hydrolyzed into lysophosphatidic acid (LPA) by autotaxin (ATX) which possesses lysophospholipase D (lyso-PLD) activity. The aim of this study was to explore the effects of LPC on proliferation and migration of human artery smooth muscle cells (HASMCs) and the involvement of LPC-ATX-LPA pathway in these processes. In vitro, we found that LPC and LPA stimulated HASMCs proliferation and migration. Knockdown of LPA1 by siRNA and inhibit Gi protein with pertussis toxin (PTX) showed the contrary results. Silencing of LPC receptor genes did not significantly affect the LPC induced proliferation and migration. We detected the higher expressed mRNA and protein of ATX in HASMCs, and measured lyso-PLD activity. In atherosclerotic rabbit model, we observed high LPC level and high lyso-D activity in blood, and high expression of LPA1 in aorta walls. We also found that neointima appeared to be thickened and mRNA expressions of LPA1 appeared to be increased. These results revealed that LPC was converted into LPA by ATX to induce the proliferation and migration in HASMCs through LPA1/Gi/o/MAP Kinase signaling pathway. Our research suggested that LPC-ATX-LPA system contributed to the atherogenic action induced by ox-LDL. LPA1 antagonist may be considered as a potential therapeutic and preventative drug for cardiovascular disease.

Overexpression of Salusin-α Inhibits Vascular Intimal Hyperplasia in an Atherosclerotic Rabbit Model.

Inhibiting vascular endothelial foam is the focus of clinical attention. Using SonoVue (an ultrasound contrast agent), the salusin-α gene was transfected into the arterial intima of an atherosclerotic rabbit model induced by a high-fat diet in this study. Subsequently the model of blood lipid indexes, the pathological structure of the intima, and changes in molecules regulating atherosclerosis were investigated. The high-density lipoprotein C and apolipoprotein A values in the salusin-α gene overexpression (P) group were higher than those in the salusin-α gene interference (RP) group (P < 0.05), whereas the total cholesterol, low-density lipoprotein C, and apolipoprotein B values were reversed. Rabbits in the P group showed significantly thinner vascular intimal thickness than that of other experimental groups (P < 0.05). The expression of positive regulators of atherosclerosis (ABCA1, ABCG1) was higher in the P group than that in the RP group (P < 0.05), and the opposite effect was observed for negative regulators (ACAT1, CD36). Thus, our results showed that the overexpression of salusin-α gene inhibited the proliferation of the vascular intima, thereby throwing some light on understanding the mechanism how salusin-α gene expression interfered with the foaming of vascular intimal cells.

Rapid inhibition of atherosclerotic plaque progression by sonodynamic therapy.

Currently, efficient regimens to reverse atherosclerotic plaques are not available in the clinic. Herein, we present sonodynamic therapy (SDT) as a novel methodology to rapidly inhibit progression of atherosclerotic plaques. In atherosclerotic rabbit and apoE-deficient mouse models, SDT efficiently decreased the atherosclerotic burden within 1 week, revealing a decrease in the size of the atherosclerotic plaque and enlarged lumen. The shrunken atherosclerotic plaques displayed compositional alterations, with a reduction in lesional macrophages and lipids. The rapid efficacy of SDT may be due to its induction of macrophage apoptosis, enhancement of efferocytosis, and amelioration of inflammation in the atherosclerotic plaque. Compared with atorvastatin, the standard of care for atherosclerosis, SDT showed more significant plaque shrinkage and lumen enlargement during 1 week treatment. Furthermore, SDT displayed good safety without obvious side effects. In a pilot clinical trial recruiting the patients suffering atherosclerotic peripheral artery disease, combination therapy of SDT with atorvastatin efficiently reduced progression of atherosclerotic plaque within 4 weeks, and its efficacy was able to last for at least 40 weeks. SDT is a non-invasive and efficacious regimen to inhibit atherosclerotic plaque progression.

Effect of pioglitazone on inflammation and calcification in atherosclerotic rabbits : An 18F-FDG-PET/CT in vivo imaging study.

We developed an atherosclerotic rabbit model and tested pioglitazone as adrug intervention for early vascular calcification. Positron emission tomography/computed tomography (PET/CT) was used to evaluate inflammation and therapeutic effects. We randomly divided 20 male New Zealand white rabbits into apioglitazone-treated group (n= 10) and acontrol group (n= 10). Atherosclerosis was induced via ahigh-cholesterol diet and endothelial denudation. The animals were maintained on ahyperlipidemic diet for 16weeks after surgery, and the treatment group received pioglitazone daily. Serum samples were obtained at 8 and 18 weeks postoperatively to assess high-sensitivity C‑reactive protein (hs-CRP) and matrix metalloproteinase-9 (MMP-9) concentrations. Sixteen rabbits underwent amid-stage PET/CT scan at week8, and 11 rabbits underwent an end-stage PET/CT scan at week18. PET/CT parameters, including the mean standardized uptake value (SUVmean) and maximum standardized uptake value (SUVmax), were measured and documented. There were significantly lower hs-CRP and MMP-9 levels in the pioglitazone group at week18 (p< 0.01). At the end of the 8thweek, no significant between-group differences in SUVmean and SUVmax were observed. From week8 to week18, the SUVmean and SUVmax decreased in the pioglitazone group but the SUVmean increased in the control group, with significant between-group differences at the end of the 18th week (p< 0.01). Histopathological examination of aortas in the control and pioglitazone groups revealed significantly smaller plaque area, macrophage density, and tissue calcification area in the latter group. Pioglitazone affects early vascular microcalcification, and pioglitazone-induced changes can be assessed using 18F-FDG-PET/CT.

Contrast-Enhanced Ultrasound Imaging Quantification of Adventitial Vasa Vasorum in a Rabbit Model of Varying Degrees of Atherosclerosis

This study used an atherosclerotic rabbit model to investigate the feasibility of quantifying adventitial vasa vasorum (VV) via contrast-enhanced ultrasound (CEUS) imaging to identify early atherosclerosis. Recent evidence has linked adventitial VV with atherosclerotic plaque progression and vulnerability. A growth in VV density has been detected preceding intimal thickening and even endothelial dysfunction. In our study, carotid atherosclerosis rabbit models were used, and animals underwent CEUS imaging at the end of the atherosclerotic induction period. Normalized maximal video-intensity enhancement (MVE) was calculated to quantify VV density. After CEUS imaging, animals were euthanized, and their carotids were processed for histopathological analysis following staining for CD31 and VEGF. Adventitial normalized MVE increased as atherosclerosis progressed (p < 0.001), and normalized MVE also progressed, demonstrating a linear correlation with histological findings (r = 0.634, p < 0.001 for VEGF-positive; r = 0.538, p < 0.001 for CD31-positive). Thus, we histologically validated that CEUS imaging can be used to quantify the development of adventitial VV associated with atherosclerosis progression. This method can be used for monitoring the VV to detect early atherosclerosis.

Heart rate lowering treatment leads to a reduction in vulnerable plaque features in atherosclerotic rabbits.

ObjectiveTo investigate the effect of a heart rate (HR) lowering agent (Ivabradine) on features of atherosclerotic plaque vulnerability with magnetic resonance imaging (MRI), ultrasound imaging, and histology.Approach and resultsAtherosclerosis was induced in the abdominal aorta of 19 rabbits. Nine rabbits were treated with Ivabradine (17 mg/kg/day) during the entire study period. At week 14, imaging was performed. Plaque size was quantified on contrast-enhanced T1-weighted MR images. Microvascular flow, density, and permeability was studied with dynamic contrast-enhanced MRI. Plaque biomechanics was studied by measuring the aortic distension with ultrasound. After, animals were sacrificed and histology was performed.HR was reduced by 16% (p = 0.026) in Ivabradine-treated animals. No differences in absolute and relative vessel wall beat-to-beat distension were found, but due to the reduction in HR, the frequency of the biomechanical load on the plaque was reduced. Plaque size (MR and histology) was similar between groups. Although microvessel density (histology) was similar between groups, AUC and Ktrans, indicative for plaque microvasculature flow, density, and permeability, were decreased by 24% (p = 0.029) and 32% (p = 0.037), respectively. Macrophage content (relative RAM11 positive area) was reduced by 44% (p<0.001) on histology in Ivabradine-treated animals.ConclusionsHR lowering treatment with Ivabradine in an atherosclerotic rabbit model is associated with a reduction in vulnerable plaque features. The current study suggests that HR reduction may be beneficial for inducing or maintaining a more stable plaque phenotype.

Ghrelin inhibits atherosclerotic plaque angiogenesis and promotes plaque stability in a rabbit atherosclerotic model

Intraplaque angiogenesis associates with the instability of atherosclerotic plaques. In the present study, we investigated the effects of ghrelin on intraplaque angiogenesis and plaque instability in a rabbit model of atherosclerosis. The rabbits were randomly divided into three groups, namely, the control group, atherosclerotic model group, and ghrelin-treated group, with treatments lasting for 4 weeks. We found that the thickness ratio of the intima to media in rabbits of the ghrelin-treated group was significantly lower than that in rabbits of the atherosclerotic model group. The number of neovessels and the levels of vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor 2 (VEGFR2) decreased dramatically in rabbits of the ghrelin-treated group compared to those of the atherosclerotic model group. Ghrelin significantly decreased the plaque content of macrophages, matrix metalloproteinase (MMP)-2, and MMP-9, in a rabbit model of atherosclerosis. In addition, the level of the pro-inflammatory factor monocyte chemoattractant protein (MCP)-1 was significantly lower in rabbits of the ghrelin-treated group than in rabbits of the atherosclerotic model group. In summary, ghrelin can inhibit intraplaque angiogenesis and promote plaque stability by down-regulating VEGF and VEGFR2 expression, inhibiting the plaque content of macrophages, and reducing MCP-1 expression at an advanced stage of atherosclerosis in rabbits.

Multimodality Imaging of Angiogenesis in a Rabbit Atherosclerotic Model by GEBP11 Peptide Targeted Nanoparticles.

Background and Aims: Angiogenesis is an important pathological process during progression of plaque formation, which can result in plaque hemorrhage and vulnerability. This study aims to explore non-invasive imaging of angiogenesis in atherosclerotic plaque through magnetic resonance imaging (MRI) and positron emission tomography (PET) by using GEBP11 peptide targeted magnetic iron oxide nanoparticles in a rabbit model of atherosclerosis.Methods: The dual-modality imaging probe was constructed by coupling 2, 3-dimercaptosuccinnic acid-coated paramagnetic nanoparticles (DMSA-MNPs) and the PET 68Ga chelator 1,4,7-triazacyclononane-N, N', N''-triacetic acid (NOTA) to GEBP11 peptide. The atherosclerosis model was induced in New Zealand white rabbits by abdominal aorta balloon de-endothelialization and atherogenic diet for 12 weeks. The plaque areas in abdominal artery were detected by ultrasound imaging and Oil Red O staining. Immunofluorescence staining and Prussian blue staining were applied respectively to investigate the affinity of GEBP11 peptide. MTT and flow cytometric analysis were performed to detect the effects of NGD-MNPs on cell proliferation, cell cycle and apoptosis in Human umbilical vein endothelial cells (HUVECs). In vivo MRI and PET imaging of atherosclerotic plaque were carried out at different time points after intravenous injection of nanoparticles.Results: The NGD-MNPs with hydrodynamic diameter of 130.8 nm ± 7.1 nm exhibited good imaging properties, high stability, low immunogenicity and little cytotoxicity. In vivo PET/MR imaging revealed that 68Ga-NGD-MNPs were successfully applied to visualize atherosclerotic plaque angiogenesis in the rabbit abdominal aorta. Prussian blue and CD31 immunohistochemical staining confirmed that NGD-MNPs were well co-localized within the blood vessels' plaques.Conclusion: 68Ga-NGD-MNPs might be a promising MR and PET dual imaging probe for visualizing the vulnerable plaques.

Abstract 12082: Radiofrequency Ablation of the Atherosclerotic Plaque: A Safety and Feasibility Study in an Atherosclerotic Rabbit Model

Introduction: Angiogenesis from the vasa vasorum plays a key role in atheroprogression and plaque vulnerability. Immature neovessels are prone to leakage, hence facilitating additional lipid and inflammatory cell accumulation and increasing intraplaque hemorrhage risk. Hypothesis: Heat-generating radiofrequency ablation (RFA) safely reduces vasa vasorum, decreases inflammatory cell influx and stabilizes the plaque. Methods: Thirteen New-Zealand White rabbits on a 0.5% cholesterol-enriched diet were subjected to balloon denudation of the infrarenal aorta. After 13 weeks, either the proximal or distal half of the infrarenal aorta was exposed to transluminal RFA, the untreated half served as an intra-individual control. Optical coherence tomography (OCT) was performed directly after RFA. Results: Within 24 hours after RFA, no plaque ruptures or adverse events were observed by OCT or histology. Treated regions showed widespread decellularization of intimal, medial and (to a lesser extent) adventitial layers, which were recellularized after 7 and 28 days. Vasa vasorum density was not different between both regions at short-term (24 hours; RFA 19.8±5.1 vs. control 29.2±8.3/mm 2 ; p=0.15) or long-term follow-up (7 days; 39.1±11.5 vs. 41.7±8.6/mm 2 ; p=0.81 and 28 days; 46.4±13.4 vs. 38.5±6.6/mm 2 ; p=0.06). After 7 days, treated regions were comparable to control areas with respect to plaque size (5.6±1.7 vs. 5.5±1.5mm 2 ; p=0.62), intimal collagen (34.6±11.9 vs. 32.7±13.0%; p=0.22) and macrophage content (14.3±2.8 vs. 18.5±5.8%; p=0.32). RFA-treated regions did show a major decrease in intimal smooth muscle cell (αSMA) content (1.8±0.4 vs. 18.0±5.9%; p=0.038), which remained decreased after 28 days (6.8±3.8 vs. 12.6±1.6%; p=0.020). Again, after 28 days no differences were observed in plaque area (4.6±0.9 vs. 4.4±1.1 mm 2 ; p=0.46), intimal collagen (39.2±9.1 vs. 40.3±12.4%; p=0.85) or macrophage content (13.2±2.5 vs. 12.6±2.0%; p=0.73). Conclusion: Radiofrequency ablation of the rabbit atherosclerotic plaque is safe and leads to a major reduction in αSMA content, but does not affect other aspects of plaque stability over time. It may thus serve as a therapeutic option for major (re)stenosis in peripheral arterial disease.