Nana Dong

A Novel Model of Atherosclerosis in Rabbits Using Injury to Arterial Walls Induced by Ferric Chloride as Evaluated by Optical Coherence Tomography as well as Intravascular Ultrasound and Histology

This study aim was to develop a new model of atherosclerosis by FeCl3-induced injury to right common carotid arteries (CCAs) of rabbits. Right CCAs were induced in male New Zealand White rabbits (n = 15) by combination of a cholesterol-rich diet and FeCl3-induced injury to arterial walls. The right and left CCAs were evaluated by histology and in vivo intravascular ultrasound (IVUS) and optical coherence tomography (OCT) examinations of 24 hours (n = 3), 8 weeks (n = 6), and 12 weeks (n = 6) after injury. Each right CCA of the rabbits showed extensive white-yellow plaques. At eight and 12 weeks after injury, IVUS, OCT, and histological findings demonstrated that the right CCAs had evident eccentric plaques. Six plaques (50%) with evident positive remodeling were observed. Marked progression was clearly observed in the same plaque at 12 weeks after injury when it underwent repeat OCT and IVUS. We demonstrated, for the first time, a novel model of atherosclerosis induced by FeCl3. The model is simple, fast, inexpensive, and reproducible and has a high success rate. The eccentric plaques and remodeling of plaques were common in this model. We successfully carried out IVUS and OCT examinations twice in the same lesion within a relatively long period of time.

Exendin-4 protects bone marrow-derived mesenchymal stem cells against oxygen/glucose and serum deprivation-induced apoptosis through the activation of the cAMP/PKA signaling pathway and the attenuation of ER stress

Exendin-4 (ex-4) is a long-acting glucagon-like peptide-1 receptor (GLP-1R) agonist which exerts beneficial effects on glycemic control and promotes cell viability. In the present study, we investigated the anti-apoptotic effects of ex-4, as well as the potential mechanisms responsible for these effects in rat bone marrow-derived mesenchymal stem cells (BM-MSCs) under conditions of oxygen, glucose and serum deprivation (OGD). The apoptosis of the MSCs was induced by subjecting the cells to OGD conditions for 4 h and was detected by Annexin V/PI and Hoechst 33258 staining. The MSCs were pre-conditioned with ex-4 for 12 h prior to being subjected to OGD conditions, and the expression levels of an apoptotic marker (cleaved caspase-3), endoplasmic reticulum (ER) stress markers [phosphorylated (p-)protein kinase RNA-like endoplasmic reticulum kinase (PERK), PERK, binding immunoglobulin protein (BIP), activating transcription factor 4 (ATF-4) and C/EBP homologous protein (CHOP)], as well as those of a survival marker (Bcl-2) were measured by western blot analysis. Furthermore, the mRNA levels of ATF-4 and CHOP were determined by RT-qPCR. ELISA was used to examine the activity of intracellular cAMP. Moreover, the GLP-1R antagonist, exendin9-39 (ex9-39), the protein kinase A (PKA) inhibitor, H89, and small interfering RNA (siRNA) targeting rat ATF-4 and CHOP were co-incubated with the MSCs. The apoptotic rate was markedly diminished following pre-conditioning with ex-4 in a dose-dependent manner (P<0.05). The ER stress markers, p-PERK, BIP, ATF-4 and CHOP, were upregulated in the cells subjected to OGD conditions. Ex-4 pre-conditioning significantly decreased the mRNA and protein levels of ATF-4 and CHOP (P<0.05), and increased the activity of intracellular cAMP (P<0.05). Furthermore, the anti-apoptotic effects of ex-4 were almost reversed by treatment with either H89 or ex9-39 (P<0.05); transfection with siRNA-CHOP significantly reduced the apoptotic rate of the MSCs and did not impair the cytoprotective effects of ex-4. Taken together, these findings suggest that ex-4 protects rat BM-MSCs from OGD-induced apoptosis through the activation of the PKA/cAMP pathway and the attenuation of the ER stress signaling pathway. Ex-4 may thus prove to be a therapeutic agent with the potential to improve the viability of MSCs in the ischemic milieu, and consequently, to optimize the therapeutic effects of MSC therapy in acute myocardial infarction.

Comparison of optical coherence tomography and intravascular ultrasound for evaluation of coronary lipid-rich atherosclerotic plaque progression and regression.

AIMS Compared with intravascular ultrasound (IVUS), optical coherence tomography (OCT) has relative merits and demerits for detecting plaque characteristics. It remains unknown whether the IVUS and OCT evaluations of plaque progression/regression are consistent. We sought to analyse the correlations between IVUS and OCT evaluations of plaques at single time points, and compare temporal changes in the IVUS and OCT data. METHODS AND RESULTS Eighty-eight lipid-rich plaques from 65 patients with coronary artery disease were analysed with IVUS and OCT at baseline and 12-month follow-up. Fibrous cap thickness on OCT was negatively correlated with total atheroma volume on IVUS (r = -0.28, P = 0.009), but not with percent atheroma volume (P = 0.84). Changes on OCT were not significantly correlated with changes on IVUS. Plaques that showed progression, regression, or no change on IVUS showed no differences in terms of changes in the OCT parameters fibrous cap thickness (P = 0.199), maximum lipid core arc (P = 0.755), mean lipid core arc (P = 0.936), and lipid index (P = 0.91). The incidence of thin-cap fibroatheroma (TCFA) was similar among the above three plaque groups at baseline (P = 0.79) and follow-up (P = 0.609). CONCLUSION Although fibrous cap thickness on OCT was negatively correlated with plaque size on IVUS at single time points, changes in OCT parameters were not correlated with changes in IVUS measures over time. Lesion progression/regression on IVUS was not associated with changes in OCT parameters (fibrous cap thickness, lipid core arc, lipid index, and TCFA).

MicroRNA-182 prevents vascular smooth muscle cell dedifferentiation via FGF9/PDGFRβ signaling

The abnormal phenotypic transformation of vascular smooth muscle cells (SMCs) causes various proliferative vascular diseases. MicroRNAs (miRNAs or miRs) have been established to play important roles in SMC biology and phenotypic modulation. This study revealed that the expression of miR-182 was markedly altered during rat vascular SMC phenotypic transformation in vitro. We aimed to investigate the role of miR-182 in the vascular SMC phenotypic switch and to determine the potential molecular mechanisms involved. The expression of miR-182 gene was significantly downregulated in cultured SMCs during dedifferentiation from a contractile to a synthetic phenotype. Conversely, the upregulation of miR-182 increased the expression of SMC-specific contractile genes, such as α-smooth muscle actin, smooth muscle 22α and calponin. Additionally, miR-182 overexpression potently inhibited SMC proliferation and migration under both basal conditions and under platelet-derived growth factor-BB stimulation. Furthermore, we identified fibroblast growth factor 9 (FGF9) as the target gene of miR-182 for the phenotypic modulation of SMCs mediated through platelet-derived growth factor receptor β (PDGFRβ) signaling. These data suggest that miR-182 may be a novel SMC phenotypic marker and a modulator that may be used to prevent SMC dedifferentiation via FGF9/PDGFRβ signaling.

Relation between baseline plaque features and subsequent coronary artery remodeling determined by optical coherence tomography and intravascular ultrasound

Atherosclerosis often leads to myocardial infarction and stroke. We examined the influence of baseline plaque characteristics on subsequent vascular remodeling in response to changes in plaque size. Using optical coherence tomography (OCT) and intravascular ultrasound (IVUS), we examined 213 plaques from 138 patients with acute coronary syndrome at baseline and repeated IVUS at the 12-month follow-up. The change in external elastic membrane (EEM) area for each 1 mm2 change in plaque area (i.e., the slope of the regression line) was calculated as a measure of vascular remodeling capacity. In plaques with static positive remodeling, the slope was smaller than in plaques without static positive remodeling. In addition, the slope of the regression line for lesions with a large plaque burden was much smaller than that for lesions with a small plaque burden. Multivariate linear regression analysis showed that diabetes, calcification and static positive remodeling were inversely and independently associated with the level of change in EEM area/change in plaque area. Lesions with a large plaque burden, calcifications or static positive remodeling had less remodeling capacity, and calcification and static positive remodeling were independent predictors of reduced subsequent remodeling. Therefore, calcifications and static positive remodeling could be used as morphological biomarkers to predict decreased subsequent arterial remodeling.

GW24-e2536 Effects of Captopril on Plaque Inflammation and Atherosclerosis Progression Evaluated by FDG-PET-CT and IVUS Imagines

Objectives Macrophages play an important role in the pathogenesis of atherosclerosis. Captopril is one kind of the effictive drugs to the atherosclerosis besides statins. However, how does macrophage change in the therapeutic process of captopril has not been described in detail. The study was designed to investigate the effects of captopril (angiotensin converting enzyme inhibitor) on the development of atherosclerosis in rabbit model. Methods Sixteen rabbits were fed with a high-cholesterol diet for 20 weeks. At week 4, all rabbits underwent ballom injury of the right common carotid artery. At week 12, 18F-fluorodeoxyglucose positron emissiontomography computed tomography (FDG-PET-CT) was performed in the right commoncarotid artery to detect macrophages and intravascular ultrasound (IVUS) was performed to evaluated the degree of plaque. Then all rabbits receives either placebo (n = 8) or 0.5 mg/kg/day of captopril (n = 8). And FDG-PET-CT and IVUS were repeatedly performed at week 20. At week 20, each 4 rabbits were sacrificed histologic evaluation. Results At week 12 and 20, the FDG-PET-CT imaging detected an increase inaverage standardised uptake value in the placebo group (from 0.41 ± 0.12 and 0.59 ± 0.07, p = 0.05), indicating progressive inflammation, whereas not in the captopril-treated group (from 0.39 ± 0.13 to 0.35 ± 0.06, p = 0.56). Atweek 12 and 20, IVUS-derived volumetric parameters showed no significantdifferences between the two groups (all p > 0.05). The volumes of the twogroups were similar in histomorphology analysis. But the macrophage content wassignificant lower in captopril-treated group (p = 0.03). Conclusions These results indicate that ACE-inhibitor has suppressed the degree of atherosclerotic inflammation but not modified the plaque size.

GW24-e3022 Erythropoietin prevent plaque disruption in apolipoprotein E-knockout mice

Objectives Erythropoietin (EPO) have received considerable interest in immunity and inflammation. The increasing number of evidences have shown that inflammation is involved in the initial and development of atherogenesis. We hypothesised that EPO treatment may stabilise atherosclerotic plaques by inhibiting inflammatory cytokine secretion and matrix metalloproteinases (MMPs) expression and promote the proliferation of smooth muscle cells (SMC) in atherosclerotic lesions. Methods We established a vulnerable carotid plaque model in apolipoprotein E- knockout mice (ApoE-/-) which were feed on high fat diet (HFD). Mice were divided into control, phosphate buffered saline (PBS) and EPO. EPO was injected in tail vein 1 week before HFD and 3 week after HFD twice a week. 8 week after HFD, the mice were sacrificed and drawn tissue after that. The levels of serum total cholesterol (TC) were detected in all mice at immediately, 8 weeks after HFD. The histopathological method was used to detect the parameters of plaque rupture. Immunohistochemical method was used to detect the quantity of macrophages and smooth muscle cells (SMC), the levels of protein expression of matrix metalloproteinase-9 (MMP-9), matrix etalloproteinase-2 (MMP-2), proinflammatory cytokines including transforming growth factor and interleukin-10 in carotid plaque of each group. The differences among the groups were analysed by statistical methods. Results Lipid levels were significantly higher after 8 weeks of HFD in each experimental group, there was significant difference compared with Lipid level immediately after HFD, but cholesterol levels between groups on day 0, 8 week were no significant differences. At 8 week after HFD, Histopathological analysis showed that the plaque disruption rate was 49%, 49% and 25% in the control, PBS, and EPO group. EPO treatment resulted in a significant decrease in the relative contents of macrophages and lipids and a substantial increase in those of SMCs and collagen in the carotid plaque, leading to an almost 48.9% reduction of plaque vulnerability index. Furthermore, EPO treatment decreased the expression of proinflammatory cytokines, MMP-2 and MMP-9. Most of these therapeutic effects of EPO were found to be mediated by transforming growth factor and interleukin-10. Statistical analysis found that plaque stable index were significantly statistically different and significantly higher in EPO group than the other groups (P < 0.01). Conclusions Adoptive transfer of EPO changed plaque composition to a stable plaque phenotype and lowered the incidence of plaque disruption in ApoE-/- mice. The major mechanisms involved reduced expression of inflammatory cytokines and MMP-2 and MMP-9, increased the contents of SMCs in the carotid plaque. EPO may provide a novel approach to the treatment of vulnerable plaques.

THE DETECTION OF VULNERABLE ATHEROSCLEROTIC PLAQUE RABBIT CAROTID MODELS BY OPTICAL COHERENCE TOMOGRAPHY

Objectives We sought to test the feasibility of imaging vulnerable atherosclerotic plaque in vivo using optical coherence tomography (OCT) to assess the value of vulnerable atherosclerotic plaque rabbit model established with a novel way. Methods Thirty Rabbits were randomly divided into two groups: Group A (n=15): Balloon injury of carotid artery followed by high cholesterol diet (HCD) for 12 weeks. Group B (n=15): In this group, a thin filter paper slip statured with 10% ferric trichloride was lay between paraffin filter paper and common carotid artery infiltrated for 3–5 mins. We analysed OCT data including plaque incidence rate, plaque type and classification, lipid-rich plaque numbers between the two groups. Histopathology logical examination of plaque were also investigated to confirm our findings. Results In each group, plaque were formation at the harvest time. In group A, lipid-rich plaque incidence rate was 76.9%, while group B was 46.2%. CD68 staining for large amount of macrophage verified that two groups had formed advanced atherosclerostic plaque. Furthermore, in group B 60% plaque had the feature of eccentricity which may get much more influence on the endothelial shear stress. Conclusions OCT can accurately detect atherosclerotic lesions in vivo and could guide the design of invasive imaging approaches for detecting vulnerable atherosclerotic plaques. The study demonstrate that both groups can produce atherosclerostic plaque, while we get a better method for inducing the eccentricity atherosclerotic plaque on animal model.

THE COMPARATIVE STUDY OF ATHEROSCLEROTIC PLAQUES BY OPTICAL COHERENCE TOMOGRAPHY AND INTRAVASCULAR ULTRASOUND

Objectives In this study we compare the detection of the value of atherosclerotic plaques by optical coherence tomography (OCT) and Intravascular Ultrasound (IVUS). Methods 60 Rabbits were gave high cholesterol diet (HCD) for 12 weeks. Balloon injury of carotid artery were made. IVUS imaging and OCT imaging were performed at baseline and 2, 4, and 6 weeks post injury to quantify the atherosclerotic plaques. On each imaging time the surgery carotid artery in which we randomly select 15 rabbits was perfusion-fixed using paraformaldehyde infused into the carotid artery, excised and paraffin-embedded, and cross-sections were stained with H&E and masson. Additional sections were frozen and stained for macrophages (RAM11) and sma. Then we analysed OCT and IVUS data including plaque incidence rate, plaque type and classification, lipid-rich plaque numbers between the two groups. Histopathologylogical examination of plaque were investigated to confirm our findings. Immunohistochemisty were investigated characterising plaque composition. Results OCT and IVUS both can detect the plaque. But OCT can distinguish the plaque composition and IVUS cannot. Conclusions The vulnerable plaque is the main reason for the acute coronary syndrome. Thus, the judgment of the plaque composition and the accurate diagnosis of vulnerable plaque is an important means of prevention and reduction of acute coronary syndrome. Compared to the traditional IVUS, OCT played a prominent advantage, that is more accurately determination of the composition of the plaque especially for the vulnerable plaque.