Watanabe Rabbits Research Articles

Long-term high-fructose high-fat diet feeding elicits insulin resistance, exacerbates dyslipidemia and induces gut microbiota dysbiosis in WHHL rabbits.

The metabolic syndrome (MetS) has become a global public health burden due to its link to cardiovascular disease and diabetes mellitus. The present study was designed to characterize the metabolic and cardiovascular disturbances, as well as changes in gut microbiota associated with high-fructose high-fat diet (HFFD)-induced MetS in Watanabe heritable hyperlipidemic (WHHL) rabbits. Twenty-one Watanabe rabbits were assigned to a control (n = 9) and HFFD (n = 12) groups, receiving a chow diet and a HFFD, respectively. During a 12-weeks protocol, morphological parameters were monitored; plasma fasting levels of lipids, glucose and insulin were measured and a glucose tolerance test (GTT) was performed. HOMA-IR was calculated. Cardiac function and vascular reactivity were evaluated using the Langendorff isolated heart and isolated carotid arteries methods, respectively. 16S rRNA sequencing of stool samples was used to determine gut microbial composition and abundance. HFFD-fed Watanabe rabbits exhibited increased fasting insulin (p < 0.03, 12th week vs. Baseline), HOMA-IR (p < 0.03 vs. Control), area under the curve of the GTT (p < 0.02 vs. Control), triglycerides (p < 0.05, 12th week vs. Baseline), TC (p < 0.01 vs. Control), LDL-C (p < 0.001 vs. Control). The HFFD group also displayed a significant decrease in intestinal microbial richness, evenness and diversity (FDR < 0.001, FDR < 0.0001, FDR < 0.01, respectively vs. Control group) and an increase in its Firmicutes/Bacteroidetes ratio (R = 3.39 in control vs. R = 28.24 in the HFFD group) indicating a shift in intestinal microbial composition and diversity. Our results suggest that HFFD induces insulin resistance and gut microbiota dysbiosis and accentuates dyslipidemia; and that, when subjected to HFFD, Watanabe rabbits might become a potential diet-induced MetS animal models with two main features, dyslipidemia and insulin resistance.

A Novel Drug to Reduce Myocardial Infarct Size, Even When Administered After Coronary Artery Reperfusion

Despite the thousands of studies on drug therapy designed to reduce myocardial infarct size (IS), almost none have been successfully translated to the clinics. A major reason for failure is that many of these drugs are only effective when administered before coronary artery reperfusion (CAR), which limits their utility in the clinical setting, where CAR must be instituted immediately in patients after the myocardial infarction (MI) diagnosis has been made, not leaving time for drug therapy. It is also important that the drug does not increase myocardial oxygen consumption. In our prior work on the adenylyl cyclase type 5 (AC5) knock out (KO) mouse model, we found that it enhanced longevity, protected against diabetes and obesity, improved exercise performance and reduced IS after coronary artery occlusion (CAO) and CAR. Based on these studies, and since it is difficult to delete genes in humans, we developed a novel pharmacological candidate to inhibit AC5, named C90. C90 reduced AC activity and cAMP in response to forskolin more than vehicle, (12.9±1.9 vs. 14.2 ± 3.1 pmol/mg/min). However, in AC5 KO C90 no longer reduced cAMP in response to forskolin, indicating its selectivity for AC5 inhibition. The goal of this investigation was to examine the new AC5 inhibitor, C90, and compare its efficacy in reducing IS when the drug was delivered after CAR with that of adenosine, the gold standard for ischemic cardioprotection. Wild type (WT) mice were subjected to 30 min CAO followed by 24 hours CAR. C90 or vehicle was then administered at 5 min after CAR by intravenous (IV) administration at doses of 0.01, 0.03, 0.06, 0.6, and 1.2 mg/kg. The area at risk was similar in all groups, determined by triphenyltetrazolium chloride (TTC) staining. IS was reduced in a dose‐response manner by C90. IS was reduced by 32% at a dose of 0.06 mg/kg of C90, whereas the same dose of adenosine reduced IS similarly when administered before CAR but failed to reduce IS when administered after CAR. C90 further reduced IS by 64% at a dose of 0.6 mg/kg and by 71% at a dose of 1.2 mg/kg. When C90 was delivered at 10 min after CAR, it still IS by 50% and 53% at doses of 0.6 mg/kg and 1.2 mg/kg, respectively. In addition, LV ejection fraction (LV EF) was also examined at 4 weeks after myocardial infarction in mice, and was preserved in the C90 group (LVEF = 65±1.6%) vs. the vehicle group (LVEF = 52±2.9%). C90 also reduced IS by 49% in the Watanabe rabbit, model of atherosclerosis. Thus, C90, an inhibitor of AC5, is a novel target for clinical development, since it reduces myocardial IS when delivered after reperfusion, a key feature for patients coming to the emergency room, where reperfusion must be carried out before additional therapy. An additional advantageous feature of C90 and inhibition of AC5, is that it reduces beta‐adrenergic receptor signaling and myocardial oxygen consumption, which are important for patients with heart disease.Support or Funding InformationNIH R21AG053514; NIH R44 HL142390; NIH R44HL112512This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Long-term atorvastatin treatment decreases heart maximal oxygen consumption and its vulnerability to in vitro oxidative stress in Watanabe heritable hyperlipidemic rabbit.

Statins are currently used in prevention of cardiovascular diseases in high-risk populations, and could be considered in primary prevention. However, few studies are available on the long-term effects of low doses of statins, especially on mitochondrial function and reactive oxygen species (ROS) metabolism at cardiac level. This study aimed to determine potential effects of a long-term atorvastatin treatment, at low-dose concentration, on the myocardium mitochondrial respiration. Thirty-four Watanabe rabbits were treated or not with atorvastatin (2.5 mg·kg-1·day-1) from the age of 3 to 12 months. Every 3 months, proton leak, basal (V0), and maximal (Vmax) mitochondrial respiration on cardiac permeabilized fibers were measured. Additionally, the vulnerability to ROS, cardiac enzymatic antioxidant defenses, and oxidative damage (lipoperoxidation) were analyzed. Proton leak increased over the duration of the experiment (up to 60% from Vmax at 12 months). Moreover, the statin treatment induced a decrease of Vmax and a decrease of ROS susceptibility of cardiac mitochondria. However, the lipoperoxidation and the antioxidant defenses were not dependent on the presence of statin treatment, or on its duration. This is the first study showing a protective effect of long-term statins treatment against the ROS susceptibility in the cardiac muscle.

Transplantation of genetically modified hepatocytes after liver preconditioning in Watanabe heritable hyperlipidemic rabbit

BackgroundHepatocyte transplantation is a potentially less invasive alternative to liver transplantation for treating inherited metabolic liver diseases. We developed an autotransplantation protocol of ex vivo genetically modified hepatocytes combining lentiviral transduction and transplantation after liver preconditioning by partial portal vein embolization. We investigated the metabolic efficiency of this approach in Watanabe rabbits, animal model of familial hypercholesterolemia. MethodsOur autotransplantation experimental protocol was used in two groups of rabbits (n = 10), experimental and sham, receiving transduced and control hepatocytes, respectively. Isolated hepatocytes from left liver lobes were transduced using recombinant lentiviruses. Median lobe portal branches were embolized under fluoroscopic control. Functional measurement of low-density lipoprotein (LDL) receptor expression was assessed by LDL internalization assays. Cholesterol level evolution was monitored. Rabbits were killed 20 wk after the procedure. ResultsThree rabbits of each group died several hours after hepatocyte transplantation; autopsy revealed portal vein thrombosis in two rabbits from each group. The protocol was therefore modified with hepatocytes being transplanted through splenic injection. Lentiviral hepatocyte transduction efficacy was 64.5%. Fluorescence microscopy revealed Dil-LDL internalization of transduced hepatocytes. Seven rabbits in each group were considered for lipid analysis. Four weeks after autotransplantation, median total cholesterol level decreased in the experimental group, without reaching statistical significance (8.9 [8.0-9.8] g/L versus 6.3 [0.5-8.3]; P = 0.171). In the experimental group, enzyme-linked immunosorbent assay detected significant antibody expression against human low-density lipoprotein receptor. ConclusionsAutotransplantation protocol allowed a nonstatistically significant improvement of the lipid profile in Watanabe rabbits. Further experiments involving a larger number of animals are necessary to confirm or refute our findings.

Abstract 18873: Al 18 F-NOTA-folate Accumulates in Atherosclerotic Plaques and Can be Detected by PET/CT

Introduction:Macrophages are a major cell type in inflamed atherosclerotic plaques. Since folate receptor β (FR-β) is highly expressed in activated macrophages, we hypothesized that it might serve as a new marker for inflamed atherosclerotic plaques. Hypothesis: We aimed at evaluating a FR-β targeted PET tracer, Al 18 F NOTA-Folate, for the detection of inflamed atherosclerotic plaques. Methods:Atherosclerotic mice deficient for low density lipoprotein receptor (LDLR -/- ApoB 100/100 , n=12), C57BL control mice (n=9), and Watanabe rabbits (n=4) with endothelial denudation-induced atherosclerosis in the aorta were used. Biodistribution of Al 18 F-NOTA-Folate (specific radioactivity 130 GMq/μmol) was investigated in vivo by PET/contrast enhanced CT and ex vivo by gamma counting and autoradiography of aortic sections. In addition, prior to Al 18 F-NOTA-Folate study, the Watanabe rabbits were PET/CT imaged with 18 F-FDG. Results:Atherosclerotic mice demonstrated large and macrophage-rich atheromatous plaques in the aorta. The in vivo PET/CT revealed significantly higher uptake of Al 18 F-NOTA-Folate in the aortic arch of atherosclerotic mice compared to controls (aorta-to-blood ratio 1.5±0.3 vs. 0.7±0.2, P &lt;0.0001), which were verified by ex vivo measurements. Autoradiography confirmed focally increased uptake of Al 18 F-NOTA-Folate in the atherosclerotic plaques (plaque-to-normal vessel wall ratio 2.6±0.7, P &lt;0.0001). Competitive study with excess of unlabelled folate reduced Al 18 F-NOTA-Folate uptake in the aorta by app. 80% and thus verified the specificity of its binding. In the rabbit aorta, the PET/CT showed a strong focal in vivo uptake of Al 18 F-NOTA-Folate co-localizing with an atherosclerotic abdominal aorta with highest aorta-to-blood ratio of 6.0. For comparison, with 18 F-FDG the ratio was 2.4 in the same area. Conclusions: Al 18 F-NOTA-Folate, targeting FR-β, accumulates in macrophage-rich atherosclerotic plaques, which can be detected in vivo by PET/CT in experimental models of atherosclerosis. Further development of the tracer for imaging of patients with atherosclerosis is warranted.

Effects of an early and low-dose hypolipidemic treatment of atherosclerosis: An OCT study in watanabe rabbits

TC Ezetimibe 224.3 (34.5) 193.3 (23.8) -12.8 (10.9)** <.001 <.001 Lomitapide 230.8 (43.5) 176.3 (43.9) -22.1 (12.8)** 0.04 0.02 Combination 225.9 (30.4) 149.8 (41.9) -32.8 (17.7)** <.001 LDL-C Ezetimibe 146.5 (27.7) 116.8 (17.6) -18.8 (12.9)** <.001 <.001 Lomitapide 150. 9 (32.9) 103.3 (31.7) -30.0 (14.2)** <0.05 0.01 Combination 149.6 (23.7) 82.2 (34.5) -43.9 (23.4)** <.001 LDL1-C Ezetimibe 30.7 (11.2) 22.4 (8.6) -24.0 (23.4)** 0.01 0.004 Lomitapide 31.3 (15.3) 21.8 (14.5) -28.3 (33.4)* 0.64 0.02 Combination 30.9 (13.5) 14.2 (8.9) -49.7 (31.4)** 0.004 LDL2-C Ezetimibe 32.1 (16.4) 26.1 (12.7) -11.4 (34.7)* 0.04 0.01 Lomitapide 30.5 (12.6) 20.4 (10.3) -28.8 (46.0)* 0.15 0.33 Combination 33.5 (13.8) 16.4 (8.8) -40.6 (35.0)** 0.01 LDL3-C Ezetimibe 41.0 (13.8) 34.8 (14.6) -14.0 (27.5)* 0.006 0.003 Lomitapide 41.0 (12.9) 23.0 (12.2) -40.0 (30.4)* 0.01 0.78 Combination 42.2 (13.5) 22.7 (15.2) -42.9 (38.5)* 0.003 LDL4-C Ezetimibe 9.5 (7.0) 8.3 (7.2) -13.3 (48.0) 0.90 0.71 Lomitapide 10.8 (10.0) 6.2 (4.0) -20.6 (45.7) 0.69 0.95 Combination 11.1 (8.7) 5.9 (3.5) -19.5 (70.2)* 0.71 Non HDL-C Ezetimibe 174.5 (31.3) 140.0 (18.9) -18.4 (12.3)** <.001 <.001 Lomitapide 180.9 (35.3) 130.0 (35.5) -26.2 (15.5)** 0.14 0.01 Combination 176.0 (25.2) 104.5 (39.8) -40.0 (21.4)** <.001 ApoB Ezetimibe 112.6 (16.1) 93.8 (11.5) -16.0 (10.1)** <.001 <.001 Lomitapide 116.8 (18.2) 88.0 (19.3) -22.9 (14.3)** 0.15 0.03 Combination 114.4 (18.0) 75.0 (22.6) -33.9 (19.7)** <.001 ApoB/AI Ezetimibe 0.75 (0.13) 0.62 (0.11) -18.0 (8.2)** 0.02 0.01 Lomitapide 0.77 (0.11) 0.63 (0.13) -17.2 (14.8)** 0.86 0.02 Combination 0.77 (0.11) 0.55 (0.16) -28.8 (19.3)** 0.01 TC/HDL-C Ezetimibe 4.60 (0.82) 3.72 (0.61) -18.5 (8.1)** 0.008 0.42 Lomitapide 4.71 (0.75) 3.92 (0.83) -15.1 (13.8)** 0.42 0.004 Combination 4.65 (0.72) 3.39 (0.95) -27.9 (16.9)** 0.02 ApoB/NonHDL-C Ezetimibe 0.65 (0.04) 0.67 (0.04) 3.6 (5.6)* 0.001 <.001 Lomitapide 0.65 (0.04) 0.69 (0.07) 5.1 (7.1)* 0.57 0.005 Combination 0.65 (0.04) 0.74 (0.09) 12.9 (12.1)** <.001

0204 : Cardiac mitochondria vulnerability to reactive oxygen species: long term effects of statins in a model of hereditary hypercholesterolemia (Watanabe rabbits)

0204 : Cardiac mitochondria vulnerability to reactive oxygen species: long term effects of statins in a model of hereditary hypercholesterolemia (Watanabe rabbits)

Point Mutation in Syntaxin-1A Causes Abnormal Vesicle Recycling, Behaviors, and Short Term Plasticity

Syntaxin-1A is a t-SNARE that is involved in vesicle docking and vesicle fusion; it is important in presynaptic exocytosis in neurons because it interacts with many regulatory proteins. Previously, we found the following: 1) that autophosphorylated Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), an important modulator of neural plasticity, interacts with syntaxin-1A to regulate exocytosis, and 2) that a syntaxin missense mutation (R151G) attenuated this interaction. To determine more precisely the physiological importance of this interaction between CaMKII and syntaxin, we generated mice with a knock-in (KI) syntaxin-1A (R151G) mutation. Complexin is a molecular clamp involved in exocytosis, and in the KI mice, recruitment of complexin to the SNARE complex was reduced because of an abnormal CaMKII/syntaxin interaction. Nevertheless, SNARE complex formation was not inhibited, and consequently, basal neurotransmission was normal. However, the KI mice did exhibit more enhanced presynaptic plasticity than wild-type littermates; this enhanced plasticity could be associated with synaptic response than did wild-type littermates; this pronounced response included several behavioral abnormalities. Notably, the R151G phenotypes were generally similar to previously reported CaMKII mutant phenotypes. Additionally, synaptic recycling in these KI mice was delayed, and the density of synaptic vesicles was reduced. Taken together, our results indicated that this single point mutation in syntaxin-1A causes abnormal regulation of neuronal plasticity and vesicle recycling and that the affected syntaxin-1A/CaMKII interaction is essential for normal brain and synaptic functions in vivo.

Adipose Tissue-Derived Multi-lineage Progenitor Cells as a Promising Tool for In Situ Stem Cell Therapy

Adipose tissue-derived cell sources are attractive for regenerative medicine due to the easy and safe accessibility of adipose tissue, lack of ethical issues, and their availability in large quantities. We have developed an isolation method for distinct stem cells, and named the cells adipose tissue-derived multilineage progenitor cells (ADMPC). ADMPC have higher potential for differentiation into adipocytic, osteocytic, and chondrocytic progeny than the widely reported adipose tissue-derived stromal/stem cells (ADSC). ADMPC can also differentiate into hepatocyte-like clusters in vitro by induction with the hepatogenic cytokines, hepatocyte growth factor, oncostatin M, and basic fibroblast growth factor. In vivo, ADMPC were reprogrammed in situ into hepatocyte-like cells that corrected the metabolic defect in hyperlipidemic Watanabe rabbits after transplantation via the portal vein. Such cells are potentially useful in regenerative medicine as sources for in situ stem cell therapy. Keywords: adipose tissue, ADMPC, in situ stem cell therapy, in situ reprogramming, in situ differentiation, ADSC, ADMSC, ASC, hepatocyte, umbilical cord.

Abstract 9201: Transplantation of Adipose Tissue-Derived Multi-Lineage Progenitor Cells Reduces Serum Cholesterol in Hyperlipidemic Watanabe Rabbits

Background : Familial hypercholesterolemia (FH) is an autosomal co-dominant disease characterized by high concentrations of pro-atherogenic lipoproteins and premature atherosclerosis. We examined the response to in situ stem cell therapy using adipose tissue-derived multi-lineage progenitor stem cells (hADMPC) in the LDL-receptor deficient Watanabe heritable hyperlipidemic (WHHL) rabbit, an animal model for homozygous FH. Methods : WHHL rabbits received either normal control rabbit-derived, GFP-rabbit-derived or WHHL rabbit-derived ADMPC (normal-ADMPC, GFP-ADMPC and diseased-ADMPC, respectively) via the portal vein. This was followed by 12-week immunosuppressive therapy to avoid allogenic rejection. In situ survival and differentiation of the ADMPC into hepatocytes was examined by flow cytometry and immunohistochemical analysis, respectively. Lipid profile was examined before-, and 4-, 8- and 12 weeks after transplantation. LDL clearance was examined at the end of the study by125I-LDL turnover. Results : In situ survival of GFP-ADMPC was confirmed after transplantation. The cells integrated into the hepatic parenchyma and co-expressed GFP and hepatocyte markers such as albumin, indicating that the cells were reprogrammed into hepatocytes-like cells in situ . Transplantation of normal-ADMPC but not diseased-ADMPC resulted in a significant reduction of serum total- and LDL- cholesterol after transplantation. 125I-LDL turnover study showed significant improvement in the rate of LDL clearance in the WHHL rabbits with transplanted normal-ADMPC but not in those transplanted with diseased-ADMPC. Conclusion : Transplantation of ADMPC but not diseased ones corrected the metabolic defects in WHHL rabbits, suggesting that ADMPC transplantation is a potentially useful therapy for FH.

HMG-CoA reductase inhibitor augments the serum total cholesterol-lowering effect of human adipose tissue-derived multilineage progenitor cells in hyperlipidemic homozygous Watanabe rabbits

Familial hypercholesterolemia (FH) is an autosomal codominant disease characterized by high concentrations of proatherogenic lipoproteins secondary to deficiency in low-density lipoprotein (LDL) receptor. We reported recently the use of in situ stem cell therapy of human adipose tissue-derived multilineage progenitor cells (hADMPCs) in lowering serum total cholesterol in the homozygous Watanabe heritable hyperlipidemic (WHHL) rabbits, an animal model of homozygous FH. Here we demonstrate that pravastatin, an HMG-CoA reductase inhibitor, augmented the cholesterol-lowering effect of transplanted hADMPCs and enhanced LDL clearance in homozygous WHHL rabbit. The results suggest the potential beneficial effects of in situ stem cell therapy in concert with appropriately selected pharmaceutical agents, in regenerative medicine.

Transplantation of Human Adipose Tissue-Derived Multilineage Progenitor Cells Reduces Serum Cholesterol in Hyperlipidemic Watanabe Rabbits

Familial hypercholesterolemia (FH) is an autosomal codominant disease characterized by high concentrations of proatherogenic lipoproteins and premature atherosclerosis secondary to low-density lipoprotein (LDL) receptor deficiency. We examined a novel cell therapy strategy for the treatment of FH in the Watanabe heritable hyperlipidemic (WHHL) rabbit, an animal model for homozygous FH. We delivered human adipose tissue-derived multilineage progenitor cells (hADMPCs) via portal vein and followed by immunosuppressive regimen to avoid xenogenic rejection. Transplantation of hADMPCs resulted in significant reductions in total cholesterol, and the reductions were observed within 4 weeks and maintained for 12 weeks. (125)I-LDL turnover study showed that the rate of LDL clearance was significantly higher in the WHHL rabbits with transplanted hADMPCs than those without transplanted. After transplantation hADMPCs were localized in the portal triad, subsequently integrated into the hepatic parenchyma. The integrated cells expressed human albumin, human alpha-1-antitrypsin, human Factor IX, human LDL receptors, and human bile salt export pump, indicating that the transplanted hADMPCs resided, survived, and showed hepatocytic differentiation in vivo and lowered serum cholesterol in the WHHL rabbits. These results suggested that hADMPC transplantation could correct the metabolic defects and be a novel therapy for inherited liver diseases.

Signal attenuation and box-counting fractal analysis of optical coherence tomography images of arterial tissue

The sensitivity of optical coherence tomography images to sample morphology is tested by two methods. The first method estimates the attenuation of the OCT signal from various regions of the probed tissue. The second method uses a box-counting algorithm to calculate the fractal dimensions in the regions of interest identified in the images. Although both the attenuation coefficient as well as the fractal dimension correlate very well with the anatomical features of the probed samples; the attenuation method provides a better sensitivity. Two types of samples are used in this study: segments of arteries collected from atherosclerosis–prone Watanabe rabbits (WHHL-MI) and healthy segments of porcine coronary arteries.

Long-term Physiologically Regulated Expression of the Low-density Lipoprotein Receptor In Vivo Using Genomic DNA Mini-gene Constructs

Familial hypercholesterolemia (FH) is a condition caused by mutations in the low-density lipoprotein receptor (LDLR) gene. Expression of LDLR is highly regulated and excess receptor expression is cytotoxic. To incorporate essential gene regulation into a gene therapy vector for FH, we generated vectors in which the expression of therapeutic human LDLR gene, or luciferase reporter gene, is driven by 10 kb of human LDLR genomic DNA encompassing the promoter region including elements essential for physiologically regulated expression. Using luciferase expression and specific LDL binding and internalization assays, we have shown in vitro that the genomic promoter element confers long-term, physiologically regulated gene expression and complementation of receptor deficiency in culture for 240 cell-generations. This was demonstrated in the presence of sterols or statins, modifiers of LDLR promoter activity. In vivo, we demonstrate efficient liver-specific delivery and expression of luciferase following hydrodynamic tail-vein injection and confirm that expression from the LDLR promoter element is sensitive to statin administration. We also demonstrate long-term LDLR expression from the 10-kb promoter element up to 9 months following delivery. The vector system that we describe provides the efficient delivery, long-term expression, and physiological regulation required for a successful gene therapy intervention for FH.

High-Affinity αvβ3 Integrin Targeted Optical Probe as a New Imaging Biomarker for Early Atherosclerosis: Initial Studies in Watanabe Rabbits

A newly developed synthetic alpha v beta 3 integrin targeted optical probe (ITOP) has been demonstrated to target cancer cells, in vivo. Compared to the commercially available cyclic peptide c[RGDfv], this optical probe has at least 20 times better binding affinity for the alpha v beta 3 receptor. The present in vitro study was designed to investigate the possibility of detecting early atherosclerotic plaque by using this ITOP. Experiments were performed on five Watanabe heritable hyperlipidemic rabbits and two New Zealand White rabbits for control. Our ITOP was used for detecting the presence of alpha v beta 3 receptors in vitro. Segments of plaque accumulation from two distinct regions of ascending and descending aortas were labeled in Watanabe rabbits. The signal was found principally in the adventitia and proximal intima of the aortic vessel, corresponding directly to the expression of integrin alpha v beta 3 as determined by antibody assay. Moreover, there was a close association between the level of labeling with the alpha v beta 3 targeted probe and the thickness of the adventitia. This high-affinity ITOP identifies the site and extent of alpha v beta 3 expression and correlates with adventitial thickness. Recent evidence associates alpha v beta 3 expression with the inflammatory process in early vulnerable plaque, making this compound a promising potential biomarker for early atherosclerotic disease.

Soluble Guanylate Cyclase

Arthur Kornberg, who shared with Severo Ochoa the 1959 Nobel Prize for Physiology or Medicine, stated in his autobiography that he had never met a dull enzyme.1 We strongly believe that soluble guanylate cyclase (sGC; E.C.4.6.1.2) is no exception to this. Article see p 2781 Nitric oxide (NO) plays an important physiological role as a signaling molecule as well as a cytotoxic agent. It is produced by 3 distinct NO synthases, specifically endothelial, neuronal, and inducible NO synthase. In the vasculature, endothelial NO synthase plays an important role in vascular homeostasis because NO generated in endothelial cells promotes vascular smooth muscle cell relaxation and inhibits platelet aggregation. Importantly, bioavailability of NO can be reduced in cardiovascular diseases, a condition which has been termed “endothelial dysfunction.” This phenomenon provides a rationale for therapeutic intervention. The primary target of NO is sGC, which is a heterodimeric enzyme consisting of an α- and β-subunit and a prosthetic heme group, which is located in the β-subunit. The heme group contains a ferrous iron atom (Fe2+). Binding of NO to this iron changes the conformation of the enzyme and leads to a >100-fold increase in catalytic rate (ie, the conversion of guanosine triphosphate to the second messenger cyclic guanosine monophosphate [cGMP]). The NO-induced cGMP signal modulates intracellularly the activity of several effector molecules: cGMP-dependent protein kinases, cGMP-regulated phosphodiesterases, and cGMP-gated ion channels. Of note, both heme iron oxidation (Fe3+) and heme removal render sGC unresponsive to NO (Figure). These 2 conditions could therefore provide a mechanism for “vascular smooth muscle cell dysfunction” in cardiovascular disease states, which are often characterized by marked vasoconstriction either systemically or in specific organs. Figure. Schematic illustrating 3 different forms of soluble guanylate cyclase and their respective responsiveness to nitrovasodilators, sGC stimulators (eg, BAY 41–2272), and sGC …

Increased plant sterol and stanol levels in brain of Watanabe rabbits fed rapeseed oil derived plant sterol or stanol esters

Foods containing plant sterol or stanol esters can be beneficial in lowering LDL-cholesterol concentration, a major risk factor for CVD. The present study examined whether high dietary intake of rapeseed oil (RSO) derived plant sterol and stanol esters is associated with increased levels of these components in brain tissue of homozygous and heterozygous Watanabe rabbits, an animal model for familial hypercholesterolemia. Homozygous animals received either a standard diet, RSO stanol or RSO sterol ester while heterozygous animals were additionally fed with 2 g cholesterol/kg to the respective diet form for 120 d (n 9 for each group). Concentrations of cholesterol, its precursor lathosterol, plant sterols and stanols in brain and additionally in liver and plasma were determined by highly sensitive GC-MS. High-dose intake of RSO derived plant sterols and stanols resulted in increased levels of these components in plasma and liver. In brain a limited uptake of plant sterols and stanols was proven, indicating that these compounds passed the blood-brain barrier and may be retained in the brain tissue of Watanabe rabbits. Plant stanol ester feeding lowered plant sterol levels in brain, liver, and plasma. Cholesterol synthesis in brain, indicated by lathosterol, a local surrogate cholesterol synthesis marker, does not seem to be affected by plant sterol or stanol ester feeding. We conclude that high dose intake of plant sterol and stanol esters in Watanabe rabbits results in elevated concentrations of these components not only in the periphery but also in the central nervous system.

1061 Atorvastatin inhibits mitral regurgitation in an experimental hypercholesterolemia rabbit model

Degenerative mitral regurgitation is the most common cause of mitral regurgitation in the United States. Recent epidemiologic studies have linked degenerative mitral valve disease and risk factors for atherosclerosis. In the current study, we studied a hypercholesterolemic rabbit model with and without atorvastatin to determine the cellular mechanisms of mitral leaflet abnormalities. Methods: 48 Watanabe rabbits were assigned to one of 3 groups: cholesterol (1%) diet, cholesterol (1%) plus atorvastatin (2.5 mg/kg), and normal diet for six months. The rabbits underwent transesophageal echocardiograms and then the mitral valves were harvested. Electron microscopy, immunohistologic staining, and Real-time PCR was performed. Immunohistologic staining of the mitral valves was performed using atherosclerotic markers macrophage (RAM11), alpha -actin smooth muscle, and Proliferating Cell Nuclear Antigen (PCNA). Bone matrix expression was determined by staining for osteopontin, osteocalcin, alizarin Red and measuring alkaline phosphatase gene expression. A 4-point grading system was used to visually describe the staining on each of the slides (0=no staining, 4=high staining). Real-time PCR was performed was performed to measure alkaline phosphatase gene expression. Results: Mitral regurgitation (3+), Macrophage (3.5+/-0.7), alpha-actin (4+/-0), osteopontin (4+/-0), osteocalcin (2.5+/-0.7), PCNA (4+/-0) and alkaline phosphatase (0.479+/-0.286) were increased the mitral valves from the cholesterol treated rabbits (p<0.05 as compared to control mitral valves). Alkaline phosphatase was unchanged in the control versus cholesterol treatment groups. Atorvastatin decreased the amount of mitral regurgitation (1+), atherosclerotic proliferative changesincluding macrophage (2.5+/-0.7), osteopontin (1.5+/-0.7), osteocalcin (2.5+/-0.7), PCNA (2+/-0) and alkaline phosphatase expression (0.479+/-0.286) in these treated valves. Conclusion: Experimental hypercholesterolemia induces proliferation and osteopontin expression in the mitral valve that may potentially be modified with the use of a lipid-lowering agent.

MRI visualized neo-intimal dissection and co-localization of novel apoptotic markers apolipoprotein C-1, ceramide and caspase-3 in a Watanabe hyperlipidemic rabbit model

Purpose Apoptotic arterial wall vascular smooth muscle cell death is known to contribute to plaque vulnerability and rupture. Novel apoptotic markers like apolipoprotein C-I have been implicated in apoptotic human vascular smooth muscle cell death via recruiting a neutral sphingomyelinase (N-SMase)-ceramide pathway. In vivo relevance of these observations in an animal model of plaque rupture has not been shown. Methods and Results Using Watanabe rabbits, we investigated three different groups (group 1, three normal Watanabe rabbits; group 2, six Watanabe rabbits fed with high cholesterol diet for 3 months; group 3, five Watanabe rabbits with similar diet but additional endothelial denudation). We followed progression of atherosclerosis to pharmacologically induced plaque rupture non-invasively using novel 3D magnetic resonance Fast-Field-Echo angiography (TR = 7.2, TE = 3.6 ms, matrix = 512 × 512) and Fast-Spin-Echo vessel wall imaging methods (TR = 3 heart beats, TE = 10.5 ms, matrix = 304 × 304) on 1.5 T MRI. MRI provided excellent image quality with good MRI versus histology vessel wall thickness correlation ( r = 0.8). In six animals of group 2/3 MRI detected neo-intimal dissection in the abdominal aorta which was accompanied by immuno-histochemical demonstration of concomitant aforementioned novel apoptotic markers, previously implicated in the apoptotic smooth muscle cell death in vitro. Conclusions Our studies suggest a potential role for the signal transduction pathway involving apolipoprotein C-I for in vivo apoptosis and atherosclerotic plaque rupture visualized by MRI.

Magnetic resonance imaging of experimental atherosclerotic plaque: Comparison of two ultrasmall superparamagnetic particles of iron oxide

To evaluate a new ultrasmall superparamagnetic particles of iron oxide (USPIO) compound, ferumoxytol, as a marker of macrophage activity in atherosclerotic plaques and to compare it to ferumoxtran-10. Ten mature heritable hyperlipidemic (WHHL) female Watanabe rabbits served as the animal model for atherosclerosis, four coeval female New Zealand White (NZW) rabbits were the control group. Five WHHL and two NZW received a single intravenous injection (250 micromol/kg) of either ferumoxtran-10 or ferumoxytol and were subjected to daily MR examinations on a 1.5T whole body scanner for the next five days. Development of signal intensity changes and susceptibility effects was assessed. Statistical analysis was based on a nonparametric Wilcoxon-Mann-Whitney-U test by using a P value at the 0.05 significance level. On day 5, the rabbits were sacrificed and the aorta was referred to histopathology, distribution of iron particles in the vessel wall was analyzed. MRI was feasible in all animals. Three days after injection of ferumoxytol the highest luminal signal intensity measurements were observed in the ferumoxytol group; the highest measurements were five days after injection in the ferumoxtran-10 group (P < 0.05). In the WHHL, susceptibility effects presented as homogeneous dark lines parallel to the aortic wall after ferumoxytol and spotted areas void of signal after ferumoxtran-10. None of these findings were observed in the NZW control groups. Ferumoxtran-10 and ferumoxytol at a respective dose of 250 mumol/kg appear well suited for atherosclerotic plaque detection with MRI in experimental atherosclerosis. Ferumoxytol warrants further analysis in humans.