Aorta Of Hypercholesterolemic Rabbits Research Articles

Protective Action of Simvastatin in the Development of Aortic and Hepatic Lesions in Hypercholesterolemic Rabbits

As simvastatin is the most prescribed and distributed drug for high cholesterol by the National Health Service the authors carry out an experimental work to study special detailed effects of this drug. The authors studied the effect of simvastatin on the development of atherosclerotic lesions in aortas of rabbits submitted to a high-cholesterol diet, at 1.5%. The authors studied 30 New Zealand rabbits, males, adults, distributed in three groups according to diet: (A) cholesterol, (B) cholesterol + simvastatin (100 mg/day) and (C) normal. We took blood samples to evaluate cholesterol and plasma triglycerides at times zero, five and nine weeks and found lower serum cholesterol levels in the treated group. After nine weeks, we sacrificed the animals and examined the aortas for the extent of the atheromatous lesion area evaluated by computerized planimetry and the height of the lesion analyzed by morphometry, in histological sections of thoracic and abdominal aorta. There was no statistically significant difference between groups A and B in relation to the percentage of the injured area. However, in rabbits treated with simvastatin, smoother and less relief lesions were observed macroscopically by us and confirmed by morphometric analyses of atheroma plaque thickness. We observed intense morphological changes in the liver of the two groups of animals with a hypercholesterolemic diet, although to a slightly lower degree in those treated with simvastatin. We concluded that simvastatin had a protective effect on the volume of atherosclerotic lesions in aortas of hypercholesterolemic rabbits.

Histological classification and collagen distribution in atherosclerotic plaques in aorta of hypercholesterolemic rabbits

Objective: To analyze the histopathological aspect of atheroma plaques in the aorta of rabbits submitted to diet modification and different evolution times. Methods: Aortic fragments of rabbits submitted to the following protocols were analyzed: all animals received a diet plus 0.5% cholesterol for 3months and/or followed by: group A (n=8) - sacrificed at 3months; group B (n=10) - followed by 3months of standard diet; group C (n=20) followed by 3months of 0.1% cholesterol diet; group D (n=12) - followed by 9months of 0.1% cholesterol diet. Histological sections of aortic arch fragment were analyzed in hematoxylin-eosin and picro-sirius red staining. The atherosclerotic plaques were analyzed, classifying them according to the predominance of cellularity (type 2) or presence of extracellular matrix and fibromuscular cap (type 3) and associated with a large amount of extracellular cholesterol crystals (type 4). Results: We did not observe a statistically significant difference in the intima/mediarelationship, although there was a tendency to larger plaques in groups with lipid diet for prolonged time (p<0.06). We found after 3months of hypercholesterolemic diet (group A), high serum cholesterol levels (1972±127 mg/dL) and plaques with higher cellularity (type 2), destructuring of the media layer, especially along the internal elastic lamina. The modification of the diet for 3months (groups B and C) reduced serum cholesterol levels, modifying the characteristics of the plaque, verifying more developed plaques (types 3 and 4) in animals with lipid diet. After 12months (group D) plaques with matrix predominance in relation to cellularity were observed, with a large amount of crystal of cholesterol and collagen fibers. Conclusion: The normalization of the diet or reduction of the lipid content modifies the histology of the plaque, evolving with higher extracellular matrix content.

Mechanical Behavior to Overexpansion of Cobalt Chromium Compared with Stainless Steel Stents in the Abdominal Aorta of Hypercholesterolemic Rabbits

Introduccion Segun un estudio publicado previamente, los stents de acero inoxidable implantados en la aorta abdominal de conejos hipercolesterolemicos, presentan un mayor grado de retraccion elastica cuando son sobreexpandidos versus aquellos que se implantan a su diametro nominal Objetivos El presente estudio tiene como objetivo general investigar cual es el comportamiento de los stents de aleacion de cromo cobalto ante la sobreexpansion comparados con los stents de acero inoxidable cuando se los implanta en aortas de conejos hipercolesterolemicos. Material y Metodos Se utilizaron 20 conejos Neozelandeses, los que fueron alimentados con colesterol al 1% agregado en la dieta convencional durante un periodo de 30 dias. Este modelo ha probado ser adecuado para el estudio del fenomeno de reestenosis luego de injuria vascular. Los animales fueron divididos en dos grupos. El grupo 1 (n= 10) recibio stents de 3.0mm x 18mm implantados a 20 atmosferas para alcanzar un diametro nominal de 3.42mm y el grupo 2 (n= 10) stents de 3.5mm x 18mm implantados a 10 atmosferas para alcanzar un diametro nominal de 3.63mm. Estos fueron comparados con una serie previa de 20 animales alimentados de igual manera a los que se le implantaron stents de acero inoxidable de igual diseno, divididos en los mismos dos grupos. Un tercer grupo con dieta convencional fue utilizado como control. Luego del implante, se realizo un estudio con ultrasonido intravascular (IVUS) en los dos grupos a fin de determinar el diametro y el area del stent. El grado de retraccion elastica se calculo comparando el diametro nominal alcanzado por el balon en el momento del implante con el diametro del stent medido por IVUS post implante. Se analizo tambien el grado de simetria utilizando los “angulos interstrut” a fin de cuantificar la separacion entre ellos, medidos considerando como vertice el centro de la circunferencia determinada por el corte transversal de la arteria. Resultados El valor promedio de colesterol en sangre del grupo 1 fue 119.6±18.2 mg, mientras que para el grupo 2 el valor promedio de colesterol en sangre fue 122.4±22.7 mg. El valor en los animales controles fue de 61,6±10.8 mg. Para el grupo 1 de stents de cromo cobalto, el valor promedio de diametro medido por ultrasonido de los stents fue 3.31 ± 0.13mm y el valor promedio de retroceso elastico de los stents fue 0.11 ± 0.13mm, correspondiendo a 3.21% de retroceso elastico. Para el grupo 2, de stents de cromo cobalto el valor promedio de diametro medido por ultrasonido de los stents fue 3.33 ± 0.12mm y el valor promedio de retroceso elastico de los stents fue 0.3 ± 0.12mm, correspondiendo a 8.26% de retroceso elastico. Para el grupo 1 de stents de acero inoxidable, el valor promedio de diametro medido por ultrasonido de los stents fue 3.41±0.01mm, y el valor promedio de retroceso elastico de los stents fue 0,28 ± 0,18mm, correspondiendo a 8.21% de retroceso elastico. Para el grupo 2 de stents de acero inoxidable el valor promedio de diametro medido por ultrasonido de los stents fue 3.58±0.01mm y el valor promedio de retroceso elastico de los stents fue 0,10 ± 0,11mm, correspondiendo a 2.79% de retroceso elastico. En terminos de simetria, en los stents de acero inxoxidabel no se observo diferencia en cuanto a la configuracion simetrica en ambos grupos ya que el 88,8% de los stents del grupo 1 y el 71,4% de los stents del grupo 2 fueron simetricos(p = 0,5). En cambio para los stents de cromo cobalto el 50% de los stents del grupo 1 y el 90% de los stents del grupo 2 fueron simetricos (p=0.05) Conclusiones En los stents de Cromo Cobalto el grupo de stents sobreexpandidos presento un retroceso elastico menor que el grupo de stents no sobreexpandidos (0.11 ± 0.13mm vs.0.3 ± 0.12mm) p= 0.002. en contraposicion con los stents de acero inoxidable donde el retroceso elastico de los stents sobreexpandidos fue mayor que en los no sobreexpandidos (0,28 ± 0,18mm vs. 0,10 ± 0,11mm) p˂ 0.001 . En terminos de simetria, no se observaron diferencias entre los grupos

Mechanical Behavior to Overexpansion of Cobalt Chromium Compared with Stainless Steel Stents in the Abdominal Aorta of Hypercholesterolemic Rabbits

Mechanical Behavior to Overexpansion of Cobalt Chromium Compared with Stainless Steel Stents in the Abdominal Aorta of Hypercholesterolemic Rabbits

Baccaurea angulata fruit juice reduces atherosclerotic lesions in diet-induced Hypercholesterolemic rabbits

BackgroundAtherosclerosis is the most common disease of large and medium-sized arteries linked to oxidative stress, dyslipidemia as well as chronic inflammation. The aim of this study was to evaluate the potential health benefits of Baccaurea angulata (BA) fruit juice on the aorta of diet-induced hypercholesterolemic rabbits, to detect an accumulation of fatty streak and evaluate the percentage of atherosclerotic lesion accrued.MethodsThirty-five healthy male adults New Zealand White rabbits were assigned to seven different groups. Four groups were fed 1% cholesterol diet and 0, 0.5, 1.0, and 1.5 mL of BA fruit juice per kg of rabbit daily (atherogenic groups), while the other three groups were fed commercial rabbit pellet and 0, 0.5, and 1.0 mL of juice per kg of rabbit daily (normocholesterolemic groups) for 90 days. The thoracic and abdominal aorta between the heart origin and bifurcation into iliac arteries of all the rabbits were carefully removed and analyzed accordingly.ResultsThe supplementation of the high-cholesterol diet of hypercholesterolemic rabbits with only 0.5 mL BA/kg rabbit per day significantly (p < 0.001) improved aortic lipid profile, attenuated aortic fatty streak development and reduced intima thickening. Higher BA doses used (1.0 and 1.5 mL/kg rabbit per day) also significantly (p < 0.001) decreased further the development of aortic fatty streaks, reduced the thickening of the tunica intima layer and preserved endothelial healing following arterial injury.ConclusionTherefore, BA fruit is a potential novel functional food with effective anti-inflammatory, anti-atherogenic and hypocholesterolemic activities.

Decreased eNOS relaxations and relation with hsp90 in hypercholesterolemic rabbit aorta

Hsp90 activates eNOS in vasculature. we investigated basal and agonist evoked eNOS dependent relaxations and the role of hsp90 on these endothelial relaxations in hypercholesterolemia, since it is not studied well.Rabbits were fed with a regular/high cholesterol diet (%2) for 6 weeks. Hypercholesterolemia almost totally abolished agonist evoked eNOS dependent KCl‐ACh relaxations (93,9 % inhibition), however inhibited phenylephrine‐ACh relaxations only by 32,8 % in aorta in krebs buffer with indomethacine suggesting increased EDHF responses in hypercholesterolemic aorta.Basal eNOS dependent LNA‐induced further contraction and papaverin‐induced relaxations were not changed in hypercholesterolemic aorta.Hsp90 inhibitor radicicol (10 μg/ml) for 15 hrs decreased ACh relaxation after pre‐contraction with KCl (11,22±1,18 vs 7,33±1,58 *p&lt;0,01 student t test, n=5) however did not cause any further inhibition in hypercholesterolemic aorta, suggesting that hsp90 inhibitors do not cause any further inhibition since hsp90 dependent eNOS activation may be already disrupted in hypercholesterolemia.We concluded that although basal eNOS dependent relaxations were not affected, since the agonist evoked eNOS relaxations were decreased which seems mainly depend on hsp90, a compansotory increase in EDHF relaxations may develop in hypercholesterolemic aorta. (Supported by TUBA GEBÝP‐2010 and FABAL).

Cholesterol diet and effect of long-term withdrawal on plaque development and composition in the thoracic aorta of New Zealand White rabbits

AimsExperimental study on plaque progression, regression and composition in atherosclerotic thoracic aorta of hypercholesterolemic rabbits after long-term withdrawal of cholesterol-enriched diet (CED). MethodsRabbits were fed 2% cholesterol for 6 weeks followed by withdrawal periods for 15, 23, 34, 68, or 78 weeks. Cholesterol, triglyceride, and phospholipids levels in blood and cholesterol concentrations in aorta were quantified. Plaque size and cellularity, phenotype of macrophages and smooth muscle cells were (immuno)histomorphometrically analyzed in segments of the thoracic aorta. ResultsAfter 6 weeks of CED, blood cholesterol levels were about 80-fold higher, whereas atherosclerosis and cholesterol content in the thoracic aorta were only minimally increased. However, the latter significantly increased within 15 weeks after cholesterol withdrawal, while serum cholesterol level was still 10-fold increased. Thereafter plaque area and cholesterol content remained almost unchanged until the end of the study despite a long-term normalization of serum cholesterol level after withdrawal of CED. Directly after 6 weeks of CED the densities of macrophages and apoptotic cells within plaques were highest, decreasing after cholesterol withdrawal, whereas, vice versa the density of smooth muscle cells (SMCs) significantly increased. ConclusionWe suggest that atherosclerotic plaques respond to long-term withdrawal of CED by decrease in number and phenotype of macrophages and increase of SMCs without regression of the lesion size. The cellular changes are suggested to considerably contribute to higher plaque stability.

D 006 ACE Inhibitor Protects Aorta, Lever and Kidney of Hypercholesterolemic and Diabetic Rabbits

D 006 ACE Inhibitor Protects Aorta, Lever and Kidney of Hypercholesterolemic and Diabetic Rabbits

Effects of overexpansion on stents' recoil, symmetry/asymmetry, and neointimal hyperplasia in aortas of hypercholesterolemic rabbits

It is not known whether overexpansion modifies stent recoil, symmetric distribution of struts, and neointimal hyperplasia. The objectives were (a) to evaluate whether stent overexpansion modifies the geometric configuration of the stent in the arterial wall, (b) to determine the relationship between overexpansion and stent recoil, and (c) to evaluate the relationship between the distribution of struts and neointimal hyperplasia. Twenty tubular stainless steel 316L stents (3.0 and 3.5 mm in diameter) were implanted at 20 and 10 atm, respectively, in the abdominal aorta of New Zealand rabbits fed a hypercholesterolemic diet (1% cholesterol). Sham operations were also performed in seven animals. Eight weeks after implantation or sham operation, an intravascular ultrasound (IVUS) study was performed to measure stent recoil and aid in stent classification (symmetric or asymmetric) according to strut distribution. The degree of injury and neointimal hyperplasia were also evaluated in hematoxylin-eosin stained sections. The symmetry/asymmetry of stents assessed by IVUS, as well as the neointimal hyperplasia, was similar in both groups. Stent recoil was significantly greater in the 3.0-mm stent (overexpanded) group (0.28+/-0.02 mm), as compared with stent recoil in the 3.5-mm stent group (0.10+/-0.01 mm, P<.05). The neointimal hyperplasia in histological slices, independent of the implant technique, was predominantly in zones with higher strut concentration as compared with zones with fewer struts. Stent overexpansion enhanced stent recoil and did not modify symmetric and asymmetric strut distribution. Neointimal hyperplasia was not modified by the implant technique. Interestingly, significant hyperplasia was observed in locations with greater strut concentration, independent of overexpansion.

Soy isoflavones reduce electronegative low-density lipoprotein (LDL−) and anti-LDL− autoantibodies in experimental atherosclerosis

Isoflavones present in soybean may contribute to soy atheroprotective effects. To investigate the effect of soy isoflavones supplementation on the formation of electronegative LDL (LDL(-)) and its autoantibodies in blood plasma and aortic atheromas of rabbits fed an atherogenic casein-based diet enriched with isoflavones. New Zealand male rabbits (n = 15) were fed an atherogenic diet (27% casein) supplemented with isoflavones (0.73 or 7.3 mg of isoflavones/kg/day, Low and High Iso groups, respectively) for 180 days. Monthly, blood samples were collected after 12-15 h fasting and at 180 days of treatment all animals were sacrificed. Isoflavones were analyzed in plasma and urine samples by HPLC. LDL(-) in plasma and atheromas was detected by ELISA and immunohistochemistry, respectively, with a monoclonal antibody reactive to LDL(-). Autoantibodies reactive to LDL(-) were analyzed in plasma and aorta by ELISA. Low and High Iso groups had decreased LDL-cholesterol, increased HDL-cholesterol and lower levels of LDL(-) in blood plasma and aortic atherosclerotic lesions than the non-supplemented Control group. IgG autoantibodies reactive to LDL(- )were higher in plasma of the Control group in comparison with the High and Low Iso groups. In contrast, the aortas from animals that consumed isoflavones showed higher levels of IgG reactive to LDL(- )than the Control group. Soy isoflavones showed hypolipidemic effects and decreased the pro-inflammatory LDL(-) subfraction in blood plasma and aorta of hypercholesterolemic rabbits.

Irinotecan‐eluting stents inhibited neointimal proliferation in hypercholesterolemic rabbit aortas

To assess the effect of irinotecan-eluting stents (IS) on neointimal growth in the aortas of hypercholesterolemic rabbits and to determine other local histopathological effects such as necrosis, fibrin, and inflammatory reaction. Phosphorylcholine-coated stents were deployed in the aortas of hypercholesterolemic rabbits. Group 1 (control; n = 8) received unloaded stents, group 2 (n = 7) and group 3 (n = 9) received IS with 0.046 mg and 1.29 mg of irinotecan, respectively. Eight weeks after implantation the rabbits were killed. Neointimal thickness (NT) was assessed by morphometry. Semiquantitative injury score (from 0 to 3+) was used to analyze inflammatory infiltrate, fibrin deposits, and necrosis in the stented segments. NT was reduced only in high-doses IS (G1, 167.4 +/- 20.8 mu; G2, 170.24 +/- 21.2 mu; G3, 111.56 +/- 12.7 mu; P < 0.05, G3 vs G1 and G2). Necrosis decreased significantly with IS [1.00 +/- 0.10 in G1 to 0.33 +/- 0.07 and 0.02 +/- 0.01 in G2 and G3, respectively] only in the media layer. The inflammatory infiltrate was present in the three layers of aortas from G1, but only decreased significantly in the intimae layer of the high-dose group [1.50 +/- 0.15 in G1 vs 1.00 +/- 0.18 in G3, P < 0.05]. Stents loaded with high-dose irinotecan inhibit NT in the aortas of hypercholesterolemic rabbits. This effect was accompanied by decreased inflammatory infiltrate and media necrosis.

Ferumoxtran-10–Enhanced MRI of the Hypercholesterolemic Rabbit Aorta

Ferumoxtran-10 is an MRI contrast agent, which accumulates in macrophages and induces magnetic susceptibility artifacts (MSAs). We evaluated the ability of ferumoxtran-10-enhanced MRI to quantify focal macrophage infiltration in the aortic wall of hypercholesterolemic rabbits. Six weeks after a double-balloon injury of the infrarenal aorta, 12 hypercholesterolemic rabbits underwent MRI of the aorta before (first MRI) and after (second MRI) intravenous injection of ferumoxtran-10 (n=10) or saline (n=2). A third MRI was performed 5 days later to detect ferumoxtran-10-induced MSA in the aortic wall. Aortas were subsequently processed for histology, immunohistochemistry, and gelatin zymography studies. Injured aortas displayed a macrophage-rich neointima with high-matrix metalloproteinase 2 and 9 activities. Iron stain of injured aortas showed massive accumulation of ferumoxtran-10 in neointimal macrophages. Five days after the injection of ferumoxtran-10, MSAs were detected only in the injured aortas by in vivo MRI and were quantified indirectly using the percentage reduction of luminal area attributable to the extension of these MSAs in the aortic lumen. This parameter correlated with macrophage infiltration on corresponding aortic cross-sections (r=0.82; P<0.05). Ferumoxtran-10-enhanced MRI allows quantitative assessment of macrophage infiltration induced by balloon angioplasty in the aorta of hypercholesterolemic rabbits.

Are Anti–Oxidized Low-Density Lipoprotein Antibodies Pathogenic or Protective?

Despite a recent decline, atherosclerosis remains the most common cause of death in the Western world. The disease course of atherosclerosis is characterized by its chronicity, with progression in its initial stages being particularly insidious. Chronic inflammation is the pathological hallmark of atherosclerosis,1–4 and inflammatory processes are instrumental at all stages of this disease. Even before the development of detectable intimal lesions, the expression pattern of the endothelium has been shown to be inflammatory in nature, conforming to the response-to-injury hypothesis first postulated by the late Russell Ross.5 Thus, in lesion-prone sites of the arterial tree, the endothelial expression of adhesion molecules is upregulated, reflecting endothelial dysfunction secondary to unfavorable blood rheology6 and/or hypercholesterolemia.7–9 Atherosclerosis is known today to be associated with the burden of infection as well.10 The presence of infectious/inflammatory pathogeneses raises the autoimmune aspect of the condition.11–14 Anti–oxidized LDL (oxLDL) antibodies are exceptional among the autoantibodies prevalent in atherosclerosis: on the one hand, a correlation was found between the existence and titers of anti-oxLDL antibodies and the extent of atherosclerosis and cardiovascular disease (CVD) (Table 1). On the other hand, experimental data indicate that anti-oxLDL antibodies may be protective. View this table: TABLE 1. Correlation Between Titers of Anti-oxLDL Abs and Atherosclerosis and CVD oxLDL frequently presents in the sera of patients with autoimmune conditions, acute coronary syndrome, or stable coronary artery disease (CAD).15–18 OxLDL has been associated with both subclinical atherosclerosis and inflammatory variables.19 A large amount of oxLDL accumulates in atherosclerotic plaques,20–22 and the serum concentrations of circulating oxLDL may correlate with the severity of CAD23 and acute coronary syndrome.24 OxLDL seems to be an immunogenic molecule that stimulates the induction of anti-oxLDL antibodies. Thus, it is not surprising that an association was found between the presence …

Vitamin E supplementation suppresses macrophage accumulation and endothelial cell expression of adhesion molecules in the aorta of hypercholesterolemic rabbits

Suppression of cell adhesion molecule expression and macrophage accumulation by the endothelium is believed to play an important role in preventing the development of atherosclerosis. Earlier, we have shown that in vitro supplementation of human aortic endothelial cells with Vitamin E dose-dependently reduced expression of adhesion molecules and monocyte adhesion. Here, we report the in vivo down-regulation of endothelial cell adhesion molecules expression and macrophage accumulation in the aortas of hypercholesterolemic rabbits supplemented with Vitamin E. To this end, New Zealand White rabbits were fed a semi-purified diet containing 30 (control) or 1000 IU/kg Vitamin E. After 4 weeks, both groups’ diets were switched to an atherogenic diet (0.3% cholesterol, 9% hydrogenated coconut oil, and 1% corn oil) containing the respective levels of Vitamin E and fed for 2, 4, and 6 weeks. Vitamin E supplemented rabbits had significantly higher levels of Vitamin E in their plasma and aortas. Frozen aorta sections were fixed and stained by an avidin–biotin complex method using Rb2/3 and Rb1/9 monoclonal antibodies against rabbit ICAM-1 and VCAM-1, respectively, and with RAM-11 for macrophage and von Willebrand factor for endothelial cells, followed by staining with secondary antibodies and counterstaining and evaluation under the microscope. At 6 weeks on atherogenic diet treatment, a trend ( P = 0.08) toward a lower score of ICAM-1 expression by endothelial cells was observed in the aorta of Vitamin E treated rabbits compared to the control. However, a decrease in the score of VCAM-1 expression by endothelial cells in Vitamin E treated rabbits did not reach to a statistical significance. At 4 and 6 weeks on atherogenic diet, Vitamin E supplementation also significantly ( P = 0.003) inhibited the accumulation of macrophages in the aorta. These results support the concept that down-regulation of adhesion molecule expression and suppression of monocyte/macrophage activation by Vitamin E in vivo is one of the potential mechanisms by which Vitamin E may suppress the development of aortic lesions in a rabbit model of atherosclerosis.

Tetrahydrobiopterin: Mediator of Endothelial Protection

During the past twenty years, the essential role of endothelial cells in preservation of vascular homeostasis has been well established.1,2⇓ Protection of the vascular endothelium against harmful influences of circulating substances including excessive levels of lipids has been a major therapeutic approach in prevention of atherosclerosis. While endothelial dysfunction, defined as a loss of biologically active nitric oxide (NO) produced in the endothelium, has been recognized as a prime target for prevention and reversal of atherosclerotic process, identification of exact molecular mechanisms responsible for the loss of endothelial NO have been more difficult to determine. In the current issue of Arteriosclerosis, Thrombosis, and Vascular Biology , Alp et al3 report a series of elegant studies on genetically modified mice supporting the concept that NO synthase cofactor, tetrahydrobiopterin (Figure 1), is an important vascular protective molecule. The beneficial effect of tetrahydrobiopterin in apolipoprotein E (ApoE)-deficient mice appears to be mediated by preservation of endothelial nitric oxide synthase (eNOS) activity and production of nitric oxide in endothelial cells. The major steps of tetrahydrobiopterin biosynthesis. GTP=guanosine 5′-triphosphate. See page 445 In the early 1990s it was recognized that in the presence of suboptimal concentrations of tetrahydrobiopterin, activation of purified neuronal nitric oxide synthase (nNOS) causes “uncoupling of NOS” with subsequent increase …

Reduced sarco/endoplasmic reticulum Ca(2+) uptake activity can account for the reduced response to NO, but not sodium nitroprusside, in hypercholesterolemic rabbit aorta.

Hypercholesterolemia (HC) impairs acetylcholine-induced relaxation but has little effect on that caused by the NO donor sodium nitroprusside (SNP), suggesting that acetylcholine releases less NO from the endothelium in HC. The relaxation to authentic NO gas, however, is also impaired in HC aortic smooth muscle, indicating an abnormal smooth muscle response. NO relaxes arteries by both cGMP-dependent and -independent mechanisms, and the response involves calcium (Ca(2+)) store refilling via the sarco/endoplasmic reticulum calcium ATPase (SERCA). We studied the involvement of cGMP and SERCA in the smooth muscle response to NO and SNP in HC rabbit aorta. A selective guanylyl cyclase inhibitor, 1H-[1,2,4]-oxadiazole-[4,3-a]quinoxalin-1-one, eliminated SNP-induced relaxation but only partially blocked NO-induced relaxation in both normal and HC aorta. The residual relaxation to NO was still less in HC and, in both normal and HC aorta, was abolished by concomitant administration of the SERCA inhibitor cyclopiazonic acid (CPA). In contrast, CPA did not affect SNP-induced relaxation in either normal or HC aorta. SERCA activity measured by (45)Ca(2+) uptake was markedly decreased in HC, although SERCA2 protein expression did not change significantly. These data suggest that NO-induced relaxation but not that to SNP is partially mediated by cGMP-independent Ca(2+) uptake into sarco/endoplasmic reticulum and that reduced sarco/endoplasmic reticulum Ca(2+) pump function can account for the impaired response to NO in HC.

Increased expression of heme oxygenase-1 and bilirubin accumulation in foam cells of rabbit atherosclerotic lesions.

Heme oxygenase-1 (HO-1) catalyzes the regiospecific oxidative degradation of heme to biliverdin IXalpha, iron, and carbon monoxide. Biliverdin IXalpha is subsequently reduced to bilirubin IXalpha by biliverdin reductase. HO-1 expression is induced under various disease conditions, including atherosclerosis, but it is unknown whether HO-1 catalyzes heme breakdown in the regions at risk. Using hypercholesterolemic rabbits fed a cholesterol-enriched diet, we attempted to demonstrate the involvement of HO-1 induction and bilirubin IXalpha production in atherosclerotic regions. Expression levels of HO-1 mRNA were elevated in the aortas of hypercholesterolemic rabbits. In situ hybridization and immunohistochemistry revealed that mRNA and protein of HO-1 are induced in endothelial cells and foam cells (lipid-filled macrophages) in atherosclerotic lesions. Furthermore, immunohistochemistry with the use of an anti-bilirubin-IXalpha monoclonal antibody, 24G7, demonstrated accumulation of bilirubin IXalpha in foam cells, indicating that heme is actually degraded in atherosclerotic lesions. Remarkably, bilirubin IXalpha, like HO-1 protein, is predominantly accumulated in the perinuclear regions of foam cells. These results provide the first in vivo evidence of the colocalization of HO-1 and bilirubin IXalpha in foam cells, suggesting a role of HO-1 induction in the modulation of macrophage activation in atherosclerosis.

Upregulation of LOX-1 Expression in Aorta of Hypercholesterolemic Rabbits: Modulation by Losartan

Angiotensin-II (Ang-II) enhances the modification of LDL and the expression of its lectin-like receptor (LOX-1) by activating type 1 (AT1) receptors. This study was designed to determine the effect of hypercholesterolemia on LOX-1 expression in aorta and its modulation by the AT1 receptor blocker losartan. Male New Zealand White rabbits were fed regular chow (Control group), chow with 1% cholesterol and 4% peanut oil (HC-diet group), or 1% cholesterol and 4% peanut oil diet plus losartan (25 mg/kg/day) (Losartan + HC-diet group) for 10 weeks. Animal body weight, serum cholesterol levels, and arterial blood pressure were measured. Aortic intimal thickening was quantitated in H&E-stained segments. LOX-1 expression in aortas was examined by immunohistochemistry and semi-quantitative RT-PCR. High-cholesterol diet did not affect body weight, but induced hypercholesterolemia and extensive intimal thickening. Aortas of rabbits in the control group showed a modest LOX-1 expression in the endothelium. Aortic intimal proliferation in HC-diet group was associated with a marked increase in LOX-1 expression (protein and mRNA) in the endothelium and neointima. Treatment with losartan attenuated aortic intimal proliferation and markedly decreased the enhanced LOX-1 expression. Thus high-cholesterol diet induces the upregulation of LOX-1 expression in neointima of aortas of rabbits. Treatment with losartan, an AT1 blocker, markedly decreases this enhanced LOX-1 expression.

Involvement of Cyclooxygenase 2 in the Protective Effect of 17β-Estradiol on Hypercholesterolemic Rabbit Aorta

The involvement of cyclooxygenase (COX) in the effects of 17β-estradiol was investigated on hypercholesterolemic rabbits aorta. Acetylsalycilic acid, nimesulide, or SQ22536 was used as respective antagonist of COX-1, COX-2, or adenylate cyclase using aortic rings precontracted with phenylephrine and exposed to cumulative concentrations of acetylcholine (ACh). The relaxation effect of ACh was impaired by hypercholesterolemia and restored by an 8-week 17β-estradiol treatment. In the control group treated with estrogen, nimesulide, acetylsalycilic acid, or SQ22536 slightly reduced the response to ACh. In hypercholesterolemic rabbits treated with estrogen, nimesulide significantly reduced the maximal relaxation and shifted to the right the relaxation curve of ACh, whereas acetylsalycilic acid did not modify the maximal response to ACh but displaced slightly the concentration–response curve. SQ22536 reduced the relaxant effect of ACh down to the level obtained in the presence of nimesulide. These results suggest that the protective effect of 17β-estradiol against hypercholesterolemia involved COX-2/adenylate cyclase pathway.

Ex vivo gene transfer of endothelial nitric oxide synthase to atherosclerotic rabbit aortic rings improves relaxations to acetylcholine

Cholesterol feeding results in impaired endothelium dependent vasorelaxation. The role of nitric oxide in this process is unclear. The aim of this study was to evaluate the role of nitric oxide in cholesterol-induced vasomotor dysfunction by examining the effect of overexpression of eNOS in the hypercholesterolemic rabbit aorta on vascular reactivity. Vascular rings from the thoracic aorta of hypercholesterolemic rabbits were exposed ex vivo either to an adenoviral vector encoding endothelial nitric oxide synthase (AdeNOS) or Escherichia coli β Galactosidase (Ad βGal). Transgene expression was examined by histochemistry for β galactosidase, immunohistochemistry for eNOS and cyclic GMP measurements and vasomotor studies were performed. Transgene expression was found to localize to the endothelium and adventitia. cGMP levels were significantly greater in AdeNOS compared to Ad βGal transduced rings. Acetylcholine mediated relaxation was significantly impaired in cholesterol fed rabbits and was markedly improved by overexpression of eNOS. These results suggest that reduced NO bioavailability observed in cholesterol-induced vascular dysfunction can be partially overcome by eNOS gene transfer.