Aortas Of apoE-deficient Mice Research Articles

Biomimetic Nanocarriers of Pro-Resolving Lipid Mediators for Resolution of Inflammation in Atherosclerosis.

Atherosclerosis (ATH) is a systemic disease characterized by a chronic inflammatory process and lipid deposition in the arterial walls. The chronic inflammation within ATH lesions results, at least in part, from the failed resolution of inflammation. This process is controlled actively by specialized pro-resolving lipid mediators (SPMs), namely lipoxins, resolvins, protectins, and maresins. Herein, biomimetic nanocarriers are produced comprising a cocktail of SPMs-loaded lipid nanoemulsions (LN) covered with macrophage membranes (Bio-LN/SPMs). Bio-LN/SPMs retain on their surface the macrophage receptors involved in cellular interactions and the "marker of self" CD47, which impede their recognition and uptake by other macrophages. The binding of Bio-LN/SPMs to the surface of endothelial cells (EC) and smooth muscle cells (SMC) is facilitated by the receptors on the macrophage membranes and partly by SPMs receptors. In addition, Bio-LN/SPMs prove functional by reducing monocyte adhesion and transmigration to/through activated EC and by stimulating macrophage phagocytic activity. After intravenous administration, Bio-LN/SPMs accumulate in the aorta of ApoE-deficient mice at the level of atherosclerotic lesions. Also, the safety assessment testing reveals no side effects or immunotoxicity of Bio-LN/SPMs. Thus, the newly developed Bio-LN/SPMs represent a reliable targeted nanomedicine for the resolution of inflammation in atherosclerosis.

Atomic force microscopy application to study of the biomechanical properties of the aortic intima in the context of early atherosclerosis.

Atherosclerosis is characterized by the infiltration of macrophages, accumulation of lipids, activation of endothelial cells and synthesis of extracellular matrix by vascular smooth muscle cells. However, there have been few atomic force microscopy (AFM) studies of the aortic intima in situ in the context of atherosclerosis. By employing a customized liquid cell for AFM, we investigated the aortic intima obtained from male C57BL/6 ApoE-deficient mice (ApoE-/- ) aged 14 weeks and male C57BL/6 ApoE-sufficient mice (ApoE+/+ ) aged between 18 and 26 weeks that were fed a high-fat and high-cholesterol diet for 4 weeks and performed force spectroscopy mapping of the biomechanical properties of the intima. In the aortas of ApoE-deficient mice, the intima became stiffer than that of ApoE-sufficient mice. In addition, the cytoskeleton of endothelial cells was enlarged, and extracellular matrix accumulated. The biomechanical properties of the aortic intima are altered in early atherogenesis, which may be induced by the enlargement of the endothelial cell cytoskeleton and the increased synthesis of extracellular matrix by activated smooth muscle cells.

Effects of icariin on long noncoding RNA and mRNA expression profile in the aortas of apoE-deficient mice.

Objective: The beneficial effects of icariin (ICA) in ameliorating atherosclerosis (AS) are well known, but the underlying protective mechanism has not been fully elucidated. The present study aimed to investigate altered long noncosing RNA (lncRNA) and mRNA expression profiles in ApoE−/− mice after ICA treatment.Method: The atherosclerotic plaque area was evaluated on high-fat diet (HFD)-induced ApoE−/− mice treated with either ICA or vehicle. LncRNA and mRNA integrated microarrays was performed on aortic tissues. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were utilized to explore the significant function and pathway of the differentially expressed (DE) mRNAs, global signal transduction network were constructed to select key mRNAs, and lncRNA–mRNA co-expression network was built to find out the interactions between lncRNA and mRNA. Quantitative real-time PCR (qPCR) was used to further validate the expressions of selected lncRNAs and mRNAs.Results: Administration of ICA significantly reduced plaque size after 12 weeks (P<0.05). A total of 1512 DE lncRNAs and 2059 DE mRNAs were identified. The mRNAs: protein kinase C, β (Prkcb), Cyp2c65, Mapk10, Calmodulin 5 (Calm5), Calmodulin-like 3 (Calml3) and Camk4 were selected as hub mRNAs, the correlated lncRNAs in co-expression network were identified as important regulatory lncRNAs. The identified target pairs such as lncRNA-NONMMUT000659/Prkcb may play critical roles in AS development mediated by ICA.Conclusion: Taken together, our study highlights a panel of DE lncRNAs and mRNAs that could explain the molecular mechanism of ICA’s anti-atherosclerotic effects. The work lays a foundation for subsequent genes functional researches, which could contribute to provide new therapeutic targets for AS.

Increased Reactive Oxygen Species Generation Contributes to the Atherogenic Activity of the B2 Bradykinin Receptor.

Atherosclerosis and ensuing cardiovascular disease are major causes of death with insufficient treatment options. In search for pathomechanisms of atherosclerosis, we investigated the impact of the B2 bradykinin receptor, Bdkrb2, on atherosclerotic lesion formation, because to date it is not clear whether the B2 bradykinin receptor is atheroprotective or atherogenic. As a model of atherosclerosis, we used hypercholesterolemic ApoE-deficient (apolipoprotein E-deficient) mice, which develop atherosclerotic lesions in the aorta with increasing age. The role of Bdkrb2 in atherosclerosis was studied in ApoE-deficient mice, which were either Bdkrb2-deficient, or had moderately increased aortic B2 bradykinin receptor protein levels induced by transgenic BDKRB2 expression under control of the ubiquitous CMV promoter. We found that Bdkrb2 deficiency led to a significantly decreased atherosclerotic plaque area whereas transgenic BDKRB2 expression enhanced atherosclerotic lesion formation in the aorta of ApoE-deficient mice at an age of 8 months. Concomitantly, the aortic content of reactive oxygen species (ROS) was higher in BDKRB2-expressing mice whereas Bdkrb2 deficiency decreased aortic ROS levels of ApoE-deficient mice. In addition, aortic nitrate as a marker of nitric oxide activity and the endothelial nitric oxide synthase (eNOS) co-factor, tetrahydrobiopterin (BH4) were reduced in BDKRB2-expressing ApoE-deficient mice. The decreased aortic BH4 content could be a consequence of increased ROS generation and down-regulated aortic expression of the BH4-synthesizing enzyme, Gch1 (GTP cyclohydrolase 1). In agreement with a causal involvement of decreased BH4 levels in the atherogenic function of BDKRB2, we found that treatment with the BH4 analog, sapropterin, significantly retarded atherosclerotic plaque formation in BDKRB2-expressing ApoE-deficient mice. Together our data show that the B2 bradykinin receptor is atherogenic, and the atherosclerosis-promoting function of BDKRB2 is partially caused by decreased aortic BH4 levels, which could account for eNOS uncoupling and further enhancement of ROS generation.

Abstract 13514: Apoptogenic Protein is a Novel Phenotypic Marker of the Contractile Vascular Smooth Muscle Cells

Introduction: Apoptogenic protein (Apop) is a mitochondrial protein originally identified in the atherosclerotic cells cultured from thoracic aortas of ApoE-deficient mice. Functional analysis revealed that the expression of Apop using an expression vector induces apoptotic death of smooth muscle cells (SMC) in culture through the release of cytochrome c from mitochondria, followed by the activation of the caspase cascade. Here, we show that Apop is expressed in the contractile SMC, and can be a novel phenotypic marker of the SMC. Methods and Results: Human aortic smooth muscle cells (HASMC) and human coronary atery smooth muscle cells (HCASMC) were cultured in HuMedia-SB2 supplanted with hEGF, hFGF, insulin and 5% FBS. Differentiated HASMC were cultured on laminin-coated dishes in HuMedia-SD2 supplemented with heparin and 1% FBS. Gene expression level was analyzed by real time PCR. SM2 and SMemb were used as differentiation marker and synthetic SMC marker, respectively. The differentiated SMC showed a higher level of expression of SM2, and the ratio of SM2/SMemb was higher compared to the synthetic SMC. Apop gene expression was higher in differentiated HASMC compared to the synthetic phase cells. Similarly, HCASMC showed a higher level of Apop and SM2 expression and SM2/SMemb expression ratio in differentiated cells compared to the cells in synthetic phase. These results indicate that Apop is expressed in differentiated SMC at a higher level than synthetic SMC. CArG box, which is often present in phenotypic marker genes expressed in differentiated SMC, exists in the intron1 region of Apop gene. Serum response factor (SRF) binds to CArG box, and regulates the expression of the genes expressed in differentiated SMC. The silencing of SRF using siRNA reduced the expression of Apop in differentiaed HASMC, indicating that SRF regulates the expression of Apop in differentiated cells. Moreover, immunostaining using human atherosclerotic coronary artery specimens revealed that Apop gene is co-expressed with the differentiation marker, SM2 in the coronary artery SMC. Conclusion: These results indicate that Apop is a novel phenotypic marker of the differentiated SMC, and may be a novel therapeutic target for the prevention of the development of atherosclerosis.

Endoglin is not expressed with cell adhesion molecules in aorta during atherogenesis in apoE-deficient mice.

Endoglin (TGF-β receptor III), has been demonstrated to affect vascular endothelium and atherosclerosis. Moreover, it was also demonstrated that endoglin is involved in inflammation and plays a role in leukocyte adhesion and transmigration in vitro and in vivo but not in atherosclerosis related vessels. In this study, we wanted to evaluate endoglin expression in two different parts of the aorta (heart aortic sinus and ascending aorta) and assess its potential simultaneous expression with cell adhesion molecules in non-atherosclerotic and atherosclerotic aortas of apoE-deficient mice. Ten-week-old female apolipoprotein E-deficient mice on a C57BL/6J background (n=24) were randomly subdivided into three groups and were fed either chow diet (for another two months) or Western type diet (for another two or four months). Immunohistochemical staining of endoglin, VCAM-1 and P-selectin in aortic sinus and ascending aorta was performed. Endoglin expression was detected only in endothelial cells and varied during atherogenic process in aorta but not in aortic sinus. Moreover, its expression seemed to be weaker in aorta when compared to aortic sinus and the positivity was detected only in endothelium covering atherosclerotic lesions but not in non-atherosclerotic endothelium regardless of the plaque size. Endoglin was not expressed with P selectin and VCAM-1 in aortic endothelium in any studied group. This study shows that endothelial expression of endoglin is related to the atherogenic process predominantly in aorta outside the heart. Moreover, endoglin is not localized with cell adhesion molecules involved in atherosclerosis, suggesting it might not participate in leukocyte accumulation in aorta of apoE-deficient mice during atherogenesis.

The evaluation of endoglin, vcam-1 and p-selectin expression and co-expression in aortas of apoe-deficient mice – An immunohistochemical study

Objectives: Endoglin, an accessory receptor of TGF-β signaling pathway is a homodimeric transmembrane glycoprotein that has been demonstrated to play a role in vascular physiology and pathology including atherosclerosis. It was also demonstrated that endoglin is involved in inflammation and plays a role in leukocyte adhesion and transmigration in vitro and in vivo. Thus, in this study, we wanted to evaluate endoglin expression in two different parts of aorta (heart aortic sinus and ascending aorta) and assess its potential co-expression with cell adhesion molecules in aortas of apoE-deficient mice.

Effects of Paricalcitol and Enalapril on Atherosclerotic Injury in Mouse Aortas

Aims: This study investigated the protective effect of vitamin D analog paricalcitol combined with angiotensin-converting enzyme inhibitor (enalapril) on aortic oxidative injury in atherosclerotic mice. Methods: Female mice were treated for 16 weeks as follows: (1) ApoE deficient + vehicle, (2) ApoE deficient + paricalcitol (200 ng 3 times a week), (3) ApoE deficient + enalapril (30 mg/l in drinking water), (4) ApoE deficient + paricalcitol + enalapril, and (5) wild-type controls. Results: ApoE-deficient mice developed hypertension which was prevented by enalapril or enalapril + paricalcitol treatment but not by paricalcitol treatment. Histology showed atherosclerotic plaque in the aorta of ApoE-deficient mice which was prevented by paricalcitol, enalapril, and paricalcitol + enalapril treatments. Aortic malondialdehyde levels, NADPH oxidase subunit p22^phox, manganese-superoxide dismutase (Mn-SOD), inducible nitric oxide synthase, monocyte chemoattaractant protein-1, tumor necrosis factor (TNF)-α, and cyclooxygenase-2 protein expressions increased, whereas glutathione levels, CuZn-SOD, and endothelial protein expressions decreased in ApoE-deficient mice compared to controls. Treatment with paricalcitol and enalapril alone or in combination protected the inflammatory and oxidative endothelial injury of the aorta in atherosclerotic mice. Conclusion: Combination therapy affords greater protection against aortic inflammatory and oxidative injury in atherosclerosis than monotherapy.

Bilberry anthocyanin-rich extract alters expression of genes related to atherosclerosis development in aorta of apo E-deficient mice

Intake of anthocyanin-rich foods has been associated with a reduced risk of cardiovascular diseases. We recently reported that a nutritional supplementation with a bilberry anthocyanin-rich extract (BE) attenuates atherosclerotic lesion development in apolipoprotein E-deficient (apoE −/−) mice. However, the mechanism(s) of their preventive action are not completely understood. Anthocyanins may alter mRNA levels of genes related to atherosclerosis in cultured macrophages and endothelial cells, but in vivo studies remain scarce. The aim of the present study was to explore the in vivo mechanisms of action of the same bilberry extract, administered by supplementation at a nutritional level, in the aorta of apo E −/− mice using a global transcriptomic approach. This study focused on the early stage of atherosclerosis development for better assessment of BE action on initiation mechanisms of this pathology. After a two week period, plasma lipid and antioxidant capacity were evaluated and the global genomic analysis was carried out using pangenomic microarrays. BE supplementation significantly improved hypercholesterolemia whereas the plasmatic antioxidant status remained unchanged. Nutrigenomic analysis identified 1261 genes which expression was modulated by BE in the aorta. Bioinformatic analysis revealed that these genes are implicated in different cellular processes such as oxidative stress, inflammation, transendothelial migration and angiogenesis, processes associated with atherosclerosis development/protection. Some of the most significantly down-regulated genes included genes coding for AOX1, CYP2E1 or TXNIP implicated in the regulation of oxidative stress, JAM-A coding for adhesion molecules or VEGFR2 implicate in regulation of angiogenesis. Other genes were up-regulated, such as CRB3, CLDN14 or CDH4 potentially associated with increased cell–cell adhesion and decreased paracellular permeability. These results provide a global integrated view of the mechanisms involved in the preventive action of bilberry anthocyanin-rich extract against atherosclerosis.

Human plasminogen kringle 1-5 reduces atherosclerosis and neointima formation in mice by suppressing the inflammatory signaling pathway.

Activation of vascular endothelial cells plays an important role in atherogenesis and plaque instability. Recent research has demonstrated that late-stage inhibition of plaque angiogenesis by angiostatin (kringle 1-4) reduces macrophage accumulation and slows the progression of advanced atherosclerosis. Kringle 1-5 (K(1-5)) is a variant of angiostatin that contains the first five kringle domains of plasminogen. To investigate whether K(1-5) has an inhibitory effect on early-stage atherosclerosis, using the apolipoprotein E (ApoE)-deficient mouse model and a carotid artery ligation model. ApoE-deficient mice received K(1-5) treatment for 4 weeks, and the severity of aortic atherosclerosis was measured. In the ligation model, the left common carotid arteries of C57BL/6 mice were ligated near the carotid bifurcation, and the mice received K(1-5) for 4 weeks. Human umbilical vein endothelial cells were pretreated with K(1-5) before tumor necrosis factor-alpha (TNF-alpha) treatment to explore the anti-inflammatory effect of K(1-5). The areas of the lesion in the aortas of ApoE-deficient mice that received K(1-5) treatment were notably decreased, and the formation of carotid neointima in the C57BL/6 mice was decreased by treatment with K(1-5). Expression of TNF-alpha-induced intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 was inhibited by K(1-5) treatment, possibly via downregulation of translocation of nuclear factor-kappaB and expression of reactive oxygen species. K(1-5) reduced atherosclerosis and neointima formation in mice, possibly through inhibition of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 expression in endothelial cells.

Cyclooxygenase inhibitors repress vascular hyaluronan‐synthesis in murine atherosclerosis and neointimal thickening

Hyaluronan (HA) is a key molecule of the extracellular matrix that is thought to be critically involved in both atherosclerosis and restenosis. Recently, it has been demonstrated that the cyclooxygenase (COX) products, prostacyclin and prostaglandin E2, induce HA synthesis in vitro by transcriptional up-regulation of HA-synthase 2 (HAS2) and HAS1. The relative roles in atherosclerotic and restenotic artery disease of tissue specifically expressed COX-1 and COX-2 are still under debate. Thus, the present study aimed to investigate the effect of COX isoform inhibition on HA-accumulation and regulation of HAS isoform expression in two models of pathologic artery remodelling in vivo. Firstly, ApoE-deficient mice were treated with a prototypic isoform non-selective inhibitor, indomethacin or with a prototypic COX-2 selective inhibitor, rofecoxib, for 8 weeks. Aortic HAS mRNA expression and HA-accumulation in atherosclerotic aortic root lesions were analyzed. Secondly, neointimal hyperplasia was induced by carotid artery ligation in ApoE-deficient mice on a high fat diet and the effects of the COX inhibitors were determined after 4 weeks of treatment. Intimal HA-accumulation was markedly reduced in both models by indomethacin and rofecoxib. This coincided with a strong inhibition of HAS1 mRNA expression in both models and with decreased HAS2 mRNA in the aorta of ApoE-deficient mice. HAS3 was not affected. The repression of HA-accumulation by both COX-2 selective and non-selective COX inhibition implicates COX-2 in the regulation of HA synthesis via stimulation of HAS1 and HAS2 expression in vivo. Modulation of vascular HA-accumulation might play a role in chronic effects of COX inhibitors on the progression of atherosclerosis.

Apple Polyphenols and Fibers Attenuate Atherosclerosis in Apolipoprotein E-Deficient Mice

Atherosclerosis, which is closely linked to nutritional habits, is a major cause of mortality in Western countries. Most of the previous investigations carried out on health effects of apples have been focused on their capacity to lower lipid concentration as well as on their antioxidant effects. The aim of the present study was to investigate the antiatherosclerotic effects of apple polyphenols and fibers. A crude apple polyphenol extract and low-viscosity apple fibers isolated from cider apples were administered separately or in association with the diet of apo E-deficient mice. After 4 months of supplementation, lipemia and oxidative stress biomarkers were measured and atheroslerotic lesions assessed by histomorphometry. Total plasmatic cholesterol and triacylgycerol levels were not affected by supplementation, and hepatic cholesterol level was lower in the group supplemented with both fibers and polyphenols. Uric acid concentrations and antioxidant capacity (FRAP) in plasma were reduced in all groups supplemented with polyphenols or fibers. The mean lesion area was reduced by 17, 38, and 38%, respectively, for the polyphenol, fiber, and polyphenol + fiber groups. Apple constituents supplied at nutritional doses therefore limit the development of atherosclerotic lesions in the aorta of apo E-deficient mice. On the basis of the results, we hypothesize that apple fibers and polyphenols may play a role in preventing atherosclerosis disease by decreasing uric acid plasma level.

Emodin Promotes Atherosclerotic Plaque Stability in Fat-Fed Apolipoprotein E-Deficient Mice

Increasing evidence indicated that plaque stabilization is attributed to the composition of the atherosclerotic plaque, and inflammation plays an important role in the formation and progress of vulnerable atherosclerotic plaque (VAP), which is prone to rupture. Emodin, an important component of traditional Chinese herb rhubarb, has obvious anti-inflammatory effect, although its effect on atherosclerotic plaque stabilization is unknown. Apolipoprotein E (ApoE) is an important component of plasma lipoprotein with anti-atherosclerosis function, and the plaque in the aorta of ApoE-deficient mice has been demonstrated with characteristics of VAP. Therefore, this study was designed to determine whether emodin can stabilize the VAP in the ApoE-deficient mice and explain the possible mechanism. After fat-fed for 13 weeks, mice were randomized into three groups (11 animals/group) and intragastrically administrated with emodin, simvastatin or distilled water for 13 weeks, respectively. The plaque stability was evaluated by the morphology and composition of atherosclerotic plaques. Additionally, the expression of peroxisomal proliferator-activated receptor-gamma (PPAR-gamma), granulocyte-macrophage colony-stimulating factor (GM-CSF), and matrix metalloproteinase 9 (MMP-9) in plaques was determined by the immunohistochemistry method. We showed that emodin could decrease the lipid core area and the ratio of lipid to collagen content in plaques. In addition, emodin significantly inhibited the expression of GM-CSF and MMP-9, whereas it induced the expression of PPAR-gamma in plaques. In conclusion, these results suggest that emodin can stabilize the VAP in the aortic root of ApoE-knockout mice, which is probably due to its anti-inflammatory effect.

Oxidative Stress and Atherosclerosis

Oxidative stress is a continuous level of oxidative damage in animal cells, which is caused by an overabundance of reactive oxygen species or a decline in antioxidant ability against them. Oxidative stress increases with individual risk factors of atherosclerosis such as obesity, hypertension, hyperlipidemia, diabetes and smoking. Thus, oxidative stress is considered to play a key role in the pathogenesis of atherosclerosis. This review discusses the relationship between oxidative stress and atherosclerosis based on findings from our research group. We have found that atherosclerotic lesions are formed in the aorta of mice fed a high-cholesterol and high-linoleic diet, in parallel with elevated serum lipid peroxide levels. This model is useful for primary screening of antiatherosclerotic agents with antioxidative activity. One notable factor in the development of atherosclerosis is oxidized low-density lipoprotein (OxLDL). In order to examine OxLDL levels in blood, we have developed anion-exchange HPLC methods using stepwise elution. Using these methods, we have found that OxLDL markedly increases in a rat model of metabolic syndrome, in animals exposed to cigarette smoke and in smokers in parallel with other oxidative stress markers. These oxidative stress markers have been attenuated by administration of several antioxidants. In addition, we have found that smoking accelerates atherogenesis in the aorta of apoE-deficient mice and this acceleration can be ameliorated by administration of vitamin E. These observations suggest that antioxidant supplementation may be an effective therapeutic strategy for metabolic syndrome and smoking-induced diseases in which elevated oxidative stress plays a pivotal role.

Increased vascular biosynthesis of tetrahydrobiopterin in apolipoprotein E-deficient mice

Previous studies suggested that loss of tetrahydrobiopterin (BH(4)) may play an important role in the pathogenesis of vascular endothelial dysfunction induced by diabetes and hypertension. In contrast, controversial results have been reported regarding BH(4) metabolism in experimental models of atherosclerosis. Therefore, the present study was designed to characterize the expression and activity of GTP-cyclohydrolase I, a rate-limiting enzyme in biosynthesis of BH(4), during atherogenesis. BH(4) levels were significantly increased in atherosclerotic aortas of apolipoprotein E (apoE)-deficient mice as compared with wild-type mice after 5 mo of Western diet treatment. This increase was further significantly enhanced in apoE-deficient mice fed for 9 and 14 mo. Removal of the endothelium almost eliminated BH(4) in wild-type mice but not in apoE-deficient mice, suggesting that a major component of increased BH(4) synthesis is localized in the vascular media of apoE-deficient mice. Oxidative products of BH(4) were low and did not differ between wild-type and apoE-deficient mice over the course of this study. Increased protein expression and enzymatic activity of GTP-cyclohydrolase I were detected in aortas of apoE-deficient mice (P < 0.05), providing molecular mechanisms responsible for elevation of vascular BH(4). In contrast to aortas, we did not detect any change in levels of BH(4) and in GTP-cyclohydrolase I expression in the brain. Our results demonstrate selective increase of intracellular BH(4) levels via elevation of GTP-cyclohydrolase I activity in vascular tissue of apoE-deficient mice.

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 …

Long-term vitamin C treatment increases vascular tetrahydrobiopterin levels and nitric oxide synthase activity.

In cultured endothelial cells, the antioxidant, L-ascorbic acid (vitamin C), increases nitric oxide synthase (NOS) enzyme activity via chemical stabilization of tetrahydrobiopterin. Our objective was to determine the effect of vitamin C on NOS function and tetrahydrobiopterin metabolism in vivo. Twenty-six to twenty-eight weeks of diet supplementation with vitamin C (1%/kg chow) significantly increased circulating levels of vitamin C in wild-type (C57BL/6J) and apolipoprotein E (apoE)--deficient mice. Measurements of NOS enzymatic activity in aortas of apoE-deficient mice indicated a significant increase in total NOS activity. However, this increase was mainly due to high activity of inducible NOS, whereas eNOS activity was reduced. Significantly higher tetrahydrobiopterin levels were detected in aortas of apoE-deficient mice. Long-term treatment with vitamin C restored endothelial NOS activity in aortas of apoE-deficient mice, but did not affect activity of inducible NOS. In addition, 7,8-dihydrobiopterin levels, an oxidized form of tetrahydrobiopterin, were decreased and vascular endothelial function of aortas was significantly improved in apoE-deficient mice. Interestingly, vitamin C also increased tetrahydrobiopterin and NOS activity in aortas of C57BL/6J mice. In contrast, long-term treatment with vitamin E (2000 U/kg chow) did not affect vascular NOS activity or metabolism of tetrahydrobiopterin. In vivo, beneficial effect of vitamin C on vascular endothelial function appears to be mediated in part by protection of tetrahydrobiopterin and restoration of eNOS enzymatic activity.

Mechanism of endothelial dysfunction in apolipoprotein E-deficient mice.

Endothelium-dependent relaxations mediated by NO are impaired in a mouse model of human atherosclerosis. Our objective was to characterize the mechanisms underlying endothelial dysfunction in aortas of apolipoprotein E (apoE)-deficient mice, treated for 26 to 29 weeks with a lipid-rich Western-type diet. Aortic rings from apoE-deficient mice showed impaired endothelium-dependent relaxations to acetylcholine (10(-)(9) to 10(-)(5) mol/L) and Ca(2+) ionophore (10(-)(9) to 10(-)(6) mol/L) and endothelium-independent relaxations to diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA-NONOate, 10(-)(10) to 10(-)(5) mol/L) compared with aortic rings from C57BL/6J mice (P<0.05). By use of confocal microscopy of an oxidative fluorescent probe (dihydroethidium), increased superoxide anion (O(2)(-)) production was demonstrated throughout the aortic wall but mainly in smooth muscle cells of apoE-deficient mice. CuZn-superoxide dismutase (SOD) and Mn-SOD protein expressions were unaltered in the aorta exposed to hypercholesterolemia. A cell-permeable SOD mimetic, Mn(III) tetra(4-benzoic acid) porphyrin chloride (10(-)(5) mol/L), reduced O(2)(-) production and partially normalized relaxations to acetylcholine and DEA-NONOate in apoE-deficient mice (P<0.05). [(14)C]L-Citrulline assay showed a decrease of Ca(2+)-dependent NOS activity in aortas from apoE-deficient mice compared with C57BL/6J mice (P<0.05), whereas NO synthase protein expression was unchanged. In addition, cGMP levels were significantly reduced in the aortas of apoE-deficient mice (P<0.05). Our results demonstrate that in apoE-deficient mice on a Western-type fat diet, impairment of endothelial function is caused by increased production of O(2)(-) and reduced endothelial NO synthase enzyme activity. Thus, chemical inactivation of NO with O(2)(-) and reduced biosynthesis of NO are key mechanisms responsible for endothelial dysfunction in aortas of atherosclerotic apoE-deficient mice.

Corresponding distributions of increased endothelin-B receptor expression and increased endothelin-1 expression in the aorta of apolipoprotein E-deficient mice with advanced atherosclerosis.

Endothelin (ET)-1 causes proliferation of vascular smooth muscle cells (VSMC). Although it has been reported that stimulation of ET(B) receptors as well as ET(A) receptors promote proliferation of VSMC, the precise distribution of each receptor subtype in atherosclerotic vessels is unknown. Previous studies demonstrated that apolipoprotein E (apoE)-deficient mice have hypercholesterolaemia and develop severe atherosclerosis. To investigate the pathophysiological roles of vascular ET system in atherosclerosis, we examined preproET-1 messenger ribonucleic acid expression in the aorta of apoE-deficient mice, and performed immunohistochemical staining for ET-1 and each ET receptor subtype (ET(A) and ET(B) receptors) in the atherosclerotic lesions of these mice. The level of preproET-1 mRNA in the aorta was significantly higher in the apoE-deficient mice than in the control mice. Strong ET-1 staining was observed in the macrophage-foam cells, intimal and medial VSMC in the atherosclerotic lesions of the apoE-deficient mice. In addition, in the atherosclerotic lesions, strong ET(B) receptor staining was observed in the macrophage-foam cells, intimal and medial VSMC, which distribution corresponded closely to that of ET-1. ET(A) receptor staining was observed in the medial VSMC of both groups, but not in the macrophage-foam cells of the apoE-deficient mice. ET(A) receptor staining in the medial VSMC was stronger in the apoE-deficient mice than in the control mice. These results suggest that the vascular ET system, including ET-1 and ET receptors, is activated in the atherosclerotic lesions of apoE-deficient mice. Since the distribution of strong ET(B) receptor staining corresponded closely to that of ET-1, it is suggested that the ET system, mediated by ET(B) receptors, has an important role in the pathophysiology of the atherosclerotic lesions of apoE-deficient mice.

OxLDL induces ceramide and apoptosis in macrophages but not in aortas of apoE-deficient mice: [formula omitted] George J, Gilburd B, Harats D, Shoenfeld Y, Barak A, Chavez C, Goldkorn T. UC Davis School of Medicine, Davis CA USA, Tel Aviv University, Israel

oxLDL induces ceramide and apoptosis in macrophages but not in aortas of apoE-deficient mice: [formula omitted] George J, Gilburd B, Harats D, Shoenfeld Y, Barak A, Chavez C, Goldkorn T. UC Davis School of Medicine, Davis CA USA, Tel Aviv University, Israel