Activation of VPAC1 receptors aggravates early atherosclerosis in hypercholesterolemic apolipoprotein E-deficient mice

Objective: Vasoactive intestinal peptide (VIP) is a 28-amino acid peptide widely expressed in the body and binding three types of receptors: VPAC₁-R, VPAC₂-R and PAC₁-R. Based on beneficial effects of VIP and VPAC₁-R agonists in mouse models of several chronic inflammatory disorders, we hypothesized that activation of VIP receptors would prevent atherosclerosis development in apolipoprotein E-deficient mice.Methods and results: Contrary to our hypothesis, administration of a VPAC₁-R agonist, (Ala^11,22,28)-VIP aggravated atherosclerotic lesion development in the aortic root of these mice compared to control mice. This was accompanied by a significant increase in the expression of MHC class II protein I-A^b, and suggests enhanced inflammatory activity in the vessel wall. The amount of macrophage-specific CD68 staining as well as serum cholesterol and triglyceride levels did not change as a result of the (Ala^11,22,28)-VIP treatment, i.e. the treatment resulted in significant changes in lipid accumulation in the lesions without changing the number of macrophages or systemic lipid levels. Interestingly, administration of VIP did not alter the course of the disease.Conclusion: Despite beneficial effects in murine models of several inflammatory disorders, VPAC₁-R activation aggravates atherosclerotic lesion formation in apolipoprotein E-deficient mice through enhanced inflammatory activity in the vessel wall.     

Neurological phenotype and reduced lifespan in heterozygous Tim23 knockout mice, the first mouse model of defective mitochondrial import

The Tim23 protein is the key component of the mitochondrial import machinery. It locates to the inner mitochondrial membrane and its own import is dependent on the DDP1/TIM13 complex. Mutations in human DDP1 cause the Mohr-Tranebjaerg syndrome (MTS/DFN-1; OMIM #304700), which is one of the two known human diseases of the mitochondrial protein import machinery. We created a Tim23 knockout mouse from a gene trap embryonic stem cell clone. Homozygous Tim23 mice were not viable. Heterozygous F1 mutants showed a 50% reduction of Tim23 protein in Western blot, a neurological phenotype and a markedly reduced life span. Haploinsufficiency of the Tim23 mutation underlines the critical role of the mitochondrial import machinery for maintaining mitochondrial function.     

Systemic markers of the redox balance and apolipoprotein E polymorphism in atherosclerosis

Prospective studies have demonstrated that an imbalance between oxidative damage and antioxidative protection can play a role in the development and progression of atherosclerosis. Also, genotypes with the apolipoprotein E ζ4 allele have been associated with an increase risk for this pathology. Based on this knowledge, the aim of this study was to evaluate indicators of the redox balance, trace elements, and apolipoprotein E allelic profile in subjects from the Lisbon population with clinically stable atherosclerosis, at risk for atherosclerotic events, and in healthy subjects for comparison. The activities of superoxide dismutase in erythrocytes and glutathione peroxidase in whole blood, plasma total thiols, and serum ceruloplasmin were kept unchanged among the three groups. Serum α-tocopherol was increased in atherosclerotic patients. Total malondialdehyde in serum and protein carbonyls in plasma, which are indicators of lipid and protein oxidative damage, respectively, reached their highest values in risk subjects. The concentrations of potassium and calcium, in plasma and in blood cells, were slightly elevated in patients and might reflect an electrolytic imbalance. Regarding the apolipoprotein E polymorphism, atherosclerotic patients had an increased incidence of the high-risk genotypes for atherogenesis (ζ3/ζ4 and ζ4/ζ4). A multivariate model applied to the general population using most of the parameters clearly separated the three groups at study (i.e., the healthy group from the steady-state group of risk disease and from the atherosclerotic one). As shown by us, the usefulness of biochemical and complementary genetic markers is warranted for a better knowledge on atherosclerosis molecular basis.     

Abundant tissue butyrylcholinesterase and its possible function in the acetylcholinesterase knockout mouse

We have described recently an acetylcholinesterase (AChE) knockout mouse. While comparing the tissue distribution of AChE and butyrylcholinesterase (BChE), we found that extraction buffers containing Triton X-100 strongly inhibited mouse BChE activity. In contrast, buffers with Tween 20 caused no inhibition of BChE. Conventional techniques grossly underestimated BChE activity by up to 15-fold. In Tween 20 buffer, the intestine, serum, lung, liver, and heart had higher BChE than AChE activity. Only brain had higher AChE than BChE activity in AChE +/+ mice. These findings contradict the dogma, based mainly on observations in Triton X-100 extracts, that BChE is a minor cholinesterase in animal tissues. AChE +/- mice had 50% of normal AChE activity and AChE -/- mice had none, but all mice had similar levels of BChE activity. BChE was inhibited by Triton X-100 in all species tested, except rat and chicken. Inhibition was reversible and competitive with substrate binding. The active site of rat BChE was unique, having an arginine in place of leucine at position 286 (human BChE numbering) in the acyl-binding pocket of the active site, thus explaining the lack of inhibition of rat BChE by Triton X-100. The generally high levels of BChE activity in tissues, including the motor endplate, and the observation that mice live without AChE, suggest that BChE has an essential function in nullizygous mice and probably in wild-type mice as well.     

Distinct pro-inflammatory properties of myeloid cell–derived apolipoprotein E2 and E4 in atherosclerosis promotion

Polymorphisms in the apolipoprotein E (apoE) gene are risk factors for chronic inflammatory diseases including atherosclerosis. The gene product apoE is synthesized in many cell types and has both lipid transport–dependent and lipid transport–independent functions. Previous studies have shown that apoE expression in myeloid cells protects against atherogenesis in hypercholesterolemic ApoE^−/− mice. However, the mechanism of this protection is still unclear. Using human APOE gene replacement mice as models, this study showed that apoE2 and apoE4 expressed endogenously in myeloid cells enhanced the inflammatory response via mechanisms independent of plasma lipoprotein transport. The data revealed that apoE2-expressing myeloid cells contained higher intracellular cholesterol levels because of impaired efflux, causing increasing inflammasome activation and myelopoiesis. In contrast, intracellular cholesterol levels were not elevated in apoE4-expressing myeloid cells, and its proinflammatory property was found to be independent of inflammasome signaling and related to enhanced oxidative stress. When ApoE^−/− mice were reconstituted with bone marrow from various human APOE gene replacement mice, effective reduction of atherosclerosis was observed with marrow cells obtained from APOE3 but not APOE2 and APOE4 gene replacement mice. Taken together, these results documented that apoE2 and apoE4 expression in myeloid cells promotes inflammation via distinct mechanisms and promotes atherosclerosis in a plasma lipoprotein transport–independent manner.     

CD34 Class III positive cells are present in atherosclerotic plaques of the rabbit model of atherosclerosis

CD34 is a positive marker for haematopoietic stem cells and endothelial cells. Recent evidence suggests that haematopoietic progenitor cells are involved in atherogenesis. CD34-positive haematopoietic progenitor cells have never been described in rabbit atherosclerotic tissues. The aim of this study is to identify CD34-positive haematopoietic progenitor cells in rabbit atherosclerotic tissues, and to compare this with macrophage (RAM-11), alpha smooth muscle cell actin and fibroblast (prolyl-4-hydroxylase) immunoreactive cells. Sixteen Male New Zealand White rabbits were divided into two groups: Group 1, control diet (Con); group 2, 0.5% cholesterol diet, and killed after 12 weeks. Immunohistochemistry was used to detect CD34 haematopoietic progenitor cells. CD34-positive haematopoietic progenitor cells were identified both within and overlying atherosclerotic plaques. As well, these haematopoietic progenitor cells also stained for RAM-11, CD45, prolyl-4 hydroxylase and alpha smooth muscle cell actin. These findings suggest that in the rabbit model of atherosclerosis, the previously identified macrophages, smooth muscle cells and fibroblasts within and overlying atherosclerotic plaques might be of haematopoietic origin.     

Complex, multimodal behavioral profile of the Homer1 knockout mouse

Proteins of the Homer1 immediate early gene family have been associated with synaptogenesis and synaptic plasticity suggesting broad behavioral consequences of loss of function. This study examined the behavior of male Homer1 knockout (KO) mice compared with wild-type (WT) and heterozygous mice using a battery of 10 behavioral tests probing sensory, motor, social, emotional and learning/memory functions. KO mice showed mild somatic growth retardation, poor motor coordination, enhanced sensory reactivity and learning deficits. Heterozygous mice showed increased aggression in social interactions with conspecifics. The distribution of mGluR5 and N-methyl-D-aspartate receptors (NMDA) receptors appeared to be unaltered in the hippocampus (HIP) of Homer1 KO mice. The results indicate an extensive range of disrupted behaviors that should contribute to the understanding of the Homer1 gene in brain development and behavior.     

Nutritional B vitamin deficiency alters the expression of key proteins associated with vascular smooth muscle cell proliferation and migration in the aorta of atherosclerotic apolipoprotein E null mice

Low B vitamin status is linked with human vascular disease. We employed a proteomic and biochemical approach to determine whether nutritional folate deficiency and/or hyperhomocysteinemia altered metabolic processes linked with atherosclerosis in ApoE null mice. Animals were fed either a control fat (C; 4 % w/w lard) or a high-fat [HF; 21 % w/w lard and cholesterol (0/15 % w/w)] diet with different B vitamin compositions for 16 weeks. Aorta tissue was prepared and global protein expression, B vitamin, homocysteine and lipoprotein status measured. Changes in the expression of aorta proteins were detected in response to multiple B vitamin deficiency combined with a high-fat diet (P < 0.05) and were strongly linked with lipoprotein concentrations measured directly in the aorta adventitia (P < 0.001). Pathway analysis revealed treatment effects in the aorta-related primarily to cytoskeletal organisation, smooth muscle cell adhesion and invasiveness (e.g., fibrinogen, moesin, transgelin, vimentin). Combined B vitamin deficiency induced striking quantitative changes in the expression of aorta proteins in atherosclerotic ApoE null mice. Deregulated expression of these proteins is associated with human atherosclerosis. Cellular pathways altered by B vitamin status included cytoskeletal organisation, cell differentiation and migration, oxidative stress and chronic inflammation. These findings provide new insight into the molecular mechanisms through which B vitamin deficiency may accelerate atherosclerosis.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-014-0446-y) contains supplementary material, which is available to authorized users.     

Deficiency of inducible NO synthase reduces advanced but not early atherosclerosis in apolipoprotein E-deficient mice

The inducible nitric oxide synthase (iNOS) is abundantly expressed by smooth muscle cells and macrophages in atherosclerotic lesions. Apolipoprotein E-deficient (apoE(-/-)) mice develop early and advanced atherosclerotic lesions. The role of iNOS in both early and advanced atherosclerotic formation was determined in apoE(-/-) mice. Mice were fed chow or a Western diet containing 42% fat, 0.15% cholesterol, and 19.5% casein. At 12 weeks of age on chow diet, iNOS(-/-)/apoE(-/-) mice developed comparable sizes of early atherosclerotic lesions in the aortic root as did iNOS(+/+)/apoE(-/-) mice (30,993+/-4746 vs. 26,648+/-6815 microm(2)/section; P=0.608). After being fed the Western diet for 12 weeks, iNOS(-/-)/apoE(-/-) mice developed significantly smaller advanced lesions than iNOS(+/+)/apoE(-/-) mice (458,734+/-14,942 vs. 519,570+/-22,098 microm(2)/section; P=0.029). This reduction in lesion formation could not be explained by differences in plasma lipid levels. To examine whether iNOS contributed to LDL oxidation, smooth muscle cells were isolated from the aorta, activated with TNF-alpha, and then incubated with native LDL in the absence or presence of N-Omega-nitro-L-arginine methyl ester (L-NAME), a specific NOS inhibitor. L-NAME significantly inhibited LDL oxidation by smooth muscle cells from iNOS(+/+)/apoE(-/-) mice (P=0.048), but it had no effect on LDL oxidation by cells from iNOS(-/-)/apoE(-/-) mice. iNOS(-/-)/apoE(-/-) mice had a significantly lower plasma lipoperoxide level on the Western diet (2.74+/-0.23 vs. 3.89+/-0.41 microM MDA; P=0.021) but not on chow diet (1.02+/-0.07 vs. 1.51+/-0.29 microM MDA; P=0.11). Thus, the absence of iNOS-mediated LDL oxidation may contribute to the reduction in advanced lesion formation of iNOS(-/-)/apoE(-/-) mice.     

Increased infarct size and exacerbated apoptosis in the glutathione peroxidase-1 (Gpx-1) knockout mouse brain in response to ischemia/reperfusion injury

Glutathione peroxidase is an antioxidant enzyme that is involved in the control of cellular oxidative state. Recently, unregulated oxidative state has been implicated as detrimental to neural cell viability and involved in both acute and chronic neurodegeneration. In this study we have addressed the importance of a functional glutathione peroxidase in a mouse ischemia/reperfusion model. Two hours of focal cerebral ischemia followed by 24 h of reperfusion was induced via the intraluminal suture method. Infarct volume was increased three-fold in the glutathione peroxidase-1 (Gpx-1) -/- mouse compared with the wild-type mouse; this was mirrored by an increase in the level of apoptosis found at 24 h in the Gpx-1 -/- mouse compared with the wild-type mouse. Neuronal deficit scores correlated to the histologic data. We also found that activated caspase-3 expression is present at an earlier time point in the Gpx-1 -/- mice when compared with the wild-type mice, which suggests an enhanced susceptibility to apoptosis in the Gpx-1 -/- mouse. This is the first known report of such a dramatic increase, both temporally and in level of apoptosis in a mouse stroke model. Our results suggest that Gpx-1 plays an important regulatory role in the protection of neural cells in response to the extreme oxidative stress that is released during ischemia/reperfusion injury.     

Amygdalin mediates relieved atherosclerosis in apolipoprotein E deficient mice through the induction of regulatory T cells

Objective: Regulatory T cells (Tregs) play a critical role in the regulation of T cell-mediated immune responses in atherosclerosis, a chronic autoimmune-like disease. Therefore, in this study, we aimed to investigate the therapeutic effect of amygdalin on atherosclerosis of apolipoprotein E deficient (ApoE^−/−) mice, and to explore its immune regulatory function by stimulation of Tregs. Methods and results: To evaluate the anti-atherosclerotic effect of amygdalin and for in vivo Treg expansion/activation analysis, ApoE^−/− mice received intraperitoneal injections of amygdalin, and this therapy resulted in a comparatively 2-fold decrease in triglyceride (TG), 1.5-fold decrease in total cholesterol (TC) and low density lipoprotein (LDL). By comparing the vessel areas, lumen areas, plaque areas, and aortic plaque coverage percentage, the effects of amygdalin on pre-existing lesions were assessed. Studies on IL-10 and TGF-β indicated that mice treated with amygdalin had increased expression of Treg-related cytokines. Meanwhile, flow cytometry and real-time PCR data showed that mice treated with amygdalin had higher percentage of CD4⁺CD25⁺Foxp3⁺ T cells than untreated mice and increased expression of forkhead box P3 (FOXP3) gene. Conclusion: Our data showed amygdalin could attenuate the development of atherosclerosis by suppressing inflammatory responses and promoting the immunomodulation function of Tregs. The effects of amygdalin ultimately resulted in the enlarged lumen area and the loss of atherosclerotic plaque. All these data indicated the therapeutic potential of amygdalin in preventing and/or treating of atherosclerosis.     

Levels, metabolism, and pharmacological activity of anandamide in CB(1) cannabinoid receptor knockout mice: evidence for non-CB(1), non-CB(2) receptor-mediated actions of anandamide in mouse brain

Anandamide [arachidonylethanolamide (AEA)] appears to be an endogenous agonist of brain cannabinoid receptors (CB(1)), yet some of the neurobehavioral effects of this compound in mice are unaffected by a selective CB(1) antagonist. We studied the levels, pharmacological actions, and degradation of AEA in transgenic mice lacking the CB(1) gene. We quantified AEA and the other endocannabinoid, 2-arachidonoyl glycerol, in six brain regions and the spinal cord by isotope-dilution liquid chromatography-mass spectrometry. The distribution of endocannabinoids and their inactivating enzyme, fatty acid amide hydrolase, were found to overlap with CB(1) distribution only in part. In CB(1) knockout homozygotes (CB(1)-/-), the hippocampus and, to a lesser extent, the striatum exhibited lower AEA levels as compared with wild-type (CB(1)+/+) controls. These data suggest a ligand/receptor relationship between AEA and CB(1) in these two brain regions, where tonic activation of the receptor may tightly regulate the biosynthesis of its endogenous ligand. 2-Arachidonoyl glycerol levels and fatty acid amide hydrolase activity were unchanged in CB(1)-/- with respect to CB(1)+/+ mice in all regions. AEA and Delta(9)-tetrahydrocannabinol (THC) were tested in CB(1)-/- mice for their capability of inducing analgesia and catalepsy and decreasing spontaneous activity. The effects of AEA, unlike THC, were not decreased in CB(1)-/- mice. AEA, but not THC, stimulated GTPgammaS binding in brain membranes from CB(1)-/- mice, and this stimulation was insensitive to CB(1) and CB(2) antagonists. We suggest that non-CB(1), non-CB(2) G protein-coupled receptors might mediate in mice some of the neuro-behavioral actions of AEA.     

Reduction of reperfusion‐induced ventricular fibrillation and infarct size via heme oxygenase‐1 overexpression in isolated mouse hearts

Heme oxygenase‐1 (HO‐1), also known as heat shock protein 32 (hsp‐32) is a stress‐induced cytoprotective protein. The present investigation evaluated the capacity of HO‐1 to reduce the incidence of reperfusion‐induced ventricular fibrillation (VF) and infarct size. HO‐1 transgenic (Tg) mice were generated using a rat HO‐1 genomic transgene. Isolated mouse hearts obtained from Tg and non‐transgenic (NTg) groups were exposed to 20 min. of global ischemia and 120 min. of reperfusion. Epicardial electrocardiogram was recorded to monitor the incidence of reperfusion‐induced VF and at the end of the reperfusion period, detection of HO‐1 by immunohistochemistry and measurement of infarct size using the tetrazolium chloride method were carried out. Results shown here provide additional support for cardioprotective effects of HO‐1 as demonstrated by the reduced infarct size. Moreover, overexpression of the HO‐1 efficiently reduced the incidence of ischemia/reperfusion induced VF in HO‐1 Tg mice.     

炎症诱导的新型大鼠动脉粥样硬化模型的建立及Rb1的防治作用

目的通过激发大鼠炎症反应建立新型动脉粥样硬化（AS）动物模型并观察人参皂苷Rb1的抗AS作用。方法模型组采用酵母多糖混悬液（20 mg/kg）每隔3d腹腔注射一次,引发大鼠持续性炎症;相同方法注射无菌石蜡液作为对照组;Rb1组同时腹腔注射Rb1（40 mg/kg）;所有大鼠均喂食高脂饲料,实验共10周。分别通过苏丹染色、透射电镜、real time PCR、免疫组化、ELISA观察大鼠主动脉壁大体标本、超微结构、NFκB、TNFα、IL6的表达。结果模型组可见红染的脂纹、斑块形成,电镜显示内膜下层出现吞噬脂滴的泡沫细胞,NFκB/P65高表达于主动脉壁的内膜层,TNFα、IL6水平均明显高于对照组,经Rb1干预后大体标本可见AS病变明显减轻,电镜下未见泡沫细胞,NFκB、TNFα、IL6水平均较模型组显著降低。结论在高脂喂饲的基础上持续炎症刺激能够成功诱导大鼠AS模型,人参皂苷Rb1能够通过抑制炎症反应抗AS。     

Resting and arecoline-stimulated brain metabolism and signaling involving arachidonic acid are altered in the cyclooxygenase-2 knockout mouse

Studies were performed to determine if cyclooxygenase (COX)-2 regulates muscarinic receptor-initiated signaling involving brain phospholipase A₂ (PLA₂) activation and arachidonic acid (AA; 20 : 4n-6) release. AA incorporation coefficients, k* (brain [1–¹⁴C]AA radioactivity/integrated plasma radioactivity), representing this signaling, were measured following the intravenous injection of [1–¹⁴C]AA using quantitative autoradiography, in each of 81 brain regions in unanesthetized COX-2 knockout (COX-2^–/–) and wild-type (COX-2^+/+) mice. Mice were administered arecoline (30 mg/kg i.p.), a non-specific muscarinic receptor agonist, or saline i.p. (baseline control). At baseline, COX-2^–/– compared with COX-2^+/+ mice had widespread and significant elevations of k*. Arecoline increased k* significantly in COX-2^+/+ mice compared with saline controls in 72 of 81 brain regions, but had no significant effect on k* in any region in COX-2^–/– mice. These findings, when related to net incorporation rates of AA from brain into plasma, demonstrate enhanced baseline brain metabolic loss of AA in COX-2^–/– compared with COX-2^+/+ mice, and an absence of a normal k* response to muscarinic receptor activation. This response likely reflects selective COX-2-mediated conversion of PLA₂-released AA to prostanoids.     

Endogenous hydrogen sulfide is involved in the pathogenesis of atherosclerosis

Atherosclerosis is a chronic, complex, and progressive pathological process in large and medium sized arteries. The exact mechanism of this process remains unclear. Hydrogen sulfide (H₂S), a novel gasotransmitter, was confirmed as playing a major role in the pathogenesis of many cardiovascular diseases. It plays a role in vascular smooth muscle cell (VSMC) proliferation and apoptosis, participates in the progress of hyperhomocysteinemia (HHCY), inhibits atherogenic modification of LDL, interferes with vascular calcification, intervenes with platelet function, and there are interactions between H₂S and inflammatory processes. The role of H₂S in atherosclerotic pathogenesis highlights the mysteries of atherosclerosis and inspires the search for innovative therapeutic strategies. Here, we review the studies to date that have considered the role of H₂S in atherosclerosis.     

Increased cyclin E expression may obviate the role of cyclin D1 during brain development in cyclin D1 knockout mice

Cyclins D and E play critical roles during the G1 phase of mammalian cell division. Cyclin D1 expression is high and expected to play an important role during mouse brain development. However, in the present study, we found no difference in CNS morphology between cyclin D1 knockout (KO) and control wild-type mice at the ages of 1, 4 and 12 months. Analysis of protein expression in embryonic brains revealed that cyclin E is obviously increased in cyclin D1 KO mice at 13.5 days post coitum. At the same age a high level of cyclin D1 expression is detected in the embryonic brain of wild-type mice. The data indicate that enhanced cyclin E protein expression in cyclin D1 KO mice may obviate the role of cyclin D1 and contribute to the normal brain development of cyclin D1 KO mice.