Colchicine Does Not Reduce Abdominal Aortic Aneurysm Growth in a Mouse Model.

Background and Aims: The nacht domain, leucine-rich repeat, and pyrin domain-containing protein 3 (NLRP3) inflammasome is upregulated in human abdominal aortic aneurysm (AAA), but its pathogenic role is unclear. The aims of this study were firstly to examine whether the inflammasome was upregulated in a mouse model of AAA and secondly to test whether the inflammasome inhibitor colchicine limited AAA growth. Methods: AAA was induced in eight-week-old male C57BL6/J mice with topical application of elastase to the infrarenal aorta and oral 3-aminopropionitrile (E-BAPN). For aim one, inflammasome activation, abdominal aortic diameter, and rupture were compared between mice with AAA and sham controls. For aim two, 3 weeks after AAA induction, mice were randomly allocated to receive colchicine (n = 28, 0.2 mg/kg/d) or vehicle control (n = 29). The primary outcome was the rate of maximum aortic diameter increase measured by ultrasound over 13 weeks. Results: There was upregulation of NLRP3 markers interleukin- (IL-) 1ß (median, IQR; 15.67, 7.11-22.60 pg/mg protein versus 6.87, 4.54-11.60 pg/mg protein, p = .048) and caspase-1 (109, 83-155 relative luminosity units (RLU) versus 45, 38-65 RLU, p < .001) in AAA samples compared to controls. Aortic diameter increase over 80 days (mean difference, MD, 4.3 mm, 95% CI 3.3, 5.3, p < .001) was significantly greater in mice in which aneurysms were induced compared to sham controls. Colchicine did not significantly limit aortic diameter increase over 80 days (MD -0.1 mm, 95% CI -1.1, 0.86, p = .922). Conclusions: The inflammasome was activated in this mouse model of AAA; however, daily oral administration of colchicine did not limit AAA growth.

Kallikrein-1 Blockade Inhibits Aortic Expansion in a Mouse Model and Reduces Prostaglandin E2 Secretion From Human Aortic Aneurysm Explants.

Background Abdominal aortic aneurysm (AAA) is an important cause of mortality in older adults. The kinin B2 receptor agonist, bradykinin, has been implicated in AAA pathogenesis through promoting inflammation. Bradykinin is generated from high- and low-molecular-weight kininogen by the serine protease kallikrein-1. The aims of this study were first to examine the effect of neutralizing kallikrein-1 on AAA development in a mouse model and second to test how blocking kallikrein-1 affected cyclooxygenase-2 and prostaglandin E2 in human AAA explants. Methods and Results Neutralization of kallikrein-1 in apolipoprotein E-deficient (ApoE-/-) mice via administration of a blocking antibody inhibited suprarenal aorta expansion in response to angiotensin (Ang) II infusion. Kallikrein-1 neutralization decreased suprarenal aorta concentrations of bradykinin and prostaglandin E2 and reduced cyclooxygenase-2 activity. Kallikrein-1 neutralization also decreased protein kinase B and extracellular signal-regulated kinase 1/2 phosphorylation and reduced levels of active matrix metalloproteinase 2 and matrix metalloproteinase 9. Kallikrein-1 blocking antibody reduced levels of cyclooxygenase-2 and secretion of prostaglandin E2 and active matrix metalloproteinase 2 and matrix metalloproteinase 9 from human AAA explants and vascular smooth muscle cells exposed to activated neutrophils. Conclusions These findings suggest that kallikrein-1 neutralization could be a treatment target for AAA.

Vitamin D deficiency promotes large rupture-prone abdominal aortic aneurysms and cholecalciferol supplementation limits progression of aneurysms in a mouse model.

Vitamin D deficiency has been associated with human abdominal aortic aneurysm (AAA); however, its role in AAA pathogenesis is unclear. The aim of the present study was to investigate the effect of vitamin D deficiency on AAA development and examine if administering cholecalciferol (CCF) could limit growth of established AAA within the angiotensin-II (AngII) infused apolipoprotein E-deficient mouse model. Mice were rendered vitamin D deficiency through dietary restriction and during AngII infusion developed larger AAAs as assessed by ultrasound and ex vivo morphometry that ruptured more commonly (48% vs. 19%; P=0.028) than controls. Vitamin D deficiency was associated with increased aortic expression of osteopontin and matrix metalloproteinase-2 and -9 than controls. CCF administration to mice with established aortic aneurysms limited AAA growth as assessed by ultrasound (P<0.001) and ex vivo morphometry (P=0.036) and reduced rupture rate (8% vs. 46%; P=0.031). This effect was associated with up-regulation of circulating and aortic sclerostin. Incubation of human aortic smooth muscle cells with 1,25-dihyroxyvitamin D3 (the active metabolite of vitamin D) for 48 h induced up-regulation of sclerostin (P<0.001) and changed the expression of a range of other genes important in extracellular matrix remodeling. The present study suggests that vitamin D deficiency promotes development of large rupture-prone aortic aneurysms in an experimental model. CCF administration limited both growth and rupture of established aneurysms. These effects of vitamin D appeared to be mediated via changes in genes involved in extracellular matrix remodeling, particularly sclerostin.

Factor XII blockade inhibits aortic dilatation in angiotensin II-infused apolipoprotein E-deficient mice.

Abdominal aortic aneurysm (AAA) is an important cause of mortality in older adults. Chronic inflammation and excessive matrix remodelling are considered important in AAA pathogenesis. Kinins are bioactive peptides important in regulating inflammation. Stimulation of the kinin B2 receptor has been previously reported to promote AAA development and rupture in a mouse model. The endogenous B2 receptor agonist, bradykinin, is generated from the kallikrein-kinin system following activation of plasma kallikrein by Factor XII (FXII). In the current study whole-body FXII deletion, or neutralisation of activated FXII (FXIIa), inhibited expansion of the suprarenal aorta (SRA) of apolipoprotein E-deficient mice in response to angiotensin II (AngII) infusion. FXII deficiency or FXIIa neutralisation led to decreased aortic tumor necrosis factor-α-converting enzyme (TACE/a disintegrin and metalloproteinase-17 (aka tumor necrosis factor-α-converting enzyme) (ADAM-17)) activity, plasma kallikrein concentration, and epithelial growth factor receptor (EGFR) phosphorylation compared with controls. FXII deficiency or neutralisation also reduced Akt1 and Erk1/2 phosphorylation and decreased expression and levels of active matrix metalloproteinase (Mmp)-2 and Mmp-9. The findings suggest that FXII, kallikrein, ADAM-17, and EGFR are important molecular mediators by which AngII induces aneurysm in apolipoprotein E-deficient mice. This could be a novel pathway to target in the design of drugs to limit AAA progression.

Development of a two-stage limb ischemia model to better simulate human peripheral artery disease.

Peripheral arterial disease (PAD) develops due to the narrowing or blockage of arteries supplying blood to the lower limbs. Surgical and endovascular interventions are the main treatments for advanced PAD but alternative and adjunctive medical therapies are needed. Currently the main preclinical experimental model employed in PAD research is based on induction of acute hind limb ischemia (HLI) by a 1-stage procedure. Since there are concerns regarding the ability to translate findings from this animal model to patients, we aimed to develop a novel clinically relevant animal model of PAD. HLI was induced in male Apolipoprotein E (ApoE-/-) deficient mice by a 2-stage procedure of initial gradual femoral artery occlusion by ameroid constrictors for 14 days and subsequent excision of the femoral artery. This 2-stage HLI model was compared to the classical 1-stage HLI model and sham controls. Ischemia severity was assessed using Laser Doppler Perfusion Imaging (LDPI). Ambulatory ability was assessed using an open field test, a treadmill test and using established scoring scales. Molecular markers of angiogenesis and shear stress were assessed within gastrocnemius muscle tissue samples using quantitative polymerase chain reaction. HLI was more severe in mice receiving the 2-stage compared to the 1-stage ischemia induction procedure as assessed by LDPI (p = 0.014), and reflected in a higher ischemic score (p = 0.004) and lower average distance travelled on a treadmill test (p = 0.045). Mice undergoing the 2-stage HLI also had lower expression of angiogenesis markers (vascular endothelial growth factor, p = 0.004; vascular endothelial growth factor- receptor 2, p = 0.008) and shear stress response mechano-transducer transient receptor potential vanilloid 4 (p = 0.041) within gastrocnemius muscle samples, compared to animals having the 1-stage HLI procedure. Mice subjected to the 2-stage HLI receiving an exercise program showed significantly greater improvement in their ambulatory ability on a treadmill test than a sedentary control group. This study describes a novel model of HLI which leads to more severe and sustained ischemia than the conventionally used model. Exercise therapy, which has established efficacy in PAD patients, was also effective in this new model. This new model maybe useful in the evaluation of potential novel PAD therapies.

Depletion of CD11c+ dendritic cells in apolipoprotein E-deficient mice limits angiotensin II-induced abdominal aortic aneurysm formation and growth.

OBJECTIVE: The role of chronic inflammation in abdominal aortic aneurysm (AAA) is controversial. CD11c+ antigen-presenting cells (APCs) (dendritic cells (DCs)) have been reported in human AAA samples but their role is unclear. The effect of conditional depletion of CD11c+ cells on experimental AAA was investigated in the angiotensin II (AngII)-infused apolipoprotein E-deficient (ApoE-/-) mouse model. APPROACH: CD11c-diphtheria toxin (DT or D.tox) receptor (DTR), ovalbumin (OVA) fragment aa 140-386, and enhanced green fluorescent protein (eGFP)-ApoE-/- (CD11c.DOG.ApoE-/-) mice were generated and CD11c+ cell depletion achieved with D.tox injections (8 ng/g body weight, i.p., every-other-day). AAA formation and growth were assessed by measurement of supra-renal aortic (SRA) diameter in vivo by serial ultrasound and by morphometry assessment of harvested aortas at the end of the study. RESULTS: Depletion of CD11c+ cells by administration of D.tox on alternative days was shown to reduce the maximum diameter of AAAs induced by 28 days AngII infusion compared with controls (D.tox, 1.58 ± 0.03 mm vs Vehicle control, 1.81 ± 0.06 mm, P<0.001). CD11c+ depletion commencing after AAA establishment by 14 days of AngII infusion, was also shown to lead to smaller AAAs than controls after a further 14 days (D.tox, 1.54 ± 0.04 mm vs Vehicle control, 1.80 ± 0.03 mm, P<0.001). Flow cytometry revealed significantly lower numbers of circulating CD44hi CD62Llo effector CD4 T cells, CD44hi CD62Llo effector CD8 T cells and B220+ B cells in CD11c+ cell-depleted mice versus controls. CD11c+ depletion attenuated SRA matrix degradation indicated by decreased neutrophil elastase activity (P=0.014), lower elastin degradation score (P=0.012) and higher collagen content (P=0.002). CONCLUSION: CD11c+ cell-depletion inhibited experimental AAA development and growth associated with down-regulation of circulating effector T cells and attenuated matrix degradation. The findings suggest involvement of autoreactive immune cells in AAA pathogenesis.

A diet enriched with tree nuts reduces severity of atherosclerosis but not abdominal aneurysm in angiotensin II-infused apolipoprotein E deficient mice.

BACKGROUND AND AIMS: Diets enriched with tree nuts have been demonstrated to reduce the risk of atherosclerosis-related cardiovascular events. Abdominal aortic aneurysm (AAA) shares common risk factors with atherosclerosis and AAA patients commonly have atherosclerosis related cardiovascular events. AAA has some distinct pathological and clinical characteristics to those of atherosclerosis. No previous study has examined the effect of a diet enriched with tree nuts on experimental or clinical AAA. This study investigated the effect of a diet enriched with tree nuts on the development and severity of AAA within an experimental rodent model. METHODS: Male apolipoprotein E deficient mice were allocated to a diet enriched with tree nuts or control diet for 56 days (n = 17 per group). After 28 days, all mice were infused with angiotensin II whilst being maintained on their respective diets. The primary outcome was AAA severity assessed by the supra-renal aortic diameter, measured by ultrasound and ex vivo morphometric analysis. The severity of atherosclerosis was assessed by computer-aided analysis of Sudan IV stained aortic arches and sections of brachiocephalic arteries prepared with Van Gieson's stain. RESULTS: The diet enriched with tree nuts did not influence aortic diameter or aortic rupture incidence. Mice receiving the diet enriched with tree nuts had significantly less atherosclerosis within the brachiocephalic artery (p = 0.033) but not in the aortic arch. CONCLUSIONS: This experimental study suggests that a diet enriched with tree nuts does not reduce the severity of AAA, but does reduce the severity of atherosclerosis within the brachiocephalic artery. The study was not powered to identify a moderate effect of the diet on the primary outcome and therefore this cannot be excluded.

Anionic nanoliposomes reduced atherosclerosis progression in Low Density Lipoprotein Receptor (LDLR) deficient mice fed a high fat diet.

Atherosclerosis is a systemic disease characterized by the deposition of cholesterol and inflammatory cells within the arterial wall. Removal of cholesterol from the vessel wall may have an impact on the size and composition of atherosclerotic lesions. Anionic phospholipids or liposome vesicles composed of a lipid bilayer such as nanoliposomes have been suggested as treatments for dyslipidemia. In this study, we investigated the effect of anionic nanoliposomes on atherosclerosis in a mouse model. Low-density lipoprotein receptor knockout mice (Ldlr-/- ) were fed with an atherosclerosis promoting high fat and cholesterol (HFC) diet for 12 weeks. Anionic nanoliposomes including hydrogenated soy phosphatidylcholine (HSPC) and distearoyl phosphatidylglycerol (DSPG) (molar ratio: 1:3) were injected intravenously into HFC-fed Ldlr-/- mice once a week for 4 weeks. Mice receiving nanoliposomes had significantly reduced atherosclerosis within the aortic arch as assessed by Sudan IV staining area (p = 0.007), and reduced intima/media ratio (p = 0.030) and greater collagen deposition within atherosclerosis plaques within the brachiocephalic artery (p = 0.007), compared to control mice. Administration of nanoliposomes enhanced markers of reverse cholesterol transport (RCT) and increased markers of plaque stability in HFC-fed Ldlr-/- mice. Reduced cholesterol accumulation was observed in the liver along with the up-regulation of the major genes involved in the efflux of cholesterol such as hepatic ATP-binding cassette transporters (ABC) including Abc-a1, Abc-g1, Abc-g5, and Abc-g8, Scavenger receptor class B, member 1 (Scarb1), and Liver X receptor alpha (Lxr)-α. Lecithin Cholesterol Acyltransferase activity within the plasma was also increased in mice receiving nanoliposomes. Anionic nanoliposome administration reduced atherosclerosis in HFC-fed Ldlr-/- mice by promoting RCT and upregulating the ABC-A1/ABC-G1 pathway.

Fenofibrate and Telmisartan in the Management of Abdominal Aortic Aneurysm.

OBJECTIVE: This mini-review provides the rationale and updated progress for ongoing randomized controlled trials assessing fenofibrate and telmisartan efficacy to limit abdominal aortic aneurysm (AAA) growth. METHODS/RESULTS: There remains an urgent need to identify a drug therapy that will limit AAA growth. Data from preclinical and human studies indicate that fenofibrate and telmisartan have the potential to slow aortic destruction. Fenofibrate has been shown to reduce serum and tissue levels of the proinflammatory protein osteopontin, as well as reducing macrophage recruitment to the aortic wall, both of which are integral processes in the development and progression of AAAs. Telmisartan acts via blockade of the angiotensin II receptor, type 1, and also as a peroxisome proliferator-activated receptor gamma agonist. In turn, this inhibits the production of a range of biomarkers associated with AAA progression, including transforming growth factor-beta one, osteoprotegerin, osteopontin and matrix metalloproteinase- 9. Based on these findings, there are currently three randomized controlled trials assessing both fenofibrate and telmisartan as potential interventions to limit aneurysm growth in AAA patients. CONCLUSION: Fenofibrate and telmisartan have potential as repurposed medications to limit AAA growth, and randomized trials for further assessment in AAA patients are ongoing.

Resveratrol Inhibits Growth of Experimental Abdominal Aortic Aneurysm Associated With Upregulation of Angiotensin-Converting Enzyme 2.

OBJECTIVE: Recent evidence suggests an important role for angiotensin-converting enzyme 2 (ACE2) in limiting abdominal aortic aneurysm (AAA). This study examined the effect of ACE2 deficiency on AAA development and the efficacy of resveratrol to upregulate ACE2 in experimental AAA. APPROACH AND RESULTS: Ace2 deletion in apolipoprotein-deficient mice (ApoE-/-Ace2-/y ) resulted in increased aortic diameter and spontaneous aneurysm of the suprarenal aorta associated with increased expression of inflammation and proteolytic enzyme markers. In humans, serum ACE2 activity was negatively associated with AAA diagnosis. ACE2 expression was lower in infrarenal biopsies of patients with AAA than organ donors. AAA was more severe in ApoE-/-Ace2-/y mice compared with controls in 2 experimental models. Resveratrol (0.05/100-g chow) inhibited growth of pre-established AAAs in ApoE-/- mice fed high-fat chow and infused with angiotensin II continuously for 56 days. Reduced suprarenal aorta dilatation in mice receiving resveratrol was associated with elevated serum ACE2 and increased suprarenal aorta tissue levels of ACE2 and sirtuin 1 activity. In addition, the relative phosphorylation of Akt and ERK (extracellular signal-regulated kinase) 1/2 within suprarenal aorta tissue and gene expression for nuclear factor of kappa light polypeptide gene enhancer in B cells 1, angiotensin type-1 receptor, and metallopeptidase 2 and 9 were significantly reduced. Upregulation of ACE2 in human aortic smooth muscle cells by resveratrol in vitro was sirtuin 1-dependent. CONCLUSIONS: This study provides experimental evidence of an important role for ACE2 in limiting AAA development and growth. Resveratrol upregulated ACE2 and inhibited AAA growth in a mouse model.

Flavonols reduce aortic atherosclerosis lesion area in apolipoprotein E deficient mice: A systematic review and meta-analysis.

Diets rich in flavonoids have been reported to have beneficial effects in the primary prevention of cardiovascular events. There are limited data, however, on the cardiovascular benefits of purified flavonoids. The aim of this systematic review and meta-analysis was to examine the reported effects of isolated flavonoids on aortic atherosclerosis in a mouse model. Medline, Pubmed, Science direct and Web of Science were searched to identify studies which examined the effect of isolated flavonoids on aortic atherosclerosis in apolipoprotein E deficient mice. A meta-analysis was performed to determine the overall effect of the flavonoids, and sub-analyses were performed to compare the effects of the flavonols and flavan-3-ols. Eleven studies, which examined a total of 208 mice receiving a flavonoid and 126 control mice, were included. Overall the flavonoids significantly reduced aortic atherosclerosis (SMD 1.10, 95% CI 0.69, 1.51). Of the 18 flavonoid interventions examined 12 were flavonols and 3 were flavan-3-ols. Sub-analyses suggested that the flavonols (SMD 1.31, 95% CI 0.66, 1.91) but not the flavan-3-ols (SMD 0.33, 95% CI -0.19, 0.85) significantly decreased atherosclerosis area. Of the eleven studies, only one examined histological markers of atherosclerosis plaque stability. Most studies did not report blinding of outcome assessors or reproducibility of the primary outcome, and did not justify the sample size used and flavonoid dose administered. Based on the included studies, the flavonols appear to be the most effective flavonoids for reducing aortic atherosclerotic lesion area in apolipoprotein E deficient mice.

Renal Denervation Promotes Atherosclerosis in Hypertensive Apolipoprotein E-Deficient Mice Infused with Angiotensin II.

Objective: To determine the effect of renal denervation (RDN) on the severity of atherosclerosis and aortic aneurysm in hypertensive mice. Methods: Hypertension, atherosclerosis and aortic aneurysm were induced by subcutaneous infusion of angiotensin II (1 µg/kg/min) for 28 days in apolipoprotein E-deficient mice. RDN was conducted using combined surgical and local chemical denervation. The norepinephrine concentration in the kidney was measured by high-performance liquid chromatography. Blood pressure was measured by the tail-cuff method. Atherosclerosis was assessed by Sudan IV staining of the aortic arch. The aortic diameter was measured by the morphometric method. The mRNA expression of genes associated with atherosclerosis and aortic aneurysm were analyzed by quantitative PCR. Results: RDN decreased the median norepinephrine content in the kidney by 93.4% (n = 5-7, P = 0.003) 5 days after the procedure, indicating that the RDN procedure was successful. RDN decreased systolic blood pressure in apolipoprotein E-deficient mice. Mice that had RDN had more severe aortic arch atherosclerosis (median percentage of Sudan IV positive area: 13.2% in control mice, n = 12, and 25.4% in mice having RDN, n = 12, P = 0.028). The severity of the atherosclerosis was negatively correlated with the renal norepinephrine content (spearman r = -0.6557, P = 0.005). RDN did not affect the size of aortic aneurysms formed or the incidence of aortic rupture in mice receiving angiotensin II. RDN significantly increased the aortic mRNA expression of matrix metalloproteinase-2 (MMP-2). Conclusion: RDN promoted atherosclerosis in apolipoprotein E-deficient mice infused with angiotensin II associated with upregulation of MMP-2. The higher MMP-2 expression could be the results of the greater amount of atheroma in the RDN mice. The findings suggest further research is needed to assess potentially deleterious effects of RDN in patients.

Parenteral administration of factor Xa/IIa inhibitors limits experimental aortic aneurysm and atherosclerosis.

Intraluminal thrombus is a consistent feature of human abdominal aortic aneurysm (AAA). Coagulation factor Xa (FXa) catalyses FII to thrombin (FIIa). We examined the effect of FXa/FIIa inhibition on experimental aortic aneurysm in apolipoprotein E-deficient (ApoE-/-) mice infused with angiotensin II (AngII). The concentration of FXa within the supra-renal aorta (SRA) correlated positively with SRA diameter. Parenteral administration of enoxaparin (FXa/IIa inhibitor) and fondaparinux (FXa inhibitor) over 14 days reduced to severity of aortic aneurysm and atherosclerosis in AngII-infused ApoE-/- mice. Enteral administration of the FIIa inhibitor dabigatran had no significant effect. Aortic protease-activated receptor (PAR)-2 expression increased in response to AngII infusion. Fondaparinux reduced SRA levels of FXa, FIIa, PAR-2, matrix metalloproteinase (MMP)2, Smad2/3 phosphorylation, and MOMA-2 positive cells in the mouse model. FXa stimulated Smad2/3 phosphorylation and MMP2 expression in aortic vascular smooth muscle cells (VSMC) in vitro. Expression of MMP2 in FXa-stimulated VSMC was downregulated in the presence of a PAR-2 but not a PAR-1 inhibitor. These findings suggest that FXa/FIIa inhibition limits aortic aneurysm and atherosclerosis severity due to down-regulation of vascular PAR-2-mediated Smad2/3 signalling and MMP2 expression. Inhibition of FXa/FIIa may be a potential therapy for limiting aortic aneurysm.

Epigenetics and Peripheral Artery Disease.

The term epigenetics is usually used to describe inheritable changes in gene function which do not involve changes in the DNA sequence. These typically include non-coding RNAs, DNA methylation and histone modifications. Smoking and older age are recognised risk factors for peripheral artery diseases, such as occlusive lower limb artery disease and abdominal aortic aneurysm, and have been implicated in promoting epigenetic changes. This brief review describes studies that have associated epigenetic factors with peripheral artery diseases and investigations which have examined the effect of epigenetic modifications on the outcome of peripheral artery diseases in mouse models. Investigations have largely focused on microRNAs and have identified a number of circulating microRNAs associated with human peripheral artery diseases. Upregulating or antagonising a number of microRNAs has also been reported to limit aortic aneurysm development and hind limb ischemia in mouse models. The importance of DNA methylation and histone modifications in peripheral artery disease has been relatively little studied. Whether circulating microRNAs can be used to assist identification of patients with peripheral artery diseases and be modified in order to improve the outcome of peripheral artery disease will require further investigation.

The association of circulating 25-hydroxyvitamin D concentration with peripheral arterial disease: A meta-analysis of observational studies.

BACKGROUND AND AIMS: The association of vitamin D deficiency with cardiovascular disease is controversial. The present meta-analysis was performed to examine if circulating levels of 25-hydroxyvitamin D [25(OH)D] were lower in patients with peripheral artery disease (PAD) when compared to non-PAD controls. METHODS: A comprehensive database search was conducted in Web of science, Scopus, PubMed, EMBASE and The Cochrane Library to identify observational studies reporting 25(OH)D concentrations in PAD patients and non-PAD participants. Data extraction and study quality assessments were conducted independently. A random-effects model was used to meta-analyse extracted data and generate standardized mean differences (SMDs) in circulating 25(OH)D levels between PAD patients and non-PAD controls. Subgroup analyses were conducted focussing on patients presenting with intermittent claudication (IC) and critical limb ischaemia (CLI). RESULTS: Six case-control studies assessing 6418 individuals fulfilled the inclusion criteria. Two studies were considered to be of moderate methodological quality and four were considered to be of high quality. A meta-analysis of data from 1217 PAD patients and 5201 non-PAD participants showed that circulating 25(OH)D concentrations were lower in PAD patients compared with non-PAD participants (SMD = -0.32, 95% CI: -0.58, -0.05; P = 0.02). Subgroup analyses showed that 25(OH)D levels were significantly lower among PAD patients with CLI, but not IC, when compared to non-PAD controls (SMD = -1.29, 95% CI: -1.66, -0.91; P < 0.001 and SMD = -0.01, 95% CI: -0.15, 0.13; P=0.88, respectively). CONCLUSIONS: This meta-analysis suggests that low levels of circulating 25(OH)D are associated with PAD presence, particularly in patients presenting with CLI. These data suggest the possibility that vitamin D insufficiency may contribute to the development of more advanced PAD although this remains to be confirmed.

A peptide antagonist of thrombospondin-1 promotes abdominal aortic aneurysm progression in the angiotensin II-infused apolipoprotein-E-deficient mouse.

OBJECTIVE: Interaction of the activating sequence in thrombospondin-1 (TSP-1) with the conserved sequence (leucine-serine-lysine-leucine [LSKL]) in the latency-associated peptide region of latent transforming growth factor (TGF)-ß complex is important in regulating TGF-ß1 activity. We aimed to assess the effect of blocking peptide LSKL on the progression of pre-established abdominal aortic aneurysm in angiotensin II-infused apolipoprotein E-deficient (ApoE(-/-)) mice. APPROACH AND RESULTS: Abdominal aortic aneurysm was established in 3-month-old male ApoE(-/-) mice with subcutaneous infusion of angiotensin II for 28 days. After this, mice received LSKL peptide or control SLLK (serine-leucine-leucine-lysine) peptide (4 mg/kg) via daily intraperitoneal injection for an additional 2 weeks. Administration of LSKL peptide promoted larger suprarenal aortic diameter, as determined by ultrasound and morphometric analysis, and stimulated more severe atherosclerosis within the aortic arch. In addition, mice receiving LSKL peptide exhibited elevated circulating proinflammatory cytokine levels and greater inflammatory cells within the suprarenal aorta compared with controls. Mice receiving LSKL peptide showed low plasma TGF-ß1 activity and low levels of aortic tissue phosphorylated to total Smad2/3. Aortic gene expression of TGF-ß receptor 1 (TGFBRI) and receptor 2 (TGFBRII), but not TGF-ß1 and thrombospondin-1, were lower in mice receiving LSKL peptide than controls. LSKL peptide administration was associated with greater aortic elastin fragmentation and lower expression and activity of the TGF-ß1-target gene lysyl oxidase like 1 (LOXL1). CONCLUSIONS: Attenuation of thrombospondin-1-directed activation of TGF-ß1 promotes abdominal aortic aneurysm and atherosclerosis progression in the angiotensin II-infused ApoE(-/-) mouse model.

Influence of apolipoprotein E, age and aortic site on calcium phosphate induced abdominal aortic aneurysm in mice.

OBJECTIVE: To assess relevant features of abdominal aortic aneurysms (AAA) induced by calcium phosphate within a mouse model. Specifically we investigated: (1) whether apolipoprotein E deficiency and older age promoted AAA formation, and (2) whether the local application of calcium phosphate affected the size of distant aortic segments. METHODS: AAA was induced by application of calcium phosphate to the infra-renal aortas of 3 and 7 month old male mice. AAA induction was assessed by calculating expansion of the infra-renal aortic diameter over 1-4 weeks. Aortic samples were assessed to quantify calcification, macrophages infiltration, elastic lamellar degradation and apoptosis. Blood pressure was measured by the tail cuff method, and plasma concentrations of total cholesterol, low density lipoprotein and very low density lipoprotein cholesterol, and pro-inflammatory cytokines were measured using commercially available kits. The maximum diameters of the aortic arch, thoracic and supra-renal aorta at sacrifice were measured by morphometry and the mean maximal diameter of these three aortic segments was calculated. RESULTS: The median expansion of the infra-renal aorta 2 weeks after AAA induction was significantly greater in apolipoprotein E deficient (ApoE(-/-)) mice than in age- and gender-matched wild type controls [275.8% (IQR 193.8%-348.5%) versus 94.7% (IQR 47.8%-163.4%), P = 0.02]. The greater aortic expansion in ApoE(-/-) mice was associated with aortic calcification, macrophage infiltration, elastic lamellar degradation and apoptosis of cells in the media and adventitia. The plasma low density lipoprotein/very low density lipoprotein cholesterol concentrations 2 weeks after AAA induction were positively correlated with the expansion of the infra-renal aorta induced by calcium phosphate. The median expansion of the infra-renal aorta 2 weeks after AAA induction was similar in 3 and 7 month old wild type mice. The local administration of calcium phosphate was associated with an increase in the mean maximal diameter of distant aortic segments, but not associated with changes in the concentrations of pro-inflammatory markers in either the plasma or the spleen. CONCLUSION: This study suggests that apolipoprotein E deficiency, but not age, predisposes to AAA induced within the calcium phosphate model. Increased AAA expansion in ApoE(-/-) mice was associated with calcification, macrophage infiltration, elastic lamellar degradation, and cell apoptosis. Local application of calcium phosphate also promoted dilation of distant aortic segments.

Proteomic and genomic analyses suggest the association of apolipoprotein C1 with abdominal aortic aneurysm.

PURPOSE: Abdominal aortic aneurysm (AAA) is an important cause of mortality in the elderly. Mouse models are widely used to investigate AAA pathogenesis but their suitability for biomarker discovery is unexplored. EXPERIMENTAL DESIGN: We conducted a three-phase study. Phase 1: Aortas from angiotensin-II-infused apolipoprotein E deficient (ApoE(-/-) ) mice with and without AAA were assessed via iTRAQ and analyzed in silico to identify potential circulating markers. Microarray data from ApoE(-/-) mice and human patients were analyzed in parallel. Phase 2: Putative markers were compared between datasets to shortlist common candidates. Phase 3: The relationship of two shortlisted markers and AAA presence was assessed. RESULTS: iTRAQ identified eight proteins with biomarker potential. Microarray data identified 72 and 96 potential biomarkers from ApoE(-/-) mice and human patients, respectively. All three datasets suggested apolipoprotein C1 (ApoC1) as a marker for AAA; microarray data identified matrix metalloproteinase 9 (MMP9) as a second potential marker. Plasma ApoC1 and MMP9 concentrations positively correlated with AAA diameter in ApoE(-/-) mice. CONCLUSIONS AND CLINICAL RELEVANCE: ApoC1 may be a novel biomarker for AAA.

Aliskiren limits abdominal aortic aneurysm, ventricular hypertrophy and atherosclerosis in an apolipoprotein-E-deficient mouse model.

Aliskiren is a direct renin inhibitor developed to treat hypertension. Several clinical studies have suggested that aliskiren has beneficial effects on cardiovascular diseases beyond its antihypertensive effect. In the present study, we examined whether aliskiren limits the progression of AAA (abdominal aortic aneurysm), VH (ventricular hypertrophy) and atherosclerosis in an AngII (angiotensin II)-infused mouse model. ApoE-/- (apolipoprotein-E-deficient) mice were infused subcutaneously with AngII (1000 ng/kg of body weight per day; 4 weeks) to induce AAA and VH. At the completion of the AngII infusion, mice were randomly allocated to three groups to receive vehicle control, low-dose aliskiren (10 mg/kg of body weight per day) or high-dose aliskiren (50 mg/kg of body weight per day) for 4 weeks. Suprarenal aortic diameter assessed by ultrasound was significantly smaller in mice administered aliskiren at days 42 and 56. Aliskiren also significantly reduced the normalized heart weight, ventricular myocyte cell width and aortic arch atherosclerosis. Aliskiren lowered PRR (pro-renin receptor) expression and MAPK (mitogen-activated protein kinase) activity in the suprarenal aorta and heart. Aortic infiltration of T-lymphocytes and macrophages was reduced by aliskiren. In conclusion, aliskiren limits the progression of AAA, VH and atherosclerosis in an AngII-infused mouse model.

Impaired acetylcholine-induced endothelium-dependent aortic relaxation by caveolin-1 in angiotensin II-infused apolipoprotein-E (ApoE-/-) knockout mice.

OBJECTIVE: The angiotensin II (AngII)-infused apolipoprotein E-deficient (ApoE(-/-)) mouse model is widely used to study atherosclerosis and abdominal aortic aneurysm. An increase in blood pressure has been reported in this model however the underlying mechanism has not been fully explored. In this study, we investigated whether vasomotor dysfunction develops in AngII-infused ApoE(-/-) mice and the underlying mechanism involved. METHODS: ApoE(-/-) mice were infused with vehicle (distilled water) or AngII subcutaneously for 14 days. Blood pressure and heart rate were measured using the non-invasive tail cuff method. Aortic vascular reactivity and expression of key proteins (endothelial nitric oxide synthase (eNOS), phospho-eNOS and caveolin-1) were assessed using tension myography and Western blotting respectively. Plasma nitric oxide (NO) level was estimated using a colorimetric assay. RESULTS: AngII infusion caused a time-dependent increase in blood pressure (P<0.001). Aortas from AngII-infused mice were significantly less responsive to acetylcholine-induced endothelium-dependent relaxation when compared to aortas from mice infused with vehicle control (P<0.05). Contractile responses to phenylephrine (P<0.01) and potassium chloride (P<0.001) were significantly enhanced in aortas from AngII-infused mice. eNOS phosphorylation was significantly decreased in the aorta of AngII-infused mice (P<0.05). Aortic caveolin-1 protein expression was significantly increased in AngII-infused mice (P<0.05). Plasma nitrate/nitrite level was significantly reduced in AngII-infused mice (P<0.05). Pharmacological disruption of caveolae using methyl-ß-cyclodextrin (MßCD) in isolated aortas from AngII-infused mice caused a significant leftward shift of the acetylcholine-induced relaxation concentration-response curve when compared to vehicle control (P<0.05). CONCLUSION: Upregulation of caveolin-1 protein expression and reduced NO bioavailability contributes to aortic endothelial dysfunction in AngII-infused ApoE(-/-) mice.