51 research outputs found

    Kallikrein-1 blockade inhibits aortic expansion in a mouse model and reduces prostaglandin E2 secretion from human aortic aneurysm explants

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    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

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

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    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

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

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    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-alpha-converting enzyme (TACE/a disintegrin and metalloproteinase-17 (aka tumor necrosis factor-alpha-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

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

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    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 inves-tigate 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

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

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    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 FXastimulated 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

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

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    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-) 1 beta (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

    Meta-Analysis of the Association between Transforming Growth Factor-Beta Polymorphisms and Complications of Coronary Heart Disease

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    Objective: To investigate the association between common transforming growth factor beta (TGF-β) single nucleotide polymorphisms (SNP) and significant complications of coronary heart disease (CHD).\ud \ud Method: We performed a meta-analysis of published case-control studies assessing the association of TGF-β SNPs with a range of CHD complications. A random effects model was used to calculate odds ratios and confidence intervals. Analyses were conducted for additive, dominant and recessive modes of inheritance.\ud \ud Results: Six studies involving 5535 cases and 2970 controls examining the association of common SNPs in TGF-β1 with CHD were identified. Applying a dominant model of inheritance, three TGF-β1 SNPs were significantly associated with CHD complications: The T alleles of rs1800469 (OR = 1.125, 95% CI 1.016–1.247, p = 0.031) and rs1800470 (OR = 1.146, 95% CI 1.026–1.279, p = 0.021); and the C allele of rs1800471 (OR = 1.207, 95% CI 1.037–1.406, p = 0.021).\ud \ud Conclusion: This meta-analysis suggests that common genetic polymorphisms in TGF-β1 are associated with complications of CHD

    Expression of VEGF as prognosticator in primary nasopharyngeal cancer and its relation to EBV status

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    Treatment failure in nasopharyngeal carcinoma (NPC) is mainly due to local and regional recurrence and at present lacks a marker to identify such cases. VEGF, a potent endothelial cell mitogen and vascular permeability-inducing agent, plays a critical role in tumor growth and neo-vascularisation. VEGF expression was assessed in 103 NPC and 26 benign adenoid lesions by immunohistochemistry and Epstein Bar Virus (EBV) presence by PCR using primers directed against EBNA-1 gene. The localization of the virus to the tumour cells was confirmed by in situ hybridization using EBER in situ hybridization. Over expression of VEGF was seen in 67% of NPC cases. Higher expression of VEGF in EBV positive tumours was related to higher rate of recurrence, nodal positivity and lower survival. This is the first report evaluating the correlation between VEGF expression levels, EBV status and recurrence in Indian NPC. The results point towards the potential of the expression pattern of VEGF as a tumor marker for the early diagnosis of metastastic NPC and also show that presence of EBV is related to up regulation of VEGF

    Down regulation of adhesion protein E-cadherin in Epstein-Barr virus infected nasopharyngeal carcinomas

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    Nasopharyngeal carcinoma (NPC) is a unique tumour due to its aetiology, incidence pattern and its consistent association with Epstein-Barr virus (EBV). EBV encodes many viral proteins, which targets crucial cell cycle regulatory proteins. Cadherins are a family of transmembrane glycoproteins, which mediates Ca2+-dependent intercellular adhesion. Epithelial (E)-cadherin is an important member of this family, which is expressed predominantly on the surface of epithelial cells. E-cadherin acts as an invasion/metastasis-suppressor gene, hence knowledge of the molecular mechanism that controls its expression or function is of primary importance in understanding the process of tumor invasion. Loss of E-cadherin function has been shown to potentiate tumor cell invasion and interestingly, a large number of invasive tumors do not involve mutation of E-cadherin, but rather down regulation of expression. Hence in this study, an attempt was made to evaluate the protein level expression pattern of E-cadherin in relation to viral involvement and to correlate it with other clinico-pathological parameters. We observed a significant down regulation of E-cadherin in NPC and its histological subsets, when compared to the controls (p<0.001). Expression of E-cadherin ranged from mild to moderate and none of the NPC showed intense expression. E-cadherin expression showed a significant down-regulation in NPC lesions with EBV infection (r=-0.436, p<0.001). The down regulation of E-cadherin observed in NPC is in line with the previous reports in E-cadherin expression in various cancers. The total lack of E-cadherin expression in neoplastic cells might be due to hypermethylation of E-cadherin promoter or its down regulation by cellular transcription repressor. Our study also shows a significant down regulation of E-cadherin in the presence of EBV, which also might involve the cellular DNA methylation machinery

    Serum EBV DNA as a biomarker in primary nasopharyngeal carcinoma of Indian origin

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    Background: Nasopharyngeal carcinoma (NPC) is a unique tumor due to its etiology and endemic distribution. Ethnic and regional factors are found to strongly influence the risk of disease; however, there have been no well-conducted studies on Indian patients. The present study assesses the relationship between Epstein-Barr Virus (EBV) and sporadic Indian NPC and the role of serum EBV DNA in NPC detection. Methods: Primers directed against non-polymorphic Epstein-Barr nuclear antigen-1 (EBNA-1) gene were used to detect the presence of EBV DNA from fresh tissue and serum in NPC, using PCR. Results: EBV DNA was detected in 69% of the biopsies and 58% of the serum of the NPC patients. With respect to histology, WHO Type III NPC, WHO Type II tumors and WHO I tumors showed 100%, 72.2% and 33% EBV positivity, respectively. EBV positivity was also observed in 23% (6/26) of benign samples. All biopsies of patients with positive serum samples were positive for EBV DNA. Conclusion: EBV infection was found in sporadic NPC of South Indian origin, which confirms the etiological role of EBV in NPC. Detection of EBNA-1 in the serum and corresponding tissues of NPC patients suggests that the serum EBV DNA originates from NPC and also indicates the benefit of circulating viral DNA as an early marker in the diagnosis of NPC. Serum DNA-PCR methods can be extrapolated to follow-up studies involving tumor regression or to assess the response to various therapies
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