The goal of this study was to investigate the role in atherosclerosis of the tumor suppressor protein ARF (human p14(ARF), mouse p19(ARF)) encoded by the CDKN2A gene.
Atherosclerosis is characterized ...by excessive proliferation and apoptosis, 2 cellular processes regulated by CDKN2A. Although recent genome-wide association studies have linked atherosclerotic diseases to a genomic region in human chromosome 9p21 near the CDKN2A locus, the mechanisms underlying this gene-disease association remain undefined, and no causal link has been established between CDKN2A and atherosclerosis.
Atherosclerosis-prone apolipoprotein E (apoE)-null and doubly deficient apoE-p19(ARF) mice were fed an atherogenic diet and sacrificed to quantify atherosclerosis burden in whole-mounted aortas and in aortic cross-sections. Proliferation and apoptosis were investigated in atherosclerotic lesions and in primary cultures of macrophages and vascular smooth muscle cells obtained from both groups of mice.
Genetic disruption of p19(ARF) in apoE-null mice augments aortic atherosclerosis without affecting body weight, plasma lipoproteins, or plaque's proliferative activity. Notably, p19(ARF) deficiency significantly attenuates apoptosis both in atherosclerotic lesions and in cultured macrophages and vascular smooth muscle cells, 2 major cellular constituents of atheromatous plaques.
Our findings establish a direct link between p19(ARF), plaque apoptosis, and atherosclerosis, and suggest that human genetic variants associated to diminished CDKN2A expression may accelerate atherosclerosis by limiting plaque apoptosis.
Abdominal aortic aneurysm (AAA) is a vascular disorder characterized by chronic inflammation of the aortic wall. Low concentrations of vitamin D3 are associated with AAA development; however, the ...potential direct effect of vitamin D3 on AAA remains unknown. This study evaluates the effect of oral treatment with the vitamin D3 receptor (VDR) ligand, calcitriol, on dissecting AAA induced by angiotensin-II (Ang-II) infusion in apoE(-/-) mice.
Oral treatment with calcitriol reduced Ang-II-induced dissecting AAA formation in apoE(-/-) mice, which was unrelated to systolic blood pressure or plasma cholesterol concentrations. Immunohistochemistry and reverse-transcription polymerase chain reaction analysis demonstrated a significant increase in macrophage infiltration, neovessel formation, matrix metalloproteinase-2 and matrix metalloproteinase-9, chemokine (CCL2 (C-C motif) ligand 2, CCL5 (C-C motif) ligand 5, and CXCL1 (C-X-C motif) ligand 1) and vascular endothelial growth factor expression in suprarenal aortic walls of apoE(-/-) mice infused with Ang-II, and all were significantly reduced by cotreatment with calcitriol. Phosphorylation of extracellular signal-regulated kinases 1/2, p38 mitogen-activated protein kinase, and nuclear factor-κB was also decreased in the suprarenal aortas of apoE(-/-) mice cotreated with calcitriol. These effects were accompanied by a marked increase in VDR-retinoid X receptor (RXR) interaction in the aortas of calcitriol-treated mice. In vitro, VDR activation by calcitriol in human endothelial cells inhibited Ang-II-induced leukocyte-endothelial cell interactions, morphogenesis, and production of endothelial proinflammatory and angiogenic chemokines through VDR-RXR interactions, and knockdown of VDR or RXR abolished the inhibitory effects of calcitriol.
VDR activation reduces dissecting AAA formation induced by Ang-II in apoE(-/-) mice and may constitute a novel therapeutic strategy to prevent AAA progression.
Previous studies indicate a role of CDKN2A/2B/2BAS genes in atherosclerosis and type 2 diabetes mellitus (T2DM). Progression of these diseases is accompanied by T-cell imbalance and chronic ...inflammation. Our main objective was to investigate a potential association between CDKN2A/2B/2BAS gene expression and T cell phenotype in T2DM and coronary artery disease (CAD) in humans, and to explore the therapeutic potential of these genes to restore immune cell homeostasis and disease progression. Reduced mRNA levels of CDKN2A (p16Ink4a), CDKN2B (p15Ink4b), and CDKN2BAS were observed in human T2DM and T2DM-CAD subjects compared with controls. Protein levels of p16Ink4a and p15Ink4b were also diminished in T2DM-CAD patients while CDK4 levels, the main target of p16Ink4a and p15Ink4b, were augmented in T2DM and T2DM-CAD subjects. Both patient groups displayed higher activated CD3+CD69+ T cells and proatherogenic CD14++CD16+ monocytes, while CD4+CD25+CD127 regulatory T (Treg cells) cells were decreased. Treatment of primary human lymphocytes with PD0332991, a p16Ink4a/p15Ink4b mimetic drug and a proven CDK4 inhibitor, increased Treg cells and the levels of activated transcription factor phosphoSTAT5. In vivo PD0332991 treatment of atherosclerotic apoE−/− mice and insulin resistant apoE−/−Irs2+/− mice augmented Foxp3-expressing Treg cells and decreased lesion size. Thus, atherosclerosis complications in T2DM associate with altered immune cell homeostasis, diminished CDKN2A/2B/2BAS expression, and increased CDK4 levels. The present study also suggests that the treatment with drugs that mimic CDKN2A/2B genes could potential be considered as a promising therapy to delay atherosclerosis.
Macronutrients represent risk factors for hyperlipidemia or diabetes. Lipid alterations and type 2 diabetes mellitus are global health problems. Overexpression of sterol regulatory element-binding ...factor (
) in transgenic animals is linked to elevated cholesterol levels and diabetes development. We investigated the impact of increased
locus expression and the effects of control and high-fat, high-sucrose (HFHS) diets on body weight, glucose and lipid metabolisms in transgenic mice (
). Wild type (
) and
were fed with both diets for 16 weeks. Plasma glucose, insulin and lipids were assessed (
= 25). Immunostainings were performed in liver, pancreas and fat (N = 10). Expression of
and
in liver was performed by RT-PCR (N = 8). Control diet:
showed reduced weight, insulin, total and HDL cholesterol and triglycerides (TG). HFHS diet widened differences in weight, total and HDL cholesterol, insulin and HOMA index but increased TG in
. In
adipocyte size was lower while HFHS diet produced lower increase, pancreatic β-cell mass was lower with both diets and
,
and
mRNA levels were higher while HFHS diet produced a rise in
and
levels.
complete gene overexpression seems to have beneficial effects on metabolic parameters and to protect against HFHS diet effects.
The mechanisms underlying accelerated atherosclerosis in metabolic syndrome (MetS) patients remain poorly defined. In the mouse, complete disruption of insulin receptor substrate-2 (Irs2) causes ...insulin resistance, MetS-like manifestations, and accelerates atherosclerosis. Here, we performed human, mouse, and cell culture studies to gain insight into the contribution of defective Irs2 signaling to MetS-associated alterations.
In circulating leukocytes from insulin-resistant MetS patients, Irs2 and Akt2 mRNA levels inversely correlate with plasma insulin levels and HOMA index and are reduced compared to insulin-sensitive MetS patients. Notably, a moderate reduction in Irs2 expression in fat-fed apolipoprotein E-null mice lacking one allele of Irs2 (apoE(-/-)Irs2(+/-)) accelerates atherosclerosis compared to apoE-null controls, without affecting plaque composition. Partial Irs2 inactivation also increases CD36 and SRA scavenger receptor expression and modified LDL uptake in macrophages, diminishes Akt2 and Ras expression in aorta, and enhances expression of the proatherogenic cytokine MCP1 in aorta and primary vascular smooth muscle cells (VSMCs) and macrophages. Inhibition of AKT or ERK1/2, a downstream target of RAS, upregulates Mcp1 in VSMCs.
Enhanced levels of MCP1 resulting from reduced IRS2 expression and accompanying defects in AKT2 and Ras/ERK1/2 signaling pathways may contribute to accelerated atherosclerosis in MetS states.
Abdominal aortic aneurysm (AAA), is a complex disorder characterized by vascular vessel wall remodeling. LIGHT (TNFSF14) is a proinflammatory cytokine associated with vascular disease. In the present ...study, the impact of genetic inactivation of Light was investigated in dissecting AAA induced by angiotensin II (AngII) in the Apolipoprotein E-deficient (Apoe−/−) mice. Studies in aortic human (ah) vascular smooth muscle cells (VSMC) to study potential translation to human pathology were also performed. AngII-treated Apoe−/−Light−/− mice displayed increased abdominal aorta maximum diameter and AAA severity compared with Apoe−/− mice. Notably, reduced smooth muscle α-actin+ area and Acta2 and Col1a1 gene expression were observed in AAA from Apoe−/−Light−/− mice, suggesting a loss of VSMC contractile phenotype compared with controls. Decreased Opn and augmented Sox9 expression, which are associated with detrimental and non-contractile osteochondrogenic VSMC phenotypes, were also seen in AngII-treated Apoe−/−Light−/− mouse AAA. Consistent with a role of LIGHT preserving VSMC contractile characteristics, LIGHT-treatment of ahVSMCs diminished the expression of SOX9 and of the pluripotency marker CKIT. These effects were partly mediated through lymphotoxin β receptor (LTβR) as the silencing of its gene ablated LIGHT effects on ahVSMCs. These studies suggest a protective role of LIGHT through mechanisms that prevent VSMC trans-differentiation in an LTβR-dependent manner.
Coexistence of insulin resistance (IR) and metabolic syndrome (MetS) increases the risk of cardiovascular disease (CVD). Genetic studies in diabetes have linked Hepatic Lipase (HL) to an enhanced ...risk of CVD while others indicate a role of HL in inflammatory cells. Thus, we explored the role of HL on atherosclerosis and inflammation in a mouse model of MetS/IR, (apoE-/-Irs2+/- mice) and in patients with MetS and IR. HL-deficiency in apoE-/-Irs2+/- mice reduced atheroma size, plaque vulnerability, leukocyte infiltration and macrophage proliferation. Compared with apoE-/-Irs2+/-HL+/+ mice, MCP1, TNFα and IL6 plasma levels, pro-inflammatory Ly6Chi monocytes and activated(CD69+)-T lymphocytes were also decreased in apoE-/-Irs2+/-HL-/- mice. The LIGHT (Tumour necrosis factor ligand superfamily member 14, TNFSF14)/Lymphotoxin β-Receptor(LTβ-R) pathway, which is involved in T-cell and macrophage activation, was diminished in plasma and in apoE-/-Irs2+/-HL-/- mouse atheromas. Treatment of apoE-/-Irs2+/-HL-/- mice with LIGHT increased the number of Ly6Chi-monocytes and lesion size. Acutely LIGHT-treated apoE-/- mice displayed enhanced proliferating Ly6Chi-monocytes and increased activation of the mitogen-activated protein kinase p38, suggesting that LIGHT/LTβ-R axis might promote atherogenesis by increasing proinflammatory monocytes and proliferation. Notably, MetS-IR subjects with increased atherosclerosis displayed up-regulation of the LIGHT/LTβ-R axis, enhanced inflammatory monocytes and augmented HL mRNA expression in circulating leukocytes. Thus, HL-deficiency decreases atherosclerosis in MetS/IR states by reducing inflammation and macrophage proliferation which are partly attributed to reduced LIGHT/LTβ-R pathway. These studies identify the LIGHT/LTβ-R axis as a main pathway in atherosclerosis and suggest that its inactivation might ameliorate inflammation and macrophage proliferation associated with atherosclerosis burden in MetS/IR.
Primary hypercholesterolemia (PH) is associated with a low grade systemic inflammation that is likely the main driver of premature atherosclerosis. Accordingly, we characterized the immune cell ...behaviour in PH and its potential consequences. Whole blood from 22 PH patients and 21 age-matched controls was analysed by flow cytometry to determine the percentage of leukocyte immunophenotypes, activation, and platelet-leukocyte aggregates. Plasma markers were determined by Enzyme-Linked ImmunoSorbent Assay (ELISA). The adhesion of platelet-leukocyte aggregates to tumor necrosis factor-α (TNFα)-stimulated arterial endothelium was investigated using the dynamic model of the parallel-plate flow chamber. PH patients presented greater percentage of Mon 3 monocytes, Th2 and Th17 lymphocytes, activated platelets, and leukocytes than controls. The higher percentages of circulating platelet-neutrophil, monocyte and lymphocyte aggregates in patients caused increased platelet-leukocyte adhesion to dysfunctional arterial endothelium. Circulating CXCL8, CCL2, CX₃CL1, and IL-6 levels positively correlated with key lipid features of PH, whereas negative correlations were found for IL-4 and IL-10. We provide the first evidence that increased platelet and leukocyte activation leads to elevated platelet-leukocyte aggregates in PH and augmented arterial leukocyte adhesiveness, a key event in atherogenesis. Accordingly, modulation of immune system behavior might be a powerful target in the control of further cardiovascular disease in PH.
•Correct modelling of age-related disease is essential.•Many mouse models of age-related disease lack an ageing component.•Discussion of the future infrastructure requirements for modelling ...age-related disease.
Ageing is a complex multifactorial process that results in many changes in physiological changes processes that ultimately increase susceptibility to a wide range of diseases. As such an ageing population is resulting in a pressing need for more and improved treatments across an assortment of diseases. Such treatments can come from a better understanding of the pathogenic pathways which, in turn, can be derived from models of disease. Therefore the more closely the model resembles the disease situation the more likely relevant the data will be that is generated from them. Here we review the state of knowledge of mouse models of a range of diseases and aspects of an ageing physiology that are all germane to ageing. We also give recommendations on the most common mouse models on their relevance to the clinical situations occurring in aged patients and look forward as to how research in ageing models can be carried out. As we continue to elucidate the pathophysiology of disease, often through mouse models, we also learn what is needed to refine these models. Such factors can include better models, reflecting the ageing patient population, or a better phenotypic understanding of existing models.
Single nucleotide polymorphisms near the Ink4/Arf locus have been associated with type-2 diabetes mellitus. Previous studies indicate a protective role of the locus in the carbohydrate metabolism ...derangement associated with ageing in wild-type mice. The present study demonstrates that the increased Ink4/Arf locus expression in 1-year-old mice, partially-deficient for the insulin receptor substrate (IRS)2 (Irs2+/−SuperInk4/Arf mice) ameliorates hepatic steatosis, inflammation and insulin resistance. Irs2+/−SuperInk4/Arf mice displayed improved glucose tolerance and insulin sensitivity compared with Irs2+/− mice which were glucose intolerant and insulin resistant compared with age-matched wild-type mice. These changes in Irs2+/− mice were accompanied by enhanced hepatic steatosis, proinflammatory macrophage phenotype, increased Ly6Chi-monocyte percentage, T-lymphocyte activation and MCP1 and TNF-α cytokine levels. In Irs2+/−SuperInk4/Arf mice, steatosis and inflammatory parameters were markedly reduced and similar to those of wild-type counterparts. In vivo insulin signalling also revealed reduced activation of the IRS/AKT-dependent signalling in Irs2+/− mice. This was restored upon increased locus expression in Irs2+/−SuperInk4/Arf mice which display similar activation levels as those for wild-type mice. In vivo treatment of Irs2+/−SuperInk4/Arf mice with TNF-α diminished insulin canonical IRS/AKT-signalling and enhanced the stress SAPK/JNK–phosphoSer307IRS1-pathway suggesting that cytokine levels might potentially affect glucose homeostasis through changes in these insulin-signalling pathways. Altogether, these results indicate that enhanced Ink4/Arf locus expression restores glucose homeostasis and that this is associated with diminished hepatic steatosis and inflammation in mice with insulin resistance. Therefore, pharmacological interventions targeted to modulate the Ink4/Arf locus expression could be a tentative therapeutic approach to alleviate the inflammation associated with insulin resistance.
•Irs2+/− mice develop insulin resistance associated with inflammation and steatosis.•Irs2+/− SuperInk4/Arf mice are protected against steatosis and inflammation.•Ink4/Arf Irs2+/− macrophages have a reduced inflammatory and proliferative phenotype.•Hepatic canonical insulin signalling is improved in Irs2+/− SuperInk4/Arf mice.•The use of Ink4/Arf mimetics might be a treatment for insulin resistance.
PDF
