Herminia González-Navarro

Control of cell proliferation in atherosclerosis: insights from animal models and human studies

Excessive hyperplastic cell growth within occlusive vascular lesions has been recognized as a key component of the inflammatory response associated with atherosclerosis, restenosis post-angioplasty, and graft atherosclerosis after coronary artery bypass. Understanding the molecular mechanisms that regulate arterial cell proliferation is therefore essential for the development of new tools for the treatment of these diseases. Mammalian cell proliferation is controlled by a large number of proteins that modulate the mitotic cell cycle, including cyclin-dependent kinases, cyclins, and tumour suppressors. The purpose of this review is to summarize current knowledge about the role of these cell cycle regulators in the development of native and graft atherosclerosis that has arisen from animal studies, histological examination of specimens from human patients, and genetic studies.

p19ARFDeficiency Reduces Macrophage and Vascular Smooth Muscle Cell Apoptosis and Aggravates Atherosclerosis

OBJECTIVES 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. BACKGROUND 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. METHODS 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. RESULTS Genetic disruption of p19(ARF) in apoE-null mice augments aortic atherosclerosis without affecting body weight, plasma lipoproteins, or plaques 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. CONCLUSIONS 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.

Molecular Mechanisms of Atherosclerosis in Metabolic Syndrome. Role of Reduced IRS2-Dependent Signaling

Objective—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. Methods and Results—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. Conclusions—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.

Insulin resistance aggravates atherosclerosis by reducing vascular smooth muscle cell survival and increasing CX3CL1/CX3CR1 axis

AIMS Insulin resistance (IR) is a major risk factor for cardiovascular disease and atherosclerosis. Life-threatening acute events are mainly due to rupture of unstable plaques, and the role of vascular smooth muscle cells (VSMCs) in this process in IR, Type 2 diabetes mellitus, and metabolic syndrome (T2DM/MetS) has not been fully addressed. Therefore, the role of VSMC survival in the generation of unstable plaques in T2DM/MetS and the involvement of inflammatory mediators was investigated. METHODS AND RESULTS Defective insulin receptor substrate 2 (IRS2)-mediated signalling produced insulin-resistant VSMCs with reduced survival, migration, and higher apoptosis than control cells. Silencing of IRS2 or inhibition of the V-akt murine thymomaviral oncogene homologue kinase (AKT)-extracellular signal-regulated kinase (ERK)-dependent pathway in VSMCs augmented expression of the inflammatory chemokine fractalkine (CX3CL1) and its receptor CX3CR1, previously involved in atheroma plaque vulnerability. Interestingly, treatment of VSMCs with CX3CL1 promoted apoptosis in the presence of other stimuli or when the AKT pathway was blocked. Analysis of a mouse model of IR-MetS and accelerated atherosclerosis, apoE-/-Irs2+/- mice, showed reduced VSMC survival, unstable plaques, and up-regulation of CX3CL1/CX3CR1 axis compared with apoE-/- mice. Human studies showed augmented soluble CX3CL1 plasma levels and CX3CR1 expression in monocytes from IR-MetS subjects compared with controls. A positive correlation between insulin levels, homeostatic model assessment (HOMA) index, carotid atherosclerosis, and CX3CR1 mRNA levels was also found in all patients. CONCLUSION IR increases plaque vulnerability by augmenting the CX3CL1/CX3CR1 axis, which is mechanistically linked to reduced VSMC survival. Thus, modulation of IRS2-dependent signalling emerges as a potential therapeutic strategy to promote VSMC survival and atheroma plaque stability and to reduce inflammatory mediators in IR-MetS.

Plasma insulin levels predict the development of atherosclerosis when IRS2 deficiency is combined with severe hypercholesterolemia in apolipoprotein E-null mice.

Atherosclerosis is increased in type 2 diabetic patients but the precise mechanisms underlying this predisposition remain vague. Mice deficient for insulin receptor substrate 2 (IRS2) develop type 2-like diabetes and thus, provide a model to explore the molecular connection between deranged carbohydrate metabolism and atherosclerosis. To explore the relationship between defective insulin signalling and atherosclerosis, we have examined the development of atherosclerosis in the following groups of fat-fed mice: wild-type, diabetic Irs2-null (Irs2-/-), atherosclerosis-prone apolipoprotein E-null (apoE-/-), and doubly-deficient apoE-/- Irs2-/-. Surprisingly, glucose levels of apoE-/- Irs2-/- mice were comparable to those seen in wild-type and apoE-/- and significantly lower than in Irs2-/- mice. Irs2-/- and apoE-/- Irs2-/- were hyperinsulinemic compared to wild-type and apoE-/- mice. Atherosclerotic lesions were barely detectable in wild-type and Irs2-/- mice, which displayed moderate hypercholesterolemia (approximately 280 mg/dL). Notably, atherosclerosis was significantly enhanced in apoE-/- Irs2-/- compared with apoE-/- mice, although both models displayed similar levels of severe hypercholesterolemia (>600 mg/dL). Circulating insulin levels predicted atherosclerotic lesion burden in apoE-/- Irs2-/- mice. Our results suggest that hyperinsulinemia as a result of Irs2 genetic ablation contributes to increased atherosclerosis when combined with severe hypercholesterolemia in the absence of hyperglycaemia (apoE-/- Irs2-/- mice), thus implicating IRS2 as an important modulator of murine hypercholesterolemia-dependent atherosclerosis. Future studies are necessary to determine whether IRS2 dysfunction may promote atherosclerosis in normoglycemic, pre-diabetic patients with clinical manifestations of hyperinsulinemia and insulin resistance.

Increased dosage of Ink4/Arf protects against glucose intolerance and insulin resistance associated with aging

Recent genome‐wide association studies have linked type‐2 diabetes mellitus to a genomic region in chromosome 9p21 near the Ink4/Arf locus, which encodes tumor suppressors that are up‐regulated in a variety of mammalian organs during aging. However, it is unclear whether the susceptibility to type‐2 diabetes is associated with altered expression of the Ink4/Arf locus. In the present study, we investigated the role of Ink4/Arf in age‐dependent alterations of insulin and glucose homeostasis using Super‐Ink4/Arf mice which bear an extra copy of the entire Ink4/Arf locus. We find that, in contrast to age‐matched wild‐type controls, Super‐Ink4/Arf mice do not develop glucose intolerance with aging. Insulin tolerance tests demonstrated increased insulin sensitivity in Super‐Ink4/Arf compared with wild‐type mice, which was accompanied by higher activation of the insulin receptor substrate (IRS)‐PI3K‐AKT pathway in liver, skeletal muscle and heart. Glucose uptake studies in Super‐Ink4/Arf mice showed a tendency toward increased 18F‐fluorodeoxyglucose uptake in skeletal muscle compared with wild‐type mice (P = 0.079). Furthermore, a positive correlation between glucose uptake and baseline glucose levels was observed in Super‐Ink4/Arf mice (P < 0.008) but not in wild‐type mice. Our studies reveal a protective role of the Ink4/Arf locus against the development of age‐dependent insulin resistance and glucose intolerance.

Arterial and Venous Endothelia Display Differential Functional Fractalkine (CX3CL1) Expression by Angiotensin-II

Objective—Angiotensin-II (Ang-II) promotes the interaction of mononuclear cells with arterioles and neutrophils with postcapillary venules. To investigate the mechanisms underlying this dissimilar response, the involvement of fractalkine (CX3CL1) was explored. Methods and Results—Enhanced CX3CL1 expression was detected in both cremasteric arterioles and postcapillary venules 24 hours after Ang-II intrascrotal injection. Arteriolar leukocyte adhesion was the unique parameter significantly reduced (83%) in animals lacking CX3CL1 receptor (CX3CR1). Human umbilical arterial and venous endothelial cell stimulation with 1 &mgr;mol/L Ang-II increased CX3CL1 expression, yet neutralization of CX3CL1 activity only significantly inhibited Ang-II–induced mononuclear cell–human umbilical arterial endothelial cell interactions (73%) but not with human umbilical venous endothelial cells. The use of small interfering RNA revealed the involvement of tumor necrosis factor-&agr; in Ang-II–induced CX3CL1 upregulation and mononuclear cell arrest. Nox5 knockdown with small interfering RNA or pharmacological inhibition of extracellular signal-regulated kinases1/2, p38 mitogen-activated protein kinase, and nuclear factor-&kgr;B also abolished these responses. Finally, when human umbilical arterial endothelial cells were costimulated with Ang-II, tumor necrosis factor-&agr;, and interferon-&ggr;, CX3CL1 expression and mononuclear cell adhesiveness were more pronounced than when each stimulus was provided alone. Conclusion—These results suggest that Ang-II induces functional CX3CL1 expression in arterial but not in venous endothelia. Thus, targeting endothelial CX3CL1–mononuclear leukocyte CX3CR1 interactions may constitute a new therapeutic strategy in the treatment of Ang-II–associated cardiovascular diseases.

Murine models to investigate the influence of diabetic metabolism on the development of atherosclerosis and restenosis.

Atherosclerosis and related forms of cardiovascular disease (CVD) are associated with several genetic and environmental risk factors, including hypercholesterolemia, diabetes mellitus (DM), hypertension, obesity and smoking. Human DM is a multi-system disorder that results from progressive failure of insulin production and insulin resistance. Most diabetic patients die from complications of atherosclerosis and CVD, and DM is also associated with increased risk of restenosis post-angioplasty. Furthermore, the incidence of DM, particularly type 2-DM, is expected to increase significantly during the next decades owing to the unhealthy effects of modern life-style habits (e.g., obesity and lack of physical exercise). Thus, it is of utmost importance to develop novel preventive and therapeutic strategies to reduce the social and health-care burden of CVD and DM. Although a number of physiological alterations thought to promote atherosclerosis have been identified in diabetic patients, the precise molecular mechanisms that link DM and atherosclerosis are largely unknown. Thus, the aim of this review is to discuss current murine models of combined DM and atherosclerosis and to explore how these experimental systems are being utilized to gain mechanistic insights into diabetes-induced neointimal lesion development, as well as their potential use in evaluating the efficacy of new therapies. Our discussion includes models generated by streptozotocin treatment and those resulting from naturally occurring or targeted mutations in the mouse.

Deficient p27 Phosphorylation at Serine 10 Increases Macrophage Foam Cell Formation and Aggravates Atherosclerosis Through a Proliferation-Independent Mechanism

Objective—Genetic ablation of the growth suppressor p27Kip1 (p27) in the mouse aggravates atherosclerosis coinciding with enhanced arterial cell proliferation. However, it is unknown whether molecular mechanisms that limit p27s protective function contribute to atherosclerosis development and whether p27 exerts proliferation-independent activities in the arterial wall. This study aims to provide insight into both questions by investigating the role in atherosclerosis of p27 phosphorylation at serine 10 (p27-phospho-Ser10), a major posttranslational modification of this protein. Methods and Results—Immunoblotting studies revealed a marked reduction in p27-phospho-Ser10 in atherosclerotic arteries from apolipoprotein E–null mice, and expression of the nonphosphorylatable mutant p27Ser10Ala, either global or restricted to bone marrow, accelerated atherosclerosis. p27Ser10Ala expression did not affect cell proliferation in early and advanced atheroma but activated RhoA/Rho-associated coiled-coil containing protein kinase (ROCK) signaling and promoted macrophage foam cell formation in a ROCK-dependent manner. Supporting the clinical relevance of these findings, human atherosclerotic coronary arteries exhibited a prominent reduction in p27-phospho-Ser10 and increased ezrin/radixin/moesin protein phosphorylation, a marker of RhoA/ROCK activation. Conclusion—Scarce phosphorylation of p27 at Ser10 is a hallmark of human and mouse atherosclerosis and promotes disease progression in mice. This proatherogenic effect is mediated by a proliferation-independent mechanism that involves augmented foam cell formation owing to increased RhoA/ROCK activity. These findings unveil a new atheroprotective action of p27 and identify p27-phospho-Ser10 as an attractive target for the treatment of atherosclerosis.

The GLP-1 analogue lixisenatide decreases atherosclerosis in insulin-resistant mice by modulating macrophage phenotype

Aims/hypothesisRecent clinical studies indicate that glucagon-like peptide-1 (GLP-1) analogues prevent acute cardiovascular events in type 2 diabetes mellitus but their mechanisms remain unknown. In the present study, the impact of GLP-1 analogues and their potential underlying molecular mechanisms in insulin resistance and atherosclerosis are investigated.MethodsAtherosclerosis development was evaluated in Apoe−/−Irs2+/− mice, a mouse model of insulin resistance, the metabolic syndrome and atherosclerosis, treated with the GLP-1 analogues lixisenatide or liraglutide. In addition, studies in Apoe−/−Irs2+/− mice and mouse-derived macrophages treated with lixisenatide were performed to investigate the potential inflammatory intracellular pathways.ResultsTreatment of Apoe−/−Irs2+/− mice with either lixisenatide or liraglutide improved glucose metabolism and blood pressure but this was independent of body weight loss. Both drugs significantly decreased atheroma plaque size. Compared with vehicle-treated control mice, lixisenatide treatment generated more stable atheromas, with fewer inflammatory infiltrates, reduced necrotic cores and thicker fibrous caps. Lixisenatide-treated mice also displayed diminished IL-6 levels, proinflammatory Ly6Chigh monocytes and activated T cells. In vitro analysis showed that, in macrophages from Apoe−/−Irs2+/− mice, lixisenatide reduced the secretion of the proinflammatory cytokine IL-6 accompanied by enhanced activation of signal transducer and activator of transcription (STAT) 3, which is a determinant for M2 macrophage differentiation. STAT1 activation, which is essential for M1 phenotype, was also diminished. Furthermore, atheromas from lixisenatide-treated mice showed higher arginase I content and decreased expression of inducible nitric oxide synthase, indicating the prevalence of the M2 phenotype within plaques.Conclusions/interpretationLixisenatide decreases atheroma plaque size and instability in Apoe−/−Irs2+/− mice by reprogramming macrophages towards an M2 phenotype, which leads to reduced inflammation. This study identifies a critical role for this drug in macrophage polarisation inside plaques and provides experimental evidence supporting a novel mechanism of action for GLP-1 analogues in the reduction of cardiovascular risk associated with insulin resistance.