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This work updates the existing list of obligate endemic serpentinophytes of the southern Iberian Peninsula. Serpentine ecosystems are developed on ultramafic outcrops which, although rare, have a worldwide distribution. The highly evolved and specialized flora that manages to inhabit these harsh ecosystems includes a very specialized and highly evolved group of plants known as serpentinophytes. Serpentinophytes are linked exclusively or almost exclusively to serpentine ecosystems. The existing list of obligate serpentinophytes (obligate endemics) which exist in the serpentine ecosystems of the southern Iberian Peninsula (Spain) consists of 22 taxa. New fieldwork has been done resulting in the description of new soil endemics and the discovery of new populations of this specialized flora. Consequently, bibliographical sources, databases such as the Global Biodiversity Information Facility (GBIF), and existing herbarium sheets have been revised. Crepis bermejana and Galatella malacitana have been added to the list and are proposed to be evaluated as threatened. Teucrium reverchonii has been excluded from the list. Euphorbia flavicoma subsp. bermejense and Armeria villosa subsp. serpentinicola required changes in nomenclature. These nomenclatural changes imply changes in population allocations. The revised list is composed of 23 obligate serpentinophyte taxa. Future conservation measures may include a complete Iberian (including Portugal) serpentinophytes checklist and corresponding revisions of States' legislation and Red Lists. ; Los ecosistemas serpentínicos se desarrollan sobre afloramientos ultramáficos y constituyen una rareza a nivel mundial. La flora que consigue habitar estos ecosistemas incluye un grupo de plantas muy especializado y evolucionado denominadas serpentinófitos, ligados exclusivamente, o casi, a ellos. El primer listado de serpentinófitos obligados (endémicos) de los ecosistemas serpentínicos del sur de la Península Ibérica (España) estaba compuesto por 22 taxa. Con este trabajo se pretende actualizar dicho listado, ya que desde entonces nuevos edafismos han sido descritos y nuevas poblaciones han sido descubiertas. Para ello se han consultado fuentes bibliográficas, bases de datos, fundamentalmente GBIF, y pliegos de herbario, así como se han hecho trabajos de campo. Como principal resultado se han añadido al listado Crepis bermejana y Galatella malacitana, propuestas como amenazadas y para ser evaluadas. Teucrium reverchonii ha sido excluido. Euphorbia flavicoma subsp. bermejense y Armeria villosa subsp. serpentinicola presentan cambios nomenclaturales que han implicado cambios en asignación de poblaciones. La lista actual queda compuesta por 23 serpentinófitos obligados.

A catalogue of ferns and lycophytes has been made from the Sierra de las Nieves Natural Area. It is a territory located in southern Iberian Peninsula (Malaga province, Spain) and it is composed of Sierra de las Nieves Natural Park and a surrounding area proposed as National Park. 38 taxa belonging to 10 families have been identified. Of the three biogeographic subsectors that make up the natural area, those with the greatest diversity of taxa are the Marbella subsector with 24 taxa and the Ronda subsector with 21, and the least diverse is the Bermeja subsector with 16. Of the total of taxa, 7 are common to all subsectors. 8 are exclusive for the Marbella subsector, 9 for Ronda and 4 for Bermeja. The most important families in diversity are Aspleniaceae and Pteridaceae with 12 taxa each. The rest of the families range from 1 to 3 taxa. Of the 38 taxa, we consider 14 to be very rare in the natural area and 10 as very frequent. Only two of the catalogued taxa are considered as threatened (vulnerable) in Andalusia and only one is protected under Andalusian legislation (Regime of Special Protection). ; Se presenta en este trabajo un catálogo de los helechos y licófitos del Espacio Natural Sierra de las Nieves, territorio localizado al sur de la Península Ibérica (Málaga, España) y del que forman parte el Parque Natural Sierra de las Nieves y el propuesto como Parque Nacional Sierra de las Nieves. Se han localizado 38 taxones pertenecientes a 10 familias. De los tres subsectores biogeográficos que conforman el espacio natural, los que presenta mayor diversidad de taxones son el Marbellí con 24 taxones y el Rondense con 21 y el de menor diversidad el Bermejense con 16. Del total de taxones 7 son comunes a todos los subsectores, siendo exclusivos 8 para el subsector Marbellí, 9 para el Rondense y 4 para el Bermejense. Las familias más importantes en diversidad son Aspleniaceae y Pteridaceae con 12 taxones cada una. El resto de las familias oscilan entre 1 y 3 taxones. De los 38 taxones consideramos 14 como muy raros en el espacio natural y como muy frecuentes 10. Solo dos taxones de los localizados se encuentran amenazados como VU (Vulnerables) a nivel de Andalucía y solo uno está protegido en la legislación andaluza como RPE (Régimen de Protección Especial).

10 páginas, 2 figuras. ; Telomeres—the specialized DNA-protein structures at the ends of eukaryotic chromosomes—are essential for maintaining genome stability and integrity and for extended proliferative life span in both cultured cells and in the whole organism. Telomerase and additional telomere-associated proteins are necessary for preserving telomeric DNA length. Age-dependent telomere shortening in most somatic cells, including vascular endothelial cells, smooth muscle cells, and cardiomyocytes, is thought to impair cellular function and viability of the aged organism. Telomere dysfunction is emerging as an important factor in the pathogenesis of hypertension, atherosclerosis, and heart failure. In this Review, we discuss present studies on telomeres and telomere-associated proteins in cardiovascular pathobiology and their implications for therapeutics. ; Work in the laboratory of V.A. is supported in part by the Ministry of Science and Technology of Spain and Fondo Europeo de Desarrollo Regional (grants SAF2001-2358 and SAF2002-1443) and from Instituto de Salud Carlos III (Red de Centros C03/01). A.L.S. is the recipient of a Marie Curie postdoctoral fellowship from the European Union. ; Peer reviewed

9 páginas, 8 figuras. ; Previous studies have demonstrated a protective effect of the cyclin-dependent kinase (CDK) inhibitor p27Kip1 against atherosclerosis and restenosis, two disorders characterized by abundant proliferation and migration of vascular smooth muscle cells and adventitial fibroblasts. These therapeutic effects might result from p27Kip1-dependent suppression of both cell proliferation and migration. However, the interplay between cell growth and locomotion remains obscure. We show here that p27Kip1 inhibits cellular changes that normally occur during cell locomotion (eg, lamellipodia formation and reorganization of actin filaments and focal adhesions). Importantly, a p27Kip1 mutant lacking CDK inhibitory activity failed to inhibit vascular smooth muscle cell and fibroblast proliferation and migration. Moreover, a constitutively active mutant of the retinoblastoma protein (pRb) insensitive to CDK-dependent hyperphosphorylation inhibited both cell proliferation and migration. In contrast, inactivation of pRb by forced expression of the adenoviral oncogene E1A correlated with high proliferative and migratory activity. Collectively, these results suggest that cellular proliferation and migration are regulated in a coordinated manner by the p27Kip1/CDK/pRb pathway. These findings might have important implications for the development of novel therapeutic strategies targeting the fibroproliferative/migratory component of vascular occlusive disorders. ; Work in the laboratory of Dr Andrés is currently supported by the Spanish Ministerio de Ciencia y Tecnología and Fondo Europeo de Desarrollo Regional (grants SAF2001-2358 and SAF2002-1143) and by Generalitat Valenciana (grants GV01-488 and CTGCA/2002/04). Dr Díez-Juan received salary support from the Spanish Government and Fondo Europeo de Desarrollo Regional (grant 1FD97-1035-C02- 02) and from Fondo Social Europeo (CSIC-Programa I3P). ; Peer reviewed

[EN] Four diets for sharpsnout sea bream juveniles (14 g body weight) with four levels of air-processed pea protein concentrate (PPC) (0, 160, 320 and 487 g/kg diet) were tested in triplicate. The experimental diets were isonitrogenous (43% crude protein) and isolipidic (19% ether extract) and the fish were fed to satiation twice a day. After 125 d, fish growth was diminished by the inclusion of PPC. Feed conversion did not show significant differences in any treatment. Neither the body analyses nor the protein and individual essential amino acid retention efficiencies were affected by the inclusion of PPC in the diet. However, histological gut examinations revealed noticeable differences. Fish fed the diet with the highest inclusion level of PPC presented the longest villous length and the most goblet cells, and the width of the lamina propria increased in the anterior intestine. Although no negative changes in nutritive parameters were detected, these alterations might affect nutrient transport, with negative consequences for fish growth. It was concluded that the PPC in the amounts tested here is an inappropriate substitute for fishmeal in diets for sharpsnout sea bream juveniles. ; This work was supported by the Generalitat Valenciana; Fisheries Unit of the Valencian Regional Government and Ministerio de Agricultura, Alimentacion y Medio Ambiente (JACUMAR, Spain). ; Nogales Mérida, S.; Tomas-Vidal, A.; Moñino López, AV.; Jover Cerda, M.; Martínez-Llorens, S. (2016). Pea protein concentrate in diets for sharpsnout seabream (Diplodus puntazzo): effect on growth and health status. Archives of Animal Nutrition. 70(6):488-502. doi:10.1080/1745039X.2016.1229456 ; S ; 488 ; 502 ; 70 ; 6

This is an, un-copyedited, author manuscript that has been accepted for publication in the Frontiers in Bioscience ; 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 multisystem 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. ; Work in the laboratory of the authors is supported by grants from the Spanish Instituto de Salud Carlos III (Red Temática de Investigación Cooperativa en Enfermedades Cardiovasculares RECAVA, Red de Grupos G03/212), and from the Spanish Ministry of Education and Science and the European Regional Development Fund (SAF2004-03057, SAF2005-06058). H. G.-N. is supported by an European Union Marie Curie postdoctoral fellowship. ; Peer reviewed

14 páginas, 4 figuras, 2 tablas.-- El documento en word es la versión post-print. ; Excessive dietary fat and cholesterol exacerbate atherosclerosis. To obtain unbiased insight into the early pathological changes induced by fat feeding in the artery wall, we used high-density microarrays to generate transcriptional profiles of aortic tissue from two groups of atherosclerosis-prone apolipoprotein E-null mice: controls maintained on standard chow and experimental animals exposed short-term to a Western-type diet, a regimen which produced severe hypercholesterolemia without significant development of atheromas. By applying rigorous selection criteria, we identified 311 genes differentially regulated by these dietary conditions. The set of diet-regulated genes exhibited striking functional relationships and represented both novel and known regulatory networks implicated in injury of the artery wall, including cell adhesion genes, histocompatibility antigen and major histocompatibility complex genes, flavin-containing monooxygenases, interferon-regulated genes, small inducible cytokines, collagen and procollagen genes, and complement system components. Further examination of genes identified by this study will provide insights into the molecular mechanisms by which high-fat cholesterol-rich dietary regime initiates pathological alterations in healthy arteries. ; Work supported by grants from Ministerio de Educación y Ciencia of Spain and Fondo Europeo de Desarrollo Regional (SAF2001-2358, SAF2004-3057), from the regional government of Valencia (GRUPOS03/072), and from Instituto de Salud Carlos III (Red de Centros RECAVA, C03/01). C. Castro is the recipient of a postdoctoral fellowship from Fundación Carolina. ; Peer reviewed

9 páginas, 9 figuras.-- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.-- El documento en word es la versión post-print. ; Excessive proliferation and migration of vascular smooth muscle cells (SMCs) participate in atherosclerotic plaque growth. In this study, we investigated whether SMCs from vessels with different atherogenicity exhibit distinct growth and migratory potential and investigated the underlying mechanisms. In fat-fed rabbits, we found increased cell proliferation and atheroma formation in the aortic arch versus the femoral artery. When examined in culture, SMCs isolated from the aortic arch (ASMCs) displayed a greater capacity for inducible proliferation and migration than paired cultures of femoral artery SMCs. Two lines of evidence suggested that distinct regulation of the growth suppressor p27Kip1(p27) contributes to establishing these phenotypic dissimilarities. First, p27 expression was comparably lower in ASMCs, which exhibited a higher fraction of p27 phosphorylated on Thr-187 and ubiquitinated. Second, forced p27 overexpression in ASMCs impaired their proliferative and migratory potential. We found that platelet-derived growth factor-BB-dependent induction of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway was comparably higher in ASMCs. Importantly, pharmacological inhibition of MAPKs increased p27 expression and attenuated ASMC proliferation and migration. In contrast, forced MAPK activation diminished p27 expression and markedly augmented femoral artery SMC proliferation and migration. We propose that intrinsic differences in the regulation of MAPKs and p27 play an important role in creating variance in the proliferative and migratory capacity of vascular SMCs, which might in turn contribute to establishing regional variability in atherogenicity. ; This work was supported by Grants PM97-0136 and SAF2001-2358 from the Spanish Government and Fondo Europeo de Desarrollo Regional. ; Peer reviewed

As orchestrators of essential cellular processes like proliferation, ERK1/2 mitogen-activated protein kinase signals impact on cell cycle regulation. A-type lamins are major constituents of the nuclear matrix that also control the cell cycle machinery by largely unknown mechanisms. In this paper, we disclose a functional liaison between ERK1/2 and lamin A whereby cell cycle progression is regulated. We demonstrate that lamin A serves as a mutually exclusive dock for ERK1/2 and the retinoblastoma (Rb) protein. Our results reveal that, immediately after their postactivation entrance in the nucleus, ERK1/2 dislodge Rb from its interaction with lamin A, thereby facilitating its rapid phosphorylation and consequently promoting E2F activation and cell cycle entry. Interestingly, these effects are independent of ERK1/2 kinase activity. We also show that cellular transformation and tumor cell proliferation are dependent on the balance between lamin A and nuclear ERK1/2 levels, which determines Rb accessibility for phosphorylation/inactivation. ; P. Crespo's laboratory is supported by grants from the Spanish Ministry of Science and Innovation (BFU2008-01728), the GROWTHSTOP project from the European Union VI Framework Program (LSHC-CT-2006-037731), and the Red Temática de Investigación Cooperativa en Cáncer (RD06/0020/0105), Spanish Ministry of Health. V. Andrés's laboratory is supported by the Spanish Ministry of Science and Innovation (SAF2007-62110), the European Regional Development Fund, and the Red Temática de Investigación Cooperativa en Enfermedades Cardiovasculares (RD06/0014/0021). The Centro Nacional de Investigaciones Cardiovasculares is supported by the Spanish Ministry of Science and Innovation and the Fundación Pro–Centro Nacional de Investigaciones Cardiovasculares. ; Peer reviewed

9 páginas, 4 figuras.-- El documento en word es la versión post-print. ; Excessive cellular proliferation is thought to contribute to neointimal lesion development during atherosclerosis and restenosis after angioplasty. Inhibition of cyclin-dependent kinase (CDK) activity by p27 inhibits mammalian cell growth. Mounting evidence indicates that p27 negatively regulates neointimal thickening in animal models of restenosis and atherosclerosis, and its expression in human neointimal lesions is consistent with such a protective role. Cell cycle progression is facilitated by cyclinE/CDK2-dependent phosphorylation of p27 on threonine 187 (T187) during late G1. The purpose of this study was to assess whether this phosphorylation event plays a role during atherosclerosis. To this end, we generated apolipoprotein E-null mice with both p27 alleles replaced by a mutated form non-phosphorylatable at T187 (apoE−/−p27T187A mice) and investigated the kinetics of atheroma development in these animals compared to apoE−/− controls with an intact p27 gene. Fat feeding resulted in comparable level of hypercholesterolemia in both groups of mice. Surprisingly, aortic p27 expression was not increased in fat-fed apoE−/−p27T187A mice compared with apoE−/− controls. Moreover, atheroma size, lesion cellularity, proliferation, and apoptotic rates were undistinguishable in both groups of fat-fed mice. Thus, in contrast to previous studies that highlight the importance of p27 phosphorylation at T187 on the control of p27 expression and function in different tissues and pathophysiological scenarios, our findings demonstrate that this phosphorylation event is not implicated in the control of aortic p27 expression and atheroma progression in hypercholesterolemic mice. ; Work in the laboratory of V.A. is supported inpart by grants fromInstituto de Salud Carlos III (Red de Centros RECAVA, C03/01), from Regional Government of Valencia (GV04B-288), and from Spanish Ministry of Education and Science and Fondo Europeo de DesarrolloRegional(SAF2004-03057).S.M.S.-G. is the recipient of a BEFI Predoctoral fellowship from the Spanish Ministry of Health. ; Peer reviewed

5 páginas, 5 figuras.-- El documento en word es la versión post-print. ; We sought to determine the relative importance of aging and hypercholesterolemia on atherosclerosis. Although plasma cholesterol levels increased similarly in young and old rabbits fed an atherogenic diet for 2 months, aortic atherosclerotic lesions were more prominent in young animals. This finding was associated with an age-dependent reduction in the DNA-binding activity of the proinflammatory nuclear factor κB (NF-κB) in aortic tissue. Atherosclerotic lesions consisted mostly of macrophages, which displayed a similar proliferative response in both age groups. Independently of the age, medial cell proliferation was low and increased as a function of intimal lesion size. Thus, higher atherogenicity in young rabbits exposed to extreme hypercholesterolemia compared to old counterparts is associated with higher activity of NF-κB in the juvenile vessel wall without apparent age-dependent changes in arterial cell proliferation. ; This work was supported by grants from the Spanish Government and Fondo Europeo de Desarrollo Regional (PM97- 0136, SAF2001-2358) and from the American Heart Association, Massachusetts Affiliate (9860022T). A.D.-J. was partially supported by the Spanish Government and Fondo Europeo de Desarrollo Regional (Grant 1FD97-1035-C02-02), and by Fondo Social Europeo (CSIC-Programa I3P fellowship). M.J.C. was supported by a CSICBancaja postdoctoral fellowship. ; Peer reviewed

6 páginas, 5 tablas. ; Atherosclerosis is characterized by excessive proliferation of neointimal leukocytes and vascular smooth muscle cells (VSMCs). In mice, the manipulation of cell cycle inhibitors such as CDKN1B (p27) and CDKN1A (p21) modifies the risk of developing atherosclerosis. In humans, CDKN1A, CDKN1B and CDKN1C (p57) are differentially expressed in normal versus atherosclerotic vessels. A DNA‑polymorphism within the CDKN1B promoter has been associated with myocardial infarction (MI). In the present study, we analyzed the effect of CDKN1A, CDKN1C and CDKN2A (p16) polymorphisms on MI‑risk. A total of 316 patients (all male, < 55 years) and 434 controls were genotyped, and the allele and genotype frequencies were compared between the two groups. Two CDKN1C polymorphisms, a promoter GT‑repeat and a variable number of repeats of the amino acid PAPA‑motif, were associated with MI. The presence of two alleles ≤ 11‑repeats (9/11, 10/11 and 11/11 genotypes) was significantly less frequent among patients (p < 0.001). This difference was also significant when analyzing the subpopulation of smokers (p = 0.004), suggesting a protective role for these low‑repeat genotypes (OR = 0.49, 95%CI = 0.32–0.73). The PAPA‑BB homozygotes were significantly less frequent in patients, but this could be attributed to a linkage disequilibrium between the 11‑repeats and B alleles. No significantly different frequencies between patients and controls for the four CDKN1A (‑1026A/G, ‑754G/C, ‑369G/C and Ser31Arg) and the three CDKN2A (‑523 G/A, +22 G/A and Ala148Thr) polymorphisms was found. In conclusion, we provide here genetic evidence for the association between DNA‑variants in the CDKN1C/p57 gene and the risk of atherosclerosis and MI. ; This work was supported by grants from the Spanish Fondo de Investigaciones Sanitarias-Fondos FEDER European Union (FIS-03/005), and Red de Investigación Renal-REDINREN (RD06/0016). I.R. is the recipient of a Ramón y Cajal Fellow. ; Peer reviewed

This is an, un-copyedited, author manuscript that has been accepted for publication in the Frontiers in Bioscience ; 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 (~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 (apoE-/-Irs2-/- mice) in the absence of hyperglycaemia, 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, prediabetic patients with clinical manifestations of hyperinsulinemia and insulin resistance. ; This work was supported by grants from the Spanish Ministry of Education and Science and the European Regional Development Fund (SAF2004-03057), from Instituto de Salud Carlos III (Red de Centros RECAVA, C03/01; Red de Grupos G03-212), and from the European Union (Marie Curie fellowship MEIF-CT-2005-024393). M.V.-C is the recipient of a fellowship from the Regional Government of Valencia. ; Peer reviewed

8 pages, PMID: 18802016 [PubMed].-- Artículo presentado a la 2ª Reunión Anual de la Red Temática de Investigación Cooperativa (RECAVA) celebrada en Madrid (España) del 1 al 2 de Noviembre de 2008. ; 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. ; Work supported by grants from the Spanish Ministry of Education and Science and the European Regional Development Fund (SAF2004-03057, SAF2007-62110, SAF2007-62533), Instituto de Salud Carlos III (RECAVA, grants RD06/0014/0008,RD06/0014/0021, CIBERDEM), Generalitat Valenciana (GV/2007/164), Sociedad Española de Cardiología (Beca Novartis 2006), European Union (InGenious HyperCare, grant LSHM-CT-2006-037093), and from the agreement between the Foundation for Applied Medical Research (FIMA) and "UTE project CIMA". H.G.-N. received salary support from the European Union (Marie Curie fellowship MEIF-CT-2005-024393). M.V.-C was a fellow from the Regional Government of Valencia and from Instituto Danone. ; Peer reviewed

This is a pre-copy-editing, author-produced PDF of an article accepted for publication in Cardiovascular Research following peer review. The definitive publisher-authenticated version Cardiovasc Res. 76 (2):340-350 is available online at: http://cardiovascres.oxfordjournals.org/cgi/content/full/76/2/340 ; OBJECTIVE: Mounting evidence suggests that activation of complement, an important constituent of innate immunity, contributes to atherosclerosis. Here we investigated the expression of complement components (CCs) in the setting of experimental and clinical hypercholesterolemia, a major risk factor for atherosclerosis, their effects on vascular smooth muscle cell (VSMC) and macrophage proliferation, and the underlying molecular mechanisms. METHODS: For this study we analyzed the mRNA and protein expression of several CCs in plasma and aorta of hypercholesterolemic atherosclerosis-prone apolipoprotein E-null mice (apoE-KO) and in plasma of normocholesterolemic subjects and familial hypercholesterolemia (FH) patients. We also carried out in vitro molecular studies to assess the role of CCs on the control of macrophage and VSMC proliferation. RESULTS: Fat-fed apoE-KO mice experiencing severe hypercholesterolemia (~400 mg/dL), but not fat-fed wild-type controls with plasma cholesterol level <110 mg/dL, displayed in aortic tissue upregulation of several CC mRNAs, including C3, C4, C1s, and C1q. In apoE-KO mice, induction of C3 mRNA was already apparent two days after fat feeding when hypercholesterolemia was manifest yet atherosclerotic lesions were absent or incipient. Rapid C3 and C4 protein upregulation was also observed in the plasma of fat-fed apoE-KO mice, and FH patients exhibited higher plasmatic C3a, C4 γ chain, C1s and C3c α chain protein levels than normocholesterolemic subjects. In vitro, C3 and C3a, but not C3a-desArg, C4 and C1q, promoted macrophage and VSMC proliferation through Gi protein-dependent activation of extracellular signalregulated kinase 1/2 (ERK1/2). We also found that C3-enriched FH plasma evoked a stronger mitogenic response in macrophages than normocholesterolemic plasma, and treatment with anti-C3 antibodies eliminated this difference. CONCLUSIONS: Both experimental and clinical hypercholesterolemia coincides with a concerted activation of several CCs. However, only C3 and C3a elicited a mitogenic response in cultured VSMCs and macrophages through Gi protein-dependent ERK1/2 activation. Thus, excess of C3/C3a in hypercholesterolemic apoE-KO mice and FH patients may contribute to atheroma growth by promoting neointimal cell proliferation. ; Work supported by Laboratorios INDAS, Instituto de Salud Carlos III - Ministerio de Sanidad y Consumo of Spain (Red Temática de Investigación Cooperativa en Enfermedades Cardiovasculares -RECAVA, and predoctoral fellowship to F.V.), Ministerio de Educación y Ciencia of Spain and European Regional Development Fund (grants SAF2004-03057 and BFU2004-01432, predoctoral FPI fellowship to D.P.), European Commission (Marie Curie postdoctoral fellowship to M.K.), Fundación Carolina (postdoctoral fellowship to C.C.), and Regional Government of Valencia (predoctoral fellowship to M.V.-C.). ; Peer reviewed