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Aging is the main risk factor for vascular disease and ensuing cardiovascular and cerebrovascular events, the leading causes of death worldwide. In a progressively aging population, it is essential to develop early-life biomarkers that efficiently identify individuals who are at high risk of developing accelerated vascular damage, with the ultimate goal of improving primary prevention and reducing the health care and socioeconomic impact of age-related cardiovascular disease. Studies in experimental models and humans have identified 9 highly interconnected hallmark processes driving mammalian aging. However, strategies to extend health span and life span require understanding of interindividual differences in age-dependent functional decline, known as biological aging. This review summarizes the current knowledge on biological age biomarkers, factors influencing biological aging, and antiaging interventions, with a focus on vascular aspects of the aging process and its cardiovascular disease related manifestations.

Aging is associated with alterations in the arterial wall that promote vascular disease development and its clinical manifestations, including myocardial infarction, stroke, and arterial dissection. The arterial wall is comprised of three layers, intima, media and adventitia, each with distinct cellular composition and function, which can therefore contribute differently to vascular disease initiation and progression. Hence, studying transcriptomic alterations, either in the entire arterial wall or separately in the three arterial layers, can aid in disentangling the etiopathology of vascular disease and thus pave the way for innovative treatments. This chapter describes protocols for total RNA extraction from complete mouse aorta and separately from intima, media, and adventitia layers for subsequent transcriptomic analysis.

Supplemental Digital Content is available in the text., Background: Hutchinson-Gilford progeria syndrome (HGPS) is a rare disorder characterized by premature aging and death mainly because of myocardial infarction, stroke, or heart failure. The disease is provoked by progerin, a variant of lamin A expressed in most differentiated cells. Patients look healthy at birth, and symptoms typically emerge in the first or second year of life. Assessing the reversibility of progerin-induced damage and the relative contribution of specific cell types is critical to determining the potential benefits of late treatment and to developing new therapies. Methods: We used CRISPR-Cas9 technology to generate LmnaHGPSrev/HGPSrev (HGPSrev) mice engineered to ubiquitously express progerin while lacking lamin A and allowing progerin suppression and lamin A restoration in a time- and cell type–specific manner on Cre recombinase activation. We characterized the phenotype of HGPSrev mice and crossed them with Cre transgenic lines to assess the effects of suppressing progerin and restoring lamin A ubiquitously at different disease stages as well as specifically in vascular smooth muscle cells and cardiomyocytes. Results: Like patients with HGPS, HGPSrev mice appear healthy at birth and progressively develop HGPS symptoms, including failure to thrive, lipodystrophy, vascular smooth muscle cell loss, vascular fibrosis, electrocardiographic anomalies, and precocious death (median lifespan of 15 months versus 26 months in wild-type controls, P

Genomic instability, the unresolved accumulation of DNA variants, is hypothesized as one of the contributors to the natural aging process. We assessed the frequency of unresolved DNA damage reaching the transcriptome of the murine myocardium during the course of natural aging and in hearts from four distinct mouse models of premature aging with established aging-related cardiac dysfunctions. RNA sequencing and variant calling based on total RNA sequencing was compared between hearts from naturally aging mice, mice with cardiomyocyte-specific deficiency of Ercc1, a component of the DNA repair machinery, mice with reduced mitochondrial antioxidant capacity, Tert-deficient mice with reduced telomere length, and a mouse model of human Hutchinson-Gilford progeria syndrome (HGPS). Our results demonstrate that no enrichment in variants is evident in the naturally aging murine hearts until 2 y of age from the HGPS mouse model or mice with reduced telomere lengths. In contrast, a dramatic accumulation of variants was evident in Ercc1 cardiomyocyte-specific knockout mice with deficient DNA repair machinery, in mice with reduced mitochondrial antioxidant capacity, and in the intestine, liver, and lung of naturally aging mice. Our data demonstrate that genomic instability does not evidently contribute to naturally aging of the mouse heart in contrast to other organs and support the contention that the endogenous DNA repair machinery is remarkably active to maintain genomic integrity in cardiac cells throughout life. F.D.M. is supported by HS-BAFTA and Kootstra fellowships of Maastricht University and a CVON-ARENA-PRIME fellowship. L.M. is supported by the fund Innovative Medical Research of the University of Münster Medical School (RÜ121510). M.R.H. is supported by a Juan de la Cierva contract from the Spanish Ministerio de Ciencia, Innovación y Universidades (IJC2019-040798-I). R.M.N. is the beneficiary of a predoctoral contract from the Spanish Ministerio de Educación, Cultura y Deporte (FPU16/ 05027). V.A. is supported by the Spanish Ministerio de Ciencia e Innovación (PID2019-108489RB-I00) and the Instituto de Salud Carlos III (ISCIII) (AC16/ 00091) as member of the ERA-CVD JCT2016 EXPERT Network (European Union’s Horizon 2020 Framework Programme), with cofunding from the European Regional Development Fund (“Una manera de hacer Europa”). The Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC) is supported by the Ministry for Research, Science and Innovation (MICIN), the ISCIII, the Pro-CNIC Foundation, and is a Severo Ochoa Center of Excellence. C.B. was supported by the Deutsche Forschungsgemeinschaft (DFG) (BA5631/ 2-1). T.T. was supported by the European Research Council (ERC) Consolidator Grant LONGHEART, by ERA-CVD JCT2016 EXPERT, and the DFG (TH903/ 22-1). D.J.D., M.S., and L.J.D.W. acknowledge support from the Dutch CardioVascular Initiative: the Netherlands Heart Foundation, Dutch Federation of University Medical Centers, ZonMW, and the Royal Netherlands Academy of Sciences (CVON-ARENA-PRIME, CVON-RACE-V, CVON-PREDICT-2). B.S. acknowledges funding by the Netherlands Heart Foundation (Dr. Dekker 2014T105 and CVON-SHE-PREDICTS-HF) and a VIDI Award 917.14.363 from the Dutch Research Council (NWO). A.S.A. was funded by Association Française contres les Myopathies (AFM 18802). F.D.M., T.T., and L.J.D.W. are supported by ERA-CVD JCT2016 EXPERT. M.S. is funded by the DFG (RTG2220, Project 281125614) and Marie Skłodowska-Curie Grant Agreement 81371. L.J.D.W. was further supported by ERC Consolidator Grant 311549 CALMIRS, a VICI Award 918-156-47 from NWO and Marie Skłodowska-Curie Grant Agreements 813716 and 765274. Sí

Lamin A, a product of the LMNA gene, is an essential nuclear envelope component in most differentiated cells. Mutations in LMNA have been linked to premature aging disorders, including Hutchinson-Gilford progeria syndrome (HGPS). HGPS is caused by progerin, an aberrant form of lamin A that leads to premature death, typically from the complications of atherosclerotic disease. A key characteristic of HGPS is a severe loss of vascular smooth muscle cells (VSMCs) in the arteries. Various mouse models of HGPS have been created, but few of them feature VSMC depletion and none develops atherosclerosis, the death-causing symptom of the disease in humans. We recently generated a mouse model that recapitulates most features of HGPS, including VSMC loss and accelerated atherosclerosis. Furthermore, by generating cell-type-specific HGPS mouse models, we have demonstrated a central role of VSMC loss in progerin-induced atherosclerosis and premature death. M.R.H. is supported by a ‘Juan de la Cierva’ postdoctoral fellowship (FJCI-2017-33299) from the Spanish Ministerio de Ciencia, Innovación yUniversidades (MCIU). Work in V.A.’s laboratory is supported by grants from the Spanish Instituto de Salud Carlos III (AC16/00091 and AC17/00067) and MCIU (SAF2016-79490-R), with co-funding from the Fondo Europeo de Desarrollo Regional (FEDER, ‘Una manera de hacer Europa’), the Progeria Research Foundation (Established Investigator Award 2014-52), and the Fundació Marató TV3 (122/C/2015). The CNIC is supported by theMCIU and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505); Instituto de Salud Carlos III [AC16/00091 and AC17/00067]; MCIU [SAF2016-79490-R]; MCIU [FJCI2017-33299]; MCIU [SEV-2015-0505]; Progeria Research Foundation [2014-52]. Sí

[EN] Ageing is the main risk factor for cardiovascular disease (CVD). The increased prevalence of CVD is partly due to the global increase in life expectancy. In this context, it is essential to identify the mechanisms by which ageing induces CVD, with the ultimate aim of reducing its incidence. Both atherosclerosis and heart failure significantly contribute to age-associated CVD morbidity and mortality. Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder caused by the synthesis of progerin, which is noted for accelerated ageing and CVD. This mutant form of prelamin A induces generalised atherosclerosis, vascular calcification, and cardiac electrophysiological abnormalities, leading to premature ageing and death, mainly due to myocardial infarction and stroke. This review discusses the main vascular structural and functional abnormalities during physiological and premature ageing, as well as the mechanisms involved in the exacerbated CVD and accelerated ageing induced by the accumulation of progerin and prelamin A. Both proteins are expressed in non-HGPS individuals, and physiological ageing shares many features of progeria. Research into HGPS could therefore shed light on novel mechanisms involved in the physiological ageing of the cardiovascular system., [ES] El envejecimiento es el principal factor de riesgo de enfermedad cardiovascular (ECV) y su prevalencia está aumentando progresivamente debido en gran parte al incremento de la esperanza de vida a nivel mundial. En este contexto, es fundamental establecer cuáles son los mecanismos por los que el envejecimiento promueve el desarrollo de ECV, con el objetivo de reducir su incidencia. La aterosclerosis y la insuficiencia cardiaca contribuyen de manera significativa a la morbimortalidad por ECV asociada a la edad. El síndrome de progeria de Hutchinson-Gilford (HGPS) se caracteriza por un envejecimiento prematuro que cursa también con ECV acelerada. Se trata de un trastorno genético raro causado por la expresión de progerina, una forma mutada de la prelamina A. La progerina induce aterosclerosis masiva y alteraciones electrofisiológicas en el corazón, promueve el envejecimiento y finalmente la muerte prematura a una edad media de 14,6 anos, ˜ principalmente por infarto de miocardio o ictus cerebral. En esta revisión se discuten las principales alteraciones estructurales y funcionales que afectan al sistema vascular durante el envejecimiento fisiológico y prematuro, así como los mecanismos que subyacen a la aterosclerosis y al envejecimiento exagerados inducidos por la prelamina A y la progerina. Dado que ambas proteínas se expresan en individuos sin HGPS y muchas de las características del envejecimiento normal se presentan en la progeria, la investigación en el ámbito del HGPS podría contribuir a la identificación de nuevos mecanismos implicados en el envejecimiento cardiovascular fisiológico., The laboratory work carried out by V.A. was funded by the Ministry of Economy, Industry and Competition (MEIC; SAF2016-79490-R) and the Instituto de Salud Carlos III (ISCIII, Spanish public health research institute) (RD12/0042/0028 and AC16/00091), with co-financing from the European Regional Development Fund (ERDF), TV3’s Fundació Marató (122/C/2015) and the Progeria research Foundation (Established Investigator Award 2014-52). The Biomedical Research Network Centres (CIBER) for Cardiovascular Diseases is an ISCIII initiative. L.C. received assistance from the ISCIII Cardiovascular Research Network’s Jordi Soler postdocotral grants programme, and M.R.H. received predoctoral assistance from the MEIC’s FPI (research staff training) programme (BES2011-043938). The CNIC (Spanish national cardiovascular research centre) receives support from the MEIC and the Fundación Pro-CNIC and has been accredited as a ‘‘Severo Ochoa’’ Centre of Excellence (MEIC SEV-2015-0505).

Hutchinson&ndash, Gilford progeria syndrome (HGPS) is among the most devastating of the laminopathies, rare genetic diseases caused by mutations in genes encoding nuclear lamina proteins. HGPS patients age prematurely and die in adolescence, typically of atherosclerosis-associated complications. The mechanisms of HGPS-related atherosclerosis are not fully understood due to the scarcity of patient-derived samples and the availability of only one atheroprone mouse model of the disease. Here, we generated a new atherosusceptible model of HGPS by crossing progeroid LmnaG609G/G609G mice, which carry a disease-causing mutation in the Lmna gene, with Ldlr&minus, /&minus, mice, a commonly used preclinical atherosclerosis model. Ldlr&minus, LmnaG609G/G609G mice aged prematurely and had reduced body weight and survival. Compared with control mice, Ldlr&minus, LmnaG609G/G609G mouse aortas showed a higher atherosclerosis burden and structural abnormalities typical of HGPS patients, including vascular smooth muscle cell depletion in the media, adventitial thickening, and elastin structure alterations. Atheromas of Ldlr&minus, LmnaG609G/G609G mice had features of unstable plaques, including the presence of erythrocytes and iron deposits and reduced smooth muscle cell and collagen content. Ldlr&minus, LmnaG609G/G609G mice faithfully recapitulate vascular features found in patients and thus provide a new tool for studying the mechanisms of HGPS-related atherosclerosis and for testing therapies.

Aging is the main risk factor for vascular disease and ensuing cardiovascular and cerebrovascular events, the leading causes of death worldwide. In a progressively aging population, it is essential to develop early-life biomarkers that efficiently identify individuals who are at high risk of developing accelerated vascular damage, with the ultimate goal of improving primary prevention and reducing the health care and socioeconomic impact of age-related cardiovascular disease. Studies in experimental models and humans have identified 9 highly interconnected hallmark processes driving mammalian aging. However, strategies to extend health span and life span require understanding of interindividual differences in age-dependent functional decline, known as biological aging. This review summarizes the current knowledge on biological age biomarkers, factors influencing biological aging, and antiaging interventions, with a focus on vascular aspects of the aging process and its cardiovascular disease related manifestations.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder caused by progerin, a mutant lamin A variant. HGPS patients display accelerated aging and die prematurely, typically from atherosclerosis complications. Recently, we demonstrated that progerin-driven vascular smooth muscle cell (VSMC) loss accelerates atherosclerosis leading to premature death in apolipoprotein E-deficient mice. However, the molecular mechanism underlying this process remains unknown. Using a transcriptomic approach, we identify here endoplasmic reticulum stress (ER) and the unfolded protein responses as drivers of VSMC death in two mouse models of HGPS exhibiting ubiquitous and VSMC-specific progerin expression. This stress pathway was also activated in HGPS patient-derived cells. Targeting ER stress response with a chemical chaperone delayed medial VSMC loss and inhibited atherosclerosis in both progeria models, and extended lifespan in the VSMC-specific model. Our results identify a mechanism underlying cardiovascular disease in HGPS that could be targeted in patients. Moreover, these findings may help to understand other vascular diseases associated with VSMC death, and provide insight into aging-dependent vascular damage related to accumulation of unprocessed toxic forms of lamin A. Work in VA’s laboratory is supported by grants from the Spanish Instituto de Salud Carlos III (RD12/0042/0028, AC17/00067 and AC16/00091) and Ministerio de Ciencia, Innovación y Universidades (MCIU) (SAF2016-79490-R), with cofunding from the Fondo Europeo de Desarrollo Regional (FEDER, “Una manera de hacer Europa”), the Progeria Research Foundation (Established Investigator Award 2014-52), and the Fundació Marató TV3 (122/C/2015). The CNIC is supported by the MCIU and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505). Work in the Lopez-Otin laboratory is supported by grants from the Ministerio de Economía y Competitividad (MINECO/FEDER), the European Research Council, and the Progeria Research Foundation. The Instituto Universitario de Oncología is supported by Obra Social Cajastur. Work in the Misteli laboratory was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, and Center for Cancer Research. The MCIU supported RVB (“Juan de la Cierva” JCI-2011-09663 and SAF-2014-60699-JIN postdoctoral contracts) and MRH (FPI predoctoral contract BES-2011-043938 and “Juan de la Cierva” FJCI-2017-33299 postdoctoral contract). RMN is supported by the Ministerio de Educación, Cultura y Deporte (FPU predoctoral contract FPU16/05027). VQ is supported by grants from the Principado de Asturias and MINECO, including FEDER funding. SV was supported by an Erwin Schroedinger Fellowship from the Austrian Science Fund (J3849-B28). Sí

Objective- Hydroxyapatite deposition on the medial layer of the aortic walls is the hallmark of vascular calcification and the most common complication in aging individuals and in patients with diabetes mellitus and those undergoing hemodialysis. Extracellular pyrophosphate is a potent physicochemical inhibitor of hydroxyapatite crystal formation. This study analyzed changes in extracellular pyrophosphate metabolism during the phosphate-induced calcification process. Approach and Results- Phosphate-induced calcification of ex vivo-cultured aortic rings resulted in calcium accumulation after 7 days. This accumulation was enhanced when aortic walls were devitalized. BMP2 (bone morphogenic protein 2) expression was associated with calcium accumulation in cultured aortic rings, as well as in cultured vascular smooth muscle cells (VSMCs) and in calcitriol-induced calcification in rats. Hydroxyapatite dose dependently induced BMP2 overexpression in VSMCs. Moreover, TNAP (tissue nonspecific alkaline phosphatase) mRNA levels and activity were found to be downregulated in early phases and upregulated in later phases of calcification in all 3 models studied. eNPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1) increased from early to later phases of calcification, whereas eNTPD1 (ectonucleoside triphosphate diphosphohydrolase 1) was downregulated during later phases. Synthesis of pyrophosphate in VSMCs increased significantly over time, in all 3 models studied. Because the rate of pyrophosphate hydrolysis was 10× slower than the rate of pyrophosphate synthesis, pyrophosphate synthesis is determined mainly by the ratio of eNPP1 to eNTPD1 activity. Hydroxyapatite also induces increments both in TNAP and eNPP1/eNTPD1 ratio in VSMCs. Conclusions- Pyrophosphate synthesis increases in VSMCs during phosphate-induced calcification because of compensatory regulation of extracellular pyrophosphate metabolism.

Progerin, an aberrant protein that accumulates with age, causes the rare genetic disease Hutchinson-Gilford progeria syndrome (HGPS). Patients who have HGPS exhibit ubiquitous progerin expression, accelerated aging and atherosclerosis, and die in their early teens, mainly of myocardial infarction or stroke. The mechanisms underlying progerin-induced atherosclerosis remain unexplored, in part, because of the lack of appropriate animal models.We generated an atherosclerosis-prone model of HGPS by crossing apolipoprotein E-deficient (We have generated the first mouse model of progerin-induced atherosclerosis acceleration, and demonstrate that restricting progerin expression to VSMCs is sufficient to accelerate atherosclerosis, trigger plaque vulnerability, and reduce lifespan. Our results identify progerin-induced VSMC death as a major factor triggering atherosclerosis and premature death in HGPS.

Background: Progerin, an aberrant protein that accumulates with age, causes the rare genetic disease Hutchinson-Gilford progeria syndrome (HGPS). Patients who have HGPS exhibit ubiquitous progerin expression, accelerated aging and atherosclerosis, and die in their early teens, mainly of myocardial infarction or stroke. The mechanisms underlying progerin-induced atherosclerosis remain unexplored, in part, because of the lack of appropriate animal models. Methods: We generated an atherosclerosis-prone model of HGPS by crossing apolipoprotein E–deficient ( Apoe –/– ) mice with Lmna G609G/G609G mice ubiquitously expressing progerin. To induce progerin expression specifically in macrophages or vascular smooth muscle cells (VSMCs), we crossed Apoe –/– Lmna LCS/LCS mice with LysMCre and SM22αCre mice, respectively. Progerin expression was evaluated by polymerase chain reaction and immunofluorescence. Cardiovascular alterations were determined by immunofluorescence and histology in male mice fed normal chow or a high-fat diet. In vivo low-density lipoprotein retention was assessed by intravenous injection of fluorescently labeled human low-density lipoprotein. Cardiac electric defects were evaluated by electrocardiography. Results: Apoe –/– Lmna G609G/G609G mice with ubiquitous progerin expression exhibited a premature aging phenotype that included failure to thrive and shortened survival. In addition, high-fat diet–fed Apoe –/– Lmna G609G/G609G mice developed a severe vascular pathology, including medial VSMC loss and lipid retention, adventitial fibrosis, and accelerated atherosclerosis, thus resembling most aspects of cardiovascular disease observed in patients with HGPS. The same vascular alterations were also observed in Apoe –/– Lmna LCS/LCS SM22αCre mice expressing progerin specifically in VSMCs, but not in Apoe –/– Lmna LCS/LCS LysMCre mice with macrophage-specific progerin expression. Moreover, Apoe –/– Lmna LCS/LCS SM22αCre mice had a shortened lifespan despite the lack of any overt aging phenotype. Aortas of ubiquitously and VSMC-specific progerin-expressing mice exhibited increased retention of fluorescently labeled human low-density lipoprotein, and atheromata in both models showed vulnerable plaque features. Immunohistopathological examination indicated that Apoe –/– Lmna LCS/LCS SM22αCre mice, unlike Apoe –/– Lmna G609G/G609G mice, die of atherosclerosis-related causes. Conclusions: We have generated the first mouse model of progerin-induced atherosclerosis acceleration, and demonstrate that restricting progerin expression to VSMCs is sufficient to accelerate atherosclerosis, trigger plaque vulnerability, and reduce lifespan. Our results identify progerin-induced VSMC death as a major factor triggering atherosclerosis and premature death in HGPS.

Aging, the main risk factor for cardiovascular disease (CVD), is becoming progressively more prevalent in our societies. A better understanding of how aging promotes CVD is therefore urgently needed to develop new strategies to reduce disease burden. Atherosclerosis and heart failure contribute significantly to age-associated CVD-related morbimortality. CVD and aging are both accelerated in patients suffering from Hutchinson-Gilford progeria syndrome (HGPS), a rare genetic disorder caused by the prelamin A mutant progerin. Progerin causes extensive atherosclerosis and cardiac electrophysiological alterations that invariably lead to premature aging and death. This review summarizes the main structural and functional alterations to the cardiovascular system during physiological and premature aging and discusses the mechanisms underlying exaggerated CVD and aging induced by prelamin A and progerin. Because both proteins are expressed in normally aging non-HGPS individuals, and most hallmarks of normal aging occur in progeria, research on HGPS can identify mechanisms underlying physiological aging. Progeria Research Foundation (Established Investigator Award 2014-52). L.d.C. is the recipient of a Jordi Soler postdoctoral fellowship from the Red de Investigacion Cardiovascular at ISCIII. The CNIC is supported by the MEIC and the Pro-CNIC Foundation and is a Severo Ochoa Center of Excellence (MEIC award SEV-2015-0505).

Purpose Phosphorus is an essential nutrient involved in many pathobiological processes. Less than 1\% of phosphorus is found in extracellular fluids as inorganic phosphate ion (Pi) in solution. High serum Pi level promotes ectopic calcification in many tissues, including blood vessels. Here, we studied the effect of elevated Pi concentration on macrophage polarization and calcification. Macrophages, present in virtually all tissues, play key roles in health and disease and display remarkable plasticity, being able to change their physiology in response to environmental cues. Methods and results High-throughput transcriptomic analysis and functional studies demonstrated that Pi induces unpolarized macrophages to adopt a phenotype closely resembling that of alternatively -activated M2 macrophages, as revealed by arginine hydrolysis and energetic and antioxidant profiles. Pi-induced macrophages showed an anti-calcifying action mediated by increased availability of extracellular ATP and pyrophosphate. Conclusion We conclude that the ability of Pi-activated macrophages to prevent calcium-phosphate deposition is a compensatory mechanism protecting tissues from hyperphosphatemiainduced pathologic calcification. The Spanish Ministerio de Economia y Competitividad (MINECO) supports R.V.-B. (Juan de la Cierva JCI-2011-09663 and SAF-201460699-JIN postdoctoral contracts) and M.R.H. (FPI predoctoral contract BES-2011-043938). This study was supported by research grants to V.A. from MINECO (SAF2013-4663-R) and the Institute de Salud Carlos III (RD12/0042/0028) with co-funding from the Fondo Europeo de Desarrollo Regional (FEDER). The CNIC is supported by the MINECO and the Pro-CNIC Foundation, and is a Severo Ochoa Center of Excellence (MINECO award SEV-2015-0505). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Sí

Calcium-phosphate deposition (CPD) in atherosclerotic lesions, which begins in middle age and increases with aging, is a major independent predictor of future cardiovascular disease morbi-mortality. Remodeling of atherosclerotic vessels during aging is regulated in part by intimal macrophages, which can polarize to phenotypically distinct populations with distinct functions. This study tested the hypothesis that classically activated macrophages (M1φs) and alternatively activated macrophages (M2φs) differently affect vascular smooth muscle cell (VSMC) calcification and investigated the underlying mechanisms. We analyzed mouse VSMC-macrophage cocultures using a transwell system. Coculture of VSMCs with M2φs significantly reduced CPD, but coculture with M1φs had no effect. The anticalcific effect of M2φs was associated with elevated amounts of extracellular ATP and pyrophosphate (PPi), two potent inhibitors of CPD, and was lost upon forced hydrolysis of these metabolites. In M2φs and VSMC-M2φs cocultures, analysis of the ectoenzymes that regulate extracellular ATP/PPi metabolism revealed increased mRNA expression and activity of ectoenzyme nucleotide pyrophosphatase/phosphodiesterase-1, which synthesizes PPi from ATP, without changes in tissue-nonspecific alkaline phosphatase, which hydrolyzes PPi. In conclusion, increased accumulation of extracellular ATP and PPi by alternatively activated mouse M2φs inhibits CPD. These results reveal novel mechanisms underlying macrophage-dependent control of intimal calcification.

Elevated serum phosphorus is a major risk factor for vascular calcification, which is characterized by the presence of calcium phosphate deposits, mainly hydroxyapatite crystals. In vitro studies of phosphate-induced calcification show that vascular smooth muscle cells undergo calcification with features similar to those observed in pathological vascular calcification in vivo, including the presence of hydroxyapatite crystals. Here, we describe the double-collagenase digestion method for isolating vascular smooth muscle cells from aorta, and a method for inducing calcification in vitro using high phosphate concentration.

The key roles of macrophages in atherosclerosis include the phagocytosis of apoptotic and necrotic cells and cell debris, whose accumulation in atherosclerotic lesions exacerbates inflammation and promotes plaque vulnerability. Evidence is accumulating that macrophage phagocytic functions peak at the early stages of atherosclerosis and that the reduced phagocytosis at the late stages of disease leads to the generation of necrotic cores and a defective resolution of inflammation, which in turn promotes plaque rupture, thrombus formation, and life-threatening acute ischemic events (myocardial infarction and stroke). The impaired resolution of inflammation in advanced lesions featuring loss of macrophage phagocytic activity may be in part due to an imbalance between M1 and M2 subsets of polarized macrophages. A better understanding of the mechanisms that regulate macrophage phagocytic activity in the context of atherosclerosis may therefore help identify novel therapeutic targets. This chapter presents a protocol for establishing primary mouse macrophage cultures, a method for polarizing macrophages to the M1 and M2 states, and a method for the in vitro study of macrophage phagocytosis of IgG-opsonized or IgM/complement component 3-opsonized erythrocytes.

The key roles of macrophages in atherosclerosis include the phagocytosis of apoptotic and necrotic cells and cell debris, whose accumulation in atherosclerotic lesions exacerbates inflammation and promotes plaque vulnerability. Evidence is accumulating that macrophage phagocytic functions peak at the early stages of atherosclerosis and that the reduced phagocytosis at the late stages of disease leads to the generation of necrotic cores and a defective resolution of inflammation, which in turn promotes plaque rupture, thrombus formation, and life-threatening acute ischemic events (myocardial infarction and stroke). The impaired resolution of inflammation in advanced lesions featuring loss of macrophage phagocytic activity may be in part due to an imbalance between M1 and M2 subsets of polarized macrophages. A better understanding of the mechanisms that regulate macrophage phagocytic activity in the context of atherosclerosis may therefore help identify novel therapeutic targets. This chapter presents a protocol for establishing primary mouse macrophage cultures, a method for polarizing macrophages to the M1 and M2 states, and a method for the in vitro study of macrophage phagocytosis of IgG-opsonized or IgM/complement component 3-opsonized erythrocytes. M.R.H. is supported by an FPI predoctoral fellowship from the Spanish Government (BES-2011-043938) and R.V-B. by a Juan de la Cierva postdoctoral contract from the Spanish Government (JCI-2011- 09663). Work in V.A.’s laboratory is supported by grants from the Spanish Ministry of Economy and Competitivity (MINECO) (SAF201346663-R), Fondo Europeo de Desarrollo Regional (FEDER), Instituto de Salud Carlos III (RD12/0042/0028), the Progeria Research Foundation (Innovator Award 2012, Established Investigator Award 2014), and the European Union (Liphos, Grant Agreement 317916). The Centro Nacional de Investigaciones Cardiovasculares (CNIC) is supported by the MINECO and the Pro-CNIC Foundation. Sí

Elevated serum phosphorus is a major risk factor for vascular calcification, which is characterized by the presence of calcium phosphate deposits, mainly hydroxyapatite crystals. In vitro studies of phosphate-induced calcification show that vascular smooth muscle cells undergo calcification with features similar to those observed in pathological vascular calcification in vivo, including the presence of hydroxyapatite crystals. Here, we describe the double-collagenase digestion method for isolating vascular smooth muscle cells from aorta, and a method for inducing calcification in vitro using high phosphate concentration.