Alessandra Stacchiotti - Academia.edu (original) (raw)

Papers by Alessandra Stacchiotti

Antioxidants

A healthy gut provides the perfect habitat for trillions of bacteria, called the intestinal micro... more A healthy gut provides the perfect habitat for trillions of bacteria, called the intestinal microbiota, which is greatly responsive to the long-term diet; it exists in a symbiotic relationship with the host and provides circulating metabolites, hormones, and cytokines necessary for human metabolism. The gut–heart axis is a novel emerging concept based on the accumulating evidence that a perturbed gut microbiota, called dysbiosis, plays a role as a risk factor in the pathogenesis of cardiovascular disease. Consequently, recovery of the gut microbiota composition and function could represent a potential new avenue for improving patient outcomes. Despite their low absorption, preclinical evidence indicates that polyphenols and their metabolites are transformed by intestinal bacteria and halt detrimental microbes’ colonization in the host. Moreover, their metabolites are potentially effective in human health due to antioxidant, anti-inflammatory, and anti-cancer effects. The aim of this...

Cells, 2019

Since the pioneering discovery of heat shock proteins in Drosophila by Ferruccio Ritossa in 1960s... more Since the pioneering discovery of heat shock proteins in Drosophila by Ferruccio Ritossa in 1960s, a long and exciting journey has been undertaken by molecular biologists and researchers worldwide. Not only lower organisms like worms, yeast, amoeba, and flies but also eukaryotes share common cellular response signals to stressful conditions that can arise from the outside but also from the inside. Moreover, extraordinary interplay between nucleus and subcellular organelles, and between different organelles, like mitochondria and the endoplasmic reticulum called mitochondria-associated endoplasmic reticulum membranes (MAMs), are involved in aging and human diseases like obesity, diabetes, inflammation, neurodegeneration, autoimmune diseases, atherosclerosis, and cancer. Actually, we know that to hit abnormal proteostasis and lipid exchanges in the endoplasmic reticulum is crucial to best guide effective therapies or discover new drugs. Indeed, restoration or impairment of endoplasmic...

Cancer Letters, 1994

ABSTRACT

Histology and histopathology, 2008

Aluminium (Al) is a neurotoxic metal and Al exposure may be a factor in the aetiology of various ... more Aluminium (Al) is a neurotoxic metal and Al exposure may be a factor in the aetiology of various neurodegenerative diseases such as Alzheimer's disease (AD). The major pathohistological findings in the AD brain are the presence of neuritic plaques containing beta-amyloid (Abeta) which may interfere with neuronal communication. Moreover, it has been observed that GRP78, a stress-response protein induced by conditions that adversely affect endoplasmic reticulum (ER) function, is reduced in the brain of AD patients. In this study, we investigated the correlation between the expression of Abeta and GRP78 in the brain cortex of mice chronically treated with aluminium sulphate. Chronic exposure over 12 months to aluminium sulphate in drinking water resulted in deposition of Abeta similar to that seen in congophilic amyloid angiopathy (CAA) in humans and a reduction in neuronal expression of GRP78 similar to what has previously been observed in Alzheimer's disease. So, we hypothesi...

Histology and histopathology, 2006

Aluminium (Al) is the third most widespread metal in the environment. It is toxic for the brain, ... more Aluminium (Al) is the third most widespread metal in the environment. It is toxic for the brain, bone and haematological system but unfortunately very little data exist for other organs. Stress proteins are induced or enhanced against metal toxicity with an essential role in the recovery of organules and other cellular proteins. This immunohistochemical study was performed to analyze the distribution of three stress proteins (HSP25, HSP72, GRP75) in rat kidney and liver orally exposed to Al sulphate daily for 3 and 6 months. Al-induced alterations were further studied by histopathology (H-E, PAS, Perl's, Masson) and ultrastructural morphometry. In the kidney: HSP25 was enhanced in proximal tubules after 6 months Al-exposure when abnormal brush borders were observed; HSP72 was induced in proximal tubules only after long Al-treatment; GRP75 was raised in midcortical area sometimes within nuclei. Furthermore, lysosomal and lipofuscins densities increased in the juxtamedullary tubul...

Frontiers in Cell and Developmental Biology, 2020

Prolonging the healthy life span and limiting neurological illness are imperative goals in geront... more Prolonging the healthy life span and limiting neurological illness are imperative goals in gerontology. Age-related neurodegeneration is progressive and leads to severe diseases affecting motility, memory, cognitive function, and social life. To date, no effective treatments are available for neurodegeneration and irreversible neuronal loss. Bioactive phytochemicals could represent a natural alternative to ensure active aging and slow onset of neurodegenerative diseases in elderly patients. Autophagy or macroautophagy is an evolutionarily conserved clearing process that is needed to remove aggregateprone proteins and organelles in neurons and glia. It also is crucial in synaptic plasticity. Aberrant autophagy has a key role in aging and neurodegeneration. Recent evidence indicates that polyphenols like resveratrol and curcumin, flavonoids, like quercetin, polyamine, like spermidine and sugars, like trehalose, limit brain damage in vitro and in vivo. Their common mechanism of action leads to restoration of efficient autophagy by dismantling misfolded proteins and dysfunctional mitochondria. This review focuses on the role of dietary phytochemicals as modulators of autophagy to fight Alzheimer's and Parkinson's diseases, fronto-temporal dementia, amyotrophic lateral sclerosis, and psychiatric disorders. Currently, most studies have involved in vitro or preclinical animal models, and the therapeutic use of phytochemicals in patients remains limited.

International Journal of Molecular Sciences, 2021

Mitophagy is a selective autophagic process, essential for cellular homeostasis, that eliminates ... more Mitophagy is a selective autophagic process, essential for cellular homeostasis, that eliminates dysfunctional mitochondria. Activated by inner membrane depolarization, it plays an important role during development and is fundamental in highly differentiated post-mitotic cells that are highly dependent on aerobic metabolism, such as neurons, muscle cells, and hepatocytes. Both defective and excessive mitophagy have been proposed to contribute to age-related neurodegenerative diseases, such as Parkinson’s and Alzheimer’s diseases, metabolic diseases, vascular complications of diabetes, myocardial injury, muscle dystrophy, and liver disease, among others. Pharmacological or dietary interventions that restore mitophagy homeostasis and facilitate the elimination of irreversibly damaged mitochondria, thus, could serve as potential therapies in several chronic diseases. However, despite extraordinary advances in this field, mainly derived from in vitro and preclinical animal models, human...

Resveratrol - Adding Life to Years, Not Adding Years to Life, 2019

Reduced calorie intake is a religious and medical practice known since very old times, but its di... more Reduced calorie intake is a religious and medical practice known since very old times, but its direct influence on life span in all organisms, included humans, has been demonstrated in the modern era. Not only periodic fasting, but also natural or synthetic compounds that mimic this phenomenon are growing to slow aging and the onset of chronic morbidities. Resveratrol (RSV), a plant polyphenol, is an elixir of longevity for simple organisms and preclinical rodent models even if a beneficial role in humans is still debated. Its main rejuvenating mechanism copes with the activation of specific longevity genes called sirtuins. Among seven known mammalian sirtuins, sirtuin 1 is the most studied. This pleiotropic nicotinamide adenine dinucleotide (NAD)-based deacetylase maintains longevity by removing acetyl group in nuclear histones, transcription factors, and other DNA repairing proteins. Actually, an exciting challenge is to discover and test novel sirtuin 1 activators to extend life span and to treat age-associated disabilities. This chapter updates on the antiaging effect of RSV and sirtuin 1 activators in experimental animals and in humans. Finally, pros and cons on RSV analogues and sirtuin 1 activators tested in preclinical and clinical trials to hamper neurological deficit, cardiovascular complications, diabetes, bone and muscle deterioration, and cancer are discussed.

Cells, 2020

Skeletal muscle disorders are dramatically increasing with human aging with enormous sanitary cos... more Skeletal muscle disorders are dramatically increasing with human aging with enormous sanitary costs and impact on the quality of life. Preventive and therapeutic tools to limit onset and progression of muscle frailty include nutrition and physical training. Melatonin, the indole produced at nighttime in pineal and extra-pineal sites in mammalians, has recognized anti-aging, anti-inflammatory, and anti-oxidant properties. Mitochondria are the favorite target of melatonin, which maintains them efficiently, scavenging free radicals and reducing oxidative damage. Here, we discuss the most recent evidence of dietary melatonin efficacy in age-related skeletal muscle disorders in cellular, preclinical, and clinical studies. Furthermore, we analyze the emerging impact of melatonin on physical activity. Finally, we consider the newest evidence of the gut–muscle axis and the influence of exercise and probably melatonin on the microbiota. In our opinion, this review reinforces the relevance of...

Nutrients, 2017

Cardiomyocytes are particularly sensitive to oxidative damage due to the link between mitochondri... more Cardiomyocytes are particularly sensitive to oxidative damage due to the link between mitochondria and sarcoplasmic reticulum necessary for calcium flux and contraction. Melatonin, important indoleamine secreted by the pineal gland during darkness, also has important cardioprotective properties. We designed the present study to define morphological and ultrastructural changes in cardiomyocytes and mainly in mitochondria of an animal model of obesity (ob/ob mice), when treated orally or not with melatonin at 100 mg/kg/day for 8 weeks (from 5 up to 13 week of life). We observed that ob/ob mice mitochondria in sub-sarcolemmal and inter-myofibrillar compartments are often devoid of cristae with an abnormally large size, which are called mega-mitochondria. Moreover, in ob/ob mice the hypertrophic cardiomyocytes expressed high level of 4hydroxy-2-nonenal (4HNE), a marker of lipid peroxidation but scarce degree of mitofusin2, indicative of mitochondrial sufferance. Melatonin oral supplementation in ob/ob mice restores mitochondrial cristae, enhances mitofusin2 expression and minimizes 4HNE and p62/SQSTM1, an index of aberrant autophagic flux. At pericardial fat level, adipose tissue depot strictly associated with myocardium infarction, melatonin reduces adipocyte hypertrophy and inversely regulates 4HNE and adiponectin expressions. In summary, melatonin might represent a safe dietary adjuvant to hamper cardiac mitochondria remodeling and the hypoxic status that occur in pre-diabetic obese mice at 13 weeks of life.

Cells, 2018

The interplay of mitochondria with the endoplasmic reticulum and their connections, called mitoch... more The interplay of mitochondria with the endoplasmic reticulum and their connections, called mitochondria-ER contacts (MERCs) or mitochondria-associated ER membranes (MAMs), are crucial hubs in cellular stress. These sites are essential for the passage of calcium ions, reactive oxygen species delivery, the sorting of lipids in whole-body metabolism. In this perspective article, we focus on microscopic evidences of the pivotal role of MERCs/MAMs and their changes in metabolic diseases, like obesity, diabetes, and neurodegeneration.

Nutrients, 2018

Taurine (TAU) is a sulfur-containing beta amino acid that is not involved in protein composition ... more Taurine (TAU) is a sulfur-containing beta amino acid that is not involved in protein composition and anabolism, conditionally essential in mammals provided through diet. Growing evidence supports a protective role of TAU supply in osmoregulation, calcium flux, and reduction of inflammation and oxidant damage in renal diseases like diabetes. Endoplasmic reticulum (ER) stress, due to abnormal proteostasis, is a contributor to nephrotic syndrome and related renal damage. Here, we investigated the effect of dietary TAU (1.5% in drinking water for 15 days) in an established rat model that mimics human minimal change nephrosis, consisting of a single puromycin aminonucleoside (PAN) injection (intraperitoneally 15 mg/100 g body weight), with sacrifice after eight days. TAU limited proteinuria and podocytes foot processes effacement, and balanced slit diaphragm nephrin and glomerular claudin 1 expressions. In cortical proximal tubules, TAU improved lysosomal density, ER perimeter, restored proper ER-mitochondria tethering and mitochondrial cristae, and decreased inflammation. Remarkably, TAU downregulated glomerular ER stress markers (GRP78, GRP94), pro-apoptotic C/EBP homologous protein, activated caspase 3, tubular caspase1, and mitochondrial chaperone GRP75, but maintained anti-apoptotic HSP25. In conclusion, TAU, by targeting upstream ER stress separate from mitochondria dysfunctions at crucial renal sites, might be a promising dietary supplement in the treatment of the drug-resistant nephrotic syndrome.

International Journal of Molecular Sciences, 2017

Fibromyalgia is a chronic syndrome characterized by widespread musculoskeletal pain and an extens... more Fibromyalgia is a chronic syndrome characterized by widespread musculoskeletal pain and an extensive array of other symptoms including disordered sleep, fatigue, depression and anxiety. Important factors involved in the pathogenic process of fibromyalgia are inflammation and oxidative stress, suggesting that ant-inflammatory and/or antioxidant supplementation might be effective in the management and modulation of this syndrome. Recent evidence suggests that melatonin may be suitable for this purpose due to its well known ant-inflammatory, antioxidant and analgesic effects. Thus, in the current study, the effects of the oral supplementation of melatonin against fibromyalgia-related skeletal muscle alterations were evaluated. In detail, 90 Sprague Dawley rats were randomly treated with reserpine, to reproduce the pathogenic process of fibromyalgia and thereafter they received melatonin. The animals treated with reserpine showed moderate alterations at hind limb skeletal muscles level and had difficulty in moving, together with significant morphological and ultrastructural alterations and expression of inflammatory and oxidative stress markers in the gastrocnemius muscle. Interestingly, melatonin, dose and/or time dependently, reduced the difficulties in spontaneous motor activity and the musculoskeletal morphostructural, inflammatory, and oxidative stress alterations. This study suggests that melatonin in vivo may be an effective tool in the management of fibromyalgia-related musculoskeletal morphofunctional damage.

Biological Trace Element Research, 2016

Your article is protected by copyright and all rights are held exclusively by Springer Science +B... more Your article is protected by copyright and all rights are held exclusively by Springer Science +Business Media New York. This e-offprint is for personal use only and shall not be selfarchived in electronic repositories. If you wish to self-archive your article, please use the accepted manuscript version for posting on your own website. You may further deposit the accepted manuscript version in any repository, provided it is only made publicly available 12 months after official publication or later and provided acknowledgement is given to the original source of publication and a link is inserted to the published article on Springer's website. The link must be accompanied by the following text: "The final publication is available at link.springer.com".

Italian journal of anatomy and embryology, 2010

Heat shock proteins and glucoseregulated proteins represent an extraordinary mechanism of defense... more Heat shock proteins and glucoseregulated proteins represent an extraordinary mechanism of defense induced in the kidney by chemicals or drugs and essential to survive. Here we resume our experience on the presence and regulation of stress proteins into acute and chronic nephro toxic models in rodents and in vitro. In acute renal damage, induced in rats by a single injection of inorganic mercury, stress pro teins enhanced in a dosedependent manner to recover cytoskeleton and mitochondria and maintain nuclear activity. When we pretreated mercury injectedrats with antioxidant melaton in or with bimoclomol, a stress proteinscoinducer, stress proteins expression was modulated together with tubular recovery. Similar data were obtained in ischemiareperfusion in rats treat ed with stannous chloride, that provided cytoprotection stimulating heme oxygenase induc tion. During nephrotoxicity induced by administration of cyclosporine A at therapeutic dos age for 12 months, stress protein overexpression well correlated with oxidative and cell death, but decreased if we counteracted renal damage using antioxidants. In aluminium intoxication through drinking water for 36 months, we detected a timedependent stress response in the rat kidney that was organ specifi c and different from the liver. In vitro studies on rat tubular proximal cells exposed to heavy metals demonstrated that stress protein expression was related to peculiar mechanisms of action of each metal. In conclusion, experimental studies on the renal chaperones can greatly contribute to understand their role, and agents able to modulate the stress response might be considered promising therapeutic tools to reduce nephrotoxicity.

PLOS ONE, 2016

Background Obesity is a common risk factor for non-alcoholic fatty liver disease (NAFLD). Current... more Background Obesity is a common risk factor for non-alcoholic fatty liver disease (NAFLD). Currently, there are no specific treatments against NAFLD. Thus, examining any molecule with potential benefits against this condition emerged melatonin as a molecule that influences metabolic dysfunctions. The aim of this study was to determine whether melatonin would function against NAFDL, studying morphological, ultrastuctural and metabolic markers that characterize the liver of ob/ob mice. Methods Lean and ob/ob mice were supplemented with melatonin in the drinking water for 8 weeks. Histology and stereology were performed to assess hepatic steatosis and glycogen deposition. Ultrastructural features of mitochondria, endoplasmic reticulum (ER) and their juxtapositions were evaluated in livers of all experimental groups. Furthermore, hepatic distribution and expression of markers of ER and mitochondria (calnexin, ATP sintase β, GRP78 and CHOP) and metabolic dysfunction (RPB4, β-catenin) and cellular longevity (SIRT1) were analyzed. Results Melatonin significantly reduced glycemia, identified also by a decrease of hepatic RBP4 expression, reversed macrosteatosis in microsteatosis at the hepatic pericentral zone, enlarged ER-mitochondrial distance and ameliorated the morphology and organization of these organelles in ob/ob mouse liver.

Mitochondria represent the key site for using and delivering energy for any cell in metabolically... more Mitochondria represent the key site for using and delivering energy for any cell in metabolically active tissues or organs such as skeletal muscles, heart, brain, and kidney. Proper nutrition, and in particular caloric restriction imitation, is a fundamental tool to retard aging, being able to stimulate mitochondria biogenesis and function. In an attempt to limit progressive organ dysfunctions associated with aging and metabolic disorders, we adopted a nutritional strategy based on the long-term supplementation to rodents of a balanced branched-chain amino acid mixture. Moreover, we tested taurine, a non essential beta-amino acid dissolved in drinking water for two weeks, in rats given puromycin aminonucleoside to reproduce podocyte damage and proteinuria. Here we resume our main morphological findings obtained by light and ultrastructural analysis and morphometry on mitochondria, associated with fibrosis and nitrosactive stress. All experiments revealed the effectiveness of amino acids supplementation to improve mitochondria feature and to reduce nephrosis, suggesting that a diet based on a single amino acid or better a mixture, may represent a promising anti-aging strategy also for humans. Keywords branched-chain amino acids; taurine; mitochondria; TEM 1. General remarks Aging induces progressive organ malfunctions and chronic diseases (including atherosclerosis, cancer, diabetes, sarcopenia) that have a large impact on social and sanitary costs all over in the world, so preventive anti-aging strategies represent a crucial issue [1]. A key event that characterizes aging is the change in metabolism and the abnormal balance between anabolic and enhanced catabolic activities, which lead to inability to fulfill basal energetic requirements in fundamental organs like heart, skeletal muscles, brain [2, 3]. Mitochondria are the favourite centre of aerobic metabolism necessary to produce a large amount of energy, associated to adenosine triphosphate molecule (ATP) via the oxidative phosphorylation reaction [4]. This biochemical reaction is responsible for converting energy from several macronutrients, like amino acids or carbohydrates to ATP molecule, through a sequence of mitochondrial reactions, called "the respiratory chain", that end with the reduction to water and adenosine diphosphate (ADP) phosphorylated to ATP [5]. It is accepted that aging is also "a mitochondrial affair", linked to a less amount of vital and efficient mitochondria in organs that develop a metabolic impairment and inability to respond properly to whole organism demands [6, 7]. Mitochondria morphology varies with aging and structural mitochondrial alterations are involved in several key pathologies associated with aging [8]. Indeed the crucial importance of mitochondria in the longevity of cells is strengthened by the evidence that mitochondria are not static organelles but they change their shape and density by using fusion and/or fission of their intrinsic DNA (mtDNA) [9, 10, 11]. Short and long-term mitochondrial dysfunctions and altered mitochondria size and density have been detected after oxidative damage and in aged organs in rodents [12, 13, 14]. During senescence the major deposition of reactive oxygen species (ROS) progressively damage mitochondria by replacing normal mtDNA with altered mtDNA, in the so called "free radical theory of aging" [15, 16]. Proteomic studies on mitochondria have recently characterized many genes, transcription factors and proteins like sirtuins, specifically associated to mitochondria activity during aging [17, 18, 19]. In this article we wish to remark that, beside molecular approach, also microscopy has a crucial role in the basic and clinical research on mitochondriopathies and aging-associated disorders. In fact any morphological approach on mitochondria may reliably report on the energyproviding system in that organism [20]. Malnutrition and micronutrient paucity, frequent in senescence, selectively impair mitochondria structure and metabolism, and vitamins, minerals or proteins deficit often induces oxidative stress, mtDNA damage and irreversible mitochondria decay [21, 22]. In this scenario, a preventive and therapeutical nutritional approach may be necessary to retard the onset of aging-related diseases and in particular to preserve mitochondria feature and activity. Current Microscopy Contributions to Advances in Science and Technology (A. Méndez-Vilas, Ed.

PLoS ONE, 2014

Obesity is a common and complex health problem, which impacts crucial organs; it is also consider... more Obesity is a common and complex health problem, which impacts crucial organs; it is also considered an independent risk factor for chronic kidney disease. Few studies have analyzed the consequence of obesity in the renal proximal convoluted tubules, which are the major tubules involved in reabsorptive processes. For optimal performance of the kidney, energy is primarily provided by mitochondria. Melatonin, an indoleamine and antioxidant, has been identified in mitochondria, and there is considerable evidence regarding its essential role in the prevention of oxidative mitochondrial damage. In this study we evaluated the mechanism(s) of mitochondrial alterations in an animal model of obesity (ob/ob mice) and describe the beneficial effects of melatonin treatment on mitochondrial morphology and dynamics as influenced by mitofusin-2 and the intrinsic apoptotic cascade. Melatonin dissolved in 1% ethanol was added to the drinking water from postnatal week 5-13; the calculated dose of melatonin intake was 100 mg/kg body weight/day. Compared to control mice, obesity-related morphological alterations were apparent in the proximal tubules which contained round mitochondria with irregular, short cristae and cells with elevated apoptotic index. Melatonin supplementation in obese mice changed mitochondria shape and cristae organization of proximal tubules, enhanced mitofusin-2 expression, which in turn modulated the progression of the mitochondria-driven intrinsic apoptotic pathway. These changes possibly aid in reducing renal failure. The melatoninmediated changes indicate its potential protective use against renal morphological damage and dysfunction associated with obesity and metabolic disease.

Current Trends in Atherogenesis, 2013

tension both in animal models and in clinical patients [11,12]. Remarkably this event is early de... more tension both in animal models and in clinical patients [11,12]. Remarkably this event is early detectable in the endothelial cells [13], sometimes concurrent with other well-known atherogenic processes, like inflammation, oxidative damage and endothelial cell death. Nevertheless considering the focal distribution of plaques and their cumulative progression during the whole lifespan [14], it is mandatory to consider the role of ER stress signaling in the circulatory bed, in order to maintain the proper ER function, so preventing or reducing the progression into irreversible cardiovascular dysfunctions, such as atherosclerosis, hypertension and ischemic heart disease [15-17]. We firmly believe that focusing integrated basic and applied research on ER stress in the artery tree and in the heart might open new avenues in the treatment and management of invalidating cardiovascular complications [18,19]. 2. The endothelium and the endoplasmic reticulum homeostasis According to the most accredited theory that indicates inflammation as the first pathogenic mechanism of atherosclerosis, the endothelium is really the crucial target of circulating molecules or cells and constitutes the main entrance for LDL during the initial step of asymptomatic artery wall changes that end into plaques or atheromata and their dramatic clinical evolution [20-23]. Recent studies have outlined that in atherosusceptible sites in the artery tree, endothelial cells acquire a proinflammatory phenotype which is permissive in the plaque development by expressing pro-inflammatory sensors such as Toll-like receptors (TLRs), that in turn attract leukocytes adhesion in the intima layer. Mainly TLR 2 and TLR4 are active in mouse in the progression of atherosclerosis and their signals stimulate a downstream adaptor molecule, called Toll/IL-1 receptor domain-related adaptor protein that induces interferon or TRIF. Indeed also in human vascular tree, by immunostaining and mRNA survey TLR2 and TLR4 have been well characterized in selected sites, including the aorta, subclavia, carotid, mesenteric, iliac and temporal arteries [24]. Nevertheless an important concept to remind here is that the relationship between the vascular endothelium and the blood is not only "passive" in receiving inflammatory or metabolic stimuli, but instead "active", with pleiotropic activities like the secretion of regulatory factors for cholesterol and lipid homeostasis, platelets recruitment, and the adaptation to local changes of blood flow and pressure [25,26]. Moreover the artery wall, in particular in healthy resistance arteries, is not a static but a dynamic and plastic structure, able to remodel its diameter and structure, adapting to rapid changes in the systemic pressure [27]. Indeed also artery geometry directly influences the athero-susceptibility and the distribution of mechanical forces associated to blood flux, that impair the endothelium [25,28,29]. In particular during unstable hemodynamic flux and changes in blood direction, mainly in arterial branches and bifurcations, it is particularly evident the heterogeneity of endothelial Current Trends in Atherogenesis 28