Current Vascular Pharmacology - Volume 12, Issue 5, 2014

In May 2012, a group of scientists and clinicians met in Athens (Greece) to consider the relevance of ageing, longevity, exceptional longevity and related genetic and non genetic markers. During this meeting, we firstly reviewed recent epidemiological and clinical studies on ageing, longevity and exceptional longevity, briefly analyzed the ageing theories and discussed successful and unsuccessful ageing also taking into account the evolutionary perspective. Secondly, we considered the three phenotypes based on the definition of ageing, longevity and exceptional longevity and the associated biomarkers. Third, we discussed proposed treatments suitable to counteract or slow down ageing. Finally, this panel produced a consensus statement to highlight the importance of ageing, longevity and exceptional longevity, since this is a rapidly increasing phenotype worldwide. We acknowledge that not all experts in this field may completely agree with this statement.

During the last decades survival has significantly improved and centenarians are becoming a fast-growing group of the population. Human life span is mainly dependent on environmental and genetic factors. Favourable modifications of lifestyle factors (e.g. physical activity, diet and not smoking) and healthcare (e.g. effective vascular disease prevention) have also increased human life span. Genetic factors contribute to the variation of human life span by around 25%, which is believed to be more profound after 85 years of age. It is likely that multiple factors influence life span and we need answers to questions such as: 1) What does it take to reach 100?, 2) Do centenarians have better health during their lifespan compared with contemporaries who died at a younger age?, 3) Do centenarians have protective modifications of body composition, fat distribution and energy expenditure, maintain high physical and cognitive function, and sustained engagement in social and productive activities?, 4) Do centenarians have genes which contribute to longevity?, 5) Do centenarians benefit from epigenetic phenomena?, 6) Is it possible to influence the transgenerational epigenetic inheritance (epigenetic memory) which leads to longevity?, 7) Is the influence of nutrigenomics important for longevity?, 8) Do centenarians benefit more from drug treatment, particularly in primary prevention?, and, 9) Are there any potential goals for drug research? Many definitions of successful ageing have been proposed, but at present there is no consensus definition. Such definitions may need to differentiate between “Longevity Syndrome” and “Exceptional Longevity”.

Some studies have shown that polymorphisms in the insulin growth factor-1 (IGF-1) signaling pathway genes could influence human longevity. However, the results of different studies are often inconsistent. Our aim was to investigate by systematic review and meta-analysis the association of the common polymorphisms defining the genetic variability of the IGF-1 signaling pathway associated with human longevity. Eleven studies investigating the association between the polymorphisms in the IGF-1 signaling pathway genes (IGF-1, IGF-1 receptor (IGF-1R), Forkhead box O3A (FOXO3A) and Silent mating type Information Regulation 1 (SIRT1) and longevity were found and analyzed. The modelfree approach was applied to meta-analyze these studies. No association was reported between the single nucleotide polymorphisms (SNPs) of IGF-1 and longevity in the only available study. The meta-analysis of available data from four studies, showed a significant association with the IGF-1R polymorphism rs2229765, suggesting that subjects with the Abearing genotype have a greater chance of longevity. Concerning the five studies on FOXO3A SNPs, for the rs2764264 a significant association with longevity was observed for C allele when only males were included in the analysis. Statistically significant results were obtained for other SNPs as well, i.e. rs2802292 (G allele), rs9400239 and rs479744 (T and A alleles, respectively). For rs9400239 the association was observed in long lived males with a lower odds ratio than in centenarians, while in rs479744 a significant association was highlighted in centenarians. Concerning SIRT1, no association between the SNPs under study and longevity was observed in the only available report. Current findings suggest that both IGF-1R and FOXO3A polymorphisms could be associated with longevity. The high degree of between-study heterogeneity and the low number of available studies underline the need for further methodologically adequate analyses to confirm this evidence.

Ageing is related to slowdown/breakdown of the somatotropic axis (i.e. the somatopause) leading to many physiological changes. The somatopause is accompanied by DNA and other macromolecule damage, and is characterized by a progressive decline in vitality and tissue function. We still do not have a definitive understanding of the mechanism( s) of ageing. Several overlapping theories have been proposed such as: 1) The free radical theory, 2) Mitochondrial Ageing, 3) The Glycation Theory, 4) Protein Damage and Maintenance in Ageing, and, 5) DNA Damage and Repair. Furthermore, several models of ageing were introduced such as genetically programmed senescence, telomere shortening, genomic instability, heterochromatin loss, altered epigenetic patterns and long lived cells. There are certain lipid modifications associated with the somatopause, characterized mainly by an increase in total cholesterol and triglyceride levels in both genders. In this review we consider the mechanisms of ageing and the associated changes in lipid metabolism according to gender.

A change in the lipoprotein profile is a metabolic hallmark of aging and has been the target for modern medical developments. Although pharmaceutical interventions aimed at lipid lowering substantially decrease the risk of cardiovascular disease, they have much less impact on mortality and longevity. Moreover, they have not affected death from other age-related diseases. In this review we focus on high density lipoprotein (HDL) cholesterol, the levels of which are either elevated or do not decrease as would be expected with aging in centenarians, and which are associated with lower prevalence of numerous age-related diseases; thereby, suggesting a potential HDL-mediated mechanism for extended survival. We also provide an update on the progress of identifying longevity-mediating lipid genes, describe approaches to discover longevity genes, and discuss possible limitations. Implicating lipid genes in exceptional longevity may lead to drug therapies that prevent several age-related diseases, with such efforts already on the way.

Cellular senescence is the state of permanent inhibition of cell proliferation. Senescent cells are characterized by several features including increased activity of senescence-associated β-galactosidase (SA-β-GAL) and senescenceassociated secretory phenotype (SASP). In vitro, 2 types of senescence have been described. One is telomere-dependent replicative senescence and the second is stress-induced premature senescence (SIPS). Despite some tissue-specific characteristics many kinds of cells, including stem/progenitor cells, can undergo senescence both in vitro and in vivo. Senescent cells were detected in murine, primate and human tissues using different markers. There is mounting evidence that senescent cells contribute to ageing and age-related disease by generating a low grade inflammation state (senescenceassociated secretory phenotype-SASP). Even though cellular senescence is a barrier for cancer it can, paradoxically, stimulate development of cancer via proinflammatory cytokines. There is evidence that senescent vascular cells, both endothelial and smooth muscle cells, participate in atherosclerosis and senescent preadipocytes and adipocytes have been shown to lead to insulin resistance. Thus, modulation of cellular senescence is considered as a potential pro-longevity strategy. This can be achieved by: elimination of selected senescent cells, epigenetic reprogramming of senescent cells, preventing cellular senescence or influencing the secretory phenotype. Some pharmacological interventions have already shown promising results.

Human longevity is a complex trait in which genetics, epigenetics, environmental and stochasticity differently contribute. To disentangle the complexity, our studies on genetics of longevity were, at the beginning, mainly focused on the extreme phenotypes, i.e. centenarians who escaped the major age-related diseases compared with cross sectional cohorts. Recently, we implemented this model by studying centenarians’ offspring and offspring of non-long lived parents. In association, during studies on many candidate genes SNPs, positively or negatively correlated with longevity have been identified. The results obtained on Insulin-like Growth Factor 1 Receptor (IGF1R) polymorphisms showed a correlation between specific genetic variants combinations and the low plasma level of IGF1 in centenarians, suggesting an impact of the IGF-I/insulin pathway on human longevity. This pathway together with mammalian target of rapamycin (mTOR) will be reviewed as being the most promising for longevity. Further, we will summarise the role of apolipoprotein E (APOE) variants in human longevity since the results of the large European project GEHA (Genetics of Healthy Aging) indicate APOE among the chromosomal loci associated with longevity. On the other hand, the identification of longevity-related genes does not explain the mechanisms of healthy aging and longevity rather pose questions on epigenetic contribution, gene regulation and the interactions with essential genomes, i.e. mitochondrial DNA and microbiota. To fully disentangle what appears to be an endless quest, all the components of the complexity of human longevity genetics are taken into account.

A main objective of current medical research is to improve the life quality of elderly people as priority of the continuous increase of ageing population. This phenomenon implies several medical, economic and social problems because of dramatic increase in number of non autonomous individuals affected by various pathologies. Accordingly, the research interest is focused on understanding the biological mechanisms involved in determining the positive ageing phenotype, i.e. the centenarian phenotype. In achieving this goal the choice of an appropriate study models is fundamental. Centenarians have been used as an optimal model for successful ageing. However, this model shows several limitations, i.e. the selection of appropriate controls and the use itself of the centenarians as a suitable model for healthy ageing. Thus, the interest has been centered on centenarian offspring, healthy elderly people. They may represent a model for understanding exceptional longevity for the following reasons: they exhibit a protective genetic background, cardiovascular and immunological profile, as well as a reduced rate of cognitive decline than age-matched people without centenarian relatives. Several of these aspects are summarized in this review based on the literature and the results of our studies.

Telomeres are DNA-protein structures that form protective caps at the end of eukaryotic chromosomes. They constitute the safeguards of chromosome degradation and are responsible for maintaining genomic integrity. The multifactorial nature of telomere length (TL) regulation increases the perplexity of studies in the field. TL is characterized by a high variability among individuals (birth and later life) and among species but it is unknown whether this is associated with their lifespan potential. TL is also highly heritable, longer in women than in men; it is highly variable between tissues and organs and inversely related to chronological age. Accelerated telomere loss has been associated with many chronic diseases of aging. Premature aging or cellular senescence, seen in early life, through increased oxidative stress and DNA damage to telomeric ends may be initiators of processes related to these diseases. During the recent decade, research around telomere biology has rapidly expanded due to its dynamic involvement in aging and longevity. However, longevity is not necessarily an indication of disability-free aging. There is substantial scientific disagreement and controversial results, regarding even the basic nature of aging and the path to longevity. We review the current evidence linking telomere biology to aging processes and mechanisms leading to longevity.

The Mediterranean diet (Mediet) is an eating pattern characterizing a lifestyle and culture that has been reported to contribute to better health and quality of life. The Mediet reflects food patterns typical of Mediterranean regions, where olive oil plays an essential role in the food pyramid. Olive oil is located in the middle and it is considered the principal source of dietary fat because of its high nutritional quality (particularly extra virgin olive oil). Several studies have shown the effect of the Mediet on healthy status by lowering the rates of coronary heart disease, certain cancers, and some other age-related chronic diseases. Although the scientific literature regarding diet and life span is complex and with different opinions, there are studies that demonstrate the beneficial effects of the Mediet on longevity. Therefore, the Mediet may be considered as including several nutraceuticals that favourably influence health. In the present review we discuss two Mediterranean populations from the island of Ikaria (Greece) and the Sicani Mounts (Sicily, Italy) whose longevity is attributed to a close adherence to the Mediet.

New onset diabetes (NOD) has been regarded as one of the factors to be considered before deciding to prescribe a statin [1- 3]. The recent Canadian Network for Observational Drug Effect Studies Investigators [2] study and an older meta-analysis [3] suggest that the administration of higher potency statins is associated with an increased risk of NOD compared with lower potency statins [1-3]. The potential increase in caloric and fat intake during statin treatment may play a role in the pathogenesis of statin-related NOD [4]. However, the exact mechanisms involved in the pathogenesis of NOD associated with statin treatment remain to be defined. Recent evidence suggests that the prevalence of NOD is significantly lower in familial hypercholesterolemia (FH) patients (n=14,296) compared with their unaffected relatives (n=24,684) [5]. For receptor-negative and receptor-defective low density lipoprotein receptor (LDLr) mutations, the odds ratio was 0.35 (0.27-0.45) and 0.51 (0.42-0.62) (p for trend <0.001), respectively, compared with unaffected relatives [5]. FH patients may be more motivated to follow lifestyle measures (diet, exercise and not smoking) which may contribute to the reported decrease in the risk of NOD in patients with FH treated with statins. Statins may cause NOD, especially in predisposed individuals, through several mechanisms [1, 6]. They may affect insulin secretion, reduce translocation of glucose transporter 4 resulting in hyperglycaemia and hyperinsulinaemia, decrease intracellular signalling through the reduction of several downstream products, and finally they may decrease leptin and adiponectin levels [6]. However, the correlation between statin intensity and risk of NOD has not been fully defined. We could not find definitive information in the literature stating if activation of sterol regulatory element-binding proteins (SREBPs) occurs in patients with FH as part of the attempt to increase hepatic LDLr numbers. In contrast, statins increase LDLr numbers through activation of SREBPs 1a, 1c and 2 [7], which are also causally related to insulin resistance [8]. Thus, the more potent the statin, the greater the possible increase in SREBPs and LDLr as well as the reduction in plasma LDL cholesterol (LDL-C); however, the “cost” may be in terms of insulin resistance and a higher incidence of NOD. Thus, it is imperative to carefully balance the risk-benefit ratio [1, 9]. However, is this the case for antibodies against proprotein convertase subtilisin kexin-9 (PCSK9), which prolong the life of the LDLr? These drugs have no apparent adverse effect on SREBPs [10]. In fact, a study using a novel antibody (1B20) against PCSK9 has shown that its combination with simvastatin in dyslipidaemic rhesus monkeys reduced LDL-C more than either agent alone, while at the same time decreasing liver mRNA levels of SREBPregulated genes [10]. This mRNA effect remains to be confirmed in humans. The phase III studies using antibodies against PCSK9 (e.g. evolocumab or alirocumab) have not reported any trend for increased NOD [11, 12]; however the issue of NOD should be carefully monitored in these on-going trials. It took some 20 years to realise that statin-induced NOD occurs. If there is a similar problem with PCSK9 inhibitors (potentially the next blockbuster in hypolipidaemic treatment [13]) we should know as soon as possible. Moreover, we need to define the risk of NOD in patients treated with a statin and PCSK9 inhibitor combination. It may be that by lowering the statin dose required to reach LDLC targets, there will also be a decrease in NOD. This potential benefit may help justify, at least in part, the cost of using PCSK9 inhibitors. A recent study reported an off-target effect of cholesterol ester transfer protein (CETP) inhibitors, a new drug category aimed at increasing high density lipoprotein cholesterol (HDL-C) levels and reduce atherosclerosis; these drugs are still under investigation in phase III trials [14]. CETP inhibitors reduce the levels of mature SREBP 2, leading to attenuated transcription of hepatic LDLr and PCSK9 [14]. Thus, it is possible that these drugs will not have the diabetogenic effects of statins. We also need to consider the evidence that currently available lipid lowering drugs, like ezetimibe and colesevelam, may also have beneficial effects on insulin sensitivity. However, the evidence for ezetimibe comes from relatively small studies [15- 19] with design limitations and the results were not always consistent [20]. This may reflect which patient category was studied. It is also of interest that ezetimibe down-regulated liver SREBP-1c and improved hepatic insulin resistance in mice fed a high-fat diet although there were also effects on other mediators [21]. Colesevelam is actually licensed (in some countries) to lower glucose levels although the mechanisms involved are not clear [22]. The above evidence suggests that ezetimibe and colesevelam as well as two new drug categories (i.e. PCSK9 and CETP inhibitors) for the treatment of dyslipidaemia might decrease/avoid the risk of NOD. This will hopefully lead to combinations with statins of lesser intensity or at lower doses which may result in a reduced risk of statin-related NOD. The key issue is whether such combinations will be as effective as statin monotherapy in terms of lowering the risk of vascular events.

The liver plays a major role in lipid metabolism, importing free fatty acids (FFA) and manufacturing, storing and exporting lipids: derangements in any of these processes can lead to non-alcoholic fatty liver disease (NAFLD) [1]. NAFLD can be seen as the result of an imbalance between lipid availability and lipid disposal resulting in hepatic steatosis [2]. NAFLD is considered by many as the hepatic manifestation of insulin resistance (IR) and is strongly associated with the metabolic syndrome [3]. The rapid increase in obesity and diabetes mellitus (DM) during the last decade is associated with an increase in the prevalence of NAFLD, making it the most common cause of chronic liver disease in the Western countries with up to 30% of the population affected [4]. Histological evaluation is the gold standard for precisely estimating the degree of liver damage caused by simple steatosis or by non-alcoholic steatohepatitis (NASH). NASH is a disturbance at the end of NAFLD spectrum characterized by hepatocellular injury/inflammation/macrophage infiltration with or without fibrosis [5]. The individuals with NAFLD develop NASH in 10% of the cases, 8-26% progress to cirrhosis and these patients are also at risk of developing hepatocellular carcinoma (HCC) [6]. As recently highlighted by the guidelines of the American Association for the Study of Liver Diseases (AASLD) [7], patients with NAFLD and NASH are at increased risk for cardiovascular disease (CVD) (their most common cause of death) [8]. Therefore, patients with NAFLD and NASH should be stratified for such risk and their CVD factors, including dyslipidemia, should be managed accordingly [9]. At the cellular level, defects in the insulin signaling pathways contribute to the increase of FFA flux in the liver which in turn activates a series of signaling cascades which lead to the phosphorylation of several substrates; in the case of IR there is a decreased insulin receptor kinase activity resulting in lower AKT activity [10]. Insulin is the main driver of the global response to nutrient ingestion, and acts on hepatic fat metabolism through the phosphatidylinositol kinase signaling pathway by accumulating the intracellular lipid small messenger phosphatidylinositol [3,4,5]-trisphosphate. This situation is reproduced faithfully by phosphatase and tensin homologue (PTEN) loss, which similarly accompanies fatty liver development [11-17]. Besides the PTEN/AKT and downstream effectors, de novo fatty acid synthesis is regulated by a plethora of other transcription factors [2]. The mechanisms behind the progression of steatosis to NASH are not completely understood; in particular the factors that lead to increasing hepatocellular damage after triglyceride (TG) accumulation. According to the ‘two-hit’ theory, the ‘first hit’ is an imbalance in hepatic lipid accumulation the likely cause being IR which leads to an increased traffic of FFA in the hepatocytes; then follows the ‘second hit’, represented by inflammatory cytokines/adipokines, mitochondrial dysfunction and oxidative stress, promoting apoptosis through several pathways that lead to inflammatory infiltrates in the liver that may progress to fibrosis [18]. This old model is now revitalized by recent studies analyzing the role of the gut microbiota. The nucleotide-binding domain and leucine-rich-repeat-containing (NLR) family of pattern-recognition molecules NLRP6 and NLRP3 take part to cytosolic protein complexes termed inflammasomes, which in turn have various roles in immune defense [19]. The NLRP6 and NLRP3 inflammasomes and the downstream cytokine interleukin-18 (IL-18) negatively regulate NAFLD/NASH progression, by regulating the activity of the gut microbiota: changed interactions between the gut microbiota and the host, produced by defective NLRP3 and NLRP6 inflammasome sensing may govern NAFLD progression [20]. Inflammasome sensing has also been proposed as a mechanism in macrophage activation in atherosclerotic lesions [21]. In this context, the hypothesis that NASH and atherosclerosis are 2 aspects of the same disease, involving the local presence of activated macrophages, has been put forward [22]. NASH may promote pro-inflammatory and pro-atherogenic factors that are likely to play a major role in CVD pathogenesis [8, 23, 24]. Oxidative stress is a strong atherogenic stimulus, associated with the secretion of inflammation biomarkers and endothelial dysfunction such as interleukin-6 (IL-6) and tumour-necrosis factor-?? (TNF-??). NASH is associated with a systemic release of pro-inflammatory mediators from the liver including C-reactive protein (CRP), fibrinogen and plasminogen activator inhibitor-1 (PAI-1) [8, 23, 24]. CRP levels and the degree of carotid atherosclerosis were found to be higher in NASH patients than in control subjects, suggesting a link between hepatic injury, inflammation and development and progression of CVD [25]. An important step in atherosclerosis progression is the transfer of oxidised (ox) low density lipoprotein (LDL) into the arterial wall [26]. OxLDL represents a variety of modifications of both the lipid and protein components of LDL and this oxidation process is thought to occur mainly in the arterial wall, rather than in plasma [27]. LDL are heterogeneous particles with several distinct subclasses that differ in physicochemical composition, metabolic and oxidative properties as well as atherogenicity [28]. Oxidative susceptibility increases and antioxidant concentrations decrease with decreasing LDL size [29], so that small dense (sd) LDL particles are those with enhanced susceptibility to oxidation and greater atherogenicity. sdLDL represent an emerging cardiovascular risk factor, as highlighted by several international guidelines [30] and expert panels [31]. It is plausible that the more sdLDL in plasma, the more oxLDL in the arterial wall; oxLDL have pro-inflammatory, pro-thrombotic and pro-apoptotic potential [27]. On this basis, sdLDL/oxLDL levels have been proposed as a novel biomarker of CVD risk beyond LDL-cholesterol concentrations [32]. Recently, studies by Bieghs et al. have shown that sdLDL receptor knock-out mice developed hepatic inflammation and liver damage upon high-fat feeding due to increased oxLDL uptake [33]. Models for atherosclerosis include the sdLDL receptor knock-out (Ldlr-/-) mouse, which display defects in the clearance of apo B- and apo E-containing lipoproteins and the apo E2 knock-in (APOE2ki) mouse, in which the murine apo E gene is substituted with the human apo E2. Apo E2 has a decreased affinity for the LDL receptor, leading to a lipoprotein profile resembling human type III hyperlipoproteinaemia [34]. Both APOE2ki mice and Ldlr-/- mice develop early hepatic inflammation and steatosis when fed a high-fat-high-cholesterol diet, whereas wild type mice only developed fatty liver [35]. The lipoprotein profile of the Ldlr-/- mouse and APOE2ki is comparable with the human, in which cholesterol is mainly present in the LDL fraction [36]. When fed a high-fat-high-cholesterol diet, both APOE2ki and Ldlr-/- mice are useful to study the factors that augment the inflammatory response during NASH. Bieghs et al. recently hypothesized that the inflammatory response in these mice models would be strong enough to induce hepatic fibrosis [33]. Upon feeding APOE2ki and Ldlr-/- mice with a high-fat-high-cholesterol diet for 3 months, a pronounced inflammatory response was observed only in Ldlr-/- mice, as well as increased apoptosis and hepatic fibrosis. These differences were dependent on an increased sensitivity for oxLDL-induced inflammation in Ldlr-/-mice compared with APOE2ki mice [33]. The Ldlr-/- model is thus a promising option to study the onset of NASH in the context of steatosis and to assess therapeutic strategies for hepatic inflammation. Accordingly, it is plausible to think that specific immunization strategies against oxLDL may reduce NASH. Interestingly, the levels of immunoglobulin M (IgM) autoantibodies to oxLDL/sdLDL are inversely correlated with atherosclerotic plaques [37-39]. Specific epitopes present in oxLDL are recognized by circulating IgM [40]. In another recent study, Bieghs et al. used the Ldlr-/- model for immunization with heat-inactivated pneumococci to investigate whether anti-oxLDL antibodies can exert a protective effect on NASH [41]. Immunized Ldlr-/- mice showed reduced NASH compared with non-immunized mice. Although no difference in liver lipids were observed between immunized and nonimmunized Ldlr-/- mice, decreased cholesterol, inflammation and expression of hepatic fibrosis-associated genes were observed upon a high-fat-high-cholesterol diet [41]. These findings in mice demonstrate the potential role of antibodies to oxLDL in the pathogenesis of NASH, in line with recent data on lipids implicated in lipotoxicity to the liver and hepatocytes [42]. Understanding the relationship between NAFLD/NASH, sdLDL/oxLDL and atherogenic processes is of clinical relevance. These recent studies suggest that vaccination strategies against sdLDL/oxLDL could be explored as an option for the prevention and treatment of NASH. Such research would help uncover new therapeutic approaches to prevent the development of CVD associated with NAFLD/NASH. Since NAFLD is often associated with obesity and IR/type 2 diabetes, lifestyle modification (diet and aerobic exercise) and weight reduction remain the cornerstones of treatment because they can ameliorate or even reverse the disease [42, 43], as indicated also by the AASLD guidelines [7]. The effects of lifestyle intervention were examined by Promrat et al. in a randomized controlled trial: patients who achieved a weight loss >7% of initial weight had significant improvement in steatosis and NASH activity score compared with patients who lost less than 7% of initial [44]. Drugs potentially effective against NAFLD are the insulin sensitizers, metformin and pioglitazone, which all reduce oxidative stress [45]. Studies on overweight or obese patients with ultrasonographic diagnosis of hepatic steatosis, metformin was found to be more effective than dietary treatment alone in ameliorating metabolic derangements and NAFLD [46]. Studies on NAFLD patients as well as on murine models have suggested some beneficial roles of natural antioxidant molecules [47, 48]. Vitamin D deficiency exacerbated NAFLD and inflammation in a Western diet rat model, suggesting that its supplementation might be beneficial [49]. Vitamin E treatment compared with placebo was associated with a significantly higher rate of improvement in NAFLD patients, and it did not cause a weight gain as observed with pioglitazone and rosiglitazone [50]. Despite these interesting findings, the impact of vitamin D supplementation in human NAFLD awaits validation and treatment with high dose vitamin E should be carefully considered due to its association with higher risk for stroke and mortality [51]. The thiazolidinediones, pioglitazone and rosiglitazone, activate peroxisome proliferator-activated receptor-gamma PPAR??, which regulates TG homeostasis, contributing to hepatic steatosis [52]. Unfortunately, the use of thiazolidinediones has been hampered by important side effects [53-55]; rosiglitazone has been taken off the market. The AASLD statement [7] suggests that pioglitazone can be used to treat steatohepatitis in patients with biopsy-proven NASH, although the long-term safety and efficacy of pioglitazone in these patients is not established. Fibrates are potent peroxisome proliferator activator receptors alpha (PPAR??) agonists and they can significantly increase the hepatic oxidation of free fatty acids [56]. A controlled trial with the gemfibrozil 600 mg daily for 4 weeks showed some lipid improvement in NAFLD [57], and similar results have been shown with fenofibrate [58]. Overall, fibrates are an attractive option to treat hypertriglyceridemia in patients with NAFLD but, as recently highlighted [9], their effect on liver histology is unclear. Another option to treat hypertriglyceridemia is supplementation with omega-3 polyunsaturated fatty acid (PUFA). Several studies have shown that PUFA supplementation can improve biochemical and ultrasonographic steatosis [59-61]. Yet, as recently highlighted in a comprehensive review [62], no clear conclusion can be made, since published human studies are of small sample size and have several methodological flaws. However, the experimental evidence strongly supports the use of PUFA in NAFLD. On the basis of this evidence, the AASLD guidelines [7] state that it is premature to recommend omega-3fatty acids for the specific treatment of NAFLD or NASH, but they may be considered as the first line agents to treat hypertriglyceridemia in NAFLD. There is reluctance to use statins in patients with suspected or established chronic liver disease (e.g. NAFLD, NASH or elevated aminotransferase activity) [63]. All statins seems to lower cholesterol levels in NAFLD patients [9] and atorvastatin showed a reduced CVD morbidity: the Greek Atorvastatin and Coronary Heart Disease Evaluation (GREACE) study showed a reduction of CVD events with statins in patients with NAFLD [64]. In addition, a review that examined all available evidence about potential statin hepatotoxicity concluded that patients with NAFLD are not at significantly increased risk of severe hepatic toxicity with standard doses of statins [65]. The AASLD statement [7] suggests that statins can be used to treat dyslipidemia in patients with NAFLD and NASH, although these drugs should not be used specifically to treat NASH. In conclusion, patients with NAFLD and NASH have increased CVD risk. NAFLD and NASH patients have several alterations in lipid metabolism that are associated with atherogenic dyslipidemia, including increased levels of TGs, high levels of sdLDL particles and decreased levels of HDL-cholesterol [66]. Lipid-lowering drugs are not fully recommended in NASH patients; therefore, the recent studies suggesting that vaccination strategies against sdLDL/oxLDL could be explored as an option for the prevention and treatment of NASH are fascinating.

Diabetic foot ulcers arise from neuropathy and/or ischaemia. The diabetic foot is associated with cardiovascular disease (CVD) and excess mortality. Lipid-lowering therapy reduces CVD morbidity and mortality in diabetic patients with foot ulcers. In particular, statins decrease CVD mortality and improve survival in diabetic foot patients, while fibrates benefit patients with a specific lipid profile. Statins reduce progression of the local disease, improving symptoms and reducing amputations, mainly due to their impact on peripheral arterial disease. Fibrates appear to reduce amputations by improving neuropathy. They also improve ulcer healing and reduce recurrence. This review assesses the role of hypolipidaemic treatment in diabetic foot patients.

Proton pump inhibitors (PPIs) are recommended in patients with prior upper gastrointestinal bleeding and considered appropriate in patients with multiple other risk factors who require dual antiplatelet treatment (DAPT). During the past few years, however, concerns have been raised about the potential for PPIs, especially omeprazole, to decrease the efficacy of clopidogrel, and both the U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA) have issued warnings regarding the concomitant use of these medications. A review of the literature revealed that the pharmacodynamic studies support an interaction, whereas the clinical evidence, which is mainly based on nonrandomized, observational studies and secondary analyses of randomized trials, is conflicting. We conclude that PPIs should be prescribed together with DAPT for patients in whom they are recommended according to the guidelines and for patients with other indications. With respect to omeprazole, current evidence does not allow clear recommendations to be provided.

There is growing evidence that Rho-kinase contributes to cardiovascular disease, which has made Rho-kinase a target for the treatment of human diseases. To date, the only Rho-kinase inhibitor employed clinically in humans is fasudil, which has been used for the prevention of cerebral vasospasm and subsequent ischemic injury after surgery for subarachnoid hemorrhage (SAH). A number of pathological processes, in particular hemodynamic dysfunctions and inflammatory reactions, are thought to be related in the pathogenesis of delayed cerebral vasospasm and subsequent ischemic injury after SAH. This review focuses on fasudil’s pleiotropic therapeutic effects: amelioration of hemodynamic dysfunction and inflammation, and discusses in detail the clinical studies on fasudil administered after the occurrence of SAH.

Clopidogrel is a thienopyridine that selectively and irreversibly inhibits the ADP purinergic receptor P2Y12 and the subsequent ADP-mediated platelet activation. Clopidogrel has been approved for clinical use as clopidogrel hydrogen sulfate (bisulfate) salt. The clinical usefulness of clopidogrel bisulfate salt has been proved in a wide variety of large scale clinical trials, thus clopidogrel bisulfate has been extensively used in a large spectrum of patients been under thrombotic risk. Recently, several generic clopidogrel formulations have been approved for clinical use. Consequently, clopidogrel is currently a cost-effective antiplatelet agent. Only small studies have compared the pharmacokinetic and pharmacodynamic properties of various clopidogrel generic salt formulations with the innovator bisulfate salt. In addition few data are available concerning the clinical efficacy and safety of these generic clopidogrel formulations in order to guide clinicians in deciding when generic substitution is appropriate. The aim of this review is to summarize the physicochemical properties as well as the pharmacokinetic and pharmacodynamic characteristics of the generic clopidogrel salts. We also critically present existing data on the clinical efficacy and safety of the generic clopidogrel formulations compared with the innovator clopidogrel bisulfate salt in patients with cardiovascular disease.