Macrophages and related tissue macrophage populations use the classical NADPH oxidase (NOX2) for the regulated production of superoxide and derived oxidants for pathogen combat and redox signaling. With an emphasis on macrophages, we discuss how sorting into secretory storage vesicles, agonist-responsive membrane trafficking, and segregation into sphingolipid and cholesterol-enriched microdomains (lipid rafts) determine the subcellular distribution and spatial organization of NOX2 and superoxide dismutase-3 (SOD3). We discuss how inflammatory activation of macrophages, in part through small GTPase Rab27A/B regulation of the secretory compartments, mediates the coalescence of these two proteins on the cell surface to deliver a focalized hydrogen peroxide output. In interplay with membrane-embedded oxidant transporters and redox sensitive target proteins, this arrangement allows for the autocrine and paracrine signaling, which govern macrophage activation states and transcriptional programs. By discussing examples of autocrine and paracrine redox signaling, we highlight why formation of spatiotemporal microenvironments where produced superoxide is rapidly converted to hydrogen peroxide and conveyed immediately to reach redox targets in proximal vicinity is required for efficient redox signaling. Finally, we discuss the recent discovery of macrophage-derived exosomes as vehicles of NOX2 holoenzyme export to other cells. Once thought upon merely as a facet of the microbicidal defense mechanisms of phagocytes, oxidants are now recognized as primary messengers in an intricate and widely arborized network of redox signaling, where the activity of target proteins is regulated by reversible oxidation and reduction cycles of low pKa thiols ,2. The cSince the discovery of the expanded NOX family of superoxide-producing NADPH oxidases in 2000 ,4, we noIn this review, we correlate the functionality and biology of NOX2 and SOD3 to delineate that these two enzymes, the oxidant producer and the dismutator, distribute and traffic in comparable ways to coordinate their activity at spatially confined microdomains of the cell surface. The result is a highly localized hydrogen peroxide production, which controls the activity of target proteins subject to redox control in the cytosol , at the 2•−), which is a radical and an anion. This process is catalyzed by the NOX family of proteins, which includes NOX2 (2•) increases the reduction potential and hence reactivity, however, at physiological pH the pKa-value of the perhydroxyl radical (4.8) dictates that less than 1% of superoxide exists in this protonated form [2• has been suggested to support the oxidation of polyunsaturated fatty acids [5 M−1 s−1) [•) to generate peroxynitrite (ONOO−). The rate constant of the reaction is at the top level of biological reactions (k ~1010 M−1 s−1) [• generated in the immediate vicinity. Peroxynitrite is a strong oxidant and will support lipid peroxidation and protein nitrosylation, and therefore, may represent some of the oxidative damage induced by superoxide generation. In addition to supporting the formation of ONOO−, this fast reaction also removes bioactive NO•, which is well described to play a central role in vasodilation as well as in bone homeostasis [The one-electron reduction of molecular oxygen generates superoxide  . DismutaM−1 s−1) . In addiM−1 s−1)  and is 1eostasis . Due to eostasis . Although superoxide has the capacity to generate hydrogen peroxide by spontaneous dismutation, a significant number of enzymes distributed in distinct subcellular compartments are known to produce hydrogen peroxide, including SOD3 . At physThe capacity of hydrogen peroxide to oxidize biological macromolecules is relatively low. However, the reactions with glutathione peroxidases (GPX) and peroxiredoxins (PRX) presents high rate constants , indicat558 complex, consisting of gp91phox and p22phox, and cytosolic subunits p40, p47, and p47phox in addition to Rac1/2. Only in response to cell stimulation will the cytosolic subunits traffic to cyt b558 in the membrane to assemble the holoenzyme and commence regulated superoxide production [In general, NOX family members constitute the most important source of oxidants in cells . Macrophoduction . In neutrophils, the majority of NOX2 is contained within granules that are formed directly from the biosynthetic pathway. However, a population of NOX2 is also present in so-called secretory vesicles, which are formed by way of endocytosis  . Human mThe NOX2-containing vesicles are mobilized upon different inflammatory stimuli, e.g., tumor necrosis factor-α (TNFα) or CD40 ligand (CD40L), to enrich the cell surface in oxidant production, or they may be mobilized to phagosomes  often foIn microglia, Rab27A and B expression seems to negatively regulate cell surface exposition of NOX2, in as much as Rab27A or B knock-down redistributes NOX2 from cell surface to the intracellular storage vesicles . Rab27A SOD3 was initially purified from human lung tissue and found to encompass structural and functional similarities to the intracellular isoenzyme SOD1, including the coordination of copper and zinc atoms as well as enzymatic activity supporting the dismutation of superoxide into hydrogen peroxide ,52. The It is an appealing thought that NOX2 and SOD3 storage compartments may traffic to the macrophage cell surface together to unite function at this location in response to cellular stimulation. Similarly, trafficking of both vesicle populations to forming phagosomes might be functionally relevant, as hydrogen peroxide is the basis for bactericidal halide formation . In thatYet another layer of spatial convergence of SOD3 and NOX2 is likely operating at the plasma membrane. NOX2 (like other NOX enzymes) is included in glycosphingolipid and cholesterol-enriched microdomains (lipid rafts) at the cell surface, which results in a focal oxidant production on the plasma membrane . As mentIn terms of autocrine cell signaling, hydrogen peroxide produced at high amplitude by the concerted action of NOX2 and SOD3 at the cell surface may either cross the plasma membrane to oxidize cytosolic target proteins  or direc558 in the membrane and commence superoxide production. Superoxide produced at the cell surface is then dismutated via SOD3 activity to hydrogen peroxide, which is able to enter cells either through diffusion through the membrane, or through select aquaporins  that alxiporins . Intriguxiporins ,72,73, axiporins . Therefoxiporins . Monocytes depend on NOX-derived oxidants for differentiation into macrophages , and theTo what extent SOD3 is necessary for autocrine redox signaling has not been addressed in detail, but in macrophages the increased presence of SOD3 on the cell surface following exocytosis of intracellular storage vesicles decreases release of TNFα when compared to cells lacking SOD3 . The capInterestingly, the low reactivity of hydrogen peroxide towards biological macromolecules in general, allows the molecule to diffuse on the scale of several hundred micrometers up to perhaps one mm in the extracellular space where reductive capacity is low ,85. WhilB- and T-lymphocytes undergo profound changes in metabolism upon activation, and both basal and activation-induced metabolism is shaped by redox signaling pathways . Antigenregs) suppress CD4+ T-cell activation in secondary immune tissues, but this mechanism fails (in part) in aging individuals. In the work of Wen et al., the authors demonstrate that this deficit is caused by down-regulation of NOX2 in Tregs, and can be countered by reconstitution of NOX2 expression levels [regs exert their suppressive effect on CD4+ T-cells by the exosomal transfer of preassembled NOX2 to the CD4+ T-cells. A tight interface reminiscent of the immunological synapse, is formed between the two cell types. Whether, exosomal transfer of NOX2 also occurs between macrophages and CD4+ T-cells in the process of antigen stimulation [3)-based cytochemical techniques to detect oxidant production in living cells at ultrastructural resolution for the unequivocal determination of oxidant production in the exosomes themselves  as a charge neutralizer in endosomes of neutrophils [Additionally, the work of Hervera et al., forwards the possibility that NOX2 transferred to neuronal endosomes from macrophage exosomes, carries out its redox signaling from late endosomes in the soma area after retrograde transport . The controphils . In the No experimental evidence for the inclusion of SOD3 into exosomes exist, but recent findings show that mesenchymal stem cells release extracellular vesicles encompassing SOD3, and importantly that these vesicles have the capacity to modulate the redox conditions of recipient cells ,111. AltThe prefix anti- in antioxidant is a word-forming element of Greek origin meaning “against, opposed to, opposite of, instead”. While antioxidants certainly can make oxidants go away, it is also clear that they have important accessory roles in shaping distinct cellular microenvironments where they orchestrate the level and type of oxidants produced and consequently support redox relays that transmit cellular ques to relevant target proteins. Specifically, the activity of SOD enzymes may both be referred to as antioxidants in their capacity to remove superoxide but also as a prooxidant in providing hydrogen peroxide. Understanding how different cells, including macrophages, dynamically redistribute their batteries of oxidant producers, modulators, and antioxidants upon cellular stimulation will require more research into molecular sorting motifs and membrane trafficking pathways that govern the spatial organization of these elements, including that of NOX2 and SOD3. Many biological responses are dependent on autocrine redox signaling, and the multitude of intracellular redox targets now constitute the “redoxome”. With offset in future research, it will in particular be interesting to realize to what extent cell surface proteins constitute meaningful targets for rapid hydrogen peroxide-mediated regulation of activity. As better and more sensitive biologically encoded sensors are continuously developed it will no doubt spur on the elucidation of direct paracrine redox signaling in living tissues as well. The concept of paracrine redox signaling has now been expanded by the revelation of macrophage exosomes as vehicles of NOX2 oxidant production, a phenomenon that could prove to be wide-spread in the different tissues of our body. The notion in the present review is that the expanding appreciation of cellular processes subject to redox regulation dictates that we must aim to further describe the intricate network of oxidants and antioxidants that supports the generation of distinct redox microenvironments. Specifically, we argue that the subcellular distribution of NOX2 and SOD3 in inflammatory cells supports the formation of such microenvironments.


Diabetes mellitus is an important risk factor for a first cardiovascular event and for worse outcomes after a cardiovascular event has occurred. This situation might be caused, at least in part, by the prothrombotic status observed in patients with diabetes. Therefore, contemporary antithrombotic strategies, including more potent agents or drug combinations, might provide greater clinical benefit in patients with diabetes than in those without diabetes. In this Consensus Statement, our Working Group explores the mechanisms of platelet and coagulation activity, the current debate on antiplatelet therapy in primary cardiovascular disease prevention, and the benefit of various antithrombotic approaches in secondary prevention of cardiovascular disease in patients with diabetes. While acknowledging that current data are often derived from underpowered, observational studies or subgroup analyses of larger trials, we propose antithrombotic strategies for patients with diabetes in various cardiovascular settings . Finally, we summarize the improvements in cardiovascular outcomes observed with the latest glucose-lowering drugs, and on the basis of the available evidence, we expand and integrate current guideline recommendations on antithrombotic strategies in patients with diabetes for both primary and secondary prevention of cardiovascular disease. Patients with diabetes mellitus have a prothrombotic status that increases the risk of cardiovascular events and worsens prognosis after these events. In this Consensus Statement, the Working Group on Thrombosis of the Italian Society of Cardiology proposes antithrombotic strategies for patients with diabetes in various cardiovascular settings. Compared with atherosclerotic plaques from individuals without diabetes, those from patients with diabetes are more vulnerable (rupture-prone), and therefore, these plaques are at increased risk of developing superimposed thrombosis because of increased amounts of soft extracellular lipids, inflammation, and prothrombotic milieu; this situation predisposes patients with diabetes to acute cardiovascular events1. Consequently, in principle, aggressive antithrombotic therapies might be associated with greater clinical benefit in patients with diabetes than in those without the condition. However, the ischaemic protection provided by antithrombotic drugs must be weighed against the drug-related bleeding risk.Diabetes mellitus is an important risk factor for a first cardiovascular event and for worse outcomes after a cardiovascular event has occurred. Cardiovascular disease in diabetes is a progressive process characterized by early endothelial dysfunction, oxidative stress, and vascular inflammation leading to monocyte recruitment and formation of foam cells and fatty streaks, which cause development of atherosclerotic plaques over years4 , acute coronary syndrome (ACS), ischaemic cerebrovascular events, peripheral artery disease (PAD), and venous thromboembolism (VTE)). We also analyse the relationship between diabetes and thromboembolic risk in atrial fibrillation (AF) and the efficacy and safety of the non-vitamin K antagonist oral anticoagulants (NOACs) in patients with diabetes and AF. On the basis of the available evidence, we propose antithrombotic strategies that expand and integrate current guideline recommendations for patients with diabetes4 Table . Finally5. Reduced insulin sensitivity causes increased signalling of P2Y purinoceptor 12 (P2Y12 receptor), the main platelet receptor for ADP6 7.In vitro studies from patients with diabetes have shown increased platelet membrane expression of P-selectin and glycoprotein IIb/IIIa (GPIIb/IIIa), as well as increased platelet response to adrenaline and thrombin8. A reduction in platelet fluidity occurs through changes in membrane lipid structure or glycation of membrane proteins5. Furthermore, platelets from patients with diabetes have an increased expression of adhesion molecules, such as CD63 antigen, integrin α2 (CD49B), platelet endothelial cell adhesion molecule 1 (CD31), platelet glycoprotein 4 (CD36), and P-selectin (CD62P), as documented by flow cytometry9.Platelets from patients with diabetes show increased turnover, resulting in a higher number of reticulated platelets, and increased adhesion to endothelial cells2 , which physiologically inhibit platelet aggregation8. Other reactive oxygen species (ROS) might also contribute to in vivo platelet hyperactivation in diabetes8.Another important determinant of platelet dysfunction in diabetes is the reduced vascular production of nitric oxide (NO) and prostaglandin I12-receptor blocker. Platelet-function studies have revealed that treatment with clopidogrel is associated with an overall modest level of P2Y12-receptor inhibition with interindividual variability, even when high loading doses are used11. ADP-induced high platelet reactivity (HPR) when taking clopidogrel has been identified as a marker of vascular risk in an international consensus document12.In patients with ACS or those undergoing coronary stent implantation, the recurrence of cardiovascular events is not infrequent despite using the current standard of care, which is dual antiplatelet therapy (DAPT) comprising acetylsalicylic acid (commonly referred to as aspirin) and a P2Y17. This finding was demonstrated in ex vivo testing of platelet reactivity and was also derived by subgroup analyses of clinical trials, in which patients with diabetes who were undergoing percutaneous coronary intervention (PCI) and receiving clopidogrel had a worse clinical course and a higher incidence of stent thrombosis than patients without diabetes16. The exact mechanism of this phenomenon remains unknown. Reduced insulin sensitivity in diabetes might cause increased signalling of P2Y12 receptors; in addition, insulin might physiologically reduce platelet aggregation by inhibiting the P2Y12 pathway through platelet insulin receptors, and this mechanism might be impaired in patients with diabetes18. Finally, diabetes is known to attenuate cytochrome P450 activity in animal models and in humans21; accordingly, in patients with diabetes who are receiving clopidogrel, HPR has been shown to be correlated with lower concentrations of the clopidogrel active metabolite22.Patients with diabetes have a more pronounced impairment in their response to clopidogrel than individuals without diabetes23. In the randomized ASPECT study24, in which multiple methods were used to assess responsiveness to aspirin (81 mg daily), patients with diabetes had greater platelet aggregability than those without diabetes, as measured by the VerifyNow assay (Accriva Diagnostics), urinary 11-dehydro-thromboxane B2 levels, and collagen-induced light transmittance aggregometry. Of note, increasing the dose of aspirin to >81 mg daily in patients with diabetes reduced platelet function and the prevalence of impaired response to the drug to values similar to those in patients without diabetes; this finding suggests that, in diabetes, low-dose aspirin does not provide adequate platelet inhibition and that higher aspirin dosing reduces the prevalence of low-responder patients24. In particular, a systematic review combining data from 31 studies showed that patients with diabetes were 36% more likely to have an impaired response to aspirin than those without diabetes and were 70% more likely to have a low drug response with aspirin 100 mg than with 101–325 mg daily25. A possible mechanism underlying HPR when receiving aspirin in patients with diabetes might be a faster recovery of platelet COX1 activity leading to incomplete thromboxane inhibition during the 24-h dosing interval. Indeed, aspirin 100 mg twice daily completely reversed the abnormal thromboxane B2 recovery in patients with diabetes27.Another area of research is investigating the possible role of HPR in response to other platelet agonists, in particular arachidonic acid, which predominantly mirrors the effects of aspirin on platelet inhibition. Diabetes has been associated with HPR in response to arachidonic acid; aspirin-naive patients with diabetes seem to have elevated levels of platelet-derived thromboxane, and aspirin might be less effective in inhibiting thromboxane synthesis in patients with diabetes than in individuals without diabetesBox 1 Mechanisms of platelet dysfunction in diabetes mellitusHyperglycaemiaIncreased expression of platelet adhesion moleculesIncreased expression of P-selectin, glycoprotein Ib, and glycoprotein IIb/IIIaReduced platelet membrane fluidity owing to changes in the lipid composition of the membrane or glycation of membrane proteinsActivation of protein kinase CInsulin resistance or deficiencyImpaired response to nitric oxide and prostacyclinIncreased formation of reactive oxygen species2+ and degranulationIncreased intracellular CaOther cellular abnormalities12 receptor) signalling, which decreases cAMP levels and lowers insulin responsivenessUpregulation of platelet P2Y purinoceptor 12  by hepatocytes and endothelial cells30.Hyperglycaemia has direct effects on gene transcription of coagulation factors, and hyperglycaemia-induced oxidative stress alters the natural vasculoprotective endothelial glycocalyx29. These changes culminate in increased thrombin generation and fibrin formation. Quantitative changes of fibrinolytic factors include increased plasma levels of PAI1 and carboxypeptidase B2 30. Levels of tissue-type plasminogen activator (t-PA) are also altered, with raised antigen concentrations and reduced t-PA activity30.Quantitative changes in coagulation factors in patients with diabetes involve higher plasma levels of fibrinogen (the soluble precursor of solid fibrin), coagulation factor VII, and coagulation factor XII; increased endothelial expression of tissue factor and tissue factor–coagulation factor VIIa complex activity; and a reduction in the anticoagulant protein tissue factor pathway inhibitor (TFPI)29. Increased thrombin generation activates coagulation factor XIII, which cross links and further stabilizes the fibrin network29. Moreover, glycation of haemoglobin in patients with diabetes alters the physiological transport and release of NO by haemoglobin itself, with impaired peripheral vasodilatation, increased insulin resistance, and pro-inflammatory and prothrombotic effects31. An overall hypercoagulable state is demonstrated by the shortening of activated partial thromboplastin time in patients with diabetes compared with individuals without diabetes29.Qualitative changes of haemostatic factors include the glycation and oxidation of fibrinogen and plasminogen, as well as the incorporation of antiplasmin, PAI1, carboxypeptidase B2, and complement C3 into the fibrin mesh, resulting in a denser clot structure and in delayed spontaneous clot lysisIn summary, our current understanding of the pathophysiological steps implicated in the increased thrombotic risk of patients with diabetes includes increased thrombin and fibrin generation, delayed clot lysis, and reduced NO bioavailability. Future therapies targeting the coagulation, fibrinolysis, and NO systems, rather than platelets alone, might open new frontiers in the prevention and treatment of cardiovascular diseases in these patients.32; therefore, aspirin was considered to be of value for primary prevention in patients with diabetes. However, a meta-analysis of 13 studies, involving a total of 45,108 patients, has subsequently shown that the cardiovascular risk of patients with diabetes without previous cardiovascular events is significantly lower than that of patients without diabetes who have previous cardiovascular disease, refuting the notion that diabetes is an equivalent risk factor to CAD33. Nevertheless, the risk of cardiovascular events in patients with diabetes is twofold to fourfold greater than in individuals of the same age and sex without diabetes35; this risk should be balanced against the bleeding risk associated with aspirin. An individual patient-level meta-analysis by the Antithrombotic Trialists’ (ATT) Collaboration indicated a similar effect of aspirin on primary prevention of major cardiovascular events in individuals with or without diabetes, with a risk ratio (RR) of 0.88 (95% CI 0.67–1.15) and 0.87 (95% CI 0.79–0.96), respectively36.Data from Haffner and colleagues supported the concept that patients with diabetes but without previous CAD have a risk of future CAD events similar to that of patients without diabetes but with previous symptomatic CAD37, individuals aged 60–85 years with hypertension, dyslipidaemia, or diabetes were randomly assigned to aspirin 100 mg daily or placebo. The 5-year primary outcome event rate ) was not different in the two groups, but treatment with aspirin significantly increased the risk of extracranial bleeding. Conversely, in the CLIPS trial38, which was performed in patients with peripheral atherosclerosis but without a history of CAD, low-dose aspirin prevented serious vascular events compared with placebo, including in the subgroups with type 2 diabetes.Other studies have involved individuals with higher cardiovascular risk profiles than those included in the ATT meta-analysis. In the JPPP trial44. Additionally, three randomized trials on primary prevention of cardiovascular disease were conducted specifically in patients with diabetes47, one of them with a 10-year follow-up48. The oldest study used higher aspirin doses (650 mg daily) versus placebo45, whereas subsequent studies used lower aspirin doses 47; none of the studies showed a significant reduction in cardiovascular events with aspirin use. A subsequent analysis of these nine studies found that aspirin treatment in patients with diabetes was associated with nonsignificant reductions in CAD (−9%) and cerebrovascular events (−11%) compared with control35. Other meta-analyses produced similar estimates50. Overall, these results suggest that aspirin, as currently prescribed, produces only a modest reduction in the risk of cardiovascular events when used for primary prevention in patients with diabetes51.Six randomized clinical studies, in which patients with diabetes were considered as subgroups , have not provided definitive results on the benefit of aspirin in primary prevention of cardiovascular disease52. Various aspirin formulations have been tested in attempts to improve its efficacy and/or limit adverse effects. An enteric-coated formulation was associated with increased rates of low responsiveness to the drug owing to reduced bioavailability compared with uncoated aspirin; this phenomenon might contribute to aspirin pseudo-resistance, with possibly lower clinical benefits in patients with diabetes receiving the coated formulation53. An extended-release, 162.5 mg formulation of aspirin was shown to provide a sustained antiplatelet effect over 24 h in patients with type 2 diabetes and multiple cardiovascular risk factors, with a favourable tolerability profile54; however, no extensive clinical data are available with this formulation. The previously mentioned increase in aspirin dose has been suggested to improve the effectiveness of the drug, but with inconsistent results and uncertain net benefit, the latter largely owing to the increased risk of adverse effects as a consequence of the reduced production of gastric prostaglandins, which protect against gastrointestinal bleeding, compared with the use of low doses of aspirin55. As discussed above, a twice-daily administration of low-dose aspirin has been tested in pharmacodynamic studies in patients with diabetes and concomitant CAD because this regimen might provide greater platelet inhibition than once-daily administration56; however, evidence of improved cardiovascular outcomes with twice-daily administration is still needed and is currently under investigation in the randomized ANDAMAN trial57.A word of caution concerns the heterogeneity and limitations of the primary prevention studies, including underpowering for sample size, differences in study designs, primary end points, risk profiles, and aspirin regimens, high rates of aspirin discontinuation during follow-up, and non-uniform reports of statin treatment prevalence. Despite these biases, the less-than-expected clinical benefit of aspirin in patients with diabetes might be explained by the presence of multiple thrombotic pathways involved in the pathogenesis of cardiovascular events and by the aforementioned platelet features 58; this finding indirectly suggests a lower suppression of platelet function by standard aspirin doses in those with diabetes than in those without diabetes. Taken together, the overall available data on aspirin in patients with diabetes indicate a modest relative reduction in the risk of cardiovascular events in primary prevention offset against a 55% relative increase in the risk of extracranial bleeding, which is mainly gastrointestinal59. Therefore, we recommend that patients aged ≥50 years with a 10-year cardiovascular risk >10% according to the Systematic Coronary Risk Estimation (SCORE) risk charts59 and without high bleeding risk should initiate aspirin. Moreover, long-term follow-up of studies of aspirin in primary prevention of cardiovascular disease showed that low doses of this drug are associated with a reduction in colorectal cancer incidence and mortality from 5 years onwards60. Although data on cancer prevention with the use of aspirin specifically in patients with diabetes are lacking, this issue needs to be addressed, because patients with type 2 diabetes have an increased risk of some types of cancer, particularly of the pancreas and the colon–rectum61. Table 62 and ACCEPT-D63) will help to assess the benefit–risk profile of low-dose aspirin in preventing multiple outcomes. The ASCEND trial62, in which adults with diabetes but no evident cardiovascular disease were randomly assigned to receive 100 mg of aspirin daily or placebo, was published in August 2018; in this study, the absolute benefits of aspirin in preventing serious vascular events were largely counterbalanced by the increased risk of bleeding.Regarding the bleeding risk of aspirin, a population-based study found a significant excess of both gastrointestinal and intracranial bleeding with aspirin compared with no aspirin use in individuals without diabetes but not in patients with diabetes64. In the setting of secondary prevention of cardiovascular disease in patients with diabetes and stable CAD, data from the ATT Collaboration on approximately 4,500 patients showed a 4.2% absolute reduction in cardiovascular events with aspirin compared with placebo at 60-month follow-up, which was similar to that observed in those without diabetes36. Clopidogrel has been extensively investigated in patients with stable CAD undergoing PCI or treated conservatively. Table 65, patients with established cardiovascular disease were randomly assigned to clopidogrel (75 mg daily) or aspirin (325 mg daily). The subgroup analysis including 3,866 patients with diabetes demonstrated that the use of clopidogrel instead of aspirin prevented 21 adverse events  per 1,000 patients per year 66. Of note, the cardiovascular protection provided by clopidogrel was even higher in the subgroup of patients with diabetes and receiving insulin therapy 66.The presence of diabetes impairs clinical outcomes in patients with cardiovascular disease, in whom meta-analyses of randomized trials indicate an overall 40% increase in major adverse cardiovascular events  compared with patients without diabetes67. At 28-month follow-up in the subgroup of patients with diabetes, protection from cardiovascular events with the use of DAPT was no greater in patients with diabetes than in those without diabetes, but the bleeding risk was higher in those with diabetes68. Of note, the use of aspirin plus clopidogrel compared with aspirin alone led to a significant reduction in the combined end point including cardiovascular death, MI, or stroke only in patients without diabetic nephropathy , whereas no significant benefit was observed in those with diabetic nephropathy 68. Therefore, the current evidence indicates that the net clinical benefit of routinely adding clopidogrel to aspirin in patients with diabetes and stable CAD treated medically is questionable because the modest ischaemic protection is outweighed by the increased risk of bleeding.The addition of clopidogrel to aspirin in stable patients was evaluated in the secondary prevention cohort  of the CHARISMA trial69 investigated the effects of preprocedural clopidogrel loading in addition to aspirin in patients undergoing elective PCI. Interestingly, in both the overall population and the subgroup with diabetes, pretreatment with 300 mg clopidogrel led to significant clinical benefit compared with downstream administration of clopidogrel, but only when the loading dose was given ≥6 h before the intervention. This finding essentially reflects the fact that several hours are needed to achieve maximal platelet inhibition after a 300 mg clopidogrel load71. Interestingly, a meta-analysis showed no reduction in MACE incidence with 12-month versus 6-month DAPT among patients with diabetes undergoing PCI with drug-eluting stent implantation for a variety of coronary syndromes72. On the basis of the available data, the antithrombotic strategies in patients with diabetes and different features of stable CAD are shown in Table The CREDO trial73 investigators explored the safety and efficacy of inhibition of thrombin generation, as provided by rivaroxaban (2.5 mg twice daily) on a background of aspirin therapy or by rivaroxaban (5.0 mg twice daily) alone in patients with stable coronary or peripheral atherosclerotic disease (91% with CAD and 62% with previous MI). This study was prematurely stopped for superiority of the rivaroxaban plus aspirin versus aspirin alone group after a mean follow-up of 23 months in terms of MACE , all-cause death, and cardiovascular death. Consistent with the overall study results, in patients with diabetes , the addition of rivaroxaban to aspirin resulted in a significantly lower incidence of MACE  with higher rates of major bleeding ; the net clinical benefit was significantly in favour of combination therapy, without increased rates of intracranial bleeding. Future guidelines on clinical practice in patients with stable CAD are likely to include recommendations derived from the COMPASS study.The COMPASS trial64. These data also indicate that, despite aspirin therapy, the recurrence of ischaemic events in patients with diabetes and an ACS is approximately 20% over a mean of 2.5 years36. Although a persistently higher platelet reactivity when taking aspirin might partly explain this elevated recurrence rate74, the randomized CURRENT-OASIS 7 trial75 demonstrated no outcome improvement with higher-dose aspirin (300–325 mg daily versus 75–100 mg daily for 30 days) in a mixed population of patients with or without diabetes who were admitted to hospital for ACS, with no significant interaction between primary outcome and diabetic status.The clinical benefit provided by antiplatelet therapy in patients with diabetes and an ACS has been clearly demonstrated. Table 12 platelet receptor on a background of aspirin therapy, aimed at further reducing adverse events in patients with ACS, was first evaluated in the CURE trial76, in which patients with non-ST-segment elevation (NSTE)-ACS already receiving aspirin were randomly assigned to clopidogrel (300 mg loading dose followed by 75 mg daily maintenance dose) or placebo for a mean duration of 9 months. A lower rate of MACE was observed with the combination of clopidogrel and aspirin regardless of diabetic status; although no significant interaction was present, the relative reduction of adverse cardiovascular events at 1 year with clopidogrel in the 2,840 patients with diabetes was numerically lower than the reduction in those without diabetes (14% versus 20% relative risk reduction). This finding might be a result of the more pronounced interindividual variability of clopidogrel responsiveness in diabetes, resulting in a higher prevalence of HPR79.Additional inhibition of the P2Y12 inhibition in patients with diabetes might include impaired drug metabolism leading to reduced active metabolite generation as well as dysregulation of the P2Y12 signalling pathway81. The OPTIMUS-3 study82 found that, in patients with diabetes, prasugrel was associated with faster and greater platelet inhibition, as well as lesser interindividual variability, than clopidogrel, even when the latter was given at high doses (600 mg loading plus 150 mg daily). Similar pharmacodynamic results were obtained with ticagrelor83. Given the pharmacodynamic drawbacks of clopidogrel in patients with diabetes, which are potentially overcome with the newer, more potent P2Y12 inhibitors, the subgroup analyses of diabetic populations from phase III studies comparing prasugrel and ticagrelor versus clopidogrel in the setting of ACS are of particular interest.As discussed above, the mechanisms behind poor clopidogrel-induced P2Y84, the use of prasugrel instead of standard-dose clopidogrel after PCI for ACS led to a higher relative risk reduction of MACE  at 15 months in patients with diabetes than in those without diabetes . Importantly, prasugrel did not increase the rates of major bleeding in the diabetes group. In this trial, the greatest reduction in adverse events with prasugrel was observed in patients receiving insulin therapy (37% relative risk reduction versus 26% in patients with diabetes who were not receiving insulin)84. The TRILOGY-ACS study85 included patients with NSTE-ACS managed without revascularization and randomly assigned to receive prasugrel or clopidogrel for up to 30 months in addition to aspirin. A subgroup analysis according to diabetes status showed no difference in the incidence of MACE  with either antiplatelet strategy in patients either with  or without  diabetes86. In the PLATO trial87, patients with ACS were randomly assigned to receive ticagrelor (180 mg loading dose and then 90 mg twice daily) or clopidogrel (300 or 600 mg loading dose and then 75 mg daily), irrespective of the subsequent therapeutic strategy. A lower rate of MACE at 1 year with ticagrelor was observed both in patients with diabetes and in those without diabetes . This ischaemic protection largely outweighed the elevation in non-CABG surgery-related major bleeding observed in the ticagrelor group. Of note, the use of ticagrelor had a strong effect in decreasing MACE, all-cause death, and stent thrombosis in patients with diabetes and haemoglobin A1c (HbA1c) ≥6%.In the TRITON-TIMI 38 trial88. A clinical comparison between prasugrel and ticagrelor is currently being investigated in the ongoing ISAR-REACT 5 trial89, in which diabetes is a predefined subgroup90. On the basis of the available evidence, we recommend consistent and highly effective platelet inhibition with the newer, more potent P2Y12 inhibitors for patients with ACS and concomitant diabetes, given their high baseline risk profile; therefore, DAPT with aspirin plus prasugrel or ticagrelor should be the first-line antiplatelet strategy up to 1 year in this setting, especially in patients without high bleeding risk (Table A pharmacodynamic comparison of prasugrel versus ticagrelor in patients with diabetes and CAD showed that ticagrelor exerts similar or greater inhibition of ADP-induced platelet reactivity than prasugrel in both the acute and the chronic phases of treatmentsk Table .91 explored the efficacy and safety of prolonging DAPT beyond 1 year with aspirin plus a thienopyridine (prasugrel or clopidogrel) versus aspirin alone for 18 more months in patients with stable CAD or ACS who had undergone implantation of a drug-eluting stent. In the subgroup analysis according to diabetes status , a significant interaction existed between MI risk reduction and prolonged DAPT in favour of patients without diabetes 92. Regardless of diabetes status, the rate of stent thrombosis was lower and the rate of bleeding events was higher in the DAPT group 92. Of note, withdrawal of thienopyridine in both groups resulted in a consistent numerical increase in early ischaemic events in patients with or without diabetes92.Given the poorer long-term cardiovascular outcomes after MI in patients with diabetes than in those without diabetes, DAPT prolongation is a particularly relevant issue in this setting. The DAPT study93 explored the efficacy and safety of DAPT prolongation  in patients with a history of MI (in the past 1–3 years). The reduction in MACE with either ticagrelor dose was consistent in patients with or without diabetes. Given their higher risk of events, patients with diabetes had a greater absolute benefit from ticagrelor, with a 3-year number needed to treat for MACE (pooling results of both ticagrelor doses) of 67 compared with 91 in those without diabetes. Additionally, in patients with diabetes, ticagrelor reduced the rate of cardiovascular death by 22%. As in patients without diabetes, ticagrelor significantly increased the rates of nonfatal major bleeding in patients with diabetes . Regardless of diabetic status, the net clinical outcome was better with the 60 mg than with the 90 mg ticagrelor dose — that is, similar ischaemic protection but lower bleeding risk.The PEGASUS-TIMI 54 trial94, where approximately 20,000 patients with type 2 diabetes and CAD, but without previous MI, are being randomly assigned to either ticagrelor (90 mg or 60 mg twice daily) or placebo for up to 4 years. As we wait for more evidence on the net clinical benefit of more aggressive antithrombotic therapies in patients with diabetes and ACS, especially in the long term, we summarize our therapeutic recommendations, which are based on currently available data, in Table The use of ticagrelor for secondary prevention of cardiovascular disease is being investigated specifically in patients with diabetes in the THEMIS trial12 inhibitor (triple therapy) to achieve a more complete, multi-pathway platelet inhibition.Given the hyperreactivity of platelets in patients with diabetes, other antiplatelet strategies have been tested to improve cardiovascular outcomes in these individuals with CAD. Specifically, additional drugs have been investigated as adjunctive treatment to aspirin alone or to aspirin plus a P2Y95, was evaluated in pharmacodynamic studies as part of a triple-therapy strategy in patients with diabetes and CAD. The OPTIMUS-2 study96 found that the addition of cilostazol to DAPT improved inhibition of platelet P2Y12 signalling. Moreover, cilostazol might decrease platelet aggregability in patients with diabetes and HPR when taking clopidogrel97. Clinical benefits of cilostazol in addition to DAPT were observed in a nonrandomized study of Asian patients undergoing PCI, mainly in terms of reduced rates of target-lesion revascularization and stent thrombosis98; in randomized studies, this improvement in outcomes was found to be greatest for patients with diabetes101. Other investigations have evaluated the effects of cilostazol in the setting of PCI for ACS. In the retrospective Korean Acute MI Registry, 4,203 patients with ST-segment elevation MI undergoing primary PCI received either DAPT (aspirin plus clopidogrel) or triple therapy (DAPT plus cilostazol)102; the latter strategy was associated with a significantly lower incidence of cardiac death, all-cause death, and MACE at 8-month follow-up. Interestingly, subgroup analysis showed that patients who were older (aged >65 years), female, or had diabetes derived greater benefit from triple therapy102. In another study, 1,212 Chinese patients with ACS undergoing PCI were randomly assigned to DAPT (aspirin plus clopidogrel) or DAPT plus cilostazol for 6 months103. Triple therapy resulted in a 35% relative reduction in MACE, without excess bleeding; the subgroup of 263 patients with diabetes derived particular benefit from the addition of cilostazol 103.Cilostazol, a selective and reversible inhibitor of phosphodiesterase 3 that has effects on various cell lines, including platelets, by increasing intracellular cAMP concentrations104. Among these drugs, terutroban and picotamide have been investigated in patients with cerebrovascular disease or PAD (see section below). Pharmacodynamic studies have evaluated EV-077, a thromboxane receptor antagonist and thromboxane synthase inhibitor, in patients with diabetes and stable CAD receiving monotherapy with aspirin or clopidogrel. In this setting, EV-077 provided further platelet inhibition through multiple signalling pathways; however, no specific in vivo data are available for EV-077105. Ridogrel, a thromboxane A2 (TXA2) synthase inhibitor and TXA2–prostaglandin endoperoxide receptor antagonist, was investigated in 907 patients with MI treated with thrombolysis and enrolled in the RAPT study106. Ridogrel was found to be not superior to aspirin in increasing the fibrinolytic efficacy of streptokinase, as assessed before discharge. In a post hoc analysis, fewer recurrent ischaemic cardiac events occurred in the ridogrel group, without an excess in serious bleeding complications. No specific data in the subgroup with diabetes are available from this investigation.Other attractive antiplatelet agents are thromboxane antagonists that potentially block the interaction of both aspirin-sensitive and aspirin-insensitive agonists, theoretically providing potent platelet inhibition, especially in patients with diabetes who show both platelet hyperreactivity and increased thromboxane-dependent platelet activation87. This situation is likely to be the direct consequence of endothelial dysfunction, oxidative stress, vascular inflammation, abnormal platelet reactivity, and decreased responsiveness to antiplatelet agents, but increased thrombin generation might also have a role107. Thrombin is a potent platelet agonist; therefore, thrombin inhibition might provide substantial suppression of platelet function in addition to reduced fibrin production. Inhibition of the effects of thrombin can be achieved by direct thrombin inhibitors, such as bivalirudin or dabigatran; by direct factor Xa inhibitors, such as apixaban, edoxaban, or rivaroxaban; or by platelet proteinase-activated receptor 1 (PAR1) antagonists, such as vorapaxar.Despite DAPT, patients with diabetes and established atherosclerosis have a high residual risk of recurrent events108 with rivaroxaban (2.5 mg twice daily) and in the TRA 2°P-TIMI 50 trial109 with vorapaxar. In both studies, the newer agent under investigation was compared with placebo on a background of DAPT with aspirin plus clopidogrel. In the ATLAS ACS–TIMI 46 trial108, no significant interaction was observed between prevention of MACE, reduction in mortality, and increase in bleeding by rivaroxaban and diabetic status. In the TRA 2°P-TIMI 50 trial109, the net clinical benefit with vorapaxar in the population with previous MI was numerically more pronounced in patients with diabetes  than in those without diabetes . Various factors have precluded the widespread integration of the findings from these two studies into clinical practice. These factors include the delayed initiation of the additional antithrombotic agent after the coronary event, the lack of comparison data between rivaroxaban or vorapaxar in addition to DAPT with aspirin plus clopidogrel or aspirin plus either prasugrel or ticagrelor, and the bleeding concerns associated with triple antithrombotic therapy.The effects of thrombin inhibition on cardiovascular outcomes in patients with ACS have been investigated in the ATLAS ACS–TIMI 46 trial110. Despite advances in pharmacotherapy, the optimal antiplatelet regimen for patients with diabetes and cerebrovascular disease (with or without concomitant CAD) remains debated because the use of more potent antithrombotic drugs after a stroke or transient ischaemic attack (TIA) might cause a considerable increase in the risk of intracranial bleeding111, casting uncertainty on the risk–benefit ratio in the setting of secondary prevention. No specific, large study on aspirin for secondary prevention of stroke in patients with diabetes exists, so the available data come from subgroup analyses of more general studies. Two large meta-analyses of cardiovascular secondary prevention trials, including patients with a previous stroke, indicate that the clinical benefit of aspirin compared with placebo is consistent in patients with or without diabetes64. As mentioned, in the CAPRIE trial65, the reduction of cardiovascular events with clopidogrel versus aspirin was more pronounced in patients with diabetes, and one-third of the patients in CAPRIE had a previous stroke or TIA.Diabetes is independently associated with an increased risk of ischaemic stroke, and the presence of diabetes after a stroke negatively affects recovery and predisposes to recurrent cerebral events12 inhibitors are associated with an increased clinical benefit in patients with diabetes and cerebral events. In randomized trials, the combination of low-dose aspirin and extended-release dipyridamole (ERDP) (400 mg daily) after a stroke showed a clinical benefit compared with aspirin alone in the ESPS-2 study112 and the ESPRIT trial113, in which patients with diabetes composed 15% and 18% of the trial cohorts, respectively. Conversely, outcomes after ischaemic stroke were similar with aspirin plus ERDP versus clopidogrel in the PRoFESS trial114 regardless of diabetic status . The use of ERDP in clinical practice is currently discouraged and limited by the high incidence of adverse effects, such as headache and gastrointestinal symptoms.The concept that the residual risk of recurrent events in patients with diabetes might be related to higher on-treatment platelet reactivity when receiving single antiplatelet therapy has led to investigations aimed at assessing whether DAPT or the newer P2Y67, >40% of patients had diabetes and approximately 36% had a previous stroke or TIA at randomization. In the subgroup analysis restricted to 4,320 patients with previous ischaemic stroke or TIA, the addition of clopidogrel was associated with a trend towards a reduction in recurrent stroke 115. Among the overall population of the CHARISMA study67, the incidence of intracranial bleeding was similar (0.3%) in patients receiving aspirin plus clopidogrel or aspirin alone, and no significant difference in severe bleeding emerged in the subgroup analysis of patients with prior cerebrovascular disease 115. The efficacy and safety profile of the aspirin plus clopidogrel combination from these analyses formed the basis for subsequent specific randomized studies.In the CHARISMA trial116 investigators randomly allocated patients with stroke or TIA and at least one cardiovascular risk factor to clopidogrel versus aspirin plus clopidogrel. No significant reduction in stroke recurrence was observed in the DAPT group, whereas bleeding complications were increased. These findings were consistent in groups defined according to diabetes status. Conversely, in the CHANCE trial117, selectively performed in a Chinese population of 5,170 patients with minor stroke or TIA, DAPT with aspirin plus clopidogrel, given for 21 days after the index event, decreased the rate of recurrent stroke at 90 days compared with aspirin alone, without a significant increase in bleeding events, irrespective of diabetes status. The POINT trial118 investigated aspirin alone (50–325 mg daily) versus aspirin plus clopidogrel in patients with minor ischaemic stroke or high-risk TIA. The primary efficacy end point was a composite of ischaemic stroke, MI, or vascular death at 90 days. In the DAPT group, a significant 25% relative reduction in the main outcome measure and a 2.3-fold increased risk of major bleeding were observed. However, no outcome data according to diabetes status are so far available.The MATCH trial119 for the prevention of cerebral and cardiovascular events in patients with previous stroke or TIA. The incidence of the primary end point  at 28 months was 11% in both groups . Similar results were obtained in the subgroup of 5,299 patients with diabetes. However, a significant increase in any bleeding events was observed in patients receiving terutroban ; therefore, this agent is not an alternative to aspirin in patients with cerebral ischaemic events.Terutroban, a selective thromboxane–prostaglandin receptor antagonist, was compared with aspirin in the PERFORM trial120. Ticagrelor was not superior to aspirin in reducing the primary end point of stroke, MI, or death at 3 months in either the overall population or the subgroup (25% of patients) with diabetes .In the context of the newer, more potent antiplatelet agents, a history of any stroke or TIA is a contraindication for prasugrel therapy. Conversely, ticagrelor, which is contraindicated only after a haemorrhagic stroke, has been tested (180 mg loading dose followed by 90 mg twice daily for 90 days) versus aspirin in 13,199 patients within 24 h of a non-severe stroke or high-risk TIA in the SOCRATES trialIn summary, the available data do not provide clear evidence in support of DAPT  or of any more potent antiplatelet agent in patients with diabetes and ischaemic cerebral events. Antiplatelet approaches in this setting are shown in Table 121. The prevalence of PAD is higher among patients with diabetes, and in these individuals, PAD is more severe, progresses more rapidly, and entails a higher risk of recurrent ischaemic events and amputation than in those without diabetes122. In addition, PAD is a potent marker of systemic atherosclerosis and increased cardiovascular risk122. Data on antiplatelet strategies in this setting are limited and are often derived from subgroup analyses of investigations devoted primarily to the management of patients with CAD, in whom PAD was often asymptomatic or not clinically apparent.Up to one-third of patients with PAD have diabetes123. This analysis was heavily driven by two studies performed on patients with diabetes, the POPADAD study47  and the VA-Cooperative trial124 (n = 231), which both showed no benefit of aspirin or of aspirin plus dipyridamole versus placebo. Various aspects might explain the apparent lower efficacy of aspirin in patients with PAD and diabetes. First, current evidence might be underpowered. Alternatively, or additionally, these patients might have phenotypic and biological differences from those with predominant CAD. For instance, the presence of PAD might render platelet activation more critically dependent on ADP than on TXA2 production104. Third, as stated above, the pharmacodynamics of aspirin might differ in patients with diabetes compared with those without diabetes24. Finally, the hypothesis that the inhibition of COX1-dependent TXA2 biosynthesis is insufficient in this setting is substantiated by data indicating that the use of picotamide, an inhibitor of both TXA2 receptors and TXA2 synthase, might increase survival versus aspirin in patients with diabetes and PAD 125.In a meta-analysis of 18 trials, involving approximately 5,000 patients with PAD, those receiving aspirin had a modest, nonsignificant 12% lower risk of cardiovascular events than those receiving placebo65, approximately one-third of whom had diabetes, clopidogrel recipients had a significant 24% and 22% relative risk reduction in cardiovascular mortality and cardiovascular events compared with aspirin, respectively, with a similar benefit in the subset of patients with diabetes. A post hoc analysis of the CHARISMA study126 on 3,096 patients with PAD demonstrated no benefit with aspirin plus clopidogrel versus aspirin alone in terms of reduction of the composite MACE end point; significant decreases in the rates of MI  and hospitalization for ischaemic events  were observed in the DAPT group at the expense of a twofold increased risk of minor bleeding. No specific outcome data are available in the subgroup of patients with PAD and concomitant diabetes in this trial.In an analysis of 6,452 patients with PAD included in the CAPRIE trial127. In the PEGASUS-TIMI 54 trial128, 1,143 patients (5%) had known PAD at randomization, and 42% of these had diabetes. Given the high baseline risk profile of this PAD subgroup, the addition of ticagrelor to aspirin  was associated with a greater absolute reduction in MACE in these patients than in patients without PAD (4.1% at 3 years and a number needed to treat of 25 versus 1.0% at 3 years and a number needed to treat of 100) without a significant increase in major bleeding128. Of note, the use of ticagrelor in patients with PAD significantly reduced major adverse limb events . No specific outcome data with ticagrelor plus aspirin versus aspirin alone have been published in the subgroup of patients with PAD and concomitant diabetes in this trial.The presence of PAD impairs cardiovascular prognosis in patients with a history of MI, leading to the rationale that multi-pathway platelet inhibition might improve clinical outcome in this setting129, ticagrelor was shown not to be superior to clopidogrel in reducing major cardiovascular and limb events, with similar rates of major bleeding. Among patients with symptomatic PAD (27% with diabetes) from the TRA 2°P-TIMI 50 trial131, the use of vorapaxar versus placebo did not significantly decrease MACE but did significantly reduced acute limb ischaemia and the rate of peripheral revascularization; the benefit in limb events was accompanied by an increased risk of bleeding, yielding an uncertain net benefit of vorapaxar in patients with PAD.In patients with symptomatic PAD (more than one-third with diabetes) from the EUCLID trial133  or CAD and occurred regardless of diabetic status; in particular, patients with PAD and diabetes receiving rivaroxaban plus aspirin had a twofold higher absolute reduction of the composite end point, including cardiovascular death, MI, and stroke, than patients with PAD but without diabetes. Of note, in the PAD population of the COMPASS study, combination therapy was also associated with a significant 46% relative reduction in major adverse limb events, including major amputation. These findings are likely to influence future guideline recommendations. The ongoing VOYAGER PAD trial135, an international, randomized, double-blind, placebo-controlled study, is evaluating the efficacy and safety of the vascular dose of rivaroxaban (2.5 mg twice daily) in patients with symptomatic PAD undergoing peripheral surgical and/or endovascular revascularization136.While acknowledging the paucity of data from specific studies, indications concerning antiplatelet therapy for cardiovascular prevention in patients with diabetes and PAD reflect the recommendations of the AHA/ACC and ESC guidelines on the general population of patients with PAD137. In the ACCORD trial138, patients with new-onset AF and diabetes had an increased risk of all-cause death (HR 2.65), MI (HR 2.10), and heart failure (HF) (HR 3.80) compared with those without diabetes. The presence of diabetes raises the incidence of thromboembolic events (stroke or systemic embolism) in patients with AF, explaining the inclusion of diabetes in contemporary scores for stroke prediction139. In a meta-analysis of seven studies including >12,000 patients with AF, an overall 70% relative increase in the risk of thromboembolic complications was observed in patients with versus those without diabetes, with a yearly incidence ranging from 3.6% to 8.6%140. This variability reflects differences in study designs, definitions of outcome measures, patients’ baseline risk profile, concomitant therapies, and types of diabetic populations included. Analyses have identified a duration of diabetes >3 years as an independent predictor of ischaemic stroke, which is a stronger predictor than glycaemic control141. A subgroup analysis from the European PREFER in AF registry, involving >5,000 patients with AF, showed that patients with diabetes receiving insulin therapy had a more than twofold higher risk of stroke or systemic embolism at 1 year than patients without diabetes, whereas diabetes not treated with insulin did not entail a significantly increased risk142.Diabetes increases the risk of developing AF and worsens the prognosis of patients with AF143. In the past decade, NOACs have revolutionized antithrombotic therapy to prevent AF-related thromboembolic events because of their favourable characteristics: predictable dose–response, rapid offset and onset, fixed doses, no interaction with food, limited interactions with other drugs, and no need for routine monitoring. A meta-analysis of four phase III trials comparing NOACs versus warfarin in patients with AF showed NOACs to be associated with a significant 19% relative reduction in the combined end point including any stroke or systemic embolism (P < 0.0001) and a 14% relative reduction in major bleeding (P < 0.06) compared with well-managed warfarin144. Among 71,683 patients receiving NOACs, the prevalence of diabetes ranged from 23.3% (in the RE-LY study) to 40.0% (in the ROCKET-AF study). Of note, in these trials, blood glucose levels and HbA1c values were not routinely available, and patients with a creatinine clearance <30 ml/min were excluded. Therefore, patients with severe diabetic nephropathy, who are at even higher risk of cardiovascular complications, were excluded145. In these phase III studies, no interaction was found between diabetes status and clinical efficacy of the NOACs versus warfarin, although the safety superiority of apixaban versus warfarin was lost among patients with AF and diabetes (P = 0.003 for interaction)145. Moreover, a study-level meta-analysis of these trials added more robust evidence on the topic146 146. Also, in patients with diabetes, use of NOACs versus warfarin significantly reduced the rate of cardiovascular death 146. To date, no data on a direct comparison between different NOACs in patients with diabetes are available; therefore, the choice of NOAC in patients with diabetes is not supported by specific evidence but should be guided by general principles and take into account the comorbidities associated with diabetes. On the basis of the available data, we recommend antithrombotic strategies for patients with AF and diabetes , and TECOS167 studies were among the first cardiovascular outcome trials evaluating a new class of glucose-lowering drugs, the dipeptidyl peptidase 4 (DPP4) inhibitors. All these studies met the primary noninferiority end point for cardiovascular safety of these drugs versus placebo in patients with diabetes and established cardiovascular disease. However, the use of DPP4 inhibitors was not associated with significant benefits on the prespecified measures of cardiovascular outcome. Conversely, a signal of increased risk of HF with saxagliptin was highlighted in the SAVOR-TIMI 53 trial166, and a post hoc analysis of the EXAMINE trial168 indicated a nonsignificant 0.9% higher absolute incidence of hospitalization for HF in the alogliptin group.The EXAMINE169 were a major breakthrough in the field, showing a significant 14% relative risk reduction in the rate of the composite primary end point, including cardiovascular death, MI, or stroke, with empagliflozin, an inhibitor of the sodium/glucose cotransporter 2 (SGLT2), causing glycosuria. Favourable cardiovascular effects have been subsequently obtained with another drug in the same class, canagliflozin170. Of note, the use of either empagliflozin or canagliflozin led to a significant decrease in hospitalizations for HF.The results of the EMPA-REG OUTCOME trial172, but not with lixisenatide173. Finally, a borderline significant (P = 0.06) 0.9% absolute reduction in the primary cardiovascular outcome measure was found with exenatide in the EXCEL study174. Of note, unlike SGLT2 inhibitors, treatment with GLP1R agonists resulted in significant protection from individual components of the composite outcome measure (MI and stroke) but was neutral in terms of rates of HF.A lower incidence of cardiovascular events has been demonstrated with two different glucagon-like peptide 1 receptor (GLP1R) agonists, liraglutide and semaglutide1c, the new glucose-lowering drugs have different effects on cardiovascular outcomes, with potential mechanisms of cardiovascular protection that are independent of glucose control (Fig. 176. Conversely, SGLT2 inhibition resulted in a consistent reduction in HF-related events, suggesting that this class of agents acts through different pathways; however, mechanistic studies are needed to clarify the pathways of cardiovascular protection of these compounds. In particular, data on the effect of SGLT2 inhibitors on platelet function and thrombosis are lacking.Taken together, these data indicate that, despite a similar control on levels of HbArol Fig. . The enc12 inhibitors in patients with ACS, DAPT prolongation beyond 1 year in patients with MI, and short-term DAPT in selected patients with acute ischaemic stroke. However, the expected ischaemic protection from antithrombotic drugs must always be weighed against the risk of drug-related bleeding. Available evidence on the efficacy and safety of antithrombotic drugs in patients with diabetes at risk of or with cardiovascular diseases often derives from underpowered observational studies or subgroup analyses of trials not specifically performed in patients with diabetes; therefore, large investigations specifically focused on patients with diabetes are needed and should be encouraged.Patients with diabetes have a prothrombotic state and a higher risk of cardiovascular events than those without diabetes. Antithrombotic therapies in these patients might be associated with a higher absolute reduction of recurrent events than in patients without diabetes. More aggressive antithrombotic strategies might be specifically indicated in patients with diabetes and cardiovascular diseases, including the use of the newer, more potent P2Y


Obesity is a major public health concern worldwide. The increased risk of certain types of cancer is now an established deleterious consequence of obesity, although the molecular mechanisms of this are not completely understood. In this review, we aim to explore the links between MAPK signalling and obesity-related cancer. We focus mostly on p38 and JNK MAPK, as the role of ERK remains unclear. These links are seen through the implication of MAPK in obesity-related immune paralysis as well as through effects on the endoplasmic reticulum stress response and activation of aromatase. By way of example, we highlight areas of interest and possibilities for future research in endometrioid endometrial cancer and hepatocellular carcinoma associated with non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH) and MAPK. Obesity is a major public health concern that develops as a result of chronic caloric excess. The excess energy is stored as lipid in adipose tissue and may accumulate in other metabolic organs (such as the liver) and skeletal muscle. Increased lipid accumulation significantly alters the normal metabolic milieu and creates an environment that chronically transmits a signal of nutrient excess to the cell, with multiple consequences, many of which are poorly understood.Global obesity rates have been increasing dramatically over the last number of years, with rates now almost triple what they were in 1975. According to the World Health Organisation, there were 1.9 billion overweight adults worldwide in 2016, with 650 million of those classified as obese, representing 13% of the world’s population. Once considered a problem confined to the developed world, obesity rates are also increasing in middle- and low-income countries with a fifty percent increase in the numbers of overweight children in Africa since the year 2000 [The effect of obesity on cardiovascular disease and diabetes is well established, however the association between obesity and increased risk of certain types of cancer is a relatively new observation and the underlying mechanisms have not been fully elucidated. Improved understanding of the molecular interactions between obesity and cancer is important if any future public health interventions are to be successful in reducing population obesity levels and the associated cancer risk.MAPK pathways are signalling modules that transduce extracellular and intracellular signals to regulatory networks within the cell via the phosphorylation of key protein targets. Each cascade is activated by extracellular signals which lead to the activation of the MAPK through successive activations of MAPK kinase kinase (MAPKKK) and MAPK kinase (MAPKK). There are three main families of MAPK—the extracellular signal regulated kinases (ERKs), the Jun amino terminal kinases (JNKs) and the stress activated protein kinases p38/SAPKs). The ERKs respond primarily to growth factors and mitogens to induce cell growth and differentiation, while the JNKs respond primarily to stress such as ionising radiation and oxidative stress and are involved in apoptosis, cytokine production, inflammation and metabolism. Similarly, the p38 MAPKs are strongly activated by cytokines and cellular stress. The activation of the p38 MAPK pathway contributes to inflammation, apoptosis, cell differentiation and cell cycle regulation [8/SAPKs. In this review, we discuss the relationship between obesity and cancer and the mechanisms underlying obesity-related carcinogenesis, with a focus on mitogen-activated protein kinase (MAPK) signalling. To do this, we have chosen endometrial cancer as a case study, for the following reasons, (a) the epidemiological association between obesity and endometrial cancer is irrefutable, and (b) weight loss reduces endometrial cancer. We also discuss the role of obesity and MAPK in hepatic steatosis.Epidemiological evidence linking obesity to cancer development is consistent and compelling for cancer of the oesophagus (adenocarcinoma), kidney, breast , colon, rectum, endometrium, liver, thyroid, gallbladder, ovary and pancreas ,4; howev2 [2, the risk of death attributable to cancer is higher than those with a normal BMI, although most of the observed decrease in life expectancy in this group is attributable to cardiovascular events [Overall there is a modest but important increase in the relative risk of developing any cancer in the order of 1.06–1.62 for each increment of body mass index BMI of 5 kg/m2 . In 2012r events . Of these confounders, metabolic dysfunction may be the most important confounder. The concept of healthy obese patients, i.e., those with no evidence of diabetes, hypertension or other co-morbidities, has gained a lot of traction in recent years. As a result, there has been an increased interest in the role of metabolic aberrations in cancer risk and outcome. A number of studies have demonstrated that individuals with metabolic syndrome and diabetes have an increased risk of cancer of the pancreas, rectum and endometrium ,9,10. InThe epidemiological links between obesity, metabolic dysfunction and cancer development are clear. What, then, is the impact of weight loss in terms of cancer risk? Observational studies have shown a decrease in cancer-related mortality for those who lose weight, to a larger extent for women than for men ,19. Largn = 6) prospective cohort study demonstrated the ability of metabolic surgery to reverse complex atypical hyperplasia of the endometrium, a known endometrial cancer precursor. Hyperplasia resolved in the majority of cases  with 3/6 (50%) resolving with metabolic surgery alone [A recent small  play a key role. The IGF1/PI3K-Akt-mTOR pathway plays an important role in the regulation of cellular growth, and the proliferation and differentiation of cells in multiple organs ,32. BindObesity is a state of chronic inflammation. This inflammation has been implicated in the development of cancer in obese individuals . Use of Adipose tissue secretes several important cytokines involved in mediating this low-grade inflammation. The most important of these are adiponectin and leptin . AdiponeAll forms of MAPK are involved in the innate immune response to pathogen infection and tissue damage in the normal, non-cancerous state. This occurs through activation of pattern recognition receptors (PRRs) on the cell surface and in the cytoplasm of immune cells. Most MAPKs are activated via Toll-like receptors (TLRs) which, through a series of downstream factors, activate MAPKKK, MAPKK and MAPK, which in turn lead to the increased production of pro-inflammatory cytokines such as TNF and IL1beta as well as recruitment of M1 macrophages , all of Obesity-related immune dysfunction may limit anti-tumour immune responses. Although obesity causes a pro-inflammatory response, as evidenced by the expansion and proliferation of pro-inflammatory macrophages in adipose tissue ,51 with A clear understanding of the mechanisms underlying immune dysfunction may improve our understanding of how the obese state allows tumours to evade immune surveillance in certain cancer types. Obesity induces both cell-specific, and global transcription changes. Chief among the global transcriptomic alterations induced by obesity are changes in intrinsic metabolic pathways ,59. In bRecent elegant studies have highlighted the crucial role of intrinsic cellular metabolism in driving immune cell functions ,64. ThesThe interaction between immunometabolism and MAPK signalling in tumour infiltrating leukocytes has not been investigated in detail, however monocyte adhesion and activation are dependent on glycolysis, which causes the activation of p38 MAPK . TherefoThe role of MAPK signalling in immune cells is further highlighted by recent findings in the MAIT cells. These are a population of non-MHC-restricted T cells which are responsible for the response to viral and bacterial infection through rapid production of various cytokines , whose eThe mTORC1 signalling complex acts as both a nutrient sensor and a metabolic regulator and is intrinsically linked to glycolytic metabolism in effector lymphocytes ,68. mTORFurther links between immune cells, obesity and MAPK have been demonstrated through examining the effects of leptin in T helper immune cells, albeit in allergic airways disease. Leptin activated mTORC1 and induced ERK and p38 MAPK phosphorylation in TH1 and TH2 cells, and both MEK and mTOR inhibitors blocked its effect on T helper cell proliferation, survival and cytokine production ,73. TakeIn addition to the evolving obesity-immunometabolism story, the role of the gut-brain axis has received a lot of attention in the obesity literature but has been relatively under-investigated in the cancer literature. The gut–brain axis consists of multidirectional communication between the enteric nervous system (ENS) and the central nervous system (CNS), linking reward centres of the brain with peripheral intestinal functions and is dependent on multiple endocrine and paracrine processes . Animal The intestinal microbiome is another important growth area highlighting the relationship between obesity, cancer and the MAPK pathway. The composition of the gut microbiome varies between individuals and in response to dietary intake. It lives in a mutualistic relationship with the host and is a key contributor to the hosts metabolism through the creation of vitamins and other substrates required for its physiology. Changes in the composition and function of the microbiota—referred to as gut microbiota dysbiosis—have been implicated in human diseases such as gastrointestinal disorders, cardiovascular disease, neurological disease and cancer . ObesityThe microbiome is also involved in changes in gut inflammation, and again the MAPK pathway is potentially involved here with dietary-induced changes in the gut affecting T cell proliferation and differentiation via the p38 and JNK pathways . This shHormonal influences also play an important role in the pro-neoplastic extra-cellular milieu associated with obesity. A report from the International Agency for Research on Cancer concluded that there was strong evidence for chronic inflammation and sex hormone metabolism mediating the relationship between obesity and cancer, whilst evidence for insulin and IGF signalling was moderate .Adipose tissue is a source of adipokines, inflammatory cytokines and hormones which can promote tumour development or growth. Amongst other actions, inflammation increases local expression and activity of aromatase ,85, the Up to 80% of endometrial and breast cancers express the oestrogen receptor ERα. The selective ERα modulator tamoxifen is a key component of treatment for ERα-positive breast cancer, however it increases the risk of endometrial cancer (reviewed in ), suggesXBP1 is an oestrogen-responsive gene ,106, howXBP1 is classically associated with ER stress. The endoplasmic reticulum is an intracellular organelle with two constituent parts—the smooth ER which has no ribosomes attached and the rough ER which has abundant ribosomes present . Rough EThe UPR is also active in many tumours. ATF6 and other upstream factors involved in ER stress signalling such as X-box-binding-protein 1 (XBP1), as well as targets of the response signalling pathway such as BiP (an ER chaperone), C/EBP homologous protein (CHOP) and others have been shown to be upregulated in cells of breast cancers , hepatocAll three MAPK pathways respond to ER stress, each with different pathways and outcomes ,123. IRECHOP links MAPK and ER stress through p38 MAPK-dependent phosphorylation. This pathway induces transcriptional changes in certain cell lines although the exact genes involved are as yet unknown . JNK MAPThere are links between ER stress and MAPK signalling in specific diseases also. In melanoma cells, hepatocellular carcinoma cells  and breaObesity is a dominant risk factor for development of non-alcoholic fatty liver disease (NAFLD), and the increase in obesity is linked to the rising rates of both NAFLD and non-alcoholic steatohepatitis (NASH) globally . ObesityThere are four isoforms of p38 MAPK—p38 alpha, beta, delta and gamma—each of which is expressed differently in individual cells and has varied and often opposite effects even within the same cell on the same substrate . P38 MAP2) population those with hepatic steatosis demonstrated higher expression of these isoforms of p38 MAPK when compared with those who did not have liver disease. A lack of these isoforms has been found to be protective against steatosis in animal models [The p38γ and p38δ isoforms are the only isoforms to have been shown to be elevated in obese people with NAFLD when compared to non-obese people without NAFLD . Furtherl models . Mice knMAPK phosphatases are a group of 10 catalytically active enzymes which are involved in dephosphorylation and inactivation of MAPK isoforms in mammalian cells and tissues. MKP1—the first of these enzymes to be discovered—antagonises p38 MAPK and JNK. Diet-induced obesity increased the hepatic expression of MKP1 in mice that developed steatosis. Liver-specific deficiency of MKP1 results in increased p38 and JNK MAPK activity in mouse models . This isThe JNK MAPK pathway consists of three proteins—JNK 1, JNK 2 and JNK 3. JNK 1 and 2 are ubiquitously expressed while JNK 3 is restricted to heart, brain and testes . SimilarAs JNK3 is not expressed in the liver, studies of the roles of JNK in hepatic metabolism have focused on the JNK1 and JNK2 isoforms. In some studies of obese humans with NASH, hepatic JNK activity has been found to be elevated ,167. TheHepatic lipid metabolism is also affected by JNK MAPK signalling. Post prandial increases in insulin levels which serve to inhibit the synthesis and storage of lipids are accompanied by a decrease in JNK activity . In miceThis pathway is the best understood of the mammalian kinase pathways. Similar to p38 and JNK, ERK 1 and 2 are activated upon phosphorylation by MAP kinase kinases called MKK1 and 2. These, in turn, are activated when phosphorylated by Raf . Raf, in turn, is recruited to the plasma membrane through Ras activity in response to several extracellular signals such as growth factors and cytokines. This pathway has downstream effects on cell proliferation, differentiation, apoptosis and migration via various transcription factors including the AP1 mentioned previously, as well as other factors such as myc.The precise impact of ERK1/2 signalling in hepatic steatosis is unclear, with conflicting studies in mice as to whether obesity alters hepatic ERK1/2 levels. Moreover, whether ERK1/2 activity is altered in obese humans that progress towards NAFLD or NASH has yet to be established . At presThe role of MAPK signalling in obesity-related cancer remains poorly understood; however, the role of MAPK signalling in immune cells and the regulation of metabolism may be the most important avenues of future research see . Longitu


The anterior pituitary gland is comprised of specialized cell-types that produce and secrete polypeptide hormones in response to hypothalamic input and feedback from target organs. These specialized cells arise during embryonic development, from stem cells that express SOX2 and the pituitary transcription factor PROP1, which is necessary to establish the stem cell pool and promote an epithelial to mesenchymal-like transition, releasing progenitors from the niche. Human and mouse embryonic stem cells can differentiate into all major hormone-producing cell types of the anterior lobe in a highly plastic and dynamic manner. More recently human induced pluripotent stem cells (iPSCs) emerged as a viable alternative due to their plasticity and high proliferative capacity. This mini-review gives an overview of the major advances that have been achieved to develop protocols to generate pituitary hormone-producing cell types from stem cells and how these mechanisms are regulated. We also discuss their application in pituitary diseases, such as pituitary hormone deficiencies. Compelling evidence during the last decades has demonstrated the existence of stem cells (SCs) within the vertebrate’s pituitary with capacity to divide and self-renew, and later to differentiate into specialized cell types. These stem cells express the transcription factor SOX2 and the pituitary transcription factor PROP1, which is necessary to establish the stem cell pool and promote an epithelial to mesenchymal-like transition, releasing progenitors from the niche .In this present review, a summary of the key findings amidst the localization of pituitary stem cells in different vertebrate model systems is presented featuring the major approaches employed for their discovery. Finally, we will discuss research advances to generate pituitary hormone-producing cell types from progenitor cells and their possible applications in regenerative therapies in hypopituitarism patients.One of the main anatomical differences between the mammalian and the teleost pituitary is the hypothalamic innervation. In mammals, a portal system carries the hypothalamic signals to the pituitary parenchyma, whereas in fish, the pituitary cells are directly innervated by the hypothalamus .Oryzias latipes) and the zebrafish (Danio rerio) have demonstrated the existence of potentially multipotent SC within the pituitary of these organisms. In Medaka, a significant number of cells expressing the marker Sox2 have been found localized in the dorsal region of the adenohypophysis in close proximity to the pars nervosa, with only a few cells dispersed throughout the parenchyma, consistent with its reported location in mammals  displays several advantages to study prenatal developmental events, as the relatively large size of its eggs allows the possibility to observe the progress of embryo development in a time-controlled environment. The avian pituitary gland contains several hormone-producing cell types distributed along the caudal and cephalic lobes of the anterior pituitary (AP). These specialized cells arise gradually during development simultaneously with the inflow of hematopoietic cells  act as both hypothalamic and potential pituitary progenitors. Interestingly, while a group of Fgf10+ progenitors are required for the development of the anterior and mammillary domains, a population of Fgf10+ cells persists in a quiescent state in the tuberal infundibulum which gives rise to the posterior pituitary. In agreement with previous reports in mice , hypothaRas-dva in the chicken embryo , microarray and sequencing analysis suggested the involvement of n embryo , which in embryo . Consistn embryo .The identification of pituitary stem cells in non-mammalian animal models is a burgeoning field of research. These cells express pluripotency markers known to have a role in mammalian pituitary development and were found in similar locations as in mammals. However, whether they are multipotent progenitors with the ability to differentiate into hormone-producing cells remains to be defined. In this regard, most convincing evidence comes from studies on mice.Several studies have reported mouse pituitary stem cell populations with focus on different markers, mainly: S100, GFRalpha2, Prop1, Sox2, Sox9, Nestin, E-Cadherin and CXCL12/CXCR4. These proteins were proposed as pituitary stem cell markers, and their expression often overlaps within the same cell population.β-Ala-Lys AMCA, a fluorescent dipeptide derivative that FS-cells incorporate. AMCA+ cells were found scattered across the anterior pituitary, consistent with the reported location of FS-cells, but also importantly in the marginal epithelial cell layer, thus indicating that a different cell-type shared the ability to import AMCA with the FS-cells. Transplantation of these AMCA+ pituitary colony-forming cells into an in-vivo system demonstrated a capacity to differentiate into growth hormone (GH) producing cells  described by Garcia-Lavandeira et al. These cells exhibit a round morphology and form scattered cultures when grown directly on the plates but form more compact colonies when grown on a feeder layer , obtained from human dermal fibroblasts, resembled human embryonic stem cells in morphology and gene expression patterns, and were able to differentiate into other cell types , some of these oral ectoderm cells began to express Lhx3 and invaginated into the cell aggregate, simulating Rathke’s Pouch development. These Lhx3+ cells later differentiated into all hormone producing cell lineages, and upon addition of Notch inhibitor DAPT, a greater proportion of ACTH producing cells was obtained. Ectopically transplanted cells into the kidney subcapsule of hypophysectomized mice, produced higher ACTH blood levels, spontaneous locomotor activities and longer overall survival in comparison to non-transplanted mice. A similar protocol was then performed for differentiating pituitary cells from human embryonic stem cells . Upon activation of the Shh pathway using em cells .A major caveat in regenerative medicine is that human endocrine cells derived from iPSCs often do not fully recapitulate the functioning levels of normal tissues . HoweverAttempts for obtaining pituitary cells in 2D cultures were also developed. A combination of BMP4, SHH, FGF8, and FGF10 stimuli was used to obtain hormone producing cells in monolayer stem cells in shorter times than 3D cultures; however, this approach relies on cell sorting based on GFP expression coupled to SIX1, challenging the process to adapt this protocol into obtaining pituitary cells from patients’ iPSCs , 49.The use of human cell lines, especially human SC, allows for an expansion in modeling human disease, particularly when there is a lack of an appropriate animal model or when the somatic cell types involved in the disease are difficult to isolate or grow in culture . Human-dPROP1, LHX3, LHX4, HESX1, POU1F1, GLI2, and OTX2 are found in patients with combined pituitary hormone deficiency. Many genetic variants found in rare diseases are novel, and found in sporadic cases, where it is not possible to show variant segregation along the family, so most of these variants remain to be of uncertain significance in relation to the pathology. Functional testing of these variants is vital then to assess pathogenicity. Matsumoto et al. 2020, used iPSCs derived from patients’ lymphocytes to functionally study a heterozygous variant in OTX2. They showed that the patient derived iPSCs were incapable of differentiating into hormone producing cells in contrast to control iPSCs. However, correcting this mutation by CRISPR/Cas-9 methodology modified the capacity of the iPSCs to fully differentiate into pituitary hormonal cells, validating that this variant was the one causing the hormone deficiency (Mutations in genes like ficiency .Finally, patient-specific iPSCs could be used for making therapeutic decisions in the context of personalized or precision medicine. Current treatments for hormone deficiencies or pituitary dysfunction mostly rely on recombinant hormone replacement. Though this has significantly reduced patient morbidity and mortality, it still presents some limitations, as drug administration cannot precisely recapitulate the hormonal changes by circadian and/or stress-induced requirements. This is mostly because the healthy pituitary responds to positive and negative regulatory feedback which regulates the amount of secreted hormones, which exogenous hormone replacement cannot address. Regenerative medicine, mostly involves the transplant of differentiated stem cells to correct the genetic or cell defects in patients, thus is a promising future treatment approach. On the other hand, patient derived iPSCs could be used to test pharmacological treatments for correcting the disease or phenotype observed .in vitro conditions to produce a specific pituitary hormone expressing cell are still underdeveloped. We need a higher insight into the molecular mechanisms that regulate pituitary development. These development-in-a-dish models have already helped us understand some of the factors involved in the process of human pituitary development, so they are sure to help us move closer into that objective.However, these approaches mentioned before for obtaining pituitary cells in both 2D and 3D cultures still need to be perfected for use broadly in the clinical setting. The main setback with both techniques is the inability to generate the specific hormone producing cells required to combat an individual patient’s pituitary hormone deficiency. Both approaches are fairly good in differentiating ACTH cells, with the other cell types varying in proportion. Gonadotropes are especially difficult to differentiate as they are often found in low frequency. In 2D culture, different proportions of BMP2 and FGF8 added to the medium can promote the change in proportion of pituitary cells. High BMP2 and low FGF8 promotes LH and FSH cells; equal concentration of both BMP2 and FGF8 has a greater proportion of GH and TSH producing cells, while high FGF8 and low BMP2 stimulate POMC+ cells . HoweverIn summary, several studies support the existence of multipotent stem cells in the adult pituitary from fish to human. Even if not all these studies arrive to the same conclusions, several features, including Sox2 expression, characterization of the side population nature, and the marginal zone configuration, seem to characterize the phenotype of pituitary stem cells. We believe that further characterization of pituitary stem cells could allow a better understanding of the biological basis of some pituitary pathologies including hypopituitarism and adenoma development. While many obstacles remain to be overcome, we envision that the therapeutic potential of iPSCs will attract further research investments in this area to resolve the challenges and allow the identification of novel targets for disease treatments.All authors planned and wrote the paper. MC, JM, and SV designed the figures. All authors contributed to the article and approved the submitted version.PICT 2017-0002 to MP-M. PICT 2018-4239 to MIPM.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The abnormalities in human metabolism have been implicated in the progression of several complex human diseases, including certain cancers. Hence, deciphering the underlying molecular mechanisms associated with metabolic reprogramming in a disease state can greatly assist in elucidating the disease aetiology. An invaluable tool for establishing connections between global metabolic reprogramming and disease development is the genome-scale metabolic model (GEM). Here, we review recent work on the reconstruction of cell/tissue-type and cancer-specific GEMs and their use in identifying metabolic changes occurring in response to liver disease development, stratification of the heterogeneous disease population and discovery of novel drug targets and biomarkers. We also discuss how GEMs can be integrated with other biological networks for generating more comprehensive cell/tissue models. In addition, we review the various biological network analyses that have been employed for the development of efficient treatment strategies. Finally, we present three case studies in which independent studies converged on conclusions underlying liver disease. The global burden of complex diseases is rising and such diseases cause millions of deaths each year worldwide, proving to be a widespread issue for not only individuals and healthcare systems but also researchers and clinicians alike. Liver diseases, including non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver cirrhosis and hepatocellular carcinoma (HCC), account for over 3 million deaths per year worldwide, with 1.3 billion adults currently overweight with a 25% lifetime risk of NAFLD  were identified as MDS nodes in HCC networks but not in non-cancer networks, indicating that inhibition of these genes would not be toxic to normal cells. Indeed, siRNA knockdown of these three genes in HepG2 and HepB3 cells led to promising reductions in cell growth by up to 35% in at least one cell line.Identifying stratifying genes or therapeutic targets based on network characteristics is an exciting emerging strand of systems biology that has already proven highly useful to researchers. By using network controllability theory, minimum driver set (MDS) nodes—those nodes required to achieve full control over a network —and indiSeveral studies employing multi-omics network analysis approaches have been congruent in identifying major pathways contributing to liver disease. For instance, the genes encoding the enzymes catalysing the conversion of acetate to acetyl-CoA—namely mitochondrial enzymes ACSS1 and ACSS3, and cytosolic enzyme ACSS2—have been identified as stratifying genes in two independent network analysis studies .iHCC2578, predicted an unusually tightly regulated FAB pathway in a background of poorly or deregulated metabolic pathways, as normally expected in cancer. On the basis that the ACSS enzymes can generate acetyl-CoA to be used as a substrate for FAB, the authors stratified 361 HCC tumours by ACSS1 and ACSS2 expression level, separately, and found that high ACSS1 expression was linked to hypoxia, suppression of fatty acid oxidation, co-expression with the proliferation-specific transcription factor (TF) Forkhead box M1 (FOXM1) and centromere protein F (CENPF)—the implications of both of which in HCC are already established [In the first study , a reconablished , 61—and iHCC1, iHCC2 and iHCC3)—each of which have distinct gene expression, biological process (BP) and clinical survival characteristics. The reconstruction of cancer GEMs differs from non-cancer GEMs of the same cell/tissue type by having the formation of biomass as an additional metabolic task to ensure cell growth. The study stratified 369 HCC tumours into three clusters on the basis of an fGGN for HCC and patient transcriptomic data: iHCC1, indicating the most favourable survival; iHCC2, indicating intermediate survival; and iHCC3, indicating the least favourable survival [iHCC1 tumours favourably expressed ACSS2, iHCC2 tumours ACSS3, whereas iHCC3 tumours ACSS1 for acetate utilisation  enrichment analysis. The DEG/GO analysis identified two transcripts (ENST00000335181 and ENST00000561609), which includes the transcript for PKM2, associated with favourable survival in TCGA KIRC datasets. Two further transcripts (ENST00000389093 and ENST00000568883), associated with unfavourable survival, were also found. These opposite prognostic effects between the sets of transcripts were validated using an independent Japanese KIRC cohort of 100 patients . This prIn an attempt to explain the differing survival rates between the sets of transcripts, the protein products of the prognostic transcripts were characterised and aligned with a template structure for PKM . Amino aHomology modelling and structure alignment with PKM1 and PKM2 revealed that ENST00000389093 lacked the catalytic site for ADP binding at residues 59–132 as a number of key contact residues within this range were missing. A newly ordered loop was found in place of the ADP binding site and it is unknown whether this loop can bind ADP in place of the active site. Meanwhile, ENST00000568883 had deletions in the A and B domains as well as the entire N-terminal domain, compared with PKM1 structure. Hence, the large deletions in the unfavourable survival transcripts corresponded to the ADP binding site, but it is still unknown whether these isoforms can bind ADP.A further example of pyruvate kinase isoforms informing on cancer survival rates includes the aforementioned study on the fGGN-assisted stratification of HCC patients. Here, it was revealed that the gene expression in the poor-survival iHCC3 cluster was enriched for genes associated with cancer hallmarks compared with the good- and intermediate-survival iHCC1 and iHCC2 clusters . In partIt is known that imbalances in redox metabolism influence proliferation and tumourigenesis, thus making redox metabolism a potential target for cancer treatment. Hence, several recent efforts have targeted redox metabolism in cancer , 69. In This recent study stratified 360 HCC patients based on the expression of 132 redox metabolism genes identified two distinct clusters of redox genes. These two groupings, named the glucose 6-phosphate dehydrogenase (G6PD) cluster and the aldehyde dehydrogenase 2 (ALDH2) cluster based on the key genes existing in each, were found to be positively co-expressed with genes in the same cluster but negatively co-expressed with genes in the opposite cluster. ALDH2 cluster genes were enriched for GO BP terms such as lipid oxidation and metabolism, amino acid metabolism and biosynthesis and carbohydrate metabolism; however, G6PD cluster genes were associated with hallmarks of cancer-related functions  such as iHCC3 also included the mammalian target of rapamycin (mTOR), the oncogene Myc, genes involved in the G-to-M–phase progression of the cell cycle and genes involved in the epithelium-to-mesenchymal transition. In particular, redox metabolism genes were once again identified as potential stratifying genes for iHCC3: these included the aforementioned G6PD and PKM, as well as ALDOA , thus strengthening the association between redox metabolism and poor prognosis of HCC  for the personalised treatment of HCC. Finally, novel methods for simulating the whole body functions should be developed analogous to a recent study that applied multi-scale, whole-systems models of liver metabolic adaptation to sugar and fat in NAFLD .With 9.4 million patients with neurological disorders in 2015  over 200Integrated multi-omics networks have been used to identify potential biomarkers and treatment strategies for the patients of complex liver diseases.Independent systems-level studies have yielded results that are consistent with one another as well as with previous knowledge.Systems biology could aid with hypothesis generation for the study of other complex diseases.