We review the evidence in support of this treatment across the spectrum of AR disease. The concept of AR control is also reviewed within the context of new European Union and Contre les Maladies Chroniques pour un VIeillissement Actif-Allergic Rhinitis and its Impact on Asthma initiatives.Allergic rhinitis (AR) poses a global health problem and can be challenging to treat. Many of the current symptomatic treatments for AR have been available for decades, yet there has been little improvement in patient quality of life or symptom burden over the years. In this review, we ask why this might be and explore the pathophysiological gaps that exist within the various AR treatment classes. We focus on the benefits and drawbacks of different treatment options and delivery routes for AR treatments and consider how, given what is known about AR pathophysiology and symptomatology, patients may be offered more effective treatment options for rapid, effective, and sustained AR control. In particular, we consider how a new AR preparation, MP-AzeFlu (Dymista Allergic rhinitis (AR) is a recognised global health problem. It is the most prevalent chronic allergic disease affecting European citizens today, with a prevalence in Sweden of 25% . AlthougThe burden of AR is also felt socioeconomically, and this, too, is often underestimated and underappreciated , 5. A reAR can be challenging to treat. Most patients presenting to their physician have moderate/severe disease , 16, 17,®, Meda, Sweden), a novel formulation of an intranasal antihistamine  and an INS [fluticasone propionate (FP)] in a single spray ., 91.5, 9g factor , may notg factor , 95, butg factor . AZE, byg factor , provideg factor . It alsog factor  and the A range of ophthalmic agents are available for control of AR-associated ocular symptoms; however, their use is limited by poor tolerance for eye drops among many patients, difficulty of administration in a sterile manner, the need for frequent application, and lack of patient compliance . Thus, pOAHs inhibit some symptoms of pruritus and erythema in laboratory models and have been shown to be superior to placebo in ocular symptom relief in clinical trials. In general, OAHs are considered superior to INSs in relief of ocular symptoms, but some studies have shown equal efficacy with these agents or even superior efficacy with INSs . HoweverPathophysiological gaps associated with the various AR treatment options mean that many patients remain symptomatic despite treatment , 108–110However, increasing the dose of the INS is not the answer, owing to its flat dose–response curve. Switching from one INS to another is also not the answer, since available INSs have comparable efficacy , 28. AltMP-AzeFlu comprises an INAH (AZE), an INS (FP), and a novel formulation in a single spray. It therefore offers the benefits of broad pathophysiological coverage due to incorporation of two agents from different medication classes with different yet complementary modes of action, antagonises both early- and late-phase allergic responses, and is convenient to use. Each of MP-AzeFlu’s components  contribute to its superior clinical effect and effectiveness over currently considered first-line therapies for AR , providip < 0.05). Furthermore, the inhibitory effect of MP-AzeFlu at a 1:102 dilution on eosinophil survival at days 3 and 4 was more than twice that induced by FP or by AZE (p < 0.05).A recent study investigated the anti-inflammatory effect of MP-AzeFlu compared with AZE and FP alone in a validated in vitro model of eosinophilic inflammation . MP-AzeFIntranasal sprays must be delivered to the nasal cavity in sufficient volume, at appropriate viscosity and droplet size, and with a technique that allows optimal retention, maximises absorption from the mucosa, and has the potential for maximum therapeutic effect. It is unlikely that concurrent use of intranasal FP and AZE would provide the same benefits as MP-AzeFlu, for two reasons. One, it is unlikely that patients would comply with a double intranasal therapy regimen in the long term, most likely choosing to use one spray only. Two, the clinical use of sequential FP and AZE requires dosing 948 μl in contrast with 274 μl for MP-AzeFlu. Application of larger volumes (more than 300–400 μl) of fluid can result in significant nasal drip (run off), as demonstrated in a human nasal cavity in vitro model . ApplicaThe effect of MP-AzeFlu on both the early- and late-phase allergic response may be observed clinically. An overview of the clinical development programme devised to study MP-AzeFlu is shown in Table Pharmacokinetic studies showed MP-AzeFlu’s formulation and device had no impact on AZE bioavailability, but did increase FP bioavailability . The bioThe efficacy and safety of MP-AzeFlu was assessed in four 14-day SAR studies , 121. Th® generic) and AZE (i.e. Astelin®) [The treatment difference was greater compared with commercially available FP (i.e. Flonasestelin®) . In thisstelin®) . Over-adstelin®) . This tistelin®) . MP-AzeFstelin®) , 121.FigThe contribution of formulation to the efficacy of MP-AzeFlu may be estimated by comparing the results of the study in which the effect of formulation was eliminated  with thep = 0.0005) [MP-AzeFlu also demonstrated a significant and speedy reduction in overall nasal symptoms compared with marketed FP in an open-label study of patients with chronic rhinitis (i.e. PAR or NAR) . Statist 0.0005) . A simil 0.0005) .Fig. 3EfMP-AzeFlu was well tolerated in clinical trials, with no safety findings that would preclude its long-term use . The mosThe efficacy and safety of MP-AzeFlu in paediatric AR has also been assessed , 130. ChThe aim of treatment is to achieve and maintain disease control, but there is a lack of consensus on what “AR control” actually is. The concept of control is well defined for other respiratory conditions such as asthma  and cateAllergy Diary) [Allergy Diary uses the VAS to track symptoms daily and categorise disease as well controlled, partly controlled, or uncontrolled according to predefined score cutoffs, thereby encouraging patients to take their AR medication and seek medical advice when appropriate. The AR CDSS uses a VAS score cutoff of 50 mm to assess control and guide treatment decisions [Many experts believe that a simple tool to both assess and define control is needed , 137. Inecisions . The oveTo date, the literature reporting use of VAS scores to quantify and assess the burden of AR and the impact of treatments is relatively sparse. The studies of OAHs, INSs, and LTRAs assess their effect on symptom severity. These studies were done prior to the introduction of the MACVIA-ARIA concept of disease control  and therBousquet and colleagues  assessedp < 0.001 for each symptom) [Ratner et al.  comparedsymptom) . Howeversymptom) .p < 0.005; midpollen season), and ended at approximately 20 mm for both groups by season’s end [Yamamoto et al.  assessedon’s end . A similThe efficacy of MP-AzeFlu in real life has been assessed in a pan-European noninterventional study conducted in Germany, Scandinavia , the United Kingdom, and Romania , 127, usThe efficacy results from Germany were later confirmed in patients from other countries in this pan-European study. Treatment with MP-AzeFlu significantly reduced the mean VAS score from 73.7 mm at baseline to 23.4 mm by the last visit and the results were consistent across countries, age, phenotype, and degrees of disease severity .MP-AzeFlu was well tolerated in real-life studies, with dysgeusia as the most frequently reported adverse drug reaction . Nausea,For patients with AR whose symptoms remain uncontrolled by pharmacotherapies, use of allergen immunotherapy may be an important treatment option . AllergeMP-AzeFlu is currently the only combination therapy available for the treatment of AR. MP-AzeFlu blocks two important pathophysiological pathways involved in the early- and late-phase reactions in the disease, providing rapid relief from all symptoms associated with AR. However, all patients are not the same, and the inflammatory pattern may vary considerably depending on what drives the disease in each individual. The new AR CDSS algorithm and use of the VAS can support management of AR in both primary and secondary care. In addition, the effectiveness of combination treatment compared with corticosteroids alone should be evaluated in broader, real-life populations, including patients with both AR and NAR, as well as those with concomitant rhinosinusitis. Further mechanism of action studies will also pinpoint more exactly the pathophysiological gaps that are plugged with MP-AzeFlu therapy.4 and LTD4 are proinflammatory mediators and potent bronchoconstrictors that induce nasal congestion. Combining an anti-leukotriene with an antihistamine has proven to be effective in some patients with AR [Another question for future research is whether it might be beneficial to block additional pathophysiological pathways that are not covered by antihistamine or corticosteroid treatment. CysLTs such as LTC with AR , 151, es


To date, allogeneic kidney transplantation remains the best available therapeutic option for patients with end-stage renal disease regarding overall survival and quality of life. Despite the advancements in immunosuppressive drugs and protocols, episodes of acute allograft rejection, a sterile inflammatory process, continue to endanger allograft survival. Since effective treatment for acute rejection episodes is available, instant diagnosis of this potentially reversible graft injury is imperative. Although histological examination by invasive core needle biopsy of the graft remains the gold standard for the diagnosis of ongoing rejection, it is always associated with the risk of causing substantial graft injury as a result of the biopsy procedure itself. At the same time, biopsies are not immediately feasible for a considerable number of patients taking anticoagulants due to the high risk of complications such as bleeding and uneven distribution of pathological changes within the graft. This can result in the wrong diagnosis due to the small size of the tissue sample taken. Therefore, there is a need for a tool that overcomes these problems by being noninvasive and capable of assessing the whole organ at the same time for specific and fast detection of acute allograft rejection. In this article, we review current state-of-the-art approaches for noninvasive diagnostics of acute renal transplant inflammation, i.e., rejection. We especially focus on nonradiation-based methods using magnetic resonance imaging (MRI) and ultrasound. Kidney transplantation (KTx) is the most preferred treatment for patients suffering from end-stage renal disease (ESRD) , as it oAR depicts a condition of sterile inflammation, either antibody or/and T cell mediated.While the T cell or cellular-mediated rejection (TCMR) usually occurs early after transplantation and is more prevalent, humoral or antibody-mediated AR (AMR) occurs later in fewer patients , 8. OverTransplantation from genetically different tissues involuntarily induces a recipients' immune response against the alloantigens. The formation of antibodies against donor-specific HLA-antigens is a key feature of AMR. Moreover, numerous non-HLA antibodies directed against both alloantigens and autoantigens have been identified to potentially initiate allograft rejection . A critiTCMR is induced by recipient's antigen-presenting cells, which present donor antigens to T-lymphocytes with following activation and differentiation of these cytotoxic T-lymphocytes (CTLs) . BesidesOwing to modern efficacious immunosuppressants, the clinical presentation of renal allograft rejection episodes has changed from fever and graft tenderness to an often subclinical manifestation characterized by increasing proteinuria, elevation of serum creatinine, or solely histological changes, which hinders early recognition .Gold standard for diagnosis of allograft rejection is the histopathologic evaluation of core needle biopsies by applying international consensus criteria, the Banff classification . The bioApart from structural details, ultrasound- and MRI-based techniques nowadays are also capable to visualize functional and biological processes to some extent in the context of the abovementioned inflammatory processes that accompany AR.As transplant recipients require active surveillance and frequent assessments of the graft's condition, we provide a review of the current nonradiation-based, noninvasive imaging techniques to detect AR that might help reduce the need for biopsies in the future. All technical approaches bare the potential to be used (in a modification) for the diagnoses of AR in other transplant organs than the kidney as well.Over the last four years, significant progress has been made particularly in the field of ultrasound- and MRI-based techniques. Sonographic examination is one of the most valuable clinical tools for patients' assessment. It is widely available and routinely utilized to monitor specific properties such as perfusion, resistance indices etc., of the graft after organ transplantation. The parameters assessed include unspecific features of AR such as enlargement of the transplant caused by swelling, abatement of corticomedullary differentiation, change in echogenicity, and distinctive structures such as medullary pyramids. Analysis of the perfusion of the grafts can be performed with Doppler ultrasound and contrast-enhanced ultrasound (CEUS) examination. Sonography is advantageous due to its innocuousness, cost-effectiveness, and wide availability. However, on the downside, it has limitations with regard to sensitivity and specificity for AR until now.Recent approaches have addressed these limitations and offered potential solutions to overcome them. One of those approaches is the assessment of the resistive index (RI) , which iA further ultrasound-based approach uses CEUS, which involves the use of either intravenously applied microbubble-based contrast agents to increase the echogenicity of blood or targeted microbubble-based contrast agents addressing specific tissues. CEUS parameters of interest are rising time, time to peak, and delta-time among other regions of interest . ConventThe acquired ultrasound signal intensities also increase with the severity of inflammation, indicating a possible measure of the degree of AR with this method. Furthermore, through targeted CEUS, it was possible to differentiate AR from ATN and acute calcineurin inhibitor toxicity. CEUS demonstrated the capability of therapeutic monitoring of the immunosuppressive treatment of manifest AR, as signal intensity distinctly decreased 24 hours after beginning of rejection therapy.Therefore, since the pathological mechanism of acute T cell mediated rejection can be observed in cases of AR in other transplanted solid organs, CEUS can potentially detect AR in other organs as well .Liao et al. demonstrated in a rat study that C4d can also serve as a target for labeled microbubbles to detect and visualize C4d deposition by means of CEUS in glomeruli and peritubular capillaries as a characteristic of antibody-mediated rejection (AMR) , which rHowever, for the translation of these approaches to the human body, the immunogenic streptavidin-based conjugation between the microbubble-based contrast agent and the appropriate antibody requires modification.t0) and 3 to 7 days before the biopsy, the SDI was calculated as RI ratio × PI ratio/CPP ratio. A retrospective analysis of 121 patients revealed that the SDI was significantly different between patients with normal graft function, acute cellular rejection, and acute vascular rejection. The RI and PI ratios were significantly different only between patients with normal graft function and acute vascular rejection. The developed SDI was able to detect acute renal transplant rejection with greater sensitivity and specificity than the RI and PI ratios, thus it might be helpful to indicate renal biopsy in the future [In another recent study, Meier et al. investigated if a newly developed serial duplex index (SDI) can be used to differentiate between AR and acute vascular rejection more effectively in comparison with the established Doppler parameters RI and pulsatility index (PI) in the first days after the transplantation . The PI e future . NeverthA different ultrasound-based technique, evaluated to detect AR, was described by Jiménez et al. in 2016. They performed real-time contrast-enhanced sonography (RT-CES) to investigate cortical capillary blood flow (CCBF) after kidney transplantation. RT-CES provides an analysis and quantification of vascular refilling in any region of interest  by depicting the destruction of injected microbubbles through an ultrasound pulse. The refilling of a certain area is an indicator of tissue perfusion. A secondary aim was to explore the influences of AR, acute tubular necrosis, and calcineurin inhibitor toxicity on CCBF, even though this study was not designed to specifically determine these differences .Although AR is associated with lower CCBF in general, CCBF did not demonstrate sufficient sensitivity to distinguish rejection due to tissue edema and cellular infiltration . UnfortuYang et al. utilized point shear wave elastography (p-SWE) based on acoustic radiation force (ARF) impulse to quantify tissue stiffness by measuring shear wave speed (SWS) in a prospective study with 115 KTx recipients . The tecA more recent prospective study to evaluate the value of SWS for the differentiation of stable allograft function from acute and chronic allograft dysfunction was performed by Ghonge et al. The study revealed that SWS can help to differentiate stable allograft function from acute and chronic dysfunction in addition to laboratory and Doppler-based parameters .Kim et al. investigated the worthwhile issue of diagnosing subclinical kidney allograft rejection (SCR) in stable functioning grafts by quantifying tissue elasticity with SWE to identify rejection episodes at a stage with better treatment options. They examined 95 patients who underwent protocol biopsies either 10 days or 1 year after Tx and exhibited a stable allograft function. 34 of them showed histological characteristics of acute rejection. The authors could demonstrate that tissue elasticity was significantly increased in patients with SCR compared to those without in univariate analysis, without being an independent predictor of SCR in multivariate analysis. No differences in tissue elasticity could be found between the histological subtypes of rejection . ImportaViscoelastic response (VisR) ultrasound is an alternative ARF-based tool using two co-localized ARF impulses to delineate tissue properties by measuring viscosity and elasticity . HossaiT1 and T2 relaxation times, water content, and diffusivity [The natural magnetic properties of hydrogen nuclei can be detected in a magnetic field with the use of magnetic resonance imaging (MRI). The strength of MRI lies in its excellent intrinsic soft tissue contrast, unlimited penetration depth, and high anatomical resolution in addition to the functional assessment of the graft. The technique is capable of differentiating tissue characteristics based on intrinsic MR properties such as fusivity . Thus, MDifferent MRI techniques have already been successfully applied to discriminate between different causes of renal allograft injury such as AR and ATN, and they allow (to some extent) the visualization of the pathophysiological processes underlying the respective type of injury , 43.A common MRI method to assess renal function is dynamic contrast enhanced MRI (DCE MRI). It depends on gadolinium-based contrast agent protocols, and it is also termed MR renography (MRR). The contrast agents used in this technique are freely filtered across the glomeruli yet not secreted or reabsorbed in the tubules ideally. Due to these specific characteristics, it optimally assesses renal perfusion, glomerular filtration rate (GFR), and tubular function that has been proved useful for discrimination between AR and ATN . One patYamamoto et al. proved the feasibility of discrimination between several types of allograft impairment using a new quantitative analysis method of MRR. They implemented a multicompartmental kinetic kidney model to determine the mean transit time (MTT) of a contrast agent through the different compartments of the kidney. Even though some significant differences in the fractional MTT values between normal grafts or grafts undergoing AR or ATN were obtained, substantial overlaps were observed when these groups were compared with themselves and with healthy control kidneys .Notably, the application of gadolinium-based MRI to patients with severely impaired renal function potentially accompanies the rare but deleterious side effect of contrast-induced nephrogenic systemic fibrosis . MoreoveTI (inversion time) after the pulsed labeling with the use of a rapid imaging technique, owing to the time required for the labeled protons to perfuse the tissue. In the control acquisition, arterial protons at the target structure are relaxed and in equilibrium. Methods for ASL and labeling pulses can be categorized into continuous (CASL), pulsed (PASL), and velocity-selective (VSASL) techniques [In contrast, arterial spin labeling (ASL) is an MRI tool that utilizes arterial blood flow as an endogenous contrast agent, and it allows the study of allograft function, particularly by longitudinal perfusion evaluation. It comprises of two acquisitions, one labeled by modifying the longitudinal magnetization of arterial blood water and a control acquisition obtained without arterial labeling. The labeled protons find their way via the arterial bloodstream to the targeted tissue, where they pass from the intra- to the extravascular compartment and reduce the equilibrium magnetization slightly by a few percent. Imaging is performed at time chniques . The subchniques ) reveal chniques . In conjchniques . HoweverT2∗. Inversely, the apparent relaxation rate R2∗(=1/T2∗) is elevated. In this context, BOLD MR is feasible in providing information about renal parenchymal oxygen concentration [R2∗ values decrease significantly, corresponding to a higher oxygenation in comparison with kidneys with ATN. Interestingly, this observation was accompanied by a reduced medullary blood flow assessed by perfusion MR which appears contradictory [Another approach for the functional examination of transplanted kidneys to distinguish between AR and ATN is exploitation of the paramagnetic properties of deoxyhemoglobin via blood-oxygen level dependent (BOLD) MR –58. Deoxntration . In AR kadictory . The incadictory .Diffusion-weighted MRI (DWI MRI) is a contrast agent-independent MRI technique that depends on the signal decay induced by relative diffusion-based displacement of water molecules. It can be quantified by calculating the apparent diffusion coefficient (ADC). The ADC is influenced by the tissue microstructure and separated from the directionality of molecular motion, thus ADC values represent a measure for tissue diffusivity , 61.T2-relaxation time (T2-mapping)—to investigate the severity and course of inflammation and edema formation following KTx during the development of AR and IRI.Hueper et al. recently performed a study wherein they investigated mice after isogenic and allogeneic KTx at day 1 and day 6 after the transplantation. They used a combination of both functional MRI techniques—DWI and mapping of The authors observed a progressive ADC reduction in allogeneic grafts when compared to isogeneic grafts and normal kidneys, which correlated to histologic findings of tissue inflammation corresponding to AR.T2 relaxation times increased as a correlate for tissue edema, and this was observed in both transplantation groups. The authors assumed that the acute kidney injury of the graft following prolonged cold ischemia time was responsible for this finding. Interestingly, only the allogeneic group showed an abrogated T2-difference between the renal compartments, which indicates a disturbance of physiological differences of tissue water content.T2-mapping. In this case, one limitation might be that edema formation interferes with diffusion and T cell invasion which is able to alter T2-relaxation.Therefore, Hueper et al., who used a 7 T MRI scanner for their study, concluded that morphological MRI clearly allowed differentiation between allogeneic kidney grafts with AR and isogenic kidney grafts with IRI and provided detailed tissue information with regard to the graft using DWI and In order to translate this into clinical practice, two issues have to be considered: first, renal anatomy and physiology of mice differ from humans, and second, clinical MRI scanners usually remain in the range of 1.5–3T in contrast to 3–9.4T frequently used in rodent studies. Thus, MRI parameters may differ .For imaging of renal structures, diffusion tensor imaging (DTI) is a more sensitive MRI-based approach that has been applied by Lanzman et al. By sampling several different diffusion directions, DTI addresses the issue of anisotropic diffusion properties due to the radial orientation of main anatomic structures like vessels and tubules. DTI is an effective tool for the assessment of the fractional anisotropy, which is a measure for directionality of diffusion in tissues , 64.Several studies have utilized nanoparticles to specifically detect immune cells or immune proteins in the kidney by using MRI to image the pathophysiological processes that occur when undergoing rejection .T2∗. Hauger et al. found a specific MR pattern for ATN that may be helpful in distinguishing ATN from AR. Furthermore, they were able to successfully discriminate between inflammatory and noninflammatory causes of kidney failure. However, the significant delay between contrast agent application and data acquisition is a strong limitation of this procedure. Moreover, imaging of phagocyte activity is unspecific, as these cells participate in different inflammatory events in the kidney. Chae et al. demonstrated macrophage homing using MRI in their allograft rejection model. However, other causes of graft failure besides AR were not investigated in their study. Unfortunately, nonphagocytic cells, such as T cells, which are more specific to AR, generally present a low SPIO-labeling efficiency and poor contrast agent uptake. Currently, this is a significant limitation to cellular MR imaging in vivo [Hauger et al. and Chae et al. proposed application of superparamagnetic iron oxide (SPIO) particle-loaded macrophages to investigate native and transplanted kidney grafts. The first study mentioned was performed in human patients, and the latter in a rat model. Imaging of macrophage infiltration was performed three and five days after the application of the contrast agent. SPIOs cause local field distortion and result in a strong reduction of the relaxation time constant  in vivo , 67.Polyethylene glycol-coated superparamagnetic nanosized iron-oxide particles constitute a new synthesized class of MRI contrast agent. These particles were tested for the labeling of T cells in a rat model of AR after heart-lung transplantation with a T cell purity of about 90% . This tein vivo studies of renal allograft injuries. This technique aims to assess regional tissue glucose content. GlucoCEST uses D-glucose as a naturally occurring biodegradable MRI contrast agent, which can be monitored by chemical exchange saturation transfer (CEST) [Recently, we published a new MRI-based approach called glucoCEST for noninvasive and differential r (CEST) .asym contrast ratio of cortex to medulla, which was found to be significantly increased in AR compared to the other subgroups  induced toxicity. The technique involved the calculation of a MTRubgroups . The difThe feasibility of clinical translation for the glucoCEST technique has already been demonstrated in patients with glioma . TherefoNew innocuous and sensitive diagnostic tools for the detection of AR assessing the whole organ, particularly in differential diagnostics of delayed graft function, are highly desired. Two particularly suited applications to address this issue are ultrasound and MRI. Advances in technology and contrast agent development have opened new possibilities in this regard. At present, all of these promising new technologies are still at an experimental stage and have yet either to be transferred from animal models to clinics or to be refined to prove an advantage over standardly applied core needle biopsy. For the moment, they are partly capable to better identify patients in need of a kidney allograft biopsy and they offer the potential to advance the clinical routine for noninvasive and specific diagnosis of AR as well as the longitudinal surveillance monitoring of the allograft. The biggest advantage of ultrasound-based diagnostics over MRI-based ones is their broad availability, their potential for bedside and real-time diagnostics, and the relatively low costs. However, in contrast to MRI, ultrasound-based diagnostics has a substantial interobserver variability and needs experienced investigators. Furthermore, most MRI sequences are already available for clinical scanners.However, detection approaches for structural, morphological, or functional features by ultrasound  as well as MRI  lack specificity for the detection of AR; imaging of characteristic molecular biological processes with both ultrasound (T cell migration and C4d deposition) and MRI  bares the potential to overcome this hurdle.Of course, rejections can occur in every allogenic transplant. Accordingly, the reviewed techniques for noninvasive detection of renal allograft rejection are principally adaptable to other allogenic transplanted solid organs.


The Forkhead box, subclass O (FOXO) proteins are critical transcription factors, ubiquitously expressed in the human body. These proteins are characterized by a remarkable functional diversity, being involved in cell cycle arrest, apoptosis, oxidative detoxification, DNA damage repair, stem cell maintenance, cell differentiation, cell metabolism, angiogenesis, cardiac development, aging and others. In addition, FOXO have critical implications in both normal and cancer stem cell biology. New strategies to modulate FOXO expression and activity may now be developed since the discovery of novel FOXO regulators and non-coding RNAs (such as microRNAs) targeting FOXO transcription factors. This review focuses on physiological and pathological functions of FOXO proteins and on their action as fine regulators of cell fate and context-dependent cell decisions. A better understanding of the structure and critical functions of FOXO transcription factors and tumor suppressors may contribute to the development of novel therapies for cancer and other diseases. IntroductionStructure of FOXO family membersPhysiological functions of FOXO proteinsImplications of FOXO proteins in pathological processes and associated diseasesCritical functions of FOXO transcription factors in stem cells and their role in cancer stem cellsExpression of FOXO family members in various tissuesFOXOs and microRNAsConclusions and future perspectives1,2. FOX proteins are well known to control the following physiological procceses: apoptosis, cell-cycle, cellular metabolism, immune response, differentiation, development (such as cardiac development) or aging. FOX proteins are also involved in various pathologies, such as cancer, diabetes and neurodegenerative diseases3,4.The Forkhead box (FOX) proteins represent a wide family of transcription factors that display an extraordinary functional diversity, regulating a variety of critical biological processesDrosophila Melanogaster forkhead mutant presenting modifications of the gut formation5. One year later, the sequence comparison between forkhead and mammalian hepatocyte-enriched transcription factor HNF-3A showed a conserved 110-amino-acid DNA binding domain, bringing evidence that forkhead proteins are a new class of transcription factors6.The first forkhead transcription factor was first identified in 1989, and its function was related to the development of the anterior and posterior gut of the Drosophila embryo. The “forkhead” name was given because of the two spiked-head structures found in the embryos of the 7.Hence, forkhead transcription factors are defined by their winged-helix DNA binding domain, a conserved structure called „Forkhead box”, the symbol FOX being assigned to all vertebrate forkhead transcription factors, according to the revised nomenclature2. Interestingly, up to now, FOX genes have not been identified in plants8. However, an update published in 2010 reported that are 50 FOX genes identified and classified in the human genome and 44 in the mouse genome9. Therefore, a standard nomenclature system was required for this extended number of discovered factors. In 2000, Daniel E. Martinez and his team established Fox Nomenclature Committee, the first step towards an unified nomenclature for the winged-helix / forkhead transcription factors. The Committee has changed the initial terms for the forkhead proteins 7. Currently, based on phylogenetic analysis, FOXO genes are classified in subclasses that range from FOXA to FOXS to yield 23 subclasses in total10-13. Each subclass has several members noted with an arabic number. While for human forkhead proteins abbreviations have all letters in uppercase , for mouse only the first letter is capitalized  and for all other chordates the first and subclass letter are in uppercase 7.Since its discovery, numerous forkhead genes have been identified in a broad range of organisms, from yeast and worms to humans1. However, FOXOs share an additional unique 5 amino acid sequence insertion within the DNA binding domain that is not present in other forkhead proteins14.One of the largest and the most important subclass of FOX family is represented by FOXO  transcription factors. FOXO transcription factors (FOXOs) are characterized by the same conserved DNA binding domain that define the family of forkhead proteins15.Only one FOXO transcription factor is known in invertebrates , while in mammals four FOXO proteins encoded by four different genes were identified: FOXO1, FOXO3, FOXO4 and FOXO616,17. A few years later, FOXO3 (previously known as FKHRL1 - forkhead in rhabdomyosarcoma like protein 1) was characterized and named, based on similarities to FKHR18. The gene for FOXO4 was described as being fused to MLL transcription factor as a result of the t chromosomal translocation in acute lymphoblastic leukemia, therefore, the initial term for FOXO4 was AFX (The acute leukemia fusion gene located on chromosome X)19.Initially, FOXO1 transcription factor (previously known as FKHR-forkhead in rhabdomyosarcoma) was identified through its involvement in chromosomal translocations t and t in alveolar rhabdomyosarcoma tumors due to PAX3/7-FKHR fusion transcript4,20. The aim of this review is to discuss the most recent advances in elucidating the functions, structure and transcriptional regulation of Forkhead Box O transcription factors, both in physiological and pathological conditions. The advances in understating the mechanism of FOXOs regulation of stem cells, cancer stem cells and how non-coding RNAs are regulated and regulate the function of FOXO genes/protein are presented.FOXO proteins are considered unique cellular targets, regulating a wide variety of critical cellular processes, such as: apoptosis, oxidative stress, DNA damage repair, cell cycle, stem cell proliferation and maintenance, metabolism, angiogenesis, vascular tone, cardiovascular development, fertility, immune response and neuronal survival21-24. All members of the FOXO family consist of four domains: a forkhead DNA-binding domain (DBD), a nuclear localization signal (NLS), a nuclear export sequence (NES) and a C-terminal transactivation domain25 .In humans, the primary structure of the FOXO proteins is characterized by a length of approximately 655-675 amino acid residues (aa) for FOXO1 (655 aa) and FOXO3 (673 aa), and a shorter sequence of approximately 500 amino acids for FOXO4 (505 aa) and FOXO6 (492 aa)26. Analysis of the amino acid sequences alignment of FOXO proteins revealed a highly conserved DNA binding domain among all the members of the broader group of forkhead transcription factors, but also among species, in eukaryotic organisms, from yeast to humans3,27.Moreover, several studies point out that forkhead DBD is not the only region in the FOXO molecule that is highly conserved. Similarities were also observed in the N-terminal region near the first AKT/protein kinase B (PKB) phosphorylation site (Thr24 for FOXO1 and Thr32 for FOXO3), the region containing NLS, and sequences from C-terminal transactivation domain25.The forkhead DNA-binding domain (DBD), also called the forkhead box, is described as a „winged helix” due to the butterfly-like aspect on X-ray crystallography and nuclear magnetic resonanceare similar to the forkhead regions of other subclasses. For example, structural similarities have been proven in the core region of FOXO3 compared to FOXA3, FOXK1 and FOXP228. The forkhead/winged helix motif is a shared sequence between subclasses of FOXO, of around 100 amino acids that folds into three alpha-helices , three beta-strands  and two wing-like loops (W1 and W2). The structure has a H1 - S1 - H2 - H3 - S2 - W1 - S3 - W2 topology and the strands S1, S2 and S3 interact with each other forming a beta-sheet. While the N-terminal part of the domain is formed by a cluster of three alpha-helices, the C-terminal half consists of beta strands S2 and S3 and two large loops (wings W1 and W2)25. The main DNA recognition site is represented by the highly conserved helix H3 of forkhead DNA-binding domain. The stability of forkhead DBD - DNA complexes is increased by the more variable regions of the DBD, including H1, the region between H2 and H3, wing W1 and C-terminal segment25.As mentioned above, the forkhead domains in FOXO subclass of FOX family28,29.Although the FOXO forkhead box is clearly related to those found in other forkhead genes, all FOXO members contain an aditionally segment of five amino acid (198-GDSNS-202) between helices H2 and H3, that creates a small extra loop, forming a coil structure in the FOXO3 DBD, and a short helix in the FOXO4 DBD30.This is important in sequence-specific interactions with DNA-binding sites31. They bind DNA through a FOXO-recognized element with a T/C-G/A-A-A-A-C-A-A consensus sequence in the forkhead DBD 26. There are fourteen protein-DNA contacts described, which mediate the activation/inhibition of FOXO’s target genes, such as Bim, TRAIL, p27, p21 and catalase. The main recognition site is the α-helix H332.FOXO proteins mediate transcriptional activation through binding to the conserved consensus core motif TTGTTTAC in the DNA20.Forkhead proteins function as transcription factors that bind to DNA through their forkhead domain in order to upregulate or downregulate the expression of a tremendous number of target genes33-38 . Moreover, FOXOs are also involved in a wide range of pathological cellular processes, such as cancer, neurodegenerative diseases , diabetus mellitus, cardiac failure, atherosclerosis, hypertension and anovulation39-46.Hence, the FOXO transcription factors control the expression of a wide spectrum of genes that regulate essential physiological cellular processes, such as cell death/cell survival, cell cycle, cell proliferation, cell differentiation/development, angiogenesis, cell metabolism, stress response and stem cell maintenance47. Several particular differences regarding the regulatory kinases, E3 ligases or other important enzymes and regulators exist. Thus, their activity is in part controlled by different mechanisms31.Differences between functions and regulatory mechanisms of FOXO1 and FOXO3 proteins are considered to be partially redundant, with several exceptions. For example, AKT phosphorylates all FOXO family members, inducing their translocation into the cytoplasm and inactivation, while PP2A dephosphorylates part of the AKT phosphorylation sites in FOXO1 and FOXO3, reactivating them48-52. Moreover, the consensus FOXO recognition element (FRE) - (G/C)(T/A)AA(C/T)AA - which differs from that of other forkhead proteins, seems to have a very important role in both apoptotic pathways, since matching functional FRE sites have been identified in the promoters of FOXO target genes encoding Fas ligand (FasL), insulin like growth factor-binding protein 1 (IGFBP1), the apoptotic regulator Bcl-2 interacting mediator of cell death (Bim) and others30.FOXO transcription factors regulate the expression of multiple pro-apoptotic and anti-apoptotic proteins, and they have the ability to induce apoptosis by activating either intrinsic or extrinsic pathways of apoptosi48-52. The expression of the anti-apoptotic protein Bcl-xL is suppressed by FOXO proteins, such as FOXO4, after increasing the expression of the transcriptional repressor Bcl-6, in order to trigger apoptosis52.FOXO triggers the mitochondria-dependent intrinsec apoptotic pathway through upregulation of multiple pro-apoptotic Bcl-2 family members, such as Puma, Bim, NOXA, BNIP3, and downregulation of the anti-apoptotic Bcl-2 family member Bcl-xL53. These mitochondrial modifications result in release of cytochrome c, Smac/DIABLO and Omi/HtrA2 from mitochondria, subsequently triggerring down-stream caspases activation54,55. Apaf 1 binds cytochrome c forming a complex named Apoptosome, required for caspase 9 activation (initiator caspase)54. Caspase 9 activation further activates the effector caspases, such 3, 6 or 7, triggering caspase cascade54.Both FOXO-induced expression of the pro-apoptotic Bcl-2 family members and FOXO-downmodulated anti-apoptotic Bcl-2 family members lead to apoptosis due to mitochondrial outer membrane permeabilization (MOMP), as a response to intracellular stress, growth factor deprivation or other factors56. Fas signaling pathway is important in apoptosis induction through the extrinsic pathway, since Jurkat cells lacking several critical components of Fas pathway fail to induce FOXO-dependent cell death57. Upregulation of tumor necrosis factor related apoptosis inducing ligand (TRAIL) by increased levels of FOXO1 or FOXO3 in cancer cells, such as prostate carcinoma cells, leads to apoptosis58. Thus, binding of Fas ligand (FasL) and TRAIL to their receptors  triggers a death-inducing signaling complex (DISC) with a subsequent activation of caspases, mainly initiator caspase 8 and effector caspase 3, leading to apoptosis59.Moreover, FOXO transcription factors directly regulate the expression of tumor necrosis factor receptor-associated death domain (TRADD). Activation of FOXO proteins by PI3K-AKT pathway inhibitors results in increased expression of TRADD protein60. TRADD is an important adaptor protein interacting with TNFR1 and Fas receptors, mediating apoptosis and NFkB pathway activation61.FOXO proteins are also able to induce apoptosis by activating the receptor-dependent extrinsic pathway of apoptosis. They induce the upregulation of the death receptor ligands FasL and TRAIL, promoting an autocrine and/or paracrine action of these death ligands on the death receptorscyclins and their associate cyclin dependent kinases (CDKs) are very important for the progression of the cell cycle62. In response to DNA damage, FOXO transcription factors increase the expression levels of the CDK inhibitors binding to cyclin/CDK complexes, such as p21  and p27 (Kip1)63,64. CDK inhibitors together with FOXO-induced inhibition of Cyclins expression act by stopping the cell cycle at different checkpoints in order to repair the DNA damage or to remove the damaged cell65. For example, a study performed on 32D murine myeloid cells and on BaF3 murine pre–B-cell lines brings evidence that endogenous FOXO proteins are required to enforce cell cycle checkpoints after DNA damage33.Under normal physiological conditions,KIP1, p57KIP2, p21CIP1/WAF1 (CIP/KIP family), p15INK, p19INK (INK family) and the retinoblastoma protein family member p13027.Also, FOXO decreases the positive regulators, such as cyclin D1, or cyclin D2, blocking the G1/S transition66. Additionally, FOXO increases the expression of negative regulators such as Gadd45alpha and cyclin G2, resulting in cell cycle arrest at G2/M66. Surprisingly, FOXO proteins act as transcription factors for PLK1 expression during cell cycle, PLK1 being critical in promoting G2-M phase transition, M phase progression and end of mitosis67. A complete knock-down of FOXO protein levels induces an arrest in cell cycle, suggesting that a basal, low levels of FOXOs activity is required for the cell cycle progression67. Surprisingly, in specific settings, FOXO may actually serve as a promoter of proliferation. For example, neutrophils isolated from FOXO3 deficient mice show high levels of FasL expression and apoptosis, revealing that FOXO3 may represses FasL expression in neutrophils, leading to proliferation in this type of cells68. FOXO1 was also shown to positively influence proliferation induction in pancreatic β cells in vitro, under low nutritional circumstances. The mechanisms implicated in this process are not completely understood. However, the induction of Ccnd1 gene transcription, which encodes Cyclin D1, may at least partially be involved. Cyclin D1 represents one of the earliest cell cycle-related events, being critical for the G1 to S phase progression during the cell cycle34.The cell cycle arrest at G1/S transition is induced by FOXO by upregulating the negative regulators of the G1/S phase, such as the CDK inhibitors: p27Thus, FOXO proteins coordinate the expression of multiple important cell cycle regulators, in order to block the G0/G1, G1/S or the G2/M transitions during cell cycle when needed, such as after DNA damage. However, a basal level of FOXO proteins is required for G2-M cell cycle transition, when FOXO-dependent PLK1 expression seems to be necessary. These results suggest that FOXO proteins have to be tightly regulated and are important and sensitive regulators of cell cycle, proliferation and other critical cellular processes.69. FOXOs can also suppress bone formation by inhibiting Wnt-β catenin-TCF signaling. Wnt signaling is known to stimulate bone formation70.FOXO proteins play an important role in regulating differentiation of a wide variety of tissues. It is well known that FOXOs can control the differentiation of precursor cells into muscle, adipose tissue or blood cells. Interestingly, FOXOs effect on differentiation is context dependent and sometimes FOXO isoform dependent. While FOXO3 promotes differentiation of erythroid cells, FOXO1 suppresses the differentiation of precursor cells in adipose and muscle tissues71 (see details in Chapter 5).Moreover, FOXO proteins are required for maintenance of somatic/adult stem cells, such as hematopoietic stem cells, and they also regulate cancer stem cells20. It was first noticed for the FOXO homolog named Dauer Formation-16 (DAF-16), in the Caenorhabditis elegans worm72.Metabolic signaling mediated by forkhead transcription factors is conserved among multiple species, including but no limited to mammals, Drosophila melanogaster and Caenorhabditis elegans73-76. In the liver, FOXO1 forms a complex with another transcription factor, the liver specific PGC1alpha, in order to induce gluconeogenesis by upregulating G6Pase and PEPCK genes. This is important for maintaining glucose homeostasis73. Confirming these results, loss of hepatic FOXO genes in mice induces a downmodulation of gluconeogenesis and an upregulation of glycolysis77. In addition, ectopic expression of FOXO1 in rat primary hepatocytes increases apocIII, an inhibitor of lipoprotein lipase, suggesting that FOXO1 is involved in regulating the lipid metabolism78. Recently, nicotinamide phosphoribosyltransferase (Nampt) gene was described to be a new transcriptional target gene of FOXOs for regulating hepatic triglyceride levels79. In pancreas, FOXO1 plays an important role in the maintenance of beta cell function, but also in the development of the pancreas, through repression of the pancreatic transcription factor Pdx174. Aditionally, FOXO1 increases the food intake by upregulating AgRP and NpY, and by downregulating POMC in hypothalamus, antagonizing the anorexigenic hormone leptin76,80. Recently, G-protein-coupled receptor Gpr17 was described to be a FOXO1 target that activates AgRP, suggesting a new mechanism for FOXO1-AgRP mediated food intake that might provide a new treatment for obesity81.FOXO proteins are involved in critical physiological processes that regulate cellular metabolic activity in many organs, such as liver, pancreas, adipose tissue and hypothalamusAll these results establish FOXO proteins as important regulators of cellular metabolism and potential target in metabolic related diseases.27. Studies on human cardiac fibroblasts revealed that FOXO also modulate the antioxidant enzyme peroxiredoxin III, fighting against cell damage induced by oxidative stress.Concomitant peroxiredoxin III knockdown and FOXO3 knockdown resulted in higher levels of hydrogen peroxide in response to serum starvation, as compared to peroxiredoxin III knockdown only82. Other FOXO transcriptional targets , such as sterrole carrier proteins (SCPs), are now known to play an important role during the defence against oxidative stress. SCPs are implicated in protecting fatty acids from peroxidation83. FOXO proteins are also able to increase DNA repair, inducing a cell cylce arrest at G2/M checkpoint, in order to provide time for repairing the DNA damage. One of the transcriptional target of FOXO, implicated not only in cell cycle arrest but also in DNA repair and cell survival in response to cellular stress, is Gadd4584. Similarly, DDB1 gene protein product is able to repair the DNA damage through upregulation by FOXO69.FOXO transcription factors function as sensors for reactive oxygen species (ROS) and play an important role in cellular resistance to stress, increasing cellular survival. FOXO proteins are able to protect the cell from oxidative damage, decreasing the availability of ROS. They stimulate the expression of certain genes responsible for the ROS inactivation, such as the antioxidant enzymes manganese superoxide dismutase (MnSOD), that catalyzes the conversion of superoxide to hydrogen peroxide, and catalase, that converts hydrogen peroxide into water and oxygen27. The lifespan extension is an important function of FOXO that is conserved among several species. A well studied example is the role of FOXO homologous DAF-16 (Caenorhabditis elegans) in aging85. Loss of FOXO3 in human skin fibroblasts results in aging specific morphological changes, suggesting that FOXO3 is necessary for maintenance or promotion of cellular longevity86. Moreover, loss of FOXO3 activity leads to decreased MnSOD and enhanced cell injury in vascular smooth muscle explanted from aged mice, due to limied ROS inactivation87. Thus, cellular lifespan maintenance requires a certain level of FOXOs activity and this is mainly achieved by inactivation of AKT activity88. FOXO proteins do not only play an important protective role during senescence/aging, but also during exercise.It was previously suggested that FOXOs may at least partially be involved in the exercise-induced beneficial cardiac effects. Interestingly, exercise induces an upregulation of FOXO3 and SIRT1 in the heart, with subsequent increased MnSOD, catalase and GAD45alpha, and decreased Cyclin D2, suggesting the protective role of FOXO proteins89. FOXOs regulate the longevity of the cell mainly by increasing resistance to stress, modulating the DNA repair process and maintaining the stem cells20. Additional reports suggest that suppression of FOXO proteins expression during oxidative stress can be protective to some extent for cells, since protein inhibition or gene knockdown of FOXO1 and FOXO3 decreases the ischemic infarct size in the brain, protects the metabotropic glutamate receptors during vascular injury, enhances pancreatic β-cell or neuronal survival through NAD + precursors and provide trophic factor protection with erythropoietin (EPO) and neurotrophins20. This suggests that while a certain level of FOXO3 activation is required in cellular stress resistance, sustained activation or activation over a threshold of FOXO3 is detrimental and may induce apoptosis. Thus, FOXO3 plays an important role in cellular decision during stress, helping the cells to survive by multiple mechanisms  and inducing apoptosis when DNA or cellular damage can’t be repaired.Interestingly, other studies revealed that, in conditions of oxidative stress, FOXO3 is also able to induce apoptosis by triggering a Fas-mediated death pathway in cultured motoneurons, by activating TRAIL, BH3-only proteins Noxa and Bim, and by promoting pro-apoptotic activity of p5390. Another study on mice with T cell-specific deletion of FOXO1 showed a decreased expression of IL-7 receptor on mature T-cells, suggesting that IL7-R is a transcriptional target of FOXO1, which is mediating through binding with IL-7 the survival and homeostatic proliferation of peripheral T cell91.FOXO proteins play a central role in maintaining the immune response of the human body. Cell type-specific deletions of FOXO1 and/or FOXO3 in mice revealed an important role of FOXO proteins in regulating immunological homeostasis and tolerance, by controlling the function and development of B and T lymphocytes. Thus, FOXO1 and FOXO3 are essential transcription factors involved in early B cell development and peripheral B cell function, since early deletion of FOXO1 blocked B cell differentiation at the pro-B cell stage. This is due to a defect in interleukin 7 receptor alpha  expression. Deletion of FOXO1 in peripheral B cells was associated with defective expression of both CD62L and AID, with subsequent failure in class-switch recombination and reduced IgG production upon immunization. Moreover, it is known that the PI3K-AKT-mediated inactivation of FOXO1 is essential for the optimal proliferation of B cells, while ectopic expression of PI3K-independent variants of FOXO1 or FOXO3 (active triple mutants at the AKT phosphorylation sites) resulted in cell cycle arrest and increased cell death in B cells20. Also, it was demonstrated that miR-182 has a central role in the late phase of clonal expansion of the helper T lymphocytes, by inducing IL-2 which is able to inactivate FOXO1. FOXO1 was found to be a suppressor of proliferation in resting helper T lymphocytes and, in order to allow proliferation, T cell activation via TCR/CD28 and IL-2R signaling must inhibit FOXO190. Other studies reported that T cells derived from Bim and Puma deficient mice were resistant to apoptosis after IL-2 deprivation, demonstrating that FOXO3, through upregulation of Bim, Puma and p27Kip1 is important for the induction of cell cycle arrest and apoptosis of T cells, in the absence of cytokines48. FOXO proteins may be benefical for autoimmune disorders by inducing a Fas mediated apoptosis that target activated T cells, followed by a decrease in cytokine stimulation in patients with autoimmune lymphoproliferative syndromes20.Excesive inflammatory cells may become harmful for the human body through the generation of ROS and through the production of cytokines. Studies performed on mice deficient of FOXO3 ilustrated lymphoproliferation, inflammation of the salivary glands, lung, and kidney, and increased activity of helper T cells. These observations demonstrate the beneficial role of FOXO proteins in human body, by preventing T cell hyperactivityFOXO proteins play an important role not only during physiological cellular processes, but also in few pathologies, such as cancer. FOXOs are well known tumor suppressor proteins.40,41,92,93.Although FOXO proteins protect the human body by playing a central role in a wide range of mainly physiological functions (as described earlier), under some circumstances, FOXO’s roles can become harmful for the human cells. This is because FOXO is also involved in a number of pathological functions, such as inflammation and muscle atrophy. For example, apoptosis places FOXO proteins on the good side when it leads to tumor suppression. However, cellular apoptosis can become itself a significant component for pathology in diseases such as neurodegenerative disease, diabetes mellitus (DM), and cardiovascular injury94,95. Inactivation of FOXO by PI3K-AKT pathway activation is a common feature of many malignancies, such as prostate cancer, breast cancer, leukemia and glioblastoma69. Conditional deletion of FOXO family members in mice leads to the lymphomas and hemangiomas, which suggests that loss of FOXOs maintains or promotes survival of tumor cells96.FOXOs loss or inactivation is known to play an important role in cancer tumorigenesis or progression, in vivo97. For example, a recent study described a significant correlation between low expression of FOXO3 and a poor prognosis for gastric cancer patients, bringing evidence that FOXO3 could be a valuable prognostic biomarker for patients with gastric cancer98. In addition, FOXO3 overexpression was shown to reduce motility, invasiveness, and aggressiveness in estrogen receptor α-positive (ERα+) breast cancer cells39.FOXO proteins are inactivated in a wide variety of malignancies, eiher posttranslational (mainly by PI3K-AKT mediated phosphorlation) or by fusion mutations 47,99. For instance, FOXO3-induced expression of Bim, a pro-apoptotic member of the Bcl-2 family of proteins induced a caspase-dependent cell death in several types of cancers, such as in chronic leukemias, breast and gastric cancers27.Similarly, FOXO-induced TRAIL and NOXA expression induces apoptosis in many malignancies, including leukemias100.FOXO proteins exert their tumor suppressor functions predominantly by promoting cell cycle arrest, apoptosis, ROS inactivation and DNA repair, through expression of their target genesFOXO3 induces the expression of Mxi1, which is a transcriptional suppressor of c-Myc, thereby inactivating Myc-dependent transcription. Mxi1 also plays a role in FOXO-induced proliferation inhibition. Similarly, FOXO-induced suppression of proliferation in colon carcinoma cell lines may involve the Mad/Mxd family of proteins, which are known Myc target genes101.Interestingly, FOXO3 can also inhibit the proto-oncogene c-myc, indirectly controlling the transcription of a wide set of target genes implicated in cell survival, cell cycle, apoptosis and tumorigenesis. Myc is a transcription factor and a well known promoter of survival, proliferation and tumorigenesis, being found upregulated in a large variety of malignancies.In chronic myeloid leukemia (CML), FOXO3 can induce CML leukemic cells differentiation, by inhibiting the expression of Id1 (Inhibitor of DNA binding 1). CML is characterized by the presence of the Bcr-Abl fusion protein, which is a constitutive active kinase, controlling many crucial downstream pathways implicated in cell cycle, proliferation, apoptosis and cell adhesion. Bcr-Abl activates PI3K-AKT pathway, inactivating FOXO proteins and their pro-apopotic and cell cycle arrest signals27. The use of the Tyrosine Kinase Inhibitors (TKIs) and of the proteasome inhibitor Bortezomib results in inhibition of Bcr-Abl activity and its downstream pathways, with a subsequent activation of FOXO proteins102. TKIs treatment induces a FOXO-dependent suppression of Id1 expression, leading to K562 Bcr-Abl positive cell line differentiation. Thus, Bcr-Abl can maintain the leukemic state not only by promoting proliferation, cell cycle progression and inhibiting apoptosis, but by also inhibiting FOXO-mediated differentiation27.Noteworthy, activation of FOXO proteins not only induces cell cycle arrest or apoptosis, but also a differentiation program.inflammatory cell activation in several disorders, with the subsequent cellular damage, through oxidative stress and excess of cytokines. Inactivation of FOXO3 in T lymphocytes, as well as inactivation of FOXO1 and FOXO4 in synovial macrophages in patients with rheumatoid arthritis and osteoarthritis result in inflammatory cell activation. In addition, loss of FOXO proteins may be a potential etiology for systemic lupus erythematosus (SLE) and rheumatoid arthritis, since FOXO1 gene transcript levels are downregulated in peripheral blood mononuclear cellsof these patients20. A link between inflammation, insulin resistance and FOXO transcription factors was previously suggested when downregulated FOXO1 decreased the levels of C/EBP beta transcription factor in adipocytes, with the subsequent reduction in expression of the pro-inflammatory cytokines CCL2 (chemokine ligand 2) and IL-6103. Thus, FOXO1 indirectly might induce an inflammatory status of the adipose tissue, responsible for the insulin resistance in type 2 diabetes. Yet, there are studies that describe the ability of FOXO proteins to directly induce inflammation through upregulation of the inflammatory cytokine IL-1b104. However, further experiments are required in order to clearly understand the context related mechanisms of FOXO-dependent modulation of inflammation.It was shown that loss of functional FOXO proteins lead to27. Studies revealed that FOXO proteins can induce muscle atrophy, characterized by decreased muscle function105. Thus, FOXO proteins have been shown to increase the transcription of key regulators of both lysosomal and proteasomal proteolysis: the autophagy followed by the lysosomal proteolysis is stimulated by upregulation of Bnip3, LC3, and Gabarapl1, while the proteasomal proteolysis is induced by increasing the ubiquitin ligase atrogin1. It was also shown that FOXO3 activity is both required and sufficient for induction of autophagy in muscle cells, since studies performed on adult muscle fibers from mice show that the ectopic expression of an active FOXO3 mutant leads to lysosomal proteolysis after formation of autophagosomes, while the knockdown of FOXO3 in these muscles fibers blocked autophagosome formation after starvation106,107.Muscle atrophy appears in a variety of diseases, including cancer, diabetes and sepsis, and is characterized by accelerated proteolysis that can be induced through two pathways: the ubiquitin – proteasome pathway and through lysosomal pathway, as a consequence of autophagy108. In cancer cells, ATP is predominantly produced through the constitutive activation of aerobic glycolysis, process that is modulated by the transcription factor HIF1α108. Since p38α is required to maintain the levels of HIF1α target genes, researchers demonstrated that in colorectal cancer cells, the inhibition of p38α causes a rapid drop in ATP levels, with an acute energy need which activates FOXO3 in an AMPK-dependent manner, in order to induce autophagy, cell cycle arrest and cell death in these cells96,108.Autophagy has a critical role in maintaining the cellular and metabolic homeostasis. It seems that the metabolic status of the cell strongly influences this process in both normal cells and cancer cells, despite the profound differences in their metabolism109. Atrogin 1, upregulated by FOXO1 and FOXO3, plays important roles in cardiovascular system, since mice lacking atrogin-1 are susceptible to cardiac hypertrophy20. FOXO proteins are inducing atrophy of differentiated cardiac and skeletal muscle cells through protein synthesis inhibition, which leads to a decrease in cell size69. In skeletal muscle, this mechanism involves myostatin, a FOXO transcriptional target and a secreted molecule that can induce atrophy by protein synthesis inhibition110.Moreover, the knockdown of FOXO3 was sufficient to induce hypertrophy in cultured neonatal rat cardiomyocytes. In these cells, stimulation with insulin inhibits FOXO3 function, with subsequent downregulation of the antioxidant enzyme catalase and increased levels of ROS, that in low levels may act as second messengers for intracellular signaling, making possible the increase in cell sizetype 2 diabetes, since increased FOXO1 expression in diabetic mouse liver is associated with increased expression of PEPCK and G6Pase, and inhibition of FOXO1 activity downregulated both PEPCK and G6Pase expression and normalized the blood glucose levels. Thus, FOXO1-mediated expression of G6Pase and PEPCK is critical for gluconeogenesis in the liver during fasting, but its deregulation may be involved in diabetes etiology111. Physiological functions of FOXO take place under certain circumstances, since sometimes the ability to maintain the proper control is overwhelmed20. Experiments on insulin-producing mouse pancreatic beta cells (betaTC-6) show that chronic exposure to high glucose activates FOXO transcription factors and leads to upregulation of endogenous inflammatory cytokines interleukin-1beta (IL-1beta) and suppressors of cytokine signalling (SOCS). These events trigger the activation of caspase-3 with subsequent apoptosis, suggesting a new mechanism that leads to the destruction of endocrine pancreas in type 2 diabetes92. Also, exposure to high glucose of the cardiac microvascular endothelial cells (CMECs) isolated from hearts of adult rats show that FOXO transcription factors leads to reactive oxygen species (ROS) accumulation and apoptosis, suggesting that FOXOs might be involved in microvascular complications of diabetes93. FOXO is also involved in insulin resistance and metabolic syndrome, since the activation of FOXO1 in cardiomyocytes leads to increased AKT activity and attenuated cellular response to insulin, followed by decreased glucose uptake112.FOXO1 might be implicated in the development or progression of 113.Interestingly, clinical studies regarding metabolic status profile on age-related diseases, fertility, fecundity and mortality revealed higher HbA1c levels and increased mortality risk associated with specific haplotypes of FOXO120.FOXO proteins are also activated in an attempt to protect the human body against the oxidative stress resulted due to hyperglycemia that leads to increased production of ROS in endothelial cells, liver cells, and pancreatic β-cells. This hyperglycemia-dependent ROS increase leads to a subsequent development of insulin resistance and significant neurodegenerative and cardiovascular diseases in the patients with diabetus mellitusangiogenesis, since mice that are deficient in FOXO1 lack development of the vascular system and die by embryonic day eleven114. In addition, FOXO1 and FOXO3 were shown to be the most abundant FOXO isoforms in mature endothelial cells, having also an important role in the regulation of postnatal vessel formation, not only in the embryogenesis36. Unfortunately, angiogenesis is not only involved in critical physiological processes, such as embryogenesis and postnatal vessel formation, but it is also involved in pathological events, such as chronic inflammation and tumor growth115. Thus, it is fascinating how the angiogenesis mediated by FOXO proteins may become a negative element for the organism, antagonizing the tumor suppresor’s main function through new vessel formation, that can lead to tumor cell growth116.FOXOs are necessary for endothelial cell development and heart muscle loss with subsequent ventricular dysfunction43,117. FOXOs are activated after AKT inhibition by insulin or other factors, leading to Atrogin-1 induction, which results in a suppression of heart muscle cell size43.FOXO3 was associated with both cardiomyocyte survival after oxidative stress and 118atherosclerosis and hypertension. Also, decreased FOXO1 expression due to high flow states in vessels leads to proliferation of vascular smooth muscle cells, vascular neointimal hyperplasia, and subsequent hypertension45. Moreover, experiments on low-density lipoprotein (Ldl) receptor knockout mice resulted in the prevention of atherosclerosis when the triple ablation of FOXO1, FOXO3 and FOXO4 was induced in endothelial cells44. Interestingly, the same experiment on myeloid cells lead to more severe atherosclerosis compared to the controls, explained by authors through increased proliferation of granulocyte-monocyte progenitors and high levels of inducible nitric oxide synthase (iNOS) and oxidative stress, which predispose to atherosclerosis119.FOXO3 proteins seem to inhibit the vascular smooth muscle cell proliferation and growth in a rat balloon carotid arterial injury model, suggesting a role of FOXOs in the regulation of vascular tone and systemic arterial blood pressure, preventing or at least lessening the effects of anovulation and luteinization of unruptured follicles, with subsequent 46infertility. In addition, FOXO3 and FOXO1 mutations were detected in a small percentage of womens with 120premature ovarian failure.Noteworthy, analysis of mouse oocytes revealed overexpressed FOXO3 transcription factors in primordial and early primary follicles, but downregulated FOXO in primary and more developed follicles, suggesting that FOXO proteins also have reproductive functions, modulating oocyte and follicular cell maturation. To confirm the hypothesis, constitutively active FOXO3 was induced in transgenic mouse oocytes in primary and more developed follicles, which affected the oocyte growth and follicular development, leading to 1,69. Recent reports show that FOXO-mediated regulation of cell cycle, oxidative stress and apoptosis plays an important role in these processes69.Involvement of FOXO family of transcription factors in stem cells self-renewal, survival, proliferation and differentiation is currently under investigation. FOXOs have been shown to play critical functions in maintaining self-renewal potential and quiescence of hematopoietic stem cells, however, the mechanisms of these processes are not yet well understood121. They are necessary in maintenance and propagation of several adult tissues, including but not limited to blood, skin and gastro-intestinal epithelium.Adult stem cells or cells with stem cell properties were also found in other critical organs/systems, such as the central nervous system and the lung69.Stem cells are characterized by the capacity of self-renewing and the ability to differentiate121. Deletion of these three FOXO family members in mice revealed their importance in controlling ROS in stem cells, in vivo27. While the number of hematopoietic stem cells in bone marrow of FOXO-deficient mice is low, an increase in myeloid progenitor cells in blood is observed. Moreover, the repopulation ability is decreased in the absence of FOXOs and the treatment of FOXO-deficient mice with N-Acetylcysteine (NAC) at least partially rescued these effects27.Previous studies suggested that hematopoietic stem cells are sensitive to reactive oxygen species (ROS) levels. FOXO family members are known to play a central role in ROS detection and in inducing an adaptative response after ROS exposure, by inducing the expression of critical enzymes that neutralise ROS, such as catalase and manganase superoxide dismutase (MnSOD). Interestingly, FOXO1, FOXO3, and FOXO4 inactivation leads to an upregulation of ROS levels in hematopoietic stem cells and their death121. Between FOXO family members, the most important regulator of hematopoietc stem cells survival and self-renewal is FOXO3, since FOXO3 knockdown induces hematopoietic stem cells depletion121. ROS neutralizing agent N-Acetylcysteine (NAC) can rescue hematopoietic stem cells quiescence and at least partially resque their ROS-induced loss121. All these results suggest that FOXO1, FOXO3 and FOXO4-induced resistance to ROS is critical in maintaining homeostasis of hematopoietic stem cells in bone marrow27.Thus, persistent AKT activation, which induces inactivation of FOXO transcription factors by AKT-mediated phosphorylation, results in ROS-induced cell death. This is due to the fact that the cells can’t synthesize the FOXO-dependent ROS neutralizing factors catalase and MnSOD121.PTEN tumor suppressor was revealed as an important modulator of hematopoietic stem cells self-renewal and survival. PTEN is a major inhibitor of AKT activation. Depletion of PTEN, induces AKT activation and subsequent FOXO inactivation. It is likely that FOXOs mediate at least a part of the PTEN effects on hematopoietic stem cells71.Although FOXO3 was established as the most important FOXO family member regulator of hematopoietic stem cell’s self-renewal, FOXO1 is critical for the human embryonic stem cells (hESC) pluripotency maintenance. FOXO1-induced SOX2 and OCT4 expression is one of the mechanisms responsible for this process. Interestingly, in embryonic stem cells AKT is not the major regulator of FOXO1Hydra vulgaris, a member of the phylogenetically old animal phyla Cnidaria, which has been suggested to be biologically immortal due to the unlimited self-renewal capacity of their stem cells38,122.Fascinating, a recent study brings evidence that FOXO is a critical regulator of stem cell maintenance in 71. Recent reports provided evidence that in many types of malignancies, such as leukemia, colon, or gastro-intestinal malignanciens, a small population of cells similar to stem cells exists121. These cells, called cancer stem cells, have the potential of forming new tumors. Notably, most of the time, the cancer stem cells are resistant to current cancer treatments, and these therapies may result in cancer stem cells enrichment. These cells serve as a starting point in cancer recurrence121. Similarities between stem cells and cancer stem cells were best described in the hematopoietic system, where similar surface markes and signal transduction patterns were described between the hematopoietic stem cells and leukemia-initiating cells121.FOXO transcription factors are not only required for maintenance of somatic stem cells, but they also play an important role in cancer stem cells71.Presented results have not only implications in uncovering the stem cells/cancer stem cells regulation, self-renewal and differentiation, but also for the development of novel therapeutic strategies in cancer, degenerative diseases and many other pathologiesincludingthe nervous system, cardiovascular system, reproductive system of males and females, lung, liver, spleen, pancreas, thymus, and skeletal muscle. However, each member of the FOXO family has its own expression pattern, since they are not equally expressed in all tissues20. FOXO1 is better represented in adipose tissue27. FOXO3 has the highest expression in liver, but it is also being predominantly expressed in heart, brain, kidneys, and ovaries, while FOXO4 is found mainly in the muscle and heart20,27. The newest member of the transcription family, FOXO6, is present in the brain. The association of this member with other tissues is still a matter of study123.FOXO family members are expressed in almost every tissue of the human body,124,125. FOXO1 is found to be expressed at high levels in IGROV 1 (human ovarian carcinoma cells), astrocyte cells, RL 7 (human follicular lymphoma cells), HT29 (human colon carcinoma cells) and HEK 293 (human embryonic kidney cells). FOXO3 high levels were especially found in SHSYSY (human neuroblastoma cell line), RPMI 8226 (human myeloma cells), MCF 7 (human breast adenocarcinoma cell line), LNCAP (human prostate adenocarcinoma cells), U251 (human glioma cells). FOXO4 is mostly expressed in CCRT CEM (human leukemia cell line), ALVA 31 (human prostate tumor cells), HL 60 (human promyelocytic leukemia cell line), TK 10 , MALME 3M  and HEP G2 (human hepatocellular carcinoma cells)126-128.Cell lines  are some of the most used and useful tools for studying the structure, function, regulation and expression of proteins in general, and FOXO family members in particular. FOXO members expression in different cell lines (including NCI 60 group of cell lines) is partially known126-128.Knowing the qualitative and quantitative expression pattern of FOXO family members is important in elucidating their functions and regulation. Thus, databases summarizing these patterns in various tissues and cell lines are very helpful and necessary. For example, BioGPS presents experimental results showing the mRNA expression levels of a wide number of genes in most of the human tissues and many (mostly human) cell lines129. Non-coding RNAs were shown to suppress translation of FOXO family members in various tissues and contexts. In particular, the microRNAs-dependent inhibition of FOXO1 and FOXO3 expression is better studied and is summariez below, in chapter 7.Regulation of FOXOs expression is not yet well understood. P300 was shown to control FOXO1 gene expression by binding to the proximal region of the FOXO1 promoter (CRE tandem sites)130.However, the main transcription factors implicated in FOXOs expression are not well understood. Interestingly, methylation of FOX promotors induces suppression of their expression. As an example, promoter methylation induced by BRAF results in inactivation of FOX genes expression131. For instance, microRNAsgained a special attention in melanoma studies and the altered pattern of miRNA in melanoma seems to be related to apoptosis (miR-15b), cell cycle (miR-193b) and invasion/metastasis (miR-182)132.The miRNAs are 18–28 nucleotide-long noncoding RNA molecules with an important role in post-transcriptional regulation of protein expression that regulate a variety of cellular processes, including cell differentiation, cell cycle progression and apoptosis. These miRNAs can function as oncogenes or tumor suppressors, and oncogenic miRNAs (oncomiRs) are upregulated in cancer cells. In cancer, miRNAs were found to be situated both upstream and downstream of the carcinogenesis process and modified expression of some miRNAs is the outcome of carcinogenic transformation or progression, revealing that miRNAs may be potential diagnostic or prognostic tools in cancer90. Thus, in melanoma cells, miR-182 modulate the expression of both FOXO3 and microphthalmia-associated transcription factor (MITF). The inhibition of miR-182 by anti-miRs (blocking antisense oligonucleotides) hindered melanoma cell migration and triggered their apoptosis90. In breast cancer cells, FOXO1 was coordinately targeted by miR-27a, miR-96, and miR-182, while the inhibition of each miRNA resulted in induced levels of FOXO1 and reduced breast cancer cell survival90. miR-182 also targets FOXO1 in osteoblasts lineage cells in order to inhibit osteoblast proliferation and differentiation, repressing the osteogenesis133.The microRNA (miRNA)-mediated regulation of FOXO transcription factors was demonstrated by several groups within the last three years. miR-182 has been shown to specifically target FOXO transcription factors irrespective of cell type, since miR-182 seems to target FOXO3 in melanoma cells, FOXO1 in breast cancer cells and FOXO1 in activated helper T (Th) lymphocytes134. Also, increased expression of miR-96 in breast cancer cells have been shown to downregulate FOXO3 transcription factor with consequent induction of cell proliferation135. Also, increased miR-96, miR-182 and miR-183 downregulate the expression of FOXO1 transcription factor in classical Hodgkin lymphoma (cHL) cell lines, suggesting that decreased FOXO1 expression is involved in lymphomagenesis136. Moreover, a recent study performed on DU145 and LNCaP human prostate cancer cells show that upregulated microRNA-370 induces proliferation due to downregulation of the FOXO1 transcription factor137. In addition, miR-155, which is highly induced in mature activated T and B cells and in Treg cells, has recently been shown to target FOXO3 in T cells, but it remains to be shown whether FOXO3 via miR-155 contributes to the observed phenotypes in B and T lymphocytes90.Many other microRNAs were shown to regulate FOXOs activity and functions, including apoptosis or cell cycle. MiR-96 has been shown to regulate FOXO1-induced apoptosis in transitional cell carcinoma90. However, some difficulties may appear in the miRNA-based therapeutic manipulation of FOXO transcription factors. For example, a single miRNA may target hundreds of genes among FOXOs, and the therapeutic manipulation of a specific miRNA could have unanticipated adverse effects by influencing whole gene networks, while having only moderate effects on FOXO genes90. Since delivery of miR-182 mimics could induce lymphoproliferative disease or other forms of cancer by systemic repression of FOXO transcription factors, another challenge in the miRNA-FOXO therapeutic manipulation is the specific delivery of the miRNA into the target cell,in order to avoid adverse effects in other cell types or tissues90.New strategies to modulate FOXO expression may now be developed since the discovery of miRNAs targeting FOXO transcription factors138.Several microRNAs were identified as being targets of FOXO transcription factors. An AKT - FOXO - miR-30d signaling pathway was recently identified. After inhibition of AKT, activated FOXO3 leads to upregulation of miR-30d. miR-30d acts as a tumor suppressor in renal cell carcinoma, further inhibiting the oncoprotein metadherin (MTDH)139.Another recent study shows that FOXO1 stimulates the expression of a microRNA cluster located on a X chromosome, in a direct manner, dependent on RNA polymerase II, but not on the de novo protein synthesis. Thus, FOXO1 upregulates miR-506, miR-508, miR-513c, miR-513a-1, miR-513a-2. Also, the same study shows that inhibition of PI3K-AKT axis in LNCaP and MCF7 human carcinoma cell lines is followed by increased miR-506. As suggested by authors, miRNAs could be valuable biomarkers of FOXO activity140. The precursors of the miR-106b~25 cluster members are located on Mcm7 gene. FOXO3 directly binds to the first intron of this gene, modulating the expression of the microRNAs. Thus, FOXO3 transcription factors could be an important tool in preventing the loss of neurogenesis during aging140. Further studies are needed to completely understand the regulation of microRNAs expression by activated FOXO proteins.A study performed on primary cultures of neural stem/progenitor cells (NSPCs) from adult mice show that the expression levels of the miR-106b~25 cluster members  is modulated by FOXO3 transcription factors in a complex mannerFOXO transcription factors and tumor suppressors are ubiquitously expressed in the human body, with some specific differences between its members. As resumed above, FOXO proteins are characterized by a remarkable functional diversity, being implicated in regulation of many critical cellular functions, such as cell cycle arrest, apoptosis, oxidative detoxification, DNA damage repair, stem cell maintenance, cell differentiation, cell metabolism, angiogenesis, cardiac development, aging and others.FOXO proteins play an important role not only during physiological cellular processes, but also in few pathologies, such as cancer. They are well known tumor suppressors proteins. Although FOXO proteins protect the human body by playing a central role in a wide range of mainly physiological functions, under some circumstances, FOXO’s roles can become harmful for the human cells. This is because FOXO is also involved in a number of pathological functions, such as inflammation, muscle atropy, and a number of physiological functions that become harmful for the organism. For example, apoptosis places FOXO proteins on the good side when it leads to tumor suppression, but in some cases cellular apoptosis can become itself a significant component for pathology in diseases such as neurodegenerative disease, diabetes mellitus (DM), and cardiovascular injury.Interestingly, while excessive FOXO levels induce cell cycle arrest and cell death, complete knock-down of FOXOs leads to cell cycle progression impairment, suggesting that certain levels of FOXO activation are required for cell cycle progression. Moreover, FOXO proteins play a variety of roles in the cells dependending on the context.In addition, FOXO have critical implications in both normal and cancer stem cell biology. FOXO proteins are required for adult stem cells maintenance. Novel strategies to modulate FOXO expression and activity may now be developed since the discovery of novel FOXO regulators and non-coding RNAs (such as microRNAs) targeting FOXO transcription factors.Thus, FOXO family of transcription factors and tumor suppressors are crucial regulators of cell fate and of critical normal or pathological cell decisions. A better understanding of their structure, critical functions and regulation may contribute to the development of novel therapies for cancer and other FOXO-related diseases.FOXO proteins are characterized by a remarkable functional diversity, controlling many critical cellular processes: cycle arrest, apoptosis, oxidative detoxification, DNA damage repair, stem cell maintenance, cell differentiation, cell metabolism, angiogenesis, cardiac development, fertility, immune response, neuronal survival,aging and others.


The transmembrane protein neuropilin-1 (NRP1) promotes vascular endothelial growth factor (VEGF) and extracellular matrix signaling in endothelial cells (ECs). Although it is established that NRP1 is essential for angiogenesis, little is known about its role in EC homeostasis. Here, we report that NRP1 promotes mitochondrial function in ECs by preventing iron accumulation and iron-induced oxidative stress through a VEGF-independent mechanism in non-angiogenic ECs. Furthermore, NRP1-deficient ECs have reduced growth and show the hallmarks of cellular senescence. We show that a subcellular pool of NRP1 localizes in mitochondria and interacts with the mitochondrial transporter ATP-binding cassette B8 (ABCB8). NRP1 loss reduces ABCB8 levels, resulting in iron accumulation, iron-induced mitochondrial superoxide production, and iron-dependent EC senescence. Treatment of NRP1-deficient ECs with the mitochondria-targeted antioxidant compound mitoTEMPO or with the iron chelator deferoxamine restores mitochondrial activity, inhibits superoxide production, and protects from cellular senescence. This finding identifies an unexpected role of NRP1 in EC homeostasis.  •A subcellular pool of NRP1 localizes in the mitochondria of endothelial cells (ECs)•NRP1 regulates mitochondrial function via ABCB8 transporter•NRP1 loss induces iron accumulation and iron-dependent oxidative stress in ECs•NRP1 protects ECs from iron-dependent premature cellular senescence Cell Biology; Functional Aspects of Cell Biology; Molecular Biology Neuropilin-1 (NRP1) is a transmembrane protein expressed by many cell types including endothelial cells (ECs) . AblatioMitochondria are emerging as regulators of EC signaling and function. ECs rely mainly on anaerobic glycolysis to meet their energy demand and to produce ATP ; thus miMitochondria are also a focal point for the regulation of cellular redox potential and iron metabolism. Mitochondria synthesize the iron-sulfur protein (Fe/S) clusters, which are essential for the electron transfer reactions and produce the heme's precursor compound protoporphyrin IX . Thus miROS are mainly generated as a by-product of the mitochondria electron transport chain during oxidative phosphorylation  or by acSenescent cells irreversibly lose the ability to replicate and thereby contribute to tissue aging by disrupting tissue homeostasis . ReplicaHere, we found that in addition to the well-established role in angiogenesis, NRP1 also has a role in EC health in non-angiogenic conditions by promoting mitochondrial function through the mitochondria-specific ATP-binding cassette transporter ABCB8 . DownregTo investigate a potential role of NRP1 in mitochondria, we first investigated whether NRP1 localizes in the mitochondria of human microvascular endothelial cells (HDMECs) by performing immunostaining for NRP1 and the mitochondrial outer membrane component TOM20. Specificity of anti-NRP1 antibody was confirmed by staining HDMECs downregulated for NRP1 expression using a previously validated small interfering RNA (siRNA)  FiguresA and S1BTo determine whether NRP1 or VEGF signaling plays a role in mitochondrial function, we downregulated NRP1 or VEGFR2 expression in HDMECs and investigated mitochondrial morphology. Thus we analyzed the mitochondrial network coverage in HDMECs transfected with NRP1 targeting, VEGFR2-targeting, or with control siRNA C and 1D.Then, we analyzed the protein level of the mitochondrial mass indicator TOM20 by immunoblotting, as a measurement of mitochondrial content . In agreBecause mitochondria are organized in a network maintained by organelle fission and fusion , we inve+ gradient across the inner mitochondrial membrane, which creates an inter-membrane mitochondrial potential (ΔΨ) functional to ATP production. Thus we measured ΔΨ by live staining HDMECs transfected with si-NRP1, si-VEGFR2, or si-control with the fluorescent dye tetramethylrhodamine methyl ester (TMRM), whose accumulation into the mitochondria depends on ΔΨ y5-labeled vasculature of nrp1asa1485/sa1485 mutant (nrp1a−/-) zebrafish embryos. Live transgenic embryos at 3 days post-fertilization (dpf) were stained with CellROX dye, which detects generalized oxidative stress  approach to analyze changes in serine phosphorylation in the proteome of HDMECs transfected with si-NRP1 or si-control. We found that NRP1 downregulation affected serine phosphorylation of several mitochondrial enzymes . Phosphoin vivo, we analyzed ABCB8 protein levels in aortas of adult conditional Nrp1-null mice lacking (Nrp1WT) or expressing a tamoxifen-inducible, endothelial-specific Cre transgene (Nrp1ECKO). NRP1 and ABCB8 staining was performed on whole-mount aortas where ECs were counterstained with PECAM  and ferric (Fe3+) content in HDMECs lacking or expressing NRP1 using a ferene S-based colorimetric assay . pression F. Then wpression F, and 4Gpression , we invepression F and 5G,pression E and 2F,pression F and 5G.To further investigate this hypothesis, we measured mitochondrial ΔΨ and ROS in HDMECs with a simultaneous knockdown for ABCB8 and NRP1. Downregulation of both NRP1 and ABCB8 H had no Cellular senescence can be prematurely induced by telomere-independent mechanisms, which include excessive oxidative stress and mitochondrial dysfunction . Thus weBecause EC proliferation was affected by NRP1 loss, we investigated whether the expression of cyclins was affected by NRP1 knockdown. In agreement with the reduced cell growth, immunoblotting analysis showed reduced cyclin D1 and cyclin B1 levels and a concomitant increase in cyclin D2 expression in HDMECs si-NRP1 compared with si-control I and 6J.Nrp1ECKO) and from control littermates (Nrp1WT). Nrp1ECKO MLECs showed significantly higher p16 protein expression  isolated from mice genetically deficient for NRP1 in ECs (pression K and 6L pression M and 6N We then investigated whether by regulating iron homeostasis the NRP1-ABCB8 pathway protects ECs from premature senescence. SA-β-gal staining showed that NRP1 or ABCB8 downregulation similarly increased the number of SA-β-gal-positive HDMECs O and 6P.Despite oxygen availability, ECs mainly rely on anaerobic glycolysis to meet their energy demand and to produce ATP . Thus alCellular senescence limits the capacity of tissues to undergo repair by cell proliferation and thereby accelerates age-related degenerative diseases such as sarcopenia , osteoarFurthermore, we show that NRP1 prevents mitochondrial superoxide production and EC senescence by promoting iron homeostasis via the mitochondrial transporter ABCB8 . As a poTreatment with the iron chelator compound deferoxamine restores the mitochondrial membrane potential of NRP1-deficient HDMECs and HUVECs and abrogates mitochondrial superoxide production. Deferoxamine has been shown to promote blood flow recovery in a hindlimb ischemia mouse model  and to iBy showing that deferoxamine restores mitochondrial membrane potential and reduces ECs senescence in NRP1-deficient ECs , our datThe function of NRP1 in regulating mitochondrial homeostasis and iron metabolism independently of VEGFR2 signaling expands the current model, which suggests that NRP1 promotes several signals in ECs and is not acting exclusively as a VEGF co-receptor . BecauseIn ECs, mitochondria function as a metabolic hub . MitochoOur data suggest that promoting NRP1-dependent signaling could be particularly relevant in age-related diseases characterized by increased oxidative stress or by deregulation of iron metabolism such as atherosclerosis, vascular dementia, and Alzheimer disease. Oxidative stress and iron-mediated free radical production mediate neuronal death and gliosis. Furthermore, oxidative stress induces endothelial dysfunction in a model of cerebral ischemia . ImportaIn conclusion, our data demonstrate an unexpected function of NRP1, which acts as a key regulator of mitochondria and iron homeostasis in ECs by suppressing iron accumulation and iron-dependent oxidative stress via ABCB8, independently of VEGF signaling. Our work suggests that the NRP1-ABCB8 pathway could protect ECs function from iron-dependent ROS-induced endothelial dysfunction and have a protective role in age-related diseases.in vitro approach that relied on siRNA-mediated transient downregulation of NRP1 and ABCB8. This approach allows neither the long-term effects of NRP1 or ABCB8 downregulation in ECs nor the role of the NRP1/ABCB8 pathway in endothelial function in vivo to be studied. Although we have established that genetic manipulation of NRP1 expression prevents oxidative stress in a zebrafish model and protects ECs from senescence in an ex vivo model, whether the NRP1-ABCB8 pathway plays a role in physiological or pathological angiogenesis, or in homeostatic EC functions such as nitric oxide synthase activation, vascular permeability, and leukocyte-EC adhesion remains to be addressed. Furthermore, the mechanisms that modulate NRP1 expression in pathological contexts, such as neurodegenerative disorders or ischemia, are currently unclear and would need to be further investigated.To identify the NRP1-ABCB8 pathway and to investigate its role in ECs, we used an All methods can be found in the accompanying