Interethnic and interindividual variability in in vivo cytochrome P450 (CYP450)-dependent metabolism and altered drug absorption via expressed transport channels such as P-glycoprotein (P-gp) contribute to the adverse drug reactions, drug–drug interaction and therapeutic failure seen in clinical practice. A cost-effective phenotyping approach could be advantageous in providing real-time information on in vivo phenotypes to assist clinicians with individualized drug therapy, especially in resource-constrained countries such as South Africa. A number of phenotyping cocktails have been developed and the aim of this study was to critically assess the feasibility of their use in a South African context. A literature search on library databases , PubMed, Scopus and TOXLINE) was limited to in vivo cocktails used in the human population to phenotype phase I metabolism and/or P-gp transport. The study found that the implementation of phenotyping in clinical practice is currently limited by multiple administration routes, the varying availability of probe drugs, therapeutic doses eliciting side effects, the interaction between probe drugs and extensive sampling procedures. Analytical challenges include complicated sample workup or extraction assays and impractical analytical procedures with low detection limits, analyte sensitivity and specificity. It was concluded that a single time point, non-invasive capillary sampling, combined with a low-dose probe drug cocktail, to simultaneously quantify in vivo drug and metabolite concentrations, would enhance the feasibility and cost-effectiveness of routine phenotyping in clinical practice; however, future research is needed to establish whether the quantitative bioanalysis of drugs in a capillary whole-blood matrix correlates with that of the standard plasma/serum matrixes used as a reference in the current clinical environment. Interindividual variability in medicine response contributes to adverse drug reactions (ADR), drug–drug interaction (DDI) and therapeutic failure ,2. The ASixty to eighty percent of commercially available drugs today are metabolized by the CYP450 enzymes with great interindividual and interethnic variability affecting therapeutic outcomes .Geographical ancestry and ethnicity influence CYP allele frequencies, resulting in worldwide variability in genotypic expression and measured phenotypes, with significant differences in treatment response, risk profile and disease prevalence . Samer eIn addition to genotype, several intrinsic and extrinsic factors influence the activity of these drug-metabolizing enzymes and transmembrane transport proteins, with a high degree of population differences in disease prevalence or outcomes. These factors include, but are not limited to, epigenetic factors regulating the expression of drug-metabolizing enzymes and transport proteins ; non-genWhen considering a more individualized approach to pharmacotherapy, it is clear that genotyping alone cannot infer altered metabolic or transport phenotypes, considering the complex interaction between genotype and extrinsic factors influencing metabolic or transport activity . GenotypThe aim of this study was therefore to critically review phenotyping cocktails aimed at assessing the real-time in vivo CYP450 metabolic activity and P-gp activity for feasibility of use in routine clinical practice within a Sub-Saharan African context and to identify challenges in the implementation thereof.A literature search conducted using the University of Pretoria’s library databases , PubMed, Scopus and TOXLINE) was limited to in vivo cocktails used in human populations consisting of five or more probe drugs to phenotype phase I metabolizing enzymes and/or the P-gp transporter with a cocktail approach. Only articles available in the English language were included. A summary of the multiple probe phenotyping cocktails is given in At present, phenotyping cocktails, containing multiple probe drugs, are used for the simultaneous assessment of drug metabolism during drug development in drug–drug interaction and toxicology studies and regulated by the EMA and the FDA . Due to During the validation of phenotyping cocktails, pilot PK studies were conducted as a proof of concept for use in human populations, and most of the reviewed cocktails included healthy non-smoking male subjects ,26,28,37Although some cocktail studies included genotyping, the objective was not to infer genotype–phenotype relationships, but rather to exclude certain genotypes or as an exploratory analysis of interindividual variation. For the Pittsburg 2006 cocktail  for examAlthough most of the 24 phenotyping cocktails in Despite the use of drug cocktails during drug development, routine phenotyping in clinical practice towards individualized pharmacotherapy has not yet become reality. The only example of routine phenotyping in clinical practice is the determination of phenylalanine in small volumes of blood (DBS) or urine in newborn infants, for phenylketonuria screening . For cliThe first main problem of current probes suggested by the FDA for phenotyping is the fact that no probe drug is completely selective for a single metabolizing enzyme or transporter. Nonetheless, the contribution of a specific pharmacokinetic pathway to the disposition of the probe drug should be primary and in addition must be indicative of changes in the phenotype when subject to an inducer or inhibitor . For exaPhenotyping drug transporter activity may also provide a useful metric to assess and predict drug absorption or excretion (depending on the location of the drug transporter protein) in vivo . The rolThe chosen probe drugs should clearly elucidate the in vivo pharmacokinetic phenotype under investigation, and overlapping substrate specificities between P-gp and CYP3A4 in particular should be considered. A higher expression of CYP3A4 in enterocytes will significantly influence the first pass bioavailability of CYP3A4 substrates and therefore if the objective is to phenotype hepatic CYP3A4 activity, probe substrates should be administered by the intravenous route . ChangesSecondly, earlier cocktails contained probe drugs at therapeutic doses, contributing to possible side effects, especially considering drugs with narrow therapeutic indexes, such as tolbutamide, warfarin and digoxin. Any small variation in enzyme or transport activity could contribute greatly to the disposition of drugs with a narrow therapeutic index, causing severe adverse reactions. Possible side effects with therapeutic probe drug doses included hypotension with debrisoquin (CYP2D6 probe), hypoglycemia with tolbutamide  (CYP2C9 0–24 h ratios for probe versus metabolite to a number of single time point plasma metabolic ratios (see 2 of 0.959). Yang et al., on the other hand, found a 4 h limited sampling AUC for midazolam and a 4 h single time point concentration to best fit a two-compartmental population PK model, derived from 2122 observations from 152 healthy subjects, for the estimation of CYP3A4 metabolic activity [A third main challenge of current proposed phenotyping cocktails is the inconvenient and impractical sample collection protocols. Multiple time point venous plasma sampling or collective urine sampling would not be feasible in a routine clinical environment. Use of a single or limited time point sampling strategy to measure metabolic or transporter activity would be advantageous especially when coupled with probe drugs with short elimination half-lives to reduce the time patients have to spend at the clinic for observation. Studies comparing the systemic clearance (AUC) of probe drugs or the clearance ratio of probe drug to metabolite to limited AUC or single time point metabolic ratios are currently underway ,86,87,88tios see  includinactivity  and limiactivity  have alsactivity , 4 h [56activity  post oraactivity ,78. Intr0–24h when administered together [An understanding of the PK and PD interaction between probe drugs used together in a cocktail approach is essential. Interactions at the target receptor sites (PD interactions) should also be considered; for example, using the antihypertensives losartan and debrisoquin together might cause hypotension. Each probe drug used in a proposed cocktail must be validated individually and then in combination to exclude interaction with other probe drugs. In the Basel cocktail, chlorzoxazone (a CYP2E1 probe) had to be excluded due to a significant interaction with CYP3A4, significantly increasing midazolam AUCtogether . To overtogether . Althougtogether . SimultaPharmacokinetic variability is caused by a complex interplay between many different factors influencing the available drug concentration in the body. Measuring specific drug concentrations of substrates for either metabolizing enzymes or drug transporter proteins provides a fingerprint of metabolic or transport activity in vivo, which is then correlated with the real-time phenotype. Unlike the functional genotype, which depends on epigenetic regulation or post-translational modifications, this approach measures biochemical activity directly correlated with functional phenotype. It considers all intrinsic and extrinsic factors influencing variability in a dynamic way, because this will change depending on pathophysiology, age, lifestyle and co-medication changing over time for an individual, and should therefore be carried out routinely in order to assist clinicians in drug selection and dosing toward personalized pharmacotherapy. This, in turn, could help to reduce the incidence of ADR, DDI and therapeutic failure seen in Africa.A number of phenotyping cocktails aimed at assessing in vivo CYP450 metabolic activity and in some instances P-gp activity have been developed, but their implementation in clinical practice has been limited by a wide variety of challenges, as set out above. Non-invasive sampling could be advantageous for implementing phenotyping in routine practice to obtain an estimation of metabolic or transport activity at baseline or for therapeutic drug management, especially in genetically diverse population groups. In this regard, dried blood spot (DBS) sampling can be used to simultaneously assess P-gp and CYP activity with a low-dose phenotyping cocktail and limited sampling to measure pharmacokinetic markers and, by extension, to measure phenotype. Before DBS sampling can be implemented in routine clinical practice, the question remains as to whether the quantitative bioanalysis of drugs in a capillary whole-blood matrix correlates with that of the standard plasma/serum matrixes used as a reference in the current clinical environment. When using alternative sampling strategies to the gold-standard plasma sampling, it is important that future studies assess the distribution of the expressed enzymes or transporters under investigation and the pharmacokinetic processes involved, i.e., absorption or excretion rates and drug distribution in different physiological compartments.


Pancreatic ductal adenocarcinoma (PDAC) is the most common form of pancreatic tumours, representing greater than 90% of all diagnosed cases of pancreatic cancer. PDAC has the lowest 5-year survival rate of all tumour malignancies with less than 9% patient survival. A unique feature of PDAC tumours is the presence of a dense fibrotic fortress that creates a physical barrier around the cancer cells, thus resulting in a reduced penetrability of drugs and a ‘sanctuary’ in which cancer cells thrive, termed desmoplasia. Extensive desmoplasia in the PDAC tumour microenvironment (TME) is a crucial factor that influences PDAC development, progression, metastasis, and resistance to treatment. This review will focus on the role of the TME in PDAC, current treatments for PDAC, and a reflection on past and current clinical trials targeting components of the TME in PDAC.An overabundance of desmoplasia in the tumour microenvironment (TME) is one of the defining features that influences pancreatic ductal adenocarcinoma (PDAC) development, progression, metastasis, and treatment resistance. Desmoplasia is characterised by the recruitment and activation of fibroblasts, heightened extracellular matrix deposition (ECM) and reduced blood supply, as well as increased inflammation through an influx of inflammatory cells and cytokines, creating an intrinsically immunosuppressive TME with low immunogenic potential. Herein, we review the development of PDAC, the drivers that initiate and/or sustain the progression of the disease and the complex and interwoven nature of the cellular and acellular components that come together to make PDAC one of the most aggressive and difficult to treat cancers. We review the challenges in delivering drugs into the fortress of PDAC tumours in concentrations that are therapeutic due to the presence of a highly fibrotic and immunosuppressive TME. Taken together, we present further support for continued/renewed efforts focusing on aspects of the extremely dense and complex TME of PDAC to improve the efficacy of therapy for better patient outcomes. Pancreatic ductal adenocarcinoma (PDAC) is the most common form of pancreatic neoplasms, representing greater than 90% of all diagnosed cases of pancreatic cancer with neuroendocrine tumours accounting for the remaining 10% . Poor suPDAC originates from the exocrine portion of the pancreas which constitutes 90% of the organ and consists of acinar cells and a ductal network. Acinar cells exhibit a degree of plasticity which allows them to contribute to both homeostasis and regeneration of pancreatic exocrine tissue. Under environmental stimuli  acinar cells transdifferentiate to a more epithelial phenotype, a process known as acinar to ductal metaplasia (ADM) ,8. DurinA unique feature of PDAC tumours is an abundance of dense fibrotic stroma that constitutes up to 90% of the tumour volume ,11. The KRAS , CDKN2A (cyclin dependent kinase inhibitor 2A), SMAD4 (Mothers against decapentaplegic homolog 4), and TP53 (Tumour protein 53) are most frequently mutated ..32].The PDAC TME consists of an abundance of stromal cells such as pancreatic stellate cells and CAFs, which are major contributors to the dense desmoplastic reaction seen in PDAC through the production of acellular components that make up the ECM ,34. StelCAFs are major orchestrators of the pancreatic TME due to their dominant production of acellular components of the ECM, e.g., collagen ,34. In tThe ECM is a non-cellular component of the TME which provides essential physical scaffolding as well as biochemical and biomechanical cues to the cells . ECM comIn PDAC progression and in pancreatitis, collagens are the most prominent component of ECM proteins, comprising more than 90% of the ECM matrisome at all stages of disease. Proteoglycans and glyco-proteins such as fibronectin make up the rest of the matrix and these ECM proteins are all overexpressed in PDAC . An analTumour stiffness caused by collagen cross-linking can also enhance cancer cell proliferation and promote cancer cell invasion via integrin signalling. Briefly, collagen cross-linking occurs via a family of enzymes, lysyl oxidases (LOX), which activate signalling factors such as yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) for cancer cell proliferation ,60. An iAs tumours increase in size, the TME becomes increasingly hypoxic, and cancer cells adapt by upregulating tissue processes that enhance access to the blood supply ,63. One Interestingly, some tumours, including PDAC, are considered ‘hypo-vascular’ and are populated with avascular stromal ‘deserts’ with substantially lower microvessel densities when compared to healthy areas of the pancreas . VasculaDespite the dysfunctional vasculature and poor blood perfusion, PDAC tumours are still able to thrive by adapting and using alternate metabolic and scavenging pathways  ,69. To tPDAC tumours are often considered immunologically ‘cold’; however, the role of the immune TME in PDAC is emerging as an important prognostic feature . The PDAH1), TH2, TH17, TH9, T follicular helper (TFH) and Tregs are found within tumours and numbers of CD4+ T cells with TH1 phenotype are associated with beneficial outcomes. In addition to recruited populations of T cells, tissue resident memory T cells (TRM) are a tissue-specific component of the TME. TRM are CD4 or CD8 positive T lymphocytes that persist in tissues long-term following primary T cell responses, and their proportion within the T cell infiltrate is related to a good prognosis . H. H115]. c cancer ,118.Historically, clinical trials with targeted treatments for PDAC have been underwhelming. Multiple clinical trials of anti-angiogenic agents that target the tumour vasculature without interfering with the normal vasculature yielded disappointing results  112,119119. EvenThe dense stromal reaction leading to the compression of blood vessels and inadequate drug penetration has also emerged as a potential target for therapeutic intervention. A preclinical study demonstrated that excessive HA present in PDAC tumours  is degraded by pegvorhyaluronidase alfa (PEGPH20), and that this degradation of HA by PEGPH20 led to an increase in drug delivery. Another clinical trial targeted ECM producing pancreatic stellate cells via all-trans-retinoic acid (ATRA) to reprogram pancreatic stroma and suppress PDAC growth. ATRA in combination with gemcitabine-nab-paclitaxel chemotherapy delivered to patients with advanced, unresectable PDAC is currently in a phase II randomised trial . AnotherGiven that PDAC tumours are intrinsically immunosuppressive and have lower immunogenic potential , targetiWhile the survival rates of most cancers have dramatically improved in the last few decades, this is not the case for PDAC where the 5-year survival rate has remained below 9% . At the As detailed above, the main challenge is the difficulty in delivering drugs to the tumour, with the presence of a densely packed ‘desmoplastic’ TME preventing most anti-PDAC drugs and immune cells from reaching the ‘heart’ of the tumour in therapeutic/effective concentration. Given that the prognosis of PDAC patients is largely determined by stage of diagnosis and tumour biology , renewed


The endothelium, the monolayer of endothelial cells that line blood vessels, is exposed to a number of mechanical forces, including frictional shear flow, pulsatile stretching and changes in stiffness influenced by extracellular matrix composition. These forces are sensed by mechanosensors that facilitate their transduction to drive appropriate adaptation of the endothelium to maintain vascular homeostasis. In the aorta, the unique architecture of the vessel gives rise to changes in the fluid dynamics, which, in turn, shape cellular morphology, nuclear architecture, chromatin dynamics and gene regulation. In this Review, we discuss recent work focusing on how differential mechanical forces exerted on endothelial cells are sensed and transduced to influence their form and function in giving rise to spatial variation to the endothelium of the aorta. We will also discuss recent developments in understanding how nuclear mechanosensing is implicated in diseases of the aorta. Summary: Endothelial cells are exposed to mechanical forces such as shear stress exerted by blood flow. We explore the emerging role of the nucleus in how these forces are sensed and transduced. The vessels of the circulatory system are lined by a continuous monolayer of endothelial cells (ECs), which are exposed to mechanical forces from circulating blood. Fluid shear stress acting on the endothelium of the aorta both maintains homeostasis and influences disease progression. Perturbation of mechanosensory pathways can impair endothelial function and eventually leads to highly prevalent diseases such as atherosclerosis . MechaniIn this Review, we discuss recent developments in the field of mechanobiology of the endothelium of the aorta. Although stretching and extracellular matrix (ECM) stiffness profoundly influence aortic EC biology, the relatively low piconewton force exerted by shear stress dominates EC phenotypes, and thus we focus on recent work describing how the aortic endothelium senses differential flow profiles. Moreover, we discuss how fluid shear stress is sensed by the cell and transduced to the nucleus either directly or indirectly, and how this informs intranuclear dynamics to shape chromatin conformation and, ultimately, transcriptional responses. The flow-dependent transcriptional response of ECs is well documented, so our focus will be on recently reported mechanosensors and mechanotransducers, and on the role of the nucleus and chromatin arrangements in shaping these transcriptional responses. Finally, we evaluate evidence of dysfunctional endothelial mechanosensing and its relevance for pathologies of the aorta.Blood ejected from the left ventricle during the cardiac cycle travels through the ascending aorta, the aortic arch and finally down the descending aorta . In the Flow patterning forms during development and is maintained throughout life. DF in the aorta occurs at postnatal day (P) 5 when the ductus arteriosus closes due to shifting of ventricular pressure induced by inflation of the lungs at birth. This was recently highlighted by en face dissections , PECAM1 and VEGFR2 (or KDR) mechanosensory complex within endothelial cell-cell junctions . This coThe junctional mechanosensory complex was further expanded to include VEGFR3 (or FLT4) through its association with the transmembrane domain of VE-cadherin. This VE-cadherin domain is critical for binding both VEGFR2 and VEGFR3, triggering downstream signal transduction and the alignment of ECs to shear stress. Activation and phosphorylation of VEGFR3 in response to shear stress occurs in both lymphatic and vascular ECs. Additionally, VEGFR3 was found to be highly expressed in the ECs of the inner aortic arch and its deletion led to reduced inflammatory markers . FurtherCCL2 and VCAM1, suggesting its differential abilities in sensing flow profiles , that binds VE-cadherin and p120 catenin (CTNND1) in ECs . The onsrphology  and, as rphology  preprintrphology .Vβ3 Vβ3 , which is localised to EC junctions under LF conditions in the aorta and exhibits cytoplasmic endosomal localisation in ECs of the DF-exposed inner aortic arch  activity  was found to regulate endothelial eNOS expression under LF conditions (G protein-coupled receptors (GPCRs) are a diverse family of multipass transmembrane receptors and are well-characterised mechanosensors . To datetic arch  Fig. 2)1 or S1PRactivity . Centralns of DF  lisation . More renditions  and NFκBnditions  2+ and remodelling of focal adhesions in response to flow (q/G11 and P2Y2 (P2RY2)  . Under D (P2RY2) . Piezo1  (P2RY2) . Interesnditions .in vitro, a phenomenon that requires HA, the glycocalyx, CD44 and the spectrin network. The same study confirmed the importance of spectrin network integrity for maintaining aortic EC alignment in vivo. Furthermore, the authors showed that shear stress altered plasma membrane tension, which subsequently triggered caveolae-localised Piezo1 activity and Ca2+ influx, which was required for downstream mechanosignalling and EC alignment  and heparan, is tethered to the apical membrane by the HA receptor CD44  and medilignment . This fiThe role of cilia as endothelial sensors of fluid shear stress is less well studied despite evidence that cilia indeed act as primary sensors of flow . Shear sThe architecture, site-specific flow dynamics and mechanosensory downstream signalling trigger the appropriate transcriptional response of the endothelium Fig. 2). KLF2  ner arch . Collectin situ staining. This study highlighted the existence of heterogeneous ECs across the aorta that exhibit specific signalling tied to their spatial location  and TAZ (WWTR1) are active due to the DF in this region . YAP/TAZOverall, several surface and junctional proteins have been shown to act as direct mechanical sensors of shear stress, which indicates how spatially distinct signalling is tuned in the endothelium of the aorta. Although recent work has shed significant light on these mechanisms, our understanding remains far from complete.KLF2 promotor , was shown to be flow sensitive and exhibited increased activity in the aortic arch compared to in the descending aorta, suggesting flow pattern-specific epigenetic regulation of histone H3 methylation . In agreKLF4 promotor methylation under DF to permit the expression of the pro-inflammatory cytokine CCL2, which would otherwise be supressed under LF  analyses of the ECs. These analyses indicated broad EC heterogeneity under LF and that exposure to DF initiated pro-inflammatory reprogramming through the modulation of KLF- and TEAD-binding motif accessibility , leading to stiffer nuclei  consisting of embedded protein complexes that bridge the cytoskeleton to the ONM and INM and directly tether chromatin. The linker of the nucleoskeleton and cytoskeleton (LINC) complex serves as a molecular bridge between the cytoskeleton, nuclear membrane and the nuclear lamina . The nucIndividual components of the nuclear membrane determine the appropriate response of ECs to mechanical force. The LINC components nesprin 1 (SYNE1) and 2 (SYNE2) regulate EC and nuclear shape as well as angiogenic capacity . Under cYAP promotor via increased repressive H3K27me3 marks to silence YAP transcription in ECs. This work also indicates that YAP/TAZ are required for the nuclear actin cap in ECs of the thoracic aorta . Laminopathies are linked to impaired mechanosensing, which has implications for the cardiovascular system .LMNA, which leads to a truncated variant of lamin A known as progerin  is caused by a single point mutation in progerin . This muprogerin . The nucprogerin , 2008. Oactivity . Interesactivity . Anotheractivity . Furthersing ECs . Defectsgnalling . Given tRegions of the aorta exposed to DF, such as the inner aortic arch, are predisposed to inflammation due to decreased anti-inflammatory and increased pro-inflammatory signalling and adhesion molecules, as discussed above. Added risk factors, such as poor diet and genetic predisposition, can trigger the influx of immune cells, accumulation of lipids and plaque development in these already insulted endothelial niches, leading to atherosclerotic lesions that pose significant risk to further pathological phenomena. A precursor step in the development of atherosclerosis is the reprogramming of the endothelium to a more mesenchymal phenotype in EndMT via the upregulation of transcription factors such as TWIST1 , which iNotch1 was in turn shown to promote atherosclerosis in regions usually ath eroprotected by LF  and Notch1 leads to atherosclerotic plaque development in normally atheroprotected regions of the descending aorta . Plexin ed by LF .Ift88 gene, which is required for ciliogenesis, specifically in ECs of the arch enhances plaque development Ift88 gen13 interactions and supresses YAP via reduced RhoA activity Jcad deficlerosis . AlthougBoth cell-cell junctions and cell-cell communication play crucial roles in maintaining the endothelium. The Notch pathway is a conserved cell-cell communication pathway with broad implications. Despite the atheroprotective role of Notch1 described by Piezo1-knockout mice, which could be due to FAK-mediated NFκB activation by DF Fig. 5). on by DF .Overall, these studies show that a number of mechanosensing apparatuses, from primary sensors to downstream signalling components of mechanotransduction, are implicated in the development of atherosclerosis. The differential role of these components in sensing DF and LF indicates that regulation of EC phenotype requires an intricate orchestration of these components to suppress pathological development, either through measured response to LF or suppressed pro-inflammatory signalling initiated by DF.Aneurysms are caused by vascular instability, where the ability of the vascular wall to contain outward forces results in bulging and ballooning of the vessel. Although the endothelium primarily senses shear flow, the role of the underlying intima, composed of the supporting parenchymal cells and vascular smooth muscle cells, in aneurysm development is far better understood. Indeed, impaired mechanosensing and genetic factors are more often associated with dysfunction of vascular smooth muscle cell contractility . HoweverC1041GFbn1 specifically into murine ECs impaired their alignment within both the ascending and descending aorta, and perturbed junctional stability under LF , presents with an increased risk of aortic aneurysm . Introduunder LF . The samunder LF . This maN-[N--L-alanyl]-S-phenylglycine t-butyl ester (DAPT) reduces AAA incidence in the angiotensin II-induced mouse model , ATACseq and Hi-C are likely to reveal further details on how signalling from sensors and transducers to the nuclear membrane informs intranuclear dynamics. Applying these advanced omics technologies will facilitate the delineation of spatially and, in the case of the aorta, mechanically unique mechanisms that program the endothelium. These will hopefully lead to important insights that could shape our understanding of vascular disease settings such as atherosclerosis, aneurysms and laminopathies.


Pulmonary arterial hypertension (PAH) is a progressive disease that is characterized by an obliterative vasculopathy of the distal pulmonary circulation. Despite significant progress in our understanding of the pathophysiology, currently approved medical therapies for PAH act primarily as pulmonary vasodilators and fail to address the underlying processes that lead to the development and progression of the disease. Endothelial dysregulation in response to stress, injury or physiologic stimuli followed by perivascular infiltration of immune cells plays a prominent role in the pulmonary vascular remodeling of PAH. Over the last few decades, our understanding of endothelial cell dysregulation has evolved and brought to light a number of transcription factors that play important roles in vascular homeostasis and angiogenesis. In this review, we examine two such factors, SOX17 and one of its downstream targets, RUNX1 and the emerging data that implicate their roles in the pathogenesis of PAH. We review their discovery and discuss their function in angiogenesis and lung vascular development including their roles in endothelial to hematopoietic transition (EHT) and their ability to drive progenitor stem cells toward an endothelial or myeloid fate. We also summarize the data from studies that link mutations in Sox17 with an increased risk of developing PAH and studies that implicate Sox17 and Runx1 in the pathogenesis of PAH. Finally, we review the results of recent studies from our lab demonstrating the efficacy of preventing and reversing pulmonary hypertension in animal models of PAH by deleting RUNX1 expression in endothelial or myeloid cells or by the use of RUNX1 inhibitors. By investigating PAH through the lens of SOX17 and RUNX1 we hope to shed light on the role of these transcription factors in vascular homeostasis and endothelial dysregulation, their contribution to pulmonary vascular remodeling in PAH, and their potential as novel therapeutic targets for treating this devastating disease. The pulmonary circulation is a low-pressure circuit with a mean pulmonary arterial pressure (mPAP) of approximately 14 mmHg. Elevation of mPAP > 20 mmHg is defined as pulmonary hypertension and is common in patients with chronic heart and lung diseases  were found to be associated with familial PAH. Mutations in BMPR2 have since been recognized as occurring in approximately 70% of HPAH , 5 and 1Although the current understanding of PAH pathophysiology remains limited, an emerging hypothesis is that vascular injury in response to a variety of factors such as hypoxia, shear stress, inflammation, oxidative stress, or growth factors, leads to abnormal proliferation or differentiation of pulmonary vascular cells including endothelial cells, smooth muscle cells, and adventitial fibroblasts. Under normal conditions, cellular stress or injury may trigger downstream activation of genetic programs involved in angiogenesis and neovascularization that are important for restoring vascular integrity. These programs are mediated by specific transcription factors which if not properly expressed may lead to abnormal pulmonary vascular remodeling .Currently approved medical therapies for PAH, have some ability to inhibit cellular proliferation and hypertrophy, but act primarily as pulmonary vasodilators and have limited ability to repress the pathogenic phenotypes of pulmonary vascular cells. Given their vital roles in angiogenesis and vascular homeostasis, several transcription factors have emerged as enticing therapeutic targets to potentially counteract and reverse the PAH vascular phenotype.In this review we focus on two transcription factors with emerging roles in PAH: SOX17 and one of its downstream targets, RUNX1. We examine their normal structure and functions, and their roles in EHT, endothelial progenitor cell differentiation, and genetic mutations associated with PAH. By examining the role of SOX17 and RUNX1 in the pathogenesis of PAH we hope to further our understanding of the cellular mechanisms that drive remodeling of damaged lung vessels in pulmonary vascular disease and propose potential future therapeutic targets for the treatment of patients with PAH.In 1990, the SRY gene—sex-determining region of the Y-chromosome was first discovered in humans and mice as a testis-determining gene , 14. TheThe high-mobility group (HMG) domain in SOX genes allows for DNA binding in a sequence-specific manner. Each of the SOX family of genes encodes an HMG domain of 79 amino acids that is highly conserved throughout eukaryotic species and enables their binding to a specific DNA sequence (A/T A/T CAA A/T) . SOX genSRY-box transcription factor 17 (SOX17) is a member of the SOXF subfamily along with SOX7 and SOX18. All members of this subfamily contain a TAD in the C-terminal and a short functional motif (DxxEFD/EQYL) thought to be involved in the interaction with Beta-catenin .SOX17 gene contains two exons expressed in human heart, lung, spleen, testis, ovary and placental and in fetal lung, and kidney tissue. Although widely expressed during development, SOX17 displays a largely endothelial-specific expression profile in healthy adult tissues . Expression of SOX17 appears to be crucial in maintenance of endothelial rather than hematopoietic cell fate, as studies have demonstrated that tissue-specific, temporally controlled, knockout of arterial genes (SOX17 and NOTCH1) causes an increase in hematopoietic cells due to loss of Sox17 dependent repression of hematopoietic transcription factors Runx1 and Gata2 in both human and mice stem cells  and fetal liver hematopoietic stem cells (HSCs) during development  signaling   . SOX17 h (hPSCs) .The signaling pathways by which SOX17 promotes progenitor cell and endothelial identities is not well understood. SOX17 appears to suppress SMAD3/TGF-β signaling—a pathway known as a potent inhibitor of epithelial cell proliferation. Interestingly, SOX17 mediated repression of hematopoietic lineage in EHT also appears to happen through TGF-β signaling . Recent SOX17 enhancer regions  (3.2% of patients) and in patients with IPAH/HPAH (0.7% of patients). Interestingly, the majority of the missense mutations occurred in the highly conserved HMG-box protein domain responsible for DNA binding   isolated from PAH patients compared to failed donor lungs  . Recent or lungs . Similaradhesion . ConversSox17 enhancer knockout mice (Sox17eKO) generated by CRISPR-mediated knockdown of the SOX17 enhancer regions initially detected in PAH patients (rs10958403 and rs765727) reduces Sox17 expression in mouse lung tissue and increases human pulmonary artery endothelial cell (PAEC) permeability while decreasing endothelial cell adhesion and VEGF-induced proliferative capacity  compared to controls when exposed to hypoxia ] were found to reverse pression .Collectively, these results point to impaired SOX17 signaling as an important contributor to the pathogenesis of PAH. The major roles of SOX17 in vascular homeostasis, neovascularization and maintenance of arterial/endothelial identity as well as its importance in response to vascular injury and stress, make it a promising therapeutic target for PAH. At the same time, SOX17 knockout models may serve as important new preclinical models for investigating PAH.Runt-related transcription factor 1 (RUNX1) is a downstream target of SOX17 and may play a role in mediating the effect of impaired Sox17 expression on PAH. The Runt gene was discovered in 1980 by Nusslein-Volhard and Wieschaus in a screen conducted to identify mutations affecting Drosophila larvae segment number, and polarity . One mutRUNX1 regulates the differentiation of HSCs into mature blood cells and is indispensable for the establishment of definitive hematopoiesis in vertebrates. RUNX1 is expressed in cells from all hematopoietic sites that contribute to formation of HSCs during development. HSCs are generated via a specialized subset of vascular endothelial cells known as the hemogenic endothelium that can differentiate into hematopoietic cells in a process called endothelial to hematopoietic transition (EHT). Developmentally, the endothelium arises in close contact with the hematopoietic system, and they share a common lineage in hemogenic endothelium. The adult hematopoietic system and long-term hematopoietic cells that give rise to life-long blood cell production are derived from hemogenic endothelial cells (HECs) in mid-gestational embryos. HECs are generated from embryonic endothelial cells after upregulation of Runx1 and activation of the Notch and TGF-β pathways during a brief developmental window in embryogenesis (Embryonic days 9.5–10.5). Runx1 is essential for EHT and its expression distinguishes HECs from non-HECs . HECs arRUNX1 are associated with Familial Platelet Disorder, a mild bleeding disorder associated with thrombocytopenia and high rates of myeloid leukemia , pancreatic cancer, myelodysplastic disorders, and a variety of other tumors (Homozygous RUNX1 mice lack hematopoiesis and are unable to survive past an early embryonic stage (E11.5-E12.5)  due to Cleukemia . Other mr tumors .Although EHT has been considered a developmental process that becomes inactive in adult life, under pathogenic conditions endothelial progenitor cell reservoirs may reactivate and contribute to adult hematopoiesis , 56. FurAlthough initially thought to only be expressed in areas of hematopoiesis, in 2011 Heley et al., found that RUNX1 is also highly expressed in the developing human lung . Future In the pulmonary vasculature, injury to the endothelium followed by upregulation of inflammatory signals and recruitment of macrophages, neutrophils and inflammatory cells are thought to be sentinel events in the development of pulmonary hypertension. In accordance with this, lung inflammation precedes pulmonary vascular remodeling in experimental models of PAH and failure to resolve progression of this inflammatory response is thought to underlie the development of PAH . PulmonaAlthough the role of RUNX1 in the pathophysiology of PAH is not yet known, one hypothesis is that disruptions in endothelial progenitor cell (EPC) fate from definitive endothelium to hematopoietic or myeloid fate may explain inadequate vascular repair and perivascular infiltrates in patients with PAH. Interestingly, increased myeloid derived cells and increased myeloid specific transcription factors are seen in the perivascular infiltrates of PAH patients and animal models of PAH . Activatin vivo and prevents both migration of EPCs from BM and the development of Su/Hx-PH- and monocrotaline-induced PH (MCT-PH) in mice  in Su/Hx mice, we demonstrated that adult endothelial cells undergo hematopoietic transformation suggesting reactivation of EHT, and migrate from the BM to the pulmonary vasculature . We also in mice  55, 56), 56in vi in mice . InteresCollectively, these results indicate that Runx1 may play a crucial pathologic role in the development and progression of PH. Considering that Runx1 is a downstream target of Sox17, it is tempting to speculate that mutations in the Sox17 enhancer that are associated with increased risk of PAH, may predispose to PH by failing to suppress Runx1 expression. Studies in our lab aimed at testing this hypothesis by examining the effect of suppressed Sox17 expression on the expression of Runx1 in the lung and in progenitor cells of animals with pulmonary hypertension and patients with PAH are ongoing. If the findings further studies support this hypothesis, Runx1 inhibition may prove an effective approach to treating PAH.Although data from our lab suggests that Sox17 and Runx1 play an important role in the pathogenesis of PAH, the mechanism by which they do so is not clear. One possibility may be their role in driving HSCs toward either an endothelial or myeloid fate. The healthy endothelial monolayer of pulmonary arteries regulates the influx of fluid, proteins, and blood cells into surrounding parenchyma and maintains vascular tone and integrity. The gradual disruption of the endothelial layer in response to stress is thought to be one of the precipitating events in PAH. Loss of endothelial integrity eventually leads to the accumulation of pro-inflammatory cells, altered cell viability, smooth muscle cell hyperplasia, fibroblast proliferation and eventually occlusive vascular lesions, and increased vascular tone. At the same time, BM-derived myeloid cells play a prominent role in the perivascular inflammation that leads to remodeling of the extracellular matrix and adventitia. Emerging data suggest that local and BM-derived progenitor cells play important roles in homeostasis of the pulmonary endothelium and circulation that may be particularly important after injury that leads to PAH. Interestingly, SOX17 and RUNX1 are closely linked in EHT, as SOX17 has been shown to directly repress RUNX1 expression and induce human embryonic stem cells towards a hemogenic fate rather than an endothelial one . The abiThe discovery of EPCs in peripheral blood during the late 1990s introduced the idea that vasculogenesis and angiogenesis did not occur only during fetal development but could arise in adulthood . The intIn 1997, Asahara et al.  charactein vitro  has shown significant overlap with markers on primitive HSCs. Furthermore, once isolated, these cells behave like hematopoietic colony forming cells and do not form endothelial cells . An altein vitro . Early EThe means by which EPCs influence PAH remains unclear. Current data support 4 main mechanisms: (1) localization to areas of vascular injury and restoration of vascular integrity; (2) paracrine effects—potentially mediated by secretions of proangiogenic growth factors; (3) support/restoration of other cells, and (4) differentiation into proinflammatory cells with subsequent immunomodulatory effects.in vitro demonstrate a hyperproliferative phenotype and an inability to form vessel networks  compared to controls (+ progenitors in human umbilical cord blood or adult BM (A pathogenic role of BM-derived EPC has been suggested by a number of studies in which these cells have been shown to induce PH when transplanted into healthy animals. For example, our lab demonstrated that whole BM obtained from mice with MCT-PH induces PH when transplanted into healthy mice , 85. Tracontrols . Collectadult BM  and we hadult BM .SOX17 gene is due to insufficient repression of its downstream target RUNX1 is not known. RUNX1 expression in the lung or circulating progenitor cells has not been examined in patients with PAH associated with SOX17 mutations, although this is an ongoing area of research in or lab and by other investigators. In addition to RUNX1, numerous other targets of SOX17 including HGF/c-Met, E2F1, BMPR2, and estrogen metabolism have been shown to play significant roles in modulating pulmonary hypertensive responses, and perturbations in their expression may play important roles in PAH associated with impaired SOX17 expression. Furthermore, if insufficient suppression of RUNX1 by impaired SOX17 expression causes pulmonary hypertension, it is not clear whether impaired SOX17 and RUNX1 signaling fails to maintain pulmonary vascular endothelial cell homeostasis or drives differentiation of BM-derived progenitor cells toward a myeloid fate. However, there is strong and accruing evidence to support the pathogenic role of SOX17 and RUNX1 in PAH and the potential of targeting the SOX17 and RUNX1 axis as a novel therapeutic approach in the management of PAH. Future studies are needed to determine if enhanced expression of SOX17 or inhibition of its downstream targets such as RUNX1, HGF/c-Met, or E2F1 can reverse PAH associated with the rare or common variants of SOX17 mutations or in patients with PAH that are not associated with impaired SOX17 expression. To this end, RUNX1 inhibitors have been developed for the treatment of hematologic malignancies and may be repurposed for the treatment of PAH and recent studies have suggested several drugs that have potential to enhance SOX17 expression despite mutations in enhancer signal 1 and 2 (Evidence is accruing to support a role for SOX17 and RUNX1 in the pathogenesis of PAH. Further research is needed to elucidate the mechanisms by which these developmentally important transcription factors may modulate pulmonary vascular remodeling and the development or progression of PAH. Whether or not the increased risk of PAH associated with mutations in the  1 and 2 . Targeti


Histone variants, which generally differ in few amino acid residues, can replace core histones  to confer specific structural and functional features to regulate cellular functions. In addition to their role in DNA packaging, histones modulate key processes such as gene expression regulation and chromosome segregation, which are frequently dysregulated in cancer cells. During the years, histones variants have gained significant attention as gatekeepers of chromosome stability, raising interest in understanding how structural and functional alterations can contribute to tumourigenesis. Beside the well-established role of the histone H3 variant CENP-A in centromere specification and maintenance, a growing body of literature has described mutations, aberrant expression patterns and post-translational modifications of a variety of histone variants in several cancers, also coining the term “oncohistones.” At the molecular level, mechanistic studies have been dissecting the biological mechanisms behind histones and missegregation events, with the potential to uncover novel clinically-relevant targets. In this review, we focus on the current understanding and highlight knowledge gaps of the contribution of histone variants to aneuploidy, and we have compiled a database (HistoPloidyDB) of histone gene alterations linked to aneuploidy in cancers of the The Cancer Genome Atlas project. H4 variant H4.G and several pseudogenes with putative regulatory functions . All other histone variants map at various cytobands . In addition to the variability in nucleosome composition conferred by the incorporation of canonical and variant histones, PTMs also add a further layer of diversity to physical properties and thus functional variability . Histonehistone variants” revealed an association with a wide range of cellular functions , and a growing association with diseases, notably cancer, in recent years .In this review, we focus on the contribution and interplay between core histones and histone variants to aneuploidy, by a comprehensive overview of histone variants alterations in cancer. By analysing publicly available cancer data from the Cancer Genome Atlas (TCGA) project, we compiled HistoPloidyDB–a database of correlations between histone gene alterations and aneuploidy  with hyperdiploid karyotypes  tail, which increases its stability and consequent protein abundance  tails  tails .H3 variants in giant cell tumour of the bone were shown to predate the development of karyotype defects  has been reported to increase genomic instability in giant cell tumour of bone stromal cells  has also been linked to the response to the DNA damage caused by the carcinogenic agents, which triggers DNA damage response mechanisms and a block of DNA synthesis leading to genomic instability . While t defects . Downregal cells . TherefoC. albicans as a resistance mechanism to antifungal drugs. The induction of numerical and structural aneuploidy is a common mechanism of stress response in the pathogen  has evolutionarily lost a key Bub1 phosphorylation site for maintenance of faithful chromosome segregation. Indeed, H2A.1 promotes the occurrence of aneuploidy through antimycotic treatment, which also results in depletion of CENP-A at centromeres  and variant histones  was identified in a mouse erythroleukaemia cell line with a tetraploid phenotype acquired through resistance to differentiation  were found to be upregulated in polyploid giant cancer cells derived from MCF7 breast cancer lines  embryos   . Certain variant . For a cH3.3 mutations produce a change from lysine to methionine (K27M and K36M) in either canonical H3 or non-canonical H3.3, causing the inability to undergo crucial methylation modifications, thus perturbing the correct epigenetic marks genome-wide , where they correlate with disease severity and the presence of copy number variations  presented mitotic aberrations with increased rates of aneuploidy, both numerical  and structural abnormalities , and a complex karyotype, when compared to H3.1-mutated cohorts  and H3.3A (variant) mutations, with the latter being associated with disease aggressiveness and dismal prognosis  syndrome, characterised by frameshift mutations in the C-terminal tail of H1.4 causative of intellectual disability and multi-organ and skeletal abnormalities  HeLa showed genomic instability in the form of micronuclei, chromatin bridges and lagging chromosomes; this role of H2AZ.2 in chromosomal stability was further shown to be dependent on PTMs, as mutated acetylation sites were unable to rescue the defects  J-related breaks and generalised DNA repairs  . It has ks (DSB) . In factgnancies  and geneocations . Even a ications . Structu repairs .H2B family have also been described in a wide range of malignancies [reviewed by Wan and Chan  of the skin  in gastric cancer , in breaOverall, this work lays the ground for research into the role of histones and their variants in the disruption of chromosomal stability in cancer. For instance, comprehensive analyses of mutations in chromosome segregation genes have shed light on their contribution to genomic stability in health and disease states , highlig


SI Appendix, Fig. S5, while SAHS1 is most intensively expressed in the storage cells, some expression was observed in the epidermis (We recently reported an in vivo expression and live imaging technology for tardigrades and an unexpected tissue specificity of anhydrobiosis-related gene expression, with the CAHS3 gene, for example, being most intensively expressed in the epidermis of tardigrades . Using iOne of the focuses of recent molecular studies of tardigrade anhydrobiosis has been the reversible filamentous gel formation of the CAHS protein, which we reported for CAHS1 , followe4Direct observation in tardigrades is an important direction, with traditional approaches like immunohistochemistry facilitated by new techniques such as our vector system. In addition, newly developed methods such as expansion microscopy and spatial transcriptomics can provide a clearer picture of anhydrobiotic tardigrades.


Transcription Factors (TFs) to establish specific gene regulatory programs. TFs also play a central role in the establishment and/or maintenance of hematological malignancies, highlighting the need to understand their functions in multiple contexts. TFs bind to specific DNA sequences and interact with each other to form transcriptional complexes that directly or indirectly control the expression of multiple genes. Over the past decades, significant research efforts have unraveled molecular programs that control HSC function. This, in turn, led to the identification of more than 50 TF proteins that influence HSC fate. However, much remains to be learned about how these proteins interact to form molecular networks in combination with cofactors (e.g. epigenetics factors) and how they control differentiation, expansion, and maintenance of cellular identity. Understanding these processes is critical for future applications particularly in the field of cell therapy, as this would allow for manipulation of cell fate and induction of expansion, differentiation, or reprogramming of HSCs using specific cocktails of TFs. Here, we review recent findings that have unraveled the complexity of molecular networks controlled by TFs in HSCs and point towards possible applications to obtain functional HSCs ex vivo for therapeutic purposes including hematological malignancies. Furthermore, we discuss the challenges and prospects for the derivation and expansion of functional adult HSCs in the near future.The functionality and longevity of hematopoietic tissue is ensured by a tightly controlled balance between self-renewal, quiescence, and differentiation of hematopoietic stem cells (HSCs) into the many different blood lineages. Cell fate determination in HSCs is influenced by signals from extrinsic factors  that are translated and integrated by intrinsic factors such as  These cells have different lifespans depending on the physiological state of each individual and must be produced continuously through life. This sustainability is maintained by self-renewal/proliferation and differentiation of a small contingent of cells called hematopoietic stem cells (HSCs), which are at the top of the hematopoietic hierarchy. The identification and characterization of HSCs began in the early 1960s with the seminal work of Till and McCulloch .HSCs exist in a specific microenvironment provided by the bone marrow, which together with extrinsic factors  contribute to determining the fate of these cells. The interaction of hematopoietic stem/progenitor cells with different microenvironments  strongly influences differentiation and proliferation, such that cells acquire specific functional and phenotypic characteristics that define each hematopoietic lineage . Historically, cells that constitute the hematopoietic system have been divided into four compartments  that are interconnected in a complex hierarchical system Figure 1Extrinsic factors influence hematopoiesis through the activation or suppression of signaling pathways, including BMP and WNT that are important for the state of HSCs such as quiescence, self-renewal and/or differentiation. These signaling pathways function through a complex mechanism of activation and/or repression of transcription factors (TFs), which in turn directly or indirectly regulate specific groups of genes necessary to promote or inhibit differentiation .Here we will review the importance and complexity of TF-based mechanisms regulating hematopoiesis, with a particular focus on HSCs. We will also highlight specific examples of how TFs can be manipulated to derive functional HSCs, which may represent key therapeutic strategies for various hematological diseases in the future.2TFs are defined as proteins that bind to DNA in a sequence-specific manner to regulate transcription. TFs have the ability to interact with each other and form stable protein complexes bound to cis-regulatory elements (CREs). By definition, CREs are short genetic elements  that act as regulatory switches to control gene expression. CREs are divided into gene promoters or distal elements called enhancers/silencers ), and insulators that form regulatory boundaries between genomic domains. Interestingly, the distal elements or enhancers/silencers are most important for activating regulatory programs that control cellular identity  and differentiation of HSCs into different cell lineages .The availability and localization of TFs in the nucleus, their relative affinity for each other’s and for specific motifs in CREs determines the complexity and composition of TF complexes and their ability to recruit cofactors, including the epigenetic regulatory machinery . Cofactors, in turn, act to activate or repress transcription of specific sets of genes through various mechanisms. These include recruitment and activation/repression of the machinery associated with RNA polymerase II , 15, altTFs can be classified according to their 3D structure and protein sequence similarity of their DNA-binding domain . TF families are well conserved throughout evolution  and in v31) aplastic anemia resulting from premature depletion of the HSC pool due to excess differentiation or massive apoptosis; (2) leukemia resulting from inhibition of differentiation and uncontrolled proliferation; (3) permanent quiescence resulting from blockade of the cell cycle, self-renewal, and differentiation.Several studies using transgenic mice  and viraComplementary to the functional studies described above, technical advances in cell purification techniques using flow cytometry have led to greater refinement of our knowledge of the different populations that make up hematopoietic tissue , 29, 30,4Gata2 at the embryonic stage showed is essential for the HSC formation from hemogenic endothelium, a process termed endothelial to hematopoietic transition (EHT)  –41. In aiability . Convers in vivo . Also inmination . In humaantation . In summAlso, perturbation of this equilibrium by extrinsic signals  through disruption of the interactions of TFs is necessary to trigger differentiation into a particular cell lineage. For example, suppression of WNT signaling in HSCs has been shown to modulate the activity of TCF TFs –47, resuAnother important aspect of differentiation mediated by TFs is the fact that certain TFs that promote differentiation to a particular hematopoietic lineage are usually also actively involved in the repression of genes from alternative lineages. An example of this is PU.1, a transcription factor that promotes granulo/macrophage differentiation and B-cell lymphopoiesis while actively repressing genes of T cell lymphopoiesis such as pre-Tα, Rag-1, and Rag-2 when ectopically expressed in T lymphocytes .5Pu.1 promoter. For example, in myeloid cells, C/EBP binds to two CRE regions located at -15Kb/-14Kb and at -12Kb upstream of the Pu.1 promoter to ensure strong expression of PU.1 in this context. In contrast, in B lymphoid cells, where Pu.1 expression is lower, the E2A and FOXO1 TFs only bind to the -15Kb/-14Kb CRE region upstream of the Pu.1 promoter  a set of TFs already expressed in each specific cellular environment , (2) by various extrinsic factors  (3), and also by certain intrinsic factors . The newly expressed TFs, in turn, interact with other TFs to recognize specific CRE regions to ensure appropriate expression levels of their target genes.6HSCs are finely regulated to maintain hematopoietic tissue homeostasis. HSCs are mainly in a quiescent state, and under various stimuli, they enter the cell cycle to self-renew and/or differentiate. The process of HSCs transitioning from the quiescent state to the cyclic state for self-renewal or differentiation is regulated by a specific set of TFs Figure 3In the next section, we review recent advances in our understanding of the role of TF networks in regulating the function of adult HSCs. Understanding the molecular networks that govern HSC biology is expected to improve therapeutic applications involving adult HSCs  and wil6.1Self-renewal and multipotency are key properties of HSCs that require the timed activation of specific gene groups. This process is largely controlled by the binding of TFs to CREs . The firde novo” CREs. Thus, CREs in HSCs can be divided into two categories: the specific CREs, which are active only in the HSC, and the “primed” CREs, which are present in different progeny of unilineage progenitors and could be responsible for binding TFs without being transcriptionally active  to generate a genome-wide profile of the landscape of the open chromatin (CREs) of any cell type. To address the problem of large amounts of starting material for rare populations such as HSCs, another method has been developed using a bacterial transposase (Tn5). This Tn5 enzyme is used to insert two short DNA fragments (a transposon) on either side of accessible chromatin regions. This assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) is comparable to DNaseI-seq in terms of specificity and signal-to-noise ratio, achieving 100- to 10000-fold with fewer cells than DNaseI-seq . Applicay active . Motif ay active . Moreovey active .Profiling of proximal CREs (at promoter level) associated with genes expressed in HSCs reveals that certain TFs (more than 20 TFs) are tightly connected by autoregulation, feedback and autoactivation (wherein a factor directly or indirectly activates its own expression). These mechanisms work together to establish a resilient transcriptional network capable of sustaining the expression of genes essential for HSC biology. During differentiation, the network of TFs expressed by HSCs gradually disappears as the corresponding TFs are no longer expressed along several lineages. At the same time, other dense TF networks emerge through the induction of other TFs associated with terminally differentiated cells, such as GATA1, LMO2, FOXO4, NFE2, and RXRA, which are involved in the TF network associated with erythroid differentiation. Certain TFs involved in TF networks associated with the functions of HSCs remain expressed and are “re-used” throughout the unilineage progeny. For example, PBX1 is involved in a TF network that regulates self-renewal of HSCs, but may later interact with other TFs to establish a TF network required for erythroid and megakaryocyte progenitor cell differentiation .Interestingly, the robustness of this strategy for predicting the TF networks controlling the functions of HSCs was tested using a perturbation-based approach. This led to validation of 17 predicted TFs involved in TF networks generated during differentiation of HSCs into different lineages (erythroid or myeloid). Specifically, by using conditioned medium that supports both erythroid and myelo-monocytic differentiation, the differentiation of enriched HSC cells into either of these lineages could be altered by simply reducing the expression of one of the predicted TFs. For example, altering the expression of 9 TFs (such as GATA1 and KLF1) affects the differentiation of HSCs toward the erythroid lineage without affecting their potential to differentiate toward the myeloid lineage. Moreover, using the CREs strategy to predict the TF network associated with the erythroid lineage reveals three novel TFs not previously associated with erythroid differentiation . In contrast, disruption of the expression of the 7 TFs (such as PU.1/SPI1 and members of the C/EBP family) involved in the TF network that drives differentiation of HSCs toward the myeloid lineage does not affect their differentiation toward the erythroid lineage but drastically reduces their potential for myelomonocytic differentiation .6.2Erg locus contains five CREs regions , of which only the +85Kb region has been validated in transgenic mice  have confirmed the central role of the interaction between GATA2 and RUNX1 and their dosage in maintaining the transcriptional activity of this TF network in HSCs and during the establishment of adult hematopoiesis  using a clinically relevant Notch-mediated ex vivo expansion system, long-term transplantation of expanded HSPCs into patients remains limited , which remains the only curative treatment for many malignant and non-malignant diseases and the most widely used cellular therapy. It has also paved the way for the concepts of gene therapy and immunotherapy as tools to combat diseases that currently have no cure . Despite limited .in vitro, confirming their “instructive” role in overcoming phenotypic and epigenetic barriers imposed by normal developmental ontogeny (Many efforts are being made to develop an efficient method to generate sufficient numbers of adult HSCs for research and therapeutic applications. The recent important discovery of somatic cell reprogramming by using TFs to induce pluripotent stem cells (iPSCs)  has stroontogeny .in vivo remains limited  into functional HSCs. Somatic cells can be considered as the optimal cell type for generating HSCs due to their availability. For example, when MEFs (mouse embryonic fibroblasts) expressing the CD34 reporter mechanism (surface marker for hematopoietic cells) are used to screen 18 TFs, only a few TFs such as GATA2, ETV6, and GFI1B are able to reactivate the expression of the CD34 reporter system and promote the generation of myeloid colony-forming cells . In anot limited .The TF reprogramming strategy has also been applied to pluripotent stem cells (PSCs) and embryonic stem cells (ESCs) to generate hematopoietic cells. Indeed, screening of 27 candidate TFs revealed the existence of two TF networks capable of inducing distinct hematopoietic programs from human PSCs: pan-myeloid (ETV2 and GATA2) and erythro-megakaryocytic (GATA2 and TAL1). Interestingly, these two transcriptional networks directly convert human PSCs (hPSCs) into endothelial cells, which subsequently transform into hematopoietic cells with a tendency to myeloid or erythro-megakaryocytic potential. Furthermore, it shows that hematopoietic differentiation from hPSCs using these two distinct sets of hematopoietic TFs proceeds through the EHT stage . Other gThus, one might conclude that TF-mediated reprogramming of committed cells to hematopoietic lineages could increase the efficiency of the conversion process, since the distance in the developmental ontogeny of these cells from HSCs is relatively small compared to nonhematopoietic cells . Indeed, ectopic expression of 6 TFs  is sufficient to reprogram adult mouse pre-Pro B cells and myeloid progenitors into induced HSCs (iHSCs) that have clonal differentiation potential for multiple cell lineages, reconstitute stem/progenitor compartments, and are serially transplantable. Moreover, single-cell gene expression analysis reveals the high similarity of iHSCs to adult HSCs. However, the stage of final hematopoiesis reached by this strategy is not yet clear. It appears that this conversion process occurs entirely within the confines of definitive hematopoiesis, without passing through the EHT stage .Taken together, these studies illustrate the feasibility and applicability of TF-mediated conversion or directed differentiation strategies for the derivation of transplantable human hematopoietic cells from a variety of cell sources. A variety of TFs has been tested, highlighting the power of the TF network strategy for cell reprogramming and hematopoietic cell derivation. However, the therapeutic value of “derived HSCs” is still subject to certain limitations.6.4-/- B cell progenitors can trans-differentiate in vitro into different hematopoietic cell types   in mice confers enhanced fitness and self-renewal properties to mature germinal center B cells, ultimately leading to aggressive lymphomas with increased repopulation potential. Interestingly, disruption of H1 function leads to a profound remodeling of genome architecture, causing a shift in chromatin from a compacted to a relaxed state in which genes related to stem cell functionality are upregulated . Thus, alteration of H1 expression could increase cell plasticity by relaxing compact chromatin in mature cells, making them potential targets for TFs and facilitating cell reprogramming. Indeed, OSKM expression in H1c-/-; H1e-/- murine embryonic fibroblasts resulted in a three- to fourfold increase in the efficiency of forming H1c/e-deficient iPSC colonies  begin earlier in the hematopoietic hierarchy. Accumulation of mutations in HSCs during the lifespan results in preleukemic HSCs, which later develop into leukemic stem cells (LSCs), 99. Howr cells)  in the pent mice  showing ent mice . Histoneent mice . Their dcolonies . Also, t in vivo .In summary, the transition of cells from one state to another  is well regulated by multiple molecular networks. Several transcriptional and epigenetic factors play a role in maintaining cell identity, and their disruption may increase cell plasticity, which could be very useful for the reprogramming of adult HSCs. Recent advances in drug development targeting proteins such as PROTACs (Proteolysis Targeting Chimaera) may be useful for transient disruption of those proteins  to increase the cell plasticity and to facilitate the reprogramming of adult HSCs from any cell type.7It is estimated that billions of hematopoietic cells are produced daily in the human body . The hemvia CRE regions that are highly enriched for motif binding of TFs such as HOXB4, which is known to be involved in self-renewal of HSCs , o of HSCs , 116. Du of HSCs  that are of HSCs Figure 5 of HSCs . For exa of HSCs , could b of HSCs . However of HSCs . Absolut of HSCs . Additio of HSCs ) could h of HSCs , 125 andThe study of TF networks in adult HSCs has laid the groundwork for the development of several strategies to obtain transplantable hematopoietic cells for research and clinical applications. Several sets of defined TFs have been tested for their efficiency in reprogramming or directly differentiating adult HSCs from a variety of cell sources. However, the potential therapeutic value of these cells remains unknown, as the developmental state of the derived HSCs is incompletely characterized.ex vivo for therapeutic purposes requires a better understanding of normal HSC biology through the use of multidisciplinary strategies. New technologies such as CRISPR/Cas could be used for high-throughput genetic screening to identify additional TFs that improve the efficiency of HSCs derivation or direct differentiation and to optimize their developmental state. This technology could also be used to test the efficiency of the different TFs previously used to derive transplantable hematopoietic cells side by side.The challenge of generating functional human HSCs ex vivo derived HSCs and promote their accumulation in the undifferentiated state. Recent studies of ZEB1/2 in adult HSCs have confirmed that those TFs play a role in repressing genes that are expressed only in differentiated cells at the HSC level , underscoring the importance of “lineage-specific” CRE repression for lineage fidelity and the multilineage potential of HSCs , 129 andAB drew up the concept of the review, wrote text, collected references, and made the first version of the Figures. JH wrote text and revised the Figures. MB drew up the concept of the review, wrote text, and revised the Figures. All the authors contributed to the article and approved the submitted version.


Numerical simulations of thoracic endovascular aortic repair (TEVAR) may be implemented in the preoperative workflow if credible and reliable. We present the application of a TEVAR simulation methodology to an 82-year-old woman with a penetrating atherosclerotic ulcer in the left hemiarch, that underwent a left common carotid artery to left subclavian artery bypass and consequent TEVAR in zone 2. During the intervention, kinking of the distal thoracic stent graft occurred and the simulation was able to reproduce this event. This report highlights the potential and reliability of TEVAR simulations to predict perioperative adverse events and short-term postoperative technical results. Numerical simulations that virtually reproduce thoracic endovascular aortic repair (TEVAR) represent innovative computational adjuncts that may potentially aid the preprocedural planning phase in the future by predicting perioperative or short-term postoperative technical events and results.A).An 82-year-old woman with hypertension and a history of heavy smoking (approximately 20 cigarettes per day) presented with a penetrating atherosclerotic ulcer (PAU) in the left hemiarch with maximum axial diameters of 38 × 37 mm on computed tomography angiography (CTA). There were no further relevant cardiovascular diseases or interventions in her medical history. Additionally, an intraluminal floating thrombus located at the outer curvature of the proximal descending aorta  was identified on CTA, that seemed to be connected to the intraluminal thrombus of the PAU anteriorly , A.Fig 1B).First, a left common carotid artery (LCCA) to left subclavian artery (LSA) bypass was performed, to obtain an adequate proximal landing zone in zone 2 for TEVAR. During the same intervention, a Valiant thoracic aortic stent graft with the Captivia delivery system   was deployed followed by a plug at the LSA origin to prevent retrograde type II endoleak. Interestingly, during the intervention, a kinking of the thoracic stent graft occurred between the fifth and sixth nitinol stent rings at the distal portion of the thoracic stent graft in zone 4, which was resolved after ballooning this portion of the stent graft , B.C). Discharge was on postoperative day 11. During follow-up, color Doppler ultrasound examination showed adequate patency and flow over the LCCA-LSA bypass. No further follow-up diagnostic imaging has been performed to date.The postoperative course was regular, without any adverse events, including neurological and peripheral thromboembolic. Postoperative CTA after 8 days showed adequate exclusion of the PAU and intraluminal thrombus, without endoleak and patency of the Valiant Captivia and LCCA to LSA bypass , C. Disc,The patient-specific ascending, arch, and descending aortic anatomy were reconstructed from preoperative CTA images, including the intraluminal thrombus in zone 3. A recently developed high-fidelity numerical methodologyThe potential impact of applying this methodological TEVAR simulation model with Valiant Captivia to patient-specific cases could be of significant value during preprocedural planning given the ability to predict both perioperative adverse events (such as kinking for the illustrated case) and short-term postoperative technical results. As an additional tool, it may serve physicians in choosing the optimal proximal and/or distal sealing zones in specific cases with challenging aortic anatomy. In fact, as depicted in The time to obtain virtual results is compatible with the time needed to plan elective TEVAR; this procedure can be performed in 1 day. Furthermore, other patient-specific components such as the presence of intraluminal thrombus (as for this case) or calcifications, can be included in the simulation, to evaluate their impact on the stent graft deployment. Virtual deployment of different thoracic aortic stent grafts of different manufacturers, if verified and validated,Similar to the present case, another illustrative case by Derycke et alThis study further highlights the potential and reliability of TEVAR simulations to be adopted in and facilitate preprocedural planning in the future. For example, they could investigate optimal proximal landing and stent graft apposition or the ideal stent graft model in demanding aortic anatomies. One of the challenges before a wider implementation of such tools in daily clinical cardiovascular practice, remains the need to further enhance simulations regarding their reliability and credibility, by providing evidence of their effectiveness and workflow, as illustrated by this case.


After being discovered over a century ago, insulin was long considered to be a hormone exclusively produced by the pancreas. Insulin presence was later discovered in the brain, which was originally accounted for by its transport across the blood-brain barrier. Considering that both insulin mRNA and insulin were detected in the central nervous system (CNS), it is now known that this hormone is also synthesized in several brain regions, including the hypothalamus, hippocampus, cerebral and cerebellar cortex, and olfactory bulb. Although many roles of insulin in the CNS have been described, it was initially unknown which of them could be attributed to brain-derived and which to pancreatic insulin or whether their actions in the brain overlap. However, more and more studies have been emerging lately, focusing solely on the roles of brain-derived insulin. The aim of this review was to present the latest findings on the roles of brain-derived insulin, including neuroprotection, control of growth hormone secretion, and regulation of appetite and neuronal glucose uptake. Lastly, the impairment of signaling initiated by brain-derived insulin was addressed in regard to memory decline in humans. A large number of review articles have already addressed the actions of insulin within the central nervous system (CNS). However, to the best of our knowledge, none of them has focused exclusively on the functions of insulin produced in the CNS. Therefore, this review article consists of two major parts; the first offers a short overview of some of the studies that documented insulin expression in various brain regions, and the second represents the collection of recently published data describing the mechanisms controlling insulin expression in the brain and its roles. The fact that neuronal insulin expression is observed not only in rodents and humans but in invertebrates as well  implies After being discovered over a century ago, insulin was long considered to be an exclusively β-pancreatic cell-derived hormone that lowered glucose levels upon its secretion in the systemic circulation . The resThe presence of insulin in the brain of adult rats was initially observed more than half a century after its initial discovery. Namely, it was reported that insulin “indistinguishable” from that typically found in the pancreas was found in extracts of the whole rat brain . MoreoveHowever, the insulin immunoreactivity in the CNS was initially accounted for by its transport across the blood-brain barrier (BBB). The transport of insulin from the blood into the cerebrospinal fluid (CSF) was documented even before its presence in the brain parenchyma. The study conducted on dogs showed that intravenous insulin administration resulted in increased CSF levels of this hormone . The traA body of evidence suggesting that in addition to the pancreas, insulin is also expressed in the brain has been growing for almost four decades. For example, insulin immunoreactivity was detected in cells cultured from rats ,12 and mIns2 mRNA expression took place in the perisomatic cytoplasm of inhibitory (GABAergic) interneurons of rat cerebral cortex and found that lowering glucose concentrations in the medium to the levels normally found in the brain extracellular fluid during hypoglycemia decreased the number of Ins2 mRNA molecules detected per neurogliaform cell (NGFC). On the other hand, the addition of glucose or glibenclamide (hypoglycemic drug) to the medium increased insulin concentration in the neocortical slices. Furthermore, the insulin secretion from NGFC was found to be dependent on dendritic Ca2+ entry.More recent in vitro studies not only reconfirmed early findings that insulin was expressed in neurons but also offered more information on the mechanisms regulating this process. Molnár et al.  showed tIns2 mRNA and Glp1r mRNA were co-expressed in these GABAergic neocortical interneurons but that the expression of both of these genes was upregulated by high extracellular glucose concentration. Functional expression of GLP-1R in NGFC was confirmed by showing that GLP-1 treatment had a reversible effect on their electrophysiological properties which was prevented by the application of exendin-4, a GLP-1R antagonist. Collectively, these findings indicate that similar to pancreatic β cells, insulin expression in neocortical inhibitory interneurons is stimulated by endogenous incretins such as GLP-1.A different study showed that a glucose-induced increase in insulin expression in NGFC occurred via the activation of glucagon-like peptide-1 receptor (GLP-1R) . It was Ins2 mRNA were significantly upregulated after the treatment of hypothalamic neurons with a hyperglycemic solution. A similar response was observed after the treatment of these cells with forskolin (an activator of adenylate cyclase) and exendin-4. However, the responses induced by forskolin and exendin-4 were biphasic, with Ins2 mRNA expression being initially increased above but ultimately reduced below the control level. These findings showed that changes in glucose and cyclic adenosine monophosphate (cAMP) levels affected insulin expression in a certain subset of hypothalamic neurons. Considering that these neurons, in addition to insulin and glucose transporters, also express glucokinase, one might hypothesize that changes in hypothalamic insulin secretion may serve as an output signal to downstream neurons involved in the regulation of energy homeostasis. However, additional analyses are needed in order to confirm this hypothesis.In addition to neocortical, glucose was shown to stimulate insulin expression in the mouse hypothalamic neuronal cell lines (mHypoE-39 and mHypoE-46) via a similar mechanism . Namely,Ins2 gene expression and insulin secretion. Moreover, neurogenic differentiation 1 (NeuroD1), one of the transcription factors that regulate insulin gene expression, was also induced by Wnt3a treatment in a time- and dose-dependent manner. Additionally, it was shown that treatment by a glycogen synthase kinase-3 (GSK3) inhibitor also increased the expression of both insulin and NeuroD1 in mHypoE-39 hypothalamic cells. These findings initially observed in cultured hypothalamic cells were subsequently validated in vivo. Namely, the knockdown of NeuroD1 not only prompted a decrease in basal Ins2 expression but also suppressed Wnt3a-induced Ins2 expression. Furthermore, the Wnt3a hypothalamic injections increased the expression of NeuroD1 and Ins2 in mice in a manner similar to that observed in vitro.Another study on a hypothalamic neuronal cell line confirmed that insulin was produced in mHypoE-39 cells but also provided additional insights into which other molecules took part in regulating insulin expression in the CNS . This stIns1 gene, the ancestral Ins2 gene was expressed in fetal, neonatal, and adult rat brains [Ins1 was retroposed from the partially processed Ins2 mRNA, while the Ins2 gene is the ortholog of the human INS gene [Despite their multitude, the studies conducted on cultured mammalian cells were not sufficient to prove that insulin expression unequivocally takes place in vivo in the brain. The first study reporting insulin presence in the rat brain was conducted by Havrankova et al. in 1978 . Later, t brains ,32. It sINS gene .Ins2 mRNA, mature insulin, and C-peptide in the cerebellum, cerebral cortex, anterior olfactory bulb, cerebellar Purkinje neurons, and most prominently in the hippocampus. Quantitative analysis showed that the rate of hippocampal insulin expression was lower than that in the pancreas. This finding, in addition to the observation of close contacts between neurites and perikaryia or neurites of neurons positively stained for insulin [Ins−/− mutants do not show significant pathological changes under physiological conditions [INS gene in several regions of the human brain, including the hippocampus, cerebral cortex, and cerebellum. A study conducted on mice showed that insulin was broadly expressed throughout developing and adult mice brains . The pre insulin  and factnditions  indicatenditions  also disThe expression of insulin receptors (IR) in the CNS was first documented almost half a century ago . It is nIn the remainder of this manuscript, we aimed to overview the data yielded mainly by more recent studies concerning specifically the actions of insulin synthesized in various brain regions and how their impairment may be associated with some neurodegenerative diseases.Ins2 mRNA and preproinsulin, a biosynthetic precursor of insulin. Subsequently, neurofilament immunoreaction was detected in the somata, dendrites, and axons of these neurons. Lastly, the treatment with either insulin antibody or the inhibitor of insulin receptor tyrosine kinase activity was shown to result in neurite retraction rendering neurons hypertrophic and vacuolated. Inhibition of MAPK, but not phosphatidylinositol 3-kinase (PI3K), also resulted in shortening and/or complete retraction of neuritis and rounding of neurons, which suggested that insulin effects on neurofilament distribution to the axons were accomplished via MAPK activation.Some of the earliest studies concerning the role of insulin produced in neurons were performed on cultured cells. One of the first roles of neural insulin to be observed was that it promoted neurofilament distribution and axonal growth via mitogen-activated protein kinase (MAPK) phosphorylation in cultured rat fetal neurons ,36. CultA study with a similar design also showed that insulin of neural origin promoted neural differentiation and growth . MoreoveIns2 mRNA expression and insulin content in the hypothalamic parenchyma, while the concentrations of circulating glucose and insulin were expectedly to decrease [Ins2 mRNA was first detected in the neurons of PEV almost four decades ago [Ins2 mRNA, mature insulin, and C-peptide were all present in EChP. Furthermore, they demonstrated that insulin secretion from primary cultured mouse EChP was stimulated by serotonin (5-HT). More precisely, it was shown that activation of the 5HT2C receptor by serotonin treatment resulted in the opening of IP3-gated Ca2+ channels in the endoplasmic reticulum. This resulted in Ca2+ being mobilized from intracellular storage, which consequently led to insulin secretion. The role of serotonin in the secretion of insulin from EChP was indirectly corroborated in vivo by showing that serotonin depletion in the dorsal raphe nucleus downregulated insulin expression in ChP. Our unpublished data showed that, such as insulin, serotonin content in the hypothalamus was also elevated following the six-hour fast. Additionally, hypothalamic insulin increment was observed following microdialysis administration of dexfenfluramine, a potent 5-HT stimulator [The distinctiveness of mechanisms controlling peripheral and central insulin expression became evident in one of our studies which examined the effects of short-term fasting on insulin expression in the rat hypothalamus. Our study showed that six-hour fasting increased both decrease . Furtherades ago , while tades ago . Namely,ades ago  found thimulator . This suIn regard to the ependymal cells in our study, strong granular proinsulin immunopositivity was observed in the apical portion of cuboidal ciliated cells in the region surrounding the lumen of the third ventricle. The intracellular location of proinsulin-containing granules implies that insulin produced in these cells was secreted into the CSF rather than being taken up from it. This conclusion was supported by the fact that, unlike circulating insulin which was lowered, the CSF insulin levels were at the control level despite the short-term food deprivation .We then sought to decipher the adaptive significance of the short-term fasting-induced increase in hypothalamic insulin expression. Considering that fasting represents metabolic strain for the organism as a whole, we wanted to investigate whether this phenomenon was related to maintaining glucose homeostasis within the hypothalamus. It was found that short-term fasting markedly increased the amount of endothelial 55 kDa isoform of glucose transporter 1 (GLUT1) and neuronal GLUT3. The levels of GLUT2, whose presence was detected in neurons, ependymocytes, and tanycytes, were also elevated . HoweverA further attempt to determine the role of fasting-promoted increase in the hypothalamic insulin expression was made by looking into how short-term fasting affected signaling pathways typically activated by this hormone . We founIns2 mRNA expression and insulin content were not elevated as previously observed in male rats, but they were not reduced either. Both of these parameters remained unaltered in both proestrus and diestrus, despite circulating insulin being significantly lowered. Similar to findings observed in male rats, insulin immunopositivity was detected in the PEV neurons and the ependymal cells at the top of the third cerebral ventricle in females. When taken together, these data indicate that control of insulin expression in the hypothalamus during short-term fasting is a sex-specific process. However, hypothalamic insulin expression in both sexes appeared to be distinctly regulated during fasting than that in the pancreatic β cells.Lastly, we wanted to examine how fasting of the same duration would affect hypothalamic insulin expression in female rats during different phases of the estrus cycle . FollowiIns2-promoter-containing cells were also found in the nucleus of the solitary tract and the dorsal vagal complex (DVC). Furthermore, this study showed that insulin expression in the brain was affected by dietary interventions in a region-specific manner. For example, eight-week exposure to a high-fat diet (HFD) decreased Ins2 mRNA expression in the hypothalamus while simultaneously increasing it in DVC. This finding suggests that the role of insulin produced in the hindbrain becomes more evident under metabolically stressful situations such as prolonged exposure to HFD. In contrast to the anorexic effects that insulin typically exhibits in the hypothalamus, this study revealed that stimulation of DVC insulin-producing neurons results in an acute increase in appetite. Namely, optogenetic stimulation of DVC insulin-producing neurons resulted in increased food intake, which peaked one hour after the onset of stimulation without affecting long-term appetite. Ventricular application of a highly specific insulin receptor antagonist blocked the aforementioned stimulating effects of DVC insulin-producing neurons on feeding.Another recent study also shed light on the complexity of centrally-produced insulin expression patterns and actions . First oIns2 mRNA, proinsulin, and mature insulin were colocalized within the same subset of neurons in the PVN. Proinsulin was mainly found in the neuronal cell bodies. In addition to that, proinsulin was also detected in the ependymal cells lining the third ventricle and, to a lesser extent, in the ameboid microglia. Unlike proinsulin, C-peptide, which connects the A and B chains of proinsulin and is secreted together with insulin, was located in neurosecretory nerve terminals in the external zone of the median eminence (ME). This suggests that C-peptide and matured insulin are transported from the somata of PVN insulin-producing neurons through axonal projections to their axon terminals in the ME.A major breakthrough in unveiling the physiological role of hypothalamus-derived insulin came from the study of Lee et al. . Not onlCrh mRNA expression proved to be biphasic, peaking half an hour and eight hours after the onset of restraint, hypothalamic Ins2 mRNA levels were continuously decreased during the exposure to restraint.Most of the insulin-producing PVN neurons (90%) examined in this study were shown to also synthesize corticotropin-releasing hormone (CRH), while somatostatin was found in only about 20% of insulin-producing neurons. Moreover, CRH was extensively colocalized with C-peptide within the same large dense-core vesicles in the axon terminals in the ME. It is noteworthy that the expression patterns of insulin and CRH genes were reciprocal during the eight-hour exposure to restraint stress. While Gh mRNA expression and the serum GH concentration were lowered after the insulin gene had been knocked down in the PVN. Considering that hypothalamic expression of growth hormone-releasing hormone (GHRH) and somatostatin was unaltered by PVN insulin knockdown, it was concluded that hypothalamic insulin-regulated GH secretion independently of these two hormones. Moreover, PVN insulin knockdown significantly suppressed the pituitary phosphorylation of Akt, the kinase known to be a major target of insulin signaling. Ultimately, the importance of PVN insulin in promoting GH secretion was confirmed in PVN insulin-knockdown young mice. Six weeks after hypothalamic Ins2 mRNA expression had been downregulated, a significant reduction in body length (without changes in food intake and body weight) of these mice was documented in comparison to the controls of the same age [The proximity of PVN insulin-producing neuronal axon terminals to the ME led the authors of this study to examine whether PVN-derived insulin might be involved in the control of anterior pituitary hormone secretion. The only pituitary hormone affected by the knockdown of PVN insulin was growth hormone (GH). Namely, both pituitary same age . This fisame age .The significance of centrally produced insulin for normal brain development was examined in a study conducted by Monte et al. . They stPTP1b mRNA levels were not changed within the cerebellum. Considering the aforementioned, decreased neuronal sensitivity to insulin following exposure to ethanol can be accounted for by PTP1b-mediated dephosphorylation of insulin receptor.It had already been known from earlier studies that ethanol-induced inhibition of insulin-stimulated tyrosine phosphorylation of insulin receptor and IRS1 and the subsequent impairment of downstream PI3K/Akt signaling results in decreased neuronal survival . Among oGlut4 mRNA [The implications of ethanol-induced decrease in insulin expression in the cerebellum were also analyzed within the context of glucose uptake and utilization. Chronic gestational exposure to ethanol reduced the levels of ut4 mRNA  and the ut4 mRNA . Consequut4 mRNA . Taken tThe effects of treatments mimicking the stress exposure on insulin and insulin receptor expression in the brain were described in the study of Osmanovic et al. . The aut1–42 (Aβ1–42) was that of Nemoto et al. [Ins2 mRNA and proinsulin were present in the neurons of the hippocampus and cerebral cortex. They also established that insulin secretion from hippocampal neurons was regulated by Ca2+-dependent activator protein for secretion 2 (CAPS2). This means that, such as the myriad of other neuropeptides, insulin is secreted from neurons via exocytosis from dense-core vesicles. The treatment of hippocampal neurons with Aβ1–42 significantly decreased the amount of proinsulin detected in these cells. Moreover, the Aβ1–42 treatment decreased Ser9-phosphorylation of glycogen synthase kinase-3β (GSK-3β). Namely, the binding of insulin to its receptor activates PI3K/Akt cascade, which results in GSK-3β inactivation through Ser9-phosphorylation. When taken together, these data suggest that downregulation of hippocampal Ins2 expression in Aβ1–42-induced model of Alzheimer’s disease occurs via activation of GSK-3β. This conclusion was backed by the fact that the effect of Aβ1–42 treatment was reduced by both GSK-3β siRNA and lithium, a mood stabilizer is known to inhibit GSK-3β activity.As previously mentioned, studies have shown that insulin signaling impairment is associated with pathologies such as Alzheimer’s disease . Moreoveo et al. . They fiIn addition to the hippocampus, the significance of insulin expression in the CNS in the context of Alzheimer’s disease was documented in the cerebral cortex as well . This elBased on the body of evidence gathered thus far, it is safe to say that the actions of insulin produced in the CNS are diverse and vary from one brain region to another. It ought to be underlined that most of the studies cited in this manuscript were performed on rodents whose genome, unlike the human, contains two nonallelic insulin genes. However, despite this limitation, the need for additional characterization of brain insulin expression in regard to human health is important, given that intranasal insulin application has been proven to be successful as a treatment not only for Alzheimer’s disease and mild cognitive impairment but for other complications such as cerebral ischemia, traumatic brain injuries, and postoperative delirium . The eff