The unique environment of the brain and retina is tightly regulated by blood–brain barrier and the blood-retinal barrier, respectively, to ensure proper neuronal function. Endothelial cells within these tissues possess distinct properties that allow for controlled passage of solutes and fluids. Pericytes, glia cells and neurons signal to endothelial cells (ECs) to form and maintain the barriers and control blood flow, helping to create the neurovascular unit. This barrier is lost in a wide range of diseases affecting the central nervous system (CNS) and retina such as brain tumors, stroke, dementia, and in the eye, diabetic retinopathy, retinal vein occlusions and age-related macular degeneration to name prominent examples. Recent studies directly link barrier changes to promotion of disease pathology and degradation of neuronal function. Understanding how these barriers form and how to restore these barriers in disease provides an important point for therapeutic intervention. This review aims to describe the fundamentals of the blood-tissue barriers of the CNS and how the use of transgenic animal models led to our current understanding of the molecular framework of these barriers. The review also highlights examples of targeting barrier properties to protect neuronal function in disease states. The central nervous system (CNS), including the brain and retina, has a relatively high energy requirement due to neuronal activity, but in order to maintain proper neuronal function, the neural micro-environment must be rigorously controlled. These organs are highly vascularized and in the case of the retina, possess a unique vasculature structure, that provide the necessary oxygen and glucose for proper neuronal activity. To safeguard the neuronal environment, the vessels in the CNS possess well developed junctional complexes in the vascular endothelium, contributing to the formation of the blood–brain (BBB) or inner blood-retinal barrier (iBRB) controlling paracellular flux , 2. In cThis review will focus on the vascular components creating the BBB and BRB although both the brain and retina possess additional membranes necessary for proper control of fluids and solute movement to the CNS. These include the choroid plexus with an epithelial layer that filters fluid generating cerebrospinal fluid (CSF) and constitutes the blood-CSF barrier  and the Brain and retina ECs possess certain properties that are unique and contribute to the integrity of the BBB and BRB . They haAnimal models have been indispensable in clarifying the underlying mechanisms of controlled transport, barrier permeability, cellular architecture, and related morphological changes, which have provided most of our basic knowledge about the BBB/BRB. Recent research utilizing transgenic animals has identified critical elements in the structural components of the BBB and BRB as well as elucidate signaling factors required for the developmental process that controls the formation of the BBB and BRB in the CNS. Furthermore, studies have begun to elucidate how changes to the BBB and BRB affect CNS function and contribute to disease pathogenesis and have begun to explore potential mechanisms to prevent loss of BBB and BRB or restore these barriers in order to intervene in disease processes. This review will specifically explore these transgenic mouse models used to study the development and functional significance of the BBB and BRB in normal physiology and disease focusing on what transgenic models have revealed about the protein components that make up the BBB and BRB and required pathways in barriergenesis. Finally, this review will highlight studies that elucidate the relationship of the BBB and BRB to proper neural function and the neurovascular unit.At the BBB and iBRB, signals from glial cells and pericytes induce vascular ECs to form well-developed TJs that restrict the passage of molecules through the paracellular space thus sealing the blood vessel lumen from the CNS parenchyma . AdditioThe TJ complex consists of numerous interacting proteins and include transmembrane claudin family members, MARVEL (MAL and related proteins for vesicle trafficking and membrane link) proteins, and junctional adhesion molecules (JAMs), which form intercellular contacts Fig.  10]. Th. Th10]. The claudin family is considered the main structural component of the TJs, forming the intramembrane strands. They have a central role in controlling paracellular transport by forming charge specific interactions between claudins that restrict or allow particular ion diffusion and restrict solute permeability . At the The claudin family is composed of 27 members  and theyExpression and localization of claudins-1, -3, -5, and -12 at the BBB and BRB have been reported, where claudin-5 seems to be the most highly enriched –24. HoweClaudin-5 remains the one claudin clearly present and required for BBB. The importance of claudin-5 at the BBB was demonstrated by Nitta et al., revealing that claudin-5 knockout mice died shortly after birth due to loss of size-selectivity permeability of the BBB, allowing the diffusion of molecules smaller than 800 Da, but not of larger molecules . SurprisThe TJ associated MARVEL proteins (TAMP) family includes occludin (MARVELD1), tricellulin (MARVELD2) and MARVELD3. Similar to the claudin family, they have 4 transmembrane domains with 2 ECL domains, however they cannot form TJ strands alone . They arOccludin was the first transmembrane TJ protein to be described  and its Tricellulin (MARVELD2) is another transmembrane protein that is present specifically in tricellular endothelial junctions in the brain and retina , 47. SevLipolysis-stimulated lipoprotein receptor (LSR) , has been shown to be necessary to recruit tricellulin to tricellular junctions . However2-terminal kinase (JNK) pathway. Loss of MarvelD3 expression in epithelial cells resulted in increased cell migration and proliferation [MARVELD3 is the third member of the TAMP family and is also a tetraspan protein; however, it lacks the C-terminus found in occludin and tricellulin  and its feration . MARVELDferation .- or trans-interaction [JAMs are a family of single span proteins with an extracellular immunoglobulin-like domain . They ineraction , 63. Dimeraction , 65.In brain and retinal ECs, JAM-A is the predominant JAM isoform , 67. JAMZonula occludens (ZO) are part of the MAGUK family and act as scaffold proteins that link transmembrane proteins to the actin cytoskeleton stabilizing TJ strands . Three iThe central role of ZO family in the formation of TJs has been demonstrated in cells deficient for ZO-1 and -2 which fail to form TJs with impaired claudin strand polymerization , 80. ZO-In ECs of the CNS small lipophilic molecules can passively diffuse across the lipid bilayer but, in many cases, are returned to the blood by efflux transporters. Larger lipophilic molecules along with hydrophilic molecules and ions are dependent on transcellular transport to cross the BBB/BRB through channels, pinocytosis, receptor and non-receptor mediated vesicular transport and excluded by efflux pumps , 86. To Some transporters are expressed early in development, such as the glucose transporter GLUT1, and its distribution pattern correlates with BBB maturation . GLUT-1 Transcytosis in ECs occurs primarily through caveola. Caveolae are plasma membrane invaginations consisting of lipid rafts and the proteins caveolin-1 or flotillin-1 and cavin. Caveolae vesicles undergo endocytosis and within the cytoplasm of cells can initiate signal transduction, be recycled to the plasma membrane, or transport their contents to the opposite plasma membrane . CaveoliAnother molecule implicated in regulating transcytosis in the CNS is the transporter major facilitator superfamily domain-containing 2a (Mfsd2a). In brain ECs, Mfsd2a transports phospholipids from the outer to the inner leaflet of the plasma membrane, including omega-3 fatty acids like docosahexaenoic acid (DHA), which is important for brain development . Gene deThe development and maintenance of the BBB and BRB results from the close contact of the vasculature with neurons, glial cells and pericytes and may also include microglia . The terPericytes are mural cells that ensheathe the abluminal surfaces of CNS capillaries and support microvessel stability and regulation of blood flow , 110. InPericytes are recruited to the CNS vasculature by release of platelet-derived growth factor-b (Pdgf-b) from nascent ECs during embryonic development, which binds to platelet-derived growth factor receptor-β (Pdgfrβ) expressed on the pericyte cell surface. This recruitment signaling was demonstrated in both Pdgfb and Pdgfrb knockout mice which completely lack CNS pericytes , 114. PeIn general ECs and pericytes are separated by a shared extracellular matrix (ECM); however, in certain areas, they form specialized junctions called peg and socket which allows for direct signaling between pericytes and ECs . After rIn addition to pericytes, glial cells also contribute to the regulation of the NVU and BBB/BRB. Recent research has clarified critical signaling pathways from astrocytes that promote barrier properties. The most prevalent glial cells in the brain and retina are astrocytes as well as Müller cells in the retina. Due to their organization and spatial arrangement, glial cells are considered to be the bridge between the neurons and the vasculature, providing immunosurveillance, nutritional and regulatory support . AstrocyEarly experiments showed that transplantation of astrocytes could induce peripheral ECs to form non-leaky vessels  implyingSonic hedgehog (Shh) signaling is involved in numerous developmental processes, including neuronal differentiation, axon guidance, and angiogenesis . Shh is Astrocytes are also known to secrete angiotensinogen , a precuAdditionally, astrocytes are one of the main sources of ECM, critical in maintaining BBB function. Astrocyte-specific deletion of laminin induces pericyte dysfunction, loss of astrocyte end-feet coverage and BBB leakage associated with decreased expression of occludin and claudin-5 , 139, suThe Wnt/beta-catenin signaling pathway has been identified as a unique CNS angiogenic program that also induces barrier properties in ECs –142. WntDifferent Wnt signaling pathways are activated when Wnt proteins connect with high affinity to Frizzled (Fzd) receptors. The canonical Wnt signaling pathway causes beta-catenin to stabilize and accumulate in the nucleus, where it binds to transcription factors from the lymphoid enhancer factor/T cell factor (Lef/Tcf) family to induce the transcription of target genes .Endothelial targeted beta-catenin gene deletion leads to specific CNS angiogenesis defects and loss of BBB properties. Besides vascular malformation, these mice show decreased expression of GLUT1, reduced border staining of claudin-5, and upregulation of PLVAP, leading to increased paracellular and transcellular BBB permeability –142. LieIn mammals, 19 Wnt proteins have been identified and ten different Frizzled receptors have been discovered. Throughout the CNS, Wnt ligands, Frizzled receptors and co-receptors are differentially expressed, with Wnt1, Wnt3, Wnt3a, Wnt4, and Wnt7a/b having a role in BBB formation , 142. ReWnt7a and Wnt7b have been identified as the dominant ligands in the brain cortex , 142 andInterestingly, BRB formation requires a different, but also specific ligand/receptor complex. In the retina, Norrin, a TGF-beta family molecule, binds with high affinity to Fzd4, , dependeWnt/beta-catenin signaling has also been shown to be necessary for BBB/BRB integrity in mature ECs. Adult mice with Fzd4 deletion or anti-Frizzled 4 blocking antibody showed similar alterations to those observed in Ndp and Fzd4 developmental knockout, with high PLVAP expression and permeability of the BBB/BRB , 161. AdImportantly, adult BBB maintenance requires a continuous and low beta-catenin signal . RecentlMultiple reviews have cited the requirement of a functional and intact BBB/BRB to maintain the microenvironment required for proper neural signaling and numerous studies correlate breakdown of BBB integrity with neurological dysfunction Fig. . For exaRecent studies have also highlighted the importance of maintaining vascular endothelial TJs in preserving visual function in models of diabetic retinopathy. In retinal ECs, occludin phosphorylation at S490  promotesOthers have demonstrated the value of preserving the BBB in stroke. Vascular endothelial conditional expression of a constitutively active mutant form of actin depolymerizing factor (ADFm) or cofilin, reducing actin polymerization leads to reduced permeability after stroke and reduced TJ disassembly at endothelial cell border . ImportaAnalysis of poststroke human and mouse blood microvessels have identified claudin-1 as highly expressed in leaky brain microvessels . This neRecent studies have also targeted microRNAs that regulate TJs expression. Specifically, miR-15a/16-1 cluster directly binds to claudin-5 3′UTR, decreasing its expression and endothelial cell-specific deletion of this cluster in mice was able to increase claudin-5 expression, leading to improvement of BBB dysfunction and post-stroke functional outcome . AdditioConsidering that barrier function also regulates the exchange of nutrients and waste with the parenchyma, a reduction in permeability may also have a detrimental impact on neuronal activity.Inducing beta-catenin signaling in the leaky vessels of the circumventricular organs converts them into BBB-like vessels by increasing claudin5 and decreasing PLVAP expression, overall reducing vascular permeability . EndotheIn Alzheimer's disease (AD), which is characterized by the accumulation of neurotoxic amyloid-β (Aβ) peptides leading to a gradual decline in cognitive function , the impRecent studies have shown that targeting the paracellular route might also enhance Aβ clearance. In a murine model of familial AD suppression of the TJ proteins claudin-5 and occludin by systemic siRNA allowed for diffusion of soluble human Aβ monomers from the brain to the blood . These aTargeting paracellular permeability has also been tested in the context of cerebral edema and brain swelling, which are associated with higher risk of permanent brain damage and mortality. Administration of siRNA against claudin-5 post induction of a cold-induced cerebral edema allowed for enhanced exchange of accumulated extra-neural water, decreasing brain-swelling, and improving the cognitive outcome after the focal injury . The incTransgenic approaches have proven to be highly valuable in understanding the complex multi-cellular interaction necessary for formation of the BBB and BRB and have provided critical insight into required factors for proper barriergenesis. The use of transgenic mice has demonstrated the importance of the BBB and BRB for proper neural function in a physiological setting. These studies have also begun to elucidate how manipulation of the barrier, either by promoting tightening of the barrier or permeability, may provide important therapeutic options to address diseases of the CNS. Future studies using stem cells and organ culture systems may provide critical information on the factors sufficient to promote formation of the BBB and provide new avenues to quickly address fundamental questions regarding the NVU. However, the use of transgenic animals in the study of the BBB and BRB promises to continue to provide important physiological insight and novel understanding of the NVU.


Insulin stores lipid in adipocytes and prevents lipolysis and the release of non-esterified fatty acids (NEFA). Excessive release of NEFA during sustained energy supply and increase in abdominal adiposity trigger systemic insulin resistance, including in the liver, a major site of insulin clearance. This causes a reduction in insulin clearance as a compensatory mechanism to insulin resistance in obesity. On the other hand, reduced insulin clearance in the liver can cause chronic hyperinsulinemia, followed by downregulation of insulin receptor and insulin resistance. Delineating the cause–effect relationship between reduced insulin clearance and insulin resistance has been complicated by the fact that insulin action and clearance are mechanistically linked to insulin binding to its receptors. This review discusses how NEFA mobilization contributes to the reciprocal relationship between insulin resistance and reduced hepatic insulin clearance, and how this may be implicated in the pathogenesis of non-alcoholic fatty liver disease. Insulin stimulates fat storage in adipose tissue in addition to increasing glucose uptake and reducing gluconeogenesis in liver. Whereas insulin signaling mediates insulin action, the process is regulated by the circulating level of insulin that is, in turn, determined not only by how much is secreted, but also by how much is cleared (reviewed in ).Insulin is secreted in pulses from pancreatic beta cells . The livMechanistically, insulin internalization via its receptor is induced by the Carcinoembryonic Antigen-Related Cell Adhesion Molecule 1 (CEACAM1) (reviewed in [Fatty acid synthase (FASN) binds to CEACAM1 to pull it off and facilitate insulin dissociation from its receptor to allow insulin degradation in the acidic milieu of late endosomes and recycling of the receptor to the plasma membrane , key insulin signaling molecules . IRS-1/2Ceacam1 gene deletion, and in mice with liver-specific inactivation of CEACAM1  plays a significant role in renal insulin clearance, its role in hepatic insulin clearance remains controversial ,14. Studlearance . Howeverlearance , deletinlearance . At the Hepatic insulin clearance serves as a major determinant of insulin sensitivity in the normal dog . Genome-Most studies in animals ,26 and hReduced insulin clearance has also been reported to occur as fat deposition increases in the liver, supporting its significant role in chronic hyperinsulinemia in non-alcoholic fatty liver disease (NAFLD), a metabolic disorder that is commonly associated with insulin resistance ,39. For In patients with type 2 diabetes, the incidence of hepatic steatosis is higher and of insulin clearance is lower than nondiabetic subjects . LowerinInsulin-resistant patients with type 2 diabetes commonly develop upper central body obesity  ,49,50,51In the Western world, high energy supply, sleep deprivation , sedentaChylomicron-triglycerides derived from excessive fat intake that escape β-oxidation are stored in adipose tissue until they are mobilized as NEFA during lipolysis under the effect of catecholamines . The risMechanistically, NEFA can activate PKC  causes systemic insulin resistance driven by a low-grade sub-acute pro-inflammatory state that develops in parallel to the activation of WAT-associated macrophages ,54,55,79As commonly observed in obese humans  and rodeGiven that insulin induces the transcriptional activity of Ceacam1 promoter , it is lThus, mobilized NEFA from WAT blunt insulin signaling and activate PPARα to lower Ceacam1 transcription . In thisNicotinic acid causes expansion of adipocytes to facilitate fat storage in WAT. Despite fat accumulation, inflammation, and blunted insulin signaling in WAT of mice fed a high-fat diet, nicotinic acid treatment protects hepatic CEACAM1 expression in parallel with insulin sensitivity . This suThat reduced hepatic CEACAM1 expression plays a key role in diet-induced hepatic insulin resistance and steatosis in addition to disturbed insulin clearance is supported by the reversal of these metabolic abnormalities upon adenoviral-mediated delivery of the wild-type, but not the phosphorylation-defective, mutants of CEACAM1 to the liver of mice at 14 days of a high-fat diet . FurtherSimilarly, high-fat feeding causes reduction in hepatic insulin clearance in parallel to reduction in the protein levels of CEACAM1 and IDE . TreatmeOf note, not all studies show reduction in insulin clearance in the presence of elevated plasma NEFA levels. For instance, high carbohydrate intake has been shown to result in a marked reduction in plasma NEFA while reducing hepatic insulin clearance and action ,100. MorThe evidence that reduced hepatic insulin clearance constitutes an immediate response to increased dietary energy is mounting in humans and rodents. Several studies have shown that reduced insulin clearance is an early adaptation to increased high energy dietary intake, and that it serves to trigger hyperinsulinemia before insulin resistance, obesity, and increased insulin secretion develop ,100,102.Ceacam1 deletion display primary reduction in insulin clearance and hyperinsulinemia at about 2 months of age, followed by hepatic insulin resistance (partly caused by downregulation of insulin receptor levels in hepatocytes) and steatohepatitis at 6–7 months of age [Our focus on CEACAM1 phosphorylation by the insulin receptor and its role in insulin clearance has provided a mechanistic underpinning for how reduced insulin clearance can cause insulin resistance and is not just a consequence thereof . Consists of age . Chronics of age . Visceras of age . AccordiCeacam1 null mice manifest reduction in insulin clearance at 2 months of age, followed by systemic insulin resistance at 6–7 months of age and hepatic steatosis. Nicotinic acid treatment stops lipolysis but fails to curb insulin resistance [Further buttressing the primary role of hepatic insulin clearance in insulin resistance, global sistance . This deCeacam1 null mice, including a higher degree of macro-steatosis in parenchyma, and more robust hepatic fibrosis and apoptosis [Reversal of the metabolic phenotype upon liver-specific reconstitution of CEACAM1 further demonstrates the primary role of hepatic insulin clearance in the pathogenesis of insulin resistance . Interespoptosis . This adpoptosis , providiReduced insulin clearance has increasingly been recognized as an integral part of insulin resistance, irrespective of whether it plays a causal or an adaptive role in insulin resistance. Dissecting out the relationship between insulin action and insulin clearance has been challenged by the dependence of both processes on insulin binding to its receptor and insulin signaling. Post-receptor mechanisms such as phosphorylation of CEACAM1 also regulate insulin signaling (by sequestering SHP2 phosphatase and activating the IRS/PI3Kinase/AKT pathway) (reviewed in [


Fibrosis can affect almost every organ and represents an increasing cause of morbidity and mortality worldwide. Despite significant progress in our understanding of the pathobiology of fibrosis, there is still a lack of putative anti-fibrotic targets to be exploited in anti-fibrosis therapies or used as biomarkers of fibrosis progression. The discovery that HIPK2 can control molecular pathways involved in fibrosis has opened a new field of study in both pathophysiology and the treatment of fibrosis.Fibrosis is an unmet medical problem due to a lack of evident biomarkers to help develop efficient targeted therapies. Fibrosis can affect almost every organ and eventually induce organ failure. Homeodomain-interacting protein kinase 2 (HIPK2) is a protein kinase that controls several molecular pathways involved in cell death and development and it has been extensively studied, mainly in the cancer biology field. Recently, a role for HIPK2 has been highlighted in tissue fibrosis. Thus, HIPK2 regulates several pro-fibrotic pathways such as Wnt/β-catenin, TGF-β and Notch involved in renal, pulmonary, liver and cardiac fibrosis. These findings suggest a wider role for HIPK2 in tissue physiopathology and highlight HIPK2 as a promising target for therapeutic purposes in fibrosis. Here, we will summarize the recent studies showing the involvement of HIPK2 as a novel regulator of fibrosis. The disproportionate accumulation of extracellular matrix (ECM) components in response to an injury, usually as a consequence of chronic inflammation, leads to fibrotic disorders that represent an increasing cause of morbidity and mortality worldwide. Fibrosis can affect nearly every organ and induce their dysfunction by disrupting the physiological tissue architecture, . FibrosiAt the molecular level, the best characterized pro-fibrotic mediator is transforming growth factor (TGF)-β, a pluripotent growth factor that activates fibroblasts into myofibroblasts to stimulate the release of ECM proteins  . The WntIn this review, we will first briefly summarize the cellular and molecular mechanisms of fibrosis that have been extensively described in many interesting reviews .α, etc.). However, when the regulatory mechanisms are disrupted, fibroblasts escape the normal control that is necessary for their inactivation, as soon as the damage is repaired, and may remain persistently activated [At the cellular level the main cell types involved in fibrosis are fibroblasts and macrophages. Physiologically, the activation of fibroblasts is required for tissue remodeling such as wound healing after a cell injury. Fibroblasts become activated into myofibroblasts by inflammatory cytokines. Myofibroblasts display up-regulated cellular migration, exaggerated ECM production and increased chemical signaling secretion and responsiveness ,14. Myofctivated . As a rectivated ,16. The ctivated . Fibroblctivated . In addictivated ,15. The Following tissue injury, an inflammatory response is activated and cytokines such as interferon (IFN)γ or tumor necrosis factor (TNF)α target macrophages that undergo marked phenotypic and functional changes to play a key role in all phases of tissue repair. Macrophages produce growth factors that promote cellular proliferation and blood vessel development . When thAt a molecular level, the central mediator of fibrogenesis is TGFβ, a pluripotent growth factor that inhibits ECM degradation and promotes the expression of pro-fibrotic genes in several cell types ,19. TGFβWnt/β-catenin is an evolutionarily conserved cellular signaling system that plays a crucial role in different biologic processes such as organogenesis and tissue homeostasis, as well as in the pathogenesis of many human diseases . The keyNotch is an evolutionarily conserved intercellular signaling pathway that regulates interactions between physically adjacent cells and may also be involved in the development of fibrosis ,27. ActiHomeodomain-interacting protein kinase (HIPK)2 is a nuclear serine–threonine kinase member of the HIPK family (numbered 1–4) that was originally identified to interact with homeodomain transcription factors  characterized by the abnormal accumulation of extracellular matrix that eventually leads to end-stage renal disease (ESRD) ,59, a maSubsequently, by analyzing several compounds using the structure–activity relationship (SAR) approach, BT173 compound was found to be the most effective HIPK2 inhibitor, particularly in reducing the interaction between HIPK2 and Smad3, thereby inhibiting Smad3 phosphorylation and activation [Recently, other molecules have been synthesized as HIPK2 inhibitors in renal fibrosis. Among several compounds containing benzimidazole and pyrimidine scaffolds, compound 15q was found to have a potent inhibitory activity against HIPK2 and, as a proof of principle, it was tested on renal cells and on mouse models of renal fibrosis . In vitrHIPK2 induction during fibrogenesis was proven to be blocked by Sirtuin 6 (SIRT6) overexpression  Figure b. SIRT6 In a mouse model of vancomycin (VAN)-induced transition of acute kidney injury (AKI) to chronic kidney disease (CKD), HIPK2 overexpression, mediated by the epidermal growth factor receptor (EGFR)/signal transducer and activator of transcription (STAT)3 pathway, was identified as a key regulatory mechanism . VAN-indFinally, in an in vitro model of human kidney (HK)-2 cells, the authors found that the overexpression of miR-141 inhibits TGF-β1-induced EMT through the repression of HIPK2 via direct interaction with the 3’-untranslated region of HIPK2 . The autFibrotic pulmonary pathologies include a group of serious and heterogeneous lung diseases such as pulmonary hypertension, chronic obstructive pulmonary disease (COPD), hypoxic/hyperoxic injury, mechanical injury, idiopathic pulmonary fibrosis (IPF), etc., which affect the respiratory system. Pulmonary fibrosis is mainly caused by the abnormal activation of alveolar macrophages and pulmonary fibroblasts. Pulmonary fibrosis scars and thickens lung tissues that do not expand as well as they should, making it hard to breath . IPF is The involvement of HIPK2 in pulmonary fibrosis has reported so far in few studies. In the fibrotic area of a mouse model of bleomycin-induced pulmonary fibrosis, HIPK2 expression was found to be downregulated . The mouInterestingly, circular (circ)HIPK2 expression was found to be increased in fibroblasts of a model of silica-induced pulmonary fibrosis . BiologiRecently, a genetic variant of HIPK2 has been found to be significantly related to radiation pneumonitis in lung cancer patients treated with radiation therapy , undersc-/- mice; in addition, HIPK2 inhibition reduces Smad3 phosphorylation in cardiac fibroblast [Heart failure is the final stage of many cardiovascular diseases and is one of the leading causes of death worldwide. It has poor prognosis as it lacks treatable clinical intervention targets. A risk factor for heart failure, following, for instance, hypertension or myocardial infarction, is pathological cardiac remodeling . Recent broblast . The latbroblast , and witbroblast . The resbroblast . The sambroblast . Mechanibroblast  , undergoing oxidative damage and apoptosis . Intrigu-/- mice, or by the fact that the protective effect of HIPK2 knockdown was only found in stressed conditions [Alternatively, HIPK2 was found reduced in human end-stage ischemic cardiomyopathy . The autnditions . AlthougLiver fibrosis is a major cause of morbidity and mortality from hepatic diseases and is characterized by the excessive deposition of ECM that eventually leads to cirrhosis and even to hepatocellular carcinoma (HCC) ,116. DurAbnormal wound healing can induce the formation of hypertrophic scars and keloids which lead to dysfunction and deformity. The mechanism of keloid formation is still under debate and, in this regard, EMT has been shown to play a key role . In a moHIPK2 was found to be involved in inflammatory response during skeletal muscle wound healing in an animal model of skeletal muscle contusion . The repRecently, it has been reported that HIPK2, Smad3 phosphorylation and p53 phosphorylation are increased in rabbit corneal keratinocytes cultured with 10% FBS  which induces the differentiation of keratinocytes into myofibroblasts . The inhCancer-associated fibrosis may contribute to cancer progression and resistance to anticancer therapies as a consequence of the interplay between cancer cells and the surrounding components embedded in the tumor microenvironment (TME), such as fibroblasts, immune system, vasculature and ECM components . Cancer Fibrotic disorders represent an increasing cause of morbidity and mortality worldwide. These diseases have a poor prognosis and are uncurable. Despite the effort in the research field of fibrosis, the absence of appropriate and fully validated biomarkers negatively impacts on the effectiveness of the therapeutic approaches. In this regard, HIPK2 is a promising biomarker for the progression of fibrosis in different tissues  and as a


Articular cartilage shows limited self-healing ability owing to its low cellularity and avascularity. Untreated cartilage defects display an increased propensity to degenerate, leading to osteoarthritis (OA). During OA progression, articular chondrocytes are subjected to significant alterations in gene expression and phenotype, including a shift towards a hypertrophic-like state  analogous to what eventuates during endochondral ossification. Present OA management strategies focus, however, exclusively on cartilage inflammation and degradation. A better understanding of the hypertrophic chondrocyte phenotype in OA might give new insights into its pathogenesis, suggesting potential disease-modifying therapeutic approaches. Recent developments in the field of cellular/molecular biology and tissue engineering proceeded in the direction of contrasting the onset of this hypertrophic phenotype, but knowledge gaps in the cause–effect of these processes are still present. In this review we will highlight the possible advantages and drawbacks of using this approach as a therapeutic strategy while focusing on the experimental models necessary for a better understanding of the phenomenon. Specifically, we will discuss in brief the cellular signaling pathways associated with the onset of a hypertrophic phenotype in chondrocytes during the progression of OA and will analyze in depth the advantages and disadvantages of various models that have been used to mimic it. Afterwards, we will present the strategies developed and proposed to impede chondrocyte hypertrophy and cartilage matrix mineralization/calcification. Finally, we will examine the future perspectives of OA therapeutic strategies. Articular cartilage has a limited capacity to self-heal owing to its avascular environment, low cellularity, and the poor proliferative activity of its resident cells, chondrocytes. Thus, untreated articular cartilage defects predispose the tissue to further degeneration leading to osteoarthritis (OA), a degenerative pathology characterized by structural, molecular, and mechanical changes in both cells and extracellular matrix (ECM) of multiple joint tissues ,2. ExistBesides an enhanced production of matrix-degrading enzymes and a state of low-grade inflammation , one of In this framework, different studies have investigated the relationship between the onset of OA and articular chondrocytes’ assumption of a hypertrophic phenotype ,6,12,16.These considerations make the case not only for the introduction of compounds targeting hypertrophic processes as new possible disease-modifying osteoarthritis drugs (DMOADs), but also for the development of new instruments  to help us in dissecting which are the pathological triggers, and ultimately to possibly discover new molecular targets for promising DMOADs. Concerning DMOADs specifically, different next-generation OA treatment drugs with the aim to modify the underlining OA pathophysiology are under development and/or are in clinical trials to test their safety and efficacy, as recently reviewed by Cho et al. . The majIn the present manuscript, therefore, we focus both on known pathways correlated with chondrocyte hypertrophic differentiation in development and in OA, but also discuss the different in vivo and in vitro models that are available or will need to be developed to better understand these processes and ultimately possibly discover new molecular targets for promising DMOADs .Healthy articular chondrocytes usually have a reduced proliferation capacity and do not experience terminal differentiation (leading to hypertrophy and apoptosis). Conversely, chondrocyte hypertrophy and cell death are natural phenomena during embryonic development, where chondrocytes undergo active proliferation with consequent enlargement and hypertrophic differentiation. While these chondrocytes grow significantly in size, the surrounding tissue becomes mineralized and remodels to bone via endochondral ossification . This phSOX9 and COL2A1 marks the first step of the endochondral ossification program. The differentiated chondrocytes then undergo transient cartilage differentiation: they start to proliferate and express Indian Hedgehog (IHH), and experience several steps of maturation, with the cells expressing COLX and RUNX Family Transcription Factor 2 (RUNX2), which terminally differentiate into hypertrophic chondrocytes expressing osteogenic factors such as ALP and Osteopontin (OPN). These steps are followed by invasion of the cartilage template by blood vessels from the subchondral bone and apoptosis of a large number of hypertrophic cells, eventually resulting in the cartilage template remodeling into trabecular bone  and p [113–132]) from the BMP7 peptide library that, even with a single dose, modulated ignaling .As previously reviewed, a second promising target for osteoarthritis therapy connected with the onset of a hypertrophic phenotype is constituted by Wnt signaling .As an example, the Wnt signaling pathway inhibitor PKF118-130 administered to MSCs directed towards chondrogenic differentiation induced upregulation in chondrogenic gene expression and a marked downregulation of hypertrophic markers .On a different note, in an in vitro chondrocyte model, the cyclooxygenase-2 (COX2) inhibitor Rofecoxib (even though COX2 is not a direct Wnt inhibitor) reduced the expression of hypertrophic markers  while also downregulating the expression of different downstream targets of the Wnt cascade  .Most notably, moreover, a small-molecule inhibitor of the Wnt signaling pathway, SM04690, (Lorecivivint) is being evaluated as a possible DMOAD in a phase III clinical trial (NCT04520607) after the success of phase I and II . LorecivCOL10A1 and MMP-13, thereby mitigating OA progression in mice [SMO, Gli2, RUNX2) [In addition, two IHH inhibitors have been proposed as therapeutic candidates: Cyclopamine and Ipriflavone. Cyclopamine is a IHH inhibitor demonstrated to downregulate the expression of hypertrophy hallmark genes  in mice . Iprifla, RUNX2) .MMP-13 and ADAMTS5, as well as a partial restoration of chondrogenic markers ACAN and COL2A1 [Finally, another small molecule, G141, a fibroblast growth factor receptor-I (FGFR-I) inhibitor, has shown promising results in mitigating hypertrophy and OA. Knockdown of FGFR-I led to a diminished expression of hypertrophic marker genes in a murine OA model, substantiating the role of FGFR-I in chondrocyte hypertrophy . Chondrod COL2A1 .Small molecules have, therefore, undeniable advantages and are showing promising results. The cellular signaling pathways that constitute the target of these compounds are, however, under strict spatio-temporal control during the development and homeostasis of articular cartilage and possibly act in multiple ways. There is, therefore, a stringent requirement to delivery modality for small molecule inhibitors in vivo in a localized manner  in order to evade possible off-target toxicity.The similarity between endochondral ossification and molecular changes witnessed in OA hint towards the involvement of angiogenic and hypoxic mediators such as VEGF and HIFs in the pathophysiology of OA.For example, a correlation has been established by genome-wide association studies between enhanced expression of VEGF and the extent of cartilage degeneration in OA , leadingMarsano et al. retrovirally transduced BMSCs to express a decoy soluble vascular endothelial growth factor (VEGF) receptor-2 (sFlk1), which efficiently sequesters endogenous VEGF in vivo, seeded them on collagen sponges, and studied their chondrogenic potential in vitro and in vivo after ectopic implantation in nude mice. sFlk-1-BMSCs inhibited angiogenesis in both in vitro and in vivo conditions and developed phenotypically stable cartilage with no evidence of expression of COLX, MMP-13, and BSP . The samCartilage is characterized by a hypoxic microenvironment. The role of hypoxia in suppressing OAC hypertrophic characteristics and the molecular mechanism of action of hypoxia have already been discussed quite well in several other reviews ,50,252. Allas et al. reported the role of dimethyloxalylglycine (DMOG), a hypoxia-inducible factor prolyl hydroxylase, in simulating hypoxia-like stabilization of HIF factors while improving their nuclear localization. DMOG reduced the expression of COLX and MMP-13 in a TGF-β1 induced in vitro OA model .2)), cells cultured under 2% hypoxia (2% O2) downregulated the expression of the hypertrophic markers COL10A1, MMP-1,-2,-3, and -13 while maintaining the expression of COL2A1 and ACAN, demonstrating that hypoxia plays a role in the suppression of hypertrophic characteristics in OA chondrocytes [2) induced chondrogenesis and suppressed hypertrophy in pellet cultures of human BMSCs even without any chondrogenic differentiation factor [Markway et al. demonstrated in a pellet culture OA chondrocytes model that in comparison to normoxic conditions  have been proposed to deliver various anti OA drugs to the desired region, but achieving the necessary local intra-articular concentration while avoiding systemic adverse effects remains challenging ,256. FurAs previously extensively reviewed , biomateMoreover, biomaterials such as chondroitin sulfate or hyaluronic acid have been shown to downregulate hypertrophy by themselves via inhibition of the Smad 1/5/8 pathway and upregulation of the p38 MAPK pathway ,260. SucIn this paragraph we will describe a series of polymeric materials that, in combination with different bioactive compounds, has been suggested for combating chondrocyte hypertrophy in OA.COL2A1, ACAN) with a high decrease in the expression of hypertrophic markers  [TGF-β3 loaded alginate microspheres, when incorporated in hyaluronic acid (HA) hydrogel discs together with hBMSCs and primed in chondrogenic medium, demonstrated a controlled release profile leading to a significant upregulation of chondrogenic gene expression. Moreover, implantation of the TGF-β3 loaded hydrogel discs into the subcutaneous pocket of nude mice led to a further increase of proteoglycan and collagen content, highlighting the chondroprotective role of the system with respect to hypertrophic maturation . Another, RUNX2) . Such ef, RUNX2) ,263.MMP-13, IL-1β, and ADAMTS5 [Similar effects were also obtained with HA conjugated with epigallocatechin-3-gallate gelatin hydrogel encapsulated with porcine ACs and primed in chondrogenic differentiation medium, which led to a significant downregulation of hypertrophic markers  ADAMTS5 .ACAN, SOX9, COL2A1) [Platelet-rich plasma (PRP) is known to exhibit anti-inflammatory potential leading to a reduced expression of MMPs and disintegrins in human OACs by inhibiting the IL-1β mediated NF-κB signaling pathway ,266. The COL2A1) . HoweverMMP-13 and ADAMTS5, an enhanced cartilage ECM deposition, and an increase in the expression of COL2A1 and ACAN in KGN supplemented chondrocytes [COL10A1 expression from hASCs using KGN [Hou et al. investigated the therapeutic effect of Kartogenin (KGN), a small molecule regulator of chondrogenesis, and demonstrated a significant decrease in the expression of drocytes . KGN wassing KGN .OA is a complex, multifactorial disease correlated with multiple environmental and genetic factors that not only leads to degradation of the articular cartilage but also affects menisci, subchondral bone, and the synovial membrane, as well as periarticular muscles and ligaments.Furthermore, while the precise pathogenic mechanism of OA has not yet been uncovered, it is expected that cartilage degeneration and joint function loss occur by more than one means, which can further vary for distinct OA subgroups depending on their pathogenesis . These observations render the individuation of a single strategy to counteract OA extremely difficult to pinpoint.One of the most accepted routes of pathological progression, however, highlights how ACs in OA are subjected to a phenotypical instability leading to differentiation processes similar to those observed in chondrocytes during endochondral ossification. Cartilage wear and tear at the joint surface leads to expansion of the calcified cartilage zone as cartilage fissures/cracks propagate. In an attempt to repair the tissue, chondrocytes show enhanced proliferation and synthetic activity, but in doing so produce matrix degradation products and proinflammatory cytokines. These factors disrupt the normal chondrocytes’ function and quiescent state while also establishing a crosstalk with the synovium that ultimately enhances proliferative and pro-inflammatory responses.In this review we not only focused on the mechanisms leading to the onset of a hypertrophic phenotype in chondrocytes in OA, but also presented and discussed both in vitro and in vivo models that have been used or might be used to increase our knowledge of these processes.Notably, we only focused on cartilage models, but more complex setups also involving synovium and subchondral bone are needed to properly understand the complex intra-tissues crosstalk that characterizes OA.In the present state, it is not known whether inflammation and cartilage degradation in general follow hypertrophy or induce it ,271. IntFor instance, it is known that following immobilization of rodent joints, hypertrophy sets in in the articular cartilage, but remobilization leads to the significant recovery of joint architecture and cellular nature . Thus, tAdditional studies will be needed to answer to these and other questions. Presenting a summary of the dysregulated pathways and of the models used to investigate them, we tried to deliver a basic compendium that could be adopted to instruct possible new research and the development of more complex models.


Puberty is a complex physiological process in females that requires maturation of the reproductive neuroendocrine system and subsequent initiation of high- frequency, episodic release of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH). Genetics and nutrition are two major factors controlling the timing of puberty in heifers. While nutrient restriction during the juvenile period delays puberty, accelerated rates of body weight gain during this period have been shown to facilitate pubertal development by programming hypothalamic centers that underlie the pubertal process. Among the different metabolic factors, leptin plays a critical role in conveying nutritional information to the neuroendocrine axis and controlling pubertal progression. Because GnRH neurons are devoid of the leptin receptor, leptin’s effects on GnRH neurons must be relayed via an afferent neuronal network. Two neuronal populations located in the arcuate nucleus (ARC) that express the orexigenic peptide neuropeptide Y (NPY), and the anorexigenic peptide alpha melanocyte-stimulating hormone (αMSH), are key components of afferent pathways that convey inhibitory (NPY) and excitatory (αMSH) inputs to GnRH neurons. In addition, ARC neurons expressing kisspeptin, a potent stimulator of GnRH release, are also involved in the nutritional regulation of puberty. Our studies have demonstrated that increased planes of nutrition during juvenile development result in morphological and functional changes in hypothalamic pathways comprising NPY, proopiomelanocortin (POMC), and kisspeptin neurons. Changes included differential expression of NPY, POMC, and Kiss1 in the ARC, and plasticity in the axonal projections to GnRH and kisspeptin neurons. Additionally, increased rates of body weight gain also promoted changes in the pattern of DNA methylation, a key epigenetic mechanism for regulation of gene expression. Finally, our most recent findings suggest that maternal nutrition during gestation can also induce structural and functional changes in hypothalamic neurocircuitries that are likely to persist long after pubertal maturation and influence reproductive performance throughout adulthood in cattle. Pubertal maturation in female mammals is an intricate physiological process that involves physical and behavioral changes associated with activation of the hypothalamic-pituitary-ovarian axis and subsequent establishment of reproductive cyclicity . Reprodue.g., Bos indicus-influenced) in which the skeletal size required to support a healthy and safe pregnancy is frequently attained well before the establishment of regular estrous cycles. Therefore, a better understanding of the neuroendocrine mechanisms controlling puberty can assist in the development of novel managerial strategies that exploit brain plasticity during critical windows of development and lead to strategies for successfully programming the onset of puberty around 12 to 14 months of age.The timing of pubertal development has important implications for livestock production. In beef heifers, lifetime productivity is heavily dependent upon their ability to reach reproductive competence, to conceive early during their first breeding season, and to calve the first time by approximately 24 months of age . HoweverThe objectives of this review are to present an overview of the neuroendocrine processes controlling puberty in heifers, discuss the effects of nutrition on these processes, and summarize recent research findings regarding the programming effects of nutrition during early development on hypothalamic pathways that control reproduction. While this review focuses primarily on pubertal development in heifers, supportive data from other species including sheep and rodents are also discussed.The onset of puberty in the heifer is characterized by a marked increase in the frequency of LH pulses that first becomes apparent at approximately 50 days before first ovulation. This characteristic increase serves as the most accurate predictor of pubertal onset . These cKiss1 gene and its receptor (KISS1R), and is expressed in a variety of tissues, including the hypothalamus. Moreover, KISS1R colocalizes with GnRH neurons and is responsible for regulating the release of GnRH , the majority of heifers weaned at approximately 3.5 months of age and fed high-concentrate diets to achieve accelerated rates of body weight gain exhibited puberty before 300 days of age (precocious puberty). Although those studies were performed in Bos taurus breeds (Angus and Hereford), similar findings were observed in our studies using Bos indicus-influenced heifers, which are later maturing. In these studies, heifers (1/2 Angus × 1/4 Hereford × 1/4 Brahman) were weaned between 3.5 and 4 months of age and fed a high-concentrate diet to promote a rate of body weight gain of approximately 1 kg/day  at rates comparable to heifers fed a high- concentrate diet continuously  are accompanied by greater adiposity and increased circulating concentrations of the metabolic hormones leptin, insulin, and insulin-like growth factor 1 (IGF1) when compared to regimens that promote growth at a slower rate  and agouti-related peptide (AgRP), both secreted predominantly in conditions of low energy balance , which is produced primarily during periods of positive energy balance . In rodeKiss1 mRNA content in the ARC , to minimal (juvenile period), followed by a postpubertal increase to maximum expression during adulthood  and growth hormone receptor (GHR). In addition, we observed differential expression of genes associated with control of feed intake and metabolism , as well as genes involved in synaptic vesicle transport, axonal growth and neuronal plasticity. These findings, in conjunction with observed changes in neuronal projections , which were hypermethylated in heifers that gained body weight at higher rates. Importantly, the hypermethylation of these genes was associated with a reduced abundance of their mRNA expression levels. Growth hormone has been previously reported to play a role in pubertal development in heifers  cows bearing female pregnancies were fed to achieve thin, moderate, or obese body condition (BC) by approximately 6 months of gestation (second trimester) and maintained at the target BC until calving. Heifer offspring from each maternal group were then weaned at approximately 3.5 months of age and assigned randomly to be fed to achieve a relatively low (0.5 kg/day) or high (1 kg/day) rate of body weight gain until 8 months of age. While results of most of our studies using this animal model remain pending, initial findings are summarized in the following sections.During the past decade, the developmental origins of health and disease (DOHaD) hypothesis by Barker and colleagues has attracted considerable attention to the concept of fetal programming . The DOHAs discussed previously, leptin is a critical metabolic hormone conveying nutritional information to the neuroendocrine axis and controlling pubertal development in females. Previous studies in sheep have shown that changes in nutritional status can modulate the transport of leptin across the blood-brain barrier. For example, in a study in which obese and lean adult sheep were fed to either gain or lose body weight, it was observed that obese animals had impaired transport of leptin across the blood-brain barrier . MoreoveTo test this hypothesis, we investigated the expression of different isoforms of the leptin receptor in the choroid plexus of heifers subjected to the different maternal  and postnatal (low and high) nutritional treatments. Importantly, the transport of leptin across the blood-brain barrier has been shown to depend on expression of the short form of the leptin receptor, which acts as a leptin transporter in endothelial cells . We founUsing hypothalamic tissue from the same group of heifers discussed above, we investigated the interactive effects of prenatal and postnatal nutrition on the extent of NPY (inhibitory) projections toward GnRH neurons. While none of the treatment combinations altered the number of GnRH neurons, reduced rates of body weight gain during postnatal development increased the proportion of GnRH neurons in close apposition to NPY-containing fibers  in specific hypothalamic nuclei, epigenetic modification , morphological development of neuronal pathways that modulate activity of the GnRH pulse generator, leptin signaling in the hypothalamus, among others. Importantly, we currently have available a large group of nutritionally- programmed heifers that will allow us to characterize the interactive effects of prenatal and early postnatal nutrition on multiple physiological processes, such as leptin transport across the blood-brain barrier, central release of neuropeptides, pulsatile secretion of GnRH and LH, responsiveness to estradiol negative and positive feedback, and estrous cycle events associated with postpubertal maturation. Moreover, because organizational changes brought about through perinatal nutritional programming are believed to be imprinted in the genome, they are likely to be associated with consistent physiological events that may, in some cases, be manifested only later in life. Thus, these nutritionally-programmed females represent a valuable resource for evaluating the long-term consequences of perinatal nutrition on reproductive function of sexually mature animals. We believe that the perinatal nutritional environment can program how the reproductive neuroendocrine axis of sexually mature heifers responds to leptin and other metabolic/hormonal factors, particularly under unfavorable metabolic conditions.In our ongoing and future studies, we will further examine the interactive effects of prenatal nutrition with nutritional treatments imposed postnatally during the early juvenile period on hypothalamic processes controlling puberty. These include changes in expression of key genes (Finally, it will be important in the future to determine the transgenerational effects of perinatal nutrition in the bovine female. Studies in rodents have shown that experimental manipulation of the nutritional plane during the perinatal period can significantly impact the reproductive and metabolic phenotypes of subsequent generations (


Since more than 3 decades, schizophrenia (SZ) has been regarded as a neurodevelopmental disorder. The neurodevelopmental hypothesis proposes that SZ is associated with genetic and environmental risk factors, which influence connectivity in neuronal circuits during vulnerable developmental periods. We carried out a non-systematic review of genetic/environmental factors that increase SZ risk in light of its neurodevelopmental hypothesis. We also reviewed the potential impact of SZ-related environmental and genetic risk factors on grey and white matter pathology and brain function based on magnetic resonance imaging and post-mortem studies. Finally, we reviewed studies that have used patient-derived neuronal models to gain knowledge of the role of genetic and environmental factors in early developmental stages. Taken together, these studies indicate that a variety of environmental factors may interact with genetic risk factors during the pre- or postnatal period and/or during adolescence to induce symptoms of SZ in early adulthood. These risk factors induce disturbances of macro- and microconnectivity in brain regions involving the prefrontal, temporal and parietal cortices and the hippocampus. On the molecular and cellular level, a disturbed synaptic plasticity, loss of oligodendrocytes and impaired myelination have been shown in brain regions of SZ patients. These cellular/histological phenotypes are related to environmental risk factors such as obstetric complications, maternal infections and childhood trauma and genetic risk factors identified in recent genome-wide association studies. SZ-related genetic risk may contribute to active processes interfering with synaptic plasticity in the adult brain. Advances in stem cell technologies are providing promising mechanistic insights into how SZ risk factors impact the developing brain. Further research is needed to understand the timing of the different complex biological processes taking place as a result of the interplay between genetic and environmental factors. Mental disorders, including schizophrenia (SZ), are the leading medical cause of years lived with disability worldwide GBD . TogetheIt has been proposed that a variety of environmental factors may interact with genetic risk factors during the pre- or postnatal period to induce symptoms of SZ in early adulthood  around 60 to 80%  in chromosome 6, which is the locus with the top association with SZ, has been shown to participate in synaptic pruning, determination of synapse density, and microglial engulfment of synapses   or rare (low frequency) genetic variation have also provided convergent evidence regarding the impact of SZ genetic risk burden on the synapse , plays a remarkable role in synapse formation at early stages of development , SETD1A (Histone-lysine N-methyltransferase SETD1A), or TRIO  , CNTN4 (Contactin-4), LRRC4B (Leucine-rich repeat-containing protein 4B), or DCC (Netrin receptor DCC)  play a prominent role in synaptic plasticity throughout development and adult life. In these cases, identifying the developmental timing of their aetiological mechanism using only genetic association studies remains a challenge. A better knowledge of the effects of genetic variation on gene regulation during pre- and postnatal development could help to clarify these questions. In this vein, some studies have integrated the results of GWAS with information on quantitative trait loci (QTLs) known to be active in the foetal brain. These approaches have shown that mechanistic effects of CNTN4, PCDHA7, PCDHA8 , and other genes on synaptic SZ risk are very likely to take place in utero, although posterior postnatal influences cannot be ruled out , with an important role both in synaptic plasticity in adult brain  and Cav1.3 (CACNA1D) mRNAs  , mapping to this region, that led to impaired synapse development in IPSC-derived excitatory neurons  with increases of cortical thickness in the upper left temporal gyrus , ANK3 (Ankyrin-3), or NCAN (Neurocan core protein), among others .Other genes that harbour genome-wide associated genetic variants have also been analysed in this context: Despite compelling evidence of the contribution to risk of genetic and environmental factors to SZ risk, their interplay within a neurodevelopmental framework has not yet been understood. Gene x Environment interactions (G x E) have been hypothesised to play a central role in the differential risk of SZ (van Os et al. Taken together, the results from genetic and environmental factors highlight the role of synaptic dysfunction an impaired myelination in the pathophysiology of SZ. Synaptic plasticity is a key biological process not only in the adult brain but also in developmental stages of the central nervous system, during the establishment and consolidation of neural networks (Forsyth and Lewis


JCI, Schaub et al. report on the changes in single-cell transcriptional profiles of young participants with type 2 diabetes who received SGLT2 inhibitors. Treatment with SGLT2 inhibitors restored metabolic perturbations in proximal tubular cells and reduced expression of the inflammatory signaling molecule mTORC1. Notably, changes in transcripts and mTORC1 were also found in the kidney of a diabetes mouse model treated with an SGLT2 inhibitor, supporting use of this model for further studies. These findings reveal cellular mechanisms of SGLT2 inhibitors and are important for advancing therapeutic targets in the treatment of DKD.The sodium-glucose cotransporter-2 (SGLT2) is expressed on the luminal side of proximal tubule epithelial cells in the kidney. While pharmacological inhibition of SGLT2 provides kidney protection in diabetic kidney disease (DKD), the molecular mechanisms remain unclear. In this issue of the  JCI in 1987 (The concept of using sodium-glucose cotransporter-2 (SGLT2) inhibitors for treatment of kidney disease has been a story of remarkable serendipity. In a sense, it has been a story line of science in reverse — clinical observations driving preclinical research — a turnaround for the usual order of inquiry for unraveling potential therapeutic mechanisms. The concept of inhibiting SGLTs in the kidney tubules to induce glucosuria, and thereby lower blood glucose and correct insulin resistance, was introduced by Rossetti, DeFronzo, and colleagues in a seminal study of diabetic rats published in the  in 1987 . SGLT2 w in 1987  and not  in 1987 , 4. Gluc in 1987 –7. Notab2, kidney failure, and death due to kidney disease  for safety after regulatory approval of glucose-lowering agents were required by the US Food and Drug Administration starting in 2008  and SGLT2 inhibitors were prescribed for clinical indications, the study observations provide detailed molecular information at the cellular level in the human kidney according to diabetes status and SGLT2 inhibitor use. Many of the structural features of early diabetic kidney disease (DKD) were diminished in the group treated with SGLT2 inhibitors along with cellular transcriptional profiles across nephron segments, reflecting restoration of metabolic perturbations toward normal. Overall, evidence of less glycolysis, gluconeogenesis, and tricarboxylic acid cycle activity was observed in proximal tubular cells, while the reverse was true in cells from distal segments, such as the medullary thick ascending limb. In all nephron segments, less expression of mTORC1, an inflammatory signaling mediator induced by metabolic and nutrient stressors, and phosphorylated S6 protein, an mTORC1 activity marker, was noted with SGLT2 inhibitor treatment. Corresponding changes in transcripts and mTORC1 were found in the kidney cortex from a mouse model of type 2 diabetes treated with an SGLT2 inhibitor, supporting use of this model for further studies. Transcriptomic changes for processes related to cellular energetics  in the proximal tubule and the opposite in the medullary thick ascending limb could be a consequence of shifts in energy requirements related to solute transport under receptor-mediated SGLT2 inhibition  and risk factors , they had little to no clinically manifest DKD at the time of the kidney biopsy. Therefore, it is unclear which of them will actually progress to DKD or what features may predict progressors versus nonprogressors. Confounding by indication is also a concern particularly with the small observational case-control study design. Notably, patients who received SGLT2 inhibitors could conceivably be either higher or lower risk for DKD based upon clinical decisions made outside the study. Although the SGLT2 inhibitors appear to have a class effect for protecting the kidney that is largely dose independent, biases may also exist due to use of various SGLT2 inhibitors in different doses. Other kidney-protective agents  were also used more commonly in the SGLT2 inhibitor users (30%) than in nonusers (17%) .In conclusion, investigations linking deep phenotyping of humans, including kidney tissue–based interrogation by advanced ‘omics and rapidly evolving methodologies, are critical for unraveling fundamental mechanisms of disease in this structurally, functionally, metabolically, and immunologically complex organ. Discovery of how SGLT2 inhibitors have unlocked previously unimagined kidney protection is an important step toward advancing therapeutic targets to meet the enormous unmet need for better treatment of patients with DKD.


Acquired vitamin B12 (vB12) deficiency (vB12D) of newborns is relatively frequent as compared with the incidence of inherited diseases included in newborn screening (NBS) of different countries across the globe. Infants may present signs of vB12D before 6 months of age with anemia and/or neurologic symptoms when not diagnosed in asymptomatic state. The possibility of identifying vitamin deficient mothers after their pregnancy during the breastfeeding period could be an additional benefit of the newborn screening. Vitamin supplementation is widely available and easy to administer. However, in many laboratories, vB12D is not included in the national screening program. Optimized screening requires either second-tier testing or analysis of new urine and blood samples combined with multiple clinical and laboratory follow ups. Our scope was to review the physiologic fate of vB12 and the pathobiochemical consequences of vB12D in the human body. Particular emphasis was put on the latest approaches for diagnosis and treatment of vB12D in NBS. VB12, also called cobalamin (Cbl), is present in several forms in nature. Cbl deficiency might be defined as absolute deficiency of vB12, or functional deficiency with the metabolite profile characteristic for inadequate cellular function of Cbl dependent enzymes . ThisFrom the chemical point of view, the corrin ring of the Cbl molecule is in covalent bond with the central cobalt ion (of various oxidation state). Above this plain, the beta-axial ligand bound to the cobalt ion determines specific reactions in which Cbl might be involved. Based on this latter ligand, molecules with distinct biochemical functions are distinguished like adenosyl-Cbl (AdoCbl), methyl-Cbl (MeCbl), hydroxy-Cbl (OHCbl), cyano-Cbl (CNCbl), and glutathionyl-Cbl (GSCbl). Most of these molecules are synthesized after intracellular processing of Cbl. AdoCbl and MeCbl are essential cofactors of methylmalonyl Coenzyme A (MMCoA) mutase (MUT) and methionine synthase (MS), respectively, enzymes in the intermediate metabolism of the human body.Cbl rich diet includes animal products such as meat, fish, dairy products, liver and egg. Strict vegan or vegetarian diet without supplementation might lead to vB12D, already after 4 weeks of dietary change from omnivore to strict vegan diet ,6. In a The process from ingestion to ileal absorption is demonstrated on During absorption , the intIntracellular processing differences between gut epithelial cells or other peripheral, non-polarized cells are barely discussed in the literature. It is suspected that Cbl either undergoes processing to the biologically active cofactor forms  that are finally used by enterocytes for specific reactions of metabolism, or passes the cell without major modifications (transcytosis) to enter the portal circulation (reviewed by ). The trSome fraction of intestinal Cbl might be bound to luminal free TC2 and absorbed via CD320 receptor according to studies on murine small and large intestine, and human polarized colorectal cancer cell lines. Another, rather small fraction of Cbl is not absorbed at all, affecting microbiota metabolism and pathogenicity in large intestines. An even smaller amount is eliminated by the stool ,16.Cytosolic processing of Cbl after release from the lysosome is a multistep procedure, in which apoenzymes bind and chaperonate different forms of Cbl . Many quTransport of Cbl in blood occurs either in form of HolotTC (~20–25% of circulating Cbl) or TCN1-Cbl (~75–80% of circulating Cbl). HoloTC is considered the biologically available (“active”) Cbl in the circulation that can enter non-polarized cells via CD320 mediated endocytosis. On the other hand, TCN1-Cbl is considered to transport Cbl to hepatocytes, and allowing the storage or mobilization from hepatic Cbl buffer depending on Cbl storage levels of the body ,12.Kidneys might also serve as a Cbl reserve. The main receptor for reabsorbing Cbl in proximal tubular cells is megalin. Apart from this, cells also exhibit a remarkable intracellular TCN2, and apical CD320 expression in murine experiments. Thus excreted TCN2 can form HoloTC taken up by their receptors at the luminal surface of epithelial cells ,17.The MUT enzyme (EC 5.1.99.1) is catalyzing the reaction in which MMCoA is converted to succinyl CoA (SuCoA); while the enzyme MS EC 2.1.1.13) catalyzes the reaction where homocysteine (HCY) is converted to methionine (Met) . AdoCbl  catalyzeConsidering the bone marrow, inadequate availability of MeCbl leads to accumulation of HCY and decreased Met, while the methyl donor methyl-THF is accumulating, thereby trapping the methyl group from other cellular methylation processes. This leads to abnormal nucleotide synthesis resulting in the histopathologic picture of macrocytic anemia (reviewed by ). ClinicNeurocognitive symptoms might be linked to impaired methylation → disturbed myelination → increased inflammatory cytokine axis, leading in extreme cases to the atrophy of the spinal cord or cerebral tissue in different degree. The spectra of childhood neurologic symptoms associated with Cbl deficiency might include convulsion, muscular hypotonia, developmental delay, intellectual disability, dizziness, tingling sensation, syncope, or blurred vision . Most ofStandardized international definition or consensus guidelines for a vB12D are lacking. Laboratory evaluation of a vB12D allows determination of at least four distinct laboratory markers: total abundance of vB12, HoloTC, MMA and HCY ,20. TherAmong analytical methods on vB12 determination, microbiological assays were the first to have found utility in the clinical laboratory, due to acceptable sensitivities and specificities . This tiSerum Cbl concentrations alone are weak indicators for functionally relevant intracellular Cbl deficiency . A functThe second marker commonly used is HoloTC, the form of vB12 that is taken up by cells to meet metabolic demand, explaining the increasing use of assays on this biomarker ,20. HoloThe first method for HoloTC was based on the enzyme-linked immunosorbent assay technique (ELISA) . CurrentThe concentration of MMA in serum reflects the availability and utilization of AdoCbl in mitochondria. MMA in fresh or optimally stored urine or blood is considered the most sensitive marker of intracellular, functionally relevant Cbl depletion  .The first techniques for the measurement of MMA applied paper chromatography, thin layer chromatography, spectrophotometry and later ELISA, but were superseded by mass spectrometric methods due to markedly improved sensitivities and specificities. Full or semi-automation of liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for MMA is also possible . DetermiAn elevated MMA is not specific for vB12 depletion or inborn errors of the Cbl metabolism. Among other disorders, a deficiency of the enzymes MUT  or acyl-CoA synthetase family member 3 (ACSF3) can also result in MMA levels increased to various magnitudes . ElevateThe total concentration of HCY in serum reflects the availability and utilization of MeCbl in the cytosol . HistoriIn addition to inborn errors of the Cbl metabolism, other congenital disorders can also manifest with elevations in serum HCY, including a cystathionine-β-synthase (CBS) or a methylenetetrahydrofolate reductase (MTHFR) deficiency . ElevateAs a complement to the determination of HCY, a recent article reported an LC-MS/MS method for the determination of SAM and S-adenosylhomocysteine (SAH) in plasma and urine. The determination of SAM and SAH is complicated by the instability of SAM under neutral and alkaline conditions and the naturally low concentration (nanomolar range) of both SAM and SAH in plasma. The clinical applicability of the assay is yet to be determined .Recommendations on the determination of vB12D include in optimal case measurement of serum vB12 or HoloTC combined with two functional markers, MMA and HCY. It is important to emphasize that HCY may also increase in folate deficiency; therefore, additional measurement of folic acid is needed for differential diagnosis . Cut-offIn most countries, screening of vB12D status is not compulsory during pregnancy ,42. EstiDuring pregnancy, concentrations of several biochemical markers are lower than those in non-pregnant women . HormonaCbl repletion of the newborn at the time of delivery is substantially determined by the Cbl status of the mother . There is positive correlation in each trimester between the Cbl status of the mother and newborn . MoreoveNBS allows early recognition and treatment of numerous inborn errors of metabolism, thus facilitating the prevention of serious morbidity and mortality . DBS is However, the characteristic alterations of an elevated propionylcarnitine (C3) and/or decreased Met may additionally be suggestive of a vB12D, that was previously considered an incidental finding and inappropriate for an inclusion in NBS until very recently ,68,69. TRecently, an increasing number of publications suggested that an inclusion of a vB12D into NBS may not only be reasonable but feasible too. According to the authors, the proper selection of diagnostic analytes, cutoffs and strategies can facilitate detection of a vB12D. Thus, an inclusion of this condition into NBS is currently under evaluation in some countries including Germany ,69. The Diagnostic specificity can be further increased by confirmatory assays utilizing chromatographic separation of diagnostically important analytes  from their isobaric substances, i.e., from those having the same molecular mass ,68,69. IThe very limited sample amount in DBS raises an analytical challenge for most diagnostic metabolites . Thus, eConsidering the diagnostic value of the first- and second-tier markers, a vB12D can present with diverse alterations ,69. In aCut-off values defined for the above biomarkers varied according to different algorithms used by NBS laboratories. A single marker (HCY high cut-off: 8 µmol/L) was only used in Norway . Most laMaternal vB12 supplementation during gravidity and post-partum (until the 6th week) could serve as primary prevention strategy not only through better Cbl values of the newborn at birth but also due to an increase of Cbl in breastmilk . MothersInfant formulas are fortified by vB12 and serve as adequate tools for vitamin administration . In the Infantile parenteral administration of Cbl might occur in the form of intramuscular injection (most common), or via intravenous injection . A single intramuscular injection (400 µg OHCbl in infants < 8 months) may correct the impaired Cbl status and improve the already evolving clinical symptoms, such as motor dysfunction and regurgitations . The lesThe vB12D is a condition that can severely affect quality of life if not diagnosed early or left untreated. In low-income regions with high prevalence of vB12D and less financial resources for NBS, primary prevention, such as mandatory food fortification, or recommendations in parallel with educations for vitamin supplementation during pregnancy could be the best choice . Recent Standardized protocols and consensus guidelines for vB12D are yet to be developed and, together with recent improvements in laboratory technology and clinical treatment, are expected to facilitate earlier diagnosis and a better quality of life of patients with vB12D.


Thoracic aortic disease affects people of all ages and the majority of those aged <60 years have an underlying genetic cause. There is presently no effective medical therapy for thoracic aneurysm and surgery remains the principal intervention. Unlike abdominal aortic aneurysm, for which the inflammatory/atherosclerotic pathogenesis is well established, the mechanism of thoracic aneurysm is less understood. This paper examines the key cell signaling systems responsible for the growth and development of the aorta, homeostasis of endothelial and vascular smooth muscle cells and interactions between pathways. The evidence supporting a role for individual signaling pathways in pathogenesis of thoracic aortic aneurysm is examined and potential novel therapeutic approaches are reviewed. Several key signaling pathways, notably TGF-β, WNT, NOTCH, PI3K/AKT and ANGII contribute to growth, proliferation, cell phenotype and survival for both vascular smooth muscle and endothelial cells. There is crosstalk between pathways, and between vascular smooth muscle and endothelial cells, with both synergistic and antagonistic interactions. A common feature of the activation of each is response to injury or abnormal cell stress. Considerable experimental evidence supports a contribution of each of these pathways to aneurysm formation. Although human information is less, there is sufficient data to implicate each pathway in the pathogenesis of human thoracic aneurysm. As some pathways i.e., WNT and NOTCH, play key roles in tissue growth and organogenesis in early life, it is possible that dysregulation of these pathways results in an abnormal aortic architecture even in infancy, thereby setting the stage for aneurysm development in later life. Given the fine tuning of these signaling systems, functional polymorphisms in key signaling elements may set up a future risk of thoracic aneurysm. Multiple novel therapeutic agents have been developed, targeting cell signaling pathways, predominantly in cancer medicine. Future investigations addressing cell specific targeting, reduced toxicity and also less intense treatment effects may hold promise for effective new medical treatments of thoracic aortic aneurysm. Thoracic aortic aneurysm (TAA) is characterized by progressive aortic dilation, often asymptomatic until dissection or rupture occurs. One in five TAA result from gene mutations affecting vascular smooth muscle cells (VSMC) or the extracellular matrix (ECM) . GeneticPathological features of TAA include ECM degradation, VSMC dedifferentiation, inflammation and endothelial cell (EC) dysregulation ,6,7. TheThe transforming growth factor β (TGF-β) signaling pathway has been the focus of investigation for genetic TAA ,12. BothDeciphering the contributions of intersecting signaling pathways to TAA is key to developing effective therapeutics. As a fundamental step towards supporting future studies, this review examines the evidence for the roles of different cell signaling pathways in TAA. The functions of each pathway in VSMC and EC, and interactions between pathways, are described. Evidence for the involvement of each pathway in the pathogenesis of TAA is reviewed and finally, potential therapeutic interventions are examined.The principal anatomical layers of the aorta are the intima, media and adventitia. The intima is a monocellular layer of ECs, separating the bloodstream from the inner aortic wall, and includes a basement membrane, subendothelium and internal elastic membrane. The intima has an essential role in vascular homeostasis, through ion and solute exchange and hemodynamic signaling. The media lies between the intima and outer adventitia, with elastin laminae, type I & III collagen, fibrillin-1 microfibrils and proteoglycans, forming the ECM that surrounds the VSMC, to form the lamellar units . The medPathological features of TAA include the degradation of the ECM, the loss of VSMC due to apoptosis, and the fragmentation of elastin laminae ,18,19,20Normal endothelium promotes vasodilation via nitric oxide (NO), contributes to the inhibition of VSMC proliferation and migration, and also inhibits platelet aggregation and adhesion . In contThe most recognized pathway associated with TAA is the TGF-β cascade ,32,33. TThere are multiple ligands in the TGF-β family, including TGF-β and bone morphogenic proteins (BMPs). Receptors are heterodimers of Type II (TGF-β and BMP subtypes) and Type I receptors, with the latter having several subtypes, notably Activin-like kinase 5 (ALK5) and Activin-like kinases 1/2/3/6 (ALK1/2/3/6). The ALK5 receptors transduce TGF-β signaling to activate SMAD2/3, whilst ALK1/2/3/6 transduce BMP signaling to activate SMAD1/5/8 . SignaliThere are several layers of regulation of TGF-β signaling . The firThe second level of regulation involves internalization and the recycling or destruction of activated receptors. Interestingly, some receptors are specifically associated with cholesterol-rich regions of the cell membrane and these receptors are particularly associated with activation of MAPK/ERK by TGF-β. Furthermore, the depletion of cholesterol in these regions appears to inhibit TGF-β induction of MAPK/ERK signaling . The degSKIL, the expression of which is increased by TGF-β -S-phenylglycine t-butyl ester (DAPT) attenuated aortic dilatation [In a murine model of MFS, Jespersen et al. have recently reported evidence of increased NOTCH3 expression and activation in ascending aortic tissue. The blockade of NOTCH signaling with the latation . There ilatation . In adulThere appears to be a role for NOTCH signaling in mediating VSMC apoptosis in TAA, with the actual outcomes likely dependent upon NOTCH subtype. It is less likely that alterations in NOTCH signaling are a primary cause of TAA, but rather that NOTCH functions as a modulator of the disease process.−/− Ldlr−/− mice, which show characteristic changes of medial necrosis in response to ANGII [−/− after ANGII exposure [The adverse remodeling of the ECM is associated with chronic ANGII/AT1R signaling, with increased collagen deposition and matrix glycosaminoglycan synthesis . In abdoto ANGII , as do texposure .FBN1 mutations), Loeys-Dietz syndrome  and familial TAA associated with ACTA2 mutations. Each of these syndromes is characterized by TAA at a young age and also by cerebral aneurysms (Loeys-Dietz and ACTA2). In a fibrillin-deficient Marfan mouse model, the loss of ANGII-AT1R signaling in EC is associated with reduced p-ERK1/2 activity and reduced TAA formation, suggesting a role for ANGII-stimulated ERK1/2 activity in pathogenesis of TAA [Fbn1C1039G/+ mouse model of MFS, through inhibition of ERK1/2 signaling mediated by AT2R signaling [Several murine models of genetic TAA have been studied, including Marfan syndrome , ACTA2, is also associated with TAA formation after ANGII. The mechanism appears to involve both the generation of ROS and the NF-κB-dependent induction of AT1R . The ANGutations . Mice deutations .Other signaling pathways, e.g., AKT, may protect from the development of TAA in response to ANGII. The stimulation of VSMC with ANGII results in increased MMP-9 synthesis, which may contribute to ECM remodeling. Although the TGF-β pathway shares the downstream elements of ERK1/2 with ANGII, the inhibition of TGF-β signaling in a murine model did not alter MMP-9 production, indicating that ANGII is the main stimulus for MMP9 upregulation in the VSMC . The expAT1R gene have been associated with increased aortic stiffness [Several lines of evidence suggest a role for ANGII/AT1R in pathogenesis of human TAA. Single nucleotide polymorphisms in tiffness . In pathtiffness . Thus, tFollowing experimental findings that the AT1R blocker may attenuate TAA formation in MFS mice , multiplThe evidence, experimental and clinical, supporting the role(s) of each of the signaling pathways in pathogenesis of TAA is summarized in Information on potential therapeutic interventions in TAA is limited, however evidence from other fields, particularly cancer treatment, points towards options for future investigation.An inhibitor of TGF-β and downstream signaling mechanisms, pirfenidone, was FDA approved in 2014 for the treatment of idiopathic pulmonary fibrosis, indicating the therapeutic potential of inhibiting TGF-β signaling in a clinical setting ,305.Several major classes of TGF-β inhibitors are currently being tested in clinical trials and include antisense oligonucleotides which cause degradation of TGF-β mRNA, TGF-β antibodies and ligand traps which prevent TGF-β binding to receptors, and small inhibitor molecules that inhibit TGF-β receptor kinases . AdditioTGF-β plays a major role in cancer progression and metastasis . IntriguTherapeutics targets of TGF-β in cancer are frequently being tested in combination with other cancer treatments to improve therapeutic potential. This need for TGF-β inhibitors in combination with other agents may also be relevant to the treatment of TAA, as the interactions between and redundancy of signaling pathways may mean the inhibition of TGF-β alone may not result in an effective outcome. This concept has been supported by the finding that TGF-β1 inhibitors combined with PI3K inhibitors are the most effective method for inhibiting the phenotype switch of human aortic VSMCs compared to the use of a single inhibitor .Several drugs targeting the WNT pathway are in the clinical trial phase of testing, predominately for the treatment of cancer. These include inhibitors of WNT protein secretion, competitive antagonists of WNT binding to Frizzled, monoclonal antibodies to Frizzled and antagonists of Dishevelled binding to Frizzled .Molecules targeting WNT secretion have shown promising results. For example, several inhibitors of porcupine, a protein that is essential for secretion of WNT, results in the inactivation of WNT ligands and the reduction of downstream signaling, arresting cell growth in preclinical models, with clinical trials now in progress for the treatment of solid tumors ,318,319.These molecules currently in development for cancer therapies also demonstrate clinical utility for the treatment of CVDs. The administration of a porcupine inhibitor after myocardial infarction in mice reduced adverse cardiac remodeling and fibrosis . AdditioWhile currently there are no FDA approved WNT pathway inhibitors, the repurposing of established agents has been an area of interest, with several FDA approved drugs shown to inhibit components of the WNT pathway . Thus, aThe PI3K/AKT pathway and the therapeutic potential of its inhibition has been extensively investigated in cancer. Numerous drugs inhibiting the PI3K/AKT pathway have been develop and progressed to human clinical trials. The inhibition of PI3K/AKT and/or mTOR strongly influence other pathways such as MAPK, downstream FOXO3a, WNT/beta-catenin and NOTCH , which mThere are two main classes of PI3K/AKT inhibitors. The first are pan-PI3K inhibitors which do not discriminate between the different PI3K isoforms and were the first generation of inhibitors to be developed. One of these drugs includes wortmannin, which reduced aneurysmal growth when administered in an experimental animal model of AAA . While tDisordered NOTCH signaling is a feature of many cancers and a variety of targeted therapies have been developed, including inhibitors of the cleavage of NOTCH receptor precursors and gamma-secretase inhibitors . The gamAs cardiovascular abnormalities appear to be a feature of impaired NOTCH signaling, NOTCH agonists may be a useful therapeutic tool. However, given the fundamental role of NOTCH in vascular homeostasis, the overall inhibition of the pathway by nonspecific agents are unlikely to be beneficial. Therefore, the more targeted inhibitors currently being investigated in the treatment of cancer may be more useful in the treatment of CVD.There have been some preclinical studies on abdominal aortic aneurysm using NOTCH inhibitors. In a murine model using ANGII infusion, aortic inflammation and elastin fragmentation were reduced by treatment with a NOTCH inhibitor ,334. WheThe ANGII pathway is a well-studied treatment target in CVD, including in TAA. Angiotensin receptor blockers (ARBs) and beta-blockers have each been proposed to reduce TAA growth rates in MFS and other genetic aortopathies ,335. TheThe magnitude of effect on slowing TAA growth is debated and available clinical studies do not all clearly differentiate individual effects of beta-blockers and ARB, as they may be used in combination. Data from experimental animal studies appear more robust than from clinical studies and the benefits of ARB use may be dependent upon the age of the treatment subject. In addition, other variables in human populations may impact the benefit of ARBs, including variability in underlying mutations causing MFS or LDS, epigenetic factors and drug dose and type . NotwithIn summary, the work done on investigating therapeutic targets of TGF-β, WNT, PI3K/AKT, NOTCH and ANGII signaling in the treatment of cancer highlight the potential for these pathways to be targeted in the treatment of TAA. However, there are a few factors that need to be considered when beginning to translate the work done in cancer to their use in the context of TAA.Firstly, several compounds which target these pathways show levels of toxicity when administrated for the treatment of cancer as the aggressive nature of the disease may require relatively high doses of a particular drug. However, this does not rule out their potential clinical utility in the treatment of TAA as it does not need to be approached in the same aggressive manner and therefore lower doses may be acceptable in TAA treatment, attenuating toxicity and potential side effects.Secondly, the aggressive nature of cancer may, on the other hand, mean certain drugs are not potent enough to show any advantageous therapeutic effect. However, a small effect on any of these pathways may be enough to influence TAA development and therefore may be beneficial in TAA treatment and are still worth pursuing.Finally, therapeutic targets of the five pathways discussed in cancer are frequently being tested in combination with other cancer treatments to improve therapeutic potential. This need for TGF-β inhibitors in combination with other agents may also be relevant to the treatment of TAA, as the interactions between and redundancy of signaling pathways may mean inhibition of TGF-β alone may not result in an effective outcome. This concept has been supported by the finding that TGF-β1 inhibitors combined with PI3K inhibitors are the most effective method for inhibiting the phenotype switch of human aortic VSMCs compared to the use of a single inhibitor .Multiple signaling pathways influence both EC and VSMC phenotype, often acting in synergy to either promote or inhibit cell proliferation and phenotype. Crosstalk between two or more pathways in determining cell fate appears to be common. Signaling outcomes consequent upon the activation of a specific pathway EC and VSMC may, however, differ and each cell type has the capacity to influence outcomes in the other.At this time a cognate integrative description of the mechanism of TAA formation remains elusive. There is evidence indicating potential for each of the major pathways described to contribute to TAA formation. Experimental and human studies support the roles of these pathways in TAA, however the evidence base remains fragmentary and further work is required, particularly in the field of signaling cross-talk.For many patients with TAA, the underlying cause remains unknown, including for many with non-syndromic h-TAA. It is quite likely that many of these patients have an underlying dysfunction/dysregulation of the signaling pathways described and an understanding of the key regulatory points in these pathways may illuminate potential new loci for the investigation of gene mutations in TAA.Despite great interest, there is very limited data on efficacy of novel treatments for prevention of TAA. The experience in cancer medicine illustrates potential new approaches, but at the same time also highlights the challenges of drug toxicity and off-target effects. The multiplicity of pathways influencing cell fate infer redundancy and, therefore, therapeutic attempts to influence a single pathway may be sub-optimal in altering TAA development. Although much work remains to be done, our improved understanding of the roles of multiple cell-signaling pathways upon cell fate in the vasculature can provide a foundation for future advances.