Arabidopsis thaliana) with pathogens systemically activates an SAR-related transcriptional reprogramming as well as a primed immune status that is strictly dependent on FLAVIN-DEPENDENT MONOOXYGENASE 1 (FMO1), an enzyme that mediates the biosynthesis of NHP. Yildiz et al. (pp. 1679–1705) now show that exogenously applied NHP is sufficient to induce upregulation of more than 1,500 SAR-related genes in Arabidopsis and primes plants for an enhanced pathogen-triggered activation of defense metabolism. Primed metabolic responses included the biosynthesis of SA, pipecolic acid (a precursor of NHP), and branched-chain amino acids, as well as the accumulation of the phytoalexin camalexin. NHP also conditioned Arabidopsis for effective SA- and pathogen-induced expression of defense-related genes. All the effects of exogenous NHP require the function of the transcriptional coregulator NONEXPRESSOR OF PR GENES1 (NPR1). These results suggest that NPR1 transduces NHP-activated immune signaling modes that involve predominantly SA-dependent and, to a lesser extent, SA-independent features. The authors propose that NHP functions as a mobile immune regulator capable of moving between leaves to systemically activate immune responses.The immune responses of previously unchallenged plants are generally not effective enough to entirely prevent infection by well-adapted pathogens. However, a localized leaf inoculation can induce systemic acquired resistance (SAR) in the foliage of the whole plant. Plants with activated SAR show broad-spectrum immunity against further microbial infestation and are primed for a timely and boosted induction of defenses in response to pathogens. The establishment of SAR requires an interplay of two immune-regulatory metabolites, salicylic acid (SA) and N-hydroxypipecolic acid (NHP), which both accumulate to substantial levels in inoculated and in distant, noninoculated leaves of pathogen-attacked plants. Previous studies have revealed that the inoculation of Arabidopsis (Striga spp.) and broomrapes (Orobanche and Phelipanche spp.), are a major biological threat to global food security, affecting cereal crops in sub-Saharan Africa, and noncereal crops in Central Asia and the Mediterranean area. A single parasitic plant can produce tens of thousands of tiny and highly viable seeds that add to a seemingly inexhaustible seed bank in the soil. The life cycle of these parasitic weeds begins with seed germination that requires—in contrast to nonparasitic plants—chemical stimulants, mainly strigolactones (SLs), released by host plants.  From an agricultural perspective, this unusual requirement for SLs is possibly the Achille’s heel of these parasitic weeds: it may be possible to control them by applying specific germination inhibitors or synthetic stimulants that induce lethal germination in the host’s absence. Current chemical screening technologies, unfortunately, are labor intensive. To address this problem, Braguy et al. (pp. 1632–1644) have developed an automatic seed census tool to count and discriminate germinated from nongerminated seeds. This new method shows an accuracy of 94% in counting seeds of Striga hermonthica and reduced the required time from 5 min to 5 s per image. The open-source software the authors have developed, SeedQuant, will enable high-throughput screening for germination stimulants/inhibitors, and can also be used to study the germination of nonparasitic plant species.Root parasitic weeds, including witchweeds (Sorghum bicolor) is generally grown in arid and semi-arid regions, and hence its productivity depends on timing and amount of rainfall. Thus, exploring the phenotypic diversity and genetic basis of stomatal traits would provide useful information to improve productivity and stress tolerance in sorghum. Stomatal size/complex area (SCA), the product of guard cell length and width, and stomatal density (SD) are two key parameters regulating gas exchange and abiotic stress response in many plants, but sorghum remains to be studied in this regard. Moreover, the diversity in stomatal traits is largely unexplored or utilized in breeding programs in general, due to cumbersome phenotyping protocols that require substantial investment of resources. Bheemanahalli et al. (pp. 1562–1579) now report success in characterizing the phenotypic diversity of 311 sorghum accessions for stomatal traits grown under two different field environments. The nearly 12,000 images collected from abaxial and adaxial leaf surfaces revealed substantial variation in stomatal traits. In sorghum, SD was 32%–39% greater on the abaxial vs. the adaxial surface, while SCA on the abaxial surface was 2%–5% lower than on the adaxial surface. By using a Genome-Wide Association Study, a powerful tool for unraveling the genetic basis of complex traits compared to biparental mapping, the authors succeeded in identifying 71 genetic loci underlying SCA and SD in sorghum. These findings provide the foundation for further studies on the genetic and molecular mechanisms controlling stomatal regulation and patterning in sorghum.Stomata, the microscopic pores that occur on the surfaces of leaves, have a huge effect on crop productivity by influencing photosynthesis and water use. Stomatal traits, therefore, are potential targets for crop improvement. Sorghum . Their findings reveal that in contrast to RBOHD-dependent systemic responses to HL stress, which were exclusively mediated through the vascular bundles of Arabidopsis, RBOHD-dependent systemic signaling during HS, or wounding, are mediated through the vascular bundles and/or mesophyll cells. They further show that propagation of the ROS wave through mesophyll cells could contribute to the stronger systemic ROS signal observed in plants subjected to HL and HS applied simultaneously to two different leaves. These findings demonstrate that ROS production in mesophyll cells is required for the propagation of rapid systemic ROS signals during responses to HS or wounding.The local perception of heat (HS), high light (HL), or mechanical injury by a single leaf triggers systemic effects, such as systemic acquired acclimation or systemic wound responses, which induce a heightened state of stress readiness in the whole plant. Among the main candidates for the systemic signals mediating these responses are electric, calcium, and reactive oxygen species (ROS). These signals propagate from the stressed or injured leaf to the rest of the plant through the plant vascular bundles. It is thought that during this process the different cells along the path of signaling chain are activated one-by-one starting at the initial  tissue and ending at the systemic tissue and that this activation process propagates and maintains the different systemic signals. It was found in Arabidopsis, for example, that the ROS produced by each cell during this process is generated by the RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD) protein and that this process is controlled by calcium-dependent activation of RBOHD. The ROS being used as a systemic signal, most likely HYDA encodes an MPK kinase involved in zygote elongation and the shootward extra-embryonic cell fate. Loss-of-function yda zygotes do not elongate and after the first asymmetric division, a short basal cell with a repressed suspensor cell fate is produced. Conversely, constitutive activation of YDA leads to opposite phenotypes manifested by very long suspensors and in extreme cases the apical cell fate is fully suppressed. By means of genetic analysis, Samakovli et al. (pp. 1526–1544) now report that HEAT SHOCK PROTEIN 90 (HSP90) proteins are potent regulators of YDA during embryo development and patterning. The genetic interactions of HSP90 with YDA affected the downstream signaling pathway to control the development of both basal and apical cell lineage of embryo. Their results demonstrate that the spatiotemporal expression of WUSCHEL-RELATED HOMEOBOX8 (WOX8) and WOX2, two major players in axis formation, is changed when the function of HSP90s or YDA is impaired, suggesting their essential role in the cell fate determination and possibly a link to auxin signaling during early embryo development. Hence, HSP90s together with the YDA signaling cascade affect transcriptional networks shaping early embryo development. Thus, HSP90s are new players involved in the main body axis formation during early embryogenesis in Arabidopsis.The formation of the main body axis is a critical first step in the embryological development of plants. During its initial asymmetric division, the zygote partitions into a small apical daughter cell and a large vacuolated basal daughter cell. The apical cell gives rise eventually to the proembryo, while the basal cell gives rise to the suspensor that connects the embryo to maternal tissue. The YODA kinase (YDA) pathway is one of the major signaling cascades controlling cell division plane orientation and cell division symmetry. It acts by targeting and phosphorylating transcription factors involved in specific developmental processes. Larix decidua), stone pine (Pinus cembra), Norway spruce (Picea abies), and silver fir  remained unaffected, while mountain pine (P. mugo), yew (Taxus baccata), and common juniper (Juniperus communis) exhibited 0.4–0.9 MPa higher vulnerability to embolism. These data demonstrate that cavitation fatigue in conifers is species-specific and depends on the intensity of the preceding LC. The lack of fatigue effects after moderate LC, and relevant effects in only three species after high LC, indicate that conifers are relatively resistant against cavitation fatigue. This is remarkable considering the complex and delicate conifer pit architecture and may be important considering climate change projections.Coping with drought is fundamentally important for tree growth and survival. Water shortage in trees impairs the long-distance water transport when critical negative xylem pressure induces cavitation and, consequently, xylem embolism and disruption of the continuity of water columns. Many cases of drought-related forest mortality have already been reported and such events are likely to become more frequent, as increases in duration, intensity, and frequency of dry spells, coupled with rising temperatures, are predicted to occur in the future. After drought-induced embolism and repair, tree xylem may be weakened against future drought events (cavitation fatigue). Feng et al. (pp. 1580–1590) have quantified vulnerability curves after embolism/repair cycles in eight European conifer species. All species demonstrated complete embolism repair and a lack of any cavitation fatigue after 50% loss of conductivity (LC). After 100% LC, European larch (


Fibrosing interstitial lung diseases (ILDs) are chronic and ultimately fatal age-related lung diseases characterized by the progressive and irreversible accumulation of scar tissue in the lung parenchyma. Over the past years, significant progress has been made in our incomplete understanding of the pathobiology underlying fibrosing ILDs, in particular in relation to diverse age-related processes and cell perturbations that seem to lead to maladaptation to stress and susceptibility to lung fibrosis. Growing evidence suggests that a specific biological phenomenon known as cellular senescence plays an important role in the initiation and progression of pulmonary fibrosis. Cellular senescence is defined as a cell fate decision caused by the accumulation of unrepairable cellular damage and is characterized by an abundant pro-inflammatory and pro-fibrotic secretome. The senescence response has been widely recognized as a beneficial physiological mechanism during development and in tumour suppression. However, recent evidence strengthens the idea that it also drives degenerative processes such as lung fibrosis, most likely by promoting molecular and cellular changes in chronic fibrosing processes. Here, we review how cellular senescence may contribute to lung fibrosis pathobiology, and we highlight current and emerging therapeutic approaches to treat fibrosing ILDs by targeting cellular senescence. For several decades, understanding lung fibrosis as a process that limits lifespan has challenged scientists. Progressive loss of lung function due to pulmonary fibrosis contributes significantly to the ever-increasing burden of chronic disease throughout the world. Around half of deaths in the developed world are attributable to fibrotic diseases, including idiopathic pulmonary fibrosis (IPF), the most common fibrotic interstitial lung disease (ILD) characterized by progressive and irreversible respiratory failure and death ,2. The pFibrosis and wound healing are essentially interwoven processes, driven by a cascade of injury, inflammation, fibroblast proliferation and migration, matrix deposition and remodelling. Pathological fibrogenesis that occurs in many diverse organs and diseases is a dynamic process involving complex interactions between epithelial cells, fibroblasts, immune cells , and/or endothelial injuries ,6. ThereCellular senescence refers to the essentially irreversible transformation that cells experience upon unrepairable damage ,10. The Over the past years, significant progress has been made in our understanding of the biology of lung fibrosis. The pathogenesis of fibrotic lung tissue remodelling is a dynamic orchestrated process and is thought to be strongly linked to aging ,22. For In this regard, a complex interplay of environmental and genetic factors, age-related processes and epigenetic reprogramming leads to profound age-related cellular alterations in alveolar epithelial cells (AECs), immune cells, and fibroblasts from fibrotic lungs  Figure . EvolutiFurthermore, recent efforts have led to the development of network theories of aging, in which the contribution of several mechanisms synergise and interact. For instance, one of the possible explanations of the key issue of the increasing number of senescent cells in the aged lung and their persistence at sites of age-related pathology is that functional and structural changes in the immune system, also termed immunosenescence, lead to a less efficient clearance of senescent cells facilitating the occurrence of lung fibrosis . NeverthUnder normal conditions, AEC2s can self-renew and differentiate into type I alveolar epithelial cells and therefore are considered as alveolar progenitor cells ,48. HoweRepair and homeostasis of the lung occurs not only in an environment with increased oxidative stress but also in one that is highly susceptible to diverse exogenous hazards such as tobacco smoke, air pollutants, environmental antigens or infections, making cellular senescence extremely likely ,52. MildAlterations in the secretome of senescent cells with a chronic increment of production of several inflammatory secretory proteins, including plasminogen activator inhibitor-1 (PAI-1), matrix remodelling proteins (MMPs), growth factors such as TGFβ1, as well as other initial inflammatory stimuli, including IL-6, IL-8 and IL-1α, are present in IPF lungs. The SASP of these cells also has the potential to induce ER stress and enhances senescence through autocrine activity, but also can promote adjacent healthy cells to undergo senescence through paracrine senescence. In IPF lungs, the SASP effect of senescent AEC2s is detrimental as it is likely to induce a myriad of profibrotic mediators that drive pathological repair responses. SASP also consist of extracellular vesicles which are major mediators of intercellular communication through proteins, mRNAs, microRNAs, or organelles such as mitochondria. Novel studies suggest that NF-κB and CEBPB (CCAAT/enhancer-binding protein-β) are essential for the inducing of SASP factors, specially by inducing an autocrine feedback through IL-6 and IL-8 . MoreoveCDKN1A (p21) induces mitochondrial dysfunction and reactive oxygen species (ROS) production, which is considered necessary for the establishment of the senescent phenotype .,90.89,90Another candidate, Navitoclax (ABT-263), which is a specific inhibitor of the anti-apoptotic proteins BCL-2 and BCL-xL, was identified from an agent library as having substantial off-target apoptotic effects on non-senescent cell types such as platelets and immune cells, making them potentially ‘panolytic’, causing apoptosis or dysfunction in multiple cell types other than senescent cells depending on the dosage ,111. IntAlthough rapalog Everolimus (an inhibitor of the mechanistic target of mTOR), seemed to worsen patient outcome in previous clinical trials in IPF, another clinical trial using Rapamycin, another mTOR inhibitor, is currently being tested in IPF patients in a double-blind placebo-controlled pilot study (NCT01462006). Rapamycin has been recently proposed as a novel therapeutic target with the potential to impact multiple IPF patho-mechanisms influencing ECM production, metabolism, autophagy and senescence .A preclinical in vivo study in aging mice with genetic and pharmacological targeting of NOX4 treated with bleomycin showed a significant decrease in senescent fibroblast and collagen deposition, and also reversed established lung fibrosis . AnotherMany other pharmaceutical drugs already used in humans have been shown to target senescence in animal models of lung fibrosis. These include antidiabetic drugs such as Metformin (prevents NF-κB nuclear translocation), cardiac glycosides (Digoxin), antihistamines (Rupatadine), other BCL-2 family protein inhibitors (ABT-737) and antibiotics . These molecules target senescent cells by affecting pro-senescence or antiapoptotic proteins and controlling several SASP factors. However, new approaches to identifying and targeting these specific senescence-related pathways are under development.Strong evidence supports a pathogenic role of cellular senescence in pulmonary fibrosis. Novel approaches have the potential to be a major milestone in our battle against fibrosing ILDs. Valuable pathogenic pathways have been recognized to have potential novel therapeutic targets that are likely to delineate the natural history of pulmonary fibrosis, and we expect that in the near future pioneering therapies targeting senescence will improve survival and quality of life in patients with this devastating disease.Furthermore, the lack of reliable and measurable biomarkers for evaluating the effectiveness of senescence interventions in patients with pulmonary fibrosis also represents a serious challenge in this field. Perhaps the application of drug cocktails to affect several pathways in a simultaneous manner is the answer to overcome the multiple obstacles in the treatment of pulmonary fibrosis.Implementing such approaches in routine clinical practice will be certainly a long-term process, but this study is a step in that direction.


Hepatocellular carcinoma (HCC) is the predominant form of primary liver cancer and one of the leading causes of cancer-related deaths worldwide. A growing body of evidence supports the hypothesis that HCC is driven by a population of cells called liver cancer stem cells (LCSCs). LCSCs have been proposed to contribute to malignant HCC progression, including promoting tumor occurrence and growth, mediating tumor metastasis, and treatment resistance, but the regulatory mechanism of LCSCs in HCC remains unclear. Understanding the signaling pathways responsible for LCSC maintenance and survival may provide opportunities to improve patient outcomes. Here, we review the current literature about the origin of LCSCs and the niche composition, describe the current evidence of signaling pathways that mediate LCSC stemness, then highlight several mechanisms that modulate LCSC properties in HCC progression, and finally, summarize the new developments in therapeutic strategies targeting LCSCs markers and regulatory pathways. Hepatocellular carcinoma (HCC) is now the second most common cause of cancer mortality due to its resistance to chemotherapy, high rates of recurrence and metastasis, and poor prognosis . AlthougCSCs have “stemness” characteristics similar to normal stem cells, including self-renewal capacity and differentiation and proliferation potential . CSCs anThe discovery of liver cancer stem cells (LCSCs) greatly enhanced our understanding of HCC development and progression. Despite these new developments, the mechanisms associated with CSC features are not clear, and the link between how CSCs maintain stemness and contribute to HCC malignancy is not known. Several studies have demonstrated how LCSCs can be differentiated from liver cancer cells by targeting the cluster of differentiation markers CD13 , CD44 Z, CD90 Y, CD133 ; epithelStem cells are defined a specific cell type with the characteristics of self-renewal, multi-directional differentiation potential, and high proliferative potential under certain circumstances . StemnesNumerous studies have reported that CSCs exist in the context of multiple human cancers including HCC . AlthougTo better understand LCSCs, distinct markers have been reported such as CD133, CD90, CD44, CD24, CD13, OV6, Delta-like 1 homolog, and EpCAM, as well as measuring Hoechst dye efflux or aldehyde dehydrogenase activities; some of these may functionally support the LCSC phenotype including highly aggressive properties and chemoresistance . For exaThe tumor niche consists of numerous extracellular matrix (ECM) components; cytokines; and many cell types including fibroblasts, endothelial cells, immune cells and cancer cells that can regulate the CSC fate during tumor development. Great progress has been made in the identification of potential LCSCs niches over the past few years. Various components in the microenvironment can maintain LCSC stemness through altering signaling pathways or disrupting the master transcriptional regulation factors that maintain embryonic stem cell self-renewal, such as Nanog, Oct4, and Sox2 . The ECMMany groups have demonstrated that CSCs release a variety of factors into the niche that stimulate the stemness of CSCs themselves and also induce cancer angiogenesis and the recruitment of immune cells  and other tumor stromal cells that secrete additional factors to promote tumor progression and chemotherapy resistance . Tumor-aMany signaling pathways have been demonstrated to control normal stem cell self-renewal and participated embryonic development and differentiation, including Wnt/β-catenin, Notch, and HH; however, persistent abnormal activation of these highly conserved pathways may underlie the characteristics of LCSCs. The activation of signaling pathways is responsible for embryogenesis, liver specification, and liver regeneration in a strictly controlled manner. Dysregulation of certain signaling pathways has been implicated in maintaining LCSC stemness. With regard to the Wnt/β-catenin pathway, STAT3 is a transcription factor that is constitutively activated in many malignancies and plays a key role in cancer growth and metastasis . The STATGF-β is a central regulator in chronic liver diseases including HCC . In HCC The Notch signaling pathway plays an important role in stem cell self-renewal and differentiation . AberranThe HH pathway has a central role in embryonic development and adult tissue homeostasis, but mutations and/or aberrant activation of the pathway involved in malignancies . Recent in vitro and in vivo and reduces LCSC proliferation (B cell-specific Moloney murine leukemia virus integration site 1 (BMI1) acts as an epigenetic chromatin modifier  and playferation , suggestThe Hippo pathway, along with its downstream transcriptional co-activator Yes-associated protein and transcriptional co-activator with PDZ-binding motif, has a decisive role in the pathogenesis of primary liver cancer . Other sThe difference between CSCs and normal stem cells lies in their ability to change their pluripotency and lineage-dependent differentiation. CSCs that are resistant to radiotherapy and chemotherapy can regenerate tumors after treatment ends. The current CSC theory that heterogeneous populations of HCC cells are dictated and maintained at least partially by LCSCs may help explain the process of HCC formation . LCSCs hvia hepatocyte growth factor/Met signaling -upregulated Notch intracellular domain expression  pathways. via the NF-κB pathway, which modulates de-methylation of miR-148a and inhibits LCSC properties  receptor 1 in endothelial cells , increase angiogenic activity (shown by up-regulation of VEGF), enhance metastasis and invasiveness , and induce EMT (marked by increased serum and hepatic levels of TGFβ1 mRNA and protein) (Extracellular vectors (EVs) derived from CSCs  are important mediators that modulate communication between CSCs and their niches. EVs are rich in enzymes, miRNAs, transcription factors, heat shock proteins, major histocompatibility complexes, cytoskeleton components, signal transducers, and ECM effectors  that medal cells . Mechaniprotein) .Following the establishment of the CSC theory and the discovery of LCSCs in HCC, autophagy was proposed as a vital mechanism driving cell fate . Remarkain vitro and induces a CD133 + LCSC subpopulation in HCC by modulating an altered genomic profile involving the FXR pathway and possibly drug metabolism. It was also previously reported that hepatitis C virus (HCV) infection of primary human hepatocytes (PHH) can induce an EMT state and extend cell lifespan (Chronic infection with hepatitis B virus (HBV) has long been linked to HCC development. Studies have found that the C-terminally truncated HBx (HBx-ΔC) plays an important hepatocarcinogenesis by conferring enhanced invasiveness and reducing the apoptotic response in HCC cells . Ng et alifespan . Compelllifespan . These dvia activation of HH signaling , Notch, Wnt and BMI1 are involved in the renewal of normal stem cells and the maintenance of tissue homeostasis. Dysregulation of these pathways is believed to be involved in driving CSC activity in a variety of cancers (including HCC) through different mechanisms . More spormation . Reporteormation . The CD1ormation . The treThere is a growing body of evidence indicating that CSCs are the root cause of cancers and are responsible for metastasis, resistance to conventional treatments, and tumor recurrence; however, the molecular mechanisms underlying the potential roles of LCSCs in HCC origin and progression have not been fully elucidated. LCSC status and survival are controlled by multiple signaling pathways, and there are several mechanisms to maintain their malignant function. This overview presented findings suggesting that LCSCs drive HCC occurrence and development. In theory, if the LCSC subpopulation can be eliminated or have reduced stemness, it would be a way to control or even cure HCC. Although a great deal of effort is being made to reduce cancer stemness by targeting stemness signaling pathways, there are still many challenges given the complex biologic properties of CSCs. Perhaps the current focus should be on clarifying maintenance mechanisms involved in the malignant function of LCSCs. Since LCSCs must be eradicated to prevent HCC progression, recurrence, or metastasis, targeting the vital CSC signaling pathways is an attractive cancer treatment strategy. A comprehensive understanding of LCSC stemness and the mechanisms involved in cancer progression may help identify potential therapeutic targets and develop more effective therapies.HT and LZ contributed to the conception of this study. DL and LC drafted the manuscript. HT, LZ, and LD revised the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Although most same-stage non-alcoholic fatty liver disease (NAFLD) patients exhibit similar histologic sequelae, the underlying mechanisms appear to be highly heterogeneous. Therefore, it was recently proposed to redefine NAFLD to metabolic dysfunction-associated fatty liver disease (MAFLD) in which other known causes of liver disease such as alcohol consumption or viral hepatitis do not need to be excluded. Revised nomenclature envisions speeding up and facilitating anti-MAFLD drug development by means of patient stratification whereby each subgroup would benefit from distinct pharmacological interventions. As human-based in vitro research fulfils an irrefutable step in drug development, action should be taken as well in this stadium of the translational path. Indeed, most established in vitro NAFLD models rely on short-term exposure to fatty acids and use lipid accumulation as a phenotypic benchmark. This general approach to a seemingly ambiguous disease such as NAFLD therefore no longer seems applicable. Human-based in vitro models that accurately reflect distinct disease subgroups of MAFLD should thus be adopted in early preclinical disease modeling and drug testing. In this review article, we outline considerations for setting up translational in vitro experiments in the MAFLD era and allude to potential strategies to implement MAFLD heterogeneity into an in vitro setting so as to better align early drug development with future clinical trial designs. The prevalence of non-alcoholic fatty liver disease (NAFLD) has risen over past decades, closely paralleling the obesity pandemic, making it the number one chronic liver disease today . NAFLD iMajor risk factors for developing NAFLD are obesity and type II diabetes mellitus (T2DM), which is why NAFLD is often considered as the hepatic manifestation of the metabolic syndrome. NAFLD patients typically exhibit serum lipid dysregulation characterized by increased levels of low-density lipoprotein (LDL) particles and a reduced amount of high-density lipoproteins (HDL) ,5. UnsurWeight loss through lifestyle modification, achieved through close follow-up by dieticians and physical activity, might be a cost-effective and efficient approach for treatment of NAFLD . ConsequThe diagnosis of MAFLD is made based on the presence of hepatic steatosis with at least one of three of the following features: (i) obesity/overweight, (ii) diabetes mellitus and/or (iii) evidence of metabolic dysregulation ,12. The In the past decades, a variety of human-based hepatic in vitro models have been developed and applied in preclinical anti-NAFLD drug development. Nevertheless, these established models, of which a non-limitative list is shown in Next to the cell source and in vitro disease triggers, each cell culture system also has its specific strengths and limitations when it comes to in vitro MAFLD modelling. These characteristics should be taken into account when selecting the most suitable in vitro model for each specific application . Table 2In this review article, we allude to the implications of the change in NAFLD nomenclature for translational in vitro research. Further, we outline strategies for integrating essential aspects of MAFLD into an in vitro setting in order to better align preclinical experiments to future clinical studies.Most research has been published in the context of the established NAFLD terminology. Although MAFLD mostly covers patients that meet the criteria for NAFLD, key differences exist in their definition and diagnostic criteria and, therefore, these terms should not be used interchangeably. Throughout this review article, we adopted the terminology used by the authors of each cited publication and only moved to the MAFLD terminology when discussing future perspectives and suggestions for applying this new nomenclature to in vitro preclinical research.NAFLD occurs more often in males, in whom higher liver pyruvate kinase levels positively correlate with NAFLD severity ,34. A reNAFLD prevalence positively correlates with aging ,41,42; h, urine cells) . Hep. Hep2]. Unlike the terminology of NAFLD, where the disease could only be diagnosed in the absence of other well-defined liver pathologies, MAFLD embraces the existence of dual etiologies. This implicates that MAFLD can coexist with concurring pathologies such as drug-induced liver injury (DILI), viral hepatitis and moderate alcohol consumption, which has been observed in clinical practice as well ,117,118.Certain pharmaceuticals are known to induce liver steatosis and inflammation . HoweverConflicting data exists regarding the effects of ethanol consumption in patients suffering from MAFLD. A prospective cohort study including 4568 NAFLD patients concluded that modest ethanol consumption  decreases all-cause mortality compared to ethanol abstinence. In contrast, drinking more than 1.5 standard drinks of ethanol per day, rapidly increases all-cause mortality . These fNTCP, infection susceptibility decreases shortly after plating, due to dedifferentiation and high variability between donors that exists due to genetic susceptibility to infection. This can somewhat be tackled with specialized cell-culture systems, e.g., by using micropatterned cocultures with stromal cells. To address variation in susceptibility to infection, iPSC-derived hepatocyte-like cells were successfully applied [NTCP, albeit at low levels in their undifferentiated state. HepaRG therefore requires lengthy differentiation, consisting of two weeks of maintaining the undifferentiated cells in culture and two to four weeks of induction using corticoids and dimethylsulfoxide, into a hepatocyte-like and biliary-like cell mixture before NTCP is significantly upregulated. The liver cell lines HepG2 and Huh7 completely lack NTCP expression, which can be addressed by inducing constitutive expression of NTCP via reprogramming [In clinical practice, MAFLD and chronic hepatitis B (CHB) are increasingly observed together, with a significant impact on clinical outcome. In patients exhibiting dual NASH/CHB etiology, disease progression is rapid, along with accelerated manifestation of advanced fibrosis, liver-related outcomes and death . Similar applied . Furthergramming .Adjustment of the NAFLD terminology has been implemented to advance anti-NAFLD drug development and mainly to redesign clinical studies. It is, however, of utmost importance to align this novel mindset to basic mechanistic and translational research in order not to generalize anti-MAFLD drug development in the early preclinical stages. A key point in translational in vitro MAFLD research is proper documentation of the used cells in culture including data on sex, age, ethnicity and lifestyle of the donor, since taking into account this variability is essential for result interpretation .Anti-NASH therapy will possibly consist of a combinatory program in which patients are actively encouraged to undertake physical activity along with pharmacological treatments to stimulate disease remission, which in turn will probably also consist of combinatorial therapy given the heterogeneity of the disease. However, particular caveats in early anti-NASH drug development can be noticed due to patient heterogeneity related to (epi)genetics, differences in diet, ethnicity, ethanol consumption, gut microbiota, sex and drug intake. Although these disease-modifiers can be deduced from translational research and patient studies, each feature will likely not contribute to disease onset and/or development in an individual manner. Moreover, one might question how the general terminology of MAFLD reflects groups of patients with seemingly different etiologies. MAFLD holds broad diagnostic criteria and might be too sensitive across the general population ,142. FroOn the other hand, since fibrosis is the most important determinant for disease-specific mortality in NAFLD , one migConsidering the multi-faced pathogenesis of MAFLD, inter-species differences are likely to bias early basic and translational studies . The useDespite the recent advancements in the expansion of primary human hepatocytes in vitro , the con


There is growing appreciation that architectural components of the nucleus regulate gene accessibility by altering chromatin organization. While nuclear membrane connector proteins link the mechanosensitive actin cytoskeleton to the nucleoskeleton, actin’s contribution to the inner architecture of the nucleus remains enigmatic. Control of actin transport into the nucleus, plus the presence of proteins that control actin structure (the actin tool-box) within the nucleus, suggests that nuclear actin may support biomechanical regulation of gene expression. Cellular actin structure is mechanoresponsive: actin cables generated through forces experienced at the plasma membrane transmit force into the nucleus. We posit that dynamic actin remodeling in response to such biomechanical cues provides a novel level of structural control over the epigenetic landscape. We here propose to bring awareness to the fact that mechanical forces can promote actin transfer into the nucleus and control structural arrangements as illustrated in mesenchymal stem cells, thereby modulating lineage commitment. Actin in the cytoplasm provides structure to the cell, dynamically remodeling cellular structure to allow cell division, compartmentalization of cellular organelles, scaffolding of signaling components, and cell motility. Actin structure also contributes to the ability of the cell to sense its microenvironment, particularly the local mechanical environment . More reThe nucleus represents the largest and most dense organelle in the cell. Its intricate structure conveys discrete mechanical properties . Extrinswithin the nucleus might contribute to both its stiffness as well as interaction with lamin, and how this might affect the heterochromatin landscape.Intrinsic aspects of the nucleus such as B- and A-type lamins  as well in vivo [Cells are attuned to perceive, respond, and employ mechanical signals to guide development and function. Sensitivity to mechanical signals is critical to sensing and balancing forces during the gastrulation phase of development in vivo  and contin vivo . While Min vivo , in adulin vivo . We notein vivo . This reThe formation of focal adhesions and actin polymers results in three interconnected structures collectively controlled by external forces. These molecular assemblies emerge under strain to connect to other focal adhesions  or from p < 0.001) [p < 0.05). While this suggests that increased cytoplasmic F-actin remodeling is the predominant determinant of the LIV-induced cell modulus change, isolated nuclei also retain some of that information as increased modulus. A large component of nuclear stiffness is determined by lamin A/C, which scales with both substrate stiffness and actin contractility during processes of differentiation [Nuclear stiffness appears to be an intrinsic property. For example, we showed that when low-intensity, high-frequency mechanical forces  were applied to MSC, F-actin contractility increased, and cell modulus measured over the nucleus increased by fourfold to 5.9 kPa (< 0.001) . The modntiation . To thisntiation . Moreoventiation .There is also evidence that nuclear stiffness can be independent of changes in lamin A/C, depending rather on heterochromatic changes in histone methylation ,36. IncrIndeed, application of mechanical force to isolated nuclei induces nuclear remodeling and stiffening  and invoActin is found within the nucleus and is critical for many nuclear processes, including gene transcription through interaction with polymerases , repair Many details of the mechanism by which actin is relocated to the nucleus have come into greater focus. Actin does not have an identifiable nuclear localization signal, but its association with cofilin depolymerizes F-actin at slow-growing ends to create new barbed ends. Because cofilin has a nuclear localization signal  and inteBecause the nucleus is very dense, visualization of actin structure inside remains a challenge, and typical phalloidin staining does not reveal many recognizable actin cables . DespiteFormins (mDia1 and miDia2) expedite primary actin filament assembly by catalyzing end-on-end actin polymerization , which iSeveral reports indicate that the polymerized state of intranuclear actin guides targeting of some transcription factors. For instance, MLK1  binds monomeric actin, thereby preventing its binding to and coactivation of serum response factor : once MLXenopus oocyte nuclei is treated with Jasplakinolide to stabilize actin filaments [Actin rods can be observed in the nuclei of a small subset of cells in response to heat shock, hypoxia, or cell toxins, depending on the degree of stress . These rilaments . These filaments . In addiilaments .Visualization of F-actin fibers in the nucleus is a challenge for the field. Phalloidin-stained actin filaments  are rarely seen in normal nuclei or only in small percentages of stressed nuclei. This may be because the polymers are too short to be recognized . FurtherThe ability to sense the environment and transmit force into the nucleus affects phenotypic endpoints of MSCs. MSCs, and certainly stem cells of other embryonic derivations, exist in many adult tissues and likely subscribe to general rules for attaining differentiated states. Our laboratories have concentrated on the output of bone marrow MSCs. In the laboratory cultures of bone marrow, MSCs can be guided to become osteoblasts, chondrocytes, adipocytes, fibroblasts, and even myocytes when grown in appropriate media and physical conditions. In developing vertebrates, however, bone marrow-derived MSCs are more limited, preferentially adopting osteoblastic or adipogenic cell fates. Dynamic loading – or exercise – of the live skeleton promotes osteoblastogenesis and formation of new bone to withstand and adapt to loading conditions. Removal of loading, for instance, during long stays in a hospital bed or lack of loading due to nerve damage or other types of restricted motion, all lead to loss of bone and increased production of bone marrow adipocytes from the MSC pool . This unCytoskeletal sensing of substrate force directs MSC differentiation: plating cells on hard substrates promotes osteoblast cell fate, and soft surfaces encourage the adipocyte phenotype . It has Inhibiting the cellular capacity to form a cytoplasmic F-actin structure during in vitro mechanical loading promotes adipogenesis and prevents osteogenesis , and alsThe load-induced assembled F-actin network in the cytoplasm transmits substrate force to the nucleus via LINC connectivity ,90. TranAnother clue to the importance of intranuclear actin structure for cell fate decisions was through effects of knockdown of mDia2, which we demonstrated to be the major diaphanous formin in the nucleus in mouse MSC, and not located in the cytoplasm . KnockinInterestingly, the actin polymerization state can, in principle, be controlled by an architectural regulatory function of β-catenin/CTNNB1 via interactions with α-catenin/CTNNA1 in cadherin junctions at points of cell-to-cell contact . α-catenAccess of proteins necessary to nuclear functions, including regulation of gene expression, requires transport through nuclear pores whose openings are subject to physical constraints provided by the cytoskeleton. For instance, the cytoplasmic actin cytoskeleton controls nuclear localization of gene trans-acting factors as evidenced study of the Yorkie-homologues YAP and TAZ. These two proteins are mechanosensitive co-regulatory factors that bind to sequence-specific transcription factors via tryptophan (W)-containing protein/protein interaction domains . In response to extracellular mechanical signals generated by substrate stiffness, both YAP and TAZ translocate to the nucleus . YAP is Moreover, polymeric actin prominently influences differentiation of MSC. Transcription factor RUNX2 mediates osteogenic lineage commitment and progression along the osteoblast lineage by interacting with a cistrome of bone-related genes during differentiation . A proliMuch attention is now being addressed to the physical structure of genomes with appreciation that spatial arrangements affect gene availability . For exaHeterochromatin, the dense and compacted nucleosomal organization of silenced, unexpressed and inaccessible genes, is generally relegated to the periphery of the nucleus. In MSCs that are in a resting or stem-like state, quiescent genes are positioned as such at the periphery of the nucleus . During Gene silencing in heterochromatin may arise through increased contact of chromosomes to the lamins that form structures at the inner nuclear membrane allowing gene partitioning . Many geFurther, alterations in intranuclear actin level and structure may be critical regulators of gene accessibility. Recent work has shown that knockdown of β-actin, resulting in an absence of nuclear actin, causes a general decrease in gene activation through associated chromatin remodeling . MoreoveThus, beyond well-accepted roles of actin to associate and promote RNA polymerase , it has In sum, while it is still difficult to characterize changes in actin structure after mechanical or pharmacological treatments, a wealth of data support that intranuclear actin structure is involved with chromatin remodeling – and maybe even that histone modifiers regulate actin structure. A recent study of differentiating T-cells indicated that EZH2ʹs methyltransferase activity initiates assembly of intranuclear actin polymers was shown using superresolution microscopy and computational modeling . The autA dynamic actin structure within the nucleus can be convincingly inferred from the functional presence of a nuclear actin tool box. The ability to transmit forces generated at the plasma membrane via F-actin cables linked into the nucleus, and over the nucleus, affects intrinsic nuclear properties. Further, in response to force, actin transport into the nucleus can rise, along with the transfer of mechanoresponse molecules that activate gene expression. Actin is necessary for gene transcription, and its partition into monomeric versus polymeric forms has been shown to affect the differentiation of stem cells. Within the nucleus, the presence of structural actin, and the ability of actin to cause shape change is predicted to regulate both chromatin organization and the activity of enzymes that mediate heterochromatin formation. As such, nuclear actin dynamics represents a new mechanism whereby architectural elements linked to actin structure influence epigenetic regulation of gene expression.


Background: Platelet function testing is a valid tool to investigate the clinical response to antiplatelet therapy in different clinical settings; in particular, it might supply helpful information in patients with cerebrovascular disease. Oral antiplatelet treatment, such as Aspirin (ASA) and Clopidogrel, is the gold standard in secondary stroke prevention of non-cardiogenic ischemic stroke; conversely, its application as a primary prevention therapy is not routinely recommended in patients with vascular risk factors. Multiple electrode platelet aggregometry (MEA) impedance aggregometer is a validated device to test platelet inhibition induced by ASA or Clopidogrel.Case Report: We report the case of a 78-year-old patient without relevant clinical history, taking ASA as primary prevention strategy, who was admitted for sudden onset of dysarthria and left facial hyposthenia during physical effort. Brain CT revealed two small subcortical bilateral spontaneous intracranial hemorrhages. Platelet aggregometry with MEA performed upon admission revealed a very strong platelet inhibition induced by ASA . MRI at longitudinal follow-up revealed the presence of two small cavernous angioma underlying hemorrhagic spots.Conclusion: The evaluation of platelet reactivity in stroke patients undergoing antiplatelet therapies, not commonly performed in clinical practice, could be useful to optimize prevention strategies; the verification of the biological effectiveness of ASA or Clopidogrel could be a valid tool in the definition of each patient's risk profile, particularly in patients with cerebrovascular disease known to be at increased risk for both hemorrhagic and thrombotic complications. International leading guidelines strongly recommended antiplatelet therapy in secondary prevention of non-cardiogenic strokes, as it is associated with an estimated reduction of relative risk of stroke or death on average by about 22% , 2. ConvTherefore, the use of Aspirin might be reasonable only for people whose 10-year vascular risk is notable (at least higher than 10%) for the benefits to outweigh the risks associated with treatment. In particular, the association of diabetes mellitus with other high-risk conditions has been considered for primary prevention strategies , 4.Platelet function testing is a valid tool to investigate the clinical response to antiplatelet therapy in different clinical settings; several clinical and biological mechanisms for antiplatelet “resistance” or, conversely, “ultra-responsiveness” have been supposed  –7. Thus,®, Roche Diagnostics International Ltd., CH-6343 Rotkreuz, Switzerland) , the platelet–platelet aggregation is induced and real-time recording starts. The ADP Test reagent contains ADP, which triggers platelet activation via different ADP receptors, the most important of which is blocked by Clopidogrel (*min)  , 12 showzerland) . Platelepidogrel . The ASPpidogrel ; TRAP agl (*min) . Cutoff l (*min) , while vl (*min) .A 78-year-old man without relevant clinical history was admitted in the Stroke Unit for sudden onset of slurred speech and left oral rhyme deviation during physical effort, without headache and/or limb weakness. Patient's past medical history reported bilateral neurosensory hypoacusis, previous cataract surgery, and carpal tunnel syndrome surgically treated. Pharmacological anamnesis revealed daily treatment with Aspirin 100 mg as a vascular primary prevention strategy, started 3 months before.Neurological examination showed paralysis of right VII cranial nerve, right deviation of protruded tongue, and mild dysarthria . Brain CT revealed multiple chronic lacunar infarctions of basal ganglia bilaterally, and two acute small intraparenchymal hemorrhages, within post-rolandic subcortical region on the right side  and pre-® platelet function analysis performed upon admission revealed a very strong platelet inhibition induced by ASA; the area under the aggregation curve (AUC) on the ASPI Test was 5 U, consistent with an ultra-responsiveness to ASA, with normal platelet aggregation induced by other agonists on the ADP Test and TRAP Test  was started, with blood pressure normalization. MultiplateRAP Test .*-weighted sequences revealed two small roundish lesions, in the anatomical site of bilateral subcortical hematomas, with minute central nucleus of methemoglobin and dark hemosiderin rim, and without surrounding edema, consistent with cavernous venous malformations (Neurological examination of patients at discharge was completely normalized. Due to the “atypical” locations of intraparenchymal hematomas, we performed a brain MRI at longitudinal follow-up in order to exclude non-hypertensive causes of bleeding. Gradient-echo T2rmations . Multiplrmations . On diffWe presented a case of a previous healthy patient, admitted for intracerebral atypical hemorrhages, taking no medications except ASA in primary prevention. Diagnostic workup revealed a condition of unrecognized arterial hypertension, and the presence of multiple intracerebral cavernous venous malformations, some of which with acute bleeding. Symptomatic hemorrhagic complication occurs as a clinical manifestation of cavernous angioma in 25% of cases , but theThe role of antiplatelet agents for the primary prevention of cardiovascular disease, including stroke, is still widely debated, due to the delicate balance between efficacy and safety in patients without established previous vascular events. Several randomized clinical trials showed that Aspirin is effective in the reduction of recurrence risk, with a tolerable increase of bleeding complications; thus, international practice guidelines strongly recommended ASA in secondary prevention of vascular diseases, as ischemic stroke or myocardial infarction . Regardi®, might supply helpful information to clinicians, primarily to assess the responsiveness to ASA or Clopidogrel in ischemic stroke patients. Nevertheless, it might be a valid tool in the stratification of patient's risk profile as well, while considering the safety of a primary prevention regimen, particularly in the presence of clinical conditions associated with an increased risk of hemorrhagic complications.The possibility to test the biological effectiveness of antiplatelet agents, with platelet function testing devices such as Multiplate AnalyzerHowever, longitudinal studies are needed to assess whether aggregometry might supply individualized information and whether it can be considered a valid tool in the development of tailored therapies, as the main limitation of its implementation in everyday clinical practice.Our report illustrates the potential clinical benefit of platelet function testing in patients undergoing antiplatelet therapy, with particularly useful application in the definition of patient's risk profile in case of primary prevention treatment with Aspirin. However, RCTs and longitudinal studies are needed to assess whether routine platelet function monitoring might be considered a decision-making tool for clinicians, both in patients with vascular diseases subjected to secondary prevention therapy and during the evaluation of safety profile of antiplatelet treatment in selected patients deserving of pharmacological primary prevention therapy.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Written informed consent was obtained from the individual for the publication of any potentially identifiable images or data included in this article.FR, GB, and AZ: manuscript drafting/revising, data acquisition, and final revision. NL: data acquisition. LV: manuscript revising and data acquisition. FN: manuscript revising. LC: manuscript revising and data acquisition. SM: manuscript drafting/revising and final revision. All authors approved the final version.AZ has received funding for speaker honoraria and consulting fees from Boehringer-Ingelheim and speaker honoraria from Cerenovus. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


This rapidly developing technology has had a significant impact on cardiovascular research, its clinical translation and education. It has expanded our understanding of the cardiovascular system resulting in better devices, tools and consequently improved patient outcomes. This review discusses the latest developments and future directions of generating medical replicas (‘phantoms’) for use in the cardiovascular field, detailing the end-to-end process from medical imaging to capture structures of interest, to production and use of 3D printed models. We provide comparisons of available imaging modalities and overview of segmentation and post-processing techniques to process images for printing, detailed exploration of latest 3D printing methods and materials, and a comprehensive, up-to-date review of milestone applications and their impact within the cardiovascular domain across research, clinical use and education. We then provide an in-depth exploration of future technologies and innovations around these methods, capturing opportunities and emerging directions across increasingly realistic representations, bioprinting and tissue engineering, and complementary virtual and mixed reality solutions. The next generation of 3D printing techniques allow patient-specific models that are increasingly realistic, replicating properties, anatomy and function. Commercially available 3D printing services100Clinical diagnosis has increasingly shifted from being invasive towards being informative. Advances in medical imaging have played a pivotal role in cardiovascular care to date, providing indispensable support in clinical settings. Yet, imaging modalities remain limited due to the lack of dynamic interaction, tactile feedback and layered structural information even when multiple views and color overlays are available. 3D printing enables physical manufacturing or printing of 3D replicas of the imaged tissue in a layer-by-layer fashion, replicating structure, behavior and even function.3D printing, also called additive manufacturing (AM), traditionally refers to the layer-by-layer build-up of non-biological material such as laser-solidified polymers or alloys.154In this review, we first introduce the general concepts and associated processes involved in 3D printing from cardiovascular imaging to phantom production. Specifically, we collated detailed information and developed significant reader guidance on all aspects of associated workflows, including image acquisition, segmentation, and registration options. We then explain and compare additive manufacturing technologies, before discussing milestone applications in the cardiovascular field across education, research and clinical practice. Then we explore promising future developments in 3D printing, and its related and competing technologies of tissue engineering, focusing on vascular bioprinting, and virtual and mixed realities leading to an overall outlook of the field and its remaining challenges.The overall workflow for cardiovascular medical image processing is shown in Fig. The required medical imaging dataset must be volumetric, which limits cardiovascular imaging modalities to 3D echocardiography, electrocardiography-gated computed tomography (ECG-gated CT), and cardiac magnetic resonance (CMR).Volumetric 3D echocardiography, either transthoracic (TTE) or transoesophageal (TEE), is a convenient modality as it is generally widely available, cost-effective and has no risk of radiation. However, as it is based on ultrasound, it is only suitable for large and clear structures, such as ventricular chambers and valve leaflets.For this reason, ECG-gated CT is the principal cardiovascular imaging modality today, providing sub-millimeter resolution and clear tissue structures such as calcium deposition. CT is able to image patients with pacemakers, pacemaker wires and general metal implants, making it a common imaging method before surgical or other structural interventions. It is therefore commonly used for idealized, patient-specific,96CMR or magnetic resonance angiography (MRA) is limited due to its comparatively lower spatial resolution (up to 1.2 mm), making it not suitable for small-scale structures such as the coronary arteries or morphological heart valve features.For all of these modalities, the derived images and hence the final phantom quality depend on sufficient signal intensity and tissue contrast whilst enabling minimal image artefacts. Thus, both the cardiac movement and breathing artefacts challenge image acquisition, and compensating techniques such as gating and breath-holding may be incorporated Table .Table 1OThe medial images acquired are 2D image stacks representing the 3D volume, requiring processing so that the region of interest within each of the images can be discriminated against the surrounding tissue. After, the 2D stacked contours require further reconstruction into a 3D representation.Several cardiovascular image segmentation and reconstruction tools exist today with varying applicability, advantages and disadvantages, including open-source software such as Seg3D, 3DSlicer, InVesalius, ITK-Snap, Osirix Lite, Horos, ImageJ, Blender, Medical Imaging Interaction Toolkit (MITK), and Vascular Modeling Toolkit (VMTK) along with popular proprietary software such as Amira, Mimics , and clinical software such as Vitrea , Intuition (Tera Recon), AW Volumeshare  Table .Table 2OSegmentation processes can be automated using centerline and boundary extraction methods, or manually using editing, extraction and cropping, or most commonly in a semi-automated workflow whereby initial automated segmentation is followed by manual expert correction.After relevant contours of the 2D regions of interest are marked or segmented, the perpendicular surface contours can then be reconstructed to form a surface using a number of available iso-surface extraction algorithms. Marching cubesin vitro mock loop compatibility. A major disadvantage of these tools is the required software expertise and challenging learning curve. The combination of these steps often requires a significant amount of learning and expertise, and thus offers opportunities to increase user-friendliness of relevant software.After segmentation, the now virtual structures require further processing for mesh smoothing, editing and improving these surface representations further. Powerful open-source tools exist for this purpose, including VMTKOnce a virtual model is created, various methods can be used to print 3D phantoms. 3D printing with a range of materials including polymers, metals or alloys has been used since the 1970s and has become a well established technology, which is increasingly affordable, convenient, and accurate in resolution and complexity.in vitro experiments respectively,51Initially, phantoms were produced by conventional methods such as casting and molding. This approach is predominantly injection molding-based where a large range of materials are available including PMDS, MoldStar 15, EcoFlex 00-30 and DragonSkin. Such materials including PDMS (poly(dimethylsiloxane)) SYLGARD elastomers often involve individualized or patient-specific phantoms. These models can be transparent, which offers opportunities for a range of functional flow assessments including Particle Image Velocimetry (PIV).Unlike casting or molding, 3D printing is fundamentally a layer-by-layer build-up technique that relies on solidification of the material between each layer. 3D printing technologies can overcome some or all of the drawbacks of casting and molding, offering patient-specific, fast and precise production of phantoms at a low cost by eliminating the need for tooling. Various 3D printing technologies are available today and an overview is provided in Fig. 3D printing for cardiovascular applications is challenging, and suitable methods should be selected depending upon many factors including resolution, materials, the required physical properties such as complexity, color/transparency, durability, biocompatibility, cost and recyclability. Similarly, post-printing requirements are an important consideration especially for some anatomies (such as vessels), which may include cleaning, removal of support materials, ultraviolet curing, sterilization and labelling.SLA uses photopolymeric resin which is solidified using a digitally guided ultraviolet laser (or sometimes a visible light source). It can achieve smallest minimum feature sizes and very high resolution, however it incurs a high cost and has a limited ability for printing complex features as often required for cardiovascular research purposes. It is capable of printing translucent prints relevant for some functional modeling.41MJ for PolyJet/MultiJet or Material Jetting refers to processes similar to SLA with orifice jetting of both a photopolymer for the actual model (solidifies through light exposure as before) and a photo-curable gel  has thus far been the procedure most intuitively suited to 3D printing from both a device and procedural planning perspective due to the variability in patient anatomy and thus the importance of appropriate device selection and sizing. With 3D printing et al.in vitro after trans-catheter aortic valve replacement (TAVR), a widely performed structural heart intervention. Other patient-specific risks of complications could potentially be minimized or even eliminated using 3D printed procedural planning, with coronary occlusion being retrospectively predicted using a 3D TAVR case model following a fatal complication.Qian A full 3D heart model was used for percutaneous structural intervention,Interventional pre-operative planning and simulations through congenital heart models have been accomplished in scenarios ranging from double outlet right ventricle,7Comparatively, the “bench-testing”, for example of coronary artery stenting has been less developed due to the use of idealized models which do not incorporate disease and use a single material,In all these scenarios, the use of such technology in procedural planning may result in reduced operating time and thus may lead to more effective cost, time and resource management and ultimately may improve treatment success rates.8Most medical phantoms are fabricated from polymeric materials with uniform tensile properties and with similar behaviors to human soft tissue when under small strain  proteins or even living cells. Whilst cardiovascular 3D bioprinting and molecular 3D printing hold revolutionary potential, they are currently not translated into clinical practice. Many challenges remain in the field of biofabrication of vascular tissues, and some of these challenges have been highlighted elsewhere.103The ultimate goal of cellularized fabricated constructs is to capture the physiology of the complex native vasculature and to enable the investigation of living processes such as basic cellular function, vessel remodeling, or even pathological pathways. The application of 3D biofabrication to the vasculature has recently been reviewed.128in vitro arteriesin vitro stent deployment in an artery containing endothelial and smooth muscle cells, allowing cellular responses to the stenting procedure to be monitored at high resolution in real time.6Biofabrication and vascular tissue engineering pose specific challenges, including dimensions and scaling, that constrain the possible technical approaches used in their production. For example, the vasculature is a hierarchical tree of vessels that spans a broad range of dimensions from several centimeters in the case of large blood vessels to a few micrometers for capillaries. Therefore, spatial control at both the macro- and microscopic scales is needed during biofabrication. Additionally, the cardiovascular tree is a large but sparse network and is thus different from small dense organs, making it harder to fabricate as a bulk material. Furthermore, the vascular wall is a heterogeneous structure that consists of a complex mix of ECM proteins as a fibrous scaffold, specialized cells with specific spatial localization, and, in the case of larger vessels, intervening structures such as the internal and external elastic laminae. Additionally, in large arteries, the outer layers of the wall have their own complex vascular network, the vaso vasora. Two additional critical constraints are cell viability and sufficiently fine spatial control. Most of the traditional fabrication technologies introduced before have been adapted for biological fabrication, aiming to meet these constraints.Beyond research applications, the ultimate goal of biofabrication is to produce constructs with translational potential in order to improve patients’ lives. The need to implant a tissue in a patient imposes significant demands on the fabrication process in terms of durability, robustness, and integration into surrounding tissues. Once implanted, artificial vessels will be subjected to prominent environmental cues that can induce remodeling and can possibly accelerate the deterioration of the construct. Therefore, engineered vessels need to be able to not only withstand but also adapt to the dynamic chemical and mechanical environment in which they will reside. Finally, to allow transplantation, immunological matching is necessary: using patient-derived induced pluripotent stem cells (iPSCs) promises to ensure this biocompatibility, and several recent studies have focused on using these cells. More broadly, iPSCs are also very useful for research applications as they allow the study of patient-to-patient variability as well as exploration of the mechanisms governing genetic diseases.in vivo values and permits the quick fabrication of larger structures.142To overcome the challenges outlined above, many technical innovations have recently emerged. We review here the most promising such innovations. The use of organoids as building blocks in the bio-ink increases cell density to better match In all of these novel approaches, the question of perfusion of large engineered tissues will likely be front and center. This perfusion is essential not only for ensuring the long-term viability of the tissue but also for providing the relevant mechanical forces which constitute important cues for remodeling and stabilization of immature vessels.64A field that will undoubtedly receive significant attention in tissue engineering in the coming years is the use of embedded sensors that will continuously monitor the structural and functional state of the engineered tissue. As in the rest of society, this push towards “smart” engineered vessels holds the tantalizing promise of providing highly personalized monitoring and tremendous amounts of data that can be mined using modern artificial intelligence algorithms. The push towards smart and communicating implantable vascular constructs raises considerable privacy concerns and ethical considerations that need to be addressed before such an approach becomes widely adopted.Whilst these novel virtual and mixed reality technologies may approximate the utility of 3D printing in education, the tangible nature of 3D printing is both intuitive and naturalistic. No training is required to hold and rotate a printed model and, as 3D printing is a more accessible technology today, it may sufficiently fulfill the main educational goals of tactile and anatomical understanding.Beyond 3D printing and physical replicas, new technologies are emerging, whereby a virtual experience is created to replace physical phantoms for training and preoperative planning. Whilst this technology is very much in its early stages for incorporation into standard clinical use, some steps towards such vision have been made in the cardiovascular context.Two of the key technologies revolutionizing this domain are Virtual Reality (VR) and mixed reality experiences. VR is a fully-immersive experience that simulates virtual experiences by controlling the user’s complete visual and auditory senses.20For the cardiovascular field, a VR simulator was developed to train intervention,Alongside medical training, preoperative preparation with holographic models may be 1 day as effective as physical models.82The improvement of visual representations of patient CT or MRI scan data used in VR and mixed reality simulations is also being explored with cinematic rendering as opposed to the traditional volume rendering technique used for post-processing. Cinematic rendering produces photo-realistic images and was found to provide greater depth perception and spatial impression in comparison to volume rendering, however at the cost of requiring significantly greater computing power.124Ultimately, mixed reality technology may replace fluoroscopy during endovascular interventions like stenting and thus eliminate radiation exposure to the patient and surgical staff.A further application incorporates the recent advancement of the digital twin in cardiovascular medicine.Whilst 3D printing is not a new industry, it is a multi-versatile tool which offers widespread impact across many different cardiovascular fields. This review gives a unique perspective on how 3D printing technologies can and will bridge the gap between engineering tools and the future of cardiovascular care.We outlined 3D printing workflows and elaborated on each step from image acquisition to processing and virtualization before introducing 3D printing technologies across non-biological and biological approaches. We then highlighted state-of-the-art work in the cardiovascular field from recent years, ranging from research, to clinical and educational efforts, pushing the boundaries of knowledge and know-how. Promising trends, complimentary technologies and future applications were also discussed in this context, providing a general outlook of the field’s directions over the next decade.in vitro research efforts, clinical practice and education with a global push for open source virtual anatomy libraries, increasingly user-friendly image processing tools and the drive for high-precision printers to be integrated into practice with economic demands of cost and physical space. Together with emerging efforts towards more realistic mechanical behavior, this offers many opportunities for rapid on-site personalized surgery planning, promises to minimize animal testing of novel cardiovascular devices in the future and may become a new standard as a readily available educational tool.Overall, 3D non-biological printing is likely to continue to rapidly penetrate Cardiovascular 3D biofabrication and tissue engineering remain comparatively more removed from clinical translation today but promise pivotal impact in the coming years by combining advanced manufacturing, cell biology, molecular biomarkers, and materials sciences. The most immediate impact is likely to be in the emerging field of vessels-on-chip for toxicity testing of cardiovascular drugs together with cellular response monitoring. Although the tantalizing prospect of whole organ printing and implantation into patients remains some time away, these approaches are already proving helpful by providing unique insight into biological mechanisms under controlled conditions, therefore yielding a platform for limited and initial device testing today.Complimenting virtual and mixed reality technologies may replace the need for some 3D printing in the future, including demonstration, education and surgical planning, especially once tactile feedback has been optimized, likely forming the pathway of assisted surgery in future. This may offer some benefits in terms of 3D printing associated material cost, increase sustainability and offer additional in-depth experiences such as anatomy virtual fly-through and scaling. Potentially it could replace 3D printing for some aspects of medical planning, education and communication purposes in the near future. However, it is not capable of replacing device testing platforms and means of functional research. Ultimately 3D printing has and will continue to significantly improve training, research and communication in the cardiovascular field with related emerging technologies increasingly taking-up complimentary functions. This will elevate the rapid development towards total patient-specific cardiovascular care in the near and far future, assisting the accomplishment of previously unmet clinical outcome success rates.


Drosophila, the major components of the Hippo pathway including the core kinase cascade, downstream effector and nuclear transcription factor are evolutionarily conserved, resulting in extensive studies from many investigators in the past decades. The Hippo pathway is now recognized as a key player in organ size control via primarily stimulating programmed cell death and restricting cell proliferation  functions as an essential intracellular messenger in a number of cellular signaling events  in this process. Uncovering additional effectors for Ca2+ signaling with relevance to the Hippo pathway will enrich our understanding of the Hippo pathway regulation from a new perspective.Calcium  (MAP3K13 in human) as a new player in the cell polarity-mediated Hippo pathway regulation. Wnd is involved in this process through nemo-like kinase, Nmo (NLK in human), and such Wnd-Nmo axis in regulating the Hippo pathway is shown conserved in evolution. Thus, it will be interesting to further examine whether dysregulation of these newly discovered regulators would cause cell polarity-associated human diseases like cancer.The tissue architecture-related cell polarity, cell-cell junctions and cell-extracellular matrix (ECM) interaction are known regulators of the Hippo pathway. Using Cho and Jiang authoritatively highlighted the recent findings of such “non-canonical” regulation for YAP and TAZ. These mechanisms highlight the complex regulation of the Hippo pathway, connect the Hippo pathway with other key cellular signaling events, and reveal novel therapeutic strategies for future investigations.Although the Hippo pathway kinase cascade transduces many upstream signaling events to YAP and TAZ, mechanisms that modulate YAP and TAZ activities independent of the Hippo kinase cascade do exist. In this Research Topic issue, Pipchuk and Yang described the application of luciferase-based biosensors to the Hippo pathway study. The authors discussed several assays using split luciferase complementation systems that have been successfully used for identifying new Hippo pathway regulators, highlighting the advantage of this luciferase-based biosensor method in studying real-time protein-protein interaction in live cells.To resolve the complex regulation of the Hippo pathway, extensive efforts have been made to identify new Hippo pathway regulators. In line with it, Zhao et al. reviewed the emerging findings of the Hippo pathway in neural crest (NC) development. This work summarized the critical role of the Hippo pathway in many aspects of NC development, including NC initiation, migration, proliferation, survival and differentiation, as well as the NC-related diseases caused by the dysregulated Hippo signaling. In terms with organogenesis. Wu et al. described how the Hippo pathway acts in pancreatic development. This review highlighted the role of the Hippo pathway in progenitor cell maintenance and normal proper cell polarization/branching during pancreatic organogenesis and morphogenesis, which involve the crosstalk with several key developmental signaling pathways, such as Notch, Wnt, and PI3K-Akt.Although the Hippo pathway is known for its crucial role in organ size control, recent studies have extended it to other biological processes, such as embryogenesis, stem cell regulation and tissue regeneration. Here, Shen et al. investigated the Hippo pathway effector TAZ in breast cancer stem cells (BCSCs) and revealed the Cyclin D1-CDK4/CDK6 axis as downstream effector for the TAZ-driven breast tumorigenesis. This work suggests a possible vulnerability for BCSCs as well as a new opportunity for breast cancer therapy.Given the critical role of the Hippo pathway in numerous physiological processes, its dysregulation has been linked to many human diseases such as cancer. For example, the Hippo pathway is implicated to control cancer stem cell expansion and maintenance against cancer development -mutated low-grade glioma (LGG). Their findings confirmed the hypermethylation of LATS2 promoter in IDH1/2-mutated LGG but failed to link this genomic alteration with YAP activation experimentally. Thus, the role of the LATS2 hypermethylation in LGG development deserves further investigation.By examining The Cancer Genome Atlas (TCGA), Studies from multiple model systems have fully established the Hippo pathway as an evolutionarily conserved signaling cascade from unicellular organisms to humans (Sebe-Pedros et al., ZZ, ZY, WH, and WW wrote the Editorial. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.