The complete polarization state of second harmonic (SH) light was measured and characterized by collagen type I and skeletal muscle fiber using a Stokes vector-based SHG microscope. The polarization states of the SH signal are analyzed in a pixel-by-pixel manner and displayed through two dimensional (2D) Stokes vector images. Various polarization parameters are reconstructed using Stokes values to quantify the polarization properties of SH light. Also, the measurements are extended for different input polarization states to investigate the molecular structure of second harmonic generation (SHG) active molecules such as collagen type I and myosin. The second harmonic generation (SHG) is a second-order nonlinear coherent optical process via virtual state transitions, widely used for imaging non-centrosymmetric molecules , 2. The The SHG signal from myosin and collagen is due to the protein crystal structure, mainly amino acids present in them . Again, Again, a skeletal muscle fiber is formed by the spatial arrangement of myofibrils which are composed of myosin and actin filaments. These myofilaments form a cross-striated-like structure called sarcomere, which is a repeating unit of Z lines. Sarcomeres are connected serially, of length ranges from 2 to 3 μm, and the myofibrils are arranged depending on the type of muscle , 37. Z lIn general, Jones calculus or Stokes algebra is used for characterizing polarization light; however, only Stokes algebra is suitable for all states of polarization, partially or unpolarized , 43, whe0 and 900 polarization states of laser light. The laser beam is scanned with 50 to 50 μm scanning area to the samples mounted on an XY stage and focused by a × 40 objective lens  [see Fig. XYZ, where laser propagates in the Z-direction and samples are scanned in XY-plane, where X is horizontal and Y is vertical axis. SHG intensity is measured as a function of the relative angles between laser beam polarization and orientation of SHG-active molecule. The polarization state of laser is rotating at an angle with respect to the samples (X-axis). In our experiment, collagen fiber and skeletal muscle are thick and arbitrary oriented and hence we assumed them to be parallel to XY-plane. The forward directed SH signal is collected using a × 20 objective lens . A 400 ± 40 nm  bandpass filter along with a 680-nm short pass (Semrock) filter was also inserted into the SHG emission path for SH signal collection. The SH light is passed through a polarization state analyzer (PSA) to reconstruct the Stokes parameters [out” is measured from the four intensity images, I, using the equation: Sout = (A4 × 4)−1 ⋅ I. MATLAB  program is used to reconstruct the 2D Stokes vector and various polarization parameters [0 and 900 polarization states of laser light. The tail of the adult mouse was removed and immediately incubated in liquid nitrogen. The tail was dissected and individual fibers were extracted by microdissection. The imaging experiment was performed immediately after fiber extraction.A four-channel Stokes polarimeter was built and integrated with a SHG microscope. The experimental arrangement is described in detail in , 36, 49.see Fig.  of workirameters . The SH r, as was derived from the four Stokes parameters from the samples, shown in Figs. Stokes parameters of SH signal from collagen type I and skeletal muscle fiber are measured with various input laser polarization states to investigate the alignment and orientation of fibers. Polarization microscopy improves the image contrast of sarcomeres in myofibrils due to the linear birefringence; the origin of SHG contrast arises from a quadratic dependence on the SHG active protein concentration. Previously, it was reported that SH contrast is enhanced with selective arrangements of collagen type I and muscle fibers , 50. Fig0 polarized excitation than the 900 polarized excitation, which is due to the strong SH signal dependency on the direction of the input polarization. DOCP values also varied significantly for two different excitation polarization since the structural arrangement of sarcomere bands are different. Furthermore, the molecular arrangement of microfibrils in skeletal muscle is measured by anisotropy “r” and ranges from − 0.5 to 1. The anisotropy images show that molecules are better aligned 900 since the “r” value is approximately − 0.5 when 900 polarized incident light. However, in the case of 00 polarized excitation light, the anisotropy value is approximately 0. Thus, it is clearly shown that the SH signal varies significantly with different excitation polarization angles. Figure 0 and 900 polarized, respectively. The SHG images are acquired at a depth of 20 μm. Stokes parameters are varied with different input polarization states which are due to the arrangement of the optical axis in collagen fibers. The polarization parameters are analyzed in each pixel of the image; the DOP value is approximately 1 indicating the SH signal is fully polarized. The DOLP values show the alignment of fiber and molecules parallel to the vertically/horizontal polarization state of the input laser beam. DOCP determines how the coil-like structure of collagen type I changes the relative polarization between the incident fundamental and SH signal. The anisotropy measurements of the SHG signal exploit the effect of birefringence, which is due to the sample heterogeneity in the Z propagation direction from collagen type I.Figure 0 polarizations in both the samples, which indicates that SH active molecules are parallel to the 900 laser polarization. The technique has the potential in the field of biomedical applications where the structure or orientation of molecules is a key factor including the progress of wound healing and tissue morphology.We discuss a Stokes polarimeter in combination with SHG microscopy to fully characterize the polarization dependence of SH signal. The polarization states of SH signal are measured, analyzed in each pixel of the 2D images, and displayed through Stokes vectors. Various polarization parameters are reconstructed using Stokes values to quantify the polarization properties of SH light. The Stokes vector-based analysis demonstrated that molecular arrangements in collagen type I and skeletal muscle fiber are different with high spatial resolution. The effect of birefringence and crystal orientation of skeletal muscle fiber and collagen type I are visualized from the polarization parameters. We observed that DOP is approximately unity at 90The developed four-channel-based Stokes vector-based SHG microscopy measures the full Stokes parameters of the SHG light in transmission geometry from thin samples such as starch, collagen, and skeletal muscle fiber. In case of thick tissue samples, the penetration depth of laser is limited and SHG light intensity is weak and therefore cannot be collected in the forward direction. In the case of in vivo imaging, SHG light is generated from the surface of the image plane and detected in epi-detection. The Stokes vector-based measurement can be implemented in an epi-detection mode for in vivo imaging with a slight modification of optical design. The Stokes polarimeter should be placed in epi-detection for SHG signal collection although fiber optics-based endoscopy integrated with SHG microscopy is demonstrated for clinical applications , 52.


CBFB-MYH11, belongs to the favorable-risk category. However, even if most patients obtain morphological complete remission after induction, approximately 30% of cases eventually relapse. While well-established clinical features and concomitant cytogenetic/molecular lesions have been recognized to be relevant to predict prognosis at disease onset, the independent prognostic impact of measurable residual disease (MRD) monitoring by quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR), mainly in predicting relapse, actually supersedes other prognostic factors. Although the ELN Working Party recently indicated that patients affected with CBFB-MYH11 AML should have MRD assessment at informative clinical timepoints, at least after two cycles of intensive chemotherapy and after the end of treatment, several controversies could be raised, especially on the frequency of subsequent serial monitoring, the most significant MRD thresholds (most commonly 0.1%) and on the best source to be analyzed, namely, bone marrow or peripheral blood samples. Moreover, persisting low-level MRD positivity at the end of treatment is relatively common and not predictive of relapse, provided that transcript levels remain stably below specific thresholds. Rising MRD levels suggestive of molecular relapse/progression should thus be confirmed in subsequent samples. Further prospective studies would be required to optimize post-remission monitoring and to define effective MRD-based therapeutic strategies.Acute myeloid leukemia (AML) carrying inv(16)/t, resulting in fusion transcript  RUNX1/RUNX1T1, while inv(16), or with significantly lower incidence t, leads to the fusion of the CBFB gene with MYH11, the smooth muscle myosin heavy chain gene, resulting in the chimeric CBFB-MYH11 gene, which occurs in approximately 8% of adults with de novo AML [The latest 2017 European Leukemia Net (ELN) recommendations for the diagnosis and management of acute myeloid leukemia (AML) in adults , containnovo AML .According to 2017 ELN risk stratification . NeverthThe effects on the long-term outcome of secondary cytogenetic abnormalities, detected in approximately 40% of inv(16) AML patients ,8, and aIn the last decades, probably depending on the accessibility of increasingly sensitive biomolecular tools, the focus has shifted to disease evaluation in terms of the dynamic quantitative assessment of molecular measurable residual disease (MRD). Indeed, the changing of MRD levels throughout cycles of therapy, in particular the reduction at a specific timepoint compared to pre-treatment baseline levels, has proved to be the most useful independent prognostic variable for survival, allowing one to identify patients at high risk of relapse, as possible candidates for more intensive therapeutic approaches, including allogeneic hematopoietic stem cells transplantation .CBFB-MYH11, with a special focus on the role of MRD monitoring in risk stratification and treatment guidance. The purpose of this manuscript is to offer an overview on the most prognostic factors affecting the clinical outcomes of patients with AML harboring In general, AML with inv16)(p13.1q22) or t, hereafter referred as inv(16), exhibits some peculiarities, which also differentiate it when compared to AML with t8;21), abbreviated t [ mutations [As reported in several studies, WBC counts and parallel bone marrow/peripheral blood (BM/PB) blast percentages are higher in inv16) than t8;21) AML, at diagnosis [ than t is less well concerted , whose  CBF AML ,28), or ometimes  OS, mainutations ,32,41,46oncerted . Differeat onset , assistipse rate ,49, rathpse rate  investign et al.  In the tn et al.  illustran et al.  documentostaurin . Howeverostaurin . MoreoveRAS genes, mainly NRAS, are observed in 17–53% of inv(16) AML, i.e., more frequently than in t AML [NRAS mutation. RAS variants, belonging to activating mutations, lead to uncontrolled proliferation, as shown by the correlation with higher WBC [In second place for incidence, mutations in the ;21) AML ,15,16,19;21) AML , suggestgher WBC .FLT3 is a member of class III tyrosine kinase receptors, and its mutations are relatively rare in CBF AML. While internal tandem duplication (ITD) is the most frequent in cytogenetically normal AML, holding an unfavorable prognostic impact depending on allelic burden, FLT3 tyrosine kinase domain (TKD) point mutations represent the most common alteration, in inv(16) AML, being associated with BM blast percentage [FLT3 mutations, because of their negative impact on relapse risk, according to some authors [FLT3-TKD high allelic variant on OS.Likewise, n, in inv AML, bein, in inv AML, bei authors , whereas authors  showed aCBFB-MYH11 promotes leukemogenesis without the need for increasing evolutionary advantage. Remarkably, recent interest is growing about the mutational landscape associated with the rearrangements of the CBF transcriptional complex. Beside the founding mutation, cooperating molecular events contribute to define a distinct gene expression profile of the specific CBF subtype . In inv AML, with an incidence increasing with age . In CBFB AML, witat onset ,30, whilat onset ,30. The at onset , accordiat onset . Recentlat onset  have retat onset , such asat onset . In the CBFB-MYH11 AML will experience relapse in nearly 30% of patients, with an estimated RFS rate of 42%, following standard care. The current definition of morphological response lacks sensitivity, and the purpose has been to more accurately detect the residual leukemic burden in BM, far below the 5% blast percentage detectable on microscopic examination [−3–10−5 [−6, quantitative reverse transcriptase PCR (qRT-PCR) is currently considered the gold standard in detecting MRD, applicable in 50–60% of AML patients showing distinct molecular lesions. The ELN MRD working party afforded clinical issues in the application of MRD monitoring in AML by updating recommendations with concepts of complete molecular remission, molecular relapse, and molecular progression [KIT and FLT3-ITD in CBF AML [Traditionally, the determinant indicators of prognosis in AML have been identified in the pre-treatment features described above, related to either the patient or the disease features. Another relevant factor impacting on prognosis is represented by the response to cytoreductive therapies, which direct subsequent disease management. Over the last two decades, efforts have been made to improve the assessment of disease response and, especially, the monitoring over time, because although being classified as a favorable risk category, mination ,58, by dmination . Availabmination ,61. Furtmination ,63. As wmination ,65, quanmination . Submicr[−3–10−5 . Furthergression . Interes CBF AML ,70. To n CBF AML . Indeed, CBF AML ,73. CBFB-MYH11 AML. Nevertheless, the lack of technique standardization and heterogeneity of available data results in non-firmly conclusive implications for clinicians, mainly regarding the indication to assign patients to intensive therapeutic approaches. In The above-mentioned ELN consensus document recommends timepoints and tools for MRD assessment in CBFB-MYH11 fusion transcripts are detected by quantitative molecular methods and normalized to endogenous reference genes. In general, no significant differences in outcomes are observed, depending on the level of transcript at diagnosis [In all the studies reported in iagnosis ,75,80,82iagnosis . Marcucciagnosis , as welliagnosis , describiagnosis ,81. Conviagnosis  brought The earlier those patients at higher risk of disease relapse are identified, the better diversified therapeutic approaches may be engaged for them. Therefore, several groups have investigated which could be the most relevant timepoint for prognosis during treatment. Some studies proposed a threshold of copy number for discriminating subgroups with shorter remission  [10/month, so that a 3-monthly assessment schedule might fail to identify a potential MRD increase up to 3 logs, impairing potential pre-emptive therapeutic strategies, including SCT [Once treatments are completed, for patients not candidate to further therapy lines, it will be necessary to set up, on a medium-long-term basis, a reliable follow-up in tracking either an ongoing response or, promptly, an impeding relapse. Even at this stage, a consensus in monitoring MRD kinetics emerges from published studies. In fact, relapse could effectively be predicted by a comparison between longitudinal sampling, rather than by overcoming a definite threshold. ELN updated recommendations reflect such an awareness: molecular relapse, as well as molecular progression, is defined by a logarithmic increase in MRD levels between two consecutive samples, underlining the dynamic interpretation of a molecular parameter. In addition, this approach makes data more comparable, despite methodological and clinical differences among studies. The use of a logarithmic increase for defining relapse and progression is therefore operational in CBF AML, because of the predominantly slow pattern of regrowth, depending on biological factors, which allows one to observe evolutive trends before morphological relapse. Some concordance exists in the proposed patterns of molecular values: as a reduction of 2–3 log after induction/consolidation ,78 is ex months) , suggest months) . Relevan months)  reportedding SCT . Puckrinding SCT  emphasizNPM1-mutated AML, in which discrimination for survival was better by using PB rather than BM [Whether PB sampling can definitely succeed in replacing bone marrow sampling for MRD testing remains an open point. Sensitive and reliable blood-based assays would be an attractive possibility, given the less invasive nature compared to BM aspirations. The two sources have been studied as alternatives in several studies, albeit the most clinically relevant findings are derived from BM specimens. As detailed in  than BM . Boeckx  than BM  reported than BM , who dem than BM , who pro than BM  revealed than BM  recommen than BM  offered 10 between BM and PB. Furthermore, additional information from PB with respect to BM could be provided, by gene expression assays and combined multiparameter flow cytometry, able to detect with more specificity, even though with less sensitivity, potential circulating leukemic cells, due to the background of less progenitor populations compared to bone marrow [Recently, Skou et al.  reinforce marrow .10 between two positive samples, confirmed by paired BM and PM assay, after 4 weeks [Hence, not only a comparable but also a better disease surveillance could be assured by monthly PB sampling, which allowed us to efficiently identify molecular relapse, as an increase by 1 log 4 weeks . p = 0.6408), due to the rapid and early tapering of immunosuppression therapy after transplantation and prophylactic donor lymphocyte infusion.Regarding the transplantation setting, although assessment is recommended before and after procedure, no specific indication is provided about the interpretation of MRD values. In consideration of the relatively satisfying long-term control of disease with repeated high or intermediate-dose cytarabine consolidation or alternatively autologous stem cell transplantation (auto-SCT), CBF AML are not usually candidated for allo-SCT in first CR, contemplating this procedure for patients in second remission , althougTo reiterate the relative significance of positive MRD pre SCT, Zhao et al.  reportedCBFB-MYH11 protein could act as neoantigen, giving rise to the potential development of a personalized adoptive TCR T-cell strategy. Because of the early and essential role in leukemogenesis and the specificity persistence in blast cells, CBFB-MYH11 should represent an optimal target, with minimal risk of off-tumor toxicity. Recent translational studies are providing the basis for future targeted therapeutic approaches. For example, advances in treatment would be warranted focusing on the molecular processes involved in leukemogenesis driven by fusion protein CBFB-SMMHC encoded by the CBFB-MYH11 founder gene, by the potential target of specific or indirect inhibitor [In recent years, the approval of several novel agents for treating AML was obtained; the most remarkable for improving outcomes in inv(16) is the addition of the anti-CD33 monoclonal antibody gemtuzumab ozogamicin (GO) to the remission induction cycle ,118,119.nhibitor , or explnhibitor . In recent years, the advances in molecular diagnostic and monitoring, with the simultaneous incoming of new therapeutic agents, have led to significant improvements in clinical AML management. Awada et al.  recentlyUltimately, further studies are needed to improve knowledge about the best employment of MRD information to improve the clinical outcomes of CBF AML patients.


Kumar summarizes the recently published literature on the AML blasts/LSC specific activated metabolic pathways and further discusses the potential therapeutic targets for the disease cure. Leukemia cells have the ability to reduce molecular oxygen utilizing electrons from carbon sources other than pyruvate as glutamine and fatty acids (FA) , acting as an important mediator of ROS  in solid tumors. It is also shown that KD can improve the response of PI3K inhibitor BKM120 in MLL-AF9 AML mouse model  in AML. This enzyme catalyzes the interconversion of acetoacetate and (R)-3-hydroxybutyrate, the two major ketone bodies produced during fatty acid catabolism. They show that low BDH1 expression predicts poor survival in AML and suggest a therapeutic potential in targeting BDH1 in the AML treatment. Recent insights into iron metabolism along with the recent discovery of ferroptosis have opened new avenues in the field of anti-tumor therapies , in the framework of precision oncology medicine and prospect how this would guide the development of new drug precisely targeting the pyrimidine metabolism in cancer.Cancer cell proliferation requires up-regulation and rewiring of metabolic pathways to feed anabolic cell growth. Oncogenic drivers directly and indirectly regulate metabolic pathways. Aberrant metabolism is central not only for leukemia cells proliferation and survival, but also mediates development of metastasis and resistance to therapies. It is now undeniable the relevant role of metabolic mechanisms in leukemogenesis with significant implications for the development of target therapies , 2. Depeids (FA) , 11. Hemids (FA) . Mitochor of ROS . These dr of ROS –15, inclr of ROS , 17 and r of ROS . Tucci ese model . Howeverherapies . Grignanherapies . Metabolherapies , 24. AmoNN and SH composed, edited, and finalized the editorial. EA and AV reviewed and finalized the editorial. All authors contributed to the article and approved the submitted version.This manuscript has been supported in part by Indian Council Medical Research (ICMR) grant 56/7/2019_hae-bms.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.


ATP) channels, plasma membrane depolarisation, Ca2+ influx, and finally the exocytosis of insulin granules. However, it has been demonstrated by the inactivation of this pathway using genetic and pharmacological means that closure of the KATP channel alone may not be sufficient to explain all β-cell responses to glucose elevation. We have previously proposed that NAADP-evoked Ca2+ release is an important step in stimulus-secretion coupling in pancreatic β-cells. Here we show using total internal reflection fluorescence (TIRF) microscopy that glucose as well as the Ca2+ mobilising messenger nicotinic acid adenine dinucleotide phosphate (NAADP), known to operate in β-cells, lead to highly localised elementary intracellular Ca2+ signals. These were found to be obscured by measurements of global Ca2+ signals and the action of powerful SERCA-based sequestration mechanisms at the endoplasmic reticulum (ER). Building on our previous work demonstrating that NAADP-evoked Ca2+ release is an important step in stimulus-secretion coupling in pancreatic β-cells, we provide here the first demonstration of elementary Ca2+ signals in response to NAADP, whose occurrence was previously suspected. Optical quantal analysis of these events reveals a unitary event amplitude equivalent to that of known elementary Ca2+ signalling events, inositol trisphosphate (IP3) receptor mediated blips, and ryanodine receptor mediated quarks. We propose that a mechanism based on these highly localised intracellular Ca2+ signalling events mediated by NAADP may initially operate in β-cells when they respond to elevations in blood glucose.Pancreatic β-cells release insulin upon a rise in blood glucose. The precise mechanisms of stimulus-secretion coupling, and its failure in Diabetes Mellitus Type 2, remain to be elucidated. The consensus model, as well as a class of currently prescribed anti-diabetic drugs, are based around the observation that glucose-evoked ATP production in β-cells leads to closure of cell membrane ATP-gated potassium (K ATP channel-mediated pathway2 is not new4. Possible mechanisms include (1) an amplification of the KATP channel dependent pathway which remains functionally silent until the latter has depolarised the membrane5,(2) an additional triggering pathway that converges with the KATP channel mediated pathway on membrane depolarisation, and (3) an independently functional pathway that may lead to insulin release in the absence of KATP channel involvement6. One potential trigger of stimulus-secretion coupling, which may be synergistic with, or independent of, KATP channel closure, is the local release of Ca2+ from intracellular stores7. Indeed, there has been some debate about the relative importance of extracellular Ca2+ influx versus release from intracellular stores during biphasic insulin secretion, with some proposing that for the first phase of insulin release, influx is dispensable8. In terms of subcellular localized Ca2+ signals, it was shown in the late 1970s using pyroantimonate precipitation that incubation of β-cells with high glucose led to a Ca2+ increase immediately beneath the cell membrane9. Levels of NAADP, a Ca2+ mobilizing intracellular messenger that releases Ca2+ from lysosomes and acidic organelles, have previously been shown to be elevated in pancreatic β-cells by increased extracellular concentrations of glucose during stimulus-secretion coupling11. Moreover, inhibition of NAADP signalling in β-cells suppresses Ca2+ spiking and membrane depolarization12. The possible role of small lysosomal Ca2+ stores in stimulus-secretion coupling7 warranted the investigation of Ca2+ signalling in β-cells at high spatial and temporal resolution. Thus the present study sought to characterise sub-membrane Ca2+ transients observed in β-cells loaded with fluo-4 in response to glucose and the membrane permeable form of the lysosomal Ca2+ mobilizing messenger, NAADP (NAADP-AM)13, using total internal reflection fluorescence (TIRF) microscopy14. Furthermore, we hypothesized that dissection of these Ca2+ transients under experimental conditions where globalized Ca2+ signals or ER-based Ca2+ sequestration15 were avoided, might reveal their substructure, akin to the sparks and puffs observed for IP3 and ryanodine receptors, respectively18.The idea that stimulus-secretion coupling involves mechanisms in addition to the K2+ signalling dynamics in pancreatic β-cells is their heterogeneity upon stimulation, with the recent proposal that some cells in the islet act as Ca2+ signalling hubs or pacemakers19, whilst others follow through gap-junctional or paracrine signalling mechanisms. However, heterogeneity is also seen in isolated cells. Indeed, Ca2+ responses to glucose in β-cells have been described as having a typical triphasic shape often seen in parallel with, and assumed to be the result of, simultaneously measured changes in membrane potential, which have a similar pattern and are in synchrony with the cytosolic Ca2+ increase15: Phase 0 consists of a ‘dip’, or initial decrease, in cytosolic free Ca2+ resulting from increased sarco-/endoplasmic reticulum Ca2+ ATPase (SERCA) activity transporting Ca2+ into the endoplasmic reticulum (ER) in response to the rising ATP concentration following glucose metabolism. Phase 1 constitutes a transient rise in Ca2+ associated with L-type Ca2+ channel activation and Ca2+-induced Ca2+ release (CICR) from intracellular stores. Phase 2 are Ca2+ oscillations superimposed on a steadily elevated plateau thought to be the result of Ca2+ influx through L-type channels. While this standardised model is useful; it has been demonstrated that β-cells display more complex responses to glucose and other stimuli such as GLP-1, insulin, and nutrients including amino acids21. A classification of primary human β-cell autocrine Ca2+ responses to insulin shows that β-cells display a variety of equally common but different Ca2+ signals and no clear ‘standard’ response22.A feature of Ca2+ transients evoked by 16.5 mM glucose in mouse primary pancreatic β-cells, measured using evanescent-wave TIRF microscopy. Recordings were made from 1,017 cells. Figure 2+ , ∼60% resembled the standardised triphasic profile, with the remaining responding cells described by 3 further classifications  resulted in similar Ca2+ responses to those observed with glucose described above. Ca2+ oscillations could be resolved that culminated in a raised plateau of elevated [Ca2+]i in 89% of cells , however, the two transients were largely superimposable   Fig. , Movie 22+ that can be seen in Fig. 28 or desensitization to signals such as NAADP29. To quantify the changes in frequency, the mean percentage of frames showing at least one spike (defined as an event 2 standard deviations above mean baseline ∆F/F0) was determined for the baseline (last 80–100 frames before stimulus) and after the addition of raised glucose or NAADP-AM. As illustrated in Fig. To resolve these individual events in more detail, a spark detection algorithm was used see  which al2+ signals in response to a high concentration of glucose (16.5 mM) after thapsigargin pre-incubation, this raises the question of whether similar responses could be evoked by a smaller increase in extracellular glucose without the use of thapsigargin. We examined whether a doubling of glucose from 3 to 6 mM  without thapsigargin pre-incubation, would still allow us to detect visible Ca2+ events with TIRF. We reasoned that at lower concentrations of glucose, less ATP would be produced, resulting in lower SERCA activity, which would otherwise obscure detection of local Ca2+ events. As shown in Fig. 2+ release events could be detected . This order of magnitude is comparable to optically-assessed Ca2+ blips through IP3 receptors (0.1 ∆F/F027) and the smallest imaged IP3-evoked Ca2+ signals in pancreatic acinar cells (< 0.1∆F/F030). To demonstrate the quantal nature of the Ca2+ responses at the level of the individual cell, responses from a representative cell cluster are shown in Fig. 2+ signalling event. These ROIs were then analysed for their mean amplitudes after subtraction of baseline fluorescence. Events were included if they lay above baseline mean + 2 SDs) of that particular cell. The frequency distribution displays modal behaviour as illustrated by the poly-Gaussian function fitted to it, and the putative unitary event amplitude for this cell cluster is 0.03 ∆F/F0.To analyse the nature of the elementary Ca2+ responses and a high level of spontaneous activity; supporting the notion of a β-cell’s ‘Ca2+ fingerprint’31. As β-cells are frequently examined in artificially amplified resting and active states during experiments32, we have shown here that stimulation with the high glucose concentrations often employed, leading to high SERCA activity33, obscures more subtle Ca2+ changes taking place within the cell. It is probable that multiple stores and channels play a part in this process34. The fact that the events occur after pre-incubation with thapsigargin, ruling out the ER as a source, are most striking when triggered with NAADP-AM which targets acidic stores, and are localised just beneath the membrane , is a strong suggestion that the source of these events are acidic Ca2+ storage organelles. However, further pharmacological studies are required to fully delineate the origin of these signals.The above experiments demonstrate that β-cells show great variability in their Ca37, and the present discovery of elementary Ca2+ release events in response to NAADP; it is likely that NAADP-evoked Ca2+ signals from acidic stores are built from elementary events via activation of TPCs. It was suggested in 1996 that NAADP-mediated Ca2+ release was quantal on the basis of its graded release from sea urchin homogenate38 and in intact sea urchin eggs localized Ca2+ responses ascribed to the osmotic lysis of acidic stores by GPN39; 25 years on, we may have evidence in a mammalian cell type that a similar principle operates. Thus with the existing sets of elementary Ca2+ signals: IP3, the IP3R, and blips; cADPR, the RyR, and quarks, respectively, elementary Ca2+ release events appear to be a governing principle of intracellular Ca2+ signalling, regardless of channels involved or Ca2+ storage organelle.With the discovery of two-pore channels (TPCs) as the putative target for NAADP2+ signals in response to NAADP for the first time, the present study also suggests a potential role for these events in stimulus-secretion coupling in β-cells. NAADP has been shown to elicit Ca2+ signals and insulin release in mouse pancreatic β-cells11. Whilst there is argument over the identity of NAADP’s target in the β-cells41, mutations in the two-pore channel gene (TPCN2), the potential principal target for NAADP, have been implied in the inheritance of diabetes type 2 in humans42. Localized Ca2+ signals from acidic stores have been proposed to cause depolarisation by activating calcium-dependent cation channels in the plasma membrane as we have previously observed43, such as TRPM47 and TRPM547. NAADP applied through a patch-pipette evoked small oscillatory cation currents which were preceded by small Ca2+ transients7 which were abolished in cells from Tpcn2−/− mice. Importantly, elevating glucose concentrations evoked similar cation currents, which along with those evoked by NAADP were inhibited by the NAADP antagonist, Ned-197. We have now imaged these localized Ca2+ events with our TIRF methodologies in the current study. However, local Ca2+ release from acidic stores is also known to trigger CICR in many cells, likely involving membrane contact sites with the ER48. In yet other cell types, NAADP was shown to induce localized Ca2+ release from secretory granules to initiate their own exocytosis49. In pancreatic β-cells, ER Ca2+ leak and subsequent uptake into mitochondria via the mitochondrial Ca2+ uniporter (MCU) complex have been proposed to prime ATP synthesis50. Since it has been suggested that TPCs can be blocked by ATP51, there may be a complex interplay between Ca2+ release from acidic stores and the dynamics of ATP concentrations at the subcellular level.In addition to demonstrating elementary CaATP channel closure induced by a rise in local ATP levels increases membrane resistance allowing small cation currents, activated by localized Ca2+ signals (a summation of elementary Ca2+ events from intracellular stores and CICR) to initiate depolarization of the plasma membrane. This in turn results in the opening of L-type Ca2+ channels whose mediation of larger globalized Ca2+ signals triggers insulin granule exocytosis. This model differs from, but contains elements of, each of the three models of a KATP channel independent pathway previously discussed. The present investigation suggests that glucose initially generates localized Ca2+ signalling events based on Ca2+ release from non-ER, likely acidic, stores prior to the influx of Ca2+ through VGCCs. We propose that some of the elementary trigger events involved are likely to be mediated by NAADP as we have previously suggested45.Thus we propose that during stimulus-secretion coupling in β-cells K52. Islets were dispersed into single cells or cell clusters and plated onto poly-L-lysine coated glass coverslips (Menzel) and incubated in cellstar dishes (Greiner) at 37 °C for 4–7 h before adding cell culture medium , supplemented with penstrep  and 10% fetal bovine serum  containing 10 mM glucose and incubated for a further 15–17 h before first use. Cells were loaded with 500 nM fluo 4-AM (Invitrogen) for 1 h in the dark, before being washed with imaging buffer  containing 3 mM glucose and left in this for 10 min before start of recording. Experiments were conducted exclusively at 37 °C, and with a baseline glucose concentration of 3 mM, simulating physiological conditions. Experiments to demonstrate localised elementary events were conducted on both single cells and small clusters; at an estimated ratio of 50% cells, 50% clusters. Clusters lend themselves to TIRF experiments, as they provide a large area positioned in the same focal-plane. Dispersed cells are usually at marginally different focal planes due to the inherent curvature of the glass coverslip. In studies using Ca2+-free medium, extracellular Ca2+ was re-admitted at the end of the experiment to visualise a global response as a verification of the cell’s viability. In experiments to visualise acidic stores, cells were preincubated for 10–30 min with LysoTracker Red (Invitrogen) at 200 nM. NAADP-AM was synthesised as previously described13. Batches varied in potency due to different efficiencies of synthesis and storage degradation as determined by HPLC analysis.Mouse pancreatic islets from 10–14 week-old WT male mice  were isolated as described previously−1 (n2/n1) , n1 > n2, where n1 is the refractive index of the cover glass (1.53), and n2 the numerical aperture of the lens (1.49). The exact angle for experimentation was determined using a Bertrand lens and the beam adjusted. This allowed for an evanescent wave illuminating the specimen to a depth of around 100 nm. All parameters were controlled using NIS Elements AR 4.0 (Nikon). Images were acquired at a rate of 3.3 Hz for single channel recordings (i.e. only TIRF or Epifluorescence) and at a pre-programmed rate for dual channel recordings (Frame Rate Epi: 1 Hz. Frame Rate TIRF: 3.3 Hz). Data in Fig. Cells were excited with an Argon-Ion laser  at 488 nm, and images were obtained using a Nikon Evanescent Wave Imaging System; an Inverted Total Internal Reflection Microscope (Nikon Eclipse Ti) equipped with 60 × and 100 × CFI Apochromat TIRF Series oil-immersion lenses. These lenses have a numerical aperture of 1.49, which allowed for a maximal incident angle of 76.87° calculated by α ≥ sin−1 n/n1 , n1 53, and while this paper was in preparation, an automated system applying a sophisticated version of it was released54. From the measured maximum intensities for each cell over time, frequency histograms were compiled to depict the quantal nature of the events, where events more than 2 standard deviations above the baseline mean were considered genuine. This is a standard estimate of minimum spark amplitude30. All analysis was conducted in NIS Elements AR 4.0 (Nikon) and MS Excel 14 (Microsoft). Figures were prepared in Illustrator CS6 (Adobe). Ca2+ traces are of fluorescence change relative to baseline mean fluorescence (∆F/F0). Baseline mean fluorescence (F0) was calculated from the fluorescence of the last 80–100 frames before stimulation. Traces are representative, as indicated in figure legends. Ca2+ fluorescence images are pseudo-coloured so that changes in colour reflect changes in fluorescence. Warmer colours represent higher levels of fluorescence. Statistical analysis was conducted in MS Excel 14 and SPSS 19 (IBM). Student’s t-tests  were used to determine the statistical significance of observed effects . Charts illustrating statistical differences between groups depict mean ± standard error of the mean (SEM) unless stated otherwise. Videos for supplementary information were prepared from Nikon .nd2 files and rendered using Premiere Pro CS6 (Adobe).For quantal analysis, fluorescence change across each whole cell  Act 1986, using exclusively Schedule 1 methods, and approved by the University of Oxford’s Local Ethical Review Committee.Supplementary Video 1.Supplementary Video 2.Supplementary Information.


These findings were supported by similar angiotensin II and attenuated endothelin-1 contractions. The involvement of voltage-gated Ca2+ channels in DOX-decreased contraction was excluded by using levcromakalim and diltiazem in PE-induced contraction and corroborated by similar K+ and serotonin contractions. Despite the evaluation of multiple blockers of transient receptor potential channels, the exact mechanism for DOX-decreased VSMC contraction remains elusive. Surprisingly, DOX reduced ex vivo but not in vivo arterial stiffness, highlighting the importance of appropriate timing for evaluating arterial stiffness in DOX-treated patients.Clinical and animal studies have demonstrated that chemotherapeutic doxorubicin (DOX) increases arterial stiffness, a predictor of cardiovascular risk. Despite consensus about DOX-impaired endothelium-dependent vasodilation as a contributing mechanism, some studies have reported conflicting results on vascular smooth muscle cell (VSMC) function after DOX treatment. The present study aimed to investigate the effects of DOX on VSMC function. To this end, mice received a single injection of 4 mg DOX/kg, or mouse aortic segments were treated ex vivo with 1 μM DOX, followed by vascular reactivity evaluation 16 h later. Phenylephrine (PE)-induced VSMC contraction was decreased after DOX treatment. DOX did not affect the transient PE contraction dependent on Ca The anthracycline doxorubicin (DOX) is one of the most efficacious chemotherapeutic drugs for the treatment of a wide array of cancers . HoweverPreviously, it was shown that DOX impairs flow-mediated vasodilation in the brachial artery in patients shortly after DOX treatment, often referred to as endothelial dysfunction . In addi2+ release from the sarcoplasmic reticulum and Ca2+ entry in VSMCs [Apart from the widely accepted toxic impact of DOX on endothelial cell (EC) function, DOX also affects vascular smooth muscle cell (VSMC) function ,24,25. Tin VSMCs . Hence, In this study, we aimed to investigate the effects of a single dose of DOX on VSMC function, including its contribution to arterial stiffness. A secondary goal was to evaluate whether the acute effects of DOX could be studied ex vivo in aortic rings. Such an ex vivo platform holds potential for mechanistic studies, as well as for future drug screening.There was a decrease in phenylephrine (PE)-induced contraction 16 h after in vivo DOX administration A. The PE2+ influx [2+ influx in VSMCs. Here, we calculated the increase in force (Δ) by subtracting the force at 800 s from the force at 100 s during the PE-elicited contraction response. This increase in contraction force (Δ) was lower in the DOX-treated group compared with the vehicle-treated group  D. Of notTo develop a drug testing platform for studying acute effects of DOX and to corroborate the in vivo DOX treatment findings, isolated aortic segments were isolated from mice and subsequently treated with DOX (1 μM) ex vivo for 16 h. Similar results were obtained under these experimental conditions. More specifically, PE-induced contraction was reduced after DOX treatment in a PE concentration response curve A and con2+ Krebs Ringer and CaCl2 (3.5 mM Ca2+) conditions to specifically evaluate the transient contraction phase, caused by Ca2+ release from the sarcoplasmic reticulum, and the tonic contraction phase, characterised by VGCC- and NSCC-mediated Ca2+ entry, respectively. To avoid the possible influence of NO, experiments were performed under L-NAME conditions. Contraction mediated by Ca2+ release from the sarcoplasmic reticulum and decline in contraction due to Ca2+ uptake and Ca2+ efflux were not altered in response to DOX  and non-selective cation channels (NSCCs), the resulting VSMC contraction was lower in the DOX-treated group compared with the vehicle group , was not altered after DOX treatment  was repeated under 0 mM Cae to DOX A. Upon ale group B. Subseqle group C. VSMC creatment D. Moreovreatment E. These Another possibility is that DOX affects NSCC-mediated contraction instead. Accordingly, further experiments were performed using several pharmacological agents blocking a variety of NSCCs, such as tranilast, SKF-96365, 2-aminoethoxydiphenyl borinate (2-APB) and 9-phenantrol. To avoid any possible inhibitory effect of NO on VGCCs and NSCCs, the following experiments were performed under L-NAME conditions.Similar to the decrease in PE-elicited contraction (with L-NAME) under levcromakalim, DOX reduced the PE-induced VSMC contraction (2 μM) in aortic segments pre-incubated with diltiazem (35 μM) A. Subseq+ . Under Krebs Ringer conditions, the Peterson’s modulus (Ep), a measure for arterial stiffness, was lower in the DOX group, especially at higher pressures A. This eIn vivo arterial stiffness was investigated by measuring the abdominal aorta pulse wave velocity (aaPWV) with ultrasound imaging as well as carotid–femoral pulse wave velocity (cfPWV) with tonometry, 16 h after injecting a single dose of DOX. Blood pressure and left ventricular ejection fraction (LVEF) were measured as well. DOX did not change aaPWV D, cfPWV It has been recently recognised that DOX impairs vascular function ,13,14,19DOX decreased PE-induced VSMC contraction and impaired ACh-induced endothelium-dependent relaxation in both the in vivo and ex vivo treatment setting. Impaired ACh-induced relaxation was in line with previous reports ,20,21. EFor the convenience of the reader, VSMC sensitivity for NO was not affected by DOX, as evidenced by similar endothelium-independent relaxation with DEANO. It is thus most likely that the cyclic guanosine monophosphate (cGMP) pathway is not targeted by DOX.3)-mediated contraction under 0 mM Ca2+ conditions, suggesting that DOX does not affect Ca2+ release from the sarcoplasmic reticulum. Moreover, the subsequent decline in force was similar in the vehicle- and DOX-treated groups. This indicates that DOX does not affect Ca2+ uptake and Ca2 efflux, mediated, for example, by the sarco-endoplasmic reticulum and plasmalemma Ca2+–ATPase pumps, respectively. However, we observed that DOX particularly reduced the tonic contraction phase, which may point to altered Ca2+ influx, either through VGCCs or NSCCs. Relative relaxation of PE-contracted aortic segments with diltiazem, a VGCC-blocker, was not altered after DOX treatment. Under 10–50 mM K+ conditions, depolarisation of the membrane potential will promote VGCC-mediated contraction. VSMC contraction with different doses of K+  was similar between the vehicle- and DOX-treated group. Moreover, after pre-incubation of aortic segments with levcromakalim and diltiazem, tonic VSMC contraction was lower in the DOX-treated group compared with the vehicle-treated group. These findings indicate that VGCCs are not the molecular mechanism explaining the reduced contractions in the presence of DOX. The attenuated VSMC contraction is therefore possibly attributable to DOX-induced alteration of NSCC function.Further experiments demonstrated that DOX did not alter the initial inositol trisphosphate  channel, a member of the extensive family of NSCCs, in cancer cells by permeating into the channel pore, thereby blocking it ,33. Tranelopment , targetielopment ,37,39. Ieostasis . TherefoTo investigate the possible role of DOX in targeting members of the NSCC family, several NSCC blockers, such as tranilast, SKF-96365, 2-APB and 9-phenantrol were evaluated. We limited our selection of NSCC blockers to inhibitors of TRP channels, which have been proven to be involved in VSMC contraction . One shoSurprisingly, DOX treatment resulted in a greater decline in VSMC contraction in the presence of tranilast, SKF-96365, 2-APB and 9-phenantrol, when expressed relative to the preceding contraction magnitude, showing the contribution of TRP channels to PE contractions. This paradoxical finding suggests that TRP channels play a relatively larger role in mediating contraction in DOX-treated aortic segments, and therefore may not be responsible for the decreased VSMC contraction after DOX. Instead, DOX may target other NSCCs that result in attenuated contraction. However, the lack of knowledge regarding the exact role of TRP channels in vascular function, the high diversity of the NSCC family and the interaction between NSCCs mutually  constitu2+ influx, while angiotensin II mediates a phasic contraction through Ca2+ release from intracellular Ca2+ stores, such as the sarcoplasmic reticulum [2+ release from the sarcoplasmic reticulum in a first transient phase and through primarily VGCC-mediated Ca2+ influx in a second tonic phase [2+ release from intracellular Ca2+ stores as well as Ca2+ influx through VGCCs are not targeted by DOX but that DOX may decrease VSMC contraction through interference with a specific, yet currently unknown, NSCC. Hence, DOX reduces VSMC contraction in a PE receptor-independent way.Other agonists, such as endothelin-1, angiotensin II and serotonin were subsequently used to determine whether our observations were specific for PE-involved pathways. Endothelin-1, angiotensin II and serotonin were selected as representative agonists for NSCC-, sarcoplasmic reticulum- and VGCC-mediated contraction, respectively. More specifically, endothelin-1 mainly induces vasoconstriction through VGCC- and NSCC-mediated Caeticulum . Serotonic phase . DOX decSurprisingly, a single dose of DOX decreased ex vivo arterial stiffness at higher pressures, which is in contrast with our previous report where two weeks DOX treatment increased VSMC tone and arterial stiffness . It is iIn contrast with our previously reported work that DOX increased arterial stiffness after two weeks , we foun3-mediated contraction, which was not observed. Given the fact that DOX also reduced the endothelin-1-elicited VSMC contraction, it further supports that DOX-decreased contraction occurs independent of α1-adrenergic receptors. Furthermore, depolarisation-induced contraction would be mitigated in the event of VSMC death [+ conditions was not affected, which excludes the occurrence of VSMC death. Hence, our data indicate that DOX decreases VSMC contraction without affecting α1-adrenergic receptor expression or contributing to VSMC death.Previous studies have reported decreased VSMC contraction after DOX treatment ,24. In tMC death ,51, yet 2, which corresponds to 12 mg/kg (assuming a body surface area of 1.9 m2 and a body weight of 70–75 kg) [50) of DOX in C57BL6/J mice are both 10 mg/kg [The difference in the used dosage of DOX in the present study and the aforementioned literature ,24,25 ma0–75 kg) ,53. Addi10 mg/kg . Previou10 mg/kg ,23,24. IAs a secondary goal, we aimed to develop an ex vivo experimental platform for mechanistic studies and for drug screening. In this experimental setting, a DOX dose of 1 μM was used, which is within therapeutic range, since plasma concentrations of DOX have been reported to fluctuate between 0.3 and 1 μM in patients ,55. Sinc2+ release or Ca2+ influx through VGCCs, but points towards a modulating role for DOX in NSCC function. Experiments with TRP blocking agents failed to pinpoint the exact mechanism causing reduced VSMC contractions, suggesting that DOX may target other NSCCs instead. Importantly, the observation of unchanged in vivo arterial stiffness or blood pressure, possibly due to counteracting effects of DOX on EC and VSMC function, may highlight the importance of timing for evaluation of DOX-induced arterial stiffness in patients.In conclusion, the present study reports decreased VSMC contraction with coinciding endothelial dysfunction after DOX treatment (4 mg/kg). The decrease in contraction does not relate to alterations in intracellular CaC57BL/6J mice  with an age of 10 to 12 weeks and a body weight between 26 and 30 g were used for all experiments. Mice were housed in the animal facility of the University of Antwerp in standard cages with 12–12 h light–dark cycles with access to regular chow and water ad libitum. To avoid the influence of female hormone confounding factors, male mice were chosen. All experiments in this work were approved by the Ethical Committee of the University of Antwerp (dossier 2019-34) and conformed with the ARRIVE guidelines, with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health  and with the Belgian Royal Decree of 2013.Male The current study composed two distinct experimental parts—namely, a part where DOX was administered to mice (in vivo DOX treatment), and a second part where aortic segments were first isolated from mice and subsequently either treated with vehicle or DOX (ex vivo DOX treatment).C57BL/6J mice received a single intraperitoneal injection of 4 mg/kg DOX (Adriamycin® (2 mg/mL), Pfizer, Puurs, Belgium) or 10 mL/kg of a 0.9% NaCl solution  as vehicle. DOX was diluted in a 0.9% NaCl solution on the day of injection. Based on other studies that investigated acute DOX effects [2 2.5, KH2PO4 1.2, MgSO4 1.2, NaHCO3 25, CaEDTA 0.025 and glucose 11.1.For in vivo DOX treatment:  effects ,23,24,56C57BL/6J mice were first sacrificed and the aorta was subsequently isolated, as described above. Following dissection, aortic segments were immediately transferred to Dulbecco’s modified Eagle medium (DMEM) medium  supplemented with 1% penicillin/streptomycin . Aortic segments were randomly divided and received either phosphate-buffered saline solution (PBS) as vehicle (1:100) or DOX (1 μM) for 16 h. DOX was diluted in PBS on the day of the experiment. After 16 h, aortic segments were mounted between two hooks of an organ bath set-up (10 mL) filled with Krebs Ringer solution  for vascular reactivity evaluation.For ex vivo DOX treatment: Healthy wild-type v/v) isoflurane  using a high-frequency ultrasound system . Images were only acquired when heart rate and body temperature met the inclusion criteria, i.e., 550 ± 50 beats/min and 37 ± 1 °C, respectively. M-mode images were obtained for determination of cardiac parameters using a 24 MHz transducer. LVEF was subsequently calculated using measurements of three consecutive M-mode cycles with Vevo LAB Software . In the same session, abdominal aorta PWV (aaPWV) was determined according to the method developed by Di Lascio et al. with a 24 MHz transducer [Ultrasound imaging was performed in anaesthetised mice under 1.5–2.5% (ansducer . Brieflyv/v) isoflurane , as previously described by our research group [cfPWV was determined in anaesthetised mice under 1.5–2.5% . To reduce stress and variability during the procedure, animals were trained for two days prior to the actual measurements.For clarity, the standard experimental protocol for the in vivo and ex vivo DOX treatment experiments is illustrated in The PE-elicited contraction response was further investigated by incubating the aortic segments in the organ bath with a single PE-dose of 2 μM for 15 min. Once the contraction was stable after 15 min, L-NAME (300 μM) was subsequently added, further increasing contraction (PE + L-NAME contraction). L-NAME is an eNOS blocker, which inhibits NO production and allows evaluation of the involvement of the endothelial cell layer in regulating VSMC contraction and tone. After 20 min, cumulative concentrations of the exogenous NO-donor DEANO (0.3 nM–10 μM) were added to the organ bath to evaluate endothelium-independent relaxation of VSMCs through the cGMP-mediated pathway.3-mediated Ca2+ release from the sarcoplasmic reticulum was investigated by incubating aortic segments with Ca2+-free Krebs Ringer solution (0 mM Ca2+) for three minutes, followed by addition of PE (2 μM) for three minutes. The 0 mM Ca2+ Krebs Ringer solution contained (in mmol/L): NaCl 118, KCl 4.7, CaCl2 0, KH2PO4 1.2, MgSO4 1.2, NaHCO3 25, CaEDTA 0.025, EGTA 1.0 and glucose 11.1. An extracellular 0 mM Ca2+ environment will prevent Ca2+ influx during PE contraction, which will shift contraction to solely Ca2+ release from the sarcoplasmic reticulum [2 (3.5 mM) was added to restore Ca2+-containing Krebs, which resulted in contraction in a similar way as the PE-elicited contraction response [2+-channel blocker. This resulted in relaxation, which offered an estimate of the amount of contraction that was attributable to VGCCs.The contraction via IPeticulum . Next, Cresponse . Finally+  was performed in the organ bath. A high dose of K+ causes depolarisation of the membrane potential, thereby activating VGCC-mediated Ca2+ influx, which eventually results in contraction [2+ influx via these channels [2+ influx through NSCCs. The protocol for investigation of NSCCs in modulating contraction is illustrated in To determine the role of VSMC membrane potential in modulating contraction, a dose–response of Ktraction ,59. Finachannels ,60. Thes0 * ΔP/ΔD, with ΔP = difference in pressure (kept constant at 40 mmHg), D0 = “diastolic” diameter and ΔD = the change in diameter between “diastolic” and “systolic” pressure. The ROTSAC protocol included the evaluation of arterial stiffness (Ep) at different pressures . The contribution of VSMC tonus was investigated by adding a high concentration (2 μM) of PE combined with L-NAME (300 μM).Ex vivo assessment of arterial stiffness was performed using the ROTSAC, as previously described ,61. In b®, 2 mg/mL) was purchased from Pfizer . PE, L-NAME, ACh, DEANO, diltiazem, tranilast, SKF-96365, 9-phenantrol, serotonin hydrochloride and angiotensin II (human) were obtained from Sigma-Aldrich . Levcromakalim and 2-APB were purchased from TOCRIS . Finally, endothelin-1 was supplied by Alexis® Biochemicals .DOX . Statistical analyses were performed using GraphPad Software . A


Extensive washing of cells or depletion of extracellular ATP levels by recombinant apyrase reduced [Ca2+]i oscillations and insulin secretion in pancreatic cell lines and primary β‐cells. Following ATP depletion, [Ca2+]i oscillations were stimulated by the replenishment of ATP in a concentration‐dependent manner. Inhibition of endogenous ecto‐ATP nucleotidases increased extracellular ATP levels, along with [Ca2+]i oscillations and insulin secretion, indicating that there is a constant supply of ATP to the extracellular space. Our combined results demonstrate that extracellular ATP is essential for β‐cell activity. The presented work suggests extracellular ATPases as potential drug targets for the modulation of insulin release. We further found that exogenous fatty acids compensated for depleted extracellular ATP levels by the recovery of [Ca2+]i oscillations, indicating that autocrine factors mutually compensate for the loss of others. Thereby, our results contribute to a more detailed and complete understanding of the general role of autocrine signaling factors as a fundamental regulatory mechanism of β‐cell activity and insulin secretion.ATP has been previously identified as an autocrine signaling factor that is co‐released with insulin to modulate and propagate β‐cell activity within islets of Langerhans. Here, we show that β‐cell activity and insulin secretion essentially rely on the presence of extracellular ATP. For this, we monitored changes of the intracellular Ca We find that ATP is an essential extracellular signaling component for calcium oscillations and insulin secretion from beta‐cells. Removal of ATP or blocking of P2Y receptors will stop oscillations. This can be partially compensated by adding fatty acids that stimulate calcium signaling via GPR40. Integration of the pancreatic β‐cell population into functional syncytia provides synchronization of [Ca2+]i oscillations along with periodic secretory bursts of insulin from the islets of Langerhans  in β‐cell medium by stringent washing or by the application of fatty acid‐free bovine serum albumin (FAF‐BSA) immediately reduced glucose‐stimulated [Ca2+]i by stimulating InsP3‐mediated Ca2+ release from the endoplasmic reticulum (ER)  provide a possible mechanism for ATP‐mediated action.In this study, we therefore aimed at (1) investigating the general role of ATP as an autocrine signaling factor for β‐cell activity and insulin secretion and (2) examining whether β‐cell activity can be modulated by the selective reduction or replenishment of extracellular ATP levels. Further, we (3) correlated increased levels of endogenous ATP in the presence of selective inhibitors of ecto‐ATPases with i imaging, 1.1B4 cells were seeded into 8‐well LabTek microscope dishes . 1.1B4 cells were transfected with a plasmid encoding the Ca2+‐sensitive red‐fluorescent protein R‐Geco1 using lipofectamine 2000  1 day prior to imaging experiments. The transfection was performed using cells attached to LabTek microscopy dishes at 60–80% confluency. After removing the medium and washing cells with DPBS, 210 μl OptiMEM medium was added to each well. Subsequently, 1.5 μl/well of lipofectamine 2000 (1 mg/ml) and 400 ng/well plasmid DNA were added to 20 μl/well OptiMEM medium, respectively. Both solutions were combined and incubated for 10 min at room temperature. 40 μl of this transfection mix were added to each well. After 6 h of incubation at 37°C in 8% CO2, OptiMEM was exchanged by 400 μl of RPMI‐1640 medium. Cells, showing a medium expression level of R‐Geco1, were used for live cell imaging the next day.1.1B4 cells were cultured in a humidified atmosphere at 37°C and 8% CO2.5l‐lysine. Animals were housed in the EMBL animal facilities under veterinarian supervision and the guidelines of the European Commission, revised directive 2010/63/EU and AVMA guidelines 2007.Female Ctrl: CD1  mice  served as donors for primary β‐cells that were isolated as previously described .The quantification of insulin secretion was based on an enzyme‐linked immunosorbent assay (ELISA) in 96‐well format (Mercodia). Experiments for the determination of insulin secretion were performed in quadruplicate per condition, following the instructions of the supplier. Insulin levels were normalized to the protein content of MIN6 cells, as determined by a BCA assay . Experiments for the determination of extracellular ATP were performed in quadruplicate per condition, following the instructions of the supplier. ATP levels were normalized to cell numbers. For normalization of ATP levels in cellular supernatants, pre‐defined numbers of MIN6 or mouse primary β‐cells were correlated to cellular protein content following cell lysis, as determined by BCA assay. Then, cell numbers were inferred from determined protein content of harvested cells.2.82. Olympus 60x Plan‐APON  or 20x UPLS APO  objectives and FluoView software (version 4.2) were applied. Images were acquired using a Hamamatsu C9100‐50 EM CCD camera. A 488‐nm laser line  in combination with a 525/50 emission mirror was employed to image the green channel. A 559‐nm laser line  and a 643/50 emission filter was used for red channel recordings. For monitoring changes of [Ca2+]i in response to external stimuli, cells were incubated with the acetoxymethyl ester of the Ca2+ indicator Fluo‐4 (Life Technologies), 5 µM in DMEM (1 g/L glucose) for 25 min at 37°C and 5% CO2. The frame time was set to 3.9 s, with images acquired in 4 s intervals. For imaging, MIN6 cells were grown as pseudoislets of ~70% confluence. Imaging experiments were performed in HEPES buffer . Washing was performed on adherent cells in LabTek imaging dishes. For this, the growth medium was removed and cells were gently washed 3× with pre‐warmed imaging buffer at respective glucose concentrations to remove extracellular signaling factors. For imaging, cells were placed in 150 µl pre‐warmed imaging buffer at indicated glucose concentrations.Live cell imaging was performed on a FluoView1200 (Olympus IX83) confocal laser scanning microscope, equipped with an environment box (made by EMBL) to allow imaging at 37°C and 5% CO2.9F/F0). Representative traces of cells within a field of view were averaged or numbers of detected high‐intensity [Ca2+]i events per 60 s interval were determined. The height of each [Ca2+]i event was determined relative to the highest detected peak per trace. This served as a criterion to group [Ca2+]i oscillations into low‐intensity (<60% of highest peak) and high‐intensity (≥60% of highest peak) events. Per condition or tested stimulus, 4–6 independent experiments were performed under identical settings. At least 50 individual representative MIN6 were picked per condition and their responses were averaged. Therefore, the number of high‐intensity [Ca2+]i oscillations refers to the sum of events from 50 representative cells within intervals of 60 s. OriginLab, version 8.5 was applied for statistical analysis and plotting.The open access software tool Fiji , with cell‐to‐cell variability in frequency and shape of oscillations. Using the Ca2+ sensitive cell‐permeant version of the fluorescent indicator Fluo‐4, we observed that MIN6 cells respond to increasing concentrations of external glucose with enhanced GSIS, along with differential levels of [Ca2+]i oscillations . Addition of KCl at the end of experiments caused cell depolarization and induced high‐intensity [Ca2+]i transients with intensities that compared well to ATP‐mediated cell depolarization. Consequently, intensity‐based normalization to the highest detected intensity peaks did not change the overall appearance of traces  in pre‐washed MIN6 and mouse primary β‐cells, as indicated by counts of high‐intensity [Ca2+]i oscillations  to pre‐washed MIN6 cells to test the immediate effects of ATP on [Ca2+]i oscillations and to exclude effects from ATP hydrolytic products. ATPγS potently induced [Ca2+]i oscillations already at 25 µM in pre‐washed MIN6 and mouse primary β‐cells  that convert ATP to adenosine  also reduced and stopped [Ca2+]i oscillations in pre‐stimulated 1.1B4 cells   was added to glucose‐stimulated oscillating MIN6 cells to efficiently reduce [Ca2+]i oscillations, as shown before  to pre‐stimulated cells reduced and partially stopped ATP‐induced [Ca2+]i oscillations  to evoke continuous i oscillations and insulin secretion. Here, we already reported the essential role of autocrine signaling factors for [Ca2+]i oscillations and insulin secretion  to reduce and stop [Ca2+]i oscillations in glucose‐stimulated MIN6 cells. Addition of oleic acid (OA) to apyrase‐treated MIN6 cells restarted [Ca2+]i oscillations  just evoked a single pronounced [Ca2+]i transient without continuous [Ca2+]i oscillations BSA to glucose‐stimulated MIN6 and mouse primary β‐cells immediately reduced and stopped [Ca2+]i oscillations, which were reduced and stopped by the application of the GPR40 antagonist GW1100 (25 µM) . Subsequent addition of OA (100 µM) to MIN6 cells partially restarted [Ca) Figure . This in42+]i transients. Further, it has been shown that this message is transmittable to neighboring cells via intermittent release of ATP without direct physical contact , but evoked only single [Ca2+]i transients in the presence of sub‐stimulatory glucose levels (3 mM), which is in line with previous reports  signaling molecules and to unravel their general role as part of a fundamental regulatory mechanism of β‐cell activity and insulin secretion.Supplementary MaterialClick here for additional data file.


I was recently invited to give a talk at a webinar organized by the World Academy of Art & Science and this prompted me to think again about a topic that has always fascinated me, namely, the similarity and difference in creativity required by these two branches of human intellectual endeavor or, as CP Snow named them, “the two cultures.”The central part of Chapter 9 is the parable “Before the Law,” told by a priest to the novel’s main figure, Joseph K. Briefly, a man from the country begs for entry to the Law. The doorkeeper tells him that he cannot be admitted at the moment, but says that it is possible that he could be allowed to enter later. In the following days, months, and years, the man repeats his question about whether he can now enter and is each time told that he cannot be admitted now. He is warned not to try to enter without permission, as the doorkeeper emphasizes that he is mighty, but that there are even mightier doorkeepers behind him. The man waits and ask and then waits again, and as the years go by, he becomes more and more fixated on the doorkeeper, noticing, for example, the fleas in his coat and even asking the fleas to help him get permission to enter. Having grown old and very weak, sensing that his end is near, he finally asks the doorkeeper why, in these many years, nobody else has come to seek entrance to the Law. The doorkeeper understands that the man is dying and answers “No one but you could gain admittance through this door, since this door was intended only for you. I am now going to shut it.”There are of course many and deeply philosophical issues in this parable that has always eluded any definitive interpretation.Nature reporting the first patch clamp whole-cell recordings (WCRs) of current from an electrically nonexcitable cell type, the pancreatic acinar cell.2+-activated K+ channels in a single cell.2+-activated channels as endogenous Ca2+ sensors to measure the cytosolic Ca2+ concentration.3) released Ca2+ from intracellular stores in permeabilized pancreatic acinar cells, which was published in Nature late in 1983.I have experienced such a situation myself. In April 1983, Yoshio Maruyama and I submitted a paper to 3 application. This experiment was finally done in my laboratory, but only 5 years later.3 evoked repetitive spikes of Ca2+-dependent current due to repetitive Ca2+ release from internal stores.2+ spiking because of IP3-elicited repetitive Ca2+ release in a mammalian cell would have been shown several years before Peter Cobbold and his collaborators demonstrated Ca2+ spiking evoked by hormonal stimulation in liver cells.The obvious experiment to do, at that point in time, would have been to use the WCR configuration to test directly the effect of intracellular IP2+ concentration would be uniform throughout the cytosol and determined by the Ca2+ concentration in the pipette solution. Ca2+ buffers, usually EGTA, were of course always present in the patch pipette solutions and in general we, and others, assumed that the cytosolic Ca2+ concentration in WCR experiments could be calculated from the concentrations of Ca2+ and EGTA, taking into account the value of the Ca2+–EGTA dissociation constant at the known pH in the pipette solution.Why did I delay doing this key experiment for such a long time? The reason is that I did not think it would work. These were still very early days in the history of the patch clamp technique and the first WCRs, which were carried out on the electrically excitable chromaffin cells by Alain Marty and Erwin Neher, indicated rapid equilibration of small ions between patch pipette and cell interiors.2+-sensitive channels to regulate Ca2+ independently of the bulk cytosolic Ca2+ concentration. A further analogy with Kafka’s parable is that, like the door to the Law that was specifically and exclusively provided for the man from the country, the IP3-WCR experiment was uniquely suited to the expertise and technology available at that time in my laboratory. In fact, and—to my luck—it turned out that even after a 5-year delay nobody else had attempted to do the experiment and so our paper, when it was finally published in 1989,2+-dependent currents and direct measurements of local changes in the cytosolic Ca2+ concentration in the subcellular region of secretory control.,The (correct) image of the very large pipette and the very small cell was always with me and, like the doorkeeper in Kafka’s story, was so strong that it seemed hopeless even to contemplate doing an experiment that would depend on the ability of regions of the cell close to the CaI grew up scientifically in a mostly very critical environment in the Institute of Medical Physiology at the University of Copenhagen, which in the late 1960s was dominated by several very clever individuals, who were very good at finding arguments against new ideas and experimental approaches. There is of course a place for such thinking, but one should never be intimidated. Even if one doubts that a key experiment is likely to work, there is merit in having the courage to try.


ADTRP expression. We discovered a critical role of ADTRP in vascular development and vessel integrity and function, manifested through Wnt signaling-dependent regulation of matrix metalloproteinase-9. ADTRP also hydrolyses fatty acid esters of hydroxy-fatty acids, which have anti-diabetic and anti-inflammatory effects and can control metabolic disorders. Here we summarize and analyze the knowledge on ADTRP and try to decipher its functions in health and disease.The novel protein ADTRP, identified and described by us in 2011, is androgen-inducible and regulates the expression and activity of Tissue Factor Pathway Inhibitor, the major inhibitor of the Tissue Factor-dependent pathway of coagulation on endothelial cells. Single-nucleotide polymorphisms in ADTRP associate with coronary artery disease and myocardial infarction, and deep vein thrombosis/venous thromboembolism. Some athero-protective effects of androgen could exert through up-regulation of  The novel protein ADTRP (androgen-dependent TFPI-regulating protein), identified and described by Lupu and colleagues in 2011, is androgen-inducible and up-regulates the expression and activity of tissue factor pathway inhibitor (TFPI), the major inhibitor of the tissue factor (TF)-dependent pathway of coagulation on endothelial cells (EC). Endothelial dysfunction, including dysregulated production and activity of the enzymes and/or inhibitors that control coagulation and fibrinolysis, impact vascular homeostasis. This favors the development of pathologies involving disruption of the vascular wall, such as myocardial infarction (MI), deep vein thrombosis (DVT)/venous thromboembolism (VTE), sepsis, and cancer.Along the ten years from its discovery, a few labs tried to address the physio-pathologic role of ADTRP and a variety of potential functions emerged for ADTRP distinct from TFPI regulation. Many pieces of evidence suggest that ADTRP is potentially beneficial in preventing cardiovascular disease (CVD). Nevertheless, the heterogeneity of roles attributed to ADTRP and the lack of mechanistic insights make it hard to connect them all into a unitary functional model at this moment. This review is aimed to bring together all the present knowledge about ADTRP in an attempt to open the field for the development of mechanistic studies designed to decipher the main function(s) of ADTRP in health and disease.TFPI is a key natural inhibitor of coagulation: it neutralizes factor (F) Xa and inhibits TF-FVIIa in the presence of FXa, thus precluding thrombin generation. Thrombin performs a number of procoagulant functions, including the activation of platelets, FV, FVIII and FXI, and cleavage of fibrinogen to fibrin.Low plasma levels of TFPIα (full-length TFPI) correlate with prothrombotic diseases, including DVT/VTE , coronarDiscovery of the role of TFPI in bleeding disorders ,12 has hOn EC, TFPIβ is glycosyl phosphatidylinositol (GPI)-anchored to the plasma membrane in caveolae/lipid rafts ,14,15,16Despite TFPI being a major endogenous inhibitor of coagulation, very few mechanisms/factors that regulate the natural expression of TFPI have been identified so far ,24,25. DC6ORF105 regulates TFPI mRNA expression, cellular distribution and EC-associated anticoagulant activity of the inhibitor, both in native conditions and in response to androgen, thus also acting as a novel androgen-controlled membrane protein. We named this protein ADTRP: androgen-dependent TFPI-regulating protein.Experimentally, we verified that the uncharacterized protein encoded by Based on sequence homology, ADTRP belongs to the androgen-inducible gene family, of which androgen-inducible gene 1 (AIG1) was cloned from human dermal papilla  and is hhttps://www.uniprot.org, accessed on 20 February 2021) C6ORF105 encodes Protein Q96IZ2. UniProtKB lists 3 described isoforms and 4 potential isoforms that are computationally mapped. Isoform 1 (Q96IZ2-1) has been chosen as the canonical sequence with 230 aminoacids and a molecular weight of 27 kDa. The protein has six predicted transmembrane domains marked as yellow highlight on the sequence (47) and Histidine (H131) are FAHFA (fatty acid esters of hydroxy fatty acids) hydrolase activity sites [7 and C62; no N-glycosylation sites, but several O-glycosylation sites possible; and, phosphorylation possible at serine residues S148 and S151, as well as threonine residues T61, T70 and T80, and tyrosine residues Y58 and Y201 , the expression of ADTRP increases during embryogenesis and then constantly into adulthood, to be followed then by abrupt decrease with aging. Whether or not this decrease correlates with the decline in androgen levels during old age remains to be determined.ADTRP mRNA and protein expression were detected in human EC in culture, human placenta and baboon lung and aorta , human mBy immunostaining, we identified ADTRP expression in human EC in both arteries and veins, lymphatics, epithelia of various organs , hepatocytes, and lung alveolar macrophages . Wang anIn mouse tissues, we detected ADTRP expression by qPCR, Western blot, immunostaining and in-situ hybridization in the skin and isolated dermal fibroblasts, lung and aorta , as welladtrp is expressed in the lateral plate mesoderm, most likely in angioblasts, and in both the dorsal aorta and cardinal vein [adtrp increased up to 50 times during zebrafish embryonic development and into adulthood [In zebrafish, we showed by in-situ hybridization that nal vein . qPCR redulthood .7 and C62, and the trans-membrane domains could play a role in targeting ADTRP to lipid rafts/caveolae. To verify the role of palmitoylation, we mutated C62 residue into A and expressed the FLAG-ADTRPmut construct in EC. Western blot after Triton X-114 extraction revealed decreased partition of ADTRPmut in the detergent fraction. Immunofluorescence illustrates less cell surface clustering, decreased membrane co- localization of TFPI and FLAG-ADTRPmut after treatment with Cholera Toxin and increased cytoplasmic localization of FLAG-ADTRPmut  in the ADTRP promoter . More reTFPI transcription start site, which contains a binding site for POU1F1. Deletion of POU1F1-binding site or knockdown of POU1F1 expression abolished ADTRP-mediated transcription of TFPI [Luo and colleagues  employed of TFPI . The pre of TFPI .Using post-transcriptional silencing and over-expression experiments in EC in vitro, we unveiled the function of novel at the time protein ADTRP in regulating the TFPI-dependent anticoagulant properties of the endothelium in both normal conditions and in response to androgen . We founmut failed to increase TFPI anticoagulant activity  is the only gene homologous to human ADTRP [adtrp1, Wang et al. [adtrp1 regulates the expression of tfpi and consequently the specification of hemangioblasts and hematopoiesis. Potential mechanisms may involve TFPI interaction with glypican-3 and inhibition of the serine protease CD26 activity, which can lead to increased proliferation and migration of hematopoietic stem cells. Although considered to be independent biological processes, hematopoiesis and coagulation could be mechanistically connected through an ADTRP-TFPI regulatory axis.Wang and colleagues  demonstran ADTRP . Using mg et al.  showed t−/− mice did not display major hemostasis defects, but we observed increased congestion and bleeding/extravascular fibrin, which associated with decreased TFPI antigen and anticoagulant activity, measured both in the plasma and in the lung. Other organs  showed similar signs of congestion, bleeding and thrombosis, as well as signs of necrosis (kidney tubules) indicating tissue damage due to ischemia/reperfusion injury that may occur post-thrombosis .Our genetic mouse model of Adtrp global deletion  revealedAdtrp deficiency in the mouse did not reduce Tfpi expression, it did affect TFPI-dependent lung-associated anticoagulant activity; therefore, we suggest that ADTRP may regulate the membrane location and anticoagulant potential of TFPI in EC in vivo similar to the described in vitro effects [Although  effects ,39.As reviewed by Wang and colleagues , CAD, a ADTRP, which could be partially responsible for pathogenesis in CAD/MI. SNP rs6903956 within the ADTRP gene is also significantly associated with CAD in ethnic groups in Singapore independently of conventional risk factors [GWAS analysis in a Chinese Han cohort  revealed factors .Guo and colleagues  analyzedMIA3/TANGO1 through positive regulation of PIK3R3 and activation of AKT signaling [MIA3/TANGO1 is a significant CAD risk variant [ADTRP associated with rs6903956 may be linked to a higher risk of CAD/MI through MIA3/TANGO1- dependent increase of ApoB that transports pro-atherogenic cholesterol. These ideas are contradictory though, since reduction of ADTRP expression correlates with decreased MIA3/TANGO, whereas increased ApoB export requires higher, not diminished levels of MIA3/TANGO. A positive association between CAD risk variants was demonstrated for ADTRP and melanoma inhibitory activity protein 3 (MIA3)/transport and Golgi organization protein 1 (TANGO1), whereby ADTRP up-regulates the expression of ignaling . SNP rs1 variant . MIA3/TA variant  and the  variant . Whereas variant  suggesteAll the data reporting associations between SNP rs6903956 in ADTRP and CAD are of potential interest, but significantly more experimental work especially in animal models will be necessary to prove causality for this mutation(s) and decipher the mechanisms that link low ADTRP expression to the increased risk of CAD/MI.ADTRP knockdown in human HepG2 and EC, where up-regulation of seven histone genes, down-regulation of cell cycle genes CCND1, CDK4, and CDKN1A, and up-regulation of apoptosis genes CASP7 and PDCD2 were found [−/− mice vs. WT  also shADTRP expression [Another beneficial effect of increasing ADTRP level could relate to the effects of androgen. Low cellular ADTRP may increase the risk of CAD/MI by affecting functions of EC that are relevant to the development of atherosclerosis, such as increasing oxidized-LDL-mediated monocyte adhesion and transmigration through EC . Androgepression . Althougpression , but thepression ,42.Besides androgen, Chinetti-Gbaguidi and colleagues showed that ADTRP is also transcriptionally controlled by PPAR- in monocytes/macrophages and up-regulated by PPAR- agonists in human atherosclerotic plaques . MacrophADTRP expression in EC and/or monocytes, this may reflect cellular dysfunction, which would indeed support the development of CAD/MI, but this needs to be experimentally demonstrated. Knowing that plasma TFPI level is significantly higher in CVD and other pathologies, including COVID-19 [Interestingly, Ooi and colleagues reported measurement of ADTRP in the plasma, and found that CAD patients had lower ADTRP levels than controls . AlthougCOVID-19 , where TUsing mouse and zebrafish models of Adtrp genetic inhibition, we showed that ADTRP deficiency leads to leaky vessels, edema, and hemorrhage involving defective EC junctions, degradation of extracellular matrix (ECM) and local inflammation . Abnorma−/− mice correlated with the degree of phenotype severity. In independently generated Adtrp mutant mice, another group [mut described above failed to down-regulate the Wnt response , indicating that the membrane localization of ADTRP is critical for its role in the inhibition of Wnt signaling.Defective vascular features associated with ADTRP deficiency include abnormal dilation, tortuosity and branching, increased permeability/microhemorrhage, EC junction defects (decreased VE-cadherin and claudin-5), loose perivascular cell coverage and degraded ECM . Neo-/peer group  found ther group  targeteder group . AccordiWnt-target genes consistently up-regulated in ADTRP-deficient mice and zebrafish include MMP-9 and IL-8, the latter being also a mediator of abnormal angiogenesis and increased MMP9 expression . One pro47 and H131 in ADTRP  family of FAHFAs have gained attention because of their beneficial effect in diabetes and inflammatory pathologies. PAHSAs could improve glucose-insulin homeostasis in insulin-resistant mice fed a high-fat diet by stimulating the release of glucagon-like peptide 1 ,63. TheyAig1, Adtrp, and double knockout (DKO) mice and a small molecule inhibitor (ABD-110207) of ADTRP and AIG1 to show that both proteins are responsible for regulating FAHFA hydrolysis primarily in the adipose tissue. They also found that ADTRP was the major driver of 9-PAHSA hydrolysis in the liver, whereas ADTRP and AIG1 had an additive effect in brown adipose tissue and kidney. Interestingly, tissues from DKO mice showed decreased hydrolytic activity towards 9-PAHSA added exogenously but no change in the overall endogenous FAHFA levels. In parallel, a significant residual 9-PAHSA hydrolytic activity was observed in the liver and kidney of DKO mice [In a recent study, Erikci Ertunc and colleagues used a combination of genetic loss-of-function and chemical approaches to demonstrate that ADTRP and AIG1 are major contributors to endogenous FAHFA hydrolysis . They usDKO mice . TogetheDKO mice  can compIntriguingly, the loss of ADTRP and/or AIG1 failed to protect the mice against high-fat diet-induced obesity and metabolic dysregulation, despite elevated FAHFA levels in adipose tissue . The lacCollectively, these studies highlight the importance of ADTRP as an endogenous FAHFA hydrolase and its potential as a drug target. Considering that ADTRP and AIG1 are androgen inducible genes, it will be interesting to investigate sex-based differences in FAHFA regulation and consecutive effects on high-fat diet-induced metabolic disorders. It would be of great interest to determine whether the hydrolase activity of ADTRP may play a role in the regulation of TFPI anticoagulant function in EC. Further studies are also required to elucidate the role of tissue-specific ADTRP mediated FAHFA hydrolysis in the context of inflammatory responses and CVD.Deficiency of ADTRP in oral cleft syndrome  and cranADTRP was down-regulated using shRNA shows significant impairment of mineralization in ADTRP-deficient cells, illustrated by Alizarin Red staining (Our own experiments addressing the role of ADTRP in human BM-MSC also showed decreased ADTRP mRNA expression after induction with osteogenesis medium. Comparison between Control BM-MSC and cells where staining .−/− mice. However, experiments like bone densitometry, alcian blue staining in embryos and neonates, and challenge models like bone-fracture and healing models are yet to be employed to properly characterize the role of ADTRP in MSC differentiation/bone formation in mice.In contrast to the human BM-MSC data, we did not observe any obvious bone/cartilage/oral cleft defects in our Adtrpβ-catenin activation, which is critically important for BM-MSC differentiation in response to androgen [It is known that androgens stimulate MSC differentiation into osteo-muscular rather than fat tissue through mechanisms involving AR and inhibition of the adipogenic factor PPAR-γ. AR controls pluripotency transcription factors and acts as negative regulator of signaling pathways that maintain the stem cell state, such as the PI3K/AKT . The decandrogen . While a significant amount of information has accumulated within the decade since its discovery, the primary function of ADTRP remains elusive. So far, data are hinting towards multiple roles, some of which are depicted in ADTRP is necessary for maintaining cell and tissue homeostasis, including regulation of TFPI-dependent anticoagulant activity, preserving vascular integrity and normal cardiovascular function, prevention of unwanted Wnt signaling, and stem cell differentiation. On the other hand, reduced ADTRP hydrolase activity may help increase the level of the metabolically beneficial FAHFAs. Apparently discrepant roles could arise from differences in organ-specific expression and possibly distinct mechanisms of regulation of ADTRP. Despite advancements, significant mechanistic work is still necessary to discriminate between the primary and secondary functional roles of ADTRP. Since ADTRP has a wide tissue expression and it is evolutionary conserved having orthologs in all classes of vertebrates, it is expected to play essential functions. However, ADTRP gene deletion in mice was compatible with survival, suggesting the existence of compensatory mechanisms. Structure-function studies may help identify the primary role of ADTRP. Proteins with six transmembrane spanning domains have a variety of functions, including enzymatic activities, membrane transporters, and channels. Membrane anchoring of ADTRP suggests a potential role as membrane microdomain organizer, which may integrate some of the described cell surface activities, such as the modulation of TFPI-dependent anticoagulant function, Wnt and androgen signaling, and hydrolysis of membrane lipids . Future cell/tissue-specific gain and loss of function studies in models of metabolic disorders, coagulation/thrombosis, and atherosclerosis, coupled with in depth multi-omics analysis will be required to unveil the primary function of ADTRP and to understand its main involvement in the mechanism of diseases.