These signals represent attractive cardioprotective targets, but the underlying mechanisms of genesis are ill-defined. Here, we investigated the role of the second messenger nicotinic acid adenine dinucleotide phosphate (NAADP), which is known in several cell types to induce Ca2+ oscillations that initiate from acidic stores such as lysosomes, likely via two-pore channels .In the heart, a period of ischaemia followed by reperfusion evokes powerful cytosolic Ca2+ oscillations and dramatically protected cardiomyocytes from cell death in vitro after ischaemia and reoxygenation, preventing opening of the mitochondrial permeability transition pore. Ned-K profoundly decreased infarct size in mice in vivo. Transgenic mice lacking the endo-lysosomal TPC1 were also protected from injury.An NAADP antagonist called Ned-K was developed by rational design based on a previously existing scaffold. Ned-K suppressed CaNAADP signalling plays a major role in reperfusion-induced cell death and represents a potent pathway for protection against reperfusion injury. Restoration of blood supply is essential to salvage the myocardium; however, this paradoxically results in further injury to the myocardium.2+ is an essential ion in the heart, coupling electrical excitation to muscular contraction. During the cardiac action potential, Ca2+ influx via the sarcolemmal L-type Ca2+ channels triggers Ca2+ release from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyRs). The resulting elevation of [Ca2+]c stimulates cardiomyocyte contraction. In healthy cardiomyocytes, therefore, [Ca2+]c is very finely regulated via homeostatic mechanisms, principally by buffering within the SR but also in other organelles such as mitochondria and lysosomes.8 When ATP becomes depleted during ischaemia, however, [Ca2+]c increases.11 Reperfusion is necessary to save the cells, but during the first few minutes of reperfusion, Ca2+ is repeatedly released from the SR and pumped back in a futile oscillatory cycle. If normal Ca2+ levels are not quickly restored, this can result in hypercontracture and cell death.13 The Ca2+ oscillations are caused by cycles of SR Ca2+ release and re-uptake, leading to mitochondrial Ca2+ overload and mPTP opening. Consequently, drugs such as ryanodine that inhibit the RyR prevent these oscillations, and protect against mPTP opening and cell death.14 Studies in cardiomyocytes have led to the proposal that the SR–mitochondrial interaction is a critical target of reperfusion injury.15 Using multiphoton microscopy of the intact heart, we have recently shown that slow [Ca2+]c waves occur in cardiomyocytes during reperfusion, and that these precede mPTP opening.3Despite its role in cell death, Ca2+-mobilizing second messenger identified to date.16 Much evidence suggests that NAADP triggers Ca2+ release from acidic organelles such as lysosomes.22 It is therefore distinct from the other known second messengers, inositol triphosphate , and cyclic ADP ribose (which stimulates Ca2+ release from the SR via the RyR). The Ca2+-mobilizing properties of NAADP were initially identified in sea urchin eggs.23 Subsequent data have shown that NAADP can stimulate Ca2+ oscillations in diverse mammalian cell types, including pancreatic beta cells, smooth muscle, and endothelium.26 Two-pore channels  are localized to endo-lysosomal membranes, and have been implicated in the release of lysosomal Ca2+ in response to NAADP,28 although their native ionic permeability is controversial.30Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca31 a role of NAADP in the cardiac inotropic response has gradually been revealed. Although NAADP does not appear to be involved in baseline cardiomyocyte contraction, there is evidence that it is involved in the increased Ca2+ transient and contraction force in response to isoproterenol.8 Delivery of NAADP into resting primary cardiomyocytes induces Ca2+ release from lysosomes, and evokes slow, cytosolic Ca2+ waves reminiscent of those we previously observed in the intact heart during reperfusion.32 Higher concentrations of NAADP also triggered spontaneous diastolic Ca2+ waves.32 Significantly, administration of an inhibitor of NAADP signalling suppressed diastolic Ca2+ waves and also prevented isoproterenol-induced arrhythmias in mice.32 Diverse stimuli can increase NAADP within seconds, whereas cardiac NAADP levels were increased 5 min after β-adrenergic stimulation.8 Recently, a cell-permeable, small-molecule inhibitor of NAADP signalling was identified in a chemical screen and shown to be effective at nanomolar concentrations in vitro.17 The compound, Ned-19, eliminated glucose-stimulated Ca2+ oscillations in mouse pancreatic beta cells.17 Ned-19 similarly inhibited Ca2+ oscillations in other tissues such as uterine smooth muscle (stimulated with oxytocin)24 and pulmonary arterial myocytes (stimulated with endothelin-1).18Since the identification of NAADP receptors in the heart over 10 years ago,2+ oscillations lead to mPTP opening and irreversible cell injury, in combination with other factors present at reperfusion, they represent an important potential cardioprotective target. However, the use of canonical Ca2+-channel inhibitors in this approach is complicated by the essential role of Ca2+ in cardiac contraction. Given the role of NAADP in the generation of Ca2+ oscillations in diverse systems including cardiac arrhythmia, we reasoned that NAADP signalling may also play a role in the generation of reperfusion-induced Ca2+ oscillations that lead to lethal injury. Using a chemically modified form of Ned-19 called Ned-K, we have demonstrated that inhibition of NAADP-dependent Ca2+ oscillations resulted in cardioprotection in both an in vitro cell-based assay and an in vivo model of ischaemia and reperfusion. Experiments demonstrating that TPC1 knockout mice were similarly protected against ischaemia and reperfusion injury validated the NAADP signalling pathway as a target for cardioprotection. Thus, inhibition of NAADP-stimulated Ca2+ oscillations represents a viable cardioprotective strategy which may have minimal effects on regular cardiac contraction.As reperfusion-induced Ca2.Supplementary material online.Methods are described in detail in 2.134 were anaesthetized by i.p. injection (0.01 mL/g) of a solution containing ketamine 10 mg/mL, xylazine 2 mg/mL, and atropine 0.06 mg/mL. Adequacy of anaesthesia was monitored by pedal response and breathing rate. Animals were euthanized by severing of the aorta. The in vivo model of myocardial infarction was performed in mice using 30 min ischaemia followed by 120 min reperfusion. Drugs were administered i.v. 5 min before reperfusion. Adult rat ventricular cardiomyocytes were prepared by standard methods.35All animals received humane care in accordance with the United Kingdom Home Office Guide on the Operation of Animal (Scientific Procedures) Act of 1986. The investigation conforms to the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes or the NIH guidelines. Male Sprague–Dawley rats were anaesthetized by i.p. injection of 160 mg/kg pentobarbitone. Male C57BL/6J and TPC1 knockout mice2.238 The time to mitochondrial depolarization provides an index of mPTP sensitivity to opening.mPTP opening was induced in a previously described model of oxidative stress in which cardiomyocytes loaded for 15 min with 5 µM tetra-methyl rhodamine methyl ester (TMRM) are scanned using the 543 nm laser line of a confocal microscope, generating reactive oxygen species (ROS).2.32+.Mitochondrial swelling was assessed by measuring the absorbance of the mitochondrial suspension (0.5 mg/mL) at 520 nm after the addition of 500 µM free Ca2.42+ sparks and transients, cardiomyocytes were loaded with the fluorescent dye Fluo4-AM, 5 µmol/L for 30 min. Rapid line scans were performed (three line scans per cell at different regions of the cell) using the HeNe 488 nm laser of an Leica SP5 confocal microscope. Ca2+ spark frequency was determined using ImageJ with the ‘Sparkmaster’ plugin. Ca2+ transients were stimulated by electrical field stimulation with platinum electrodes . Addition of 10 mmol/L-Caffeine was used to trigger Ca2+ release from the SR and measure SR Ca2+ content. Cells were subjected to simulated ischaemia by incubating in a glucose-free, anoxic buffer (pH 6.4), before reoxygenation in normoxic buffer. Cell death was determined by staining with propidium iodide (PI).To detect Ca2.513 The synthesis of Ned-K is described in Supplementary material online.The trans-form of Ned-19 was synthesized as described previously.2.6post hoc analysis using Dunnett's test for comparisons solely against control values, or Tukey's test for multiple comparisons.All values are expressed as mean ± SEM. Data were analysed by one-way ANOVA followed, where significant, by 3.2+-containing acidic organelles.39 Cardiomyocytes contain abundant lysosomes, as detected by staining with the fluorescent lysosomotropic dye lysosensor green . Although rhod2-AM is typically used as a probe of mitochondrial Ca2+, it also accumulates in lysosomes as has been previously noted.40 There was a high degree of overlap between lysosensor and rhod2-AM fluorescence , indicating that most of the lysosomes in cardiomyocytes contain Ca2+. Sequestration of Ca2+ in lysosomes depends on their low luminal pH. Consequently, treatment of cells with bafilomycin A, an inhibitor of vacuolar H+-ATPases,41 resulted in gradual loss of both the pH-dependent, lysosensor probe, and rhod2 fluorescence from the lysosomes . Pretreatment with glycyl-l-phenylalanine-beta-naphthylamide (GPN), which selectively permeabilizes lysosomes by osmotic lysis, also caused gradual disappearance of lysosensor green staining , in parallel with loss of Rhod-2 fluorescence. Interestingly, GPN also caused a transient increase in the frequency of spontaneous Ca2+ sparks . Thus, lysosomes contain Ca2+ and are a potential source of Ca2+ release in ventricular cardiomyocytes.Lysosomes are CaFigure ). This modification improved its cLogP value, a measure of aqueous solubility calculated by the Cresset Fieldview System, and its topological polar surface area (TPSA), . Indeed, this new compound, which we called Ned-K, is noticeably more soluble than Ned-19, and it was possible to dissolve it at concentrations up to 300 mmol/L in stock solutions of DMSO, compared with a maximal limit of 100 mmol/L solutions of Ned-19. We compared the potency of Ned-K with Ned-19 using the sea urchin egg homogenate assay system that was used in the original characterization of Ned-19.17 Addition of NAADP to this system causes a rapid release of Ca2+ from acidic organelles, and compounds such as Ned-19 inhibit this NAADP-stimulated Ca2+ release. According to this assay, Ned-K retained an almost identical inhibitory capacity as Ned-19 in vitro .Next, with the aim of improving the biological effectiveness and selectivity of Ned-19, we developed a new analogue of Ned-19, in which the fluoride was replaced with a cyano group . Adult rat cardiomyocytes were loaded with the fluorescent Ca2+ sensor, Fluo4-AM, and imaged during a period of 60 min simulated ischaemia followed by reoxygenation for 20 min (sIR) on the stage of a confocal microscope. Before ischaemia, [Ca2+]c was stable except for occasional spikes during spontaneous cellular contractions, which occur in unstimulated cultures . As expected, [Ca2+]c had risen markedly after ∼40 min simulated ischaemia . After reoxygenation, most cells underwent rapid periodic contractions corresponding with large [Ca2+]c oscillations . The frequency of these Ca2+ oscillations reached a maximum at ∼3 min, then decreased gradually over the next 7–12 min , during which time some cells underwent Ca2+ overload and hypercontracture , and others regained [Ca2+]c homeostasis, recovering normal rod-shaped morphology.Since lysosomal Ca2+]c oscillations during reoxygenation. Ned-19 (10 µmol/L) decreased the frequency of [Ca2+]c oscillations . The chemical modification in Ned-K appears to greatly improve its effectiveness at preventing Ca2+ oscillations in this model, since 10 µmol/L of Ned-K almost completely eliminated [Ca2+]c oscillations , and 0.1 µmol/L of Ned-K was as effective at suppressing [Ca2+]c levels as 10 µmol/L of Ned-19 . The average frequency of Ca2+ oscillations in each cell was analysed statistically  and was found to be significantly suppressed by the treatments . These results demonstrate that Ned-19 and, particularly, Ned-K were effective at dampening sIR-induced Ca2+ oscillations in cardiomyocytes.We evaluated the potential of the Ned drugs to inhibit c oscillations at reperfusion to a similar degree .To determine whether inhibition of NAADP-stimulated Ca2+ oscillations observed with Ned-19 and Ned-K was not due to off-target inhibition of the RyR directly, we evaluated their effect on the frequency of spontaneous Ca2+ sparks, and on efficiency of excitation–contraction coupling. Ca2+ sparks occur spontaneously in unstimulated primary cardiomyocytes primarily as a consequence of spontaneous Ca2+ release from the SR via the RyR,43 as demonstrated by the complete inhibition of Ca2+ sparks by ryanodine . The frequency of Ca2+ sparks in cardiomyocytes treated with different concentrations of Ned-K was not significantly altered, even at concentrations up to 300 µmol/L . However, at the highest concentration used (100 µmol/L), Ned-19 inhibited Ca2+ spark frequency from 2.7 ± 0.4 Ca2+ sparks/s in controls to 0.9 ± 0.2 Ca2+ sparks/s . Similarly, while Ca2+ transient amplitude in response to electrical pacing was unaltered by the presence of 100 µmol/L of Ned-K , their amplitude was significantly decreased by 100 µmol/L of Ned-19 , with Ca2+ transients completely eliminated in 30% of cells analysed. These results suggested that 100 µmol/L of Ned-19 affects SR Ca2+ handling either by inhibiting the RyR or decreasing SR Ca2+ content, and subsequent experiments measuring the release of SR Ca2+ by treatment with caffeine indicated that the latter is the case . Ned-K, however, appears to be more specific as it has no such direct effect on SR Ca2+ handling.To confirm that the protection and suppression of Cain vivo mouse model of ischaemia and reperfusion injury. The left anterior descending coronary artery of anaesthetized mice was occluded for 30 min, followed by a period of reperfusion for 2 h, after which time the heart was removed and the extent of infarction in the area at risk measured by tetrazolium chloride staining and Evans blue. In control mice, injected with the vehicle 5 min before reperfusion, infarct size measured 51 ± 9% (n = 5) of the ischaemic area ‘at risk’ . The infarct size in mice injected with Ned-19 was slightly lower but not significantly different from controls . Injection of Ned-K, however, caused a significant reduction in infarct size .Next, we investigated whether Ned-19 or Ned-K would protect against cardiac ischaemia and reperfusion injury in an 2+ channel in the lysosomal membrane that is regulated by NAADP.27 We therefore probed the role of TPC1 in injury using mice lacking TPC1.33 TPC1 knockout mice have no obvious phenotype, but after subjecting them to experimental myocardial infarction they were revealed to be protected against cardiac ischaemia and reperfusion, with infarct sizes of 32 ± 4% of the area at risk, compared with 51 ± 5% of the area at risk in littermate wild-type controls . Taken together, the data in Figure  and B show that both chemical and molecular inhibition of NAADP signalling in vivo reduces ischaemia and reperfusion injury.We tested whether genetic suppression of the NAADP signalling pathway would be similarly cardioprotective. TPC1 has been proposed to form a Ca44 was used to quantify NAADP in hearts subjected to global ischaemia and reperfusion. NAADP levels in control perfused mouse hearts were 8.3 ± 2.0 fmol mg−1 protein . NAADP was significantly reduced after a period of 30 min global ischaemia to 2.0 ± 0.3 fmol mg−1. Upon reperfusion, NAADP levels recovered, though they did not increase above baseline levels. However, these data show that NAADP levels are dynamically regulated during ischaemia and reperfusion further supporting a role for NAADP signalling in injury at reperfusion.To test whether ischaemia and reperfusion injury was associated with changes in NAADP levels, mouse hearts were isolated and perfused using a Langendorff apparatus. An improved enzymatic cycling assay46 in which primary adult cardiomyocytes are loaded with the fluorescent dye TMRM, which accumulates in mitochondria and generates ROS in response to confocal laser-induced phototoxicity. This results in Ca2+ overload culminating in mPTP opening and cell death.46 We measured the time taken under continual confocal laser scanning before mPTP opening occurs, visualized by the rapid redistribution of TMRM from the mitochondria to the cytosol, and by an abrupt increase in fluorescence as the dye dequenches in the cytosol . As a positive control, ciclosporin A (CsA) significantly delayed the time to mPTP opening compared with vehicle by 55 ± 7% . There was a significant delay in mPTP opening in the presence of 10 or 100 µmol/L of Ned-K  or Ned-19 . In this model, ROS exposure caused a 2.4-fold increase in [Ca2+]cyto, detected using Fluo4-AM , and this increase was suppressed in the presence of 10 µmol/L of ryanodine or, 1–100 µmol/L of Ned-K . Since this is the first time that a link between Ca2+ release from acidic stores and mPTP opening has been demonstrated, we performed a further experiment in which cardiomyocytes were pretreated for 30 min with bafilomycin A to assess whether this would have any effect on mPTP opening. In line with our hypothesis, pretreatment with 100 nM bafilomycin A significantly delayed the time to mPTP opening by 41 ± 25% . The delay in mPTP opening was not due to a direct effect of the Ned drugs on the mPTP, since Ca2+-induced swelling of isolated mitochondria was unaffected by their presence .Finally, we investigated the mechanism of cardioprotection by Ned-K by examining whether it could prevent opening of the mPTP. We used a previously validated model,4.2+ oscillations in primary adult cardiomyocytes can be suppressed by treatment with Ned-19, or, more effectively, with an improved form of Ned-19 called Ned-K. Although Ned-19 decreased SR Ca2+ content at higher concentrations, Ned-K had no effect on SR Ca2+ content, and did not alter spontaneous Ca2+ spark frequency or electrically stimulated Ca2+ transient amplitude. While both drugs protected primary cardiomyocytes against sIR injury in vitro, Ned-K was uniquely protective against IR injury in vivo, in accordance with its improved pharmacological properties. Genetic deletion of TPC1 was similarly protective in mice. The protective effects of Ned-K were mirrored in vitro by treatment with GPN to eliminate lysosomes. Mechanistically, protection is likely via prevention of reperfusion-induced Ca2+ oscillations that trigger mPTP opening in cardiomyocytes.Our results demonstrate that reperfusion-induced Ca2+ from lysosomes which then triggers SR/ER Ca2+ release,49 in much the same way as sarcolemmal Ca2+ entry triggers SR Ca2+ release during excitation–contraction coupling.47 Indeed, in recent years, lysosomes have been gaining attention, with the growing realization that their function extends beyond the ‘garbage disposal’ role in protein degradation originally ascribed to them. They are now recognized as an important store of Ca2+, containing ∼400–600 µmol/L in macrophages, and ∼550 µmol/L in fibroblasts.39 Our observation that pretreatment of cardiomyocytes with bafilomycin A to deplete lysosomal Ca2+, both delayed mPTP opening and protected against sIR, supports a mechanism in which NAADP stimulates the release of lysosomal Ca2+, which then triggers Ca2+ oscillations during reperfusion.The mechanism of action of NAADP is not entirely clear. Increasing evidence supports a ‘trigger mechanism’ in which NAADP causes release of Ca2+ channel during reperfusion may sensitize it to NAADP, in a manner similar to the redox regulation of RyR2.50 We have previously shown that intracellular oxidation and cytosolic Ca2+ overload occur together in highly localized regions during reperfusion of the myocardium, leading to mPTP opening.3Interestingly, there was a significant reduction in total cardiac NAADP content after a period of 30 min global ischaemia which returned to basal levels by 5 min reperfusion. We speculate that oxidation of the lysosomal Ca2+ oscillations are caused by the cyclic release and uptake of Ca2+ from the SR. While we have shown that inhibition of lysosomal release channels reduces these oscillations, other mechanisms could affect Ca2+ SR load and triggering of SR Ca2+ release during ischaemia or reperfusion, such as ATP depletion which prevents Ca2+ pumps from functioning. Other transporters, such as the plasmalemmal Na+/Ca2+ exchanger, contribute indirectly to Ca2+ oscillations by increasing cytosolic Ca2+ overload during reperfusion.52 Notably, in these studies, the complete absence of extracellular Ca2+ during reperfusion only reduced the oscillation frequency by ∼50%,51 which reflects the fact that internal Ca2+ sources must contribute to the oscillations.Reperfusion-induced Ca2+ release activity. Similarly, Ned-19 inhibited NAADP-induced Ca2+ release in mouse pancreatic beta cells, and reduced glucose-induced Ca2+ increase in islets. By replacing the fluoride in Ned-19 with a cyano group in Ned-K , we improved its cLogP value from 4.6 to 4.05. Although the change in cLogP value may appear marginal, this is a tangible outcome given that values are logarithmic. The TPSA is a measure of the polar contribution of groups such as O, N, and –CN and is related to how hydrated a molecule can become in aqueous environments. This value was improved from 80.8 in Ned-19 to 105 in Ned-K. More importantly, it seems that the receptor sites or ion channels involved in cardioprotection may have water molecules that can bridge between the peptide residues and the cyano group in Ned-K.Some differences were observed between Ned-19 and Ned-K, which may be ascribed to the changes in chemical structure. Ned-19 was previously identified in a virtual screen using NAADP as the query ligand. Using the sea urchin egg homogenate assay, it was verified as inhibiting NAADP-stimulated Ca53 Given that NAADP stimulates Ca2+ oscillations, and Ca2+ oscillations cause mitochondrial Ca2+ overload and mPTP opening, we hypothesized that the mechanism of protection of NAADP inhibitors would be via inhibition of the mPTP. We used a model of ROS-induced mPTP opening in primary cardiomyocytes. Ca2+ was shown to increase in this model and contribute to mPTP opening. The involvement of lysosomes in this model was confirmed by the delay in mPTP opening observed after pretreatment with bafilomycin A to empty acidic stores . In accordance with our hypothesis, we found that Ned-19 and Ned-K inhibited mPTP opening in cells , but that this effect was indirect, since they did not prevent Ca2+-induced swelling in isolated mitochondria . The mPTP represents a common end target of many pharmacological cardioprotective agents, although often the exact mechanism by which they prevent mPTP opening is not known.54 It may be interesting to evaluate whether any of these agents affect lysosomal Ca2+ release.Opening of the mPTP is a major step on the path to cell necrosis and IR injury, as shown by experiments with mice lacking the cypD component of the mPTP or treated with CsA, which binds to and inhibits cypD.in vitro, sea urchin egg homogenate assay of NAADP inhibition, only Ned-K was found to be cardioprotective in vivo. The exact reason for this difference is not known, but may be related to differences in pharmacokinetic profile. An alternative possibility is that Ned-19 has an additional detrimental off-target effect—perhaps one related to the effect it was observed to have in vitro on depleting SR Ca2+. We attempted to measure tissue content of Ned compounds by HPLC after administration in vivo, but the levels were at or below the lower limit of detection. A further limitation of the study is that we did not test cardioprotection of Ned-K in the TPC1 knockout mice, which would have helped to confirm its mechanism of action.Despite Ned-19 and Ned-K having similar activities in an 2+-permeable channels.28 NAADP-mediated Ca2+ signals are thus potentiated when TPCs are overexpressed and inhibited by siRNA, gene knockout, and dominant-negative TPC constructs (reviewed in Hooper and Patel48). In two thorough, but highly controversial publications, data have been presented indicating that TPCs are not Ca2+ channels, but rather Na+-selective ion channels that are insensitive to NAADP and instead activated by the phosphoinositide PIP2 and ATP depletion.55 These discrepancies have been partly reconciled by a recent study which defined ionic conditions uncovering co-regulation of TPCs by NAADP, PIP2, and ATP.56 Thus, during ischaemia, ATP depletion might contribute to activation of TPCs by NAADP and/or PIP2. Further work, however, is required to determine whether NAADP-dependent Ca2+ oscillations reported here are a direct consequence of Ca2+ flux through TPCs. Nevertheless, recent pharmacological57 and genetic59 approaches reaffirm the importance of TPCs in NAADP-mediated Ca2+ release.28 In support, both NAADP antagonism and TPC1 knockout afford protection against reperfusion injury highlighting the therapeutic potential of this pathway.Deletion of TPC1 protected against ischaemia and reperfusion injury, suggesting that it is functionally important in the ventricular myocardium. Much evidence has accumulated recently identifying TPCs as NAADP-regulated CaCardiovascular Research online.Supplementary material is available at MRC (EAA/17568), theBritish Heart Foundation (PG-10-005), and by theBiotechnology and Biological Sciences Research Council grant numbers (BB/G013721/1 and BB/K000942/1). The work was undertaken at UCLH/UCL who received a proportion of funding from the Department of Health's NIHR Biomedical Research Centres funding scheme. Funding to pay the Open Access publication charges for this article was provided by the MRC and BHF.This work was supported by


Mbnl1 abrogates transformation and impairs the fibrotic phase of wound healing in mouse models of myocardial infarction and dermal injury. Mechanistically, MBNL1 directly binds to and regulates a network of differentiation-specific and cytoskeletal/matrix-assembly transcripts to promote myofibroblast differentiation. One of these transcripts is the nodal transcriptional regulator serum response factor (SRF), whereas another is calcineurin Aβ. CRISPR-Cas9-mediated gene-editing of the MBNL1-binding site within the Srf 3′UTR impairs myofibroblast differentiation, whereas in vivo deletion of Srf in fibroblasts impairs wound healing and fibrosis. These data establish a new RNA-dependent paradigm for myofibroblast formation through MBNL1.The differentiation of fibroblasts into myofibroblasts mediates tissue wound healing and fibrotic remodelling, although the molecular programme underlying this process remains poorly understood. Here we perform a genome-wide screen for genes that control myofibroblast transformation, and identify the RNA-binding protein muscleblind-like1 (MBNL1). MBNL1 overexpression promotes transformation of fibroblasts into myofibroblasts, whereas loss of  Fibroblast-to-myofibroblast differentiation is crucial for wound healing and regeneration. Davis et al. describe a new regulatory mechanism underlying myofibroblast differentiation via the RNA-binding protein MBNL1, which promotes the maturation of certain mRNA transcripts that are integral nodes in fibroblast differentiation. Most forms of cardiac injury and disease elicit a universal wound healing-like process similar to that described in the skin whereby extracellular matrix (ECM) is generated during a fibrotic response to modify the structural characteristics of the tissue124567822351232+ signalling mediated by calcineurinA fundamental initiator of myofibroblast differentiation is the cytokine transforming growth factor β (TGFβ), which is part of a cytokine milieu that is generated within an injury environment. TGFβ initiates intracellular signalling through both canonical SMAD and non-canonical mitogen-activated protein kinase (MAPK)-p38 kinase signalling4MBNL1 is a highly conserved RNA-binding protein that modulates alternative splicing, alternative polyadenylation, mRNA stability and mRNA localization by directly binding to select transcripts1213151617192116Here, we demonstrate that MBNL1 is induced within the fibroblast during an injury response to bind and regulate a selective population of mRNAs that directly programme myofibroblast differentiation. MBNL1 regulated the maturation of the mRNA transcript for SRF and calcineurin Aβ, which increased their apparent activity and augmented myofibroblast differentiation. Indeed, deletion of the MBNL1-binding site in the 3′ untranslated (UTR) of the SRF transcript reduced the efficiency of myofibroblast differentiation.Postn gene), which was also induced at the promoter level by MBNL1  under serum-reduced conditions with an αSMA promoter-containing luciferase reporter plasmid as a transcriptional readout for myofibroblast transformation . Baselinby MBNL1 . To funcby MBNL1 . MBNL1-eby MBNL1 .Mbnl1−/− and wild-type (Mbnl1+/+) mice were treated with TGFβ and examined for αSMA stress fibre expression  mouse model in which expression of MBNL1 can be increased by Cre-dependent recombination  cassette inserted within the Tcf21 locus  had minimal fibrotic staining. These results indicate that induction of MBNL1 expression in quiescent tissue-resident fibroblasts induces a fibrotic response likely by initiating myofibroblast differentiation that mimics the injured state.To achieve MBNL1 expression in quiescent fibroblasts pression . With foand lung . At thisand lung . In compTcf21MCM and NTG littermates were given 2 weeks of tamoxifen and then subjected to a chronic fibrotic stimulus in which a mixture of AngII and phenylephrine (PE) were delivered by osmotic mini-pump for 2 weeks. Immediately after pump removal, the hearts were excised and fibrosis was determined by Masson's trichrome staining  or treated with TGFβ  from ic genes . The mRNreatment . Consistntiation . We werentiation .To identify RNAs that are directly regulated by MBNL1, RNA immunoprecipitation (RIP) assays were performed as illustrated in In comparing MBNL1-bound transcripts (MBNL1 Rip-Seq) to upregulated genes from the input (MBNL1 OE Upregulated), only 77 genes were overexpressed, but remarkably all except three of those genes are bound by MBNL1 . We also17shown here) as well as SRF, which were all reconfirmed here (Fn1) as well as the splice variant (FnEDa) characteristic of myofibroblast transformationThe RIP data identified mRNA targets corresponding to several signalling nodes that positively influence myofibroblast differentiation . We usedmed here . These oMBNL1 RIP target RNAs were also analysed by Gene Ontology biological networks and there was a clear enrichment in differentiation and developmental processes similar to the total RNAseq gene expression profile described earlier. These processes included vasculature development, regulation of development, cardiovascular system development, regulation of cell migration, tube development and epithelial-to-mesenchymal transition, in addition to strong enrichment in signatures of late mesoderm-associated genes. These MBNL1-bound transcripts included SRF, Twist1, FoxO3, Runx1, Cxcl12 and Il6st, which were all validated and quantified by RT–PCR here .We have previously shown the requirement for TGFβR2, SRF and CnAβ in fibroblast to myofibroblast differentiationMbnl1−/− mice after MI  null MEFs, which are devoid of all calcineurin functionMbnl1−/− MEFs transformed ∼70% of the fibroblast population into myofibroblasts similar to the level of conversion achieved in Mbnl1+/+ MEFs, demonstrating that calcineurin functions downstream of MBNL1 to promote myofibroblast differentiation  infection (Srffl/fl+AdCre) were refractory to MBNL1-mediated myofibroblast differentiation when compared with MEFs replete with SRF , suggesting that SRF is also a critical downstream mediator of MBNL1-directed differentiation, and that the ability of MBNL1 to augment SRF transcript stability is another critical mechanism for myofibroblast differentiation regulated through MBNL1  to map potential MBNL1-binding sites in the 3′UTR of SRF, followed by CRISPR-Cas9 gene editing to delete putative sites (SrfMBNL CRISPR). A control cell line that contains an innocuous green fluorescent protein (GFP) cassette with Cas9 was also used for comparison. Of the predicted binding sites, five of them had sequences consistent with those identified as preferred MBNL1 targetsSrf gene was mutated, compared with a Cas9-GFP control cell line that had a wild-type Srf gene  when compared with control fibroblasts . Loss of Srf from activated skin fibroblasts not only prevented wound healing over 6 days, but also it caused wound rupture and expansion  and focal adhesion regulation  micePostnMCM (C57BL/6-sv129) mice were generated by inserting the MerCreMer cDNA cassette into the first coding exon of the Postn gene by standard homologous recombination in embryonic stem cells, followed by generation of germline-targeted mice. Srf-LoxP, CD-1 background-targeted mice were described previouslyTcf21MCM knock-in allele was activated by feeding the mice tamoxifen citrate chow  for at least 2–4 weeks unless otherwise stated. For the dermal wound-healing model, fully anaesthetized mice received two dorsal 6 mm excision punch biopsies, and the wounds were subsequently assessed for size and health at the time of injury for up to 6 days31−1 of collagenase. Fibroblasts were isolated by allowing the total cell mix from the hearts to settle by gravity for 10 min. The supernatant was centrifuged for 5 min at 1,000g, resuspended in DMEM with 10% fetal bovine serum (FBS), and then plated on gelatin-coated dishes for primary cell culture. A minimum of three passages was used to achieve a pure fibroblast populationMbnl1+/+, Mbnl−/− , Ppp3r1−/− ref.  MEFs wer2 section of the dorsal skin was dissected free down to the muscle layer. The skin explant was digested in 0.25% trypsin overnight at 4 °C. The skin was then minced and digested for 2–4 h in collagenase type I at 37 °C. The skin was triturated and resuspended in DMEM with 10% FBS. Cell lines were not tested for mycoplasma.Primary dermal fibroblast were isolated by enzymatic digestion of mouse skin explants−1, R&D System), AngII  or adenoviral gene transfer and analysed 48 h later by immunocytochemistry for αSMA stress fibres  and gene expression as described previouslyMyofibroblast transformation was induced using recombinant porcine TGFβ . The recombinant adenovirus was plaque purified, expanded and cesium chloride gradients were used to purify and concentrate the virus43143Postn)-luciferase promoter reporter was also used to confirm activityPostn promoter was cloned upstream of luciferase . This construct was co-transfected  into fibroblasts with either an MBNL1 or control (pcDNA) plasmid and treated with vehicle in media or recombinant TGFβ (10 ng ml−1). Luciferase activity was measured 48 h post transfection.The Mammalian Genome Collection library, which contains ∼18,400 human and rodent cDNAs, was seeded with one clone per well, 40 ng each, in a 384-well format . This library was co-transfected  with an equal amount of αSMA-luciferase reporter construct into SV40-transformed MEFs and luciferase activity was assessed 2 days after transfectionTRIzol extraction. Total RNA was reverse transcribed using Superscript III first-strand synthesis kit (Invitrogen). The primer sequences used for all PCR reactions are as follows: IL6st: 5′- CAGATCGAGCAGAATGTGTATGGAATC-3′ , 5′- ATTGGATGTCCGACTTCAGCC-3′ ; Runx1: 5′- CGAGATTCAACGACCTCAGGTT-3′ , 5′- ACAGGAGGCGAGTAGGTGAA-3′ ; Cxcl12: 5′- CCAACGTCAAACATCTGAAAATCCTCA-3′ , 5′- GTGGAAGGTTGCTACTCCCC-3′ ; FnEDa: 5′- CCCACCGTGGAGTATGTGG-3′ , 5′- AGCCCTGACACAATCACGGA-3′ ; Rock2: 5′- ATGCAGATTCACTTGTAGGAACCTACAG-3′ , 5′- TGTTCCAGTTCCTCCTTGGA-3′ ; SRF: 5′- GGGAAACCAAGGACACACTGA-3′ , 5′- ATAAGTGGTGCCGTCCCTTG-3′ ; α1Catenin: 5′- CGACTTCACCCGAGGCAAA-3′ , 5′- CCCCACAGTAGGGCTAAAGG-3′ ; Arhgef11: 5′- CACGCTACGGGGAACTGTTT-3′ , 5′- CTGTCTTCCAGGGCGATGTC-3′ ; Twist1: 5′- GTCGCTGAACGAGGCATTTG-3′ , 5′- GACATCTAGGTCTCCGGCCT-3′ ; FoxO3: 5′- TCATCTGTTTGTACCCGTCGAA-3′ , 5′- CTCTTGCTCTCTCCTCTCGC-3′ ; TGFβR1: 5′- TGGCAGAGCTGTGAGGCCTTGA-3′ , 5′- CCCTAAGGGCTGCCGGTCGT-3′ ; TGFβR2: 5′- CAGGGGTGCTCCACT-3′ , 5′- CCAACAGCGGGCAGGTGGGA-3′ ; CnAβ: 5′- GATGTAGGTTCAGCTGCAGCC-3′ ; 5′- GTCCCGTGGTTCTCAGTGGTA-3′ ; CnAβ1: 5′- GATGTAGGTTCAGCTGCAGCC-3′ , 5′- CCAGTCGATCTGAGGCACAGC-3′ ; MBNL1: 5′- TAGTGTCACACCAATTCGGGACACAAA-3′ , 5′- CCCTTGATGTAATCCATGCAGACAGTGA-3′ ; Gapdh: 5′- GACATGCCGCCTGGAGAAAC-3′ , 5′- AGCCCAGGATGCCCTTTAGT -3′ (rat); Gapdh: 5′- ATGTGCCGGACCTTGGAAG-3′ , 5′- CCTCGGGTTAGCTGAGAGATCA-3′ (mouse).For fibroblast experiments, RNA was isolated and prepared with an RNAeasy kit (Qiagen), and for skin wounds, RNA was prepared using de novo junction discovery. Alternative splicing, alternative polyadenylation and differential gene expression analyses along with all visualized pathways, heat maps and Venn diagrams were obtained using AltAnalyze version 2.0.9  overnight at 4 °C. The resin was washed and RNA extracted using a Magna RIP kit (Millipore) and phenol-chloroform precipitation. Eluted RNAs were detected by RNAseq analysis using paired-end 100 nucleotide (nt) sequencing and 40 million reads for input-RNAs and RIP-RNAs. FASTQ sequence files were aligned to the rat genome version 5 using TopHat2 with 0.9 ref. , and theSrf gene of NIH 3T3 fibroblasts (American Type Culture Collection). Stable cell lines for the mutant Srf (SrfMBNL) and a GFP-Cas9n control were created using puromycin selection (1.0 μg ml−1). MBNL1-binding sites in SRF's 3′ UTR were predicted using RBPmap software that further ranks the motif based on local clustering of sites and its conservationUGCUGUG -3′. We created a CRISPR-mediated deletion, which excised both sites using the following guide and protospacer-associated motif (PAM) sequences: 5′- CAACGTGTGCTGTGTGATTG-3′ and TGG. A reverse sequence left shifted by nine base pairs was used to make the other deletion on the sense strand. The single guide RNA (sgRNA) was delivered using the pX462 expression plasmid that contains both the D10A mutant Cas9 nickase and a puromyocin selection cassette. For clarity, the CRISPR-targeted Srf sequence is illustrated below with the location of the two sgRNAs (blue brackets), the PAM (red text), and the mutated MBNL1-binding sites (green text).CRISPR-Cas9 technology was used to mutate predicted MBNL-binding sites in the The deletion was confirmed by sequencing across the deletion using genomic DNA obtained from the CRISPR cell line and the new sequence was as follows:Analysis of the excised region by miRanda software indicated there was no disruption of known microRNA target sites. For orientation, the three bases of the PAM sequence is shown in red again, and the guide sequence is shown in yellow.SrfMBNL CRISPR-mutated fibroblasts were infected or transduced with MBNL1 for 36 h at which time transcription was inhibited with 2 μg ml−1 Act.D (Sigma), and RNA was isolated 0, 2, 4 and 8 h later and analysed by real-time PCR.Neonatal cardiac fibroblasts or t-tests were used to compare between two groups, and a one-way analysis of variance was used for multivariate analysis with subsequent pairwise comparisons made using Newman–Keuls tests (Prism software). The mean±the standard error of the mean (s.e.m.) is reported in all figures unless otherwise noted, statistical significance was set a P<0.05 and tests for normality were not performed. In vitro experiments were repeated three times and animals from multiple litters were used for in vivo studies to ensure repeatability. Sample size for animal experiments was determined based on previously reported standards in the field for achieving statistical power, no samples were excluded from the analysis and experiments were not randomized. Data were collected and analysed blinded for both in vitro and in vivo studies.To determine statistical significance, unpaired Accession Codes: RNA-Seq data have been deposited in GEO (Gene Expression Ominbus) of NCBI under accession code GSE74185.How to cite this article: Davis, J. et al. MBNL1-mediated regulation of differentiation RNAs promotes myofibroblast transformation and the fibrotic response. Nat. Commun. 6:10084 doi: 10.1038/ncomms10084 (2015).Supplementary Figures 1-7 and Supplementary Tables 1-2A list of MBNL1-bound RNAs (MBNL1 RIP-Seq) as categorized by whether they were alternative spliced, alternatively polyadenylated, or bound with undetermined regulation. Also listed here are RNAs that were alternatively spliced or polyadenylated secondarily in the AdMBNL1 input but not targeted by MBNL1. Transcripts validated by real-time PCR are represented by blue text. Refers to Figure 4.


However, the augmentation of exocytosis induced by an elevation of the free cytosolic [Ca2+] to ~1.5 μM (9 mM Ca2+ + 10 mM EGTA) was potent at 32°C or 37°C but less distinct at 27°C and abolished at 23°C. Adiponectin secretion stimulated by 30 min incubations with the membrane permeable cAMP analogue 8-Br-cAMP (1 mM) or a combination of 10 μM forskolin and 200 μM IBMX was unaffected by a reduction of temperature from 32°C to 23°C. At 32°C, cAMP-stimulated secretion was 2-fold amplified by inclusion of the Ca2+ ionophore ionomycin (1μM), an effect that was not observed at 23°C. We suggest that cooling affects adipocyte exocytosis/adiponectin secretion at a Ca2+-dependent step, likely involving ATP-dependent processes, important for augmentation of cAMP-stimulated adiponectin release.We investigated the effects of temperature on white adipocyte exocytosis (measured as increase in membrane capacitance) and short-term adiponectin secretion with the aim to elucidate mechanisms important in regulation of white adipocyte stimulus-secretion coupling. Exocytosis stimulated by cAMP (included in the pipette solution together with 3 mM ATP) in the absence of Ca Adiponectin is a protein hormone secreted exclusively from white adipose cells and involved in modulation of glucose and lipid metabolism i) was recorded with dual-wavelength ratio imaging as previously described [2+]i using equation (5) of [d of 224 nM. Values of maximum (Rmax) and minimum (Rmin) ratios as well as Sf2/Sb2 were determined using solutions with a high Ca2+ concentration (12 mM CaCl2 + 10 mM EGTA) or lacking Ca2+ . Measurements were carried out at 23°C or 32°C as indicated.The intracellular Caescribed . Brieflyn (5) of  and a Kd3T3-L1 adipocytes were grown and differentiated in 6- or 12-well plates  and secretion studies were carried out as previously described . BrieflyC/Δt) was measured by applying a linear fit to the curve at specified time points as shown in The rate of capacitance increase (Δ2+-free pipette solution containing 0.1 mM cAMP (10 mM EGTA and 3 mM ATP included) known to trigger adipocyte exocytosis [C/Δt) was measured at the indicated time intervals as shown in 2 (free [Ca2+] ~1.5 μM). In agreement with what we have previously reported i resulting in a peak at t = ~3 min, after which [Ca2+]i declined towards baseline before stabilising at a plateau at 10 min .3T3-L1 adipocytes secrete substantial quantities of adiponectin  and we ht 10 min . The magse sites . Inclusi2+-dependent step involved in 3T3-L1 adipocyte stimulus-secretion coupling and further underscore the strong association between 3T3-L1 adipocyte membrane capacitance increases and release of adiponectin.As can be seen in 2+. We have recently shown that an elevation of cytosolic Ca2+ is necessary in order to achieve robust cAMP-stimulated adiponectin secretion as well as to maintain secretion over longer time-periods . Thus, 2+. ATP-dependent processes are involved in maintenance of exocytosis in a magnitude of secreting cell types  and hydiewed in ,20.10 value of 3.3 and the energy of activation (53 kJ mol-1) derived from the capacitance recordings are lower than that reported in pancreatic beta-cells (5 and 145 kJ mol-1 respectively) [Our calculated Qctively) . Howeverctively) . Subcutactively) ,23. In aTaken together, the results of this study show that white adipocyte exocytosis/adiponectin secretion encompasses energy-dependent processes. It is conceivable that control of vesicle recruitment signifies an essential mechanism by which the white adipocyte adjusts secretion of adiponectin in response to increased physiological requirements. The fact that adiponectin levels are reduced in obese individuals combined with observations that elevated levels provide protection towards development of type-2 diabetes in normal-weight individuals  emphasis


Renal angiomyolipoma (AML) is a benign renal tumor with a risk of rupture in intratumoral aneurysms. Although renal AML in pregnancy is rare, risk of rupture is greater. Management for AML and childbirth is important during pregnancy; however, it is undefined yet. We present a case of hemorrhagic angiomyolipoma in pregnancy that is effectively managed by immediate cesarean section and elective transcatheter arterial embolization. Renal AML can spontaneously rupture and cause retroperitoneal hemorrhage. Although renal AML in pregnancy is relatively rare, due to changes in blood flow and pressure, compression by the enlarged uterus, and hormonal influence, it has a greater risk of rupture, consequently leading to medical emergency such as maternal shock or fetal death. Therefore, it is difficult to establish therapeutic strategies to manage both AML rupture and safe delivery. We present the case of a hemorrhagic AML in pregnancy managed effectively by immediate cesarean section and elective transcatheter arterial embolization (TAE).RA 30-year-old woman in her first 19th week of pregnancy with asymptomatic gross hematuria was referred to our department for the treatment of a left renal mass 12 cm in diameter, which was confirmed by computed tomography (CT) in a previous hospital . Gross hAt the time of the consultation, the patient and fetus were hemodynamically stable. However, considering the AML rerupture, we decided to perform an immediate cesarean section earlier than the previously scheduled date and then a selective TAE to treat the reruptured AML. A healthy infant was delivered by a cesarean section, which was followed by an angiography and selective TAE. Angiography showed two AML feeding arteries and two aneurysms, one on each of the arteries . Althoug1 These benefits are even more important for management of AML in pregnancy. However, only a few cases of AML rupture during pregnancy treated with TAE were reported so far. When considering the potential risk of carcinogenesis, the radiation exposure to the fetus must be minimized. Furthermore, a consensus on the modifications of TAE-related dose limits for radiation exposure when treating AML in pregnancy has not yet been reached. Recently it has been suggested that the decision to perform TAE should be based on hemodynamic stability of the mother and fetus.2 If they are stable, conservative management including close follow-up or elective TAE is considered first. In our case, we decided for a conservative management of the first AML rupture, because hemorrhage was not significant and both the patient and fetus were hemodynamically stable. However, when the AML rerupture occurred, we decided for invasive management of both the hemorrhagic AML and pregnancy. At that time, hemodynamic stability in both the patient and the fetus was recognized. However, CT findings clearly showed a retroperitoneal hemorrhage caused by an AML rupture. Furthermore, the pregnancy was close to the date of the scheduled elective cesarean section. Thus, considering several factors including the hemodynamic stability of mother and fetus, number of weeks in pregnancy (possibility of cesarean section), and the extent of hemorrhage, active treatment for ruptured AML should be considered in case of reruptured AML in pregnancy.The most severe problem related to renal AML is spontaneous hemorrhage. Because it is minimally invasive and also provides an opportunity for renal preservation, TAE has been the standard intervention for treatment of AML rupture and prevention of rerupture.


It has previously been shown that SDF1/CXCR4-induced migration is dependent on activation of downstream BTK in chronic lymphocytic leukaemia (CLL) and multiple myeloma. Here we show that SDF-1 induces BTK phosphorylation and downstream MAPK signalling in primary AML blast. Furthermore, we show that ibrutinib can inhibit SDF1-induced AKT and MAPK activation. These results reported here provide a molecular mechanistic rationale for clinically evaluating BTK inhibition in AML patients and suggests that in some AML patients the blasts count may initially rise in response to ibrutinib therapy, analgous to similar clinical observations in CLL.Pharmacological targeting of BTK using ibrutinib has recently shown encouraging clinical activity in a range of lymphoid malignancies. Recently we reported that ibrutinib inhibits human acute myeloid leukemia (AML) blast proliferation and leukemic cell adhesion to the surrounding bone marrow stroma cells. Here we report that in human AML ibrutinib, in addition, functions to inhibit SDF1/CXCR4-mediated AML migration at concentrations achievable  Despite recent significant progress in the understanding of the biology of AML the clinical outcomes for the majority of patients diagnosed with AML presently remain poor. AML is primarily a disease of the elderly, and forBruton’s Tyrosine Kinase (BTK) is a non-receptor tyrosine kinase that belongs to the Tec family and clearly has an important function in a number of benign and malignant cells of the haematopoietic system -7. MoreoInhibition of BTK has been shown to regulate CLL, MCL and MM cell migration by inhibiting SDF1  induced CXCR4 regulated cell trafficking -22. SDF1Previously we have shown that ibrutinib inhibits AML proliferation and adhesion to BMSC. In myeloma and CLL ibrutinib inhibits CXCR4/SDF1-mediated migration , 25. Morin vivo  [Since SDF1 has been shown to mediate migration of AML in a CXCR4 dependent mechanism and that SDF1/CXCR4-induced migration is dependent on activation of downstream BTK in CLL, myeloma and normal B cells , 27, we e a day) , 16. TheSince CXCR4 is a Gi-coupled receptor and pertussis toxin inhibits Gi-coupled receptor activation , we usedIt has been shown that ibrutinib has multiple kinase targets including interleukin-2-inducible kinase , as wellBTK is a cytoplasmic tyrosine kinase widely expressed in hematopoietic cells and long known to be critical in B cell differentiation and survival pathways. BTK is a member of the BTK/Tec family of tyrosine kinases . BTK actIn CLL and other B cell malignancies ibrutinib inhibits SDF1/CXCR4-induced tumor cell migration , 25. We Ibrutinib has been shown to be well tolerated with limited grade 3 and 4 toxicity across a broad age range of patients with CLL and MCL , 15, 16.In patients with CLL treated with ibrutinib an initial and sometimes persistent rise in the circulating lymphocyte count may be seen, even in the context of clinically responding disease . This isHere we show for the first time that in human AML ibrutinib inhibits SDF1/CXCR4 pathway and functions to disrupt migration signals from the microenvironment This study further validates an emerging focus of targeting kinases critical for the survival of malignant cells and gives us a clearer understanding of how inhibition of BTK may optimally be harnessed therapeutically in AML. In summary, we provide additional scientific support for clinical trials of ibrutinib in patients with AML.Anti-phosphorylated and pan AKT, BTK and MAPK antibodies were purchased from Cell Signaling Technology . Anti-CD34-PE and anti-CXCR4-FITC antibodies were purchased from Miltenyi Biotec . Ibrutinib was obtained from Selleck Chemicals. SDF1 was purchased from Miltenyi Biotec. Pertusis toxin was purchased from R&D systems . LY294002 and U0126 were purchased from Cell Signalling Technology. All other reagents were obtained from Sigma-Aldrich , unless indicated.AML cells were obtained from patients’ bone marrow or blood following informed consent and under approval from the UK National Research Ethics Service (LRECref07/H0310/146). For primary cell isolation, heparinized blood was collected from volunteers and human peripheral blood mononuclear cells (PBMCs) isolated by Histopaque  density gradient centrifugation. AML samples that were less than 80% blasts were purified using the CD34 positive selection kit (denoted by * in Table The AML-derived cell lines were obtained from the DMSZ (German Collection of Microorganisms and Cell Cultures) and European Collection of Cell Cultures where they are authenticated by DNA-fingerprinting. In the laboratory they are used at low passage number for a maximum of 6 months post-resuscitation, testing regularly for Mycoplasma infectionwww.invitrogen.com/rnai) and plasmid pcDNATM6.2-GW/EmGFP-miR-neg (Invitrogen) was used as source for negative control as previously described [MicroRNA sequence miRNA-BTK (5′-TTCACTGGACTCTTCACCTCT-3′) targeting human BTK was selected with Invitrogen Block-iT RNAi Designer software . The lower compartment contained 500μL of serum free media supplemented with 100 ng/ml SDF1 and the cells were applied to the upper compartment and allowed to migrate for 3 h. The amount of viable migrated cells was determined by counting using trypan blue exclusion and expressed as a percentage of the input. The bars represent the means ± SD of at least three independent experiments, each assayed in triplicate.Student’s T test was performed to assess statistical significance from controls. Results with P < 0.05 were considered statistically significant (*). Results represent the mean ± SD of 3 independent experiments. For Western blotting, data are representative images of 3 independent experiments.


Recent studies have revealed localized and dynamic endothelial Ca2+ events comprising an intricate signaling network along the vascular intima. Discrete Ca2+ transients emerging from both internal stores and plasmalemmal cation channels couple to specific membrane K+ channels, promoting endothelial hyperpolarization and vasodilation. The spatiotemporal tuning of these signals, rather than global Ca2+ elevation, appear to direct endothelial functions under physiologic conditions. In fact, altered patterns of dynamic Ca2+ signaling may underlie essential endothelial dysfunction in a variety of cardiovascular diseases. Advances in imaging approaches and analyses in recent years have allowed for detailed detection, quantification, and evaluation of Ca2+ dynamics in intact endothelium. Here, we discuss recent insights into these signals, including their sources of origination and their functional encoding. We also address key aspects of data acquisition and interpretation, including broad applications of automated high-content analysis.Although acute and chronic vasoregulation is inherently driven by endothelial Ca New findings have exposed a complex mosaic of physiologic endothelial Ca2+ signals , a Ca2+-binding protein containing a high-affinity EF-hands motif that allows for cellular responses with nanomolar Ca2+ changes. Key Ca2+-CaM dependent endothelial effectors include nitric oxide synthase (eNOS) that produces the diffusible vasorelaxing factor nitric oxide (NO) , KCa2.3, and KCa3.1  as well as asynchronous and synchronous Ca2+ waves control vascular tone through coordination of cellular activation and feedback regulation of constriction . Various strategies have been employed for vascular endothelial imaging including intravital microscopy , Ca2+ pulsars release intermittently from the endoplasmic reticulum through clusters of inositol 1,4,5-trisphosphate receptors (IP3Rs). Pulsars are similar to Ca2+ puffs, localized Ca2+ events previously described in Xenopus oocytes , and these ongoing events are blocked by inhibiting phospholipase C  increases the number of Ca2+-emitting sites along the intima and augments the frequency of events occurring at previously active sites. Thus, Ca2+ pulsars can be tuned acutely by Gq protein-coupled receptor (GqPCR) stimulation.Basal endothelial Ca2+ pulsar events occur predominantly around the nucleus and at distinct myoendothelial junction (MEJ) sites where endothelial cell projections form close contacts (and often heterocellular gap junctions) with smooth muscle cells through holes in the internal elastic lamina  may be communicated across MEJs, augmenting endothelial Ca2+ dynamics  non-selective cation channels  primarily target KCa3.1 channels concentrated in densities along the endothelial basolateral membrane and myoendothelial junctions. However, related KCa2.3 channels are distributed quite differently, residing primarily along endothelial cell–cell borders, associated with the plasma membrane protein caveolin , and once on, the attributes of the events are tunable over some range . In addition to frequency, analog signaling components include magnitude, duration, and spatial spread of Ca2+ events, all of which could affect the type and extent of effector recruitment. Ultimately, definitive tracking of discrete Ca2+ signaling patterns will be needed to reveal the nature and capacity of parameter expansion and decipher the idiosyncrasies of endothelial function and dysfunction.Expansion or redirection of inherent Ca2+ signals, a key challenge moving forward will lie in our ability to adequately and comprehensively characterize Ca2+ activity along the intact intima. Disparate approaches have been employed to measure and analyze Ca2+ data, often applying arbitrary, if any, selection criteria and providing little explication of spatial and temporal parameters. Regardless of experimental preparation and approach, some crucial criteria for acquisition and analysis should be considered. First, spatial or temporal under-sampling of Ca2+ fluorescent signals washes out discrete dynamics or misses them altogether. High-speed confocal imaging systems, particularly spinning disk platforms with high-quantum efficiency cameras, offer sub-micron spatial resolution with fast acquisition rates and high signal to noise ratios. Analysis of dynamic Ca2+ activity typically involves hand selection of an event within an image time-series and manual placement of a region of interest  around each event site for measurement of average fluorescence. Albeit straight-forward, this approach is tedious, time-consuming and prone to user-bias and error. Recent efforts have produced automated detection and analysis algorithms to extract signals from continuous image sequences. Evidence suggests that in addition to saving time and resources, automated analyses can avoid inconsistencies of manual analysis and identify signaling signatures within complex fluorescence data. Multiple software applications, particularly in neurobiology, have employed independent component analysis and watershed image segmentation to define individual cells within dense fields and to track region-specific deflections of Ca2+-dependent fluorescence . The algorithm also allows “batch” analysis of multiple parallel data sets. Such analysis is particularly useful because it can generate complete parameter distributions and provide practical quantification of replicate data sets or complex signal changes following perturbation . For instance, relative changes in binary  and analog signals  can be automatically calculated, plotted, and statistically evaluated in a series of experiments without intermediate data processing by the user. Regardless of specific approach, stringent analysis of large data sets will be a necessary step in decoding Ca2+ dynamics.Given the inherent complexity of endothelial CaOverall, automated analysis approaches have become useful for defining cell boarders, discerning cellular/subcellular fluorescence signals from statistical noise, and providing comprehensive quantitation of component signal parameters. Current limitations of such approaches primarily stem from narrowly targeted applications and disparate processing algorithms that can contribute to false-negatives or false-positives when data fall outside an optimal range. As discussed below, extended initiatives should promote more widely applicable tools capable of reducing complex and heterogeneous data sets to intuitive indices of functional signaling.Looking forward, careful consideration should be given to the limits and liabilities of experimental approaches. While high-speed confocal imaging is valuable for signal resolution, it imposes certain experimental restrictions, including thin-plane sampling (~ 1 μm) and low tolerance for tissue movement. Wire-mounted or pressurized arteries can be studied as intact segments, but endothelial exposure is very limited. On the other hand, open vessel preparations expose vast endothelial fields in a single plane but sacrifice tubular structure and functional assessment. Imposing rigid acquisition criteria may be impractical. Rather, strategies should be implemented and optimized to ensure adequate spatial and temporal resolution and prevent image artifact. Importantly, Nyquist sampling criteria should be satisfied  to ensure reliable signal quantification without signal aliasing or distortion. Automated detection software such as LC_Pro may be useful for optimization by identifying which spatial and temporal acquisition conditions achieve convergence of parameter values while avoiding oversampling. Stack registration software can also be employed as a data processing step to correct for spatial drift (x-y movement)  compared to a broad wave passing through the same ROI, even if both have the same absolute signal intensity. Also, a single fixed ROI may detect spill-over signal from nearby events over time, distorting quantification of site-specific activity. In addition, fixed ROI sampling can promote artifact due to x-y drift by allowing hot spots or even regions of high or low background fluorescence to move into and out of the measured region  over time. Such issues may be resolved by tracking each event individually in space and time, allowing a signal to define its own transient polygonal ROI without establishing a permanent sampling window. Finally, dynamic Ca2+ events are often represented as ratios of relative fluorescence change within an ROI . Defining appropriate base values can be challenging, particularly when photobleaching causes signal drift or high dynamic activity obscures the background. In addition to background correction algorithms, linear regression of time-course data can be applied for F0 designations (Francis et al., 0 values.While ROIs are convenient for assessing spatially discrete Ca2+ dynamics are complex, data are typically represented by a profile of parameters or parameter distributions rather than a single scalar value. This multidimensional description has the capacity to distinguish Ca2+ signaling modalities, such as responses to distinct stimuli or among different vascular beds. Notably, because perturbations can increase some parameters and decrease others, quantification, comparison, and interpretation of data can be quite complex. Future analysis and meta-analysis approaches will need to address this complexity, perhaps by tracking trends in global distribution profiles or by defining cumulative metrics that combine parameters into standard indices. Additional indices might also include site distribution, cell heterogeneity, and event synchrony. A growing number of analysis algorithms are available as open source packages and plug-ins, making them not only widely accessible but amenable to customization. The hope is that eventually, a suite of analysis modules could be employed universally for parameter compilation, data mining, and pattern recognition. This would allow a standard analysis scheme for comparison of data sets across labs and preparations. Still, the onus ultimately falls on investigators to extract data or composite parameters germane to their specific experimental questions.Because Ca2+ signaling modalities and effector distributions likely underlie the distinct functions of different circulations. Further dissection of this diverse activity will allow for identification of sub-modalities, and potentially distinct cell phenotypes within the intima. We submit that shifts in prevailing Ca2+ dynamics necessarily impact blood pressure and flow and may predict disease. Indeed, endothelial dysfunction is an overarching feature of cardiovascular pathology. It is therefore particularly imperative that future studies shift away from assumptions based on global Ca2+ changes and broad cellular protein concentrations and focus on spatially and temporally relevant aspects of real-time signaling. Ultimately, the development of a definitive and predictive model of endothelial function should allow for elucidation of specific control points and therapeutic targets.New insights suggest the endothelium functions as a continuum of dynamically regulated influences that are always engaged and are constantly adjusted. The prevailing CaThe 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.


The transport of small molecules between the cells occurs through gap junctions comprising connexin 43. Examples of cells coupled into networks include astrocytes, keratinocytes, chondrocytes, synovial fibroblasts, osteoblasts, connective tissue cells, cardiac and corneal fibroblasts, myofibroblasts, hepatocytes, and different types of glandular cells. These cells are targets for inflammation, which can be initiated after injury or in disease. If the inflammation reaches the CNS, it develops into neuroinflammation and can be of importance in the development of systemic chronic inflammation, which can manifest as pain and result in changes in the expression and structure of cellular components. Biochemical parameters of importance for cellular functions are described in this review.Several organs in the body comprise cells coupled into networks. These cells have in common that they are excitable but do not express action potentials. Furthermore, they are equipped with Ca In conditions that lead to inflammation, changes in several cellular parameters of coupled cell networks occur throughout many organs in the body. Inflammation is a physiological response to injury that is designed to remove dangerous stimuli, kill bacteria and viruses, remove cell debris, and initiate healing. It can persist or become exaggerated and may cause undesirable negative effects. Inflammation can be induced by several substances produced or released by tissues or by environmental factors, including circulating glucose, gut microflora, interleukins, endotoxins or other toxins, all of which have an impact on immune receptor expression. The underlying molecular processes are just beginning to be elucidated.When an injury occurs in peripheral tissue, pro-inflammatory mediators are released into the bloodstream, and white blood cells are attracted to the injury site. The endothelium lining the blood vessels becomes permeable, allowing leukocytes to migrate from the blood vessels to the injury site , 2. The Neuroinflammation can also be initiated when a local peripheral injury gives rise to inflammatory activation in the CNS at the site of the damaged or affected nerve(s) –9. The iIf this dysfunction persists for a long time, it can lead to pathogenic chronic neuroinflammation and can transition into long-term pain , 9, 17. 2+-based excitability [2+ changes are important due to their influence on many cell functions, including matrix synthesis and degradation [2+ levels can lead to the release of signaling molecules such as transmitters, cytokines, prostaglandins, proteins, and peptides via regulated exocytosis [2+, adenosine triphosphate (ATP), nicotinamide adenine dinucleotide (NAD+), glutamate, prostaglandins, and glutathione, which are less than 1.5 kDa in size, between contiguous cells [Similarities exist between different types of coupled cell networks in different body organs with respect to several cellular parameters. Examples of cells coupled into networks include astrocytes, keratinocytes, chondrocytes, synovial fibroblasts, osteoblasts, connective tissue cells, cardiac and corneal fibroblasts, myofibroblasts, hepatocytes, and different types of glandular cells Fig. . Intercetability . Intraceradation . An incrocytosis . The dynocytosis . For thiocytosis . Gap junus cells . Connexius cells . Cytoskeus cells  Fig. 2)2+-based The well-studied cells coupled into networks are astrocytes in the CNS , 21. Ast2+ concentration. Stimuli such as transmitters released from neurons and glial cells can evoke Ca2+ elevation in single astrocytes, which passes to adjacent astrocytes and leads to a Ca2+ wave that can propagate over long distances, albeit much more slowly than the propagation of action potentials in neurons [Because they do not communicate via action potentials, astrocytes are not electrically active; however, they display a form of excitability that manifests as an increased intracellular Ca neurons , 29, 30.2) and cause the release of inositol 1,4,5-trisphosphate (IP3) into the cytosol. IP3 receptors located on the endoplasmic reticulum respond to this elevation of IP3 by releasing Ca2+. Cytosolic Ca2+ plays a key role as a second messenger; thus, the control of Ca2+ signals is critical. This control involves coordinating Ca2+ entry across the plasma membrane, Ca2+ release from the endoplasmic reticulum, endoplasmic reticulum store refilling, and Ca2+ extrusion across the plasma membrane [+-Ca2+ exchanger, a Ca2+ transporter that controls the intracellular Ca2+ concentration, is driven by the Na+ electrochemical gradient across the plasma membrane.This Na+ pump, the Na+/K+-ATPase, indirectly modulates Ca2+ signaling [2+ homeostasis in astrocyte networks [Incoming stimuli activate G protein-coupled receptors that hydrolyze phosphatidylinositol 4,5-bisphosphate ergic neurons. Glutamine is then converted to glutamate in neurons and released into the synaptic space. The majority of the released glutamate is taken up by astrocytes through the glutamate transporters; glutamate/aspartate transporter , and glial glutamate transporter-1 (GLT-1/EAAT2) and then metabolized by glutamine synthetase to glutamine , 40.2+ signaling. G protein-coupled receptors, which activate phospholipase C (PLC) and convert PIP2 into diacylglycerol and IP3, trigger the release of Ca2+ from intracellular Ca2+ stores [The epidermis is a dynamic, stratified structure formed by continually proliferating and differentiating keratinocytes that surround the sensory nerve endings of several C- and Aδ-fiber subtypes. The skin and buccal membrane primarily comprise keratinocytes, the epidermis. The cells are connected by well-developed intercellular junctions such as gap junctions. Within these gap junctions, Cx43 is associated with the regulation of cell proliferation and mediates forms of intercellular communication in which ions and small molecules are allowed to pass from one cell to another. Cx43 is primarily localized to the lower epithelial layer, the stratum basale and stratum spinosum . Cx43 de+ stores , 43.2) receptors are then activated, resulting in increased intracellular Ca2+ [In response to stress, injury or even chronic pain, keratinocytes can release ATP through hemichannels, resulting in Cx43 upregulation. Metabotropic purinergic , and corneal stroma because the cells of these tissues are coupled to form networks. Two adjacent cells join through Cx43, allowing direct cell-to-cell communication via Cary genes . Tenocyt+ or IP3 . The mec+ or IP3 . Some of+ or IP3 . Throughlar Ca2+ . Cx43 aslar Ca2+ .2+ signaling that causes the release of Ca2+ from the endoplasmic reticulum in response to ATP, histamine, 5-hydroxytryptamine (5-HT) [+/K+-ATPase activity levels in the extracellular matrix [Cardiac fibroblasts are the most abundant cell type in the heart, play a key role in the myocardial maintenance and repair, and can transform into cardiac myofibroblasts, which are present in valve leaflets in the adult heart. These cells express α-smooth muscle actin (α-SMA) and are referred to as α-SMA-containing stress fibers . The cele (5-HT)  (Lundqvie (5-HT) . These cr matrix , and alsr matrix .2+ signals are found in the liver and are manifested as the propagation of intercellular Ca2+ waves through liver cells called hepatocytes. Agonist binding to plasma membrane receptors stimulates Gq proteins, which activate PLC and cause Ca2+ mobilization from internal stores [Agonist-evoked Cal stores . The intl stores . ATP rell stores .2+ waves through gap junctions and induces Ca2+ release via the IP3 receptor [2+ signaling between cells [Intercellular signaling in salivary glands has been observed when 5-HT triggers intercellular Careceptor . Pancreaen cells .During inflammation, the expression and affinities of several receptors are changed. In astrocytes, Toll-like receptor 4 (TLR4) expression increases , 73, and2+ signaling in the astrocyte network is elevated, resulting in increased ATP production and release through the opening of hemichannels. ATP stimulates purinoceptors through autocrine or paracrine mechanisms and results in increased Ca2+ release from internal stores; this release occurs in the form of Ca2+ oscillations, which may change the balance of Ca2+-regulating processes [2+ signaling attenuates intercellular Ca2+ signaling, causing reduced communication via gap junctions [+/K+-ATPase are downregulated [Furthermore, the cytoskeleton is disrupted into more diffuse and ring-structured actin filaments. Lipopolysaccharide (LPS) exposure alters the actin cytoskeleton in astrocytes , macrophrocesses . This exunctions . Sodium egulated . Neuronaegulated . Glutamaegulated . The incegulated , 82, 83 Inflammation plays a part in most, if not all, CNS insults. Examples of inflammatory diseases where some of these cellular parameters have been shown to be changed are in the Alzheimer’s disease , ParkinsChanges in cellular parameters due to inflammation are also observed in other cellular networks.The cellular location of TLR4 in keratinocytes has a key role in the initiation of innate immunity and in the regulation of adaptive immune responses. TLR4 may also be an important regulator of wound inflammation  and derm2+ signaling [In rheumatoid arthritis, the synovial lining cells exhibit an infiltration of macrophages, increased levels of pro-inflammatory cytokines, and upregulation of catabolic matrix-degrading enzymes such as matrix metalloproteinases, which leads to the activation and destruction of cartilage and bone cells. An interaction between LPS, TLR4, and collagen type II in chondrocytes has a role in initiating this pro-inflammatory activity and can lead to the inhibition of cartilage extracellular matrix production and chondrocyte inflammation and apoptosis . The stiignaling . Alteredignaling . The conignaling .The increased extracellular glutamate concentration is regulated by glutamate transporters in rat chondrocytes  as well Cardiac fibroblasts likely have important roles in the inflammatory process in the heart. These cells transform into myofibroblasts in conditions of reduced ventricular function; extracellular matrix production is increased, chemokine production is upregulated, and inflammatory pathways are changed. Furthermore, these fibroblasts recruit monocytes into cardiac tissue . MechaniDuring pathophysiological inflammation in salivary gland cells, LPS induces the secretion of IL-β into the saliva. TLR4 is upregulated via the LPS signaling pathway . InflammTLR4 may be involved in the immune response in liver disease caused by hepatitis B, which can lead to inflammation, cirrhosis, and hepatocellular carcinoma .Airway inflammation is often caused by Gram-negative bacteria or by the presence of endotoxins, which can lead to the development of asthma. TLR4 also seems to play an important role in this process .Do coupled cell networks in different organs possess a signaling system that can spread or propagate from the coupled cell networks of one organ to those in other organs on either the contralateral or ipsilateral side? Can chronic, low-grade, systemic inflammation influence coupled cell networks in one or several organs, leading to chronic tissue degradation?2+ entry and membrane depolarization. They contribute to cell volume regulation and are involved in diseases such as osteoarthritis, cardiovascular disorders, type 2 diabetes mellitus etc. [Obesity promotes chronic, low-grade, systemic inflammation that contributes to the development of type 2 diabetes, cardiovascular disease and metabolic disorders such as liver steatosis , 104. Botus etc. , 111. Sttus etc. . Osteoartus etc. .Innate immune dysregulation can be the driver for autoinflammatory diseases. It is efforts to define overlapping, maybe genetically determined inflammatory responses in autoimmunity and infection diseases . These q2+ signaling by altering the balance between Ca2+-regulating processes [CNS glia, especially microglia and astrocytes coupled into networks, appear to be capable of action at a distance. The nervous system can also initiate signals that alter the function of glial cells. Activated glia release immunomodulatory products, which can be key mediators of the modulation of neuronal activity. It may lead to low-grade inflammation at the site of the damaged or affected peripheral nerve(s). Inflammatory receptors are affected, and interactions between these receptors may induce immune signaling changes, which may be of importance in neuroinflammation. Such immune-mediated inflammation can be underlying mechanisms of persistent or long-term pain. Astrocytes and microglia become activated after pain-activating substances are released from neurons located in the spinal cord and brain in response to peripheral and/or CNS trauma, which can lead to over-activation  that resrocesses .Changes in the release of pro- and anti-inflammatory cytokines are observed in many clinical studies , 115. Th2+ signaling systems. These cells are targets in conditions that can lead to immune-mediated inflammation. Several basal cellular parameters are then changed. One hypothesis is that coupled cell networks use a signaling mechanism between the cells within their own networks. The signaling can be transferred to other coupled cell networks in other organs. This form of communication may give rise to inflammatory systemic diseases. Such signaling may facilitate the spreading of pain to sites both contralateral and ipsilateral to the original injury site; this spreading is sometimes observed with long-term pain.Coupled cell networks throughout the body are coupled by gap junctions expressing Cx43 and Ca


Acute myeloid leukaemia (AML) blasts misuse these bone marrow niches for protection by the local microenvironment, but also might create their own microenvironment. Here we identify IL-8 as a hypoxia-regulated cytokine in both AML cell lines and primary AML samples that is induced within 48 hours of severe hypoxia (1% O2). IL-8 lacked effects on AML cells but induced migration in mesenchymal stromal cells (MSC), an integral part of the bone marrow. Accordingly, MSC were significantly increased in AML bone marrow as compared to healthy bone marrow. Interestingly, mononuclear cells obtained from healthy bone marrow displayed both significantly lower endogenous and hypoxia-induced production of IL-8. IL-8 mRNA expression in AML blasts from 533 patients differed between genetic subgroups with significantly lower expression of IL-8 in acute promyelocytic leukaemia (APL), while in non APL-AML patients with FLT ITD had the highest IL-8 expression. In this subgroup, high IL-8 expression was also prognostically unfavourable. In conclusion, hypoxia as encountered in the bone marrow specifically increases IL-8 expression of AML, which in turn impacts niche formation. High IL-8 expression might be correlated with poor prognosis in certain AML subsets.The bone marrow microenvironment is physiologically hypoxic with areas being as low as 1% O Recent evidence suggests that in acute myeloid leukaemia (AML) the microenvironment is an important part of disease progression and pathophysiologythe microenvironment in the context of AML. They are rather well understood, and they influence AML blasts in a variety of ways, e.g. by induction of metabolic changes22). Due to the low solubility of O2 in tissue the bone marrow is hypoxic , which has been shown beyond doubt562 however is less clear, but hypoxia has been shown to impact differentiation of normal haematological progenitors56Cellular components are haematological (monocytes or blasts to name a few) or non-haematological cells  residing in the bone marrow. MSC are a part of the stem cell niche, which is commonly referred to as While the effects of the microenvironment and parts thereof on AML have been extensively investigated, little attention has been paid to the effects AML exerts on the microenvironment. There are several studies of cytokine production of AML10Hence, this project aimed to elucidate a factor that interlinks the cellular with the non-cellular microenvironment. This factor would most likely be a cytokine produced by AML, and to effectively combine the different components of the microenvironment, it would need to fulfil 2 requirements: (I) it has to be regulated by hypoxia and (II) in turn it should influence niche formation (i.e. MSC). Preliminary data suggested IL-8 (CXCL8) to be upregulated in AML by hypoxia, fulfilling the first prerequisite, while published data pointed on its influence on MSC  are an integral part of the microenvironment in the bone marrow. Hence, in a first step we examined whether AML bone marrow differs in MSC presence from normal bone marrow. We investigated 8 random normal bone marrows and compared these to 8 random AML bone marrows for the staining of CD90+, CD10+ cells with a distinct morphology. As shown in 5 viable cells/mL]) after 48 hours IL-8 secretion was significantly increased in both cell lines at 1% O2 only, while the effects of both 12% and 6% O2 did not differ from IL-8 secretion at standard laboratory conditions of 21% O2 . Similarly, both cell lines maintained this increased production of IL-8 even when adapted to the respective level of hypoxia for up to 10 days. We also observed that at 6% O2 IL-8 production was already slightly increased in OCI-AML3 . However, we were unable to detect any differences for HGF and hence abandoned further investigation of this cytokine, focusing on IL-8 instead for further experiments.As AML blasts and MSC might interact via cytokines, we performed a screening assay for 80 cytokines in the supernatant of AML cell lines. In this screening, only IL-8 and HGF showed a significant (semi quantitative) increase at hypoxic conditions and were therefore further investigated (data not shown). As shown in 2 were chosen based on the previous results. Patient samples from 22 patients with AML were available . IL-8 secretion was profoundly higher in primary AML samples as compared to AML cell lines , and induction of IL-8 expression at hypoxia of 1% O2 was also confirmed in the primary samples  nor did they differ in hypoxia mediated induction of IL-8  and beta (CXCR2) on AML cell lines was investigated. CXCR1, considered more specific for IL-8 was absent on all 9 cell lines  investigated, and “promiscuous” CXCR2  was also absent on the majority (6 out of 9) of these cell lines  and hypoAccording to this low to absent expression of IL-8 receptors in AML, cell line MV4/11 (displaying the highest CXCR2 expression) did not migrate towards increasing doses of chemokine IL-8 in transwell experiments . These dHowever, when serum levels of IL-8 in patients with AML (n = 4 with at least 3 serum specimen at 1 time point available) that were undergoing intensive chemotherapy , higher levels of IL-8 were found to profoundly impact overall survival (OS) and relapse free survival (RFS). Hazard ratio (HR) was 1.11  for OS and 1.13  for RFS. However, this effect was lost when only non APL-AMLs were analysed , the reason being a significant lower expression of IL-8 in APL than non APL-AML . HoweverThe microenvironment of the bone marrow emerges as a critical player in cancer biology. However, mostly isolated parts of the microenvironment have been investigated, in the case of AML e.g. either MSC45CXCL8 has long been identified as a pro-inflammatory cytokine and a strong chemoattractant for neutrophilsin vitro by different AML subgroups, but this is most likely owed to the fact that the number of patient samples was too small and hence underpowered for the detection of this difference. In addition, we found that IL-8 mRNA in AML blasts correlated to initial blast count, an old but well established risk factor24conditio sine qua non, it is not quite so surprising that no strong impact on prognosis is observed as potentially all AML are dependent on it. The course of serum levels of IL-8 in AML patients - correlating with leukocyte burden (but not with AML blasts) - suggests however that IL-8 is not solely produced by AML but rather related to healthy myelopoiesis. However, questions remain concerning which cells actually produce this serum IL-8 in vivo and whether there are differences in IL-8 concentrations between the bone marrow and the peripheral blood. It should of course not be forgotten that IL-8 is an inflammatory cytokine in the first place, and hence this increase might simply reflect the fact that patients in aplasia after chemotherapy suffer from infections due to neutropenia, which are known to increase IL-8 levels26However, little is known about the role of IL-8 in AML. The overexpression of the chemokine in AML has been known for over 20 years2 is considered physiological in the stem cell nicheThe induction of IL-8 by hypoxia has been described in solid tumoursThe role of IL-8 receptors remain elusive: one recent publication found CXCR2 to be overexpressed in AML on a mRNA level, but did not show direct expression by e.g. flow cytometry or microscopy. They additionally found a significantly higher expression of CXCR2 mRNA than CXCR1 mRNA and also a prognostic impact in 200 AML samplesin vitro, confirming previous reports2 present in the stem cell niche one would assume that MSC density in AML bone marrow is increased when compared to normal bone marrow and this is exactly what we observed. The effects of MSC on AML have been well established meanwhile and need not be reviewed here again in detail. They are mostly chemo-protective, e.g. by upregulation of anti-apoptotic proteins2Indeed, we found that bone marrow derived MSC (I) expressed CXCL1 but not CXCL2 and that (II) primary MSC migrated towards an IL-8 gradient As the CXCL8-IL8 receptor axis has been identified as an drugable target in e.g. diseases with chronic inflammation, several mechanisms have been developed to inhibit it. One way is direct inhibition via neutralizing antibodies for CXCL8, which were mainly developed for the treatment of inflammatory diseases like chronic obstructive pulmonic disease, but which also did show promising results in pre-clinical melanoma modelsIn conclusion, this data supports the importance of IL8 in AML but provides an additional novel mechanism: IL-8 could be the missing link that ties MSC and hypoxia in AML together. As the means of inhibition of the CXCL8-IL-8 receptor axis are at hand, further studies on the therapeutic intervention seem warranted and may utilize a novel concept in AML therapy: to aim at the crucial AML microenvironment interaction rather than the leukemic cell itself.μm and stained for CD90 , CD10  and CD31  by the indirect immunoperoxidase-staining technique as described elsewhereImmunohistochemical analyses were performed on archival bone marrow trephine biopsies taken from the iliac crest after informed consent had been obtained from the patients. Diagnosis of AML was confirmed according to WHO criteria (≥20% myeloid blasts with >3% positivity for peroxidase). Eight samples of patients with AML were used; these samples were completely anonymized for analysis, hence no further information on these patients is available. Eight normal controls consisted of patients without bone marrow involvement; again, these patients were anonymized and no further information was made available. After formalin-fixation, these specimens had been mildly decalcified overnight in acetic acid, and embedded in paraffin wax. Sections were cut at 3 2, 5% CO2, and 37 °C. For experiments in a reduced oxygen environment, the hypoxic Workstation INVIVO2 400 from Ruskinn Technology  was used. Cells were incubated at 12%, 6% or 1% O2, 5% CO2, and 37 °C. Live cell numbers were evaluated by trypane blue exclusion .Standard laboratory (normoxic) conditions comprised 21% O5/ml in RPMI 1640 containing 10% FCS , 2 mM glutamine and 1% penicillin-streptomycin . Primary AML samples were collected from bone marrow aspirates or from peripheral blood from AML patients who underwent routine diagnostics. Mononuclear cells were separated by Ficoll-Hypaque  density-gradient centrifugation and cultured at a density of 106/ml in RPMI 1640 containing 10% FCS, 2 mM glutamine and 1% penicillin-streptomycin. Experiments were performed on fresh and frozen cells. All experiments were carried out in accordance with approved guidelines and regulations. All experimental procedures were approved by the local ethics committee .AML cell lines were all obtained from the DSMZ . Cell lines were maintained at a density of 2.0 × 106/ml) or AML cell lines KG-1a and OCI-AML-3 (2.0 × 105/ml) were collected after 48 h of in vitro cultivation at the indicated oxygen concentration by centrifugation . For long term cultures cell lines were pre-cultured under hypoxic conditions for 8 days prior to this treatment. IL-8 and HGF protein in the supernatant was quantified with the human IL-8 ELISA Kit II  and human HGF ELISA  according to the manufacturer’s instructions. For quantification of IL-8 protein in patients with AML, serum samples were collected during routine surveillance at the indicated time points and spun down at 2000 rpm for 10 minutes. Serum was stored at −80 °C and IL-8 quantification was performed by ELISA as described.Supernatant of primary AML cells  according to the manufacturer’s instructions with appropriate isotype controls. Flow cytometry was performed with a FACSCalibur cytometer  and data was analysed with BD CellQuest software.20 primer was applied. Quantitative PCR amplifications were performed using Fast SYBR Green Master Mix  on the 7900HT Fast Real Time PCR system from the same company. For IL-8 cDNA amplification the following primers were used: human IL-8 forward 5′-TAGCAAAATTGAGGCCAAGG-3′ and reverse 5′-AGCAGACTAGGGTTGCCAGA-3′ . ß-actin was used as housekeeping gene: human ß-actin forward 5′-CCGAGGACTTTGATTGCACA-3′ and reverse 5′-AGTGGGGTGGCTTTTAGGAT-3′. . PCR conditions were: 95 °C for 15 min followed by 40 cycles of 95 °C for 15 sec, 60 °C for 30 sec, and 72 °C for 30 sec. Fold of RNA induction relative to normoxic conditions was calculated as the mean 2eΔΔCt of n = 4 independent experiments.AML cell lines KG-1a and OCI-AML-3 were collected after 5 days of culture at the appropriate oxygen conditions and total RNA was extracted with the Absolutely RNA Mini Prep Kit  following the manufacturer’s instructions. For RT-PCR the First-Strand cDNA Synthesis System  with the supplied oligo-(dT)5 cells were placed in the upper compartment. After 24 hrs the amount of viable cells migrated to the lower compartment was determined by trypan blue exclusion cell counting in at least three independent experiments. Migration of MSCs was assessed in Transwell permeable 96 well plates with 8.0 μM pores using the fluorimetric QCM Cell Migration Assay Kit . The lower compartment contained 150 μL of serum free media supplemented with IL-8. 5 × 104 primary MSC were applied to the upper compartment and allowed to migrate for 16 hrs. The amount of migrated cells was determined according to the manufactures instructions at 480/520 nm using Cytofluor 4000 Fluorescence/Bioluminescence Plate Reader . MYR PKCζ pseudosubstrate was from Life Technologies .Migration of cell lines was assessed in Transwell permeable 24 well plates with 5.0 μM pores . The lower compartment contained 500 μL of serum free media supplemented with indicated concentrations of IL-8 , while 10Pre-treatment bone marrow samples of 562 patients at time of diagnosis were analysed using Affymetrix U133A + B and Affymetrix U133 Plus2.0 microarrays  as published previouslyt test. If other tests were used, they are specified at the according section. For survival analyses, Cox regression and the method of Kaplan-Meier using Log Rank test with SPSS Statistics®  were used. Microsoft Excel was used for data acquisition and storage. A P value < 0.05 was considered statistically significant.Results are shown as the mean ± SEM or SD of at least 3 experiments each. Paired data were analysed using the paired Student How to cite this article: Kuett, A. et al. IL-8 as mediator in the microenvironment-leukaemia network in acute myeloid leukaemia. Sci. Rep.5, 18411; doi: 10.1038/srep18411 (2015).