Hypoxia enhances anti-fibrotic properties of extracellular vesicles derived from hiPSCs via the miR302b-3p/TGFß/SMAD2 axis.

BACKGROUND: Cardiac fibrosis is one of the top killers among fibrotic diseases and continues to be a global unaddressed health problem. The lack of effective treatment combined with the considerable socioeconomic burden highlights the urgent need for innovative therapeutic options. Here, we evaluated the anti-fibrotic properties of extracellular vesicles (EVs) derived from human induced pluripotent stem cells (hiPSCs) that were cultured under various oxygen concentrations. METHODS: EVs were isolated from three hiPSC lines cultured under normoxia (21% O2; EV-N) or reduced oxygen concentration (hypoxia): 3% O2 (EV-H3) or 5% O2 (EV-H5). The anti-fibrotic activity of EVs was tested in an in vitro model of cardiac fibrosis, followed by a detailed investigation of the underlying molecular mechanisms. Sequencing of EV miRNAs combined with bioinformatics analysis was conducted and a selected miRNA was validated using a miRNA mimic and inhibitor. Finally, EVs were tested in a mouse model of angiotensin II-induced cardiac fibrosis. RESULTS: We provide evidence that an oxygen concentration of 5% enhances the anti-fibrotic effects of hiPS-EVs. These EVs were more effective in reducing pro-fibrotic markers in activated human cardiac fibroblasts, when compared to EV-N or EV-H3. We show that EV-H5 act through the canonical TGFß/SMAD pathway, primarily via miR-302b-3p, which is the most abundant miRNA in EV-H5. Our results show that EV-H5 not only target transcripts of several profibrotic genes, including SMAD2 and TGFBR2, but also reduce the stiffness of activated fibroblasts. In a mouse model of heart fibrosis, EV-H5 outperformed EV-N in suppressing the inflammatory response in the host and by attenuating collagen deposition and reducing pro-fibrotic markers in cardiac tissue. CONCLUSIONS: In this work, we provide evidence of superior anti-fibrotic properties of EV-H5 over EV-N or EV-H3. Our study uncovers that fine regulation of oxygen concentration in the cellular environment may enhance the anti-fibrotic effects of hiPS-EVs, which has great potential to be applied for heart regeneration.

Driver Mutations of Pancreatic Cancer Affect Ca2+ Signaling and ATP Production.

Glandular pancreatic epithelia of the acinar or ductal phenotype may seem terminally differentiated, but they are characterized by remarkable cell plasticity. Stress-induced trans-differentiation of these cells has been implicated in the mechanisms of carcinogenesis. Current consensus links pancreatic ductal adenocarcinoma with onco-transformation of ductal epithelia, but under the presence of driver mutations in Kras and Trp53, also with trans-differentiation of pancreatic acini. However, we do not know when, in the course of cancer progression, physiological functions are lost by mutant acinar cells, nor can we assess their capacity for the production of pancreatic juice components. Here, we investigated whether two mutations-KrasG12D and Trp53R172H-present simultaneously in acinar cells of KPC mice (model of oncogenesis) influence cytosolic Ca2+ signals. Since Ca2+ signals control the cellular handling of digestive hydrolases, any changes that affect intracellular signaling events and cell bioenergetics might have an impact on the physiology of the pancreas. Our results showed that physiological doses of acetylcholine evoked less regular Ca2+ oscillations in KPC acinar cells compared to the control, whereas responses to supramaximal concentrations were markedly reduced. Menadione elicited Ca2+ signals of different frequencies in KPC cells compared to control cells. Finally, Ca2+ extrusion rates were significantly inhibited in KPC cells, likely due to the lower basal respiration and ATP production. Cumulatively, these findings suggest that driver mutations affect the signaling capacity of pancreatic acinar cells even before the changes in the epithelial cell morphology become apparent.

Molecular mechanisms of pain in acute pancreatitis: recent basic research advances and therapeutic implications.

Although severe abdominal pain is the main symptom of acute pancreatitis, its mechanisms are poorly understood. An emerging body of literature evidence indicates that neurogenic inflammation might play a major role in modulating the perception of pain from the pancreas. Neurogenic inflammation is the result of a crosstalk between injured pancreatic tissue and activated neurons, which leads to an auto-amplification loop between inflammation and pain during the progression of acute pancreatitis. In this review, we summarize recent findings on the role of neuropeptides, ion channels, and the endocannabinoid system in acute pancreatitis-related pain. We also highlight potential therapeutic strategies that could be applied for managing severe pain in this disease.

Activation of pancreatic stellate cells attenuates intracellular Ca2+ signals due to downregulation of TRPA1 and protects against cell death induced by alcohol metabolites.

Alcohol abuse, an increasing problem in developed societies, is one of the leading causes of acute and chronic pancreatitis. Alcoholic pancreatitis is often associated with fibrosis mediated by activated pancreatic stellate cells (PSCs). Alcohol toxicity predominantly depends on its non-oxidative metabolites, fatty acid ethyl esters, generated from ethanol and fatty acids. Although the role of non-oxidative alcohol metabolites and dysregulated Ca2+ signalling in enzyme-storing pancreatic acinar cells is well established as the core mechanism of pancreatitis, signals in PSCs that trigger fibrogenesis are less clear. Here, we investigate real-time Ca2+ signalling, changes in mitochondrial potential and cell death induced by ethanol metabolites in quiescent vs TGF-ß-activated PSCs, compare the expression of Ca2+ channels and pumps between the two phenotypes and the consequences these differences have on the pathogenesis of alcoholic pancreatitis. The extent of PSC activation in the pancreatitis of different aetiologies has been investigated in three animal models. Unlike biliary pancreatitis, alcohol-induced pancreatitis results in the activation of PSCs throughout the entire tissue. Ethanol and palmitoleic acid (POA) or palmitoleic acid ethyl ester (POAEE) act directly on quiescent PSCs, inducing cytosolic Ca2+ overload, disrupting mitochondrial functions, and inducing cell death. However, activated PSCs acquire remarkable resistance against ethanol metabolites via enhanced Ca2+-handling capacity, predominantly due to the downregulation of the TRPA1 channel. Inhibition or knockdown of TRPA1 reduces EtOH/POA-induced cytosolic Ca2+ overload and protects quiescent PSCs from cell death, similarly to the activated phenotype. Our results lead us to review current dogmas on alcoholic pancreatitis. While acinar cells and quiescent PSCs are prone to cell death caused by ethanol metabolites, activated PSCs can withstand noxious signals and, despite ongoing inflammation, deposit extracellular matrix components. Modulation of Ca2+ signals in PSCs by TRPA1 agonists/antagonists could become a strategy to shift the balance of tissue PSCs towards quiescent cells, thus limiting pancreatic fibrosis.

Transcriptomics and Network Pharmacology Reveal the Protective Effect of Chaiqin Chengqi Decoction on Obesity-Related Alcohol-Induced Acute Pancreatitis via Oxidative Stress and PI3K/Akt Signaling Pathway.

Obesity-related acute pancreatitis (AP) is characterized by increasing prevalence worldwide and worse clinical outcomes compared to AP of other etiologies. Chaiqin chengqi decoction (CQCQD), a Chinese herbal formula, has long been used for the clinical management of AP but its therapeutic actions and the underlying mechanisms have not been fully elucidated. This study has investigated the pharmacological mechanisms of CQCQD in a novel mouse model of obesity-related alcohol-induced AP (OA-AP). The mouse OA-AP model was induced by a high-fat diet for 12 weeks and subsequently two intraperitoneal injections of ethanol, CQCQD was administered 2 h after the first injection of ethanol. The severity of OA-AP was assessed and correlated with changes in transcriptomic profiles and network pharmacology in the pancreatic and adipose tissues, and further docking analysis modeled the interactions between compounds of CQCQD and their key targets. The results showed that CQCQD significantly reduced pancreatic necrosis, alleviated systemic inflammation, and decreased the parameters associated with multi-organ dysfunction. Transcriptomics and network pharmacology analysis, as well as further experimental validation, have shown that CQCQD induced Nrf2/HO-1 antioxidant protein response and decreased Akt phosphorylation in the pancreatic and adipose tissues. In vitro, CQCQD protected freshly isolated pancreatic acinar cells from H2O2-elicited oxidative stress and necrotic cell death. The docking results of AKT1 and the active compounds related to AKT1 in CQCQD showed high binding affinity. In conclusion, CQCQD ameliorates the severity of OA-AP by activating of the antioxidant protein response and down-regulating of the PI3K/Akt signaling pathway in the pancreas and visceral adipose tissue.

When healing turns into killing - the pathophysiology of pancreatic and hepatic fibrosis.

Disorders such as pancreatic or hepatic fibrosis are a cruel reminder that disruption of the delicate physiological balance could result in severe pathological consequences. Fibrosis is usually associated with chronic diseases and manifests itself as excessive deposition of the extracellular matrix, which gradually leads to the replacement of the cellular components by fibrotic lesions, significantly compromising normal tissue functions. The main cellular mediators of fibrosis are different populations of tissue fibroblasts, predominantly hepatic and pancreatic stellate cells in the liver and pancreas, respectively. These cells undergo a phenotypic switch in response to (bio)chemical or physical stimuli and acquire a myofibroblast-like phenotype characterised by increased contractile and adhesive properties, elevated expression of certain cytoskeletal and membrane proteins, and prominent production of extracellular matrix components. In the past few decades, a substantial scientific effort has been undertaken to investigate the pathogenesis of fibrosis. Here, cellular mechanisms of hepatic and pancreatic fibrosis, their aetiological factors, associated diseases and prospective therapies are discussed. New therapies against fibrosis are likely to be focused on regulation of hepatic/pancreatic stellate cell physiology as well as normalisation of the organ mechanostasis.

A microRNA checkpoint for Ca2+ signaling and overload in acute pancreatitis.

Acute pancreatitis (AP) is a common digestive disease without specific treatment, and its pathogenesis features multiple deleterious amplification loops dependent on translation, triggered by cytosolic Ca2+ ([Ca2+]i) overload; however, the underlying mechanisms in Ca2+ overload of AP remains incompletely understood. Here we show that microRNA-26a (miR-26a) inhibits pancreatic acinar cell (PAC) store-operated Ca2+ entry (SOCE) channel expression, Ca2+ overload, and AP. We find that major SOCE channels are post-transcriptionally induced in PACs during AP, whereas miR-26a expression is reduced in experimental and human AP and correlated with AP severity. Mechanistically, miR-26a simultaneously targets Trpc3 and Trpc6 SOCE channels and attenuates physiological oscillations and pathological elevations of [Ca2+]i in PACs. MiR-26a deficiency increases SOCE channel expression and [Ca2+]i overload, and significantly exacerbates AP. Conversely, global or PAC-specific overexpression of miR-26a in mice ameliorates pancreatic edema, neutrophil infiltration, acinar necrosis, and systemic inflammation, accompanied with remarkable improvements on pathological determinants related with [Ca2+]i overload. Moreover, pancreatic or systemic administration of an miR-26a mimic to mice significantly alleviates experimental AP. These findings reveal a previously unknown mechanism underlying AP pathogenesis, establish a critical role for miR-26a in Ca2+ signaling in the exocrine pancreas, and identify a potential target for the treatment of AP.

Generation of microRNA-378a-deficient hiPSC as a novel tool to study its role in human cardiomyocytes.

microRNA-378a (miR-378a) is one of the most highly expressed microRNAs in the heart. However, its role in the human cardiac tissue has not been fully understood. It was observed that miR-378a protects cardiomyocytes from hypertrophic growth by regulation of IGF1R and the expression of downstream kinases. Increased levels of miR-378a were reported in the serum of Duchenne muscular dystrophy (DMD) patients and female carriers of DMD gene-associated mutations with developed cardiomyopathy. In order to shed more light on the role of miR-378a in human cardiomyocytes and its potential involvement in DMD-related cardiomyopathy, we generated two human induced pluripotent stem cell (hiPSC) models; one with deletion of miR-378a and the second one with deletion of DMD exon 50 leading to the DMD phenotype. Our results indicate that lack of miR-378a does not influence the pluripotency of hiPSC and their ability to differentiate into cardiomyocytes (hiPSC-CM). miR-378a-deficient hiPSC-CM exhibited, however, significantly bigger size compared to the isogenic control cells, indicating the role of this miRNA in the hypertrophic growth of human cardiomyocytes. In accordance, the level of NFATc3, phosphoAKT, phosphoERK and ERK was higher in these cells compared to the control counterparts. A similar effect was achieved by silencing miR-378a with antagomirs. Of note, the percentage of cells with nuclear localization of NFATc3 was higher in miR-378a-deficient hiPSC-CM. Analysis of electrophysiological properties and Ca2+ oscillations revealed the decrease in the spike slope velocity and lower frequency of calcium spikes in miR-378a-deficient hiPSC-CM. Interestingly, the level of miR-378a increased gradually during cardiac differentiation of hiPSC. Of note, it was low until day 15 in differentiating DMD-deficient hiPSC-CM and then rose to a similar level as in the isogenic control counterparts. In summary, our findings confirmed the utility of hiPSC-based models for deciphering the role of miR-378a in the control and diseased human cardiomyocytes.

A Novel, Pan-PDE Inhibitor Exerts Anti-Fibrotic Effects in Human Lung Fibroblasts via Inhibition of TGF-ß Signaling and Activation of cAMP/PKA Signaling.

Phosphodiesterase (PDE) inhibitors are currently a widespread and extensively studied group of anti-inflammatory and anti-fibrotic compounds which may find use in the treatment of numerous lung diseases, including asthma and chronic obstructive pulmonary disease. Several PDE inhibitors are currently in clinical development, and some of them, e.g., roflumilast, are already recommended for clinical use. Due to numerous reports indicating that elevated intracellular cAMP levels may contribute to the alleviation of inflammation and airway fibrosis, new and effective PDE inhibitors are constantly being sought. Recently, a group of 7,8-disubstituted purine-2,6-dione derivatives, representing a novel and prominent pan-PDE inhibitors has been synthesized. Some of them were reported to modulate transient receptor potential ankyrin 1 (TRPA1) ion channels as well. In this study, we investigated the effect of selected derivatives (832-a pan-PDE inhibitor, 869-a TRPA1 modulator, and 145-a pan-PDE inhibitor and a weak TRPA1 modulator) on cellular responses related to airway remodeling using MRC-5 human lung fibroblasts. Compound 145 exerted the most considerable effect in limiting fibroblast to myofibroblasts transition (FMT) as well as proliferation, migration, and contraction. The effect of this compound appeared to depend mainly on its strong PDE inhibitory properties, and not on its effects on TRPA1 modulation. The strong anti-remodeling effects of 145 required activation of the cAMP/protein kinase A (PKA)/cAMP response element-binding protein (CREB) pathway leading to inhibition of transforming growth factor type ß1 (TGF-ß1) and Smad-dependent signaling in MRC-5 cells. These data suggest that the TGF-ß pathway is a major target for PDE inhibitors leading to inhibitory effects on cell responses involved in airway remodeling. These potent, pan-PDE inhibitors from the group of 7,8-disubstituted purine-2,6-dione derivatives, thus represent promising anti-remodeling drug candidates for further research.

Electron paramagnetic resonance spectroscopy reveals alterations in the redox state of endogenous copper and iron complexes in photodynamic stress-induced ischemic mouse liver.

Divalent copper and iron cations have been acknowledged for their catalytic roles in physiological processes critical for homeostasis maintenance. Being redox-active, these metals act as cofactors in the enzymatic reactions of electron transfer. However, under pathophysiological conditions, owing to their high redox potentials, they may exacerbate stress-induced injury. This could be particularly hazardous to the liver - the main body reservoir of these two metals. Surprisingly, the involvement of Cu and Fe in liver pathology still remains poorly understood. Hypoxic stress in the tissue may act as a stimulus that mobilizes these ions from their hepatic stores, aggravating the systemic injury. Since ischemia poses a serious complication in liver surgery (e.g. transplantation) we aimed to reveal the status of Cu and Fe via spectroscopic analysis of mouse ischemic liver tissue. Herein, we establish a novel non-surgical model of focal liver ischemia, achieved by applying light locally when a photosensitizer is administered systemically. Photodynamic treatment results in clear-cut areas of the ischemic hepatic tissue, as confirmed by ultrasound scans, mean velocity measurements, 3D modelling of vasculature and (immuno)histological analysis. For reference, we assessed the samples collected from the animals which developed transient systemic endotoxemic stress induced by a non-lethal dose of lipopolysaccharide. The electron paramagnetic resonance (EPR) spectra recorded in situ in the liver samples reveal a dramatic increase in the level of Cu adducts solely in the ischemic tissues. In contrast, other typical free radical components of the liver EPR spectra, such as reduced Riske clusters are not detected; these differences are not followed by changes in the blood EPR spectra. Taken together, our results suggest that local ischemic stress affects paramagnetic species containing redox-active metals. Moreover, because in our model hepatic vascular flow is impaired, these effects are only local (confined to the liver) and are not propagated systemically.

Pancreatic Cancer and Its Microenvironment-Recent Advances and Current Controversies.

Pancreatic ductal adenocarcinoma (PDAC) causes annually well over 400,000 deaths world-wide and remains one of the major unresolved health problems. This exocrine pancreatic cancer originates from the mutated epithelial cells: acinar and ductal cells. However, the epithelia-derived cancer component forms only a relatively small fraction of the tumor mass. The majority of the tumor consists of acellular fibrous stroma and diverse populations of the non-neoplastic cancer-associated cells. Importantly, the tumor microenvironment is maintained by dynamic cell-cell and cell-matrix interactions. In this article, we aim to review the most common drivers of PDAC. Then we summarize the current knowledge on PDAC microenvironment, particularly in relation to pancreatic cancer therapy. The focus is placed on the acellular stroma as well as cell populations that inhabit the matrix. We also describe the altered metabolism of PDAC and characterize cellular signaling in this cancer.

Signaling in the Physiology and Pathophysiology of Pancreatic Stellate Cells - a Brief Review of Recent Advances.

The interest in pancreatic stellate cells (PSCs) has been steadily growing over the past two decades due mainly to the central role these cells have in the desmoplastic reaction associated with diseases of the pancreas, such as pancreatitis or pancreatic cancer. In recent years, the scientific community has devoted substantial efforts to understanding the signaling pathways that govern PSC activation and interactions with neoplastic cells. This mini review aims to summarize some very recent findings on signaling in PSCs and highlight their impact to the field.

Experimental Acute Pancreatitis Models: History, Current Status, and Role in Translational Research.

Acute pancreatitis is a potentially severe inflammatory disease that may be associated with a substantial morbidity and mortality. Currently there is no specific treatment for the disease, which indicates an ongoing demand for research into its pathogenesis and development of new therapeutic strategies. Due to the unpredictable course of acute pancreatitis and relatively concealed anatomical site in the retro-peritoneum, research on the human pancreas remains challenging. As a result, for over the last 100 years studies on the pathogenesis of this disease have heavily relied on animal models. This review aims to summarize different animal models of acute pancreatitis from the past to present and discuss their main characteristics and applications. It identifies key studies that have enhanced our current understanding of the pathogenesis of acute pancreatitis and highlights the instrumental role of animal models in translational research for developing novel therapies.

ABT-199 (Venetoclax), a BH3-mimetic Bcl-2 inhibitor, does not cause Ca2+ -signalling dysregulation or toxicity in pancreatic acinar cells.

BACKGROUND AND PURPOSE: Many cancer cells depend on anti-apoptotic B-cell lymphoma 2 (Bcl-2) proteins for their survival. Bcl-2 antagonism through Bcl-2 homology 3 (BH3) mimetics has emerged as a novel anti-cancer therapy. ABT-199 (Venetoclax), a recently developed BH3 mimetic that selectively inhibits Bcl-2, was introduced into the clinic for treatment of relapsed chronic lymphocytic leukaemia. Early generations of Bcl-2 inhibitors evoked sustained Ca2+ responses in pancreatic acinar cells (PACs) inducing cell death. Therefore, BH3 mimetics could potentially be toxic for the pancreas when used to treat cancer. Although ABT-199 was shown to kill Bcl-2-dependent cancer cells without affecting intracellular Ca2+ signalling, its effects on PACs have not yet been determined. Hence, it is essential and timely to assess whether this recently approved anti-leukaemic drug might potentially have pancreatotoxic effects. EXPERIMENTAL APPROACH: Single-cell Ca2+ measurements and cell death analysis were performed on isolated mouse PACs. KEY RESULTS: Inhibition of Bcl-2 via ABT-199 did not elicit intracellular Ca2+ signalling on its own or potentiate Ca2+ signalling induced by physiological/pathophysiological stimuli in PACs. Although ABT-199 did not affect cell death in PACs, under conditions that killed ABT-199-sensitive cancer cells, cytosolic Ca2+ extrusion was slightly enhanced in the presence of ABT-199. In contrast, inhibition of Bcl-xL potentiated pathophysiological Ca2+ responses in PACs, without exacerbating cell death. CONCLUSION AND IMPLICATIONS: Our results demonstrate that apart from having a modest effect on cytosolic Ca2+ extrusion, ABT-199 does not substantially alter intracellular Ca2+ homeostasis in normal PACs and should be safe for the pancreas during cancer treatment. LINKED ARTICLES: This article is part of a themed section on Mitochondrial Pharmacology: Featured Mechanisms and Approaches for Therapy Translation. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.22/issuetoc.

BH4 domain peptides derived from Bcl-2/Bcl-XL as novel tools against acute pancreatitis.

Biliary acute pancreatitis (AP) is a serious condition, which currently has no specific treatment. Taurolithocholic acid 3-sulfate (TLC-S) is one of the most potent bile acids causing cytosolic Ca2+ overload in pancreatic acinar cells (PACs), which results in premature activation of digestive enzymes and necrosis, hallmarks of AP. The inositol 1,4,5-trisphosphate receptor (IP3R) and the ryanodine receptor (RyR) play major roles in intracellular Ca2+ signaling. Inhibition of these endoplasmic reticulum-located channels suppresses TLC-S-induced Ca2+ release and necrosis, decreasing the severity of AP. Anti-apoptotic B-cell lymphoma (Bcl)-2-family members, such as Bcl-2 and Bcl-XL, have emerged as important modulators of IP3Rs and RyRs. These proteins contain four Bcl-2 homology (BH) domains of which the N-terminal BH4 domain exerts critical roles in regulating intracellular Ca2+ release channels. The BH4 domain of Bcl-2, but not of Bcl-XL, binds to and inhibits IP3Rs, whereas both BH4 domains inhibit RyRs. Although clear cytoprotective effects have been reported for these BH4 domains, it remains unclear whether they are capable of inhibiting pathological Ca2+-overload, associated with AP. Here we demonstrate in PACs that the BH4 domains of Bcl-2 and Bcl-XL inhibit RyR activity in response to the physiological agonist cholecystokinin. In addition, these BH4 domains inhibit pathophysiological TLC-S-induced Ca2+ overload in PACs via RyR inhibition, which in turn protects these cells from TLC-S-induced necrosis. This study shows for the first time the therapeutic potential of BH4 domain function by inhibiting pathological RyR-mediated Ca2+ release and necrosis, events that trigger AP.

On BH3 Mimetics and Ca2+ Signaling.

Preclinical Research BH3 mimetics are anticancer agents that reproduce the spatial arrangement of the BH3 domain of Bcl-2 family proteins. Just like the BH3-only proteins, these compounds bind to the hydrophobic cleft of the pro-survival Bcl-2 members such as Bcl-2 or Bcl-xL, and disrupt their heterodimerization with pro-apoptotic Bax or Bak, sensitizing cells to chemotherapy. In recent years, it has become clear that Bcl-2 family proteins are engaged in regulation of intracellular Ca2+ homeostasis, including Ca2+ release from the intracellular stores as well as Ca2+ fluxes across the plasma membrane. Given that BH3 mimetics shift the balance between the prosurvival and proapoptotic Bcl-2 members, they might indirectly exert effects on intracellular Ca2+ signals. Indeed, it has been reported that some BH3 mimetics release Ca2+ from the intracellular stores causing Ca2+ overload in the cytosol. Therefore, the effects of any new BH3 mimetics on cellular Ca2+ homeostasis should be tested before these compounds progress to clinical trials. Drug Dev Res 78 : 313-318, 2017. © 2017 Wiley Periodicals, Inc.

Biology of pancreatic stellate cells-more than just pancreatic cancer.

Pancreatic stellate cells, normally quiescent, are capable of remarkable transition into their activated myofibroblast-like phenotype. It is now commonly accepted that these cells play a pivotal role in the desmoplastic reaction present in severe pancreatic disorders. In recent years, enormous scientific effort has been devoted to understanding their roles in pancreatic cancer, which continues to remain one of the most deadly diseases. Therefore, it is not surprising that considerably less attention has been given to studying physiological functions of pancreatic stellate cells. Here, we review recent advances not only in the field of pancreatic stellate cell pathophysiology but also emphasise their roles in physiological processes.

BH3 mimetic-elicited Ca2+ signals in pancreatic acinar cells are dependent on Bax and can be reduced by Ca2+-like peptides.

BH3 mimetics are small-molecule inhibitors of B-cell lymphoma-2 (Bcl-2) and Bcl-xL, which disrupt the heterodimerisation of anti- and pro-apoptotic Bcl-2 family members sensitising cells to apoptotic death. These compounds have been developed as anti-cancer agents to counteract increased levels of Bcl-2 proteins often present in cancer cells. Application of a chemotherapeutic drug supported with a BH3 mimetic has the potential to overcome drug resistance in cancers overexpressing anti-apoptotic Bcl-2 proteins and thus increase the success rate of the treatment. We have previously shown that the BH3 mimetics, BH3I-2' and HA14-1, induce Ca2+ release from intracellular stores followed by a sustained elevation of the cytosolic Ca2+ concentration. Here we demonstrate that loss of Bax, but not Bcl-2 or Bak, inhibits this sustained Ca2+ elevation. What is more, in the absence of Bax, thapsigargin-elicited responses were decreased; and in two-photon-permeabilised bax-/- cells, Ca2+ loss from the ER was reduced compared to WT cells. The Ca2+-like peptides, CALP-1 and CALP-3, which activate EF hand motifs of Ca2+-binding proteins, significantly reduced excessive Ca2+ signals and necrosis caused by two BH3 mimetics: BH3I-2' and gossypol. In the presence of CALP-1, cell death was shifted from necrotic towards apoptotic, whereas CALP-3 increased the proportion of live cells. Importantly, neither of the CALPs markedly affected physiological Ca2+ signals elicited by ACh, or cholecystokinin. In conclusion, the reduction in passive ER Ca2+ leak in bax-/- cells as well as the fact that BH3 mimetics trigger substantial Ca2+ signals by liberating Bax, indicate that Bax may regulate Ca2+ leak channels in the ER. This study also demonstrates proof-of-principle that pre-activation of EF hand Ca2+-binding sites by CALPs can be used to ameliorate excessive Ca2+ signals caused by BH3 mimetics and shift necrotic death towards apoptosis.