Inhibition Of Epac1 Research Articles

EPAC1 and 2 inhibit K+ currents via PLC/PKC and NOS/PKG pathways in rat ventricular cardiomyocytes.

The exchange protein directly activated by cAMP (EPAC) has been implicated in cardiac proarrhythmic signaling pathways including spontaneous diastolic Ca2+ leak from sarcoplasmic reticulum and increased action potential duration (APD) in isolated ventricular cardiomyocytes. The action potential (AP) lengthening following acute EPAC activation is mainly due to a decrease of repolarizing steady-state K+ current (IKSS) but the mechanisms involved remain unknown. This study aimed to assess the role of EPAC1 and EPAC2 in the decrease of IKSS and to investigate the underlying signaling pathways. AP and K+ currents were recorded with the whole cell configuration of the patch-clamp technique in freshly isolated rat ventricular myocytes. EPAC1 and EPAC2 were pharmacologically activated with 8-(4-chlorophenylthio)-2'-O-methyl-cAMP acetoxymethyl ester (8-CPTAM, 10 µmol/L) and inhibited with R-Ce3F4 and ESI-05, respectively. Inhibition of EPAC1 and EPAC2 significantly decreased the effect of 8-CPTAM on APD and IKSS showing that both EPAC isoforms are involved in these effects. Unexpectedly, calmodulin-dependent protein kinase II (CaMKII) inhibition by AIP or KN-93, and Ca2+ chelation by intracellular BAPTA, did not impact the response to 8-CPTAM. However, inhibition of PLC/PKC and nitric oxide synthase (NOS)/PKG pathways partially prevents the 8-CPTAM-dependent decrease of IKSS. Finally, the cumulative inhibition of PKC and PKG blocked the 8-CPTAM effect, suggesting that these two actors work along parallel pathways to regulate IKSS upon EPAC activation. On the basis of such findings, we propose that EPAC1 and EPAC2 are involved in APD lengthening by inhibiting a K+ current via both PLC/PKC and NOS/PKG pathways. This may have pathological implications since EPAC is upregulated in diseases such as cardiac hypertrophy.NEW & NOTEWORHTY Exchange protein directly activated by cAMP (EPAC) proteins modulate ventricular electrophysiology at the cellular level. Both EPAC1 and EPAC2 isoforms participate in this effect. Mechanistically, PLC/PKC and nitric oxide synthase (NO)/PKG pathways are involved in regulating K+ repolarizing current whereas the well-known downstream effector of EPAC, calmodulin-dependent protein kinase II (CaMKII), does not participate. This may have pathological implications since EPAC is upregulated in diseases such as cardiac hypertrophy. Thus, EPAC inhibition may be a new approach to prevent arrhythmias under pathological conditions.

Abstract 3075: Different roles of EPAC1 and EPAC2 in ovarian cancer: Implications for precision targeting in cancer therapy

Abstract There are two distinct functional isoforms of Exchange proteins directly activated by cAMP (EPACs), EPAC1 and EPAC2, which exhibit fundamental distinctions in structure, expression patterns, signaling specificity, and subcellular localization. This diversity suggests that EPAC1 and EPAC2 can operate independently, synergistically, or antagonistically, contributing to the wide range of reported functions and impacts in different cancers and studies. We compared the function of EPAC1 and EPAC2 in ovarian cancer using in vitro and in vivo models and further explored the underlying mechanism in the regulation of metabolism reprogramming. Selective inhibition of EPAC1 and EPAC2 markedly reduced proliferation in ovarian cancer cell lines, leading to cell death and cycle arrest. RNA-seq analysis indicated different downstream signaling pathways triggered by EPAC1 and EPAC2, with EPAC2 specifically linked to functions in energy metabolism. Treatment with an antagonist or knockdown of EPAC2 resulted in attenuation of both glycolysis and oxidative phosphorylation (OXPHOS), while EPAC1 did not induce such effects. Furthermore, inhibition of EPAC2 induced mitochondrial dysfunction, manifested by reduced ATP production, decreased mitochondrial membrane potential, and increased burden of reactive oxygen species (ROS). Our data also revealed that EPAC2 governs the MAPK pathway and mediates the mitochondrial unfolded protein response (UPRmt) in ovarian cancer cells. By delineating the distinctions between EPAC1 and EPAC2 within ovarian cancer cells, this project revised the prevailing understanding of EPACs and suggests the potential for precise targeting of EPAC isoforms as a viable strategy in cancer therapy. Citation Format: Zhengyin Gao, Wanjun Yuan, Leo T.O. Lee. Different roles of EPAC1 and EPAC2 in ovarian cancer: Implications for precision targeting in cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3075.

Upregulation of Epac1 Promotes Pericyte Loss by Inducing Mitochondrial Fission, Reactive Oxygen Species Production, and Apoptosis.

The pathogenic mechanisms behind the development of ischemic retinopathy are complex and poorly understood. This study investigates the involvement of exchange protein directly activated by cAMP (Epac)1 signaling in pericyte injury during ischemic retinopathy, including diabetic retinopathy, a disease that threatens vision. Mouse models of retinal ischemia-reperfusion injury and type 1 diabetes induced by streptozotocin were used to investigate the pathogenesis of these diseases. The roles of Epac1 signaling in the pathogenesis of ischemic retinopathy were determined by an Epac1 knockout mouse model. The cellular and molecular mechanisms of Epac1-mediated pericyte dysfunction in response to high glucose were investigated by specific modulation of Epac1 activity in primary human retinal pericytes using Epac1-specific RNA interference and a pharmacological inhibitor. Ischemic injury or diabetes-induced retinal capillary degeneration were associated with an increased expression of Epac1 in the mouse retinal vasculature, including both endothelial cells and pericytes. Genetic deletion of Epac1 protected ischemic injury-induced pericyte loss and capillary degeneration in the mouse retina. Furthermore, high glucose-induced Epac1 expression in retinal pericytes was accompanied by increased Drp1 phosphorylation, mitochondrial fission, reactive oxygen species production, and caspase 3 activation. Inhibition of Epac1 via RNA interference or pharmacological approaches blocked high glucose-mediated mitochondrial dysfunction and caspase 3 activation. Our study reveals an important role of Epac1 signaling in mitochondrial dynamics, reactive oxygen species production, and apoptosis in retinal pericytes and identifies Epac1 as a therapeutic target for treating ischemic retinopathy.

EPAC1 inhibition protects the heart from doxorubicin-induced toxicity.

Anthracyclines, such as doxorubicin (Dox), are widely used chemotherapeutic agents for the treatment of solid tumors and hematologic malignancies. However, they frequently induce cardiotoxicity leading to dilated cardiomyopathy and heart failure. This study sought to investigate the role of the Exchange Protein directly Activated by cAMP (EPAC) in Dox-induced cardiotoxicity and the potential cardioprotective effects of EPAC inhibition. We show that Dox induces DNA damage and cardiomyocyte cell death with apoptotic features. Dox also led to an increase in both cAMP concentration and EPAC1 activity. The pharmacological inhibition of EPAC1 (with CE3F4) but not EPAC2 alleviated the whole Dox-induced pattern of alterations. When administered in vivo, Dox-treated WT mice developed a dilated cardiomyopathy which was totally prevented in EPAC1 KO mice. Moreover, EPAC1 inhibition potentiated Dox-induced cell death in several human cancer cell lines. Thus, EPAC1 inhibition appears as a potential therapeutic strategy to limit Dox-induced cardiomyopathy without interfering with its antitumoral activity.

EPAC1 Pharmacological Inhibition with AM-001 Prevents SARS-CoV-2 and Influenza A Virus Replication in Cells.

The exceptional impact of the COVID-19 pandemic has stimulated an intense search for antiviral molecules. Host-targeted antiviral molecules have the potential of presenting broad-spectrum antiviral activity and are also considered as less likely to select for resistant viruses. In this study, we investigated the antiviral activity exerted by AM-001, a specific pharmacological inhibitor of EPAC1, a host exchange protein directly activated by cyclic AMP (cAMP). The cAMP-sensitive protein, EPAC1 regulates various physiological and pathological processes but its role in SARS-CoV-2 and influenza A virus infection has not yet been studied. Here, we provide evidence that the EPAC1 specific inhibitor AM-001 exerts potent antiviral activity against SARS-CoV-2 in the human lung Calu-3 cell line and the African green monkey Vero cell line. We observed a concentration-dependent inhibition of SARS-CoV-2 infectious viral particles and viral RNA release in the supernatants of AM-001 treated cells that was not associated with a significant impact on cellular viability. Furthermore, we identified AM-001 as an inhibitor of influenza A virus in Calu-3 cells. Altogether these results identify EPAC1 inhibition as a promising therapeutic target against viral infections.

The P2Y11 receptor of human M2 macrophages activates canonical and IL-1 receptor signaling to translate the extracellular danger signal ATP into anti-inflammatory and pro-angiogenic responses

The cytoprotective ATP receptor P2Y11 is upregulated during M2 macrophage differentiation and contributes to the anti-inflammatory properties of this macrophage subset. Here, we studied P2Y11-induced reprogramming of human M2 macrophages at the level of mRNA and protein expression. Upregulation of IL-1 receptor (IL-1R) and its known downstream effectors VEGF, CCL20 and SOCS3 as well as downregulation of the ATP-degrading ecto-ATPase CD39 emerged as hallmarks of P2Y11 activation. The anti-inflammatory signature of the P2Y11 transcriptome was further characterized by the downregulation of P2RX7, toll-like receptors and inflammasome components. P2Y11-induced IL-1R upregulation formed the basis for reinforced IL-1 responsiveness of activated M2 macrophages, as IL-1α and IL-1ß each enhanced P2Y11-induced secretion of VEGF and CCL20 as well as the previously reported shedding of soluble tumor necrosis factor receptor 2 (sTNFR2). Raising intracellular cyclic AMP (cAMP) in M2 macrophages through phosphodiesterase 4 inhibition enhanced P2Y11-driven responses. The cAMP-binding effector, exchange protein activated by cAMP 1 (Epac1), which is known to induce SOCS3, differentially regulated the P2Y11/IL-1R response because pharmacological Epac1 inhibition enhanced sTNFR2 and CCL20 release, but had no effect on VEGF secretion. In addition to cAMP, calcium and protein kinase C participated in P2Y11 signaling. Our study reveals how P2Y11 harnesses canonical and IL-1R signaling to promote an anti-inflammatory and pro-angiogenic switch of human M2 macrophages, which may be controlled in part by an Epac1-SOCS3 axis.

Skin/muscle incision and retraction regulates the persistent postoperative pain in rats by the Epac1/PKC-βII pathway

Persistent postoperative pain causes influence the life quality of many patients. The Epac/PKC pathway has been indicated to regulate mechanical hyperalgesia. The present study used skin/muscle incision and retraction (SMIR) to induce postoperative pain in rats and evaluated the Epac/PKC pathway in postoperative pain. Mechanical allodynia was assessed by paw withdrawal threshold before and after incision. The levels of Epac, PKC, proinflammatory cytokines, and blood-nerve barrier-related proteins were assessed using Western blotting. We found that SMIR induced the activation of the Epac/PKC pathway, mechanical allodynia, and upregulation of Glut1, VEGF, and PGP9.5 proteins in dorsal root ganglia. Under the influence of agonists of Epac/PKC, normal rats showed mechanical allodynia and increased Glut1, VEGF, and PGP9.5 proteins. After inhibition of Epac1 in rats with SMIR, mechanical allodynia was alleviated, and proinflammatory cytokines and Glut1, VEGF, and PGP9.5 proteins were decreased. Moreover, dorsal root ganglia neurons showed abnormal proliferation under the activation of the Epac/PKC pathway. Using Captopril to protect vascular endothelial cells after SMIR had a positive effect on postoperative pain. In conclusion, SMIR regulates the persistent postoperative pain in rats by the Epac/PKC pathway.

Pharmacological Inhibition of Epac1 Averts Ferroptosis Cell Death by Preserving Mitochondrial Integrity.

Exchange proteins directly activated by cAMP (Epac) proteins are implicated in a wide range of cellular functions including oxidative stress and cell survival. Mitochondrial-dependent oxidative stress has been associated with progressive neuronal death underlying the pathology of many neurodegenerative diseases. The role of Epac modulation in neuronal cells in relation to cell survival and death, as well as its potential effect on mitochondrial function, is not well established. In immortalized hippocampal (HT-22) neuronal cells, we examined mitochondria function in the presence of various Epac pharmacological modulators in response to oxidative stress due to ferroptosis. Our study revealed that selective pharmacological modulation of Epac1 or Epac2 isoforms, exerted differential effects in erastin-induced ferroptosis conditions in HT-22 cells. Epac1 inhibition prevented cell death and loss of mitochondrial integrity induced by ferroptosis, while Epac2 inhibition had limited effects. Our data suggest Epac1 as a plausible therapeutic target for preventing ferroptosis cell death associated with neurodegenerative diseases.

Exchange proteins directly activated by cyclic adenosine monophosphate inhibitor reverses mechanical allodynia via the modification of astrocytes activity in the spinal cord.

BackgroundGliosis and inflammation are pivotal in the development of acute and chronic pain. Here, we demonstrated a previously unidentified molecular mechanism in which the activation of exchange proteins directly activated by cyclic adenosine monophosphate (Epac)1 accelerated the activation of astrocytes in the spinal cord, thereby promoting chronic postsurgical pain (CPSP).MethodsWe established a rat model of CPSP induced by skin/muscle incision and retraction (SMIR). Pain behaviors were assessed using mechanical withdrawal threshold (MWT) at different times. The lumbosacral enlargement of the spinal cord was isolated to detect the expression of Epac1 and the activity of astrocytes. They were assessed using western blot and immunofluorescence staining.ResultsSMIR induced persistent mechanical hyperalgesia after surgery. This hyperalgesia response was prolonged to more than 21 d after surgery. The time course of spinal Epac1 upregulation was correlated with SMIR-induced pain behaviors. Meanwhile, Epac1 immunoreactivity was colocalized primarily with astrocytes but not with microglial cells or neurons on 7 d after surgery. Intrathecal injection of Epac1 inhibitor CE3F4 significantly suppressed SMIR-induced mechanical allodynia and activation of astrocytes in the spinal cord. This analgesic effect of single-dose administration of CE3F4 lasted up to 6 h and wore off at 12 h after injection.ConclusionsSpinal Epac1-mediated activation of astrocytes may facilitate CPSP. Inhibition of Epac1 may effectively prevent CPSP.

Intracellular receptor EPAC regulates von Willebrand factor secretion from endothelial cells in a PI3K-/eNOS-dependent manner during inflammation

Coagulopathy is associated with both inflammation and infection, including infections with novel severe acute respiratory syndrome coronavirus-2, the causative agent Coagulopathy is associated with both inflammation and infection, including infection with novel severe acute respiratory syndrome coronavirus-2, the causative agent of COVID-19. Clot formation is promoted via cAMP-mediated secretion of von Willebrand factor (vWF), which fine-tunes the process of hemostasis. The exchange protein directly activated by cAMP (EPAC) is a ubiquitously expressed intracellular cAMP receptor that plays a regulatory role in suppressing inflammation. To assess whether EPAC could regulate vWF release during inflammation, we utilized our EPAC1-null mouse model and revealed increased secretion of vWF in endotoxemic mice in the absence of the EPAC1 gene. Pharmacological inhibition of EPAC1 in vitro mimicked the EPAC1-/- phenotype. In addition, EPAC1 regulated tumor necrosis factor-α–triggered vWF secretion from human umbilical vein endothelial cells in a manner dependent upon inflammatory effector molecules PI3K and endothelial nitric oxide synthase. Furthermore, EPAC1 activation reduced inflammation-triggered vWF release, both in vivo and in vitro. Our data delineate a novel regulatory role for EPAC1 in vWF secretion and shed light on the potential development of new strategies to control thrombosis during inflammation.

Interspecies comparison of plasma metabolism and sample stabilization for quantitative bioanalyses: Application to (R)-CE3F4 in preclinical development, including metabolite identification by high-resolution mass spectrometry

The CE3F4 is an inhibitor of the type 1 exchange protein directly activated by cAMP (EPAC1), which is involved in numerous signaling pathways. The inhibition of EPAC1 shows promising results in vitro and in vivo in different cardiac pathological situations like hypertrophic signaling, contributing to heart failure, or arrhythmia. An HPLC-UV method with a simple and fast sample treatment allowed the quantification of (R)-CE3F4. Sample treatment consisted of simple protein precipitation with 50 µL of ethanol and 150 µL of acetonitrile for a 50 µL biological sample. Two wavelengths were used according to the origin of plasma (220 or 250 nm for human samples and 250 nm for murine samples). Accuracy profile was evaluated for both wavelengths, and the method was in agreement with the criteria given by the EMA in the guideline for bioanalytical method validation for human and mouse plasma samples. The run time was 12 min allowing the detection of the (R)-CE3F4 and a metabolite. This study further permitted understanding the behavior of CE3F4 in plasma by highlighting an important difference between humans and rodents on plasma metabolism and may impact future in vivo studies related to this molecule and translation of results between animal models and humans. Using paraoxon as a metabolism inhibitor was crucial for the stabilization of (R)-CE3F4 in murine samples. HPLC-UV and HPLC-MS/MS studies were conducted to confirm metabolite structure and consequently, the main metabolic pathway in murine plasma.

Cyclic AMP-binding protein Epac1 acts as a metabolic sensor to promote cardiomyocyte lipotoxicity

Cyclic adenosine monophosphate (cAMP) is a master regulator of mitochondrial metabolism but its precise mechanism of action yet remains unclear. Here, we found that a dietary saturated fatty acid (FA), palmitate increased intracellular cAMP synthesis through the palmitoylation of soluble adenylyl cyclase in cardiomyocytes. cAMP further induced exchange protein directly activated by cyclic AMP 1 (Epac1) activation, which was upregulated in the myocardium of obese patients. Epac1 enhanced the activity of a key enzyme regulating mitochondrial FA uptake, carnitine palmitoyltransferase 1. Consistently, pharmacological or genetic Epac1 inhibition prevented lipid overload, increased FA oxidation (FAO), and protected against mitochondrial dysfunction in cardiomyocytes. In addition, analysis of Epac1 phosphoproteome led us to identify two key mitochondrial enzymes of the the β-oxidation cycle as targets of Epac1, the long-chain FA acyl-CoA dehydrogenase (ACADL) and the 3-ketoacyl-CoA thiolase (3-KAT). Epac1 formed molecular complexes with the Ca2+/calmodulin-dependent protein kinase II (CaMKII), which phosphorylated ACADL and 3-KAT at specific amino acid residues to decrease lipid oxidation. The Epac1-CaMKII axis also interacted with the α subunit of ATP synthase, thereby further impairing mitochondrial energetics. Altogether, these findings indicate that Epac1 disrupts the balance between mitochondrial FA uptake and oxidation leading to lipid accumulation and mitochondrial dysfunction, and ultimately cardiomyocyte death.

EPAC1 inhibition as a new therapeutic target in anthracyclines induced cardiotoxicity

Doxorubicin (Dox) is an Anthracycline commonly used to treat many types of cancer; unfortunately this chemotherapeutic agent often induces side effects such as cardiotoxicity leading to cardiomyocyte death and dilated cardiomyopathy (DCM). This cardiotoxicity has been related to reactive oxygen species generation, DNA intercalation, topoisomerase II inhibition and bioenergetics alterations resulting in DNA damages leading to cardiomyocyte death. There is a need for new therapeutic agents and strategies aiming at reducing Dox side effects in the heart. Among these mechanisms, the role of Epac (exchange protein directly activated by cAMP) could be worth investigating. Therefore we have investigated the time/dose-dependent Dox effect on Epac signaling in both in vivo mice model (C57BL/6 vs. Epac1 KO mice, iv injections, 12 mg/kg) and in vitro (primary culture of neonatal rat cardiomyocytes (NRVM), Dox 1 μM). In vivo, Dox-treated mice developed a DCM associated with Ca 2+ homeostasis dysfunction. In vitro, as measured by flow cytometry and western blot, Dox induced DNA damage and cell death. This cell death is associated with apoptotic features including mitochondrial membrane permeabilization, caspase activation, cell size reduction and relative plasma membrane integrity. We also observed that Dox led to a modification of the protein level of Epac1 and Epac2 isoforms. The inhibition of Epac1 (ESI09, CE3F4), but not of Epac2 (ESI05), prevented DNA/TopIIβ complexes, decreased Dox-induced DNA damage, loss of mitochondrial membrane potential, apoptosis and finally cardiomyocyte death. These results were confirmed in vivo since Dox-induced cardiotoxicity was prevented in Epac1 KO mice as evidenced by unaltered cardiac function (no DCM) 15 weeks post-treatment. Inhibition of Epac1 could be a valuable therapeutic strategy to limit Dox-induced cardiomyopathy during cancer chemotherapy.

Epac1 Signaling Pathway Mediates the Damage and Apoptosis of Inner Ear Hair Cells after Noise Exposure in a Rat Model

To investigate the role of the exchange protein directly activated by cAMP (Epac) signaling pathway in inner ear hair cell damage and apoptosis after noise exposure, we analyzed the expression level of Epac1 in a rat model of noise-induced hearing loss (NIHL), based on rat exposure to a 4-kHz and 106-dB sound pressure level (SPL) for 8 h. Loss of outer hair cells (OHCs), mitochondrial lesions, and hearing loss were examined after treatment with the Epac agonist, 8-CPT, or the Epac inhibitor, ESI-09. The effects of 8-CPT and ESI-09 on cell proliferation and apoptosis were examined by CCK-8 assays, holographic microscopy imaging, and Annexin-V FITC/PI staining in HEI-OC1 cells. The effects of 8-CPT and ESI-09 on Ca2+ entry were evaluated by confocal Ca2+ fluorescence measurement. We found that the expression level of Epac1 was significantly increased in the cochlear tissue after noise exposure. In NIHL rats, 8-CPT increased the loss of OHCs, mitochondrial lesions, and hearing loss compared to control rats, while ESI-09 produced the opposite effects. Oligomycin was used to induce HEI-OC1 cell damage in vitro. In HEI-OC1 cells treated with oligomycin, 8-CPT and ESI-09 increased and reduced cell apoptosis, respectively. Moreover, 8-CPT promoted Ca2+ uptake in HEI-OC1 cells, while ESI-09 inhibited this process. In conclusion, our data provide strong evidence that the Epac1 signaling pathway mediates early pathological damage in NIHL, and that Epac1 inhibition protects from NIHL, identifying Epac1 as a new potential therapeutic target for NIHL.

EPAC1 inhibition prevents adverse cardiotoxicity induced by anticancer treatment

Anticancer treatments (chemo- and radiation therapies), such as Doxorubicin (Dox), used to treat many types of cancer are associated with many side effects such as cardiotoxicity mainly leading to a dilated cardiomyopathy (DCM) in chronic. There is a need for new treatment options and strategies aiming at reducing Doxorubicin side effects in the heart. Among these mechanisms Epac (exchange protein directly activated by cAMP) signaling could be worth investigating. We have investigated the time/dose-dependent Dox effect on Epac signaling in both in vivo mice model (C57Bl63/Knock-out Epac1 mice, iv injections, 12 mg/kg cumulative dose) and in vitro using primary culture of neonatal rat cardiomyocytes (NRVM, 24 h, Dox 1 μM). Then we investigated the effect of Epac inhibition on human cancer cell lines (24 h, Dox 1-10 μM). Dox-treated mice developed a DCM associated with Ca2+ homeostasis dysfunction. In vitro, as measured by flow cytometry and western blot, Dox induced DNA damages and cell death. This cell death is associated with apoptotic features including mitochondrial membrane permeabilization, caspase activation, cell size reduction and relative plasma membrane integrity. The inhibition of Epac1 (ESI09, CE3F4), but not Epac2 (ESI05), prevented DNA/TopIIβ complexes, decreased Dox-induced DNA damage, loss of mitochondrial membrane potential, apoptosis and finally cardiomyocyte death. These results were confirmed in vivo since Dox-induced cardiotoxicity was prevented in Epac1 KO mice as evidenced by unaltered cardiac function (no DCM) 15 weeks post-treatment. Interestingly, the protection conferred by Epac1 inhibition was not transferred to human cancer cell lines treated by Dox. Inhibition of Epac1 may thus represents a novel strategy to prevent Anthracyclines-induced cardiotoxicity. Moreover, Epac1 inhibition seems to be a valuable therapeutic strategy to treat cancer by potentiating Dox toxicity.

Epac1 inhibition ameliorates pathological angiogenesis through coordinated activation of Notch and suppression of VEGF signaling.

In this study, we investigated the roles of Epac1 in pathological angiogenesis and its potential as a novel therapeutic target for the treatment of vasoproliferative diseases. Genetic deletion of Epac1 ameliorated pathological angiogenesis in mouse models of oxygen-induced retinopathy (OIR) and carotid artery ligation. Moreover, genetic deletion or pharmacological inhibition of Epac1 suppressed microvessel sprouting from ex vivo aortic ring explants. Mechanistic studies revealed that Epac1 acted as a previously unidentified inhibitor of the γ-secretase/Notch signaling pathway via interacting with γ-secretase and regulating its intracellular trafficking while enhancing vascular endothelial growth factor signaling to promote pathological angiogenesis. Pharmacological administration of an Epac-specific inhibitor suppressed OIR-induced neovascularization in wild-type mice, recapitulating the phenotype of genetic Epac1 knockout. Our results demonstrate that Epac1 signaling is critical for the progression of pathological angiogenesis but not for physiological angiogenesis and that the newly developed Epac-specific inhibitors are effective in combating proliferative retinopathy.

EPAC1 and EPAC2 promote nociceptor hyperactivity associated with chronic pain after spinal cord injury

Chronic pain following spinal cord injury (SCI) is associated with electrical hyperactivity (spontaneous and evoked) in primary nociceptors. Cyclic adenosine monophosphate (cAMP) signaling is an important contributor to nociceptor excitability, and knockdown of the cAMP effector, exchange protein activated by cAMP (EPAC), has been shown to relieve pain-like responses in several chronic pain models. To examine potentially distinct roles of each EPAC isoform (EPAC1 and 2) in maintaining chronic pain, we used rat and mouse models of contusive spinal cord injury (SCI). Pharmacological inhibition of EPAC1 or 2 in a rat SCI model was sufficient to reverse SCI-induced nociceptor hyperactivity, indicating that EPAC1 and 2 signaling activity are complementary, with both required to maintain hyperactivity. However, EPAC activation was not sufficient to induce similar hyperactivity in nociceptors from naïve rats, and we observed no change in EPAC protein expression after SCI. In the mouse SCI model, inhibition of both EPAC isoforms through a combination of pharmacological inhibition and genetic deletion was required to reverse SCI-induced nociceptor hyperactivity. This was consistent with our finding that neither EPAC1−/− nor EPAC2−/− mice were protected against SCI-induced chronic pain as assessed with an operant mechanical conflict test. Thus, EPAC1 and 2 activity may play a redundant role in mouse nociceptors, although no corresponding change in EPAC protein expression levels was detected after SCI. Despite some differences between these species, our data demonstrate a fundamental role for both EPAC1 and EPAC2 in mechanisms maintaining nociceptor hyperactivity and chronic pain after SCI.

P3116New role of EPAC1 in Anthracycline-induced cardiotoxicity and anticancer therapy

Abstract Introduction Doxorubicin (Dox) is an anthracycline commonly used to treat many types of cancer; unfortunately this chemotherapeutic agent induces many side effects such as cardiotoxicity leading to dilated cardiomyopathy (DCM). The cardiotoxicity of Dox has been related to reactive oxygen species generation, DNA intercalation, topoisomerase II inhibition and bioenergetics alterations leading to cardiomyocyte death. Objective Nowadays the challenge is to find new treatment options aiming at reducing Dox cardiotoxicity. Epac (exchange protein directly activated by cAMP) signaling could be worth investigating as Epac activates small G proteins which are known to be involved in Dox-induced cardiotoxicity. Methods We investigated the time/dose-dependent Dox effect on Epac signaling in both in vivo mice model (C57Bl63/ Knock-out Epac1 mice, iv injections, 12mg/kg cumulative dose) and in vitro (primary culture of neonatal rat cardiomyocytes (NRVM, 24h, Dox 1μM). Results In vivo, Dox-treated mice developed a DCM associated with Ca2+ homeostasis dysfunction (increase of Ca2+ waves and Ca2+ leaks). In vitro, as measured by flow cytometry and western blot, Dox (1μM) induced DNA damages and cell death in NRVM. This cell death is associated with apoptotic features including mitochondrial membrane permeabilization, caspase activation and cell size reduction. The inhibition of Epac1 (ESI09, CE3F4) decreased Dox-induced DNA damage, loss of mitochondrial membrane potential, apoptosis and finally cardiomyocyte death. Moreover, in vivo, Epac1 KO mice were protected against Dox-induced cardiotoxicity by unaltered cardiac function (no DCM) and calcium homeostasis at 15 weeks post-treatment. Conclusion Inhibition of Epac1 could be a valuable therapeutic strategy to limit Dox-induced cardiomyopathy during cancer chemotherapy. Indeed, preliminary data show also that preventing Dox-induced cardiotoxicity, the inhibition of Epac1 can also potentiate cancerous cells death. Acknowledgement/Funding Labex Lermit (ANR 0033), Torino and Inserm

Epac1 inhibits PKR to reduce NLRP3 inflammasome proteins in retinal endothelial cells.

Purpose: Inflammation has been strongly associated with retinal damage in diseases such as diabetic retinopathy. Several studies have reported that high glucose exposure induces damage to the retinal vasculature. We and others have shown that high glucose can activate the NOD-like receptor family, pyrin domain containing family member 3 (NLRP3) pathway, leading to increased levels of cleaved caspase 1 and IL-1β to activate a number of inflammatory pathways in the retina.Methods: We used retinal endothelial cells grown in normal (5 mM) or high (25 mM) glucose or retinal lysates from endothelial cell-specific knockout mice for exchange protein activated by cAMP 1 (Epac1). Human recombinant protein kinase R (PKR) or C16, a PKR inhibitor, was used on the cells to dissect PKR and NLRP3 signaling.Results: Using retinal endothelial cells (REC) in high glucose and whole retinal lysates from endothelial cell-specific knockout of Epac1, we demonstrate that Epac1 regulates PKR phosphorylation. Using an Epac1 agonist or PKR inhibition with C16, we demonstrated that loss of PKR resulted in reduced NLRP3, cleaved caspase 1, and IL-1β levels. Furthermore, despite the addition of recombinant human PKR, Epac1 was still able to significantly reduce NLRP3 signaling.Conclusion: Overall, these studies demonstrated that PKR regulates the NLRP3 inflammasome in REC, and that Epac1 inhibition of PKR can reduce activation of the NLRP3 inflammasome.

Anticancer therapy induced cardiotoxicity: Involvement of EPAC1 in cardioprotection and anticancer therapy

Doxorubicin (Dox) is an anthracycline commonly used to treat many types of cancer; unfortunately this chemotherapeutic agent induces many side effects such as cardiotoxicity leading to dilated cardiomyopathy (DCM). The cardiotoxicity of Dox has been related to reactive oxygen species generation, DNA intercalation and topoisomerase II inhibition, bioenergetics alterations leading to cardiomyocyte death. Nowadays the challenge is to find new treatment options aiming at reducing Dox cardiotoxicity. Epac (exchange protein directly activated by cAMP) signaling could be worth investigating as Epac activates small G proteins which are known to be involved in Dox-induced cardiotoxicity. We investigated the time/dose-dependent Dox effect on Epac signaling in both in vivo mice model (C57Bl63/Knock-out Epac1 mice, iv injections, 12mg/kg cumulative dose) and in vitro (primary culture of neonatal rat cardiomyocytes (NRVM, 24h, Dox 1μM). In vivo, Dox-treated mice developed a DCM associated with Ca2 + homeostasis dysfunction (increase of Ca2 + waves and Ca2 + leaks). In vitro, as measured by flow cytometry and western blot, Dox (1μM) induced DNA damage and cell death in NRVM. This cell death is associated with apoptotic features including mitochondrial membrane permeabilization, caspase activation and cell size reduction. The inhibition of Epac1 (ESI09, CE3F4) decreased Dox-induced DNA damage, loss of mitochondrial membrane potential, apoptosis and finally cardiomyocyte death. Moreover, in vivo, Epac1 KO mice were protected against Dox-induced cardiotoxicity by unaltered cardiac function (no DCM) and calcium homeostasis at 15 weeks post-treatment. Inhibition of Epac1 could be a valuable therapeutic strategy to limit Dox-induced cardiomyopathy during cancer chemotherapy. Indeed, preliminary data show also that preventing Dox-induced cardiotoxicity, the inhibition of Epac1 can also potentiate cancerous cells death.