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  • EPAC induces SOCS gene expression in VECs resulting


    EPAC1 induces SOCS3 gene expression in VECs, resulting in suppression of the JAK–STAT activation initiated by the sIL-6Rα/IL-6 trans-signalling complex [67]. EPAC1 regulates SOCS3 gene induction through the activation of C/EBP and c-Jun transcription factors, which interact directly with the SOCS3 promoter 68, 69 (Figure 3). The pathway leading to SOCS3 induction requires Rap1 GTPase and occurs independently of PKA [67]. Another key role of EPAC1 in VECs is the stabilisation of vascular endothelial cadherin (VE-cadherin) complexes between adjacent cells to maintain barrier function 70, 71 (Figure 3). EPAC1-mediated barrier protection involves reciprocal regulation of the Rho GTPase family members Rac and RhoA, which exert opposing effects on endothelial barrier function. Rac activation by EPAC1 promotes junction stability [72], whereas RhoA activation disrupts VE-cadherin junctions through microtubule destabilisation [73]. The importance of EPAC-activated Rac in these processes has been demonstrated by the use of the EPAC inhibitor ESI-09 (Table 1), which inhibits Rac activation and prevents the recovery of endothelial barrier function in response to thrombin treatment [74]. Intriguingly, alterations in cytoskeletal stability are also thought to underlie the effects of EPAC1 in VSMCs, where EPAC1 has been shown to synergise with PKA to suppress the VSMC proliferation that is normally associated with NH [75]. In this case EPAC1 is thought to suppress Rac activity, leading to cytoskeletal remodelling, nuclear export of ERK1/2, and inhibition of the transcription factor Egr1 [76]. Rac activation normally promotes VSMC proliferation and neointima formation, whereas inhibition of Rac by PKA and EPAC1 leads to upregulation of the Plumbagin inhibitor p27(KIP1) through suppression of Skp2, an F-box protein component of the Skp–Cullin–F-box(Skp2) ubiquitin ligase, which normally targets p27(KIP1) for proteolytic degradation during S phase [77] (Figure 3). Clearly, small-molecule activators of EPAC1 have the ability to induce SOCS3 and inhibit proinflammatory IL-6 signalling in VECs and suppress the proliferation of VSMCs, an event normally associated with neointima formation, and therefore may form the basis of novel therapeutic agents to combat the localised inflammation associated with atherosclerosis and NH. Also related to vascular function is recent work demonstrating that knockout or pharmacological inhibition of EPAC1 blocks adhesion to, and subsequent invasion of, endothelial cells by Rickettsia bacteria, demonstrating that EPAC1 may be a promising target for the treatment of rickettsioses [78]. Caution should be taken, however, particularly in light of the study conducted by Yokoyama et al. demonstrating that EPAC1 levels are upregulated during neointima formation and EPAC activation promotes VSMC migration, independently of PKA [79]. Moreover, while EPAC can negatively regulate proinflammatory JAK–STAT signalling in VECs, it has also been reported to promote the exocytosis of Weibel–Palade bodies, which contain inflammatory mediators, from endothelial cells [80]. Furthermore, while EPAC1 expression appears to be elevated, expression of the EPAC1 target gene SOCS3 within proliferating VSMCs in the neointima may be reduced [81]. In vitro studies suggest that this is due to DNA methyltransferase-I-mediated hypermethylation of the CpG island within the SOCS3 promoter, which blocks gene induction [82]. As a result, it would be anticipated that the capacity of EPAC1 to limit proinflammatory responses is compromised, which would aggravate the pathological effects of EPAC1 activation in VSMCs. Clearly, further genetic and pharmacological studies will help to further define the contribution of EPAC1 to atherosclerosis and vascular remodelling.
    EPAC-selective cAMP analogues The role of EPAC in the regulation of multiple physiological processes highlights how manipulation of EPAC isoforms could be exploited for treatment of diseases like T2D (EPAC2) and atherosclerosis and NH (both EPAC1). Initial attempts to develop EPAC-selective regulators focused on attempts to produce analogues of cGMP, which is a known antagonist of EPAC 15, 83, 84. Despite this, there are no cyclic nucleotide inhibitors of EPAC in current use. Rather, work has focused on the development of cAMP analogues able to activate EPACs independently of PKA (Table 1). In particular, the addition of a methyl group to the oxygen of the second carbon of the ribose moiety was observed to promote EPAC1 and 2 activation while greatly reducing the affinity of the 007 cAMP analogue for PKA [85]. This specificity arose due to a single amino acid difference within the cAMP-binding pocket of the otherwise highly conserved CNBD of PKA and EPAC (Figure 5). The substitution of a bulky glutamic acid residue within PKA for glutamine or lysine, in EPAC1 and EPAC2 respectively, allowed the EPACs, but not PKA, to accept the 2′O-methylated cAMP analogue [85] (Figure 5). 007, along with its improved, cell-permeable analogue 007-AM (Figure 5) [86], has greatly facilitated the study of the cellular actions of EPAC, by allowing the PKA-independent effects of cAMP signalling to be observed directly 70, 85, 87. However, in vivo use has been limited by its high effective dose and low cell permeability and the induction of cardiac arrhythmia, fibrosis, and hypertrophy [88]. Furthermore, various off-target effects limit its specificity, such as its inhibitory effect over PDEs [89] and off-target activation of the P2Y12 purinergic receptors present in platelets [90].