The cyclic 3′,5′-adenosine monophosphate (cAMP) sensor enzyme, EPAC1, is a candidate drug target in vascular endothelial cells (VECs) due to its ability to attenuate proinflammatory cytokine signalling normally associated with cardiovascular diseases (CVDs), including atherosclerosis. This is through the EPAC1-dependent induction of the suppressor of cytokine signalling gene, SOCS3, which targets inflammatory signalling proteins for ubiquitinylation and destruction by the proteosome. Given this important role for the EPAC1/SOCS3 signalling axis, we have used high throughput screening (HTS) to identify small molecule EPAC1 regulators and have recently isolated the first known non-cyclic nucleotide (NCN) EPAC1 agonist, I942. I942 therefore represents the first in class, isoform selective EPAC1 activator, with the potential to suppress pro-inflammatory cytokine signalling with a reduced risk of side effects associated with general cAMP-elevating agents that activate multiple response pathways. The development of augmented I942 analogues may therefore provide improved research tools to validate EPAC1 as a potential therapeutic target for the treatment of chronic inflammation associated with deadly CVDs.

Pulmonary arterial hypertension, group 1 of the pulmonary hypertension disease family, involves pulmonary vascular remodelling, right ventricular dysfunction and cardiac failure. Oxidative stress, through activation of mitogen-activated protein kinases is implicated in these changes. Inhibition of apoptosis signal-regulating kinase 1, an apical mitogen-activated protein kinase, prevented pulmonary arterial hypertension developing in rodent models. Here, we investigate apoptosis signal-regulating kinase 1 in pulmonary arterial hypertension by examining the impact that its inhibition has on the molecular and cellular signalling in established disease. Apoptosis signal-regulating kinase 1 inhibition was investigated in in vivo pulmonary arterial hypertension and in vitro pulmonary hypertension models. In the in vivo model, male Sprague Dawley rats received a single subcutaneous injection of Sugen SU5416 (20 mg/kg) prior to two weeks of hypobaric hypoxia (380 mmHg) followed by three weeks normoxia (Sugen/hypoxic), then animals were either maintained for three weeks on control chow or one containing apoptosis signal-regulating kinase 1 inhibitor (100 mg/kg/day). Cardiovascular measurements were carried out. In the in vitro model, primary cultures of rat pulmonary artery fibroblasts and rat pulmonary artery smooth muscle cells were maintained in hypoxia (5% O2) and investigated for proliferation, migration and molecular signalling in the presence or absence of apoptosis signal-regulating kinase 1 inhibitor. Sugen/hypoxic animals displayed significant pulmonary arterial hypertension compared to normoxic controls at eight weeks. Apoptosis signal-regulating kinase 1 inhibitor decreased right ventricular systolic pressure to control levels and reduced muscularised vessels in lung tissue. Apoptosis signal-regulating kinase 1 inhibition was found to prevent hypoxia-induced proliferation, migration and cytokine release in rat pulmonary artery fibroblasts and also prevented rat pulmonary artery fibroblast-induced rat pulmonary artery smooth muscle cell migration and proliferation. Apoptosis signal-regulating kinase 1 inhibition reversed pulmonary arterial hypertension in the Sugen/hypoxic rat model. These effects may be a result of intrinsic changes in the signalling of adventitial fibroblast.

Cardiac magnetic resonance-derived ventricular variables are predictive of mortality in pulmonary arterial hypertension. Rodent models which emphasize ventricular function, allowing serial monitoring, are needed to identify pathophysiological features and novel therapies for pulmonary arterial hypertension. We investigated longitudinal changes in the Sugen–hypoxia model during disease progression. Sprague Dawley rats ( n = 32) were divided into two groups. (1) Sugen–hypoxia: a dose of subcutaneous Sugen-5416 and placed in hypobaric hypoxia for two weeks followed by normoxia for three weeks. (2) Normoxia: maintained at normal pressure for five weeks. Rats were examined at five or eight weeks with right-heart catheter, cardiac magnetic resonance, and autopsy. Compared to normoxic controls (23.9 ± 4.1 mmHg), right ventricular systolic pressure was elevated in Sugen–hypoxia rats at five and eight weeks (40.9 ± 15.5 mmHg, p = 0.026; 48.9 ± 9.6 mmHg, p = 0.002). Right ventricular end-systolic volume index was increased in eight weeks Sugen–hypoxia (0.28 ± 0.04 µlcm–2, p = 0.003) compared to normoxic controls (0.18 ±0.03 mlcm–2). There was progressive dilatation of the right ventricular at eight weeks Sugen–hypoxia compared to normoxic controls (0.75 ± 0.13 µlcm–2 vs 0.56 ± 0.1 µlcm–2 p = 0.02). Ventricle mass index by cardiac magnetic resonance at five weeks (0.34 ± 0.06, p = 0.003) and eight weeks Sugen–hypoxia (0.34 ± 0.06, p = 0.002) were higher than normoxic controls (0.21 ± 0.04). Stroke volume, right ventricular ejection fraction, and left ventricular variables were preserved in Sugen–hypoxia. Ventricular changes during the course of illness in a pulmonary arterial hypertension rodent model can be examined by cardiac magnetic resonance. These changes including right ventricular hypertrophy and subsequent dilatation are similar to those seen in pulmonary arterial hypertension patients. Despite the persisting pulmonary hypertension, there are features of adaptive cardiac remodeling through the study duration.

An exchange protein directly activated by cAMP 1 (EPAC1) is an intracellular sensor for cAMP that is involved in a wide variety of cellular and physiological processes in health and disease. However, reagents are lacking to study its association with intracellular cAMP nanodomains. Here, we use non-antibody Affimer protein scaffolds to develop isoform-selective protein binders of EPAC1. Phage-display screens were carried out against purified, biotinylated human recombinant EPAC1ΔDEP protein (amino acids 149–811), which identified five potential EPAC1-selective Affimer binders. Dot blots and indirect ELISA assays were next used to identify Affimer 780A as the top EPAC1 binder. Mutagenesis studies further revealed a potential interaction site for 780A within the EPAC1 cyclic nucleotide binding domain (CNBD). In addition, 780A was shown to co-precipitate EPAC1 from transfected cells and co-localize with both wild-type EPAC1 and a mis-targeting mutant of EPAC1(K212R), predominantly in perinuclear and cytosolic regions of cells, respectively. As a novel EPAC1-selective binder, 780A therefore has the potential to be used in future studies to further understand compartmentalization of the cAMP-EPAC1 signaling system.

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