Endothelial junctional protein associated with coronary artery disease (JCAD) as a potential therapeutic target for acute ischemic stroke

Abstract Introduction Acute ischemic stroke (AIS) is a leading cause of mortality and long-time disability worldwide. Despite the increasing availability of early reperfusion, the neurological outcome of patients with AIS is still poor. Junctional protein associated with Coronary Artery Disease (JCAD) is a novel protein, associated to coronary artery disease, arterial thrombosis, atherosclerosis and inflammation. Yet, its role in acute ischemic stroke is unclear. Purpose To investigate the role of JCAD in the pathophysiology of brain acute ischemia and to explore its potential as a pharmacological target in AIS. Methods Transient Middle Cerebral Artery Occlusion (tMCAO) was inducede in mice with either global or endothelial-specific JCAD deletion, and in littermate controls. Stroke size was assessed ex-vivo 48 hours after reperfusion. For neurological assessment, RotaRod Test and Bederson score were recorded 24 and 48 hours after reperfusion. In vivo silencing of JCAD was achieved by intravenous injection of a JCAD small interfering RNA (siRNA) after tMCAO. JCAD silencing was performed in vitro in human brain microvascular endothelial cells (HBMVECs) using siRNA transfection, followed by hypoxia/reoxygenation (H/R) injury. Cell death and monolayer integrity were measured by LDH assay and trans-endothelial electrical resistance (TEER), respectively. Molecular mechanisms were investigated in vivo by immunohistochemistry and in vitro by Western blot, respectively. JCAD plasma levels were measured by ELISA in two independent cohorts of patients with AIS. Results The expression of JCAD was up-regulated in the ipsilateral hemisphere of stroke in wild-type mice. Both global and endothelial-specific JCAD knockout mice displayed reduced stroke size after tMCAO and an improved neurological outcome, similar to mice treated with post-ischemic JCAD silencing (Figure 1). In vitro, JCADsilenced HBMVECs showed a reduced cell death and an improved monolayer integrity after H/R injury, paralleled by a reduced expression of cellular adhesion molecules (CAMs). The intracellular messenger Akt was hyperphosphorylated in JCAD-silenced HBMVECs (Figure 2). The treatment with the Akt/PI3K inhibitor Wortmannin, retrieved the non-silenced phenotype in HBMVECs, following H/R. Circulating levels of JCAD were persistently increased in patients with AIS and higher levels of JCAD at the time of hospitalization were associated with higher inflammation and an increased risk of death 90 days after the event. Conclusions JCAD expression is associated with a larger brain damage in mice in vivo and with a higher mortality in patients. Mechanistic results suggest that JCAD plays a pivotal role in regulating the integrity of endothelium after a H/R injury, inducing cellular death and inflammation through the inhibition of the Akt/PI3K pathway. Thus, in vivo silencing of JCAD may represent a therapeutic target for the treatment of acute ischemic stroke, on top of early reperfusion.Figure 1Figure 2