Endothelial colony-forming cell NOX4 signalling promotes vasoreparative capacity of coronary microvascular endothelial cells and reduces adverse cardiac remodelling

Objective
Endothelial dysfunction is a central driver of adverse myocardial remodelling, whilst cord blood-derived endothelial colony-forming cells (CB-ECFCs), which we have reported to be positively regulated by NOX4 expression, show clear potential to modulate pathological cardiac signalling. This study aimed to investigate effects of CB-ECFC NOX4 signalling on mature endothelial cells exposed to experimental hypoxia, as a relevant remodelling stress, and impact on cardiac structural and functional changes in a mouse model of hypertension.

Methods
Human coronary microvascular endothelial cells (HCMECs; n=3) were treated with conditioned media collected from NOX4 overexpressing (OE) or empty vector (EV) transfected human CB-ECFCs prior to exposure to hypoxia (1% O2) or normoxia (21% O2) for 48h and assessment of barrier function and gene expression (qRT-PCR, western blot, RNA sequencing). Adult C57BL6/J mice were infused with angiotensin II (1.5 mg/kg/day; n=6) for 1 week prior to intramyocardial injection with NOX4OE or EV mouse ECFCs and terminal echocardiography and histology analysis after a further 3 weeks.

Results
HCMECs showed impaired barrier integrity in hypoxia which was associated with decreased expression of key barrier proteins. Co-incubation with conditioned medium from NOX4OE CB-ECFCs rescued claudin-5 expression but did not affect barrier function. RNA sequencing analysis of hypoxic HCMECs highlighted several impacts of CB-ECFC NOX4 paracrine signalling, including activation of cell migration and decreased inflammatory associated signalling (e.g. glycoprotein VI and IL-4). In vivo studies demonstrated that intra-myocardial injection of NOX4 OE ECFC in hypertensive mice increased myocardial vascular density (vascular area: EV 11.25+0.2, OE 13.11+0.4 µm2; p<0.001) and extracellular matrix gene expression (e.g. α-SMA, CTGF), with a tendency towards improved left ventricular function (e.g. increased global longitudinal strain).

Conclusions
Taken together, these findings highlight NOX4 ECFC paracrine signalling as an important regulator of HCMEC function in hypoxia which also enhances ECFC vasoreparative capacity in vivo with potential to positively impact on pathological cardiac remodelling.