Background: Myocardial endothelial cells (ECs) are able to modulate the cardiac stress response during health and disease and thereby exert a major impact on heart growth and function. While so far mainly endothelial derived proteins were studied, growing evidence suggests that long non-coding RNAs (lncRNAs) can also contribute to these effects.

Aim of the study was to investigate how endothelial cell derived lncRNAs contribute to angiogenesis, endothelial cell survival and fibrotic/mesenchymal gene expression during cardiac pressure overload.

Methods and Results: In a screen for differentially regulated lncRNAs in cardiac endothelial cells during pressure overload, we identified the so far largely unstudied intergenic lncRNA downstream of Cdkn1b, Lockd. It is significantly upregulated in heart tissue during cardiac pressure overload induced by transverse aortic constriction (TAC). We found that Lockd expression peaks in cardiac tissue one week after TAC operation (approx. 11-fold increase vs. Sham, p<0.05, n=5), and gradually declines back to basal levels, indicating that Lockd is induced during the compensatory phase of cardiac remodelling.

Separation of cardiac cells with MACS/Langendorff one week after TAC and Sham revealed that Lockd upregulation is happening in cardiomyocytes and endothelial cells, but not in fibroblasts.

Lockd expression in cardiac ECs also peaked one week after TAC surgery (approx. 6-fold increase compared to Sham, p<0.005, n=6) and then gradually fell back to basal values. After two weeks, the upregulation of Lockd in ECs is on average three times as high and 16 weeks after TAC, Lockd expression is on average twice as high as in sham-operated mice.

Our results show that upregulation of Lockd in ECs is regulated by the growth factor FGF2. On the other hand, we observed a negative effect of TGF-β on Lockd expression.

We next investigated the functional impact of Lockd on endothelial cells in vitro. Upregulation of Lockd in ECs utilizing adenoviral infection reduced angiogenesis as shown by sprouting assay. In contrast, down-regulation of Lockd with siRNA triggered a stronger sprouting of endothelial cell spheroids. Silencing of Lockd with siRNA resulted in 90% reduction of Lockd levels.

Furthermore, down-regulation of Lockd caused an increased ability of ECs to proliferate while its overexpression reduced proliferation, demonstrated with BrdU proliferation assay.

Interestingly, down-regulation of Lockd in ECs increased the Caspase3/7 activity, suggesting that Lockd may play a role in the negative regulation of apoptosis and hence contributes to cell survival.

RNA sequencing in siRNA control and siLockd treated endothelial cells showed that Lockd downregulation inhibited the expression of genes related to the pentose phosphate pathway (G6pd2, Notch1, Esd) and genes involved in the negative regulation of apoptotic processes (Mt1, Myc, Ada). On the other hand, it increased the expression of genes associated with mesenchyme development (Fgf9, Hif1a, Wnt11) and extracellular matrix organization (Ahr, Col2a1, Fbn2, Eln).

Conclusion: Lockd expression increases in cardiac ECs in the early phase of induced pressure overload. As suggested by our in vitro results, Lockd upregulation might counteract angiogenesis, apoptosis and mesenchymal gene expression in endothelial cells.