Background: Cardiac endothelial cells (ECs) modulate the cardiac stress response during health and disease and thereby exert a major impact on heart growth and function. To date, mainly endothelial derived proteins were studied; however, growing evidence suggests that long non-coding RNAs (lncRNAs) can also contribute to these effects. The 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 hitherto largely unstudied intergenic lncRNA downstream of Cdkn1b, Lockd. Lockd is significantly upregulated in heart tissue during cardiac pressure overload induced by transverse aortic constriction (TAC). We found that Lockd expression peaks in the cardiac tissue one week after TAC operation approximately 11-fold in comparison to Sham. Then Lockd gradually declines back to basal levels. This observation indicates 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 occurs in cardiomyocytes and endothelial cells, but not in fibroblasts.

Lockd expression in cardiac ECs also peaked one week after TAC surgery, on average 6-fold increase compared to Sham. One week post-TAC, endothelial cells contribute the most to Lockd upregulation 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 10 weeks after TAC, Lockd expression is on average twice as high as in Sham-operated mice.

To further elucidate the role of endothelial Lockd, we performed RNA sequencing following siRNA Lockd knockdown. Silencing of Lockd with siRNA resulted in 90% reduction of Lockd levels. This revealed that Lockd downregulation inhibits the expression of genes related to regulation of blood vessel development (Pdgfb, Mtor, Foxc2) and genes involved in the extracellular matrix organisation (Col4a3, Col4a4, Prg4). On the other hand, Lockd knock-down increased the expression of genes associated with cell-cycle progression (Uhmk1, Cdc25b, Ccnd3) and positive regulation of cell death (Hyal2, Ccar2, Htt).

Next, single cell sequencing screen from cardiac ECs one week after TAC and Sham revealed that Lockd is almost exclusively present in one representative cell cluster associated to ECs undergoing mitosis.

Functional investigation showed that upregulation of Lockd in ECs via 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.

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.

Conclusion: Lockd expression increases in cardiac ECs in the compensatory phase of induced pressure overload. Lockd upregulation seems to reduce angiogenesis by modulating cell cycle and mesenchyme development related gene expression in endothelial cells.