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Circular RNAs (circRNAs) are generated by back-splicing and control cellular signaling and phenotypes. Pericytes stabilize the capillary structure and play an important role in the formation and maintenance of new blood vessels. Here, we characterized hypoxia-regulated circRNAs in human pericytes and showed that circPLOD2 is induced by hypoxia and regulates pericyte function. Silencing of circPLOD2 increased pericyte proliferation, endothelial-pericyte interaction and tube formation. Transcriptional profiling of circPLOD2-depleted cells and epigenomic analyses revealed widespread changes in gene expression and identified the circPLOD2-dependent regulation of the transcription factor KLF4 as a key effector of these changes. Importantly, overexpression ofKLF4was sufficient to reverse the effects on pericyte proliferation and endothelial-pericyte interactions observed after circPLOD2 depletion. Together, these data revealed a novel function of circPLOD2 in the control of pericyte proliferation and capillary formation and showed that circPLOD2-mediated regulation of KLF4 significantly contributes to the transcriptional response to hypoxia.HighlightscircPLOD2 is upregulated in hypoxia in human vascular pericytesSelective depletion of circPLOD2, but not linearPLOD2mRNA, changes pericyte migration and endothelial-pericyte interactioncircPLOD2 depletion triggers widespread changes in gene expression that are mirrored in the transcriptional hypoxia responseEpigenomic analyses pinpoint the transcription factor KLF4 as a central player in circPLOD2-mediated expression changesKLF4overexpression is sufficient to rescue the changes in pericyte function caused by circPLOD2 depletion

Cardiac resident macrophages (crMPs) were recently shown to exert pivotal functions in cardiac homeostasis and disease, but the underlying molecular mechanisms are largely unclear. Long non-coding RNAs (lncRNAs) are increasingly recognized as important regulatory molecules in a number of cell types, but neither the identity nor the molecular mechanisms of lncRNAs in crMPs are known. Here, we have employed deep RNA-seq and single cell RNA sequencing to resolve the crMP lncRNA landscape from healthy and diseased murine myocardium. CrMPs express previously unknown and highly cell type-specific lncRNAs, among which one lncRNA, termedSchlafenlnc, was particularly abundant and enriched in crMPs. We foundSchlafenlncto be necessary for migration-associated gene expression in macrophagesin vitroandin vivoand essential for their adhesion and migration. Collectively, our data provide a basis to the systematic characterization of lncRNAs in crMPs and establishSchlafenlncas a critical regulator of macrophage migratory functions.

AimsAdvanced age is unequivocally linked to the development of cardiovascular disease, however, the mechanisms leading to loss of endothelial cell regenerative capacity during aging remain poorly understood. Here we aimed to investigate novel mechanisms involved in endothelial cell senescence, that impact on endothelial cell transcription and the vascular repair response upon injuryMethods and resultsRNA sequencing of a unique collection of native endothelial cells from young and aged individuals, showed that aging (20 vs. 80 years) is characterized by p53- mediated reprogramming to promote the expression of senescence-associate genes. Molecular analysis revelead that p53 accumulated and acetylated in the nucleus of aged human endothelial cells to suppress glycolysis. Metabolic flux analysis identified an associated reduction in glucose uptake and ATP availability that inhibited the assembly of the telomerase complex, which was essential for proliferation. Nuclear translocation of p53 in aged endothelial cells was attributed to the loss of the vasoprotective enzyme, cystathionine γ-lyase (CSE), which physically anchored p53 in the cytosol. In mice, loss of endothelial cell CSE activated p53 and arrested vascular repair upon injury, while the AAV9 mediated re-expression of an active CSE mutant retained p53 in the cytosol, maintained endothelial glucose metabolism and proliferation, and prevented endothelial cell senescence. Adenoviral overexpression of CSE in human native aged endothelial cells maintained low p53 activity and re-activated telomerase to revert endothelial cell senescence.ConclusionOur data identified the interaction between CSE and p53 as a promising target to preserve vascular regeneration during aging.Key QuestionTo identify the mechanisms that regulate endothelial cell senescence under native conditions and their impact on vascular repair in aging.Key FindingLack of a physical interaction between CSE and p53 metabolically reprogrammes endothelial cells to reduce telomerase activity and halt endothelial cell regeneration.Take home messageInterventions to increase CSE expression represent a novel therapy against p53-induced endothelial cell cycle arrest and senescenseTranslational perspectiveEndothelial rejuvenation strategies could serve as promising therapies against age-related cardiovascular diseases. By investigating human native endothelial cells from young and aged individuals, we identified that the age-related nuclear accumulation of p53 reprograms endothelial cell metabolism, regulates telomerase activity and inhibits endothelial cell regeneration. Nuclear localization of p53 resulted from a loss of its interaction with the cysteine catabolizing enzyme cystathionine γ-lyase in the cytoplasm. Enhancing the physical interaction of p53 with CSE by gene therapy could revert endothelial cell senescence and activate endothelial reparative responses.

Long non-coding RNAs (lncRNAs) can act as regulatory RNAs which, by altering the expression of target genes, impact on the cellular phenotype and cardiovascular disease development. Endothelial lncRNAs and their vascular functions are largely undefined. Deep RNA-Seq and FANTOM5 CAGE analysis revealed the lncRNA LINC00607 to be highly enriched in human endothelial cells. LINC00607 was induced in response to hypoxia, arteriosclerosis regression in non-human primates and also in response to propranolol used to induce regression of human arteriovenous malformations. siRNA knockdown or CRISPR/Cas9 knockout of LINC00607 attenuated VEGF-A-induced angiogenic sprouting. LINC00607 knockout in endothelial cells also integrated less into newly formed vascular networks in an in vivo assay in SCID mice. Overexpression of LINC00607 in CRISPR knockout cells restored normal endothelial function. RNA- and ATAC-Seq after LINC00607 knockout revealed changes in the transcription of endothelial gene sets linked to the endothelial phenotype and in chromatin accessibility around ERG-binding sites. Mechanistically, LINC00607 interacted with the SWI/SNF chromatin remodeling protein BRG1. CRISPR/Cas9-mediated knockout of BRG1 in HUVEC followed by CUT&RUN revealed that BRG1 is required to secure a stable chromatin state, mainly on ERG-binding sites. In conclusion, LINC00607 is an endothelial-enriched lncRNA that maintains ERG target gene transcription by interacting with the chromatin remodeler BRG1.