The evolutionary conserved Taurine Upregulated Gene 1 (TUG1) is a ubiquitously expressed gene that is one of the highest expressed genes in human and rodent endothelial cells (ECs). We here show that TUG1 expression decreases significantly in aging mouse carotid artery ECs and human ECs in vitro, indicating a potential role in the aging endothelial vasculature system. We therefore investigated if, and how, TUG1 might function in aging ECs, but despite extensive phenotyping found no alterations in basal EC proliferation, apoptosis, barrier function, migration, mitochondrial function, or monocyte adhesion upon TUG1 silencing in vitro. TUG1 knockdown did slightly and significantly decrease cumulative sprout length upon vascular endothelial growth factor A stimulation in human umbilical vein endothelial cells (HUVECs), though TUG1-silenced HUVECs displayed no transcriptome-wide mRNA expression changes explaining this effect. Further, ectopic expression of the highly conserved and recently discovered 153 amino acid protein translated from certain TUG1 transcript isoforms did not alter angiogenic sprouting in vitro. Our data show that, despite a high expression and strong evolutionary conservation of both the TUG1 locus and the protein sequence it encodes, TUG1 does not seem to play a major role in basic endothelial cell function.

Circadian clocks control rhythms in physiology and behavior entrained to 24 h light-dark cycles. Despite of conserved general schemes, molecular circadian clockworks differ between insect species. With RNA interference (RNA i) we examined an ancient circadian clockwork in a basic insect, the hemimetabolous Madeira cockroach Rhyparobia maderae. With injections of double-stranded RNA (dsRNA) of cockroach period (Rm´per), timeless 1 (Rm´tim1), or cryptochrome 2 (Rm´cry2) we searched for essential components of the clocḱ s core negative feedback loop. Single injections of dsRNA of each clock gene into adult cockroaches successfully and permanently knocked down respective mRNA levels withiñ two weeks deleting daytime-dependent mRNA rhythms for Rm´per and Rm´cry2. Rḿ per RNAi or Rm´cry2 RNAi affected total mRNA levels of both genes, while Rm´tim1 transcription was independent of both, also keeping rhythmic expression. Unexpectedly, circadian locomotor activity of most cockroaches remained rhythmic for each clock gene knockdown employed. It expressed weakened rhythms and unchanged periods for Rm´per RNAi and shorter periods for Rm´tim1 RNAi and Rm´cry2 RNAi .As a hypothesis of the cockroach´s molecular clockwork, a basic network of switched differential equations was developed to model the oscillatory behavior of clock cells expressing respective clock genes. Data were consistent with two synchronized main groups of coupled oscillator cells, a leading (morning) oscillator, or a lagging (evening) oscillator that couple via mutual inhibition. The morning oscillators express shorter, the evening oscillators longer endogenous periods based on core feedback loops with either PER, TIM1, or CRY2/PER complexes as dominant negative feedback of the clockwork. We hypothesize that dominant morning oscillator cells with shorter periods express PER, but not CRY2, or TIM1 as suppressor of clock gene expression, while two groups of evening oscillator cells with longer periods either comprise TIM1 or CRY2/PER suppressing complexes. Modelling suggests that there is an additional negative feedback next to Rm´PER in cockroach morning oscillator cells.

Vascular ageing is a key risk factor for cardiovascular diseases and is characterised by a continuous decline in endothelial function. Despite progress in recent years, the molecular mechanisms for this deterioration remain incompletely understood. Long non-coding RNAs (lncRNAs) are a heterogeneous class of RNAs that have been shown to regulate gene expression and protein function, however, little is known about their role in the ageing-associated dysregulation of endothelial cell (EC) function. In this study, we aimed to identify and functionally characterise a novel ageing-regulated lncRNA in ECs. Using RNA sequencing data of cardiac ECs from 12 weeks young and 20 months old mice, we identified Mirial as an ageing-induced lncRNA (1.32-fold, p=0.ehab724.33565). MIRIAL is conserved between mice and humans and has no obvious coding potential. GapmeR-mediated silencing of MIRIAL in human umbilical vein ECs (HUVECs) decreased cell proliferation by 50%, migration by 24% (p=0.045) and basal angiogenic sprouting by 53% (p=0.0029), while increasing VEGF-A-stimulated sprouting by 50% (p=0.0139) and not affecting apoptosis or senescence. Subcellular fractionation of HUVECs revealed that MIRIAL was predominantly associated with the chromatin (80%). Pathway analysis of RNA sequencing data showed an overrepresentation of upregulated p53 target genes upon MIRIAL knockdown in HUVECs which was validated using qRT-PCR (1.8–5.2-fold increased). Using siRNA against p53 we showed that this effect is fully dependent on the presence of p53. Moreover, p53 and its phosphorylated form (Ser15) were both increased (1.8-fold, p=0.01 and 2.9-fold, p=0.02) after MIRIAL silencing. Intriguingly, RNA immunoprecipitation revealed that MIRIAL physically interacts with p53 (3.75-fold enriched, p=0.0067). To further study the interactome of MIRIAL, we performed RNA pulldown assays followed by mass spectrometry analysis of bound proteins, which identified the ageing-associated prohibitin (PHB) 1 and 2 to potentially interact with MIRIAL. Similar to MIRIAL knockdown, siRNA-mediated PHB 1 or 2 silencing caused proliferative defects. Further, PHBs are known to physically interact with p53 and control mitochondrial metabolism, a key factor in cellular ageing. Interestingly, silencing of MIRIAL in HUVECs increased mitochondrial mass (1.8-fold, p=0.0008) and spare respiratory capacity (1.95-fold) with the latter being decreased in isolated aged murine ECs. Taken together, MIRIAL is an ageing-induced lncRNA in ECs acting as a key regulator of metabolic and cellular function. MIRIAL promotes cell proliferation, migration and basal angiogenic sprouting while decreasing mitochondrial function and VEGF-A-stimulated sprouting. We hypothesise that MIRIAL influences p53 signalling and mitochondrial respiration through PHB 1 and 2. The present study suggests that modulation of MIRIAL expression may be a promising strategy to prevent or even reverse ageing-induced functional decline of ECs. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – EU funding. Main funding source(s): European Research Council (ERC) Starting Grant: Non-coding RNA in Vascular Ageing (NOVA)

Background Cardiovascular diseases (CVDs) remain the leading cause of death worldwide. Hypoxia induces significant changes in cardiovascular control mechanisms potentially resulting in pathophysiology. Recently, an increasing number of long non-coding RNAs (lncRNAs) was reported to participate in the regulation of Hypoxia-inducible factors (HIF). Analysis of single-cell RNA-sequencing of human Abdominal Aortic Aneurysms pinpointed the endothelial-enriched lncRNA LINC01235. LINC01235 was previously correlated with tumour progression in gastric cancer and worse patient prognosis in breast cancer. Globally, the role of LINC01235 in the cardiovascular system remains unknown. Purpose The objective of this study is to unravel the function of LINC01235 in endothelial cells (ECs). Methods and results LINC01235 levels were elevated in human umbilical vein ECs (7.66 fold, p<0.05), human aortic ECs (16.84 fold, p<0.05) and human dermal microvascular ECs (639.73 fold, p<0.05) over other human cardiovascular cells like vascular smooth muscle cells, aortic fibroblasts and cardiomyocytes. Severe hypoxia (0.2% O2 for 24h) reduced LINC01235 expression significantly (0.33 fold, p<0.05). SiRNA-mediated LINC01235 silencing in HUVECs (0.12, p<0.05) resulted in decreased proliferation (0.76 fold, p<0.05) and vascular endothelial growth factor A (VEGFA)-stimulated angiogenic sprouting (0.39 fold, p<0.05). Loss of LINC01235 did not affect apoptosis, metabolism or barrier function. Analysis of RNA-sequencing data revealed that many hypoxia-responsive genes were downregulated after knockdown of LINC01235 (siCtrl vs. siLINC01235). These included HIF-3α (0.24 fold, p<5.86e-28) as a potential key regulator of the cellular feedback to hypoxia. Phenotypically, knockdown of HIF3A using siRNAs (0.07 fold, p<0.05) resulted in decreased proliferation (0.82 fold, p<0.05) and VEGFA-stimulated angiogenic sprouting (0.50 fold, p<0.05). Accordingly, hypoxia response and LINC01235 knockdown exhibit a negative correlation based on transcriptomics data (R=−0.157, p<2.2e-16), further emphasizing a role of LINC01235 in hypoxia response. Conclusion In summary, the EC-enriched lncRNA LINC01235 is likely required for the suppression of hypoxia-induced gene expression under normoxic conditions potentially mediated by HIF-3α. Functionally, loss of LINC01235 decreased proliferation and VEGFA-stimulated angiogenic sprouting without an effect on cell death, metabolism or barrier integrity. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): DFG - TRR267

Vascular ageing is a key risk factor for cardiovascular diseases and is characterised by a continuous decline in endothelial cell function. Despite progress in recent years, the molecular mechanisms for this deterioration remain incompletely understood. Long non-coding RNAs (lncRNAs) are a heterogeneous class of RNAs that have been shown to regulate gene expression and protein function, however, little is known about their role in the ageing-associated dysregulation of endothelial cell (EC) function. In this study, we aimed to identify and functionally characterise a novel ageing-regulated lncRNA in ECs. Using RNA sequencing data of cardiac ECs derived from 12 weeks young and 20 months old mice, we identified Mirial as an ageing-induced lncRNA (1.32-fold, p=0.00005). Mirial is conserved between mice and humans and has no obvious coding potential. GapmeR-mediated silencing of MIRIAL in human umbilical vein ECs (HUVECs) decreased cell proliferation by 50%, migration by 24% (p=0.045) and basal angiogenic sprouting by 53% (p=0.0029), without affecting apoptosis or senescence. Additionally, silencing of MIRIAL increases mitochondrial mass (1.8-fold, p<0.01) and spare respiratory capacity (1.95-fold). Preliminary data from the hearts of Mirial knockout mice confirm the elevated mitochondrial mass after Mirial ablation (1.26-fold, p=0.05). In HUVECS, MIRIAL is mainly associated with the chromatin (80%), suggesting a role in the regulation of gene expression. Pathway analysis showed an overrepresentation of p53 target genes that were upregulated upon MIRIAL knockdown, which was validated using qRT-PCR (1.8–5.2-fold increases). Interestingly, this effect is fully dependent on the presence of p53. Moreover, p53 and phospho-p53 (Ser15) were both increased (1.8-fold, p=0.01 and 2.9-fold, p=0.02, respectively) after MIRIAL silencing. Pulldown of MIRIAL identified DDX5 and MRPL41 as direct p53 interactors and RNA immunoprecipitation revealed that MIRIAL physically interacts with p53 (3.75-fold enrichment, p<0.01). Gene set enrichment analysis of RNA sequencing data revealed that 10% of deregulated genes after MIRIAL knockdown have a binding site for Forkhead Box O (FoxO) transcription factors. In particular, FoxO1 is known as one of the key players in endothelial proliferation and regulation of angiogenesis as well as in mitochondrial biogenesis. Taken together, MIRIAL is an ageing-induced lncRNA in endothelial cells acting as a key regulator of metabolic and cellular function. MIRIAL promotes cell proliferation, migration and basal angiogenic sprouting while decreasing mitochondrial function. We hypothesise that MIRIAL influences these cellular functions by affecting the p53 pathway and mitochondrial respiration through FoxO signalling. The results from the present study suggest that modulation of cellular MIRIAL expression may be a promising strategy to prevent or even reverse ageing-induced functional decline of ECs, both in vitro and in vivo. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft - Collaborative Research Centre (SFB) 834 - Project B9Deutsche Forschungsgemeinschaft - Collaborative Research Centre/Transregio (TRR) 267 - Project B4