Role of the LINC00961 locus in vascular endothelial cell function
Sanders, Rachel Louise
Over 17 million yearly deaths are caused by cardiovascular diseases worldwide, and up to 80% are due to heart attacks and strokes caused by atherosclerosis: fatty plaque build-up within artery walls restricting blood flow. Atherosclerotic plaque can also build up in arteries supplying the extremities such as the arms and legs; this is called peripheral arterial disease and is the 3rd most common atherosclerotic disease following that of the coronary arteries and cerebral arteries. With no cure for chronic ischemic diseases, clinical management includes reducing risk factors and utilising drug therapies to help with ailments that exacerbate disease such as diabetes and hypertension. Surgical intervention is a last resort with a high number of peripheral arterial disease patients requiring limb amputation. To avoid this, many clinical trials have attempted to increase patients’ blood flow by targeting endothelial cells and stimulating angiogenesis, the development of new blood vessels from pre-existing vessels. However, none of these attempts have led to a curative therapy yet. To prevent ischemic disease from escalating to amputation, heart attacks, or strokes, it is vital we find a way to combat them at early stages. Endothelial dysfunction, the aberrant or extended activation of adaptive endothelial behaviours, is an early event in atherosclerotic development, hence further understanding of endothelial molecular mechanisms is required. Long non-coding RNAs (lncRNAs) regulate many cell functions but are not well characterised and are poorly understood due to their previous categorisation as ‘junk DNA’. Several lncRNAs have been identified in aspects of cardiovascular pathophysiology, however, the human genome is estimated to possess ~ 270,000 lncRNAs, ergo many remain undiscovered. High-throughput RNA-sequencing in a human embryonic stem cell to endothelial cell differentiation protocol identified the lncRNA LINC00961 as endothelial enriched. This locus houses a micropeptide, small peptide of amino acid regulation (SPAAR), and has a mouse homologue; unique factors suggesting an important and evolutionary conserved function. Therefore, this project sought to investigate the role of the LINC00961 locus in the endothelium. Knock down of LINC00961 expression by ~90% was achieved in human umbilical vein endothelial cells which significantly reduced several endothelial functions; tubule formation, proliferation, adhesion, migration, and barrier integrity. The LINC00961 locus knock out mouse line showed no lethality; however, a foetal growth restriction like phenotype was identified in male LINC00961-/- animals; these offspring were significantly smaller and lighter with an increased brain weight to body ratio at 9 weeks of age. Cardiac ultrasound at 8 weeks of age found no differences in cardiac output between female LINC00961-/- and wildtype controls. However, reduced left ventricular wall diameter, slower mitral valve deceleration, and isovolumetric contraction time were observed in these mice. This restricted heart filling and compromised myocardial relaxation indicates the early stages of diastolic dysfunction. Adult male LINC00961-/- and wild type control mice underwent surgically induced hind limb ischemia. Comparable to in vitro data, LINC00961 deletion caused transient changes to capillary number during early hypoxic injury, and a lack of mature α- smooth muscle actin vessels at baseline, indicating underlying issues with vessel physiology. Crucially, lentiviral overexpression cassettes showed LINC00961 acted independently of SPAAR in human umbilical vein endothelial cells, and LINC00961 and SPAAR were linked to the actin binding proteins thymosin β-4 and SYNE1, respectively. LINC00961 and SPAAR are encoded by the same locus but have opposing effects on angiogenesis. Reduction of locus expression also affected other endothelial behaviours; thus, this locus contributes to maintaining proper endothelial function. This refinement of angiogenic control may be in part due to actin cytoskeletal regulation via thymosin β-4 and SYNE1 interactions. Murine LINC00961 contributes to blood vessel physiology and may also have a role in heart physiology given the altered parameters in LINC00961-/- hearts. Therefore, this locus has important roles in several aspects of cardiovascular biology and is a potential novel target for therapeutic regulation of angiogenesis in patients with compromised blood flow.