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
