Introduction:
Genome-wide association studies have identified rs2107595, a non-coding locus on chromosome 9 between HDAC9 and
Twist1
genes, as a risk allele for several vascular phenotypes, including Coronary Artery Disease (CAD). Rs2107595 has, more specifically, been associated with stable CAD over myocardial infarction phenotypes. Recent work has shown that rs2107595 risk allele increases
Twist1
expression in smooth muscle cells (SMCs) by creating an RBPJ binding site. In other cell types,
Twist1
is known to maintain cells in a de-differentiated state and to promote epithelial to mesenchymal transformation, driving tumor progression and metastasis.
Hypothesis:
Twist1
modulates SMC differentiation to promote an immature proliferative state over a differentiated (osteoblastic) state. This shift in phenotype promotes features of plaque stability
in vivo
.
Methods:
Twist1
expression plasmid (pCMV6-TWIST1) was transfected into A7r5 rat smooth muscle cells. To assess proliferation, cells were counted at 24, 48, 72, and 96 hours. To assess calcification, A7r5 cells were cultured in calcification media (2mM NaPhos) for 10 days and stained with Alizarin Red.
In vivo
studies were performed in Twist1 fl/fl tamoxifen-inducible MYH11-Cre C57BL/6 mice on ApoE-/- background fed a Western diet for 16 weeks to induce atherosclerotic lesions. Immunohistochemistry with SM22a identified lesion SMCs, and alizarin red was used to identify calcifications.
Results:
Ectopic overexpression of
Twist1
in A7r5 SMCs decreased proliferation at 48h and 72h (80%, p=0.014).
Twist
1 overexpression also decreased the total area of calcification (33% reduction, p=0.007).
In vivo
, both control and
Twist
1 KO mice show similar burden of atherosclerosis. However, there is a decrease in sub-endothelial SMCs in atherosclerotic lesions by SM22a staining in the
Twist1
KO. Additionally,
Twist1
KO mice have more prominent and larger focal calcifications.
Conclusions:
Twist1
promotes SMC proliferation and decreases calcification
in vitro
, and may affect the presence of subendothelial SMCs and calcification
in vivo
. This provides a compelling link that rs2107595 may promote plaque stability in CAD by increasing
Twist1
to modulate SMC phenotypes.
PS210. Inhibition of VEGFR2 Reduces Angiogenic Microvessel Leakiness in Murine Vein Graft Atherosclerotic Lesions and Increased Plaque Stability
