Vascular Smooth Muscle Cell PIEZO-1 in Abdominal Aortic Aneurysm Formation

Background
An Abdominal Aortic Aneurysm (AAA) is a potentially life-threatening condition with no current disease-specific pharmacological therapies targeting aneurysm growth. Aortic vascular smooth muscle cells (VSMCs) maintain aortic vascular tone, due to their mature contractile differentiated phenotype. However, they can phenotypically switch to a de-differentiated cells which has shown to play a role in the aneurysmal disease process. PIEZO-1 is a calcium-permeable shear stress sensor previously shown to contribute to hypertension-induced wall remodelling and has recently AAA disease. Despite this, evidence potentially linking PIEZO-1 to AAA pathophysiological processes remains limited.

Methods
The hypothesis that activation of the PIEZO-1 mechanosensor in aortic VSMCs by stressors drives vascular remodelling (i.e. cell proliferation and stiffness) was assessed using in-vitro and in-vivo models. Non-diseased aortic VSMCs were exposed to constant oscillatory shear force, uni-axial stretch and high and low matrix stiffnesses. Additionally, cells were physiologically stressed by undergoing multiple cell passages, treated with the PIEZO-1 agonist Yoda-1 and cultured in serum-deprived media. Calcium imaging, using Yoda-1 to quantify PIEZO-1-dependent intracellular calcium entry, was performed. Cell behaviour was characterised using proliferation and migration assays. C57BL6/J [wild-type] and PIEZO-1 smMHCCre-ER(T2) [transgenic] male mice underwent aneurysm induction surgery to assess for changes in PIEZO expression. Serial ultrasound imaging was performed to quantify the anatomical changes. Quantitatively PCR was used to assess the expression of PIEZO mechanosensors, krüppel-like transcription factor 2 (KLF-2), krüppel-like transcription factor 4 (KLF-4) and transglutaminase 2 (TG2).

Results
PIEZO-1 was expressed in both non-diseased VSMCs as well as VSMCs obtained from aneurysmal tissue. PIEZO-1 mRNA was shown to be elevated in aortic VSMCs following treatment with the PIEZO-1 agonist Yoda-1, the application of constant oscillatory shear stress, and cell culture with serum-deprived media. Proliferation was significantly slower following prolonged stimulation with PIEZO-1. Although there was no physiological evidence linking vessel distensibility or matrix stiffness to PIEZO-1, there is a suggestion that PIEZO-1 influences the mRNA expression of TG2. Functional knockdown of PIEZO-1 in VASCs could not be reliably achieved. Furthermore, the knockdown of VSMC PIEZO-1 in transgenic was inconclusive. Never the less the was a significant increase in both PIEZO mechanosensors at day 7 following surgery in C57BL6/J mice, however only PIEZO-2 remained significantly elevated at day 14.

Conclusions
In this thesis, VSMC PIEZO mechanosensors were shown to be able to detect and adapt to common mechanical stimuli and stressors likely to be experienced in the AAA microenvironment. The role of PIEZO-1 in aneurysmal disease may be one that is transient and potentially of limited clinical significance. However, further investigation is required to confirm the findings of this thesis and interrogate the role of PIEZO-2.