Shear Stress Induced Arterial and Venous Specification through Piezo1

Periodic Reporting for period 1 - SAVE (Shear Stress Induced Arterial and Venous Specification through Piezo1)

Reporting period: 2018-07-01 to 2020-06-30

Summary of the context and overall objectives of the project

Arteriovenous malformations (AVMs) are congenital disorders, which consist of an abnormal connection between arteries and veins, and are caused by defects during vascular remodelling. If arteries and veins do not differentiate and remodel properly, the result is congenital disorders, such as AVMs, with an estimated prevalence of 18 in 100,000, of which an astounding two-thirds occur before the age of 40. Although most patients with AVM appear asymptomatic, they have a significantly increased risk of stroke and aneurysm. This occurs because the vein of an AVM is exposed to abnormally high arterial blood pressure, causing it to expand and eventually rupture. Moreover, AVMs can form anywhere in the body and some of them can be quite large and difficult to remove with conventional surgery. My innovative investigation of the involvement of the ion channel Piezo1 in AV specification provides a new perspective in gaining knowledge to understand the molecular mechanism underlying vascular development and the onset of diseases such AVMs, possibly providing future new potential therapeutic avenues in order to reduce the area affected by AVMs prior to surgical operation.
The overall objectives of this SAVE research proposal are:
1) Show that Piezo1 activation by shear stress leads to calcium signal that activates Notch signalling.
2) Demonstrate that Piezo1 mediates arterial-venous differentiation by activating Notch signalling in response to haemodynamic forces generated by the passage of blood flow i.e. shear stress.
3) Demonstrate that ablation of Piezo1 results in AVM development due to impairment of Notch signalling activation.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

With the execution of the SAVE programme we identify Piezo1 as a new molecular player linking mechanical stimuli, such as blood flow, to Notch activation and vascular remodelling. To do so, with a peristaltic pump, we tried to mimic the force created by the passage of blood flow on microvascular endothelial cells (HMvECs) or we cultured the cells in presence of a Piezo1 activator called Yoda1. We demonstrated that activation of Piezo1 by Yoda1 or shear stress resulted in increased intracellular Ca2+; activation of ADAM10, a metalloprotease involved in Notch signalling stimulation; upregulation of Notch target genes i.e. Delta-like 4 (DLL4), Hairy and enhancer of split1 (HES1) and Hairy/enhancer-of-split related with YRPW motif protein1 (HEY1) and increase of NICD release. When Piezo1 was depleted by transfecting HMvECs with siRNA targeting Piezo1, all Yoda1 and shear stress effects were suppressed. We also tried to ablate the expression of Piezo1 specifically in endothelial cells in embryos and neonatal pups in order to assess AVMs in the brain, retina and heart. However, the high lethality due to tamoxifen injection (used to inducibly disrupt the Piezo1 gene) in animals of a such young age brought us to eventually end these experiments. In order to investigate the biological importance of these molecular interactions in vivo, we analysed the expression of Notch members and arterial venous makers in freshly isolated liver endothelial cells. We were able to show that specific ablation of Piezo1 in liver endothelial cells was reflected in a decrease of Notch member expression (Hes1, Dll4, Hey1, HeyL and Jag1) and arterial marker expression Efnb2. Altogether these results identify a previously unrecognised determinant of vascular morphology downstream of shear stress, the Piezo1-Notch1 axis and were recently published in Elife journal.

In order to exploit and disseminate the results of this research, the experienced researcher (ER), Dr Caolo has presented her data in scientific conferences such as the “Cardio Metabolic Retreat” held in Ullswater (England, UK). The ER also regularly attended the weekly department seminars organized at LICAMM.
As mentioned above, the data obtained with the execution of the SAVE fellowship have been successfully published in Elife scientific journal.
In order to generate possible media interest in outreach journals and local newspapers, Dr Caolo communicated the research outcome through the LICAMM press office. The publication of this work was also mentioned on the LICAMM eNewsletter and Leeds School of Medicine twitter account, and on Vincenza’s twitter and Linkedin accounts.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Fluid shear stress and Ca2+ signalling have been recently reported to induce the activation Notch signalling, a fundamental molecular pathway in vascular biology and arterial venous specification. However, the molecular mechanism underlying this process was completely unknown. With the execution of the SAVE programme we unravel the molecular mechanism, involving Piezo1, that links the mechanical stimulus generated by blood flow to Notch activation and vascular remodelling.

These new findings will definitely help to develop new strategies and diagnostic tools to prevent and/or reverse pathological arterial venous specification and remodelling. Also, the optimisation of the protocols used during this research project could be employed in the future for other vascular research applications.