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Biomechanics

Biomechanics - Poster Session E

Poster D3 - Piezo1 Activation in Vascular Smooth Muscle Cells lead to Alteration of Synthetic Gene Expression

Saturday, October 26, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  In recent years, Piezo1 and Piezo2 have been heavily investigated within the context of vascular mechanobiology. Both mechanically-gated calcium channels have implications with aortic aneurysm and stenosis, and thus should be scrutinized within the context of vascular remodeling during arterial diseases. Overactivation of Piezo1 has been correlated with upregulation of matrix remodeling markers as well as genes indicative of inflammation and senescence. In recent studies, Luu et al. and Qian et al. have identified overactivation of Piezo1 as a critical mediator of pathological vascular remodeling during abdominal aortic aneurysm and impaired mechanosensitivity [3, 4]. We hypothesize that Piezo1 overactivation accelerates vascular remodeling in arterial disease by promoting a transition to a more synthetic phenotype. Real time calcium imaging has been conducted to confirm Piezo1 activation in osmotically loaded vascular smooth muscle cells (VSMCs) and qPCR has been run to investigate changes in cell proliferation (CTGF), upstream matrix remodeling markers (MMP3), and matrix stiffness markers (Col6α) [4].

Materials and

Methods:  Current methods for studying Piezo1 mechanosensation typically employ osmotic loading to induce membrane stretch and activate mechanically gated calcium channels. Savadipour et al. proposed a schematic for the mechanisms involved in Piezo1 activation by which media osmolarity determines the number of exposed piezo channels [5]. As such, the VSMC line, MOVAS (ATCC), was osmotically loaded via the addition of hypertonic (~500 mOsm), isotonic (~300 mOsm), or hypotonic (~100 mOsm) media. Media osmolarity was adjusted by adding either sucrose or deionized water. Yoda1, a specific agonist of Piezo1, was used in addition to osmolarity changes. To confirm activation of mechanically gated calcium channels, MOVAS were stained with Fluo4 and Fura Red dyes before osmotic loading and stimulation with Yoda1. Calcium imaging was conducted for 10 minutes at 0.33 fps on a confocal microscope (n = 2). Controls included addition of vehicle (9% DMSO) during osmotic loading before Yoda1 stimulation at 100 uM.
For long-term activation trials, MOVAS were osmotically loaded for one hour a day for three days. MOVAS were either osmotically loaded only (n = 4), or osmotically loaded with vehicle (0.1% DMSO) or 1 uM Yoda1 (n = 3). RNA for qPCR was extracted 24 hours following final treatment to give time for all downstream signaling to occur. Probes for CTGF, MMP3, and COL6A were normalized to GAPDH expression and fold change was analyzed by ΔΔCt. IL -6 ELISA was performed on media obtained before and after treatment to gauge an inflammatory response among experimental groups.

Results, Conclusions, and Discussions: Results from real time calcium imaging (Fig. 1) support the mechanism of Piezo1 action asserted by Savadipour et al [5]. Quantified ratio for green (high calcium)-to-red (low calcium) MOVAS fluorescence ratio in hypotonic media showed greater increase in calcium flux over time compared to isotonic media, indicating little to no activation of mechanically gated calcium channels in vehicle control. Once Yoda1 is added to the media, however, both isotonic and hypotonic groups experience an increase in calcium flux. Both groups, once stimulated, reach a max ratio, possibly due to the saturation of Piezo1 with Yoda1.
RT-qPCR of osmotically loaded MOVAS demonstrated an average increase in CTGF as osmolarity decreased (Fig. 2a). This may allude to Piezo2 activation of CTGF via the YAP/TAZ pathway; however, further work is needed to investigate this statement. Interestingly, averaged CTGF, COL6A, and MMP3 expression increased upon addition of Yoda1 to hypotonic and hypertonic groups but demonstrated no discernable change in expression upon addition of Yoda1 to isotonic groups (Figs. 2b,c,d). Statistical testing of expression datasets via two-way ANOVA demonstrated no significant difference between osmolarity or drug groups. In the future, more samples will be gathered to obtain a more robust data set.
ELISA of MOVAS media following prolonged osmotic conditioning with Yoda1 (Fig. 3) suggest increases in IL-6 may be correlated with the availability of Piezo1 channels available for Yoda1 stimulation. Two-way ANOVA conducted on ELISA data demonstrated that IL-6 quantity for hypotonic and isotonic groups were significantly different, though the presents of drug or vehicle had no significant effect. It is worth noting that IL-6 quantity may fluctuate with the number of cells. If repeated, IL-6 quantity could be normalized to the number of cells.
Differences in calcium flux between hypertonic and isotonic groups allude to an increase in the availability of Piezo1 channels to Yoda1 upon membrane stretch (Fig. 4). Testing of synthetic phenotype markers and inflammation in response to Piezo1 activation warrants further investigation. The study of Piezo1/2 mechanosensation in VSMC presents a novel perspective for investigating matrix remodeling and changes in cell behavior in arterial disease pathology.

Acknowledgements (Optional):  1. Urbán, Z., et al., Supravalvular aortic stenosis: genetic and molecular dissection of a complex mutation in the elastin gene. Hum Genet, 2001. 109(5): p. 512-20.
2. Lin, C.J., A.J. Cocciolone, and J.E. Wagenseil, Elastin, arterial mechanics, and stenosis. Am J Physiol Cell Physiol, 2022. 322(5): p. C875-c886.
3. Luu, N., et al., Aging-associated Decline in Vascular Smooth Muscle Cell Mechanosensation is Mediated by Piezo1 Channel. bioRxiv, 2023.
4. Qian, W., et al., Microskeletal stiffness promotes aortic aneurysm by sustaining pathological vascular smooth muscle cell mechanosensation via Piezo1. Nature Communications, 2022. 13(1): p. 512.
5. Savadipour, A., et al., Membrane stretch as the mechanism of activation of PIEZO1 ion channels in chondrocytes. Proc Natl Acad Sci U S A, 2023. 120(30): p. e2221958120.

Special thanks to all the members of the Wagenseil Vascular Mechanics Laboratory for all their guidance and assistance with this project.

Funding provided by the
the National Science Foundation Science and Technology Center for Engineering Mechano-Biology (NSF award number #1548571) and NIH Grant RO1HL166448