J23 - Mimicking Wall Shear Stress Patterns using Optogenetics

Introduction: : The lymphatic system is a crucial network of vessels and nodes that maintains tissue fluid homeostasis and immune surveillance. Blockages in the system can cause the atrophy of lymphatic valves, which ensure unidirectional fluid flow, by disrupting the mechanical cues required to maintain valve health. Currently, severe lymphedema remains incurable because no treatment can regenerate the lost lymphatic valves. This is in part because the precise role of biomechanical and biochemical stimuli in lymphatic valve formation has yet to be elucidated. The method most researchers use to investigate the effect of shear stress on lymphatic endothelial cells (LECs) is culturing the cells on rockers or in microfluidic channels. These methods either lack the spatiotemporal resolution necessary for controlled valve formation or are difficult to scale up for clinical-scale tissue engineered therapeutics. We have previously developed a light-operated calcium channel and used it to control hematopoietic stem cell self-renewal and gene expression (PMID: 33431868). Intracellular calcium is also a key second messenger in the endothelial mechanotransduction pathway that regulates valve gene expression. Therefore, we hypothesized that optogenetic calcium channels can mimic some aspects of mechanical shear stress signals by reproducing shear stress-induced calcium dynamics and upregulating lymphatic valve gene expression and valve matrix deposition.

Materials and

Methods: : LECs were cultured to confluency on 24-well plates before being transduced with lentivirus encoding light-operated Ca2+ channel version 3 (LoCa3), which was created by inserting a plant-derived photosensory module into the intracellular loop of an engineered ORAI1 channel. 3 wells were transduced, while 6 wells were left as wildtype controls. After waiting one day for transgene expressions, LECs were exposed to blue light for repeating cycles of 33.5 s on/ 216.5 s off for a total of 12 minutes of light exposure per day for 4 days. This light pattern mimics the calcium oscillations induced by wall shear stress gradients and pulsatile flow (WSSG+PF) that upregulate valve-forming genes. After 4 days of culture, the cells were immunostained for PROX1, a lymphatic valve transcription factor, and the valve core matrix protein fibronectin.

Results, Conclusions, and Discussions::

Results:
LoCa3-LECs exposed to the WSSG+PF blue light pattern had higher PROX1high nuclei % and higher mean fibronectin fluorescence after 4 days of daily exposure compared with light-exposed wildtype LECs and wildtype LECs in the dark. Light-exposed wildtype LECs had higher percentage of PROX1high nuclei than wildtype LECs in the dark, but there was no significant increase in the mean fibronectin fluorescence. LoCa3-LECs also had more yellow media than wildtype LECs by the end of the fourth day.

Discussion:
Exposing LECs expressing a light-activatable calcium channel to a light pattern that mimics WSSG+PF has successfully upregulated valve genes. The slight upregulation of PROX1 in the wildtype LECs exposed to the light may be due to light-induced reactive oxidative species activating the VEGF-C/VEGFR-3 pathway, although it was not sufficient to increase fibronectin deposition. This shows that the calcium profile induced by blue light was necessary for the observed valve gene upregulation and valve matrix deposition. The yellower media color also suggests increased glycolysis, which is known to be activated by inflammatory activation of endothelial cells. Future research will limit blue light exposure to certain areas to spatially control matrix deposition.

Conclusion:
To our knowledge, this is the first study that used optogenetic stimulation to mimic physical stimulation. This novel application of optogenetics offers high spatiotemporal resolution interrogation of the effect of intracellular calcium dynamics on valve formation by endothelial cells and can model various shear stress-related diseases such as atherosclerosis. The knowledge will ultimately contribute to the fabrication of lab grown valves for the treatment of lymphedema and heart valve disease.

Acknowledgements and/or References (Optional)::