(J-397) Pharmaceutical activation of Wnt signaling pathway in a bifurcated microfluidic lymphatic channel promotes localized valve gene expression.

Introduction:: Sub-track: Developmental Biology and Morphogenesis of Engineered Tissues

The lymphatic system drains excess interstitial fluids containing nutrients and waste and recirculates them back to the vascular system through the subclavian vein. In 2020 alone, 5 million Americans suffered from breast cancer-related lymphedema, which is caused by lymph node dissections intended to prevent cancer from metastasizing through the lymphatic vessels. The resulting blockage of lymph fluid can lead to inflammation, fibrosis, adipogenesis, infections, and disfigurement, representing a significant human health problem. Currently, early to mild stage lymphedema can be managed or even reversed by using elevation, compression, or manual lymphatic drainage. Unfortunately, these techniques are not effective on moderate and late-stage lymphedema due to pathological remodeling of lymphatic vessels to the obstructed lymph flow and increased intraluminal pressure.

                Several therapeutics have been developed to ameliorate lymphedema symptoms, but they are unable to regenerate the lost lymphatic valves. Regenerative scaffolds, vascular growth factor-C (VEGF-C) therapy, and mesenchymal stem cell therapy have some success in inducing the sprouting of new valveless lymphatic capillaries. However, lymphatic valve regeneration, or any type of valve regeneration, has not yet been achieved. The lack of valves severely limits the lymph fluid transport capacity of these new vessels, and a tissue engineered construct consisting of valved lymphatic vessels is critically needed for a permanent cure to lymphedema. The objective of this study is to recapitulate lymphatic valve development via biochemical and biophysical cues in a microfluidic model to gain insight into fabricating implantable valved lymphatic constructs.

Materials and Methods::

During embryonic valve development, the lymphatic endothelial cells (LECs) at valve formation sites express higher levels of the transcription factors FOXC2 and PROX1, which lead to the expression of downstream genes such as ITGA9 that drive leaflet matrix core deposition and leaflet formation. Several biochemical and biophysical stimuli have been shown to upregulate valves genes in vitro. The small molecule WNT activator 6-bromo-indirubin-3′-oxime (BIO) upregulates PROX1, FOXC2 and ITGA9 in adult LECs. Oscillatory shear stress (OSS) and shear stress gradients (SSG) generated by a vessel bifurcation geometry similarly upregulate FOXC2 and ITGA9, but not PROX1.

To test the combined effects of BIO, OSS and SSG on valve induction, we developed a bifurcation vessel model that can be cultured with human dermal LECs and subjected to controlled flow patterns. Using our gravitational lumen patterning (GLP) technique that coats a microfluidic channel in evenly thick collagen, we patterned a bifurcating vessel lumen in 5 mg/mL type 1 rat tail collagen using a 500 µm wide bifurcation microfluidic model (Fig. 1A). Post-GLP, the average lumen diameter in the main vessel was ~350 µm. Once a bifurcating design was achieved, we seeded LECs at a concentration of 15 x 10⁶/mL overnight. Once a confluent vessel was formed, we combinatorically and independently subjected the model to different flow conditions (laminar and oscillatory; shear stress ranging from 1-4 dynes/cm²) and BIO treatment (0.5 μM) for 3 days. Flow rates were controlled using a programmable syringe pump.

Results, Conclusions, and Discussions::

Results

When the cells were exposed to OSS, we saw a significant increase in the coverage area of ITGA9-high LECs (Fig. 1C,D). This expression was mostly in cells after the bifurcation. The coverage of PROX1 and ITGA9-high LECs further increased with BIO treatment (Fig. 1C,E). BIO increased the expression of valve markers at and after the bifurcation under both steady and oscillatory shear stress, except for IGA9 in the oscillatory +BIO group. No morphological changes were observed.

Discussions

The bifurcation design was expected to have increased shear activation by SSGs at the highlighted regions (Fig. 1B). As both SSGs and OSS have been shown to activate FOXC2 and ITGA9, this suggests a synergistic effect these two biophysical stimuli, which explains the high ITGA9 expression at or after but not before the bifurcations. In utero, valves preferentially form at bifurcations along the vessels. It has long been believed that localized OSS form at these sites, which lead to valve formation at these locations. However, recent studies are showing that the vessel diameter and flow rates in embryonic lymphatic vessels would not allow for the existence of OSS. Our observation of valve markers at bifurcations under laminar flow and BIO treatment suggests that SSGs, instead of OSS controls valve distribution. This supports a new model of lymphatic valve formation where OSS occurs throughout the entire vessel and SSGs further determine valve locations.

Unfortunately, we did not observe valve leaflet formation. In utero, lymphatic valve formation takes about 2 days, where the localized valve gene expression pattern like the one in BIO treated groups lead to the invagination of the LECs and the formation of a leaflet core. This is likely due to the lack of other morphogenic cues such as VEGF-C, suggesting that the upregulation of valve genes was insufficient.

Conclusions

This study supports the feasibility of regenerating lymphatic valves using a chemical cocktail that actives valve-forming genes in conjunction with spatio-temporal shear stress patterns. This data advances our understanding of lymphatic valve formation and aids in future endeavors regenerating other valves such as cardiac and venous valves.

Acknowledgements (Optional): :

References (Optional): :