Measurement of Nitric Oxide Production from Lymphatic Entothelial Cells Under Mechanical Stimuli

Abstract

The lymphatic system plays an important role in fluid and protein balance within the interstitial spaces. Its dysfunction could result in a number of debilitating diseases, namely lymphedema. Lymphatic vessels utilize both intrinsic and extrinsic mechanisms to pump lymph. Intrinsic pumping involves the active contraction of vessels, a phenomenon that is regulated in part by nitric oxide (NO) produced by lymphatic endothelial cells (LECs). NO production by arterial endothelial cells has been shown to be sensitive to both shear stress and stretch. Therefore, because of the unique mechanical environment of the LECs, we hypothesize that mechanical forces play an important role in regulation of the lymphatic pumping. Parallel-plate flow chambers and indenter-based cyclic stretch devices were constructed and used to apply mechanical loads to LECs. In addition, high-throughput micro-scale channels were developed and tested for shear experiments to address the need to increase the productivity and high- resolution imaging. Twenty-four hours treatment of LECs with different shear stress conditions showed a shear-dependent elevation in NO production. Moreover, 2.5 folds increase in cumulative NO was observed for stretched cells compared to the unstretched cells over six hours period. In conclusion, the upregulation observed in NO production under mechanical stimuli suggest new regulatory mechanisms that can be pharmaceutically targeted. These results provide an unprecedented insight into lymphatic pumping mechanism.