(394c) Investigating the Effect of Spatially Varying Wall Shear Stress on Lymphatic Endothelial Cell Alignment and Transcriptional Regulation

The
lymphatic system is an essential though sometimes overlooked part of the
cardiovascular system. The best-known role of the lymphatic system is to drain interstitial
tissues. In addition, both recent and historical evidence demonstrate a central
role for lymphatic vessels in lipid absorption, immune surveillance, and in regulating
salt-dependent blood pressure. Failure to establish adequate tissue drainage
results in lymphedema, which is painful and debilitating, and is a common
outcome of cancer treatment. At present there is no cure for lymphedema.

Lymphatic
valves are necessary for unidirectional lymph flow and thus for the
physiological function of the lymphatic circulatory system. The inability to
treat valvular dysfunction constitutes a critical bottleneck in the treatment
of lymphatic diseases including lymphedema. Unfortunately, adults have only a
limited ability to generate new lymphatic valves. There is likewise little
progress in developing lymphatic valves, or any other cardiovascular valve, in
vitro.

In
preliminary results, we have built a novel 2D in vitro assay that reproduces
key aspects of the fluid flow environment near valves, such as flow through a
constriction, and discovered that lymphatic endothelial cells seem to recapitulate
the first steps in valve formation when exposed to this flow environment. We
show that human lymphatic microvascular endothelial cells (hLMVECs) align
perpendicular to the flow direction at the region of the maximum wall shear
stress (WSS) (Fig. 1), i.e. at the maximum constriction site, and
exhibit a highly nuclear localization of Prox1, a transcription factor required
for valve formation (Fig. 2). Further experiments demonstrated that chemical
inhibition of the G-Protein Coupled Receptor, Sphingosine 1-Phosphate Receptor
1 (S1PR1), which we have previously showed to be important in hLMVEC response
to fluid flow, with antagonist 5 μM W146 blocks both the perpendicular
alignment of hLMVECs with respect to flow (Fig. 1B) and the upregulation
of Prox1 (Fig. 2B).

Ongoing work aims to build
an in vitro 3D assay to guide lymphatic valve formation, and to further characterize
the interaction of spatiotemporal flow-based stimuli with signaling pathways
that are known to be required for valvulogenesis. If successful, this approach
will allow us to precisely define the molecular and biomechanical factors that
are required for valve formation. Moreover, it will lay the groundwork for
valve growth in vitro, which may impact the understanding and treatment
not only of lymphatic dysfunction, but also valvulopathies of the heart and
venous systems.