An in vitro study of lymphatic cell alignment and transcriptional regulation under spatiotemporally varying wall shear stress
Keywords: Chemical Engineering.

The lymphatic system is an essential though sometimes overlooked part of the cardiovascular system. During embryonic development, lymphatic endothelial cells (LECs) form one-way valves within lymphatic vessels that prevent lymph backflow and are critical for proper function of the lymphatic system. Fluid flow is proposed to be a key cue in determining the location and timing of valve formation. In particular, local gradients in wall shear stress (WSS) have been proposed to play an essential role in the development of lymphatic valves. Although several studies have identified key components of a molecular flow sensor that converts the physical stimulus provided by WSS into downstream signaling, how exactly LECs that line the lymphatic vessels sense and respond to the physical stimulus provided by fluid flow remains poorly understood. Previous studies in mouse embryonic development have identified that lymphatic valves preferentially form near vessel branches, junctions and constrictions, sites that LECs experience local gradients in WSS (WSSGs). Same in vivo studies have showed that future lymphatic valve cells display perpendicular alignment and a subsequent increase in nuclear localization of PROX1 and FOXC2, two transcription factors that have been recognized as lymphatic valve markers, and upregulation of nuclear factor of activated T cells 1 (NFATc1). To investigate how LECs respond to gradients in WSS, our labs developed a constricted channel that simulates the flow environment found at sites of lymphatic valve formation. We examined the effect of spatially and spatiotemporally varying WSS in human lymphatic microvascular EC (HLMVEC) phenotypical and transcriptional changes. We found that spatial variations in WSS in the constricted channel trigger HLMVEC perpendicular alignment and high nuclear localization of FOXC2, key phenotypic changes observed during lymphatic valve formation in vivo. Also, we demonstrated that the constricted channel complete with physiologically relevant oscillatory flow (spatiotemporally varying WSS) provides the semblance of a stabilized vessel as being demonstrated by the parallel alignment and elevated nuclear levels of FOXC2, since HLMVECs seem to display the biochemical changes reminiscent of a valve site without morphological changes, which are thought to supersede this. Next, we sought to reveal how flow stimuli are connected to the transcriptional changes observed in HLMVECs. We found that a combination of spatially and temporally varying WSSs led to upregulated transcription of PROX1 and FOXC2, recapitulating an early step of lymphatic valve initiation. In addition, we observed that combined spatial and temporal variations in WSS modulated Ca2+ signaling and led to increased cellular levels of NFATc1, a transcription factor required for valve formation and maintenance. Finally, we investigated the potential role of canonical and non-canonical WNT, and planar cell polarity signaling pathways in the spatiotemporal WSSG-mediated responses observed in our microfluidic system. We found that key components of these signaling pathways may be implicated in a mechanosensory mechanism that can detect localized gradients in WSS as occur at sites of lymphatic valve formation.