Biochemical and mechanical signals in the lymphatic vasculature

Abstract

Lymphatic vasculature is an integral part of the cardiovascular system where it maintains interstitial fluid balance. Additionally, lymphatic vasculature regulates lipid assimilation and inflammatory response. Lymphatic vasculature is composed of lymphatic capillaries, collecting lymphatic vessels and valves that function in synergy to absorb and transport fluid against gravitational and pressure gradients. Defects in lymphatic vessels or valves leads to fluid accumulation in tissues (lymphedema), chylous ascites, chylothorax, metabolic disorders and inflammation. The past three decades of research has identified numerous molecules that are necessary for the stepwise development of lymphatic vasculature. However, approaches to treat lymphatic disorders are still limited to massages and compression bandages. Hence, better understanding of the mechanisms that regulate lymphatic vascular development and function is urgently needed to develop efficient therapies. Recent research has linked mechanical signals such as shear stress and matrix stiffness with biochemical pathways that regulate lymphatic vessel growth, patterning and maturation and valve formation. The goal of this review article is to highlight these innovative developments and speculate on unanswered questions.

Keywords: FOXC2; GATA2; Lymphatic endothelial cells; Lymphatic vascular development; Mechanotransduction; PROX1; Shear stress; Valve; Wnt; YAP/TAZ.

Fig. 1

Molecular mechanisms of endothelial mechanotransduction. Endothelial cells interact with each other through cell junction molecules such as VE-Cadherin and with ECM through integrins. The intracellular domains of VE-Cadherin and integrins are connected to the cytoskeletal machinery. The various mechanical forces such as shear stress, stretch, and ECM stiffness affect cell–cell and cell-ECM interactions, which in turn affect the cytoskeleton

Fig. 2

Architecture of the lymphatic vasculature. A Collection and transportation of interstitial fluid by capillaries: The interstitial fluid (blue circle) is taken up and transported upstream by several lymphatic capillaries. The capillaries converge into one or two thicker collecting lymphatic vessels that transport the fluid further upstream. B Drainage of lymph into blood circulation: lymphatic valves (red structures) regulate the direction of lymph flow within lymphatic vessels. Lymph is filtered by series of lymph nodes (LN) before it drains into blood through either the thoracic duct (TD) or right lymphatic duct (RLD) at the junction of jugular (JV) and subclavian veins (SCV) that are upstream of superior vena cava (SVC). Two pairs of lymphovenous valves (LVV) regulate lymph return to blood circulation while preventing the entry of blood into the lymphatic vessels. SVC and inferior vena cava (not shown) drain into the right atrium (RA) of the heart. The diastolic suction force generated by the heart is likely critical for lymphatic drainage. LA left atrium, LV left ventricle, RV right ventricle, Ao aorta, PA pulmonary artery

Fig. 3

Interstitial fluid uptake by lymphatic capillaries. LECs of lymphatic capillaries interact with each other at discrete button-like junctions (red dots). The lymphatic capillaries are attached to the surrounding connective tissue through anchoring filaments (fibers). A When the interstitial pressure is low the anchoring filaments are in a relaxed confirmation resulting in the closure of oak leaf-shaped overlapping flaps. Lymph flows intraluminally towards collecting lymphatic vessels (blue dotted line). B An increase in interstitial fluid pressure causes the anchoring filaments to stretch and pull the oak leaf-shaped overlapping flaps allowing the interstitial fluid to percolate into the capillary lumen (blue dotted lines). (This Figure was modified from Leak and Burke [119])

Fig. 4

Lymph transport by collecting lymphatic vessels. A High pressure causes the lymphatic valves to open and allow upstream flow of the fluid. LECs of the lymphatic vessels and the upstream side of lymphatic valves are exposed to laminar shear stress during this phase of the lymphatic valve cycle. B A drop in the upstream fluid pressure combined with retrograde flow on the lymphatic valve sinus results in valve closure. Red arrows indicate the direction of lymph flow in both panels with the thickness of arrows correlating with the magnitude of fluid force

Fig. 5

Specification of LECs and the onset of lymphatic drainage. A PROX1⁺ LEC progenitors are specified in embryonic veins through the action of SOX18 and COUP-TFII. The PROX1⁺ cells upregulate VEGFR3 and podoplanin as they migrate out of the vein as clusters of differentiated LECs. B The LECs quickly aggregate to form the lymph sacs (LS) and peripheral longitudinal vessel (PLLV) that are connected anteriorly. The entire lymphatic structure is connected to the blood vasculature through 2-pairs of lymphovenous valves (red arrow). C Lymphovenous valves are made up of two layers of cells. The PROX1⁺ VEGFR3⁺ podoplanin⁺ LECs interact with PROX1^high FOXC2^high GATA2^high valvular endothelial cells to form the valves through which lymph drainage occurs

Fig. 6

Laminar shear stress regulates lymphatic vessel growth and patterning. A Laminar shear stress (LSS) promotes the polarization of LECs in the direction of lymph flow. GATA2 and FAT4 are necessary for the polarization of LECs in the direction of lymph flow. In addition, LSS regulates LEC proliferation by controlling VEGF-C and Notch signaling pathways. LSS induces the secretion of VEGF-C from LECs and also enhances the sensitivity of LECs to VEGF-C. S1PR1 specifically antagonizes LSS-enhanced VEGF-C signaling in the lymphatic plexus. LSS inhibits Notch signaling by antagonizing the expressions of the Notch receptor DLL4 and Notch Intracellular Domain. B Tip cells are devoid of fluid flow or LSS. Therefore, DLL4 and Notch signaling are restricted to the tip cells where they sustain VEGF-C signaling. DLL4 expression is positively regulated by VEGF-C signaling. S1PR1 activity is observed in the lymphatic plexus where it antagonizes LSS-enhanced VEGF-C signaling to prevent excessive sprouting. Vessels that do not carry lymph are less sensitive to VEGF-C signaling and therefore regress

Fig. 7

Models for the stepwise development of valves within lymphatic vessels. Valves could develop within the lymphatic vasculature by three types of mechanisms. Type A valves develop when two vessels converge. Type A valve rudiments experience oscillatory shear stress (OSS, red double edged arrows) after the connection between the two vessels is established. Type B valves form at branch points that experience OSS. Type C valves develop within a single vessel. Drop in fluid pressure (blue dotted arrows) could generate OSS, which in turn triggers the formation of valves. This figure was modified with permission from Kampmeier [209]. American Journal of Anatomy

Fig. 8

Oscillatory shear stress (OSS) is a central regulator of lymphatic valve development. The expression of lymphedema-associated transcription factors FOXC2 and GATA2 is enhanced by OSS. PROX1, Wnt and HDAC3 are critical mediators of OSS-response in LECs. FOXC2 and GATA2 regulate each other and additional molecules such as connexin-37 (CX37), ITGA9 and ephrin-B2. GATA2 could also regulate its own expression and that of PROX1. CX37 promotes Ca²⁺ influx and the nuclear localization of NFATC1. FOXC2 forms a transcriptional complex with NFATC1. FOXC2 inhibits the OSS-promoted activity of YAP/TAZ. However, YAP/TAZ activity could also be enhanced by VEGF-C and this pathway is necessary to maintain the expression of PROX1 in valves