Simplified method to quantify valve back‐leak uncovers severe mesenteric lymphatic valve dysfunction in mice deficient in connexins 43 and 37

Abstract

Key points Lymphatic valve defects are one of the major causes of lymph transport dysfunction; however, there are no accessible methods for quantitatively assessing valve function. This report describes a novel technique for quantifying lymphatic valve back‐leak. Postnatal endothelial‐specific deletion of connexin 43 (Cx43) in connexin 37 null (Cx37−/−) mice results in rapid regression of valve leaflets and severe valve dysfunction. This method can also be used for assessing the function of venous and lymphatic valves from various species, including humans. Abstract The lymphatic system relies on robust, spontaneous contractions of collecting lymphatic vessels and one‐way secondary lymphatic valves to efficiently move lymph forward. Secondary valves prevent reflux and allow for the generation of propulsive pressure during each contraction cycle. Lymphatic valve defects are one of the major causes of lymph transport dysfunction. Genetic mutations in multiple genes have been associated with the development of primary lymphoedema in humans; and many of the same mutations in mice result in valve defects that subsequently lead to chylous ascites or chylothorax. At present the only experimental technique for the quantitative assessment of lymphatic valve function utilizes the servo‐null micropressure system, which is highly accurate and precise, but relatively inaccessible and difficult to use. We developed a novel, simplified alternative method for quantifying valve function and determining the degree of pressure back‐leak through an intact valve in pressurized, single‐valve segments of isolated lymphatic vessels. With this diameter‐based method, the competence of each lymphatic valve is challenged over a physiological range of pressures (e.g. 0.5–10cmH2O) and pressure back‐leak is extrapolated from calibrated, pressure‐driven changes in diameter upstream from the valve. Using mesenteric lymphatic vessels from C57BL/6J, Ub‐CreERT2;Rasa1fx/fx, Foxc2Cre/+, Lyve1‐Cre;Cx43fx/fx, and Prox1‐CreERT2;Cx43fx/fx;Cx37−/− mice, we tested our method on lymphatic valves displaying a wide range of dysfunction, from fully competent to completely incompetent. Our results were validated by simultaneous direct measurement of pressure back‐leak using a servo‐null micropressure system. Our diameter‐based technique can be used to quantify valve function in isolated lymphatic valves from a variety of species. This method also revealed that haplodeficiency in Foxc2 (Foxc2Cre/+) is not sufficient to cause significant valve dysfunction; however, postnatal endothelial‐specific deletion of Cx43 in Cx37−/− mice results in rapid regression of valve leaflets and severe valve dysfunction.

Fluid pumping of peristaltic vessel fitted with elastic valves

“…2015; Castorena-Gonzalez et al. 2020) and abnormal conduction of contraction waves (Castorena-Gonzalez et al. 2018).…”

Lymphatic Connexins and Pannexins in Health and Disease

“…As signs of retrograde lymph flow were also observed in Cx37 −/− Cx43 +/− mice, the authors proposed that the chylothorax may be due to a failure to move lymph in an unidirectional (anterograde) manner [ 29 ]. Finally, postnatal LEC-specific deletion of Cx43 in Cx37 −/− mice resulted in rapid regression of valve leaflets and severe valve dysfunction [ 34 ].…”

“…Under an imposed gravitational load (near-vertical body position), these mice showed inhibition of in vivo lymph transport, which could not be observed in a horizontal position. Interestingly, lymphatic vessels from mice with smooth muscle-specific ablation of Cx45 displayed a higher number of pacemaking sites, but none carried a dominant role, leading to inefficient contractions and reduced ejection fractions from their lymphangions [ 34 ]. This pattern further worsened in these lymphatic vessels when they were exposed to elevated downstream pressure.…”

“…This pattern further worsened in these lymphatic vessels when they were exposed to elevated downstream pressure. The authors concluded that efficient lymph transport not only needs strong entrained contractions of all SMCs across multiple lymphangions, but also requires a dominant pacemaker for which Cx45-mediated electrical coupling between lymphatic SMCs is critical [ 34 ].…”

Role of RASA1 in cancer: A review and update (Review)

“…The inhibition of Ras signal transduction by RASA1 has been revealed to negatively affect the maintenance of lymphatic vasculature by VEGFR-3 in a steady-state situation ( 14 ). RASA1 has also been revealed to directly interact with mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) for lymphatic vascular development ( 15 – 17 ).…”