Hierarchical Requirement for Endothelial Cell Connexins Cx37, Cx47, Cx43, and Cx45 in Lymphatic Valve Function

The proper functioning of lymphatic valves is critical for unidirectional lymph transport. Valve development and maintenance depends on multiple genes in lymphatic endothelium, including those controlling the expression of 4 connexin (Cx) isoforms-Cx37, Cx47, Cx43, and Cx45. The relative importance of these isoforms for valve function is undefined, but primary human lymphedema is linked to loss-of-function mutations in Cx47 or Cx43, while deficiencies in Cx43 or Cx45 produce functional valve defects in mice. Tests of back leak and closure for single lymphatic valves from mice with selective deficiency of each Cx isoform revealed defects associated with the loss of Cx37 or Cx43, but not Cx47. Combined deletion of multiple isoforms, including Cx45 but not Cx47, produced even more severe valve defects in certain genotypes, sometimes with nearly complete regression of valves within 6 d. Back leak across connexin-deficient LVs correlated highly with gaps between the commissures formed by leaflet insertion into the vessel wall, indicating that connexin function may be critical for the formation and/or maintenance of leaflet commissures. Our results reveal the following hierarchy of Cx importance in valve function: Cx37 = Cx43 > Cx45 > Cx47 and predict that patients with loss of function mutations in Cx37 (GJA4) should develop lymphedema. We propose a general classification scheme describing 4 stages of progressive valve dysfunction.