CHD4-regulated plasmin activation impacts lymphovenous hemostasis and hepatic vascular integrity
Patrick L. Crosswhite, … , R. Sathish Srinivasan, Courtney T. Griffin
Patrick L. Crosswhite, … , R. Sathish Srinivasan, Courtney T. Griffin
Published May 3, 2016
Citation Information: J Clin Invest. 2016;126(6):2254-2266. https://doi.org/10.1172/JCI84652.
View: Text | PDF
Research Article Vascular biology

CHD4-regulated plasmin activation impacts lymphovenous hemostasis and hepatic vascular integrity

Abstract

The chromatin-remodeling enzyme CHD4 maintains vascular integrity at mid-gestation; however, it is unknown whether this enzyme contributes to later blood vessel or lymphatic vessel development. Here, we addressed this issue in mice harboring a deletion of Chd4 specifically in cells that express lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1), which include lymphatic endothelial cells (LECs) and liver sinusoidal endothelial cells. Chd4 mutant embryos died before birth and exhibited severe edema, blood-filled lymphatics, and liver hemorrhage. CHD4-deficient embryos developed normal lymphovenous (LV) valves, which regulate the return of lymph to the blood circulation; however, these valves lacked the fibrin-rich thrombi that prevent blood from entering the lymphatic system. Transcripts of the urokinase plasminogen activator receptor (uPAR), which facilitates activation of the fibrin-degrading protease plasmin, were upregulated in Chd4 mutant LYVE1+ cells, and plasmin activity was elevated near the LV valves. Genetic reduction of the uPAR ligand urokinase prevented degradation of fibrin-rich thrombi at the LV valves and largely resolved the blood-filled lymphatics in Chd4 mutants. Urokinase reduction also ameliorated liver hemorrhage and prolonged embryonic survival by reducing plasmin-mediated extracellular matrix degradation around sinusoidal blood vessels. These results highlight the susceptibility of LV thrombi and liver sinusoidal vessels to plasmin-mediated damage and demonstrate the importance of CHD4 in regulating embryonic plasmin activation after mid-gestation.

Authors

Patrick L. Crosswhite, Joanna J. Podsiadlowska, Carol D. Curtis, Siqi Gao, Lijun Xia, R. Sathish Srinivasan, Courtney T. Griffin

×

Figure 7

The extracellular matrix component laminin is diminished in Chd4fl/fl Lyve1-Cre+ left liver lobes.

Options: View larger image (or click on image) Download as PowerPoint
The extracellular matrix component laminin is diminished in Chd4fl/fl Ly...
(A) Expression of fibrinogen or of the extracellular matrix components laminin, fibronectin, and collagen IV were assessed by immunoblotting lysates generated from the left lobes of E13.5 control and Chd4fl/fl Lyve1-Cre+ livers. (B) Laminin, which is directly cleaved by plasmin, was significantly reduced in Chd4fl/fl Lyve1-Cre+ livers compared with controls. n = 3 embryos per genotype; experiments were repeated twice. Values expressed as relative normalized expression are mean ± SEM. P = 0.0002 as calculated by Student’s t test. (C) Expression of CHD4 and LYVE1 was assessed by immunoblotting lysates generated from the left lobes of E13.5 control and Chd4fl/fl Lyve1-Cre+ livers. (D) CHD4 expression was significantly reduced in Chd4fl/fl Lyve1-Cre+ left liver lobe samples compared with controls, while LYVE1 expression remained unchanged. n = 3 embryos per genotype; experiments were repeated twice. Values expressed as relative normalized expression are mean ± SEM. P = 0.043 as calculated by Student’s t test. (E) Expression of CHD4 was analyzed in liver samples taken from the left lobe and remaining lobes of E13.5 and E19.5 control embryos. Lanes were run on the same gel but were noncontiguous. (F) CHD4 expression was higher in the left lobe than in the remaining lobes of E13.5 livers (P = 0.007), but CHD4 expression was dramatically decreased throughout the liver by E19.5 (P = 0.002 for left lobe and P = 0.004 for remaining lobes). n = 3 embryos per genotype; experiments were repeated twice. Values expressed as relative normalized expression are mean ± SEM. P values were calculated by one-way ANOVA using Holm-Šidák’s multiple comparisons test.