Endothelial Mitogen-Activated Protein Kinase Kinase Kinase Kinase 4 Is Critical for Lymphatic Vascular Development and Function

Abstract

The molecular mechanisms underlying lymphatic vascular development and function are not well understood. Recent studies have suggested a role for endothelial cell (EC) mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) in developmental angiogenesis and atherosclerosis. Here, we show that constitutive loss of EC Map4k4 in mice causes postnatal lethality due to chylothorax, suggesting that Map4k4 is required for normal lymphatic vascular function. Mice constitutively lacking EC Map4k4 displayed dilated lymphatic capillaries, insufficient lymphatic valves, and impaired lymphatic flow; furthermore, primary ECs derived from these animals displayed enhanced proliferation compared with controls. Yeast 2-hybrid analyses identified the Ras GTPase-activating protein Rasa1, a known regulator of lymphatic development and lymphatic endothelial cell fate, as a direct interacting partner for Map4k4. Map4k4 silencing in ECs enhanced basal Ras and extracellular signal-regulated kinase (Erk) activities, and primary ECs lacking Map4k4 displayed enhanced lymphatic EC marker expression. Taken together, these results reveal that EC Map4k4 is critical for lymphatic vascular development by regulating EC quiescence and lymphatic EC fate.

FIG 1

Mice constitutively lacking EC Map4k4 develop chylothorax. (A) Map4k4 (M4K4) Cdh5 Cre animals die at a median of p8.5 (n = 26; P < 0.0001). (B) Map4k4 Cdh5 Cre animal at p7 displaying chylothorax. (C) Fluid from the thoracic cavity of a representative Map4k4 Cdh5 Cre animal.

FIG 2

Lymphatic abnormalities in Map4k4 Cdh5 Cre mice. (A to C) Ear skin from p6 Flox/Flox or Map4k4 Cdh5 Cre animals was stained with Lyve-1 as a marker of lymphatic capillaries. (A) Representative images. Scale bars, 100 μm. (B) Vessel density as a measure of percent stained area normalized to the Flox/Flox area. (C) Average vessel diameter as normalized to the Flox/Flox diameter. (D and E) Mesenteries from p2 Flox/Flox or Map4k4 Cdh5 Cre animals were stained with Prox-1 as a marker of lymphatic valves. (D) Representative images. The arrowheads indicate valves. Scale bars, 100 μm. (E) Numbers of valves per millimeter mesentery as normalized to Flox/Flox animals. (F and G) p16 pups were injected with Evans blue dye in the footpad, and the mice were sacrificed to visualize dye in lymph nodes 1 h later. The images are representative of at least 4 animals per genotype. (F) Inguinal lymph nodes; the arrowhead indicates enhanced capillary visualization in the skin of Map4k4 Cdh5 Cre mice. (G) Iliac lymph nodes; the arrowheads indicate a lack of dye in iliac lymph nodes of Map4k4 Cdh5 Cre mice. The error bars represent standard errors of the mean. **, P < 0.005; n = 4 to 6.

FIG 3

Vascular abnormalities in Map4k4 Cdh5 Cre mice. (A to C) Retinas were isolated from p6 Flox/Flox or Map4k4 Cdh5 Cre pups and stained with isolectin B4. (A) Representative images (×5 magnification, scale bars = 250 μm [top]; ×20 magnification, scale bars = 50 μm [bottom]). (B) Quantitation of retinal outgrowth as the diameter from the optic nerve to the perimeter as normalized to Flox/Flox (*, P < 0.05; n = 5 or 6). (C) Vessel density quantified as percent stained area and normalized to Flox/Flox (*, P < 0.05; n = 6 or 7). (D to I) Intestines were isolated from p18 Flox/Flox or Map4k4 Cdh5 Cre pups. (D to F) Intestinal cross sections were stained with Lyve-1 as a lymphatic capillary marker and with 4′,6-diamidino-2-phenylindole (DAPI) to visualize villi. (D) Representative images; scale bars, 50 μm. (E) Quantitation of Lyve-1-stained area as normalized to Flox/Flox animals. (F) Quantitation of Lyve-1-stained-vessel diameter as normalized to Flox/Flox animals (n = 4 to 6). (G to I) Intestinal cross sections were stained with vWF as a blood endothelial marker and with DAPI to visualize villi; scale bars, 50 μm. (G) Representative images. (H) Quantitation of vWF-stained area as normalized to Flox/Flox animals. (I) Quantitation of vWF-stained-vessel diameter as normalized to Flox/Flox animals (n = 4 to 6). The error bars represent standard errors of the mean.

FIG 4

Enhanced proliferation in ECs lacking Map4k4. (A to D and F) Primary MLECs were derived from chow-fed Flox/Flox or Map4k4 iECKO mice. (A) MLECs were plated at subconfluence and counted daily. The cell counts were normalized to that of day 1 and expressed as fold change. (ANOVA; *, P < 0.05; n = 7 to 11). (B) Ki67 staining in MLECs as assessed by flow cytometry (*, P < 0.05; n = 6 or 7). (C) Annexin V staining in serum-fed or serum-starved MLECs as assessed by flow cytometry (n = 6 or 7). (D) The cell cycle was assessed by BrdU and 7-AAD staining using flow cytometry (G₀/G₁, BrdU⁻ 7-AAD⁻; S, BrdU⁺; G₂/M, 7- AAD⁺; *, P < 0.05; n = 6). (E) HUVECs were transfected with scrambled or Map4k4 siRNA, the cells were serum starved overnight, and a microarray analysis was performed. The heat map represents 30 of the most up- or downregulated genes after Map4k4 knockdown (|fold change| > 1.5). (F) Ccnd2 expression was assessed in MLECs by qRT-PCR and normalized to that of 36b4 (**, P < 0.005; n = 6). The error bars represent standard errors of the mean.

FIG 5

MAP4K4 binds to RASA1 and regulates basal RAS-ERK signaling. (A) Diagram of MAP4K4 and RASA1 domains that directly bound in a yeast 2-hybrid screen. (B) Coimmunoprecipitation of endogenous RASA1 and overexpressed MAP4K4 in HUVECs. The immunoblots are representative of the results of at least 3 independent experiments. (C) Immunoprecipitation of endogenous RASA1 and endogenous MAP4K4 in HDLECs. The immunoblots are representative of the results of at least 3 independent experiments. (D to G) ECs were treated with scrambled or MAP4K4 siRNA for 48 h. (D) RASA1 protein levels were assessed in HUVECs and normalized to β-actin (representative results; n = 4). (E) Ras-GTP levels were measured in serum-starved HUVECs by assessing GST-RAF1-bound RAS in pulldowns (top) as normalized to total RAS levels in cell lysates (bottom) by densitometry (*, P < 0.05; n = 4). The immunoblots were run on the same gel but in noncontiguous lanes. (F to H) HUVECs (F), HDLECs (G), and MLECs (H) were serum starved overnight and stimulated with 100 ng/ml VEGF-C in a time course. The lysates were immunoblotted for MAP4K4, p-ERK, and total ERK, and p-ERK levels were normalized to total ERK by densitometry (*, P < 0.05; **, P < 0.005; n = 3 or 4 HUVECs or HDLECs; n = 7 or 8 MLECs). (I) MLECs were isolated from chow-fed Flox/Flox or Map4k4 iECKO mice at least 16 weeks after tamoxifen injections, and the indicated genes were assessed by qRT-PCR and normalized to 36b4 (*, P < 0.05; ***, P < 0.0005; n = 8 to 13). (J) Model of the hypothesis. Map4k4 directly binds Rasa1 to negatively regulate Ras-GTP and Erk activity; thus, Map4k4 loss drives EC proliferation and a lymphatic endothelial cell fate switch, which promotes lymphatic dysfunction. The error bars represent standard errors of the mean.