eNOS Regulates Lymphatic Valve Specification by Controlling β-Catenin Signaling During Embryogenesis in Mice

Abstract

Background: Lymphatic valves play a critical role in ensuring unidirectional lymph transport. Loss of lymphatic valves or dysfunctional valves are associated with several diseases including lymphedema, lymphatic malformations, obesity, and ileitis. Lymphatic valves first develop during embryogenesis in response to mechanotransduction signaling pathways triggered by oscillatory lymph flow. In blood vessels, eNOS (endothelial NO synthase; gene name: Nos3) is a well-characterized shear stress signaling effector, but its role in lymphatic valve development remains unexplored.

Methods: We used global Nos3^-/- mice and cultured human dermal lymphatic endothelial cells to investigate the role of eNOS in lymphatic valve development, which requires oscillatory shear stress signaling.

Results: Our data reveal a 45% reduction in lymphatic valve specification cell clusters and that loss of eNOS protein inhibited activation of β-catenin and its nuclear translocation. Genetic knockout or knockdown of eNOS led to downregulation of β-catenin target proteins in vivo and in vitro. However, pharmacological inhibition of NO production did not reproduce these effects. Co-immunoprecipitation and proximity ligation assays reveal that eNOS directly binds to β-catenin and their binding is enhanced by oscillatory shear stress. Finally, genetic ablation of the Foxo1 gene enhanced FOXC2 expression and partially rescued the loss of valve specification in the eNOS knockouts.

Conclusions: In conclusion, we demonstrate a novel, NO-independent role for eNOS in regulating lymphatic valve specification and propose a mechanism by which eNOS directly binds β-catenin to regulate its nuclear translocation and thereby transcriptional activity.

Keywords: cadherins; catenins; lymphedema; mechanotransduction; nitric oxide.

Figure 1:. eNOS is upregulated and activated in response to OSS.

(A) Whole-mount immunostaining of PROX1 (red) and eNOS (green) in WT mesenteries at E18.5. The bottom images are high-magnification images of the white boxed areas. (B) Whole-mount immunostaining of GFP (green) and phospho-Ser1177 eNOS (red) in WT mesenteries at E18.5 showing a mature valve and valve forming area. The images on the right are high magnification images of the white boxed areas. (C) Western blot for indicated proteins using lysates from hdLECS exposed to static conditions or OSS conditions for 5, 10 and 15 mins. (D) Western blot for indicated proteins using lysates from hdLECS treated with vehicle or SC-79. Scale bars are 50μm in A and 25μm in B.

Figure 2:. Nos3^−/− embryos have a defect in lymphatic valve development.

(A) Whole-mount immunostaining of PROX1 (green) and FOXC2 (red) in WT and Nos3^−/− E16.5 mesenteries. (B) PROX1 clusters per millimeter from each E16.5 mesentery. (C) Total length of lymphatic vessels from each E16.5 mesentery. (B,C) All values are means ± SEM of n=9 littermates per genotype. (D) Fluorescence imaging of Prox1-GFP (green) mesenteries at E18.5. The bottom images are high-magnification images of the white boxed areas. (E) Valves per millimeter from each E18.5 mesentery. (F) Total length of lymphatic vessels from each E18.5 mesentery. (E,F) All values are means ± SEM of n=6 littermates per genotype. *P<0.05, unpaired Student’s t-test. Scale bars are 150μm in A and 300μm in D. Sex was not determined and data from both sexes were combined.

Figure 3:. Nos3^−/− animals have fewer lymphatic valves postnatally.

(A,B) Fluorescence imaging of Prox1-GFP (green) mesenteries at P3. The bottom images are high-magnification images of the white boxed areas. (C) Valves per millimeter from each mesentery at P3. (D) Total length of lymphatic vessels from each mesentery at P3. (E) Total number of branchpoints from each mesentery at P3. (F) Percentage of total branchpoints containing valves from each mesentery at P3. (G) Fluorescence imaging of Prox1-GFP (green) mesenteries at P7. The bottom images are high-magnification images of the white boxed areas. (H) Valves per millimeter from each mesentery at P7. (I) Total length of lymphatic vessels from each mesentery at P7. (J) Total number of branchpoints from each mesentery at P7. (K) Percentage of total branchpoints containing valves from each mesentery at P7. All values are means ± SEM of n=6 littermates per genotype. *P<0.05, unpaired Student’s t-test. Scale bars are 300μm in A, B and G. Sex was not determined and data from both sexes were combined.

Figure 4:. eNOS regulates PROX1 and FOXC2 expression.

(A) Whole-mount immunostaining of PROX1 (green), FOXC2 (red) and VE-Cadherin (magenta) in WT and Nos3^−/− P3 mesenteries. (B) Mean pixel intensity measurements of PROX1 and FOXC2 in lymphangion or valve LECs from each mesentery. (C) Western blot for indicated proteins using lysates from hdLECS cultured under static or OSS conditions for 48 hours and transfected with a control scramble or shRNA against NOS3. (D) Quantification of the indicated proteins probed by western blot. All values are means ± SEM of (B) n=4–6 lymphangion or valve areas from N=5–6 littermates per genotype and (D) n=3–4 independent experiments. *P<0.05, unpaired Student’s t-test in (B) and Two-way ANOVA followed by Tukey’s multiple-comparison test in (D). Scale bars are 50μm in A. Sex was not determined and data from both sexes were combined.

Figure 5:. eNOS regulates β-catenin signaling.

(A) Whole-mount immunostaining of PROX1 (green) and Non-phosphorylated β-catenin (referred to as Non-P β-catenin) (red) in WT and Nos3^−/− E18.5 mesenteries. The images on the right are high-magnification images of the white boxed areas. (B) Immunostaining of β-catenin (green) in hdLECs cultured under static or OSS conditions for 48 hours and transfected with a control scramble or shRNA against NOS3. (C) Western blot for indicated proteins using lysates from scramble or shNOS3 hdLECS cultured under static or OSS conditions for 48 hours. (D) Quantification of the indicated proteins probed by western blot. All values are means ± SEM of n=3–4 independent experiments. *P<0.05, **P<0.01, ***P<0.001, Two-way ANOVA followed by Tukey’s multiple-comparison test. (E) Lysates from hdLECS cultured under static or OSS conditions were immunoprecipitated (IP) using anti-β-catenin antibody or mouse IgG control antibody followed by western blot for indicated proteins. (F) PLA was carried out in hdLECs exposed to static or OSS conditions using antibodies against β-catenin and eNOS or using no antibodies as a negative control. The images on the right are high-magnification images of the white boxed areas. Scale bars are 25μm in A and, 50μm in B and F.

Figure 6:. Postnatal β-catenin deletion does not lead to a complete loss of valves.

(A) Tamoxifen injection schedule for postnatal deletion of Ctnnb1. (B) Whole-mount immunostaining of GFP (green) and β-catenin (red) in Ctnnb1^fl/fl;Prox1-GFP and Ctnnb1^LEC-KO;Prox1-GFP (Jscal) P8 mesenteries. (C-D) Fluorescence imaging of Prox1-GFP (green) mesenteries at P8. The bottom images are high-magnification images of the white boxed areas. (E-G) Valves per millimeter from each mesentery at P8. (H-J) Total length of lymphatic vessels from each mesentery at P8. All values are means ± SEM of n=6 littermates per genotype. *P<0.05, unpaired Student’s t-test. Scale bars are 50μm in B and, 500μm in C and D. Sex was not determined and data from both sexes were combined.

Figure 7:. Foxo1 deletion partially rescues valve loss in Nos3^−/− embryos.

(A) Tamoxifen injection schedule for embryonic deletion of Foxo1. (B) Whole-mount immunostaining of E18.5 Prox1-GFP mesenteries from controls (Foxo1^fl/fl or Foxo1^+/fl), Nos3^−/−;Foxo1^fl/fl and Nos3^−/−;Foxo1^LEC-KO (Jscal). (C) Valves per millimeter from each mesentery at E18.5. (D) Total length of lymphatic vessels from each mesentery at E18.5. All values are means ± SEM of n=6–7 embryos per genotype. *P<0.05, **P<0.01, ***P<0.001, One-way ANOVA followed by Tukey’s multiple-comparison test. Scale bar is 300μm in B. Sex was not determined and data from both sexes were combined.