Figure 9.
S1PR1 regulates OSS response and the expression of valve regulatory genes in HLECs. (A) HLECs were transfected with siControl or siS1PR1 and grown for 24 h under static conditions to knockdown S1PR1. Subsequently, cells were cultured under static or OSS for 24 h. Cells were immunostained for F-actin or VE-cadherin. F-actin was primarily located along the cell wall (cortical actin) of control cells under both static and OSS conditions. Control HLECs became more spherical, and cortical actin expression appeared to be increased by OSS. In contrast, siS1PR1-transfected HLECs appeared elongated and had increased expression of stress fibers. The percentage of VE-cadherin+ overlapping cell junctions was increased by OSS, and this enhancement was abolished by siS1PR1. (B) HLECs were cultured as described above, and western blotting was performed for the indicated proteins. OSS induced the expression of the shear stress-responsive transcription factor KLF4 and the valve-regulatory molecules active β-catenin, FOXC2, and CX37. Knockdown of S1PR1 significantly inhibited the expression of these molecules. (C) HLECs were transfected with siControl or siS1PR1 and grown for 48 h under static conditions to knockdown S1PR1. Subsequently, cells were cultured under static or OSS for 10 min. Cell lysates were western blotted for the indicated antibodies, and quantified. pAKT, pERK, and pFOXO1 were upregulated by OSS. siS1PR1 significantly downregulated the expression of pAKT and pFOXO1. (D) Schematic summary of OSS response in LECs. S1PR1 preserves VE-cadherin and cortical actin and promotes the phosphorylation of AKT. Phosphorylated AKT promotes the phosphorylation and nuclear exclusion of FOXO1 and prevents the phosphorylation and degradation of β-catenin. The later two processes are likely responsible for the expression of valve-regulatory molecules FOXC2 and CX37 (encoded by GJA4) and the shear-stress responsive transcription factor KLF4. In the absence of S1PR1, LECs lose VE-cadherin, gain stress fibers, become elongated, and do not upregulate valve-regulatory genes or KLF4 in response to OSS. (E) The mesenteric tissues from 1-year-old control and Foxc2+/− mice were analyzed by IHC for the indicated markers to identify and quantify TLOs. A representative TLO from a Foxc2+/− mouse is shown. A significantly higher number of TLOs were observed in the Foxc2+/− mice. Statistics: (A) The axis was measured in 30 cells, and the junction was analyzed in 20 cells in a single field from each of the three experiments. Each dot represents one cell in the graphs; (B and C) the blot is representative of three independent experiments. The data from all experiments were used to prepare the graphs; (E) each dot in the graph indicates an individual mouse. n = 5 controls, n = 9 Foxc2+/− mice. The graphs are shown as mean ± SD. Two-way ANOVA with Tukey’s post hoc test (A–C) and unpaired t test with Welch’s correction (E) were performed to determine statistical significance. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Source data are available for this figure: SourceData F9.

S1PR1 regulates OSS response and the expression of valve regulatory genes in HLECs. (A) HLECs were transfected with siControl or siS1PR1 and grown for 24 h under static conditions to knockdown S1PR1. Subsequently, cells were cultured under static or OSS for 24 h. Cells were immunostained for F-actin or VE-cadherin. F-actin was primarily located along the cell wall (cortical actin) of control cells under both static and OSS conditions. Control HLECs became more spherical, and cortical actin expression appeared to be increased by OSS. In contrast, siS1PR1-transfected HLECs appeared elongated and had increased expression of stress fibers. The percentage of VE-cadherin+ overlapping cell junctions was increased by OSS, and this enhancement was abolished by siS1PR1. (B) HLECs were cultured as described above, and western blotting was performed for the indicated proteins. OSS induced the expression of the shear stress-responsive transcription factor KLF4 and the valve-regulatory molecules active β-catenin, FOXC2, and CX37. Knockdown of S1PR1 significantly inhibited the expression of these molecules. (C) HLECs were transfected with siControl or siS1PR1 and grown for 48 h under static conditions to knockdown S1PR1. Subsequently, cells were cultured under static or OSS for 10 min. Cell lysates were western blotted for the indicated antibodies, and quantified. pAKT, pERK, and pFOXO1 were upregulated by OSS. siS1PR1 significantly downregulated the expression of pAKT and pFOXO1. (D) Schematic summary of OSS response in LECs. S1PR1 preserves VE-cadherin and cortical actin and promotes the phosphorylation of AKT. Phosphorylated AKT promotes the phosphorylation and nuclear exclusion of FOXO1 and prevents the phosphorylation and degradation of β-catenin. The later two processes are likely responsible for the expression of valve-regulatory molecules FOXC2 and CX37 (encoded by GJA4) and the shear-stress responsive transcription factor KLF4. In the absence of S1PR1, LECs lose VE-cadherin, gain stress fibers, become elongated, and do not upregulate valve-regulatory genes or KLF4 in response to OSS. (E) The mesenteric tissues from 1-year-old control and Foxc2+/− mice were analyzed by IHC for the indicated markers to identify and quantify TLOs. A representative TLO from a Foxc2+/− mouse is shown. A significantly higher number of TLOs were observed in the Foxc2+/− mice. Statistics: (A) The axis was measured in 30 cells, and the junction was analyzed in 20 cells in a single field from each of the three experiments. Each dot represents one cell in the graphs; (B and C) the blot is representative of three independent experiments. The data from all experiments were used to prepare the graphs; (E) each dot in the graph indicates an individual mouse. n = 5 controls, n = 9 Foxc2+/− mice. The graphs are shown as mean ± SD. Two-way ANOVA with Tukey’s post hoc test (A–C) and unpaired t test with Welch’s correction (E) were performed to determine statistical significance. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Source data are available for this figure: SourceData F9.

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