Journal: Molecular medicine reports

Article Title: Effects of S1PR2 antagonist on blood pressure and angiogenesis imbalance in preeclampsia rats.

doi: 10.3892/mmr.2021.12095

Figure Lengend Snippet: Figure 1. S1PR2 is increased in the serum and placental tissue of PE rats. (A) Measurement of S1P in serum via ELISA. (B) The expression of S1PR1, S1PR2 and S1PR3 in serum of control and PE rats was analyzed by reverse transcription‑quantitative PCR analysis. (C) The effect of S1P on the expression of S1PR2 in the plasma of PE rats was analyzed via ELISA. (D) The effect of S1P on the expression of S1PR2 in the placental tissues of PE rats was analyzed by western blotting. n=3. *P<0.05, **P<0.01 and ***P<0.001 vs. Control group; #P<0.05 and ###P<0.001 vs. Model group; ∆P<0.05 and ∆∆∆P<0.001 vs. S1P group. S1P, sphingosine‑1‑phosphate; S1PR2, sphingosine‑1‑phosphate receptor 2; PE, preeclampsia; ELISA, enzyme‑linked immunosorbent assay.

Article Snippet: The membranes were incubated with primary antibodies against S1PR2 (cat. no. 21180‐1‐AP; 1:1,000; ProteinTech Group, Inc.), VEGF (cat. no. 66828‐1‐AP; 1:1,000; ProteinTech Group, Inc.), Flt‐1 (cat. no. 13687‐1‐AP; 1:1,000; ProteinTech Group, Inc.), endo‐ thelial (e)NOS (cat. no. ab76198; 1:1,000; Abcam) and β‐actin (cat. no. ab8226; 1:1,000; Abcam) overnight at 4 ̊C.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Control, Clinical Proteomics, Western Blot

Journal: Molecular medicine reports

Article Title: Effects of S1PR2 antagonist on blood pressure and angiogenesis imbalance in preeclampsia rats.

doi: 10.3892/mmr.2021.12095

Figure Lengend Snippet: Figure 2. Inhibition of S1PR2 with JTE‑013 decreases BP in PE rats. Effect of JTE‑013 on (A) SBP and (B) DBP in PE rats in tail‑cuff measurement. n=3. *P<0.05, **P<0.01 and ***P<0.001 vs. Control group; #P<0.05, ##P<0.01 and ###P<0.001 vs. Model group; ∆P<0.05 vs. Model + S1PR2 antagonist low group. S1PR2, sphingosine‑1‑phosphate receptor 2; PE, preeclampsia; BP, blood pressure; SBP, systolic blood pressure; DBP, diastolic blood pressure.

Article Snippet: The membranes were incubated with primary antibodies against S1PR2 (cat. no. 21180‐1‐AP; 1:1,000; ProteinTech Group, Inc.), VEGF (cat. no. 66828‐1‐AP; 1:1,000; ProteinTech Group, Inc.), Flt‐1 (cat. no. 13687‐1‐AP; 1:1,000; ProteinTech Group, Inc.), endo‐ thelial (e)NOS (cat. no. ab76198; 1:1,000; Abcam) and β‐actin (cat. no. ab8226; 1:1,000; Abcam) overnight at 4 ̊C.

Techniques: Inhibition, Control

Journal: Molecular medicine reports

Article Title: Effects of S1PR2 antagonist on blood pressure and angiogenesis imbalance in preeclampsia rats.

doi: 10.3892/mmr.2021.12095

Figure Lengend Snippet: Figure 3. Effect of JTE‑013 on serum NO and iNOS and the expression of eNOS in placental tissues. (A) Summarized data showing the inhibitory effect of JTE‑013 on serum NO levels in PE rats. (B) Summarized data showing the inhibitory effect of JTE‑013 on serum iNOS levels in PE rats. (C) Summarized data showing that JTE‑013 prevented the decreased expression of eNOS in placental tissues of PE rats. n=3. *P<0.05, **P<0.01 and ***P<0.001 vs. Control group; ##P<0.01 and ###P<0.001 vs. Model group; ∆P<0.05 and ∆∆∆P<0.001 vs. Model + S1PR2 antagonist low group. S1PR2, sphingosine‑1‑phosphate receptor 2; PE, preeclampsia; iNOS, inducible nitric oxide synthase; eNOS, endothelial nitric oxide synthase; NO, nitric oxide.

Article Snippet: The membranes were incubated with primary antibodies against S1PR2 (cat. no. 21180‐1‐AP; 1:1,000; ProteinTech Group, Inc.), VEGF (cat. no. 66828‐1‐AP; 1:1,000; ProteinTech Group, Inc.), Flt‐1 (cat. no. 13687‐1‐AP; 1:1,000; ProteinTech Group, Inc.), endo‐ thelial (e)NOS (cat. no. ab76198; 1:1,000; Abcam) and β‐actin (cat. no. ab8226; 1:1,000; Abcam) overnight at 4 ̊C.

Techniques: Expressing, Control

Journal: Molecular medicine reports

Article Title: Effects of S1PR2 antagonist on blood pressure and angiogenesis imbalance in preeclampsia rats.

doi: 10.3892/mmr.2021.12095

Figure Lengend Snippet: Figure 4. JTE‑013 inhibits the PE model‑induced expression levels of VEGF and Flt‑1 receptor in PE rats. (A) Summarized data showing the inhibitory effect of JTE‑013 on the PE model‑induced mRNA levels of VEGF and Flt‑1 in reverse transcription‑quantitative PCR assay. (B) Representative western blotting images and summarized data showing the inhibitory effect of JTE‑013 on the PE model‑induced protein levels of VEGF and Flt‑1 in the placental tissues of PE rats. n=3. *P<0.05 and ***P<0.001 vs. Control group; #P<0.05, ##P<0.01 and ###P<0.001 vs. Model group; ∆∆P<0.01 and ∆∆∆P<0.001 vs. Model + S1PR2 antagonist low group. S1PR2, sphingosine‑1‑phosphate receptor 2; PE, preeclampsia; VEGF, vascular endothelial growth factor; Flt‑1, fms‑like tyrosine kinase 1.

Article Snippet: The membranes were incubated with primary antibodies against S1PR2 (cat. no. 21180‐1‐AP; 1:1,000; ProteinTech Group, Inc.), VEGF (cat. no. 66828‐1‐AP; 1:1,000; ProteinTech Group, Inc.), Flt‐1 (cat. no. 13687‐1‐AP; 1:1,000; ProteinTech Group, Inc.), endo‐ thelial (e)NOS (cat. no. ab76198; 1:1,000; Abcam) and β‐actin (cat. no. ab8226; 1:1,000; Abcam) overnight at 4 ̊C.

Techniques: Expressing, Western Blot, Control

Journal: Molecular medicine reports

Article Title: Effects of S1PR2 antagonist on blood pressure and angiogenesis imbalance in preeclampsia rats.

doi: 10.3892/mmr.2021.12095

Figure Lengend Snippet: Figure 5. JTE‑013 attenuates pathological changes in placental tissues and decreases inflammation in PE rats. (A) Summarized data showing the inhibitory effect of JTE‑013 on the increased expression of serum TNF‑α, IL‑1β and IL‑6, as determined by an enzyme‑linked immunosorbent assay. (B) Summarized data showing JTE‑013 attenuated the infiltration of inflammatory cells in placental tissues, as detected by hematoxylin and eosin staining (magnification, x100 or 200). n=3. *P<0.05, **P<0.01 and ***P<0.001 vs. Control group; ##P<0.01 and ###P<0.001 vs. Model group; ∆P<0.05 and ∆∆P<0.01 vs. Model + S1PR2 antagonist low group. PE, preeclampsia; TNF‑α, tumor necrosis factor‑α; IL‑, interleukin; S1PR2, sphingosine‑1‑phosphate receptor 2; lab, labyrinth; JZ, junctional zone; de, decidua.

Article Snippet: The membranes were incubated with primary antibodies against S1PR2 (cat. no. 21180‐1‐AP; 1:1,000; ProteinTech Group, Inc.), VEGF (cat. no. 66828‐1‐AP; 1:1,000; ProteinTech Group, Inc.), Flt‐1 (cat. no. 13687‐1‐AP; 1:1,000; ProteinTech Group, Inc.), endo‐ thelial (e)NOS (cat. no. ab76198; 1:1,000; Abcam) and β‐actin (cat. no. ab8226; 1:1,000; Abcam) overnight at 4 ̊C.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Staining, Control

Journal: Circulation

Article Title: Tumor necrosis factor-α-mediated downregulation of the cystic fibrosis transmembrane conductance regulator drives pathological sphingosine-1-phosphate signaling in a mouse model of heart failure.

doi: 10.1161/CIRCULATIONAHA.111.047316

Figure Lengend Snippet: Figure 1. The cystic fibrosis transmembrane conductance regulator (CFTR) mediates cellular fluorescein isothiocyanate (FITC)– sphingosine-1-phosphate (S1P) uptake. A, Western blots confirm CFTR protein expression in isolated mouse cerebral and mesenteric arteries, as well as primary vascular smooth muscle cells (VSMCs) isolated from mouse mesenteric arteries. No CFTR signal was detected from mesenteric VSMCs isolated from CFTR knockout (CFTR/) mice. The blots displayed are representative of at least 3 separate experiments. B, A standard fluorescence-activated cell sorting analysis technique reveals a shift in the median fluorescence intensity at 525 nm for VSMCs incubated with FITC-S1P compared with cells incubated with unlabeled S1P (background [Bkgd]). This median shift represents a measure of FITC-S1P uptake. C, In wild-type littermate VSMCs (CFTR/), the median fluorescence intensity shift (n6) is abolished by CFTR inhibition [100 nmol/L CFTR(inh)-172 for 30 minutes; n5]. In VSMCs isolated from CFTR knockout mice, no shift in median fluorescence intensity is observed after FITC-S1P treatment (n6), nor is there an effect of CFTR inhibition (n5). Con indicates control. D, The S1P uptake deficit in CFTR/ VSMCs was rescued by transfecting the cells with a wild-type CFTR construct (CFTRwt; n6); the rescued S1P uptake was abolished by CFTR inhibition (n5). E, In baby hamster kidney (BHK) cells, stable expression of CFTRwt significantly increased S1P uptake compared with nontransfected cells (n4). F, Maintaining BHK cells that express CFTRF508 at 27°C for 24 hours (a procedure that increases plasma membrane abundance of CFTR) also significantly increased S1P uptake (n6). *P0.05 for multiple, unpaired comparisons within each genotype in C; multiple, unpaired comparisons in D; and single, unpaired comparisons in E and F. E and F display data normalized to their respective backgrounds.

Article Snippet: Sphingosine-1-phosphate (S1P) was purchased from Biomol International (Plymouth Landing, USA), fluorescein-labeled S1P (FITC-S1P) from Echelon Biosciences (Salt Lake City, USA), JTE013 from Tocris Bioscience (Ellisville, USA) and etanercept from Amgen (Thousand Oaks, USA).

Techniques: Western Blot, Expressing, Isolation, Knock-Out, FACS, Incubation, Inhibition, Control, Construct, Clinical Proteomics, Membrane

Journal: Circulation

Article Title: Tumor necrosis factor-α-mediated downregulation of the cystic fibrosis transmembrane conductance regulator drives pathological sphingosine-1-phosphate signaling in a mouse model of heart failure.

doi: 10.1161/CIRCULATIONAHA.111.047316

Figure Lengend Snippet: Figure 2. The cystic fibrosis transmembrane conductance regulator (CFTR) modulates sphingosine-1-phosphate (S1P)–dependent responses in cultured vascular smooth muscle cells and isolated arteries. A, S1P (100 nmol/L) significantly inhibits wild-type (CFTR/) vascular smooth muscle cell proliferation, an effect that is enhanced by CFTR inhibition [100 nmol/L CFTR(inh)-172; n5 for all groups]. B, CFTR knockout (CFTR/) vascular smooth muscle cell proliferation is lower under control (con) conditions than CFTR/ vascular smooth muscle cells (P0.05 comparing white bars in A and B; n5). S1P inhibits cell proliferation in CFTR/ vascular smooth muscle cells; however, CFTR inhibition has no further effect (n5). C, S1P-stimulated vasoconstriction is stronger in posterior cerebral arteries iso- lated from CFTR/ compared with CFTR/ littermate controls (n6 for both groups). Posterior cerebral artery responses to phenylephrine (D) and acetylcholine (E) are similar in the 2 genotypes (n4 for CFTR/; n3 for CFTR/). F, Posterior cerebral artery myogenic tone is stronger at all transmural pressures 20 mm Hg (ie, 40–100 mm Hg); a similar pattern is observed for mesenteric arteries (G) (for both cere- bral and mesenteric arteries: n6 for CFTR/; n8 for CFTR/). The enhanced myogenic tone in CFTR/ cerebral (F) and mesenteric (G) arteries is abolished by S1P2 receptor inhibition (1 mol/L JTE013; 30 minutes; n4 for both artery types). Maximal vessel diameters at 45 mm Hg (diamax) were as follows: posterior cerebral artery CFTR/ 1668 m (n8), posterior cerebral artery CFTR/ 1625 m (n6), PNS; and mesenteric CFTR/ 24011 m (n8), mesenteric CFTR/ 30134 m (n6), PNS. In A and B, *P0.05 relative to control; P0.05 relative to S1P for multiple, unpaired comparisons; in C through E, *P0.05 after 2-way repeated-measures ANOVA; in F and G, *P0.05 relative to the CFTR/ genotype after 2-way ANOVA.

Article Snippet: Sphingosine-1-phosphate (S1P) was purchased from Biomol International (Plymouth Landing, USA), fluorescein-labeled S1P (FITC-S1P) from Echelon Biosciences (Salt Lake City, USA), JTE013 from Tocris Bioscience (Ellisville, USA) and etanercept from Amgen (Thousand Oaks, USA).

Techniques: Cell Culture, Isolation, Inhibition, Knock-Out, Control

Journal: Circulation

Article Title: Tumor necrosis factor-α-mediated downregulation of the cystic fibrosis transmembrane conductance regulator drives pathological sphingosine-1-phosphate signaling in a mouse model of heart failure.

doi: 10.1161/CIRCULATIONAHA.111.047316

Figure Lengend Snippet: Figure 3. Heart failure (HF) downregulates the cystic fibrosis transmembrane conductance regulator (CFTR) but not sphingosine-1- phosphate (S1P) signaling components. HF (4–6 weeks after myocardial infarction) is associated with the downregulation of posterior cerebral artery CFTR protein expression (n4) (A), which negatively correlated with the extent of the myocardial infarction (B). C through E, HF does not affect the mRNA expression of mesenteric artery sphingosine kinase 1 (SphK1; n12), S1P phosphohydrolase 1 (SPP1; n7), or the S1P2 receptor subtype (S1P2R; n6). *P0.05 for single, unpaired comparisons.

Article Snippet: Sphingosine-1-phosphate (S1P) was purchased from Biomol International (Plymouth Landing, USA), fluorescein-labeled S1P (FITC-S1P) from Echelon Biosciences (Salt Lake City, USA), JTE013 from Tocris Bioscience (Ellisville, USA) and etanercept from Amgen (Thousand Oaks, USA).

Techniques: Expressing

Journal: Circulation

Article Title: Tumor necrosis factor-α-mediated downregulation of the cystic fibrosis transmembrane conductance regulator drives pathological sphingosine-1-phosphate signaling in a mouse model of heart failure.

doi: 10.1161/CIRCULATIONAHA.111.047316

Figure Lengend Snippet: Figure 6. Tumor necrosis factor- (TNF-) downregulates cystic fibrosis transmembrane conductance regulator (CFTR) expression and stimulates a reduction in plasma membrane–associated CFTR. A, TNF- (10 ng/mL; 24 hours) stimulates the downregulation of mesen- teric vascular smooth muscle cell CFTR protein expression (n6), with (B) a concomitant reduction in fluorescein isothiocyanate– sphingosine-1-phosphate (S1P) uptake (n6). Both the reduced CFTR expression (n4) (C) and attenuated fluorescein isothiocyanate– S1P uptake (n4–10) (D) were completely reversed 24 hours after TNF- washout. E, TNF- treatment (10 ng/mL) stimulates a steady decline in CFTR protein expression (n4). Analysis of the 165-kDa (immature, nonglycosylated CFTR; band B) and 170-kDa (mature, glyco- sylated CFTR; band C) bands indicated that only the mature form of CFTR declined. F, Analysis of plasma membrane and intracellular frac- tions (n6) indicated that membrane-associate CFTR was reduced without a concomitant accumulation of intracellular-associated CFTR. In A and B, *P0.05 for unpaired comparisons with control (con)/background (Bkgd); in C through F, *P0.05 for multiple, unpaired compari- sons with the control value at time0 hours; P0.05 for difference between pre–TNF- removal and TNF- removal.

Article Snippet: Sphingosine-1-phosphate (S1P) was purchased from Biomol International (Plymouth Landing, USA), fluorescein-labeled S1P (FITC-S1P) from Echelon Biosciences (Salt Lake City, USA), JTE013 from Tocris Bioscience (Ellisville, USA) and etanercept from Amgen (Thousand Oaks, USA).

Techniques: Expressing, Clinical Proteomics, Membrane, Control

Journal: Circulation

Article Title: Tumor necrosis factor-α-mediated downregulation of the cystic fibrosis transmembrane conductance regulator drives pathological sphingosine-1-phosphate signaling in a mouse model of heart failure.

doi: 10.1161/CIRCULATIONAHA.111.047316

Figure Lengend Snippet: Figure 8. Schematic representation of the proposed relationship between cystic fibrosis transmembrane conductance regu- lator (CFTR) and sphingosine-1-phosphate (S1P) signaling. S1P produced by sphin- gosine kinase 1 (Sphk1) is released to the extracellular compartment, where it (1) ac- tivates S1P receptor (S1PR)–dependent signaling pathways or (2) is transported by CFTR across the plasma membrane for degradation by S1P phosphohydrolase 1 (SPP1). During heart failure, tumor necro- sis factor- (TNF-) stimulates the down- regulation of CFTR (3), thus inhibiting S1P degradation and concomitantly enhancing S1P receptor signaling. sER indicates smooth endoplasmic reticulum.

Article Snippet: Sphingosine-1-phosphate (S1P) was purchased from Biomol International (Plymouth Landing, USA), fluorescein-labeled S1P (FITC-S1P) from Echelon Biosciences (Salt Lake City, USA), JTE013 from Tocris Bioscience (Ellisville, USA) and etanercept from Amgen (Thousand Oaks, USA).

Techniques: Produced, Protein-Protein interactions, Clinical Proteomics, Membrane