Journal: Pharmacology Research & Perspectives

Article Title: Involvement of S1P 1 receptor pathway in angiogenic effects of a novel adenosine-like nucleic acid analog COA-Cl in cultured human vascular endothelial cells

doi: 10.1002/prp2.68

Figure Lengend Snippet: S1P receptor antagonists attenuate phosphorylation responses of ERK1/2 in HUVEC induced by COA-Cl and S1P. The figure shows the results of immunoblot (IB) analyses in which HUVEC were treated with COA-Cl or S1P either in the presence or absence of pretreatment with antagonists specific for S1P receptors. (A) Effects of the S1P 1 antagonist W146. HUVEC were treated with 10 μ mol/L of W146 for 30 min, followed by 100 μ mol/L of COA-Cl for 15 min. They were then subjected to IB analyses for phospho- and total-ERK1/2. The upper half of the panel shows the representative results of four independent experiments, which yielded equivalent data. The results are summarized in the lower half graphs. (B) VPC23019, a dual antagonist for S1P 1 /S1P 3 (5 μ mol/L for 15 min), was used instead of W146 ( n = 6). (C and D) Cells were treated with 1 μ mol/L of S1P for 5 min instead of COA-Cl ( n = 4), respectively. * P < 0.05 versus vehicle alone. † P < 0.05 versus cells not treated with W146 or VPC23019.

Article Snippet: Rabbit polyclonal anti-S1P 2 antibody was from Alomone (Jerusalem, Israel).

Techniques: Western Blot

Journal: Pharmacology Research & Perspectives

Article Title: Involvement of S1P 1 receptor pathway in angiogenic effects of a novel adenosine-like nucleic acid analog COA-Cl in cultured human vascular endothelial cells

doi: 10.1002/prp2.68

Figure Lengend Snippet: Immunoblot assay and RT-PCR analysis of HUVEC for S1P receptor subtypes and effects of siRNA. (A) Results of immunoblot (IB) analyses using lysates derived from HUVEC and tissue homogenates derived from healthy male rats. Equal quantities of cellular proteins were separated and probed using antibodies directed to S1P receptor subtypes S1P 1–3 or to MAP kinases ERK1/2, as indicated. HUVEC expressed abundant S1P 1 and detectable S1P 3 immunoreactive signals but did not express S1P 2 ( n = 3). (B) Effective knockdown of S1P receptor proteins in HUVEC transiently transfected with siRNA directed to S1P 1 or S1P 3 . In the left half of the figure, the cells were transfected with siRNA specific for S1P 1 (Qiagen Hs_EDG1_1, 40 nmol/L) or negative control siRNA. Forty-eight hours after transfection, the cells were harvested and subjected to a series of IB assays using antibodies directed to S1P 1 , S1P 3 , ERK1/2, VEGFR2, GAPDH and cyclophilin-B (Cy-B). In the right half, the cells were transfected with siRNA specific for S1P 3 (Qiagen Hs_EDG3_6, 20 nmol/L) or control siRNA. For both halves, six independent cultures were tested that yielded similar results. siRNA directed to S1P 1 or S1P 3 leads to significant and specific knockdown of their target protein. (C) Results of RT-PCR assays. HUVEC were transiently transfected with Qiagen siRNA directed to human S1P 1 receptor or with negative control. They were then subjected to RNA isolation and RT-PCR assays using primer pairs as indicated. Note that these cells express detectable levels of S1P receptor subtypes S1P 1−3 along with VEGFR2 and GAPDH. Densitometric analysis revealed that the expression level of S1P 1 transcript relative to GAPDH in cells treated with siRNA specific to S1P 1 decreased to 24.5% ± 5.5% of the control, while those of S1P 3 , S1P 2 , and VEGFR2 remained unchanged ( n = 3).

Article Snippet: Rabbit polyclonal anti-S1P 2 antibody was from Alomone (Jerusalem, Israel).

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Transfection, Negative Control, Isolation, Expressing

Journal: Pharmacology Research & Perspectives

Article Title: Involvement of S1P 1 receptor pathway in angiogenic effects of a novel adenosine-like nucleic acid analog COA-Cl in cultured human vascular endothelial cells

doi: 10.1002/prp2.68

Figure Lengend Snippet: Effects of siRNA directed to S1P receptors on HUVEC stimulated with COA-Cl or S1P. The results of immunoblot (IB) analyses using lysates derived from HUVEC treated with COA-Cl or S1P that had been transfected with siRNA directed to S1P receptor subtypes are shown. (A) HUVEC transfected with siRNA specific for S1P 1 or control oligonucleotides were treated with COA-Cl (100 μ mol/L, upper half) for the times indicated, followed by IB analyses for phosphorylated and total forms of ERK1/2. (B) Transfection was performed using siRNA specific for S1P 3 . (C and D) Cells were treated with S1P (100 nmol/L) instead of COA-Cl ( n = 4) for each subset. * P < 0.05 versus vehicle alone. † P < 0.05 versus control siRNA.

Article Snippet: Rabbit polyclonal anti-S1P 2 antibody was from Alomone (Jerusalem, Israel).

Techniques: Western Blot, Derivative Assay, Transfection

Journal: Pharmacology Research & Perspectives

Article Title: Involvement of S1P 1 receptor pathway in angiogenic effects of a novel adenosine-like nucleic acid analog COA-Cl in cultured human vascular endothelial cells

doi: 10.1002/prp2.68

Figure Lengend Snippet: Promotion of tube formation activity by COA-Cl in HUVEC and inhibitory effects of S1P 1 antagonists and a Src tyrosine kinase. The results of tube formation analyses are shown. (A) HUVEC were cocultured with normal human fibroblasts for 10 days in the presence or absence of COA-Cl (100 μ mol/L), with/without W146 (20 μ mol/L) or VPC23019 (5 μ mol/L), as indicated. Endothelial tube formation was then identified as CD31-positive signals in microphotographs, which were subsequently quantified in the graph. The right half of the figure summarizes the results derived from four independent assays, representative images of which are shown in the left half. (B) Certain cells were treated with PP2 (2.5 μ mol/L). In both panels, scale bars correspond to 500 micrometer ( n = 4).

Article Snippet: Rabbit polyclonal anti-S1P 2 antibody was from Alomone (Jerusalem, Israel).

Techniques: Activity Assay, Derivative Assay

Journal: Pharmacology Research & Perspectives

Article Title: Involvement of S1P 1 receptor pathway in angiogenic effects of a novel adenosine-like nucleic acid analog COA-Cl in cultured human vascular endothelial cells

doi: 10.1002/prp2.68

Figure Lengend Snippet: Displacement of [ 3 H]S1P by COA-Cl in receptor ligand-binding competition assay using membrane preparation derived from S1P 1 -overexpressing chem-1 cells. The figure shows the results of receptor ligand-binding competition assay in which [ 3 H]S1P was bound to membrane preparation derived from chem-1 cells overexpressing S1P 1 . Increasing concentrations of COA-Cl, unlabeled S1P, or unlabeled adenosine were included along with [ 3 H]S1P (A−C, respectively). In each panel, 100% and 0% correspond to the [ 3 H]S1P binding values obtained in the presence of the vehicle and excess S1P, respectively ( n = 6−9). (D) Degrees of [ 3 H]S1P binding obtained in the presence of the vehicle, COA-Cl (500 μ mol/L), S1P (10 μ mol/L) and adenosine (2.5 mmol/L). COA-Cl displaced [ 3 H]S1P but adenosine did not.

Article Snippet: Rabbit polyclonal anti-S1P 2 antibody was from Alomone (Jerusalem, Israel).

Techniques: Ligand Binding Assay, Competitive Binding Assay, Derivative Assay, Binding Assay

Journal: Pharmacology Research & Perspectives

Article Title: Involvement of S1P 1 receptor pathway in angiogenic effects of a novel adenosine-like nucleic acid analog COA-Cl in cultured human vascular endothelial cells

doi: 10.1002/prp2.68

Figure Lengend Snippet: Characterization of ERK1/2 responses to COA-Cl in CHO-EDG1 cells. Shown are the results of protein immunoblot (IB) assay of cell lysates derived from CHO-EDG1 cells and their parental CHO-K1 cells. They were treated with increasing concentrations of COA-Cl (A) and S1P (B). They were then subjected to IB with antibodies directed to phosphorylated and total forms of ERK, as indicated. Left side shows representative images and right side shows graphic summaries, where closed and open circles represent values derived from CHO-K1 and CHO-EDG1 cells, respectively ( n = 4). * P < 0.05 and ** P < 0.01 versus vehicle-treated cells. †† P < 0.01 versus CHO-K1 cells.

Article Snippet: Rabbit polyclonal anti-S1P 2 antibody was from Alomone (Jerusalem, Israel).

Techniques: Western Blot, Derivative Assay

Journal: Mediators of Inflammation

Article Title: Sphingosine-1-Phosphate/Sphingosine-1-Phosphate Receptor 2 Axis Can Promote Mouse and Human Primary Mast Cell Angiogenic Potential through Upregulation of Vascular Endothelial Growth Factor-A and Matrix Metalloproteinase-2

doi: 10.1155/2016/1503206

Figure Lengend Snippet: Role of S1P/S1PR2 signaling in VEGF-A secretion. (a) Blood was collected from allergenically challenged WT (open bar, n = 5-6 mice) and S1PR2-null (black bar, n = 5-6 mice) mice, euthanized 2 hours after Ag challenge, and serum VEGF-A levels were measured in duplicate determination for each animal. (b) Bone marrow-derived mast cells (BMMC, three independent populations) from both genotypes were stimulated for 24 hours with vehicle (DMSO/PBS/4 mg/mL fatty acid-free BSA), S1P (100 nM), IgE/Ag, or ionomycin (Iono) and VEGF-A was measured in MC supernatants, in duplicate determinations. Error bars show standard error of means. ∗ p < 0.05, ∗∗ p < 0.005.

Article Snippet: S1PR2 knockout mice (Taconic Biosciences, Inc., Hudson, NY) and corresponding wild-type (WT) mice were on a mixed 129/SvEV-C57BL/6 background.

Techniques: Derivative Assay

Journal: Mediators of Inflammation

Article Title: Sphingosine-1-Phosphate/Sphingosine-1-Phosphate Receptor 2 Axis Can Promote Mouse and Human Primary Mast Cell Angiogenic Potential through Upregulation of Vascular Endothelial Growth Factor-A and Matrix Metalloproteinase-2

doi: 10.1155/2016/1503206

Figure Lengend Snippet: Human MC secrete proangiogenic factors upon exposure to S1P in a S1PR2-dependent manner. (a) VEGF-A levels were measured in the supernatants of activated human MC in the absence (open bars, vehicle (DMSO/PBS/4 mg/mL fatty acid-free BSA)) or presence of a 30-minute pretreatment with CYM-5442 (filled bars, S1PR1 agonist, 1 μ M) or with JTE-013 (gray bars, S1PR2 antagonist, 1 μ M), followed by the indicated stimuli similar to . (b) Matrix metalloproteinase- (MMP-) 2 levels were measured in supernatants of MC activated exactly as described in . All supernatants were collected 24 hours after stimulation. Activation experiments were conducted using five independent human skin MC populations generated from five donors. Activation was conducted in triplicate determinations and measurements were conducted in duplicate determinations for each individual determination. When reaching significance, statistics are indicated in each figure. Error bars show standard error of means. ∗∗∗ p < 0.0001.

Article Snippet: S1PR2 knockout mice (Taconic Biosciences, Inc., Hudson, NY) and corresponding wild-type (WT) mice were on a mixed 129/SvEV-C57BL/6 background.

Techniques: Activation Assay, Generated

Journal: Frontiers in Immunology

Article Title: Hyperammonemia increases the release of pathological extracellular vesicles from monocytes by impairing lysosomal function and autophagy through the TNFα–cAMP–PKA–LC3 pathway

doi: 10.3389/fimmu.2025.1724800

Figure Lengend Snippet: Ex vivo incubation with EV from CD4 + of hyperammonemic rats (HA-M-EV) does not induce TNFα–TNFR1–S1PR2–IL-1β–BDNF pathway activation in hippocampal slices from control rats. Protein content of (A) TNFα (n= 9-15), (B) TNFR1 (n= 9-14), (C) IL-1β (n= 11-15), (D) BDNF (n= 9-14), (E) CCL2 (n= 8), and (F) TrkB (n= 10-13) in hippocampal slices, assessed by Western blot. Representative images of the blots of each protein are shown. All data are presented as mean ± SEM. One-way ANOVA followed by Tukey’s (TNFα, TNFR1, IL-1β, BDNF, TrkB) post-hoc tests were performed to compare all groups. The data corresponding to CCL2 content were non-normally distributed and were analyzed using Kruskal–Wallis tests. Values significantly different from control group are indicated by asterisk (*p<0.05, **p<0.01).

Article Snippet: The hippocampal slices were homogenized and analyzed by Western blot as described above, using the following primary antibodies: GluaA1 (Glutamate A1 Subunit of Ampa Receptors, 1:1,000, Millipore, #04-855), GluA2 (Glutamate A2 Subunit of AMPA Receptors, 1:2,000, Proteintech, #11994-1-AP), IL-1R (Interleukin 1-Beta Receptor, 1:500, Abcam, AB106278 ), NR2A (2A subunit of NMDA receptors, 1:1,000, Millipore, #04-901), NR2B (2B subunit of NMDA receptors, 1:1,000, Millipore, #06-600), TNFR1 (1:1,000, Abcam, #ab19139), TrkB (1:500, Abcam, #ab18987), and S1PR2 (1:1,000, Proteintech, #21180-1-AP).

Techniques: Ex Vivo, Incubation, Activation Assay, Control, Western Blot

Journal: Frontiers in Immunology

Article Title: Hyperammonemia increases the release of pathological extracellular vesicles from monocytes by impairing lysosomal function and autophagy through the TNFα–cAMP–PKA–LC3 pathway

doi: 10.3389/fimmu.2025.1724800

Figure Lengend Snippet: Ex vivo incubation with EV from monocytes of hyperammonemic rats (HA-M-EV) induces TNFα–TNFR1–S1PR2–IL-1β–BDNF pathway activation in hippocampal slices from control rats. Protein content of (A) TNFα (n= 8-21), (B) TNFR1 (n= 8-15), (D) S1PR2 (n= 8-18), (F) IL-1β (n= 5-14), (H) CCL2 (n= 8-21), and (I) BDNF (n= 6-14) and membrane expression of (C) TNFR1 (n= 5-15), (E) S1PR2 (n= 6-12), and (G) IL1R (n= 3-20) in hippocampal slices, assessed by Western blot. For the analysis of membrane expression, the sections were incubated in the presence (+) or absence (−) of the cross-linker BS3. Samples in the absence of BS3 represent the total amount of each protein, whereas samples incubated in the presence of BS3 represent the non-membrane fraction of each protein. Representative images of the blots of each protein are shown. All data are presented as mean ± SEM. Statistical analysis was determined using four separate one-way ANOVAs, each comparing the four baseline groups (C, HA, C-M-EV, and HA-M-EV) with one of the treatment groups (C-M-EV + Forsk, HA-M-EV + PKAi, C-M-EV + TNFα, or HA-M-EV + aTNFα). One-way ANOVA followed by Fisher’s post-hoc tests were performed to compare all groups. Values significantly different from the C group are indicated by asterisk (*p<0.05, **p<0.01, ***p<0.001), values significantly different from the C-M-EV group are indicated by a (a=p<0.05, aa=p<0.01), and values significantly different from the HA-M-EV group are indicated by b (b=p<0.05, bb=p<0.01, bbb=p<0.001, bbbb=p<0.0001).

Article Snippet: The hippocampal slices were homogenized and analyzed by Western blot as described above, using the following primary antibodies: GluaA1 (Glutamate A1 Subunit of Ampa Receptors, 1:1,000, Millipore, #04-855), GluA2 (Glutamate A2 Subunit of AMPA Receptors, 1:2,000, Proteintech, #11994-1-AP), IL-1R (Interleukin 1-Beta Receptor, 1:500, Abcam, AB106278 ), NR2A (2A subunit of NMDA receptors, 1:1,000, Millipore, #04-901), NR2B (2B subunit of NMDA receptors, 1:1,000, Millipore, #06-600), TNFR1 (1:1,000, Abcam, #ab19139), TrkB (1:500, Abcam, #ab18987), and S1PR2 (1:1,000, Proteintech, #21180-1-AP).

Techniques: Ex Vivo, Incubation, Activation Assay, Control, Membrane, Expressing, Western Blot

Journal: Frontiers in Immunology

Article Title: Hyperammonemia increases the release of pathological extracellular vesicles from monocytes by impairing lysosomal function and autophagy through the TNFα–cAMP–PKA–LC3 pathway

doi: 10.3389/fimmu.2025.1724800

Figure Lengend Snippet: Schematic representation of the proposed mechanism by which (1) hyperammonemia induces the formation of pathological EV containing TNFR1-TNFα in monocytes, (2) how TNFα and PKA modulate this process, and (3) how these EV induce pathological effects in the hippocampus. Hyperammonemia increases TNF α and activation of its receptor TNFR1 in monocytes, leading to activation of adenylate cyclase and increased cAMP levels and PKA activation. PKA alters both LC3, leading to increased lysosomal pH and impaired expression of lysosomal proteins such as cathepsin-L and LAMP2. This results in lysosomal dysfunction and altered autophagic flux enhances the release from multivesicular bodies (MVBs) of EV containing increased levels of TNFR1 and TNFα. These pathological HA-M-EV enhance activation of the TNFα–TNFR1–S1PR2–IL-1β–BDNF pathway and the TrkB–NR2B–GluA1–GluA2 pathway in hippocampal slices from control rats, inducing neuroinflammation and altered glutamatergic neurotransmission. All these effects are reversed by blocking TNFα with anti-TNFα or inhibiting PKA with H69 in the cultures of monocytes from hyperammonemic rats (green symbols). Activation of TNFR1 with recombinant TNFα (rTNFα) or of adenylate cyclase with forskolin in monocyte cultures from control rats induce essentially the same effects (red arrows). This figure was created using Biorender.com .

Article Snippet: The hippocampal slices were homogenized and analyzed by Western blot as described above, using the following primary antibodies: GluaA1 (Glutamate A1 Subunit of Ampa Receptors, 1:1,000, Millipore, #04-855), GluA2 (Glutamate A2 Subunit of AMPA Receptors, 1:2,000, Proteintech, #11994-1-AP), IL-1R (Interleukin 1-Beta Receptor, 1:500, Abcam, AB106278 ), NR2A (2A subunit of NMDA receptors, 1:1,000, Millipore, #04-901), NR2B (2B subunit of NMDA receptors, 1:1,000, Millipore, #06-600), TNFR1 (1:1,000, Abcam, #ab19139), TrkB (1:500, Abcam, #ab18987), and S1PR2 (1:1,000, Proteintech, #21180-1-AP).

Techniques: Activation Assay, Expressing, Control, Blocking Assay, Recombinant

Journal: Acta Histochemica et Cytochemica

Article Title: Effect of Hepatic Lipid Overload on Accelerated Hepatocyte Proliferation Promoted by HGF Expression via the SphK1/S1PR2 Pathway in MCD-diet Mouse Partial Hepatectomy

doi: 10.1267/ahc.24-00046

Figure Lengend Snippet: SphK1/S1P signaling in mouse liver during regeneration. qPCR analysis of ( A ) SphK1 , ( B ) SphK2 , ( C ) S1PR3 , and ( D ) ABCC1 mRNA expression in the liver of chow- and MCD-diet fed mice after PHx. ( E ) Immunofluorescence analysis of S1PR2 (green) and DAPI (blue) in mouse liver after PHx. Bar = 20 μm; 40× objective lenses were used. ( F ) Quantitative analysis of S1PR2-positive cells in the liver of chow- and MCD-diet fed mice after PHx. Data represent the mean ± SEM for 6–8 mice per group. Asterisks indicate statistically significant differences (*p < 0.05, **p < 0.01 and ***p < 0.001).

Article Snippet: S1PR2-expressing cells were stained with rabbit polyclonal antibody against S1PR2 (Novus Biologicals) followed by treatment with Alexa-633 conjugated goat anti-Rabbit IgG.

Techniques: Expressing, Immunofluorescence

Journal: Acta Histochemica et Cytochemica

Article Title: Effect of Hepatic Lipid Overload on Accelerated Hepatocyte Proliferation Promoted by HGF Expression via the SphK1/S1PR2 Pathway in MCD-diet Mouse Partial Hepatectomy

doi: 10.1267/ahc.24-00046

Figure Lengend Snippet: Expression of S1PR2 in mouse liver. Double immunofluorescence analysis of S1PR2 (green) and HNF-4α (red staining in upper panel), F4/80 (red staining in middle panel), Ly6G (red staining in lower panel), and DAPI (blue) in the liver of MCD-diet fed mice at 12hr after PHx. Arrows indicate double-positive cells. Bar = 20 μm.

Article Snippet: S1PR2-expressing cells were stained with rabbit polyclonal antibody against S1PR2 (Novus Biologicals) followed by treatment with Alexa-633 conjugated goat anti-Rabbit IgG.

Techniques: Expressing, Immunofluorescence, Staining

Journal: Acta Histochemica et Cytochemica

Article Title: Effect of Hepatic Lipid Overload on Accelerated Hepatocyte Proliferation Promoted by HGF Expression via the SphK1/S1PR2 Pathway in MCD-diet Mouse Partial Hepatectomy

doi: 10.1267/ahc.24-00046

Figure Lengend Snippet: HGF and FGF2 mRNA expression in S1PR2-positive cells after PHx. ( A ) Macrophages (F4/80) and neutrophils (Ly6G) were isolated from the liver of chow- and MCD-diet fed mice at 0, 6, and 12 hr after PHx. The percentage of macrophages ( B ) and neutrophils ( C ) in the liver of chow- and MCD-diet fed mice. HGF and FGF2 mRNA expression levels in ( D , F ) macrophages and ( E , G ) neutrophils isolated from the liver of chow- and MCD-diet fed mice at 0, 6 and 12 hr after PHx. Data represent the mean ± SEM for 3 mice per group. Asterisks indicate statistically significant differences (*p < 0.05, **p < 0.01 and ***p < 0.001).

Article Snippet: S1PR2-expressing cells were stained with rabbit polyclonal antibody against S1PR2 (Novus Biologicals) followed by treatment with Alexa-633 conjugated goat anti-Rabbit IgG.

Techniques: Expressing, Isolation