Journal: Circulation

Article Title: Role for Substance P-Based Nociceptive Signaling in Progenitor Cell Activation and Angiogenesis During Ischemia in Mice and in Human Subjects

doi: 10.1161/CIRCULATIONAHA.111.089763

Figure Lengend Snippet: Bone marrow progenitor cells express substance P and calcitonin gene-related peptide receptors. A, Immunostaining of lineage-negative (Lin−) cells (selected with the use of magnetic beads and a cocktail of antibodies against committed hematopoietic cells) expressing neurokinin 1 (NK1) (a), receptor activity-modifying protein 1 (RAMP-1) (b), and calcitonin receptor-like receptor (CRLR) (c) (green). Nuclei are stained with 4′, 6-diamidino-2-phenylindole (blue). Immunoreactivity was not detected when primary antibodies were omitted (negative control, d). B through D, Flow cytometry confirms the expression of neuropeptide receptors in progenitor cells. Typical scatterplots and bar graphs show the analyzed data. A substantial fraction of NK1+, RAMP-1+, and CRLR+ cells are Lin− and express the progenitor cell markers c-Kit (c-Kit+) and Sca-1 (Sca-1+).

Article Snippet: Mouse Bone Marrow Cell Isolation Bone marrow cells were depleted of mature hematopoietic cells by magnetic cell sorting with the use of a lineage cell depletion kit and a cocktail of lineage marker antibodies (MACS, Miltenyi Biotec) and then processed for immunocytochemistry or in vitro functional assays.

Techniques: Immunostaining, Magnetic Beads, Expressing, Activity Assay, Staining, Negative Control, Flow Cytometry

Journal: Circulation

Article Title: Role for Substance P-Based Nociceptive Signaling in Progenitor Cell Activation and Angiogenesis During Ischemia in Mice and in Human Subjects

doi: 10.1161/CIRCULATIONAHA.111.089763

Figure Lengend Snippet: Substance P (SP) and calcitonin gene-related peptide (CGRP) exert chemoattractant effects on mouse bone marrow cells. A, SP and CGRP induce migration of lineage-negative (Lin−) bone marrow (BM) cells. Data are expressed as fold increase of vehicle (veh). B, SP (100 nmol/L for 15 minutes) induces phosphorylation/activation of Akt in Lin− cells, which is prevented by the phosphoinositide-3 kinase antagonist LY 294002 (LY) (15 μmol/L). *P<0.05 vs control; n=6 to 9. LY 294002 pretreatment also prevents SP-induced progenitor cell migration. *P<0.05 vs control; #P<0.05 vs SP only; n=3. C and D, Primary culture of mouse dorsal root ganglia (DRG) and respective conditioned medium (CM) induces cell migration (C) leading to an enrichment of Lin− c-Kit+Sca-1+ PC in the migrated fraction (D). E, The migratory effect induced by DRG is reduced by antagonists for CGRP (CGRP8–37, 1000 nmol/L) and NK1 (RP67580, 100 nmol/L). *P<0.05 vs vehicle; n=6; #P<0.05 vs DRG alone; n=5.

Article Snippet: Mouse Bone Marrow Cell Isolation Bone marrow cells were depleted of mature hematopoietic cells by magnetic cell sorting with the use of a lineage cell depletion kit and a cocktail of lineage marker antibodies (MACS, Miltenyi Biotec) and then processed for immunocytochemistry or in vitro functional assays.

Techniques: Migration, Phospho-proteomics, Activation Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Insulin-like Growth Factor Binding Protein-2 (IGFBP2) Is a Key Molecule in the MACC1-Mediated Platelet Communication and Metastasis of Colorectal Cancer Cells

doi: 10.3390/ijms222212195

Figure Lengend Snippet: Dependency of platelet activation by SW620 cells from the MACC1 expression. ( A ) Representative curves of platelet aggregation by light transmission measurement after contact with MACC1-positive SW620 Ctrl cells or the MACC1 KO variant. ( B ) ATP release from platelet dense granule induced by SW620 Ctrl and MACC1 KO cells. ( C ) Platelet adhesion to a cell layer of SW620 Ctrl or MACC1 KO cells exclude a diminished cell contact formation as the reason for lower platelet activation by the MACC1-positive cells. ** p < 0.01, *** p < 0.001.

Article Snippet: The total number of samples analyzed was six: three replicates for cells with high endogenous MACC1 expression after stable gene transfer of control shRNA and three replicates for cells that showed reduced MACC1 gene expression after stable gene transfer of a MACC1-specific shRNA (both shRNAs: OriGene Technologies, Rockville, MD, USA).

Techniques: Activation Assay, Expressing, Transmission Assay, Variant Assay

Journal: International Journal of Molecular Sciences

Article Title: Insulin-like Growth Factor Binding Protein-2 (IGFBP2) Is a Key Molecule in the MACC1-Mediated Platelet Communication and Metastasis of Colorectal Cancer Cells

doi: 10.3390/ijms222212195

Figure Lengend Snippet: The cell supernatant of SW620 cells interferes with platelet activation. ( A ) Representative traces showing platelet aggregation in response to TRAP-6 (black) and its inhibition by supernatant of SW620 MACC1 Ctrl cells (solid grey line) and a lower inhibition by MACC1 KO cells (dashed grey line), respectively. ( B ) Quantification of ATP release from resting platelets, platelets activated with TRAP-6, and co-incubated with supernatant of SW620 MACC1 Ctrl (s. MACC1 Ctrl) and MACC1 KO (s. MACC1 KO) cells, respectively. Data indicate a soluble inhibitory compound in the cell supernatant that is more potent in the SW620 Ctrl cells. *** p < 0.001.

Article Snippet: The total number of samples analyzed was six: three replicates for cells with high endogenous MACC1 expression after stable gene transfer of control shRNA and three replicates for cells that showed reduced MACC1 gene expression after stable gene transfer of a MACC1-specific shRNA (both shRNAs: OriGene Technologies, Rockville, MD, USA).

Techniques: Activation Assay, Inhibition, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Insulin-like Growth Factor Binding Protein-2 (IGFBP2) Is a Key Molecule in the MACC1-Mediated Platelet Communication and Metastasis of Colorectal Cancer Cells

doi: 10.3390/ijms222212195

Figure Lengend Snippet: MACC1 activity is directly related to IGFBP2 expression, which interferes with platelet activation. The downregulation of IGFBP2 in the MACC1 KO variant of SW620 cells was indicated at the mRNA level by qPCR ( A ) and confirmed at the protein level by ELISA ( B ). ELISA data confirm the lower expression of IGFBP2 by the MACC1 KO variant of SW620 cells, either in supernatant (s. MACC1 Ctrl vs. s.MACC1 KO, left columns) and at a lower level in the cell lysate (right columns). ( C ) IGF-I is costimulatory for platelet activation and accelerates the effect of 7.5 µM TRAP-6 to induce platelet aggregation. ( D ) IGF-I is associated with platelets and found in platelet plasma or supernatant after activation, but not in considerable amounts in supernatant of SW620 cells. ( E ) Recombinant IGFBP2 is able to block concentration-dependently the activity of TRAP-6 to induce platelet aggregation and ( F ), significantly, the platelet ATP release. *** p < 0.001; **** p < 0.0001.

Article Snippet: The total number of samples analyzed was six: three replicates for cells with high endogenous MACC1 expression after stable gene transfer of control shRNA and three replicates for cells that showed reduced MACC1 gene expression after stable gene transfer of a MACC1-specific shRNA (both shRNAs: OriGene Technologies, Rockville, MD, USA).

Techniques: Activity Assay, Expressing, Activation Assay, Variant Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Blocking Assay, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Insulin-like Growth Factor Binding Protein-2 (IGFBP2) Is a Key Molecule in the MACC1-Mediated Platelet Communication and Metastasis of Colorectal Cancer Cells

doi: 10.3390/ijms222212195

Figure Lengend Snippet: IGFBP2 controls platelet activation by SW620 cells. ( A ) The shRNA-mediated knockdown of IGFBP2 in SW620 cells was confirmed by Western blot, compared to nontargeted knockdown IGFBP2 Ctrl, and quantified by IGFBP2 ELISA using cell supernatants (left columns) and cell lysates (right columns) ( B ). ( C ) The knockdown of IGFBP2 in (MACC1-positive) SW620 cells restored the activation potential to platelets in the aggregation assay, or in ATP release ( D ). * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The total number of samples analyzed was six: three replicates for cells with high endogenous MACC1 expression after stable gene transfer of control shRNA and three replicates for cells that showed reduced MACC1 gene expression after stable gene transfer of a MACC1-specific shRNA (both shRNAs: OriGene Technologies, Rockville, MD, USA).

Techniques: Activation Assay, shRNA, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Insulin-like Growth Factor Binding Protein-2 (IGFBP2) Is a Key Molecule in the MACC1-Mediated Platelet Communication and Metastasis of Colorectal Cancer Cells

doi: 10.3390/ijms222212195

Figure Lengend Snippet: The impact of IGFBP2 as a functional downstream component of MACC1 on SW620 cell dynamics. ( A,B ) Detection of cell migratory dynamics in a 2D wound healing assay comparing the impact of MACC1 ( A ) and IGFBP2 ( B ) on migration and the effect of platelets to accelerate dynamic properties. ( C ) Analyzing cell invasion in a transmigration assay and the impact of MACC1 knockout or IGFBP2 knockdown. ( D,E ) Analyzing the impact of MACC1 KO or IGFBP2 KD on cell capability to induce coagulation indicates no differences in thrombin formation upon MACC1 KO ( D ) or IGFBP2 KD ( E ) when compared to the respective control cells. ( F ) Flow cytometry data confirm that the knockout of MACC1 in SW620 cells has no impact on tissue factor expression. * p < 0.05; ** p < 0.01, ns = non-significant.

Article Snippet: The total number of samples analyzed was six: three replicates for cells with high endogenous MACC1 expression after stable gene transfer of control shRNA and three replicates for cells that showed reduced MACC1 gene expression after stable gene transfer of a MACC1-specific shRNA (both shRNAs: OriGene Technologies, Rockville, MD, USA).

Techniques: Functional Assay, Wound Healing Assay, Migration, Transmigration Assay, Knock-Out, Coagulation, Flow Cytometry, Expressing

Journal: Journal of neuroinflammation

Article Title: Angiotensin AT 1 and AT 2 receptor heteromer expression in the hemilesioned rat model of Parkinson's disease that increases with levodopa-induced dyskinesia.

doi: 10.1186/s12974-020-01908-z

Figure Lengend Snippet: Fig. 1 Human AT1 and AT2 receptors interact in a heterologous expression system. a–c Immunocytochemistry assays were performed in HEK- 293T cells expressing AT1R-YFP (1 μg cDNA), which was detected by its own yellow fluorescence (green), and AT2R-Rluc (1 μg cDNA), which was detected by a mouse anti-Rluc antibody and a secondary Cy3 anti-mouse antibody (red). Colocalization is shown in yellow. Cell nuclei were stained with Hoechst (blue). Scale bar: 20 μm. d BRET assays were performed in HEK-293T cells transfected with a constant amount of cDNA for AT2R-Rluc (0.9 μg) or σ1R-Rluc (0.75 μg) (as negative control) and increasing amounts of cDNA for AT1R-YFP (0.5 to 4 μg) or AT2R-YFP (0.1 to 4 μg) (as negative control). Values are the mean ± S.E.M. of 8 independent experiments performed in duplicates. e Schematic representation of BRET assay: the occurrence of energy transfer depends on the distance between the BRET donor (Rluc) and the BRET acceptor (YFP)

Article Snippet: After 1-h incubation at 37 °C with the blocking solution in a pre-heated humidity chamber, samples were incubated overnight at 4 °C with a mixture of a mouse monoclonal anti-AT1R antibody (1/100, sc-515884, Santa Cruz Biotechnology, Texas, USA), a rabbit monoclonal antiAT2R antibody (1/100, ab92445, Abcam, Cambridge, UK), and Hoechst (1/100 from stock 1 mg/mL; SigmaAldrich) to stain the nuclei.

Techniques: Expressing, Immunocytochemistry, Fluorescence, Staining, Transfection, Negative Control, Bioluminescence Resonance Energy Transfer

Journal: Journal of neuroinflammation

Article Title: Angiotensin AT 1 and AT 2 receptor heteromer expression in the hemilesioned rat model of Parkinson's disease that increases with levodopa-induced dyskinesia.

doi: 10.1186/s12974-020-01908-z

Figure Lengend Snippet: Fig. 2 Functional characterization in HEK-293T cells expressing the AT1R-AT2R heteromer. HEK-293T cells were pretreated with selective receptor antagonists (300 nM candesartan for AT1R or 1 μM PD123319 for AT2R) and subsequently treated with selective agonists (100 nM angiotensin II for AT1R and 300 nM CGP-42112A for AT2R) a–c Cytosolic calcium detection assay were performed in HEK-293T cells transfected with the cDNAs for an engineered calcium sensor, 6GCaMP (1 μg), AT1R (1 μg), and/or AT2R (1 μg). Values are the mean ± S.E.M. of 5 independent experiments performed in duplicates. d–f Intracellular cAMP levels were determined by TR-FRET as described in Methods. HEK-293T cells were transfected with cDNAs for AT1R (1 μg) and/or AT2R (1 μg). When Gi coupling was assessed, decreases in [cAMP] were determined using 0.5 μM forskolin added 15 min after the agonists stimulation. Values are the mean ± S.E.M. of 6 independent experiments performed in triplicates. In cAMP one-way ANOVA followed by Bonferroni’s multiple comparison post-hoc test were used for statistical analysis. Signaling output was the dependent variable and the different treatments were the independent variables. (*p < 0.05, **p < 0.01, ***p < 0.001 versus forskolin treatment; +++p < 0.001 versus Ang II treatment; &&&p < 0.001 versus CGP-42112A treatment)

Article Snippet: After 1-h incubation at 37 °C with the blocking solution in a pre-heated humidity chamber, samples were incubated overnight at 4 °C with a mixture of a mouse monoclonal anti-AT1R antibody (1/100, sc-515884, Santa Cruz Biotechnology, Texas, USA), a rabbit monoclonal antiAT2R antibody (1/100, ab92445, Abcam, Cambridge, UK), and Hoechst (1/100 from stock 1 mg/mL; SigmaAldrich) to stain the nuclei.

Techniques: Functional Assay, Expressing, Detection Assay, Transfection, Comparison

Journal: Journal of neuroinflammation

Article Title: Angiotensin AT 1 and AT 2 receptor heteromer expression in the hemilesioned rat model of Parkinson's disease that increases with levodopa-induced dyskinesia.

doi: 10.1186/s12974-020-01908-z

Figure Lengend Snippet: Fig. 4 AT1R-AT2R heteromer functionality in primary cultures of striatal neurons. For cAMP (a) or ERK1/2 phosphorylation (b), cells were pretreated (15 min) with selective receptor antagonists (300 nM candesartan for AT1R or 1 μM PD123319 for AT2R) and subsequently treated with selective agonists (100 nM angiotensin II for AT1R and/or 300 nM CGP-42112A for AT2R). Values are the mean ± S.E.M. of 5 independent experiments performed in triplicates. One-way ANOVA followed by Bonferroni’s multiple comparison post-hoc test were used for statistical analysis. Signaling output was the dependent variable and the different treatments were the independent variables. (&p < 0.05 versus CGP-42112A treatment; *p < 0.05, **p < 0.01, ***p < 0.001 versus forskolin treatment in cAMP determinations or versus vehicle treatment (basal) in pERK determinations)

Article Snippet: After 1-h incubation at 37 °C with the blocking solution in a pre-heated humidity chamber, samples were incubated overnight at 4 °C with a mixture of a mouse monoclonal anti-AT1R antibody (1/100, sc-515884, Santa Cruz Biotechnology, Texas, USA), a rabbit monoclonal antiAT2R antibody (1/100, ab92445, Abcam, Cambridge, UK), and Hoechst (1/100 from stock 1 mg/mL; SigmaAldrich) to stain the nuclei.

Techniques: Phospho-proteomics, Comparison

Journal: Journal of neuroinflammation

Article Title: Angiotensin AT 1 and AT 2 receptor heteromer expression in the hemilesioned rat model of Parkinson's disease that increases with levodopa-induced dyskinesia.

doi: 10.1186/s12974-020-01908-z

Figure Lengend Snippet: Fig. 3 Functional characterization of AT1R-AT2R heteromer in HEK-293T cells. HEK-293T cells were transfected with cDNAs for AT1R (1 μg) and/or AT2R (1 μg). Cells were pretreated (15 min) with selective receptor antagonists (300 nM candesartan for AT1R or 1 μM PD123319 for AT2R receptors) and subsequently treated with selective agonists (100 nM angiotensin II for AT1R and 300 nM CGP-42112A for AT2R receptors). a–c ERK1/2 phosphorylation was analyzed using an AlphaScreen®SureFire® kit (Perkin Elmer). Values are the mean ± S.E.M. of 5 independent experiments performed in duplicates. One-way ANOVA followed by Bonferroni’s multiple comparison post-hoc test were used for statistical analysis. Signaling output was the dependent variable and the different treatments were the independent variables (*p < 0.05, **p < 0.01, ***p < 0.001; versus vehicle treatment (basal)). d–f DMR tracings represent the picometer-shifts of reflected light wavelength over time. Values are the mean ± S.E.M. 8 independent experiments performed in triplicates

Article Snippet: After 1-h incubation at 37 °C with the blocking solution in a pre-heated humidity chamber, samples were incubated overnight at 4 °C with a mixture of a mouse monoclonal anti-AT1R antibody (1/100, sc-515884, Santa Cruz Biotechnology, Texas, USA), a rabbit monoclonal antiAT2R antibody (1/100, ab92445, Abcam, Cambridge, UK), and Hoechst (1/100 from stock 1 mg/mL; SigmaAldrich) to stain the nuclei.

Techniques: Functional Assay, Transfection, Phospho-proteomics, Amplified Luminescent Proximity Homogenous Assay, Comparison

Journal: Journal of neuroinflammation

Article Title: Angiotensin AT 1 and AT 2 receptor heteromer expression in the hemilesioned rat model of Parkinson's disease that increases with levodopa-induced dyskinesia.

doi: 10.1186/s12974-020-01908-z

Figure Lengend Snippet: Fig. 5 AT1R-AT2R heteromer functionality in microglial primary cultures treated with LPS and IFN-γ. a–c Expression of AT1R/AT2R heteromers in primary microglial cultures were determined by PLA, which was performed using specific primary antibodies against AT1 and AT2 receptors (confocal microscopy images (stacks of 3 consecutive planes) show heteroreceptor complexes as red clusters and Hoechst-stained nuclei (blue)). Scale bar: 20 μm. d Bar graph showing the percentage of red dots/cell respect non-treated cells; mean ± S.E.M of counts in 5–7 different fields (n = 5; **p < 0.01; Student’s t test versus the control condition). e, f Microglial cultures were incubated for 48 h in the absence (left) or in the presence (right) of 1 μM LPS and 200 U/mL IFN-γ. Microglial cells were pretreated (15 min) with selective receptor antagonists (300 nM candesartan for AT1R or 1 μM PD123319 for AT2R receptors) and subsequently with the specific agonists (100 nM angiotensin II for AT1R and 300 nM CGP-42112A for AT2R receptors). cAMP (e-f) and ERK1/2 phosphorylation (g-h) were subsequently measured. Values are the mean ± S.E.M. of 5 independent experiments performed in triplicates. One-way ANOVA followed by Bonferroni’s multiple comparison post-hoc test were used for statistical analysis. Signaling output was the dependent variable and the different treatments were the independent variables. (+p < 0.05 versus Ang II treatment in pERK determinations; and *p < 0.05, **p < 0.01, ***p < 0.001; versus forskolin treatment in cAMP measurements or versus vehicle treatment (basal) in pERK measurements)

Article Snippet: After 1-h incubation at 37 °C with the blocking solution in a pre-heated humidity chamber, samples were incubated overnight at 4 °C with a mixture of a mouse monoclonal anti-AT1R antibody (1/100, sc-515884, Santa Cruz Biotechnology, Texas, USA), a rabbit monoclonal antiAT2R antibody (1/100, ab92445, Abcam, Cambridge, UK), and Hoechst (1/100 from stock 1 mg/mL; SigmaAldrich) to stain the nuclei.

Techniques: Expressing, Confocal Microscopy, Staining, Control, Incubation, Phospho-proteomics, Comparison

Journal: Journal of neuroinflammation

Article Title: Angiotensin AT 1 and AT 2 receptor heteromer expression in the hemilesioned rat model of Parkinson's disease that increases with levodopa-induced dyskinesia.

doi: 10.1186/s12974-020-01908-z

Figure Lengend Snippet: Fig. 6 AT1R-AT2R heteromer expression in brain striatal sections of Parkinson’s disease (PD) rat model. a–d PLA assays in striatal sections from the 6-OH-dopamine PD rat model, non-lesioned (a), lesioned (b), and lesioned plus chronically treated with L-DOPA and either lacking (c) or displaying (d) dyskinesias. Confocal microscopy images (stacks of 3 consecutive planes) show heteroreceptor complexes as red clusters and Hoechst-stained nuclei (blue). Scale bar: 20 μm. e Bar graph showing the percentage of red dots/cell. Data are the mean S.E.M . of counts in 9–12 different fields per animal (n = 4 per group). One-way ANOVA followed by Bonferroni’s post-hoc multiple comparison tests were used to compare the red dots/cell values. The number of clusters (r) was the dependent variable and the four animal groups treatments were independent variables (***p < 0.001; versus lesioned condition, ++p < 0.01; versus L-DOPA non-dyskinesia condition)

Article Snippet: After 1-h incubation at 37 °C with the blocking solution in a pre-heated humidity chamber, samples were incubated overnight at 4 °C with a mixture of a mouse monoclonal anti-AT1R antibody (1/100, sc-515884, Santa Cruz Biotechnology, Texas, USA), a rabbit monoclonal antiAT2R antibody (1/100, ab92445, Abcam, Cambridge, UK), and Hoechst (1/100 from stock 1 mg/mL; SigmaAldrich) to stain the nuclei.

Techniques: Expressing, Confocal Microscopy, Staining, Comparison