Journal: Biomolecules

Article Title: Phosphomimetic Thrombospondin-1 Modulates Integrin β1-FAK Signaling and Vascular Cell Functions

doi: 10.3390/biom16010084

Figure Lengend Snippet: TSP1 S93D inhibits EC migration but not proliferation. ( A ) Representative images from a wound healing scratch assay using control, wild-type TSP1, and phosphomutant TSP1-transfected BPAECs. Scratches were made in a confluent EC monolayer 24 h post-transfection. Images were captured at 0, 8, and 24 h post-scratch. Scale bar = 100 µm. ( B ) Wound closure was evaluated by measuring the open area at each time point, normalized to the 0 h image (**** p < 0.0001). Statistical analysis was completed using one-way ANOVA followed by Tukey’s post hoc test. Data are presented as mean ± SD ( n = 5). ( C ) Representative measurement of an in vitro wound healing assay performed using ECIS. Wounding was applied at 1 h. Each line represents the mean of three replicates ± SD. ( D ) Statistical analysis of EC migration rate was performed using one-way ANOVA with Tukey’s test (**** p < 0.0001). Data are represented as mean ± S.D ( n = 8). ( E ) BPAEC were either untransfected (ctr) or transfected with TSP1 WT -, TSP1 S93A -, or TSP1 S93D -expressing constructs. Cell proliferation was measured by an MTT assay. Absorbance values were normalized to 0 h. No significant differences in proliferation were observed between groups. Data represent SD ( n = 6).

Article Snippet: TSP1 or IL-6 levels in the cell culture supernatant were quantified using a Human TSP1 ELISA Kit from Elabscience Biotechnology (E-EL-H1589) or a Mouse IL-6 ELISA Kit from Fine Test (EM0121), respectively.

Techniques: Migration, Wound Healing Assay, Control, Transfection, In Vitro, Expressing, Construct, MTT Assay

Journal: Biomolecules

Article Title: Phosphomimetic Thrombospondin-1 Modulates Integrin β1-FAK Signaling and Vascular Cell Functions

doi: 10.3390/biom16010084

Figure Lengend Snippet: TSP1 S93D inhibits FAK signaling and downstream targets during cell migration. ( A ) Control or TSP1-transfected confluent monolayers of BPAECs were scratched 24 h post-transfection. Samples were collected at the indicated time points (0, 4, and 8 h). Overexpression of TSP1 and the levels of signaling protein were analyzed by Western blot. ( B ) Quantitative analysis was performed by densitometry of the Western blot bands. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test ( n = 3–5) (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Article Snippet: TSP1 or IL-6 levels in the cell culture supernatant were quantified using a Human TSP1 ELISA Kit from Elabscience Biotechnology (E-EL-H1589) or a Mouse IL-6 ELISA Kit from Fine Test (EM0121), respectively.

Techniques: Migration, Control, Transfection, Over Expression, Western Blot

Journal: Biomolecules

Article Title: Phosphomimetic Thrombospondin-1 Modulates Integrin β1-FAK Signaling and Vascular Cell Functions

doi: 10.3390/biom16010084

Figure Lengend Snippet: TSP1 S93D shows enhanced binding to ITGB1. ( A ) Bacterially expressed GST and GST-TSP1 1–221 WT, GST-TSP1 1–221 S93A, and GST-TSP1 1–221 S93D recombinant proteins immobilized on glutathione Sepharose beads were incubated with BPAEC lysate for pull-down assays. EC lysates and the eluted proteins were analyzed by Western blot using ITGB1- and TSP1-specific antibodies. ( B ) Quantitative analysis of pull-down samples. Statistical analysis was performed using one-way ANOVA ( n = 4) (** p < 0.01). ( C ) Control and TSP1-transfected BPAECs were subjected to immunoprecipitation using c-myc antibody to purify recombinant TSP1 proteins. Total cell lysates and immunocomplexes were tested for c-myc and ITGB1 by Western blot. ( D ) Quantitative analysis of IP. Statistical analysis was performed using one-way ANOVA ( n = 4) (*** p < 0.001).

Article Snippet: TSP1 or IL-6 levels in the cell culture supernatant were quantified using a Human TSP1 ELISA Kit from Elabscience Biotechnology (E-EL-H1589) or a Mouse IL-6 ELISA Kit from Fine Test (EM0121), respectively.

Techniques: Binding Assay, Recombinant, Incubation, Western Blot, Control, Transfection, Immunoprecipitation

Journal: Biomolecules

Article Title: Phosphomimetic Thrombospondin-1 Modulates Integrin β1-FAK Signaling and Vascular Cell Functions

doi: 10.3390/biom16010084

Figure Lengend Snippet: TSP1 S93D alters ITGB1 clusterization. ( A ) Representative images of control cells and cells expressing TSP1 recombinant variants, immunostained for ITGB1 (green) and nuclei (DAPI, blue). Images were acquired using the Opera Phenix HCS (PerkinElmer, Inc., Shelton, CT, USA. Scale bar: 100 μm. ( B ) Quantitative analysis of ITGB1 clusters was performed using the built-in Harmony software (version 4.8, Perkin Elmer). Statistical analysis was performed using one-way ANOVA. Data are presented as means ± S.D, and >10,000 cells were analyzed per condition. (**** p < 0.0001).

Article Snippet: TSP1 or IL-6 levels in the cell culture supernatant were quantified using a Human TSP1 ELISA Kit from Elabscience Biotechnology (E-EL-H1589) or a Mouse IL-6 ELISA Kit from Fine Test (EM0121), respectively.

Techniques: Control, Expressing, Recombinant, Software

Journal: Biomolecules

Article Title: Phosphomimetic Thrombospondin-1 Modulates Integrin β1-FAK Signaling and Vascular Cell Functions

doi: 10.3390/biom16010084

Figure Lengend Snippet: The S93A mutation of TSP1 increases its secretion via N-glycosylation. ( A ) Cell culture supernatants were analyzed for TSP1 levels using ELISA. Statistical analysis of TSP1 concentration was performed using one-way ANOVA followed by Tukey’s post hoc test ( n = 7). Data are reported as means ± S.D. ( B ) TSP1 glycosylation was assessed by immunoprecipitating c-myc–tagged TSP1 proteins from the supernatant of transfected cells. Total cell lysates, supernatants, and IP complexes were tested for c-myc. IP complexes were further analyzed for glycosylation using lectin and O-GlcNac antibody by Western blot. ( C ) Quantification of glycosylation of immunoprecipitated recombinant TSP1 proteins. O-GlcNAc levels were not significantly different among groups. Data are shown as normalized signal intensity. (** p < 0.01 and **** p < 0.0001).

Article Snippet: TSP1 or IL-6 levels in the cell culture supernatant were quantified using a Human TSP1 ELISA Kit from Elabscience Biotechnology (E-EL-H1589) or a Mouse IL-6 ELISA Kit from Fine Test (EM0121), respectively.

Techniques: Mutagenesis, Glycoproteomics, Cell Culture, Enzyme-linked Immunosorbent Assay, Concentration Assay, Transfection, Western Blot, Immunoprecipitation, Recombinant

Journal: Biomolecules

Article Title: Phosphomimetic Thrombospondin-1 Modulates Integrin β1-FAK Signaling and Vascular Cell Functions

doi: 10.3390/biom16010084

Figure Lengend Snippet: TSP1 S93D enhances SMC migration and proliferation and induces morphological changes. ( A ) MOVAS cells were seeded at 10% confluence and treated with conditioned media from non-transfected (ctr) or TSP1-transfected BPAECs. Proliferation was assessed using MTT, with absorbance measured at 540 nm at 24, 48, and 72 h. Data are shown as means ± SEM ( n = 12). Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test (* p < 0.01). ( B ) Migration of treated MOVAS cells was measured using an ECIS-based wound healing assay. Data presented mean ± S.D. from three chambers per condition. ( C ) Statistical analysis of migration rates was performed using one-way ANOVA with Tukey’s post hoc test ( n = 5; means ± S.D.; **** p < 0.0001). ( D ) Representative images of control and TSP1-treated MOVAS cells analyzed by HCS. Actin filaments were stained with Texas Red phalloidin (red) and nuclei with DAPI. White arrows indicate filopodia of cells. Scale bars: 50 μm. ( E ) Morphological parameters of MOVAS cells were analyzed using Harmony software on the Opera Phenix HCS system. Data are presented as mean ± SD (1000–1600 cells per well, n = 4). Statistical analysis was performed using ANOVA (**** p < 0.0001).

Article Snippet: TSP1 or IL-6 levels in the cell culture supernatant were quantified using a Human TSP1 ELISA Kit from Elabscience Biotechnology (E-EL-H1589) or a Mouse IL-6 ELISA Kit from Fine Test (EM0121), respectively.

Techniques: Migration, Transfection, Wound Healing Assay, Control, Staining, Software

Journal: Biomolecules

Article Title: Phosphomimetic Thrombospondin-1 Modulates Integrin β1-FAK Signaling and Vascular Cell Functions

doi: 10.3390/biom16010084

Figure Lengend Snippet: TSP1 S93D induces a shift toward a synthetic-like state in SMCs and increases IL-6 secretion. ( A ) MOVAS were treated with conditioned media from TSP1 overexpressing ECs. Expression levels of indicated proteins were tested by Western blot. Actin was used as a loading control. ( B ) Densitometric analysis of Western blot signals. Statistical analysis was performed using one-way ANOVA with Tukey’s test ( n = 4). ( C ) Representative images of control and TSP1-treated MOVAS cells analyzed by HCS. Actin filaments were stained with Texas Red phalloidin (red) and nuclei with DAPI. Scale bars: 50 μm. ( D ) Conditioned media treated and untreated MOVAS cell supernatants were analyzed by ELISA for IL-6. Statistical analysis was performed using one-way ANOVA ( n = 9). Data are reported as means ± S.D. (** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Article Snippet: TSP1 or IL-6 levels in the cell culture supernatant were quantified using a Human TSP1 ELISA Kit from Elabscience Biotechnology (E-EL-H1589) or a Mouse IL-6 ELISA Kit from Fine Test (EM0121), respectively.

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

Journal: EMBO Molecular Medicine

Article Title: Endothelial MT 1‐ MMP targeting limits intussusceptive angiogenesis and colitis via TSP1/nitric oxide axis

doi: 10.15252/emmm.201910862

Figure Lengend Snippet: A, B Representative maximum‐intensity projection images of staining for TSP1 (red) and CD31 (gray) in (A) and also for MT1‐MMP (green) in (B) in colon sections obtained from MT1 f/f and MT1 iΔEC mice left untreated (A) or treated with 1% DSS for 3 days (B). Magnified views are shown to the right. Scale bars, 100 μm in main panels and 10 μm in the magnified views. C TSP1 mean fluorescence intensity (MFI) in the perivascular area of large vessels present in the colon mucosa of treated mice, as depicted in (B); n = 4 mice per genotype. Data are shown as mean ± SEM and were tested by t ‐test; * P < 0.05. Data information: Please see Appendix Table S3 for exact P ‐values. Source data are available online for this figure.

Article Snippet: Soluble TSP1 was measured in serum by enzyme‐linked immunosorbent assay (Mouse TSP1 ELISA kit; Cusabio, CSB‐E08765m).

Techniques: Staining, Fluorescence

Journal: EMBO Molecular Medicine

Article Title: Endothelial MT 1‐ MMP targeting limits intussusceptive angiogenesis and colitis via TSP1/nitric oxide axis

doi: 10.15252/emmm.201910862

Figure Lengend Snippet: A Representative maximum‐intensity projection images of HUVEC expressing control or MT1‐MMP siRNA and stained for MT1‐MMP (gray), TSP1 (red), and F‐actin (phalloidin, green); nuclei are stained with Hoechst (blue). Scale bar, 20 μm. B DAF‐FM mean fluorescence intensity (MFI) in HUVEC expressing control or MT1‐MMP siRNA and left untreated or treated with blocking anti‐CD47 or anti‐αv integrin antibodies (1 and 10 μg/ml, respectively, for 24 h) or their corresponding IgG isotype controls (IgG); n = 149–360 cells analyzed per condition in three independent experiments. C DAF‐FM mean fluorescence intensity (MFI) in HUVEC expressing control or MT1‐MMP siRNA and left untreated or treated with 2 nM or 200 μM RGDS or its control peptide RADS; n = 147–385 cells analyzed per condition in three independent experiments. D DAF‐FM mean fluorescence intensity (MFI) in HUVEC expressing control or MT1‐MMP siRNA and left untreated or treated with 2 nM or 20 μM of the RGD cyclic peptide cilengitide; n = 100–360 cells analyzed per condition in three independent experiments. E DAF‐FM mean fluorescence intensity (MFI) in HUVEC expressing control or MT1‐MMP siRNA and left untreated or treated with 2 nM or 200 μM of the nonamer GDGRGDACK or its control peptide GDGRADACK; n = 149–337 cells analyzed per condition in three independent experiments. Data information: Data are shown in all panels as individual cell values and mean ± SEM and were tested by the Kruskal–Wallis test; **** P < 0.0001, #### P < 0.0001. * indicates comparison with Ctrl‐siRNA, and # indicates comparison of the condition with its corresponding control. Please see Appendix Table S3 for exact P ‐values. Source data are available online for this figure.

Article Snippet: Soluble TSP1 was measured in serum by enzyme‐linked immunosorbent assay (Mouse TSP1 ELISA kit; Cusabio, CSB‐E08765m).

Techniques: Expressing, Control, Staining, Fluorescence, Blocking Assay, Comparison

Journal: EMBO Molecular Medicine

Article Title: Endothelial MT 1‐ MMP targeting limits intussusceptive angiogenesis and colitis via TSP1/nitric oxide axis

doi: 10.15252/emmm.201910862

Figure Lengend Snippet: A Western blot of TSP1 expression (developed with an antibody against an epitope nearby the N‐terminus; Lee et al , ) in lysates from siCtrl and siMT1‐silenced HUVEC. MT1‐MMP and tubulin are included as silencing and loading controls, respectively. The arrowhead marks full‐length TSP1 and the asterisk the N‐terminal TSP1 fragment. B Western blot of in vitro digested TSP1 (developed with the same antibody as in A) incubated with increasing amounts of recombinant human MT1‐MMP catalytic domain. rhMT1‐MMP catalytic domain is also included. The arrowhead marks full‐length TSP1 and the asterisk the N‐terminal TSP1 fragment generated by MT1‐MMP cleavage. C In silico model of the membrane‐anchored MT1‐MMP dimer (blue/orange) and TSP1 type 1 repeat domains 2 and 3 (green). Yellow marks the catalytic pocket in the MT1‐MMP protease, and red indicates the two selected cleavage sites in TSP1. D Scheme depicting the TSP1 domain structure with the binding sequences to CD36, CD47, and αvβ3 integrin, as well as the positions of the identified cleavage sites for MT1‐MMP. E DAF‐FM mean fluorescence intensity (MFI) in HUVEC expressing MT1‐MMP siRNA and left untreated or treated with 200 ng of full‐length TSP1 or the E123CaG‐1 fragment; n = 128–184 cells analyzed per condition in two independent experiments. Data are shown as individual cell values and mean ± SEM and were tested by the Kruskal–Wallis test; * P < 0.05, *** P < 0.001. F Representative maximum‐intensity projection images of CD31 staining (green) in whole‐mount colon mucosal plexus of MT1 f/f mice injected intraperitoneally with 1 μg full‐length TSP1 or the E123CaG‐1 fragment at day 0 and day 2 during 1% DSS treatment for 3 days. Scale bar, 40 μm. Arrows, arrowheads, and asterisks indicate duplications, loops, and pillars, respectively; n = 6 and 7 mice per condition in two independent experiments. G Quantification of IA events in mice treated as in (F). Data are shown as mean ± SEM and were tested by t ‐test; * P < 0.05. Data information: Please see Appendix Table S1 for exact P ‐values. Source data are available online for this figure.

Article Snippet: Soluble TSP1 was measured in serum by enzyme‐linked immunosorbent assay (Mouse TSP1 ELISA kit; Cusabio, CSB‐E08765m).

Techniques: Western Blot, Expressing, In Vitro, Incubation, Recombinant, Generated, In Silico, Membrane, Binding Assay, Fluorescence, Staining, Injection

Journal: EMBO Molecular Medicine

Article Title: Endothelial MT 1‐ MMP targeting limits intussusceptive angiogenesis and colitis via TSP1/nitric oxide axis

doi: 10.15252/emmm.201910862

Figure Lengend Snippet: A Representative maximum‐intensity projection images of staining for CD31 (green), MT1‐MMP or TSP1 (red), and Hoechst (blue, nuclei) in colon sections obtained from an IBD patient. Boxed areas are shown in magnified views below with merged images of green and red channels in the middle and single channels to the bottom. Non‐affected and affected mucosa areas from the same patient are shown for comparison. Scale bar, 50 μm (upper panel) and 20 μm (magnified views). V, vessel. B ELISA analysis of serum TSP1 in healthy individuals and patients affected by ulcerative colitis (left) or Crohn's disease (right). n = 12–37 individuals per condition. LA and HA indicate patients with low and high active disease based on clinical score. Data are shown as individual values and mean ± SEM and were tested by one‐way ANOVA with Benjamini and Hochberg post‐test; *** P < 0.001. C Representative maximum‐intensity projection images of CD31 staining (green) in whole‐mount colon mucosal plexus of MT1 f/f mice injected intraperitoneally with 3 mg/kg of the anti‐MT1‐MMP inhibitory antibody LEM‐2/15 or its isotype control at day 0 and day 2 during the 3 days of treatment with 1% DSS. Scale bar, 40 μm. Arrows, arrowheads, and asterisks indicate duplications, loops, and pillars, respectively. D Quantification of IA events in mice treated as in (C); n = 8 mice per condition in two independent experiments. Data are shown as mean ± SEM and were tested by t ‐test; ** P < 0.01. E Representative second‐harmonic generation (SHG) microscopy images of whole‐mount colons from MT1 f/f mice treated as in (C). Scale bar, 40 μm. F Representative maximum‐intensity projection images of CD31 staining (green) in whole‐mount colon mucosal plexus of MT1 f/f mice treated with the TSP1 nonamer GDGRGDACK or its control peptide GDGRADACK by continuous minipump delivery (up to 2.4 mg/mouse/day) for 3 days during 1% DSS treatment. Scale bar, 40 μm. Arrows, arrowheads, and asterisks indicate duplications, loops, and pillars, respectively. Mice/ n independent experiments. G Quantification of IA events in mice treated as in (F); n = 5 and 6 mice per condition in two independent experiments. Data are shown as mean ± SEM and were tested by t ‐test; * P < 0.05. H Representative second‐harmonic generation (SHG) microscopy images of whole‐mount colons from MT1 f/f mice treated as in (F). Scale bar, 40 μm. Data information: Please see Appendix Table S3 for exact P ‐values. Source data are available online for this figure.

Article Snippet: Soluble TSP1 was measured in serum by enzyme‐linked immunosorbent assay (Mouse TSP1 ELISA kit; Cusabio, CSB‐E08765m).

Techniques: Staining, Comparison, Enzyme-linked Immunosorbent Assay, Injection, Control, Microscopy

Journal: EMBO Molecular Medicine

Article Title: Endothelial MT 1‐ MMP targeting limits intussusceptive angiogenesis and colitis via TSP1/nitric oxide axis

doi: 10.15252/emmm.201910862

Figure Lengend Snippet: The scheme depicts the role that endothelial cell MT1‐MMP (left) exerts in IA during colitis by processing TSP1 and binding of the generated TSP1 C‐terminal fragment to CD47/αvβ3 integrin, thus inducing nitric oxide production. This signaling cascade ultimately leads to arteriole vasodilation and endothelial cell intussusceptive remodeling in the capillary plexus of the inflamed intestinal mucosa. When MT1‐MMP is absent from endothelial cells (right), this pathway is impaired, IA reduced, and colitis ameliorated. EC, endothelial cell; P, pericyte; FB, fibroblast; Col, collagen.

Article Snippet: Soluble TSP1 was measured in serum by enzyme‐linked immunosorbent assay (Mouse TSP1 ELISA kit; Cusabio, CSB‐E08765m).

Techniques: Binding Assay, Generated