Journal: Frontiers in Endocrinology

Article Title: Thrombospondin 1 (THBS1) Promotes Follicular Angiogenesis, Luteinization, and Ovulation in Primates

doi: 10.3389/fendo.2019.00727

Figure Lengend Snippet: Thrombospondin production by the ovulatory follicle. Granulosa cells were aspirated from monkey ovarian follicles after ovarian stimulation before (0), 12, 24, or 36 h after administration of an ovulatory dose of hCG; additional monkeys received hCG and the PTGS2 inhibitor celecoxib (36+C) 36 h before follicle aspiration. Granulosa cells were assessed by qPCR for mRNA levels of THBS1 (A) , THBS2 (B) , and THBS4 (C) . All THBS mRNA levels are expressed relative to BACT . Granulosa cell lysates were assessed for THBS1 (D,E) and THBS4 (D,F) by western blotting and are expressed relative to pan-actin (D) . For (A–C,E,F) , data are expressed as mean + SEM, n = 3–7 samples/treatment. Within each panel, groups with no common letters are different by ANOVA and Duncans post hoc test, p < 0.05.

Article Snippet: On the next day, intrafollicular injection of an antibody against THBS1 (R&D Systems, Minneapolis, MN; AF3074; n = 4) or control IgG antibody (Abbiotec, San Diego, CA; n = 4) was performed during aseptic surgery; an estimated 10 μg of antibody protein was delivered to each follicle at injection ( ).

Techniques: Western Blot

Journal: Frontiers in Endocrinology

Article Title: Thrombospondin 1 (THBS1) Promotes Follicular Angiogenesis, Luteinization, and Ovulation in Primates

doi: 10.3389/fendo.2019.00727

Figure Lengend Snippet: THBS1 and THBS4 immunodetection in monkey ovarian follicles. Immunocytochemical detection (brown) of THBS1 (A–G) and THBS4 (H–N) was localized to the granulosa cell layer of ovulatory follicles obtained before (0; A,H) and 12 (B,I) , 24 (C,J) , and 36 (D,K) hours (h) after hCG administration as well as 36 h after administration of hCG and celecoxib (36+C h; E,L ). Images shown are representative of n-3-6 ovaries/treatment. Granulosa cell immunodetection of THBS1 (F) and THBS4 (M) was reduced after preabsorption of the primary antibody with recombinant human THBS1 or THBS4 and similar to staining observed with no primary antibody (G,N) . Nuclei are counterstained blue. All panels are oriented as shown in (A) , with stroma (st) in lower left, granulosa cell (gc, arrow) layer central, and follicle antrum (an) in upper right. Arrowheads indicate stromal staining with antibody against THBS1 (A,E) , THBS4 (J) , and no primary antibody (G,N) . Images in (A–N) are at the same magnification; bar in (A) is 50 μm. Lower magnification images shows immunocytochemical detection of THBS1 (O) and no primary antibody (P) stained sections of an ovary obtained after ovarian stimulation and 36 h hCG. Staining is apparent in stromal vessels ( O , arrows). Non-specific staining is indicated near the granulosa cell basement membrane and in vessel lumens in (O,P) (arrowheads); bar in (A) is 100 μm for images in (O,P) .

Article Snippet: On the next day, intrafollicular injection of an antibody against THBS1 (R&D Systems, Minneapolis, MN; AF3074; n = 4) or control IgG antibody (Abbiotec, San Diego, CA; n = 4) was performed during aseptic surgery; an estimated 10 μg of antibody protein was delivered to each follicle at injection ( ).

Techniques: Immunodetection, Recombinant, Staining, Membrane

Journal: Frontiers in Endocrinology

Article Title: Thrombospondin 1 (THBS1) Promotes Follicular Angiogenesis, Luteinization, and Ovulation in Primates

doi: 10.3389/fendo.2019.00727

Figure Lengend Snippet: THBS1 is pro-angiogenic in vitro . Monkey ovarian microvascular endothelial cells (mOMECs) were treated with human THBS1 protein at concentrations of 0.001–1 nM or no THBS1 (0 nM) as indicated. (A–C) Migration was assessed 24 h after plating on a porous membrane and treatment with THBS1. mOMECs (arrow) which migrated through pores (arrowhead) were stained with hematoxylin and eosin, photographed, and counted. Representative membranes from 0 nM (B) and 1 nM (C) treatment groups are shown. (D–F) Proliferation was assessed by Ki67 immunodetection in mOMECs cultured for 24 h with THBS1. Ki67 positive (arrows) and negative (arrowhead) cells are indicated in representative images from mOMECs treated with 0 nM (E) and 1 nM (F) THBS1. Data are expressed as a percentage of Ki67 positive cells among all cells counted. (G–M) Sprout formation in response to THBS1 treatment was determined after 1 day (G,I) and 2 days (H,J,L,M) in vitro . mOMECs coating a polymer bead in vitro before THBS1 treatment (Day 0; K ) shows absence of sprouts. Arrows indicate representative sprouts on Day 2 of treatment with no THBS1 (L) and 0.1 nM THBS1 (M) . Images were quantified for both the number of sprouts (sprouts/bead; G,H ) and sprout length in μm (H,J) . For (A,D,G–J) , data are expressed as mean + SEM, n = 3–5 samples/treatment. Within each panel, groups with no common letters are different by ANOVA and Duncans post hoc test, p < 0.05.

Article Snippet: On the next day, intrafollicular injection of an antibody against THBS1 (R&D Systems, Minneapolis, MN; AF3074; n = 4) or control IgG antibody (Abbiotec, San Diego, CA; n = 4) was performed during aseptic surgery; an estimated 10 μg of antibody protein was delivered to each follicle at injection ( ).

Techniques: In Vitro, Migration, Membrane, Staining, Immunodetection, Cell Culture, Polymer

Journal: Frontiers in Endocrinology

Article Title: Thrombospondin 1 (THBS1) Promotes Follicular Angiogenesis, Luteinization, and Ovulation in Primates

doi: 10.3389/fendo.2019.00727

Figure Lengend Snippet: Confirmatory experiments for the intrafollicular antibody injections. Serum levels of estradiol (A) and progesterone (B) were not different between animals receiving intrafollicular injection of the THBS1 antibody and animals receiving control IgG. For each day, hormone levels were compared by unpaired t -test. Data are expressed as mean + SEM, n = 4/group. (C) THBS1 antibody reduces THBS1 (1 nM)-stimulated mOMEC migration in vitro . Within each panel, groups with no common letters are different by ANOVA with 1 repeated measure and Duncans post hoc test, p < 0.05. Data are expressed as mean + SEM, n = 3/group.

Article Snippet: On the next day, intrafollicular injection of an antibody against THBS1 (R&D Systems, Minneapolis, MN; AF3074; n = 4) or control IgG antibody (Abbiotec, San Diego, CA; n = 4) was performed during aseptic surgery; an estimated 10 μg of antibody protein was delivered to each follicle at injection ( ).

Techniques: Injection, Control, Migration, In Vitro

Journal: Frontiers in Endocrinology

Article Title: Thrombospondin 1 (THBS1) Promotes Follicular Angiogenesis, Luteinization, and Ovulation in Primates

doi: 10.3389/fendo.2019.00727

Figure Lengend Snippet: Follicle rupture and oocyte release are compromised after intrafollicular injection with an antibody against THBS1. Ovarian surface at the time of ovary removal after intrafollicular injection of control IgG (A) or THBS1 antibody (B–D) . Oocytes were located within a THBS1 antibody-injected follicle (E) and on the surface of a THBS1 antibody-injected follicle (F) . Rupture site after intrafollicular injection of control IgG (G) . Rupture sites in follicles injected with the THBS1 antibody were either small (H) or absent (I) . Tissues shown in (E–I) were stained with hematoxylin and eosin. Oocytes (E,F) shown at approximately maximal diameter. Images in (G–I) are at the same magnification; bar in (I) = 0.5 mm. (J) Rupture site area after intrafollicular injection with control IgG (IgG) or THBS1 antibody. Groups are different by two-tailed unpaired t -test as indicated by * p < 0.05. Data are expressed as mean + SEM, n = 4/group.

Article Snippet: On the next day, intrafollicular injection of an antibody against THBS1 (R&D Systems, Minneapolis, MN; AF3074; n = 4) or control IgG antibody (Abbiotec, San Diego, CA; n = 4) was performed during aseptic surgery; an estimated 10 μg of antibody protein was delivered to each follicle at injection ( ).

Techniques: Injection, Control, Staining, Two Tailed Test

Journal: Frontiers in Endocrinology

Article Title: Thrombospondin 1 (THBS1) Promotes Follicular Angiogenesis, Luteinization, and Ovulation in Primates

doi: 10.3389/fendo.2019.00727

Figure Lengend Snippet: Oocyte retention and follicle rupture.

Article Snippet: On the next day, intrafollicular injection of an antibody against THBS1 (R&D Systems, Minneapolis, MN; AF3074; n = 4) or control IgG antibody (Abbiotec, San Diego, CA; n = 4) was performed during aseptic surgery; an estimated 10 μg of antibody protein was delivered to each follicle at injection ( ).

Techniques:

Journal: Frontiers in Endocrinology

Article Title: Thrombospondin 1 (THBS1) Promotes Follicular Angiogenesis, Luteinization, and Ovulation in Primates

doi: 10.3389/fendo.2019.00727

Figure Lengend Snippet: Angiogenesis and luteinization are compromised after intrafollicular injection with an antibody against THBS1. Histological sections of ovaries shown in were immunostained for VWF (brown) to assess angiogenesis; hematoxylin counterstain. (A,B) Endothelial cell invasion into the granulosa cell layer of a control IgG-injected follicle (A) and THBS1 antibody-injected follicle (B) shows VWF+ cells within the granulosa cell (gc) layer. Arrows indicate VWF+ cells which have invaded furthest from the stroma into the granulosa cell layer. Single arrowheads indicate stromal vessels. Double arrowheads indicate capillary luminal spaces with red blood cells in the control-IgG injected follicle only. (C) shows representative measurements of granulosa cell layer thickness (brown lines) and endothelial cell invasion (green lines). (A–C) are oriented as shown in (A) , with stroma (st) at bottom, granulosa cell (gc) layer central, and follicle antrum (an) at top. Images in (A–C) are at the same magnification; bar in (B) is 100 μm. Granulosa cell layer thickness (D) , endothelial cell invasion (E) , and the percent of the granulosa cell layer penetrated by endothelial cells (F) after intrafollicular injection with control IgG (IgG) or THBS1 antibody. Groups are different by two-tailed unpaired t -test as indicated by * p < 0.05. Data are expressed as mean + SEM, n = 4/group. (H,J) 3D modeling of endothelial cells (white on black background) is shown alongside (G,I) representative VWF immunostained ovarian sections after intrafollicular injection with control IgG (G,H) or THBS1 antibody (I,J) . Green arrows indicate stromal vessels, green arrowheads indicate capillary-like structures that connect to a stromal vessel, and yellow arrowheads indicate endothelial cells that lack connect to a stromal vessel. (G–J) are oriented with antrum at the top, granulosa cells central, and stroma at the bottom of each image/model; (G,I) at the same magnification.

Article Snippet: On the next day, intrafollicular injection of an antibody against THBS1 (R&D Systems, Minneapolis, MN; AF3074; n = 4) or control IgG antibody (Abbiotec, San Diego, CA; n = 4) was performed during aseptic surgery; an estimated 10 μg of antibody protein was delivered to each follicle at injection ( ).

Techniques: Injection, Control, Two Tailed Test

Journal: eLife

Article Title: Paracrine signalling between intestinal epithelial and tumour cells induces a regenerative programme

doi: 10.7554/eLife.76541

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-THBS1 (mouse monoclonal) , Novus Biologicals , 2059SS , (1:100).

Techniques: Transduction, Control, Derivative Assay, Cell Recovery, Plasmid Preparation, Multiplex sample analysis, RNAscope, Multiplex Assay, Recombinant, Sequencing, Software, Transfection, Construct

Journal: Methods and Protocols

Article Title: Efficient Ex Vivo Screening of Agents Targeting Thrombospondin1-Induced Vascular Dysfunction Using a Digital Multiwire Myograph System

doi: 10.3390/mps4040074

Figure Lengend Snippet: Native thrombospondin 1 (TSP1) from different sources inhibited endotheliumdependent vasorelaxation with different efficacies. ( A ) Representative force vs. time traces recordings of isolated murine thoracic aorta responses to phenylephrine followed acetylcholine stimulations in the presence of Pierce TM —TSP1 or vehicle control, i.e., Krebs buffer (PE: phenylephrine; Ach: acetylcholine; the numbers marked in the recording are the logarithm value of concentrations of the stimulants). Concentration response curves of isolated mouse thoracic aorta exposed to Pierce TM —TSP1 ( B ) or Athens—TSP1 ( C ) at 2.2, 4.4, or 6.6 nM. Pierce TM —TSP1 induced impairment of vasodilation in a slightly different pattern than Athens—TSP1 (*, p < 0.05, TSP1 vs. control; ***, p < 0.0001, TSP1 vs. control). ( D ) The same concentration TSP1, e.g., 4.4 nM, from three sources inhibited vasorelaxation, two of which produced greater suppression of vasorelaxation induced by acetylcholine 1 × 10 −7 and 3 × 10 −7 M (*, p < 0.05, Athens—TSP1 vs. control; ***, p < 0.0001, Pierce TM —TSP1 or Novus—TSP1 vs. control). ( E ) An equivalent inhibition of vasorelaxation was reached when Novus—TSP1 or Athens—TSP1 at 2.2 nM and Pierce TM —TSP1 at 4.4 nM was applied (***, p < 0.0001, TSP1 vs. control).

Article Snippet: Therefore, it was essential to examine the bioactivity of native human TSP1 available from different commercial vendors (Thermo Scientific, Novus Biologicals, and Athens Research & Technology), specifically in terms of modulation of vasoactivity, in order to determine which TSP1 protein products can be used for the candidate drug screening assays afterwards.

Techniques: Isolation, Control, Concentration Assay, Produced, Inhibition

Journal: Methods and Protocols

Article Title: Efficient Ex Vivo Screening of Agents Targeting Thrombospondin1-Induced Vascular Dysfunction Using a Digital Multiwire Myograph System

doi: 10.3390/mps4040074

Figure Lengend Snippet: Thrombospondin 1 (TSP1)−impaired endothelium-dependent vasorelaxation was prevented by treatment with the proposed drug candidate, but not by blocking signal-regulatory protein α (SIRP α). Representative force vs. time traces recording from endothelium intactness test to acetylcholine-induced vasorelaxation by isolated murine thoracic aorta pre-treated with the proposed drug candidate 6.6 nM ( A ), CD47 antibody (CD47 ab) 1 µg/mL ( B ), or SIRPα antibody (SIRPα ab) 2 µg/mL ( C ) in the presence of Pierce TM —TSP1 4.4 nM (PE: phenylephrine; Ach: acetylcholine; the numbers marked in the recording are the logarithmic values of stimulant concentrations). Prior to receiving any treatment, the vessel segments were pre-contracted with 1 × 10 −6 M PE followed by 1 × 10 −6 M Ach-stimulated endothelium-dependent vasorelaxation to confirm endothelial intactness. ( D ) Concentration response curves of isolated mouse thoracic aorta exposed to TSP1, in the presence of vehicle, CD47-based drug candidate, or CD47 antibody. ( E ) Concentration response curves of isolated mouse thoracic aorta exposed to TSP1, in presence of vehicle or SIRPα antibody (***, p < 0.0001, TSP1, TSP1 + drug candidate, or TSP1 + SIRPα ab vs. control; ###, p < 0.0001, TSP1 + drug candidate or TSP1 + CD47 antibody vs. TSP1).

Article Snippet: Therefore, it was essential to examine the bioactivity of native human TSP1 available from different commercial vendors (Thermo Scientific, Novus Biologicals, and Athens Research & Technology), specifically in terms of modulation of vasoactivity, in order to determine which TSP1 protein products can be used for the candidate drug screening assays afterwards.

Techniques: Blocking Assay, Isolation, Concentration Assay, Control

Journal: Methods and Protocols

Article Title: Efficient Ex Vivo Screening of Agents Targeting Thrombospondin1-Induced Vascular Dysfunction Using a Digital Multiwire Myograph System

doi: 10.3390/mps4040074

Figure Lengend Snippet: Acute TSP1 challenge-induced reduction in endothelium-dependent vasorelaxation was significantly restored by therapeutic treatment with the proposed CD47-based drug candidate. Representative force vs. time traces recording from endothelium intactness test to acetylcholine-induced vasorelaxation by isolated murine thoracic aorta exposed to acute TSP1 (Pierce TM —TSP1 4.4 nM) challenge for 15 min, followed by treatment with the proposed CD47-based drug candidate 13.2 nM ( A ) or CD47 antibody (CD47 ab) 1 µg/mL ( B ) for 45 min (PE: phenylephrine; Ach: acetylcholine; the numbers marked in the recording are the logarithmic values of stimulant concentrations). Prior to receiving any treatment, the vessel segments were pre-contracted with 1 × 10 −6 M PE followed by 1 × 10 −6 M Ach-stimulated endothelium-dependent vasorelaxation to confirm endothelial intactness. ( C ) Concentration response curves of isolated mouse thoracic aorta exposed to TSP1, in the presence of vehicle, CD47-based drug candidate, or CD47 antibody (***, p < 0.0001, TSP1 or TSP1 + drug candidate vs. control; #, p < 0.05, TSP1 + drug candidate vs. TSP1; ###, p < 0.0001, TSP1 + CD47 antibody vs. TSP1).

Article Snippet: Therefore, it was essential to examine the bioactivity of native human TSP1 available from different commercial vendors (Thermo Scientific, Novus Biologicals, and Athens Research & Technology), specifically in terms of modulation of vasoactivity, in order to determine which TSP1 protein products can be used for the candidate drug screening assays afterwards.

Techniques: Isolation, Concentration Assay, Control