Journal: Journal of Extracellular Vesicles

Article Title: CD73 + extracellular vesicles inhibit angiogenesis through adenosine A 2B receptor signalling

doi: 10.1080/20013078.2020.1757900

Figure Lengend Snippet: TIMP-1 carried by pEVs affects tube formation, but not endothelial migration in vitro . (a) Western Blot and relative quantification showing TIMP-1 overexpression in pEVs. Unpaired T test; * P < 0.05. (b) Analysis of the relative tube lengths in the tube formation assay. The treatment of SVEC4-10 with the anti-TIMP-1 5 µg/mL (AF980 R&D) rescued the tube formation. (c) Conversely, the analysis of relative migration in the scratch assay in the presence of the same anti-TIMP-1 antibody did not shown any restoration of the endothelial migration ability in the presence of pEVs. Data are expressed as means ± SEM (3 independent experiments). Kruskal–Wallis test with Dunn’s multiple comparisons test; * P < 0.05.

Article Snippet: The treatment of SVEC4-10 with the anti-TIMP-1 5 µg/mL (AF980 R&D) rescued the tube formation. (c) Conversely, the analysis of relative migration in the scratch assay in the presence of the same anti-TIMP-1 antibody did not shown any restoration of the endothelial migration ability in the presence of pEVs.

Techniques: Migration, In Vitro, Western Blot, Over Expression, Tube Formation Assay, Wound Healing Assay

Journal: bioRxiv

Article Title: Circuit to target approach defines an autocrine myofibroblast loop that drives cardiac fibrosis

doi: 10.1101/2023.01.01.522422

Figure Lengend Snippet: (A) Phase plot where the axes represent the amount of cells. The separatrix (dashed lines) separates the basins of attraction of healing (gray area) and cold fibrosis (white). The separatrix is calculated with reference (WT) parameters (black), and with a 30% decrease in the production rates of the macrophage paracrine growth factor (green), myofibroblast paracrine growth factor (blue), and myofibroblast autocrine growth factor (red). (B) Simulations of the cell circuit response (red lines) to acute injury that leads to cold fibrosis with WT parameters (left panel). Healing is seen with a 42% or larger decrease in the myofibroblast autocrine growth factor production rate. (C) Schematic representation of NicheNet analysis to identify ligand-receptor interactions between fibroblasts (F), macrophages (M) and myofibroblasts (mF) at day 3 and 28 following MI. NicheNet performed on the scRNAseq data of . S1A-B. (D) Heatmap of potential ligands based on F, M and mF differentially expressed (DE) genes at days 3 (right) and 28 (left) post-MI. Ligand scores (Pearson correlation) and average ligand expression per cell type are presented as mean± SD (by either white-orange scale or blue-red scale, respectively). (E) Pie charts presenting the % of total interactions as sender per cell type and time point post-MI (day 3 or 28). Presented ligands are predicted by NicheNet analysis. (F) Weighted interactions (where interaction strength is normalized to the total number of sender and receiver cells) between F, M and mF following-MI (day 3 and 28) (G) Representative immunofluorescence images of TIMP1 (green), LRP1 (red), alpha smooth muscle actin (ɑSMA, grey) and DAPI (blue) in primary adult cardiac myofibroblasts cultures. (n = 5 biological replicates, 2510±515 SEM cells per replicate). Scale bars = 50μm, white frame represent the inset on the right. Quantification of images (H) based on their TIMP1, LRP1 and ɑSMA protein expression (as % of total cells) presented as mean± SEM. (I) Top panels- Timp1 and Lrp1 spatio-temporal distribution after MI. Positive spots (either Timp1 , Lrp1 or both) were defined as spots with expression, of each gene, higher than the median expression over all slices. Middle panels- zone distribution. Bottom panels- quantification of Timp1 - Lrp1 spot distribution, divided per zone. % of spots (based on Timp1 - Lrp1 expression) per zone are presented in a percentile graph. (J) Pig TIMP1 normalized mRNA expression at day 28 post-MI, in infarct and remote zones of rhAgrin (blue) and saline (red) treated hearts. Circles denote biological replicates. (K) Schematic of flow cytometry experiment for EdU incorporation and representative images of primary cardiac myofibroblasts cultures (day 4 of culture). Fraction of myofibroblasts at day 4 (n=4 relicates, mean cells=4253±749 SEM). Scale bar = 50μm. (L) Cardiac myofibroblast proliferation was assessed in cultures by means of delta EdU + (%) cells of control (either PBS or IgG) and 48hrs treated cells (either rmTIMP1 or ɑTIMP1, respectively). In rmTIMP1 and ɑTIMP1 experiments n = 4 and 5 biological replicates, respectively). Data is represented as mean± SEM. Avg = Average. (M) Schematic experimental plan by which adult mice underwent MI and treated with either TIMP1 neutralizing antibodies (ɑTIMP1, n = 12 biological replicates) or IgG control (n = 11 biological replicates) immediately after injury onset and 3 days after injury. At 35 days left ventricular (LV) fibrosis was assessed by picro-sirius red staining. (N) Representative sequential picro-sirius red staining images along the the base to apex axis of ɑTIMP1/IgG control treated hearts. Scale bars: 2 mm. (O) Fibrosis parameters presented as % of total sections that are either transmural, non-transmural or sections with no scar (methods), and scar area out of the LV quantification.

Article Snippet: For primary cardiac myofibroblasts proliferation assay, cells were incubation with 10uM EdU (5-ethynyl-2’ -deoxyuridine) (Invitrogen, C10424) for 48hours together with either recombinant-mouse TIMP1 protein (1µg/ml, ab206786, Abcam), PBS, anti-TIMP1 (1ug/ml, AF980, R&D) antibodies or IgG control (1µg/ml, AB-108-C, R&D).

Techniques: Expressing, Immunofluorescence, Flow Cytometry, Staining

Journal: bioRxiv

Article Title: Circuit to target approach defines an autocrine myofibroblast loop that drives cardiac fibrosis

doi: 10.1101/2023.01.01.522422

Figure Lengend Snippet: (A) Heatmap of potential receptors based on cardiac fibroblast (F), macrophage (M) and myofibroblast (mF) on days 3 (left) and 28 (right) post-MI. This data is complementary to . Receptor interaction score and average receptor expression per cell type are presented as mean± SD (by either white-orange scale or blue-red scale, respectively). (B) single-cell mRNA expression of Timp1 by cardiac interstitial cells shows dominant expression in cardiac myofibroblasts. Scale denotes normalized mRNA expression. Dataset used is described in . S1A-B (C) Timp1 mRNA expression dynamics after MI quantified between day 3 and day 28 in cardiac myofibroblasts cluster. (D) Timp1 mRNA expression in the publicly available bulk mRNA-sequencing ((Z. ). P1 (blue) and P8 (red) mice underwent Sham (squares) or MI (circles) injury by LAD ligation and ventricles were sampled on days 1.5, 3 and 7 post-MI for sequencing. Data is shown as normalized mRNA expression. (n = 3 per time-point, per age/ treatment). Statistical analysis was performed using DESeq2 (methods), Raw P-values were adjusted for multiple testing using the procedure of Benjamini and Hochberg. (E) single-cell mRNA expression of Lrp1 by cardiac interstitial cells shows dominant expression in cardiac myofibroblasts. (F) Lrp1 mRNA expression dynamics after MI quantified between day 3 and day 28 in cardiac myofibroblasts cluster. (G) Lrp1 mRNA expression in P1 and P8 sham and MI operated mice. (H) The dynamic of cardiac myofibroblasts proliferation was assessed using the scRNAseq data described in . S1A-B (methods). ES- effect size; FC- fold change.

Article Snippet: For primary cardiac myofibroblasts proliferation assay, cells were incubation with 10uM EdU (5-ethynyl-2’ -deoxyuridine) (Invitrogen, C10424) for 48hours together with either recombinant-mouse TIMP1 protein (1µg/ml, ab206786, Abcam), PBS, anti-TIMP1 (1ug/ml, AF980, R&D) antibodies or IgG control (1µg/ml, AB-108-C, R&D).

Techniques: Expressing, Sequencing, Ligation