CXCL6 is an important paracrine factor in the pro-angiogenic human cardiac progenitor-like cell secretome

Figures

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  Figure 1. Proteomic analysis of the CPC secretome. (a) CPC secretome inferred from whole label-free (LF) analysis. Distribution of the CPC secretome (465 proteins) by subcellular localization (top) and by enzymatic activity (bottom). (b) Most relevant cytokines (left) and growth factors (right) in the CPC secretome as inferred from whole LF analysis. (c) Comparison of whole LF proteomics secretome and secretome obtained from CPC-conditioned medium (CM); only 19% (104; blue) of the proteins described are common. (d) Distribution of the CPC CM secretome by subcellular localization. (e) The majority of the CPC secretome (>50%) is included in the Exocarta database, indicating association with the exosome compartment.

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  Figure 2. Comparative validation of the CPC secretome. (a) Conditioned medium from MSC, CPC and HDF were analyzed using an antibody array (122 cytokines). A color-scale of expression levels is included; proteins expressed specifically in CPC are in red. (b) Conditioned medium from MSC (red), HDF (black) and CPC (green) were analyzed using the Milliplex-MAP Human Cytokine/Chemokine Panel (Luminex); chemokine/cytokines are expressed in pg/ml. Duplicates were analyzed for two independent isolates from each cell lineage. (c) ELISA of CM from CPC1 (dark grey), CPC3 (light grey), HDF (black) and MSC (red). Duplicates were analyzed for each cell sample (n = 3). (d) Immunofluorescence analysis of IGF1R and HGFR (cMET) expression (red) in CPC1-3 compared with HDF and MSC; nuclei were DAPI-counterstained (blue). Bars, 20 μm.

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  Figure 3. Validation of the CPC secretome inferred from the transcriptomic analysis. (a) Comparative GO analysis of the CPC, MSC and HDF secretomes, carried out and visualized using the GOplot application. The representation shows up- (yellow) and downregulated (blue) genes differentially expressed in CPC (p < 0.02) relative to HDF (outer circumference) or MSC (inner circumference); non-differentially expressed genes are indicated (black). Colored network connects each gene(s) with main categories analyzed. (b) Comparison of the CPC secretome defined by mRNAseq (682 gene/proteins) with the direct proteomics secretome obtained from CM (185 proteins). (c) Venn diagram of the comparison of data from mRNAseq studies (682 gene/proteins), whole LF proteomics (465 proteins) and direct secretome analysis (185 proteins); only 79 gene/proteins were found in all three data sets.

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  Figure 4. Functional evaluation of the CPC secretome. (a) CPC wound healing capacity. All CPC isolates (CPC1-3) showed enhanced repair capacity compared with HDF cells; expressed as the percentage of scratch closure. (b) HDF cells in CM from the three CPC isolates showed improved repair capacity compared with the negative control (DMEM); expressed as percentage of closure. (c,d) Angiogenic activity in CM from the three CPC isolates compared with negative (C+) and positive controls (C+), using HUVEC (see Methods); after 6 h incubation, tube formation was analyzed using ImageJ software. Bar, 500 μm. Data are expressed as mean ± SD; n = 3.

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  Figure 5. CXCL6 is an important paracrine factor in CPC CM. (a) ELISA evaluation of CXCL6 concentration in CPC (CPC1, CPC3), HDF and MSC CM. Data are shown as mean ± SD (n = 3). (b) Western blot analysis of CXCL6 (11.9 kDa) in CPC (CPC1-3), HDF and MSC CM; β-actin was used as loading control. (c) Western blot analysis of CXCR1 (53 kDa) and CXCR2 (45 kDa) in CPC (CPC1, CPC3), HDF and MSC lysates; β-actin was used as loading control. (d) Immunocytochemical analysis of CXCR1 and CXCR2 expression in CPC (CPC1-3) and MSC; cells were counterstained with laminin (green) and nuclei with DAPI (blue); bars, 20 μm. (e) Role of CXCL6 in pro-repair activity (migration) of CPC CM. Culture with CPC3 CM (1:50 dilution) induced migration of THP-1 cells in comparison with control medium. Coincubation with anti-CXCR1, anti -CXCR2 or anti -CXCL6 antibodies were tested for their ability to interfere with THP-1 cell migration. (f) CXCL6 in the angiogenic activity of CPC CM. HUVEC were incubated with CPC1-CPC3 CM, alone or with anti-CXCL6 antibody (50 nM). After 6 h, cultures were analyzed using ImageJ. Data expressed as mean ± SD (n = 3); black lines indicate p-values (***< 0.002, **< 0.02, *< 0.05; one-way…

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  Figure 6. In vivo evaluation of CXCL6 function in a doxorubicin-induced cardiac toxicity model. (a) Experimental scheme. Full CPC-conditioned medium (CM; from isolate CPC3) was compared with CXCL6-depleted conditioned medium (depCM) for their capacity to counteract DOX toxicity (at 5 mg/Kg). Both CM were injected into the pericardial cavity 6 days after the first DOX dose and compared with mice that received vehicle alone (saline). Four weeks after the initial DOX dose, mice were sacrificed, the heart isolated and analyzed by RT-qPCR or in cardiac sections. Drawings design by Vicente Díaz. (b) ELISA quantification of CXCL6 in CM, depCM and CXCL6 bound to agarose beads. (c,d) All mouse groups were analyzed by RT-qPCR for cardiac expression of myosin light chain 7 (Myl7) and cardiac muscle alpha actin (Actc1) (c), and for Des, Bnp, Casp9, Col1a, Ctgf and Actc1, which are compared with values for mice treated with DOX alone (d). (e) TUNEL evaluation of apoptosis levels in cardiac sections from all mouse groups relative to the negative control. (f) Comparative estimation of collagen I deposition in cardiac sections, after Sirius Red staining. (g) Comparative quantification of angiogenic areas…