Fig. 1. Characterization of human cardiac fibroblasts. cFbs were cultured either in EGM-2 (A–D) or in DMEM:F12 1:1 containing 10% FBS (E–H). Contrast images (A, E) and immunofluorescence analysis to detect vimentin (B, F), fibroblast (Fb) antigen (C, G) and α-Smooth Muscle Actin (α-SMA) (D, H). (I) Western blot analysis of total extracts from cFbs cultured in EGM-2 and DMEM:F12 with 10% FBS, respectively to detect α-SMA. The same filter was probed with anti α-tubulin to show equal proteins loading. Lower panel: average results, normalized to α-tubulin, of α-SMA densitometric analyses of western blots (n = 3, p < 0.05). (L) CFbs and MyoFbs proliferation assay. Time-dependent changes in the number of cFbs and MyoFbs cultured in EGM and DMEM:F12 with 10% FBS, respectively (n = 3, p < 0.05).

Fig. 2. Effect of HMGB1 on cFbs. (A) RAGE expression in cFbs and MyoFbs. Western blot analysis of total extracts from cFbs and MyoFbs. Filter was also probed with α-tubulin, to demonstrate equal loading. Lower panel: average results, normalized to α-tubulin, of RAGE densitometric analyses of western blots (n = 3, p < 0.05). (B) HMGB1 exerts a chemotactic effect on cFbs. EBM and EGM-2 were used as negative (Ctrl⁻) and positive controls (Ctrl⁺), respectively. HMGB1 was added in EBM medium at the indicated concentrations. The data are expressed as the fold increase in the number of migrated cells relative to the number of migrated cells in the absence of factor (migration index) and represent the mean ± SE of 4 independent experiments performed in duplicate (p < 0.05 vs the negative control). (C) HMGB1 has no effect on cFb proliferation. cFbs (2 × 10⁵) were plated in EBM and either left untreated (−) or treated with 10 ng/ml HMGB1 (+) for 3 days. (D) HMGB1 has no effect on α-SMA expression. Western blot analysis of total extracts from EGM-2-cultured fibroblast untreated and treated for 3 days with the indicated concentrations of HMGB1. Lower panel: average results of α-SMA densitometric analyses normalized to α-tubulin, of western blots (n = 3, p < 0.05).

Fig. 3. HMGB1 enhances growth factors, cytokines and chemokines release from cFbs. CFbs were cultured in EGM-2 and then transferred in starvation medium (EBM). HMGB1 was added at 10 and 100 ng/ml. Supernatants were collected after 3 days and analysed by Bio-plex assay. Data were obtained from 4 independent experiments performed with cFbs isolated from 4 different patients and expressed as fold increase. p < 0.05 vs untreated cFbs (−).

Fig. 4. HMGB1 enhances VEGF and PlGF secretion by cFbs. CFbs were cultured in EGM-2 and then transferred in starvation medium (EBM). HMGB1 was added at 10 and 100 ng/ml. Supernatants were collected after 3 days and analysed by ELISA. (A) HMGB1 (10 ng/ml) treatment significantly increased VEGF concentration in cFbs supernatants compared to untreated cFbs (−); (n = 6 p < 0.05). (B) PlGF secretion was significantly higher in 10 and 100 ng/ml HMGB1-stimulated cFbs, compared to unstimulated; n = 4, p < 0.05 vs untreated cFbs.

Fig. 5. Conditioned medium of HMGB1-treated cFbs induces murine CSC migration and proliferation. (A) 2 × 10⁴ murine c-kit⁺ cells were placed in EBM in the upper compartment of the modified Boyden chambers. In the lower compartment of the chamber were added: EBM without (−) and with 10 ng/ml HMGB1 (+), CM from untreated (−) and 10 ng/ml HMGB1-treated (+) cFbs. The data are expressed as the fold increase in the number of migrated cells relative to the number of migrated cells in the absence of factor (migration index) and are the means SE of at least 4 independent experiments performed in duplicate. p < 0.05 vs EBM (−); †p < 0.05 vs EBM (+) and CM (−). (B) Murine c-kit⁺ were cultured in the presence of CM from untreated (−) and 10 ng/ml HMGB1-treated (+) cFbs. After 48 h cells were harvested and counted. (C) Quantification of BrdU expressing c-kit⁺ cells. Cells were cultures as in (B) and BrdU added the last 24 h (n = 3; p < 0.05) (D) Representative BrdU staining. Blue light fluorescence (arrowheads), BrdU⁺ cells; blue fluorescence, Hoechst 33342 of nuclei. Bar = 50 μm. (E) Bar graph of flow cytometric measurements of c-kit⁺ cells in the total non-myocyte cardiac fraction after 48 h co-culture with untreated (−) and HMGB1-treated (+) cFbs. The percentage of c-kit⁺ cells in the non-myocytes fraction of cardiac murine cells detected by FACS analysis was 0.36 ± 0.27% before co-culture experiments. n = 3, p < 0.05.

Fig. 6. Conditioned medium of HMGB1-treated cFbs enhances human c-kit⁺ cells recovery from human cardiac explants. (A) Phase micrograph of human auricle fragments. CFbs (white arrows) and small bright round cells (black arrows) emerging from the sample are indicated. (B) FACS analysis of c-kit cells in the explant cultures untreated (−) and treated with 10 ng/ml HMGB1 (+); (n = 7, p < 0.05).

Fig. 7. Conditioned medium of HMGB1-treated cFbs promotes endothelial differentiation of murine c-kit⁺ cells. Purified c-kit⁺ cells were cultured in EBM for 6 days either with CM from untreated (−) and 10 ng/ml HMGB1-treated (+) cFbs or with EBM either in absence (−) or in presence of HMGB1 (+). Bar graph indicates the percentage of c-kit⁺ derived-Ac-LDL-DiI⁺ cells on total nuclei (n = 5 p < 0.05 vs all culture conditions). (B) Representative immunofluorescence of c-kit⁺ derived-Ac-LDL-DiI⁺ cells (red fluorescence) cultured in CM from untreated (−) and 10 ng/ml HMGB1-treated (+) cFbs. Blue fluorescence, Hoechst 33342 of nuclei.

Cytokines chemokines and growth factors released in the supernatant of untreated and HMGB1-treated cFbs and MyoFbs

CFbs and MyoFbs were cultured in EGM-2 and in DMEM:F12 containing 10% FBS, respectively. Then cells were transferred in starvation medium (EBM for cFbs and DMEM for MyoFbs). HMGB1 was added at 10 and 100 ng/ml. Supernatants were collected after 3 days and analysed by Bio-plex assay. Data were expressed both in pg/ml normalized for cell number and as fold increase of HMGB1-treated vs untreated cells. Results were obtained from 4 independent experiments performed with cFbs and MyoFbs isolated from 4 different patients. p < 0.05 vs untreated cFbs (−).