Exposure of endothelial cells to Doxorubicin inhibits Extracellular matrix production by dermal fibroblasts in a paracrine manner

Figures

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  Fig. 1.. Experimental design. Shown is a graphical outline of the experimental design of this study. For evaluating Dox-induced EndMT, HUVECs were exposed to Dox, (10 µM for 1 h; or 1 µM for 24 h), followed by a 7-day recovery in Dox-free medium to assess EndMT using immunofluorescent microscopy (IF). To assess the paracrine effects of Dox-exposed EC on dermal fibroblasts, conditioned medium (CM) was collected from HUVEC cultures, 24 h after exposure to Dox or DMSO. This CM was then applied to primary human dermal fibroblasts up to 21 days of culture, with or without supplemented magnesium ascorbyl phosphate (VitC). The effects of CM on fibroblast activation and senescence were assessed at 3 and 21 days (WB or/and IF); on collagen synthesis (WB, collagen assay, eosin staining) was evaluated at 21 days; and on MMP1 and MMP9 gene expression (qPCR) at 2 days.

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  Fig. 2.. Dox treatment-induced effects on endothelial-to-mesenchymal transition (EndMT). A) Representative immunofluorescence images of S100A4 and CD31 staining in HUVECs show increased S100A4 expression 7 days after Dox treatment (10 µM, 1 h), indicating induced EndMT. White arrows indicate CD31-positive cell membranes (red), whereas double-head white arrows indicate cytoplasmic S100A4 positivity (green). Nuclei were counterstained with Hoechst in blue, as shown in the merged images. B, C) Quantification of S100A4 and CD31 expression in HUVECs treated with Dox (10 µM for 1 h or 1 µM for 24 h), assessed by quantifying the relative fluorescence intensity from immunofluorescence images. Fifteen images per condition were analyzed across 3 individual experiments (n = 3), using identical imaging settings. Data are represented as Mean ± SD. *P<0.05, ns indicates not significant.

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  Fig. 3.. Paracrine effects of Dox-exposed EC on fibroblast activity and myofibroblast transition. A) Representative immunofluorescence images showing FAP (red) and α-SMA (green) in human dermal fibroblasts treated with conditioned medium (CM) harvested from HUVECs that were exposed to Dox (10 µM, 1 h) for 3 days. White arrows indicate cytoplasmic FAP; double-headed white arrows indicate filamentous α-SMA. Nuclei were counterstained with Hoechst in blue, as shown in the merged images. B) Representative Western blot (WB) images showing bands for FAP, α-SMA, S100A4, and β-actin in human dermal fibroblasts treated with CM harvested from HUVECs exposed to Dox (Dox-CM) or equivalent doses of DMSO (DMSO-CM) for 3 days or 21 days (with or without magnesium ascorbyl phosphate (VitC)). C, D) Quantification of WB results for FAP, α-SMA and S100A4 expression, relative to β-actin, in human dermal fibroblasts from 7 individual donors (n = 7), treated with CM harvested from HUVECs exposed to Dox (Dox-CM) or equivalent doses of DMSO (DMSO-CM), with/without VitC, for 3 or 21 days. The results for CM of HUVECs exposed to 10 µM Dox (or DMSO) for 1 h are shown in (C), and for CM of HUVECs exposed to 1 µM Dox…

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  Fig. 4.. Paracrine effects of Dox-exposed EC on fibroblast senescence. (A) Left: Representative bright field images of human dermal fibroblasts treated with conditioned medium (CM) harvested from HUVECs exposed to Dox (Dox-CM; either 10 µM for 1 h or 1 µM Dox for 24 h) or equivalent doses of DMSO (DMSO-CM), for 21 days (with or without magnesium ascorbyl phosphate (VitC)). The differences in cellular morphology are visible. Right: Quantification of the elongation factor, calculated as the ratio of the maximum to minimum cell diameter in the treatments described under (A) left. B) Representative Western blot (WB) images showing bands of Lamin B1, p21, and β-actin in human dermal fibroblasts treated with conditioned medium (CM) from HUVECs exposed to Dox (Dox-CM; either 10 µM for 1 h or 1 µM Dox for 24 h) or equivalent doses of DMSO (DMSO-CM), for 3 days or 21 days (with or without magnesium ascorbyl phosphate (VitC)). C, D) Quantification of WB results for p21 and Lamin B1 expression, relative to β-actin, in human dermal fibroblasts from 7 individual donors (n = 7), treated with CM harvested from HUVECs exposed to Dox (Dox-CM) or equivalent doses of DMSO (DMSO-CM), with/without VitC, for 3…

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  Fig. 5.. Paracrine effects of Dox-exposed EC on fibroblast collagen production and ECM formation. A) Left: Representative eosin staining images of human dermal fibroblasts treated with conditioned medium (CM) harvested from HUVECs exposed to Dox (Dox-CM; either 10 µM for 1 h or 1 µM Dox for 24 h) or equivalent doses of DMSO (DMSO-CM), for 21 days [with or without magnesium ascorbyl phosphate (VitC)]. Formed ECM stains red. Right: Quantification of the eosin staining intensities in the treatments described under (A) left. B, C) Quantification of insoluble collagen and soluble collagen levels in the harvested fibroblasts + ECM after the treatment described under (A) left. The results for CM of HUVECs exposed to 10 µM Dox (or DMSO) for 1 h are shown in (B) and for CM of HUVECs exposed to 1 µM Dox (or DMSO) for 24 h are shown in (C). D) Representative Western blot (WB) images showing Collagen I expression in the supernatants of human dermal fibroblasts treated as described in (A). Supernatant was collected daily and from each donor the samples from days 1 to 7, 8 to 14, and 15 to 21 were combined to form 3 groups over time. E, F) Quantification of supernatant collagen I levels from human…

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  Fig. 6.. Dox effects on fibroblast collagen production and ECM formation. A) Left: Representative eosin staining images of human dermal fibroblasts were exposed to Dox (Dox: either 10 µM for 1 h or 1 µM for 24 h) or equivalent doses of DMSO (DMSO), followed by 21-day culture in Dox-free medium with or without magnesium ascorbyl phosphate (VitC). Right: Quantification of the eosin staining intensities in the treatments described under (A) left. B) Left: Representative Western blot (WB) images showing collagen I expression in the supernatants of human dermal fibroblasts treated as described in (A). Supernatant was collected daily, for each donor, samples from days 1 to 21 were pooled to generate a single sample. Right: Quantification of supernatant collagen I levels from human dermal fibroblasts, from 7 individual donors (n = 7), treated as described in (A). Data are normalized to the supernatant of fibroblasts cultured in complete DMEM with DMSO. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 and ns indicates not significant.

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  Fig. 7.. Paracrine effects of Dox-exposed EC on the transcriptional levels of MMP1 and MMP9. A—B) Quantification of MMP1 and MMP9 transcriptional expression relative to YWHAZ and GPI in human dermal fibroblasts, from 7 donors (n = 7), treated with conditioned medium (CM) harvested from HUVECs exposed to Dox [Dox-CM; either (A) 10 µM for 1 h or (B) 1 µM Dox for 24 h] or equivalent doses of DMSO (DMSO-CM), for 2 days [with or without magnesium ascorbyl phosphate (VitC)]. Data are shown normalized to fibroblasts treated with DMSO-CM. *P < 0.05 and ns indicates not significant.

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  Fig. 8.. Cytokine measurements in conditioned media and IL-6 effects on ECM formation. A, B) Cytokine [CXCL10 (IP-10), IL-1β, CCL2 (MCP-1), IL-6, CXCL8 (IL-8)] measurements in conditioned medium (CM) from HUVECs treated with 10 µM Dox for 1 h (CMDox10) or 1 µM Dox for 24 h (CMDox1) or equivalent DMSO concentrations (CMDMSO10 or CMDMSO1), data from 3 individual experiments (n = 3). C) Left: Representative eosin staining images of human dermal fibroblasts cultured for 21 days in complete DMEM containing recombinant human IL-6 (330 or 3300 pg/ml) or vehicle control (complete DMEM without IL-6) with or without magnesium ascorbyl phosphate (VitC). Formed ECM stains red. Right: Quantification of the eosin staining intensities in the treatments described under (A) left. D) Left: Representative Western blot (WB) images showing collagen I expression in the supernatants of human dermal fibroblasts treated as described in (C). Supernatant was collected daily, for each donor, samples from days 1 to 21 were pooled to generate a single sample. Right: Quantification of supernatant collagen I levels from human dermal fibroblasts, from 7 individual donors (n = 7), treated as described in (C). Data are…