Smad3 promotes adverse cardiovascular remodeling and dysfunction in doxorubicin-treated hearts

Figures

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  Figure 1.. Accumulation of doxorubicin (Dox) in cardiac endothelial cells of treated mice. Mice were intravenously injected with 5 mg/kg doxorubicin under general anesthesia with isoflurane. Heart tissues were harvested 1 h after the injection. Isolectin B4-DyLight 649 conjugate (ILB4) was intravenously injected 5 min before tissue harvesting to label endothelial cells (ECs). A: accumulation of doxorubicin in endothelial and non-endothelial nuclei 1 h after its intravenous injection. Intrinsic fluorescence of doxorubicin was pseudocolored green. Control mice did not receive doxorubicin. Nuclei were stained with 2 µM Hoechst 33342, whereas endothelial cells were identified using the ILB4 conjugate. Scale bar = 20 µm. B: fluorescence intensity profiles of endothelial cell (EC) and non-endothelial cell (non-EC) nuclei. Endothelial cells were identified using the ILB4 conjugate (shown in red); nuclei were identified using Hoechst 33342 dye (shown in blue). C: analysis of doxorubicin accumulation in endothelial vs. non-endothelial nuclei 1 h after doxorubicin injection. Twenty nuclei were analyzed per heart and averaged to present the results of n = 5 independent experiments. The P value for the…

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  Figure 2.. Doxorubicin enhances activity of the canonical TGF-β pathway in endothelial cells. A: mice were intravenously injected with doxorubicin (5 mg/kg) or saline, and heart tissues were harvested 24 h after the injections and processed for immunohistochemical staining. Cardiac sections were stained with the TGF-β pan-specific antibody and analyzed using color bright-field microscopy. Scale bar = 100 µm. B: TGF-β expression in cardiac tissues was measured by density of horseradish peroxidase substrate 3,3′-diaminobenzidine (DAB) deposits. n = 3 hearts per group. Eight to ten randomly selected fields per heart were analyzed. P value for the unpaired two-tailed t test is shown. C: human cardiac microvascular endothelial cells were pretreated with 16 nM doxorubicin (Dox) for 72 h, starved for 4 h, and incubated with 0.3 ng/mL TGF-β1 for 0 to 90 min. Protein extracts were then processed according to the immunoblotting protocol and incubated with the indicated antibodies. pSmad3, phospho-Smad3. D: pSmad3 band intensity in cells lysed at the indicated time points after treatment with TGF-β1 is presented as a ratio of phospho- to total Smad3. Results of 3 independent experiments are shown. P…

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  Figure 3.. Smad3 deficiency suppresses the TGF-β pathway and promotes sprouting by doxorubicin (Dox)-treated mouse aortic ring explants. A: Smad2 and Smad3 expression in Scramble shRNA (Scrmb) and Smad3 shRNA (Smad3) human cardiac microvascular endothelial cell lines. Cell protein lysates were processed according to the immunoblotting protocol and incubated with the indicated antibodies. A representative immunoblot (of 3 independent experiments performed) is shown. B and C: human cardiac microvascular endothelial cells were pretreated with 16 nM doxorubicin (Dox) for 72 h, starved for 4 h in growth factor-free endothelial basal media, and treated with 0.3 ng/mL TGF-β1 for 16 h. Total RNA was isolated and sequenced, and transcriptomic analysis was performed. Volcano plots for the Scrmb Dox vs. Scrmb (B) and Smad3 Dox vs. Scrmb Dox (C) comparisons are shown. D: activation Z-scores for the upstream regulators identified using Scrmb Dox vs. Scrmb (in blue) and Smad3 Dox vs. Scrmb Dox (in orange) pairwise comparisons analysis. P < 0.05 for all presented Z-scores. E: heatmap of the TGF-β1 pathway-related transcripts in untreated and doxorubicin-treated scrambled shRNA and Smad3 shRNA endothelial…

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  Figure 4.. Cardiac inflammatory response to doxorubicin is mediated by Smad3. Human cardiac microvascular endothelial cells were pretreated with 16 nM doxorubicin (Dox) for 72 h, starved for 4 h in growth factor-free endothelial basal media, and treated with 0.3 ng/mL TGF-β1 for 16 h. A: Venn diagram quantifying genes overlapping for comparisons shown in Fig. 3, B and C (in blue and orange, respectively). One thousand and sixty-two genes were significantly differentially expressed and shared between the 2 comparisons. B: heatmap of significantly differentially expressed 1,062 transcripts in response to TGF-β1 in pretreated with Dox Scrmb shRNA and Smad3 shRNA endothelial cell lines. C: heatmap of significant cytokine signaling-related gene expression in response to TGF-β1 in pretreated with Dox Scrmb shRNA and Smad3 shRNA endothelial cell lines. D: IL-6 expression in cardiac tissues of wild-type (WT) and Smad3-knockout (KO) mice 48 h after intravenous injections of 5 mg/kg doxorubicin (Dox) or saline. Cardiac sections were stained using anti-IL-6 antibodies and counterstained with hematoxylin. Scale bar, 50 µm. E: quantitative analysis of IL-6 expression in cardiac tissues of treated mice.…

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  Figure 5.. The role of Smad3 in cardiac microvascular remodeling after completion of doxorubicin therapy. Wild-type (WT) and Smad3-knockout (KO) mice were intravenously injected with 4 weekly injections of doxorubicin (Dox, 5 mg/kg each, for a total dose of 20 mg/kg) or saline. Animals were euthanized 9 wk after the last injection of the drug or vehicle. A: cardiac sections from treated mice were stained with ILB4 to visualize microvasculature. Scale bar = 20 µm. B: quantification of individual microvessel area. n = 4 to 6 animals per treatment group. P value for the one-way ANOVA test is shown. C and D: transcriptomic analysis was performed on endothelial cells isolated from hearts of treated mice 9 wk after the last injection. Volcano plots for the WT Dox vs. WT saline (C) and Smad3 KO Dox vs. WT Dox (D) comparisons. E: Venn diagram quantifying genes overlapping for comparisons shown in C and D (in blue and orange, respectively). Seven hundred and sixty-six genes were significantly differentially expressed and shared between the 2 comparisons. F: activation Z-scores for the upstream regulators identified using overlapping differentially expressed genes analysis for the WT Dox vs. WT…

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  Figure 6.. Smad3 deficiency prevents contractile decline and adverse cardiac remodeling in doxorubicin-treated hearts. Wild-type (WT) and Smad3-knockout (KO) mice were intravenously injected with four weekly injections of doxorubicin (Dox, 5 mg/kg each, for a total dose of 20 mg/kg) or saline, and underwent transthoracic echocardiography under light anesthesia with isoflurane 9 wk after the last injection. Experiments on three to seven animals per group were performed. P values for the one-way ANOVA tests are shown. A: left ventricular systolic function was assessed using ejection fraction (EF) and end-systolic diameter (LVESD). B: global radial strain (RS) was examined as an additional measure of left ventricular systolic function. The representative images on the left show separate strain curves generated for each of the six standard myocardial regions, with each cardiac segment represented with a designated color, and the seventh line (black) denoting average strain for the heart at each time point. Quantitative RS analysis is presented in the right panel. C: cardiac remodeling was evaluated using left ventricular end-diastolic diameter (LVEDD).