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 cell lines. Significantly upregulated and downregulated genes for which TGF-β1 is an upstream regulator are indicated in red and blue, respectively. F: sprouting of aortic ring explants from wild-type (WT) and Smad3-knockout (KO) mice in collagen matrix visualized using isolectin B4 (ILB4) staining. Scale bar = 1,000 µm. G: quantification of aortic ring sprouting. Results of 6 independent experiments are presented with 4 rings per experimental group each. P values for the two-way ANOVA tests are shown.