Fig. 9: ITGA5 signaling mediates synthesis of angiogenic mediators in infarct and in bone marrow macrophages.

In order to identify specific angiogenic mediators regulated by ITGA5 integrin, we performed an angiogenesis PCR array using RNA extracted from CD11b + /Ly6G- macrophages harvested from ITGA5 fl/fl and Myα5KO infarcts (7 days after infarction) A–H Of the 84 angiogenesis regulators assessed, Vegfa (A), Cxcl1 (B), Cxcl2 (C), Csf3 (D), Tgfb2 (E), were significantly decreased in Myα5KO infarct macrophages. In contrast, the expression of other critical angiogenesis mediators, including Vegfb (F), Vegfd (G), and Igf1 (H) was not significantly affected by ITGA5 loss. I–P In order to examine whether ITGA5 directly modulates macrophage synthesis of angiogenic mediators, we studied the effects of an anti-ITGA5 neutralizing antibody (clone HMa5-1, Biolegend) in bone marrow macrophages. RNA-seq showed that only Vegfa was markedly downregulated in bone marrow macrophages upon ITGA5 neutralization (I). Reduced Vegfa mRNA levels upon ITGA5 blockade were associated with a decrease in VEGFA protein levels, assessed through an ELISA (J). In contrast the angiogenesis regulators Cxcl1 (K), Cxcl2 (L), Csf3 (M), Tgfb2 (N), Vegfb (O), Vegfd (P) and Igf1 (Q) were not significantly affected by ITGA5 blockade (****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, n = 3 biologically independent experiments/group). Thus, the in vitro and in vivo experiments suggest that the angiogenic actions of ITGA5 in macrophages involve VEGFA synthesis. Data are shown as mean values +/- SEM Statistical analysis was performed using unpaired two-tailed Student’s t test. Source data are provided as a Source Data file. Fpkm fragments per Kilobase Million, IgG immunoglobulin G.