Fig 2: Confocal imaging and fluorescence quantification of intra- and extra-cellular molecules. (A) Confocal images of aortic rings labeled with endothelial markers (IB4 and CD31) in red. Bar ~100 μm. (B) Images of aortic ring labeled with HABP (green) and IB4 (red). White arrows point to areas of HABP clearly identified outside the endothelium. (C) Images of aortic rings labeled for intracellular proteins P-PERK and HAS2 (green) and endothelial marker IB4 (red). Inset from the dotted rectangle (right), with white arrows pointing to intracellularly labeled proteins. (D) Aortic ring images labeled with IB4 (cyan). Yellow dotted line outlines the sprouting area. (E) Quantification of number of main sprouts and sprouting area of rings incubated from 0 to 7 days. Mean ± SEM. Ring marks the aortic ring.

Fig 3: HAS2 expression is dependent on nSMase2/Cer and is involved in the resistance of fro/fro to nutrient deprivation-induced apoptosis. (A) Western blot of HAS2 in wt and fro/fro fibroblasts grown in complete medium (compl) and nutrient deprivation conditions (depriv). (B) HAS2 mRNA expression evaluated by q-PCR experiments, and normalized to HPRT, in fro/fro and wt fibroblasts grown in complete medium (compl), nutrient derivation conditions (depriv). The effect of C2-ceramide (C2Cer, 5 µM), was tested in fro/fro fibroblasts, as indicated. (C) Western blot of HAS2 in fro/fro fibroblasts grown in complete medium and treated with scrambled siRNA or with specific HAS2 siRNA (upper panel) or in complete (compl) or nutrient deprivation (depriv) medium with or without methylumbelliferone (MU, 1 mM) (lower panel). (D) After 48 h incubation, the cytotoxicity was evaluated by the MTT test. Data are mean±SEM of 5 separate experiments, ⁎p<0.05.

Fig 4: Hsp72 expression is increased in a HAS2-dependent manner in fro/fro fibroblasts and is involved in their resistance to nutrient deprivation. (A) Expression of Hsp72 in wt and in fro/fro fibroblasts, after 48 h incubation in complete medium (compl) or in nutrient deprivation conditions (depriv). (B) Effect of scrambled (scr) siRNA or HAS2-specific siRNA on Hsp72 expression in fro/fro fibroblasts. (C,D) Effect of the specific Hsp72 inhibitor KNK437 (KNK, 5 µM), on the viability of fro/fro fibroblasts, in nutrient starvation conditions (St) evaluated by the MTT assay (C), and by counting the apoptotic cell number, after syto13/PI staining (D). These data are expressed as percent of the results obtained in complete medium (C), and are a mean±SEM of 5 separate experiments, ⁎p<0.05.

Fig 5: Has2 expression in VSMCs and femoral artery. a Starved WT, SMIRKO, or SMIGF1RKO VSMCs were stimulated with 10 nM insulin, and the gene expression of Has2 was determined by qPCR (0 h, n = 4 for each group; 0.5 h, n = 3 for each group; 1 h, n = 3 for each group; 2 h, n = 4 for each group; 4 h and 8 h, n = 4 for WT and SMIGF1RKO, n = 3 for SMIRKO; 24 h, n = 3 for WT and SMIGF1RKO; n = 2 for SMIRKO). b Has2 protein expression in VSMCs treated with insulin. c Hyaluronan in cultured medium. The hyaluronan content before and 12 h after 10 nM insulin stimulation was determined by ELISA. d The femoral arteries were harvested at 9 days after wire injury, and the expression of Has2 gene was determined by qPCR (IRflox/flox sham, n = 6; IRflox/flox injury n = 7; SMIRKO sham, n = 4; SMIRKO injury, n = 8; IGF1Rflox/flox sham, n = 6; IGF1Rflox/flox injury, n = 9; SMIGF1RKO sham, n = 3; SMIGF1RKO injury, n = 5). e, f Hyaluronan staining in wire-injured femoral artery. Hyaluronan staining was performed on feoral arteries at 4 weeks after wire injury (arrow indicates neointima. IRflox/flox, n = 8; SMIRKO, n = 8; IGF1Rflox/flox, n = 9; SMIGF1RKO, n = 7). g VSMCs were infected with AdFoxO1 CA or AdGFP and then stimulated with insulin for 4 h. The expression of Has2 gene was determined by qPCR (n = 3 per group). h VSMCs were treated with rapamycin (10 ng/ml) for 1 h and then stimulated with insulin for 4 h. Expression of Has2 gene was determined by qPCR (n = 3 per group). i The expression of Has2 in VSMCs was knockdown by siRNA, and cellular proliferation was determined by EdU incorporation (n = 8 for each group). j Starved VSMCs were pretreated with 1 mM 4-MU for 4 h and then stimulated with 100 nM insulin for 24 h. The cell proliferation was determined by measuring EdU-positive cells by flow cytometry (n = 3 per group). The data are mean ± SEM. Two-tailed t test or two-way ANOVA with a post hoc test. Source data are provided as a Source Data file