Fig 2: Reperfusion and new vessel formation following ischemia in mice lacking EPCR in Tie2-expressing cells.(A) Representative laser Doppler perfusion images in EPCRfl/fl Tie2.Cre mice (n = 11) and EPCRfl/fl littermate controls (n = 10) immediately before and after, as well as on, day 7, day 14, day 21, and day 28 after induction of hindlimb ischemia. For technical reasons, 1 animal in each group could not be imaged at day 21. (B) Quantitative analysis of the laser signal (expressed as % of the contralateral, nonischemic site). *P < 0.05, ***P < 0.001, and ****P < 0.0001 versus values immediately after surgery; ##P < 0.01 versus EPCRfl/fl controls at the same time point; 2-way ANOVA, Sidak’s multiple-comparison test. (C) Representative immunofluorescence microscopy images after visualization of CD31 expression (red) in nonischemic and ischemic hindlimbs of EPCRfl/fl Tie2.Cre mice and control littermates. DAPI+ cell nuclei appear blue. Scale bar: 10 µm. (D) Quantitative analysis of CD31-immunopositive cells per muscle fiber; n = 7 biological replicates. *P < 0.05 and ***P < 0.001 versus nonischemic control muscle; #P < 0.05 versus EPCRfl/fl littermates; 2-way ANOVA, Sidak’s multiple-comparison test. (E) Representative bright-field images of CD31+ endothelial cells isolated from EPCRfl/fl and EPCRfl/fl Tie2.Cre littermate mice and subjected to the spheroid assay; n = 4 biological replicates per 2 experimental repeats. Scale bar: 10 µm. (F and G) Quantitative analysis of the number of sprouts (F) and total length of the sprouts (G) migrated out of the spheroids. **P < 0.01 and ****P < 0.0001 versus EPCRfl/fl littermate control mice. Data were analyzed using Student’s t test.

Fig 3: Endothelial EPCR and hemoglobin expression and soluble EPCR plasma levels in patients with chronic peripheral artery ischemia.(A) Representative IHC images of human skeletal muscle specimens from nonischemic controls (top panel) and patients with peripheral artery disease (PAD) showing expression of EPCR, HBA, and eNOS+ endothelial cells; n = 4 biological replicates. Scale bars: 10 µm. (B) Representative IHC images of human PAD specimens showing expression of HBA, CD31, and EPCR on serial cross sections. Scale bars: 10 µm (upper row). Lower row shows enlarged areas of interest. Images are taken with 200× (upper row) and 400× (lower row) magnification. (C) Results of quantitative analysis of the number of positive cells residing in vascular specimens isolated from patients with PAD; n = 4 biological replicates. **P < 0.01; 1-way ANOVA, Bonferroni’s multiple-comparison test. (D) Plasma levels of soluble EPCR (sEPCR) in 19 patients with PAD and 19 age- and sex-matched individuals with similar risk factors (RF) and no PAD diagnosis (controls with RF). (E) Plasma levels of nitrite/nitrate in patients with PAD and age-and sex-matched controls. *P < 0.05 and **P < 0.01. Student’s t test.

Fig 4: ABO increased ANXA7 level in the endothelium of apoE-/- mice. (a) Double-stained images of co-localization (yellow) of ANXA7 with CD31-positive VECs. Scale bars, 60 µm. (b) The relative fluorescent intensity of ANXA7 in the endothelium of apoE-/- mice. Data are mean±S.E.M. #P<0.05 versus control, n=6

Fig 5: LDN effect on Dullard mutants.(A) Analysis of phosho-Smad intensity at different levels of the OFT at E11.5 after 48h exposure to LDN. (B) Transversal sections of the distal OFT stained for PECAM and GFP after 48h treatment with LDN or vehicle of mutant E11.5 embryos.