Figure-1:
Increased VEGF165b-expression inversely correlates with decreased VEGFR1-activation and directly with M1-like macrophage phenotype. A) VEGF165b ELISA in human normal, intermittent claudication (IC) and critical limb ischemia (CLI) patients’ plasma samples. Control n=24, IC n=20, CLI n=19. Non-parametric one-way ANOVA Kruskal-Wallis test with Dunn’s multiple comparisons test and Geisser-Greenhouse correction was used to determine statistical significance. B, C) VEGF165b levels in conditioned-medium (medium) and lysates from control and hypoxia serum starved (HSS) (B) human peripheral blood monocyte derived macrophages (HPBMDM) and (C) mouse bone marrow derived macrophages (BMDM). VEGF165b levels in conditioned-medium was normalized to the corresponding cell lysate total protein levels. n=4. A-C) One way ANOVA with Dunnett’s multiple test correction was used to determine statistical significance. D) VEGF165b levels in conditioned-medium and lysates from bone marrow monocytes from non-ischemic leg (NIL-Mo) and monocytes from ischemic leg (IL-Mo). VEGF165b levels in conditioned-medium was normalized to the corresponding cell lysate total protein levels. n=4. E-F) Flowcytometric analysis of E) VEGF165b and F) pVEGFR1Y1333 expression in macrophages from non-ischemic gastrocnemius (NGA) and ischemic gastrocnemius (IGA) muscle. n=4. G-H) Flowcytometric analysis of pVEGFR1 expression in G) VEGF165b+ve (VEGF165b positive) and H) VEGF165b−ve (VEGF165b negative) macrophages from NGA and IGA. n=4. I-J) Flowcytometric analysis of I) CD80high M1-like macrophages and J) Arg-1+ M2-like macrophages from NGA and IGA. n=4. D-J) Unpaired T-test was used to determine statistical significance. Increased VEGF165b-expression correlate with higher CD68+CD80+ M1-like macrophages in human PAD. Immunostaining VEGF165b, CD68, CD206 and CD80 in normal (NL) and peripheral arterial disease (PAD) muscle-biopsies. Quantification of K) VEGF165b expressing (VEGF165b+ve) cells, L) total macrophages (CD68+), M) macrophages expressing CD80 (CD68+CD80+) and N) macrophages expressing CD206 (CD68+CD206+). Normal n=5, PAD n=7. K-N) Unpaired T-test was used to determine statistical significance. *P<0.05 considered significant.