Figure 4.

Histologic and immunofluorescence evaluation of the efficacy of EndMT inhibition by SMAD3 knockdown in preclinical large animal AVF model at 30 days (Phase 2). (A) Representative images stained using Masson’s trichrome stain with analyses of inner perimeter, calculated lumen area, and collagen content of the vessel wall. Lumen area was calculated from the inner perimeter (i.e. inner circumference) and assuming the vessel was circular in cross-section. For this panel, all images are from the narrowest portion of the venous limb of the AVF. Scale bar = 1 mm. (B) Representative images stained using EVG stain to identify the inner elastic lamina that demarcates the intima-media boundary (arrows),²⁴ with quantitation of overall neointimal thickness (from the intima-media boundary to the intima) for each AVF determined by averaging the neointimal thickness measurement from three sites per AVF from a single section. For B, images are from close to the narrowest portion of the venous limb of the AVF (within 1–2 mm). Scale bar = 0.5 mm. (C) Representative immunofluorescence staining for CD31 (green), eNOS (red), and DAPI-stained nuclei (blue) with quantifications. Scale bar = 50 µm. (D) Representative immunofluorescence staining for VE-Cad (green), eNOS (red), and DAPI-stained nuclei (blue) with quantifications. Scale bar = 50 µm. Images in C and D are from the venous limb of the AVF, within 5–10 mm of the narrowest portion. Analyses were performed as follows: (A) inner perimeter with unpaired Student’s t-test and both calculated lumen area and collagen content with Mann–Whitney test; (B) neointimal thickness with unpaired Student’s t-test; (C) CD31⁺ cells/DAPI⁺ cells and DAPI⁺ cells with unpaired Student’s t-test, CD31⁺eNOS⁺/DAPI⁺ cells with Mann–Whitney test; (D) DAPI⁺ cells with unpaired t-test; other analyses in D with Mann–Whitney test. *P < 0.05; **P < 0.01; ns, not significant. n = 8 pigs per group for all analyses.