Reversal of diabetic impairments in the blood flow recovery and neovascularization of ischemic areas by ANG-(1-7): A: Representative laser Doppler images.

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Reversal of diabetic impairments in the blood flow recovery and neovascularization of ischemic areas by ANG-(1-7): A: Representative laser Doppler images of blood flow in different treatment groups before and after HLI. B: Blood flow was quantified by RBC flux (blood × area−1 × time−1) expressed as a percentage of the respective contralateral limb. Reversal of diabetic impairments in the blood flow recovery and neovascularization of ischemic areas by ANG-(1-7): A: Representative laser Doppler images of blood flow in different treatment groups before and after HLI. B: Blood flow was quantified by RBC flux (blood × area−1 × time−1) expressed as a percentage of the respective contralateral limb. Blood flow recovery was lower in STZ-diabetic mice, which was restored to normal by ANG-(1-7) treatment (n = 6). *P < 0.05, **P < 0.01, and ***P < 0.001 vs. control-vehicle; #P < 0.05, ##P < 0.01, and ###P < 0.001 vs. STZ-diabetic-vehicle using two-way ANOVA. C: Immunohistochemistry for tissue necrosis and capillary density. Shown were representative light microscopic images of hematoxylin-eosin (H-E) staining, and fluorescence images of isolectin-B4 (IB4) or CD31 staining of gastrocnemius muscle sections. Muscle samples were fixed in 3.8% paraformaldehyde and embedded in paraffin blocks to obtain 8 micron sections. For H-E staining, sections were deparaffinized, hydrated, and stained with Harris Hematoxylin Solution (Electron Microscopy Sciences), followed by counterstaining with Eosin Y Phloxine B Solution (Electron Microscopy Sciences). Then, the sections were dehydrated and mounted in Permount Mounting Medium (Electron Microscopy Sciences). For determining the capillary density, sections were deparaffinized in histoclear and hydrated. Antigen retrieval was performed in citrate buffer of pH 6.0. Nonspecific receptors were blocked by using normal horse serum (Vector Laboratories), then stained with CD31 primary antibody (Santa Cruz Biotechnology) followed by secondary antibody (Vector Laboratories) and IB4 (Enzo Life Sciences, Inc.). Imaging was performed in DAPI mounting media (Vector Laboratories) using a fluorescence microscope (Olympus). D–F: Capillary density was quantified by IB4, CD31, or dual positive capillaries in the muscle sections. Capillary density decreased in STZ-diabetic mice and was normalized by ANG-(1-7) treatment (n = 6). Scale bar, 25 μm. Goutham Vasam et al. Diabetes 2017;66:505-518 ©2017 by American Diabetes Association