Coordinated Activation of VEGF/VEGFR-2 and PPARδ Pathways by a Multi-Component Chinese Medicine DHI Accelerated Recovery from Peripheral Arterial Disease in Type 2 Diabetic Mice

Figures

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  Fig 1. DHI improved glucose homeostasis. (A) In KKAy mice, the levels of blood glucose in DHI-treated and rosiglitazone-treated groups were significantly lower than that in vehicle-treated group. (B) Intraperitoneal glucose tolerance testing was performed in KKAy mice at the end of the feeding course, and the areas under the glucose tolerance tests curves were shown in (C). (D) In STZ-induced diabetic mice, the level of blood glucose in DHI-treated and metformin-treated groups were significantly lower than that in the vehicle-treated group. (E) Intraperitoneal glucose tolerance testing was performed in STZ-induced diabetic mice at the end of the feeding course, and areas under the glucose tolerance tests curves were shown in (F). Values are mean ± SEM. *P<0 .05, **P<0 .01, ***P<0 .001 vs vehicle.

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  Fig 2. DHI improved perfusion of ischemic limbs in KKAy mice. (A) Representative images of laser Doppler perfusion analysis for WT control mice, KKAy mice treated with AMD3100, KKAy mice treated with or without DHI before surgery and at different time points after surgery. Low perfusion signals (dark blue) were observed in the ischemic hind limb, whereas high perfusion signals (red) were detected in KKAy mice treated with DHI on postoperative day 8 through 35 and in KKAy mice treated with AMD3100 on postoperative days 8, 21, 28, and 35. (B) Hind-limb perfusion recovery was impaired in untreated KKAy mice. The mean hind-limb blood flow was calculated as the ratio of ischemic (left) side to non-ischemic (right) side. (C) DHI or AMD3100 significantly improved perfusion recovery after HLI surgery. *P<0 .05, **P<0 .01, ***P<0 .001 vs. KKAy+vehicle.

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  Fig 3. DHI improved perfusion of ischemic limbs in STZ-induced diabetic mice. (A) Representative images of laser Doppler perfusion analysis for non-diabetic mice, STZ-induced diabetic mice treated with or without DHI before surgery and at different time points after surgery. Low perfusion signals (dark blue) were observed in the ischemic hind limb, whereas high perfusion signals (red) were detected in diabetic mice treated with DHI on postoperative days 14, 21, 28 and 35. (B) Perfusion recovery was impaired in untreated STZ-induced diabetic mice. The mean hind-limb blood flow was calculated as the ratio of ischemic (right) side to non-ischemic (left) side. DHI showed a significantly improved perfusion recovery after HLI surgery. **P<0.01, ***P<0 .001 vs. DM+vehicle.

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  Fig 4. DHI increased EPC mobilization in KKAy mice. EPC (defined as Sca-1+/Flk-1+cells) mobilization after tissue ischemia was determined by flow cytometry in C57BL/6J mice and KKAy mice after administration of saline, DHI or AMD3100. DHI and positive-control AMD3100 both showed a significantly improved EPC mobilization after HLI surgery. *P<0 .05 vs. KKAy+vehicle.

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  Fig 5. In vivo expression of VEGFR-2 in diabetic VEGFR-2-luc mice. (A-C) Representative Bioluminescent images of HLI mice were obtained at 0, 7 and 10 days under the same imaging conditions. (D) The dynamic measurement of bioluminescent intensities in non-DM, DM+vehicle and DM+DHI groups. Regions of interest (ROI) from displayed images were identified on the HLI sites and quantified as photons per second (p/s). Data is shown as mean ± SEM. n = 5 in each group. *P<0.05, vs. DM+vehicle.

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  Fig 6. DHI decreased incidence of limb necrosis in diabetic mice post HLI. Representative images of hind limbs showing evidence of tissue necrosis. In DM+vehicle group, six out of seven ischemic limbs showed necrosis (86%), while in DM+DHI group, four out of seven ischemic limbs showed necrosis (57%).

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  Fig 7. DHI increased capillary density in diabetic mice post HLI. Representative sections of ischemic limb muscle 5 weeks after DHI treatment. Capillaries were identified by Tie-2 or VEGFR-2 antibody staining. Ischemic limbs of KKAy mice injected with saline (A1) or DHI (A2) were shown where capillaries were identified by an anti-Tie-2 antibody. Ischemic limbs of STZ-induced diabetic mice injected with saline (B1) or DHI (B2) were shown where capillaries were identified by an anti- VEGFR-2 antibody. Quantitative analysis of capillary density of the ischemic region was performed at the end of week 5 for KKAy (A3) and STZ-induced (B3) diabetic mice. *P<0 .05, **P<0 .01.

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  Fig 8. Increase of angiogenic factors in DHI-treated ischemic muscle. (A) Quantitative PCR showed DHI increased the expression of VEGF-A and Flk-1 in KKAy mice. (B) Quantitative PCR showed DHI increased the expression of VEGF-A and Flk-1 in STZ-induced diabetic mice. Data represent the means ± SEM, C57BL/6J: n = 5, KKAy+vehicle: n = 4, KKAy+DHI: n = 6; Non-DM: n = 3, DM+vehicle: n = 5, DM+DHI: n = 5; vs. vehicle, ***P<0 .001.

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  Fig 9. IPA indicated the overlap between angiogenesis and glucose tolerance networks. (A) Red colored genes are related to glucose tolerance and green colored genes are related to angiogenesis. The arrows indicate effects of regulated genes on other genes. (B) Orange colored are predicted to be activation and blue colored are predicted to be inhibited.

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  Fig 10. DHI increased PPARδ expression in ischemic muscle tissue. (A) Quantitative PCR showing increased PPARδ expression in genetic diabetic mice treated with DHI. (B) Quantitative PCR showing increased expression of PPARδ in chemically induced diabetic mice treated with DHI. Data represent the mean ± SEM, C57BL/6J: n = 5, KKAy+vehicle: n = 4, KKAy+DHI: n = 6; Non-DM: n = 3, DM+vehicle: n = 5, DM+ DHI: n = 5; vs. vehicle, **P<0 .01, *P<0.05.