The Impact of Maternal Fructose Exposure on Angiogenic Activity of Endothelial Progenitor Cells and Blood Flow Recovery After Critical Limb Ischemia in Rat Offspring

Figures

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  Figure 1. Maternal fructose exposure impaired angiogenic activity of bone marrow-derived endothelial progenitor cells. (A,B) In vivo angiogenic activity analysis (matrigel) on bone marrow-derived endothelial progenitor cells (BMDEPC) of 3-month-old offspring. (C) Calculation of tube formation in matrigel (red arrows). (D) Calculation of network formation in matrigel (yellow dotted line). (E) Calculation of cluster formation in matrigel (blue arrows). (F) Calculated of accumulated tube length. (G,H) Trans-well analysis to determine the migratory activity of BMDEPC. (I) Calculation of migrated cells in the trans-well assay. Results showed that the numbers of tube, network, and clusters in the MFE group were significantly lower than those in the NC group. NC, maternal control; MFE, fructose exposure. Scale bar in the lower-right corner indicates 100 μm. Error bars represent the standard deviation (SD). * Indicates statistical significance between NC and MFE group. n = 8 for each group.

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  Figure 2. Maternal fructose exposure reduced the number of circulating endothelial progenitor cells. (A–C) The number of c-Kit+/CD31+, Sca-1+/KDR+, and CXCR4+/CD34+ in the peripheral bloods of the offspring prior to induction of critical limb ischemia (CLI). (D–F) The number of c-Kit/CD31, Sca-1/KDR, and CXCR4/CD34 in the peripheral bloods of the offspring 18 h post induction of CLI. (G–I) The number of c-Kit/CD31, Sca-1/KDR, and CXCR4/CD34 in the peripheral bloods of the offspring 14 days post induction of CLI. In normal condition, compared with the NC group, the number of circulating c-Kit+/CD31+ and Sca-1+/KDR+ EPCs were lower in the offspring with MFE. However, the number of CXCR4+/CD34+ cells was not affected by maternal fructose exposure. Eighteen hours and 14 days after induction of CLI. The circulating levels of c-Kit+/CD31+ and Sca-1+/KDR+ cells in MFE group was lower than that in the NC group, while levels of CXCR4+/CD34+ cells showed no difference between the two groups. NC, normal control; MFE, maternal fructose exposure; CLI, critical limb ischemia. Error bars represent the standard deviation (SD). * Indicates statistical significance between NC and MFE group. n = 8 for each…

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  Figure 3. Maternal fructose exposure reduced blood flow recovery after induction of critical limb ischemia. (A) Laser Doppler scanning to determine the blood flow recovery after induction of CLI. (B) Quantitation and calculation of blood flow. The parameter ratio of ischemia to normal blood flow (INBF) was used to evaluate the blood flow recovery initiated by neovascularization after CLI. On day 2 after CLI and day 0 prior to CLI, the ratio of INBF showed no difference between NC and MFE groups. However, by day 14, the blood flow recovery in the NC group was significantly higher than the MFE group. NC, normal control; MFE, maternal fructose exposure; CLI, critical limb ischemia. Error bars represent the standard deviation (SD). * Indicates statistical significance between NC and MFE group. n = 8 for each group.

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  Figure 4. Capillary density in ischemic quadriceps was reduced by maternal fructose exposure. (A–D) Immunofluorescent staining to detect the distribution of CD31+ capillaries in quadriceps. (E) Calculation of capillary density in quadriceps. In normal condition, the capillary density showed no difference between the NC and MFE groups. By 14 days after induction of CLI, the capillary density in MFE group was significantly lower than that in NC group. NC, normal control; MFE, maternal fructose exposure; CLI, critical limb ischemia. Scale bar in the lower-right corner indicates 200 μm. Error bars represent the standard deviation (SD). * Indicates statistical significance between NC and MFE group. n = 8 for each group.

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  Figure 5. Maternal fructose exposure increased the fibrotic area in ischemic quadriceps after induction of critical limb ischemia. (A) Hematoxylin and eosin (H&E) staining for quadriceps. (B) Quantitation and calculation of the fibrotic area in the quadriceps. In normal conditions, no fibrotic area was found in quadriceps in both NC and MFE group. However, by 14 days after induction of CLI, the fibrotic area in MFE group was significantly higher than that in the NC group. NC, normal control; MFE, maternal fructose exposure; CLI, critical limb ischemia. Scale bar in the lower-right corner indicates 200 μm. Error bars represent the standard deviation (SD). Groups with different symbols (*, †, ‡), p< 0.05. n = 8 for each group.

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  Figure 6. Maternal fructose exposure regulated expression and phosphorylation levels of angiogenesis-associated signaling in bone marrow cells. (A) Western blots to detect the expression and phosphorylation levels of several intracellular signaling proteins, including Akt, ERK1/2, p38-MAPK, and JNK in peripheral mononuclear cells (PBMC) of offspring 14 days post induction of CLI. (B) Quantitative data represent the ratio of phosphorylated p38-MAPK expression to total p38-MAPK expression. (C) Quantitative data represent the ratio of phosphorylated ERK1/2 expression to total ERK1/2 expression. (D) Quantitative data represent the ratio of phosphorylated Akt expression to total Akt expression. (E) Quantitative data represent the ratio of phosphorylated JNK expression to total JNK expression. NC, normal control; MFE, maternal fructose exposure; CLI, critical limb ischemia. Error bars represent the standard deviation (SD). Groups with different symbols (*, †), p< 0.05. n = 8 for each group.

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  Figure 7. Maternal fructose exposure reduced the endothelium-mediated vasorelaxant response. (A,B) Hematoxylin & Eosin (H&E) staining for a cross-section of the aorta in NC offspring. (C,D) H&E staining for a cross-section of aorta in MFE offspring. (E) Endothelium-mediated relaxation to acetylcholine was assessed in aortic rings from 3-month-old offspring. Data were calculated as changes from the contraction induced by phenylephrine. NC, normal control; MFE, maternal fructose exposure; Ach, acetylcholine. Scale bar in the upper-left corner of (A) and (C) indicates 500 μm. Scale bar in the upper-left corner of (B) and (D) indicates 100 μm. Error bars represent the standard deviation (SD). * Indicates statistical significance between NC and MFE group. n = 8 for each group.