Deletion of TXNIP Mitigates High-Fat Diet-Impaired Angiogenesis and Prevents Inflammation in a Mouse Model of Critical Limb Ischemia

Figures

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  Figure 1. HFD-induced obesity and dyslipidemia in WT and TKO mice. (A) Body weight measured weekly for seven weeks in both WT and TKO mice (* p-value < 0.05 vs WT-ND or TKO-ND); (B) AUC analysis showed an increase in mice weight in WT-HFD and TKO-HFD groups compared to WT-ND and TKO-ND respectively; (n = 9–13, * p-value < 0.05 vs. WT-ND and # vs. TKO-ND) (C) Plasma total cholesterol and (D) triglyceride levels showed higher levels in both WT-HFD and TKO-HFD groups, compared to WT-ND. TKO-ND group showed no change in total cholesterol levels, but increased higher basal levels of triglycerides compared with WT-ND (n = 8, * p-value < 0.05 vs. WT-ND, # vs. TKO-ND). 2 × 2-way ANOVA analysis showed that diet exerted significant effect (p < 0.05) on body weight, total cholesterol and triglycerides level and that deletion of TXNIP exerted significant effect (p < 0.05) on triglycerides level. HFD: High-fat diet, WT: wild type, TKO: TXNIP knockout, AUC: area under the curve, ND: normal diet, TXNIP: thioredoxin interacting protein.

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  Figure 2. HFD-induced obesity impaired glucose tolerance at 4 weeks and 8 weeks in WT mice but not TKO. IPGTT was performed where peripheral blood glucose levels recorded over time after an IP bolus dose of 2 grams of glucose/kg mouse body weight (zero) and up to 120 min. (A) Blood glucose levels (mg/dL) are shown from various groups after 4 weeks of HFD; (B) AUC analysis showed that after 4 weeks, HFD significantly impaired peripheral blood glucose response in the WT-HFD group but no significant effect on TKO-HFD. TKO mice showed lower blood glucose level in both ND and HFD groups that were significant compared to WT-ND (n = 8, * p < 0.05 vs. WT-ND, # p < 0.05 vs. WT-HFD). (C) Blood glucose levels (mg/dL) are shown from various groups after 8 weeks of HFD; (D) AUC analysis showed that HFD impaired peripheral blood glucose response in WT-HFD group and TKO-HFD groups compared to WT-ND and TKO-ND groups, respectively. TKO mice showed lower blood glucose level compared to WT-mice that were significant compared to WT-HFD (n = 11, * p < 0.05 vs. WT-ND, $ p < 0.05 vs. TKO-ND, # p < 0.05 vs. WT-HFD). 2 × 2-way ANOVA analysis showed that both diet and TXNIP gene deletion exerted significant effect…

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  Figure 3. HFD significantly impaired post-ischemic vascular recovery in WT but not in TKO mice. (A) Representative image of blood flow of both limbs from WT-ND immediately following unilateral hindlimb ischemia (zero time-point); (B) Representative image of blood flow of both limbs from WT-ND 3 weeks post unilateral hindlimb ischemia; (C) Representative image of blood flow of both limbs from WT-HFD 3 weeks post unilateral hindlimb ischemia; (D) Relative Blood flow measured at baseline and monitored weekly after hindlimb ischemia from various groups; (E) AUC analysis showed that HFD significantly reduced blood flow in WT-HFD compared to WT-ND. In contrast, AUC levels from TKO-HFD were not significant from TKO-ND (n = 9–12, * p-value < 0.05 vs. WT-ND). 2 × 2-way ANOVA analysis showed diet exerted significant effect (p < 0.05) on vascular recovery.

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  Figure 4. HFD significantly impaired post-ischemic angiogenesis and vascular density in WT but not in TKO. (A–C) Representative images of skeletal muscles from ischemic side of various groups stained for CD31 (red), DAPI (blue) and Merge CD31 and DAPI (purple); (D) 2 × 2-way ANOVA statistical analysis showed a significant interaction among the groups, in which HFD significantly reduced CD31 count in WT-HFD compared to WT-ND. There was no change in CD31 count in TKO-HFD compared to TKO-ND. TKO mice showed lower CD31 count level in both ND and HFD groups that were significant compared to WT-ND (n = 6, * p < 0.05 gene diet interaction). DAPI: 4,6-Diamidino-2-phenylindole, dihydrochlorid

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  Figure 5. HFD triggered systemic inflammation and significantly increased post-ischemic infiltration of CD68+ cells in WT but not TKO. (A) Levels of plasma IL-1β were measured using ELISA and statistical analysis showed a strong trend of HFD to increase levels of plasma IL-1β in WT-HFD but it did not reach significance compared to WT-ND (n = 17–24, p = 0.059). 2 × 2-way ANOVA statistical analysis showed that TXNIP gene deletion exerted significant effect (p < 0.05) on plasma IL-1β level; (B) Representative images of skeletal muscles from the ischemic side from WT-HFD stained for CD68+ cells (red), and DAP (blue) from all groups; (C) 2 × 2-way ANOVA statistical analysis showed a significant interaction among the groups. HFD-induced CD68+ cells infiltration after ischemia in WT-HFD compared to ND-WT, TXNIP deletion abolished this effect. (n = 4–6, * p < 0.05 gene diet interaction).

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  Figure 6. Ischemia increased NLRP3 expression in WT but not in TKO. (A) Representative images of Western blots of NLRP3 and Tubulin in gastrocnemius muscles isolated from WT and TKO mice; (B) 2 × 2-way ANOVA statistical analysis showed a strong trend of increase in NLRP3 expression following ischemia in WT compared to control (n = 4–5, p = 0.06 vs. ND CT). HFD tended to increase NLRP3 expression in WT-HFD compared to WT-ND. 2 × 2-way ANOVA statistical analysis showed lack of gene effect where deleting TXNIP did not alter NLRP3 expression following ischemia or HFD compared to WT-ND CT or TKO-ND CT (n = 5).

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  Figure 7. TXNIP deletion prevented HFD-induced NLRP3-inflammasome activation in skeletal muscles; (A) Representative images of Western blots of cleaved caspase-1, cleaved IL-1β and Tubulin in gastrocnemius muscles isolated from WT and TKO mice; (B) 2 × 2-way ANOVA statistical analysis showed that HFD increased activation of NLRP3 inflammasome evident by significant increases in cleaved caspase-1 in WT-HFD compared to WT-ND but not in TKO mice compared to WT-ND or TKO-ND (n = 3–5, * p < 0.05 vs. ND CT); (C) 2 × 2-way ANOVA statistical analysis showed that HFD increased cleaved IL-1β in WT-HFD compared to WT-ND CT but not in TKO mice compared to WT-ND CT or TKO-ND CT (n = 3–5, * p < 0.05 vs. ND CT).

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  Figure 8. TXNIP deletion prevented ischemia/HFD-induced oxidative stress in skeletal muscles. (A) Representative images of slot blots of nitrotyrosine levels in gastrocnemius muscles isolated from WT and TKO, respectively, were extracted from the same gel and same exposure; (B) 2 × 2-way ANOVA statistical analysis showed that HFD from both control (CT) and ischemic (IS) muscles significantly increased nitration in WT-HFD compared to WT-ND (n = 4–5, * p < 0.05 vs. ND CT). Overall, diet exerted significant effect but ischemia showed a trend of increase in nitration compared to control in WT mice but it did not reach statistical significance (n = 4). (C) Deleting TXNIP abolished the increase in nitration following ischemia or HFD compared to TKO-ND (n = 4).

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  Figure 9. HFD impaired post-ischemic VEGFR2 activation but not levels of VEGF in WT mice. (A) Representative images of Western blots of VEGF and Tubulin in gastrocnemius muscles isolated from WT and TKO mice; (B) 2 × 2-way ANOVA statistical analysis showed a trend of increase in VEGF expression following ischemia in WT and TKO compared to control but it did not reach statistical significance (n = 7); (C) Representative images of Western blots of pVEGFR2 and VEGFR2 in gastrocnemius muscles isolated from WT and TKO mice; (D) 2 × 2-way ANOVA statistical analysis showed a significant increase in phosphorylation of VEGFR2 following ischemia in WT-ND that was mitigated in the HFD group compared to non-ischemic control (n = 5–6, * p < 0.05 vs. ND CT). 2 × 2-way ANOVA statistical analysis showed lack of gene effect where deleting TXNIP had minimal effect of altering pVEGFR2 in response to ischemia or HFD compared to WT-ND or TKO-ND (n = 6).