Angiogenic Factor AGGF1-Primed Endothelial Progenitor Cells Repair Vascular Defect in Diabetic Mice

Abstract

Hyperglycemia-triggered vascular abnormalities are the most serious complications of diabetes mellitus (DM). The major cause of vascular dysfunction in DM is endothelial injury and dysfunction associated with the reduced number and dysfunction of endothelial progenitor cells (EPCs). A major challenge is to identify key regulators of EPCs to restore DM-associated vascular dysfunction. We show that EPCs from heterozygous knockout Aggf1^+/- mice presented with impairment of proliferation, migration, angiogenesis, and transendothelial migration as in hyperglycemic mice fed a high-fat diet (HFD) or db/db mice. The number of EPCs from Aggf1^+/- mice was significantly reduced. Ex vivo, AGGF1 protein can fully reverse all damaging effects of hyperglycemia on EPCs. In vivo, transplantation of AGGF1-primed EPCs successfully restores blood flow and blocks tissue necrosis and ambulatory impairment in HFD-induced hyperglycemic mice or db/db mice with diabetic hindlimb ischemia. Mechanistically, AGGF1 activates AKT, reduces nuclear localization of Fyn, which increases the nuclear level of Nrf2 and expression of antioxidative genes, and inhibits reactive oxygen species generation. These results suggest that Aggf1 is required for essential function of EPCs, AGGF1 fully reverses the damaging effects of hyperglycemia on EPCs, and AGGF1 priming of EPCs is a novel treatment modality for vascular complications in DM.

Figure 1

Aggf1 is required for essential functions of EPCs in capillary tube formation, proliferation, TEM, and migration as in a diabetic mouse model (db/db mice). EPCs were isolated from bone marrow of WT, heterozygous Aggf1^+/− KO mice, db/db mice, and Aggf1^+/−db/db mice and characterized. A: Aggf1 haploinsufficiency inhibits angiogenesis mediated by EPCs. B: Images from panel A were analyzed, quantified, and plotted. C: Aggf1 haploinsufficiency inhibits EPC proliferation. D: Aggf1 haploinsufficiency inhibits TEM of EPCs in transwell assays. E: Images from panel D were quantified and plotted. F: Aggf1 haploinsufficiency inhibits EPC migration in scratch wound migration assays. G: Representative images from EPC migration assays. Data are mean ± SD. *P < 0.05, **P < 0.01 (n = 6 mice/group).

Figure 2

AGGF1 dramatically improves essential functions of EPCs impaired by HG. A: Western blot analysis showing increased AGGF1 expression in EPCs. B: AGGF1 treatment reversed the impairment of angiogenic function of EPCs by HG. C: Images from panel B were analyzed, quantified, and plotted. D: AGGF1 treatment reversed the reduced cell proliferation of EPCs by HG. E: AGGF1 treatment reversed the HG-impaired TEM of EPCs. F: Images from panel E were quantified and plotted. G: AGGF1 treatment reversed the HG-impaired migration of EPCs in scratch wound migration assays. H: Representative images from EPC migration assays. Data are mean ± SD. **P < 0.01 (n = 6 mice/group).

Figure 3

AGGF1 protein treatment robustly potentiates the therapeutic effects of EPCs on peripheral vascular complications in a hindlimb ischemia model in db/db mice. A hindlimb ischemia model was created in db/db mice. A: Transplantation of AGGF1-pretreated EPCs dramatically improved blood perfusion compared with EPCs without AGGF1 pretreatment in db/db mice. B: Therapeutic effects of AGGF1-pretreated EPCs on necrosis compared with EPCs without AGGF1 pretreatment. C: Therapeutic effects of AGGF1-pretreated EPCs on ambulatory impairment compared with EPCs without AGGF1 pretreatment. D: Effects of AGGF1-pretreated EPCs on the density of CD31⁺ capillary vessels compared with EPCs without AGGF1 pretreatment. *P < 0.05, **P < 0.01. EB, elution buffer; NC, negative control.

Figure 4

AGGF1 regulates the nuclear accumulation of Nrf2. A: Western blot analysis for the effect of AGGF1, HG, and HG + AGGF1 on the expression levels of n-Nrf2 and its downstream signaling molecules, including HO-1, NQO-1, and CAT. GAPDH was used as loading control. B: AGGF1 protein treatment induces expression of Nrf2 downstream antioxidative genes in EPCs. Data are mean ± SD. **P < 0.01 (n = 3/group).

Figure 5

AGGF1 activates AKT-Fyn-Nrf2 signaling in EPCs. A and B: Western blot analysis showing the effect of siRNA for AKT (siAKT) and wortmannin on AGGF1-activated phosphorylation of AKT (p-AKT) and n-Fyn. C–E: Western blot analysis showing the effect of siAKT, wortmannin, and siRNA for Nrf2 (siNrf2) on AGGF1-induced increases of n-Nrf2 and its downstream signaling molecules. Data are mean ± SD. *P < 0.05, **P < 0.01 (n = 3/group). t-AKT, total AKT.

Figure 6

Knockdown of AKT expression, wortmannin treatment, and knockdown of Nrf2 expression attenuate the protective effects of AGGF1 on EPCs. A: Western blot analysis for AGGF1 in EPCs. B–E: Effects of AKT siRNA on AGGF1-mediated rescue of HG-impaired cell proliferation (B), tube formation (C), TEM (D), and cell migration (E) by EPCs. F: Western blot analysis for AGGF1 in EPCs. G–J: Effects of wortmannin on AGGF1-mediated rescue of HG-impaired cell proliferation (G), tube formation (H), TEM (I), and cell migration (J) by EPCs. K: Western blot analysis for AGGF1 in EPCs. L–O: Effects of Nrf2 siRNA on AGGF1-mediated rescue of HG-impaired cell proliferation (L), tube formation (M), TEM (N), and cell migration (O) by EPCs. Data are mean ± SD. **P < 0.01 (n = 6 mice/group).

Figure 6

Knockdown of AKT expression, wortmannin treatment, and knockdown of Nrf2 expression attenuate the protective effects of AGGF1 on EPCs. A: Western blot analysis for AGGF1 in EPCs. B–E: Effects of AKT siRNA on AGGF1-mediated rescue of HG-impaired cell proliferation (B), tube formation (C), TEM (D), and cell migration (E) by EPCs. F: Western blot analysis for AGGF1 in EPCs. G–J: Effects of wortmannin on AGGF1-mediated rescue of HG-impaired cell proliferation (G), tube formation (H), TEM (I), and cell migration (J) by EPCs. K: Western blot analysis for AGGF1 in EPCs. L–O: Effects of Nrf2 siRNA on AGGF1-mediated rescue of HG-impaired cell proliferation (L), tube formation (M), TEM (N), and cell migration (O) by EPCs. Data are mean ± SD. **P < 0.01 (n = 6 mice/group).

Figure 6

Knockdown of AKT expression, wortmannin treatment, and knockdown of Nrf2 expression attenuate the protective effects of AGGF1 on EPCs. A: Western blot analysis for AGGF1 in EPCs. B–E: Effects of AKT siRNA on AGGF1-mediated rescue of HG-impaired cell proliferation (B), tube formation (C), TEM (D), and cell migration (E) by EPCs. F: Western blot analysis for AGGF1 in EPCs. G–J: Effects of wortmannin on AGGF1-mediated rescue of HG-impaired cell proliferation (G), tube formation (H), TEM (I), and cell migration (J) by EPCs. K: Western blot analysis for AGGF1 in EPCs. L–O: Effects of Nrf2 siRNA on AGGF1-mediated rescue of HG-impaired cell proliferation (L), tube formation (M), TEM (N), and cell migration (O) by EPCs. Data are mean ± SD. **P < 0.01 (n = 6 mice/group).