Elevating CXCR7 Improves Angiogenic Function of EPCs via Akt/GSK-3β/Fyn-Mediated Nrf2 Activation in Diabetic Limb Ischemia

Abstract

Rationale: Endothelial progenitor cells (EPCs) respond to stromal cell-derived factor 1 (SDF-1) through chemokine receptors CXCR7 and CXCR4. Whether SDF-1 receptors involves in diabetes mellitus-induced EPCs dysfunction remains unknown.

Objective: To determine the role of SDF-1 receptors in diabetic EPCs dysfunction.

Methods and results: CXCR7 expression, but not CXCR4 was reduced in EPCs from db/db mice, which coincided with impaired tube formation. Knockdown of CXCR7 impaired tube formation of EPCs from normal mice, whereas upregulation of CXCR7 rescued angiogenic function of EPCs from db/db mice. In normal EPCs treated with oxidized low-density lipoprotein or high glucose also reduced CXCR7 expression, impaired tube formation, and increased oxidative stress and apoptosis. The damaging effects of oxidized low-density lipoprotein or high glucose were markedly reduced by SDF-1 pretreatment in EPCs transduced with CXCR7 lentivirus but not in EPCs transduced with control lentivirus. Most importantly, EPCs transduced with CXCR7 lentivirus were superior to EPCs transduced with control lentivirus for therapy of ischemic limbs in db/db mice. Mechanistic studies demonstrated that oxidized low-density lipoprotein or high glucose inhibited protein kinase B and glycogen synthase kinase-3β phosphorylation, nuclear export of Fyn and nuclear localization of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), blunting Nrf2 downstream target genes heme oxygenase-1, NAD(P)H dehydrogenase (quinone 1) and catalase, and inducing an increase in EPC oxidative stress. This destructive cascade was blocked by SDF-1 treatment in EPCs transduced with CXCR7 lentivirus. Furthermore, inhibition of phosphatidylinositol 3-kinase/protein kinase B prevented SDF-1/CXCR7-mediated Nrf2 activation and blocked angiogenic repair. Moreover, Nrf2 knockdown almost completely abolished the protective effects of SDF-1/CXCR7 on EPC function in vitro and in vivo.

Conclusions: Elevated expression of CXCR7 enhances EPC resistance to diabetes mellitus-induced oxidative damage and improves therapeutic efficacy of EPCs in treating diabetic limb ischemia. The benefits of CXCR7 are mediated predominantly by a protein kinase B/glycogen synthase kinase-3β/Fyn pathway via increased activity of Nrf2.

Keywords: CXC chemokine receptor type 7; angiogenesis; chemokine CXCL12; endothelial progenitor cells; hindlimb ischemia.

Figure 1

Diabetes attenuates CXCR7 expression and impairs the angiogenic function of EPCs. A-C, Early EPCs were isolated from bone marrow of WT (WT-EPCs) and db/db (db/db-EPCs) mice at 10-12 weeks of age and assayed within 7 days of isolation. Expression of CXCR7 and CXCR4 was detected by Western blot (A) and flow cytometry (B). Angiogenic function of EPCs was evaluated by tube formation assay (C). D, Early WT-EPCs were transfected with specific siRNA against mouse CXCR7 (CXCR7-siRNA) or Silencer Select Negative Control (Ctrl-siRNA), and the transfection reagent (Lipofectamine 2000, Lipo 2000) was used as a blank control; early db/db-EPCs were infected with purified lentivirus carrying recombinant CXCR7 (Lv-CXCR7) or control vector (Lv-Ctrl), and phosphate buffered saline (PBS) was used as vehicle control (Vehicle); Efficiency of CXCR7 knockdown or upregulation was determined by Western blot (shown in Online Figure II) and angiogenic function of EPCs was evaluated by tube formation assay. n=6 mice per group for A, B and C, and three independent experiments were performed for D. Data shown in graphs represents the Means ± SD. *p<0.05, vs WT-EPC for A-C or WT-EPC with Lipo 2000 treatment for D, # P< 0.05, vs db/db-EPCs with vehicle treatment; & P<0.05, vs db/db-EPCs with Lv-Ctrl infection.

Figure 2

Upregulation of CXCR7 expression rescues angiogenic function of db/db-EPCs in ischemic limb of db/db diabetic mice. A, Time course of blood perfusion shown in images and quantitative analysis after hind limb ischemia (HLI) surgery with or without EPC transplantation. Blood perfusion is the ratio of ischemic to non-ischemic limb perfusion measured by a Pericam Perfusion Speckle Imager (PSI). B & C, Images and quantitation of immunofluorescent isolectin- and/or GFP-positive capillaries in transverse sections of soleus muscle (B) and gastrocnemius muscle (C) tissue from ischemic hind limbs. Capillary density was expressed as isolectin-positive capillaries per muscle fiber. The exogenous EPC incorporation (white arrows) was expressed as the percentage of GFP positive capillaries. D, Representative images and quantitation of alpha smooth muscle actin (α-SMA) positive arteriole in the ischemic adductor muscle tissue. Arteriogenesis was expressed as α-SMA positive arteriole area per fiber normalized by PBS control. Data shown in graphs represents the Means ± SD. n=8 mice per group. * p<0.05, vs PBS; # p<0.05, vs Lv-Ctrl.

Figure 3

Upregulating CXCR7 expression protects EPCs from ox-LDL-induced apoptosis and angiogenic dysfunction. A, The apoptosis of EPCs was analyzed by flow cytometry analysis using Annexin V/propidium iodide (PI) staining after exposure to ox-LDL (50μg/mL, 24 h). Apoptotic cells were defined as Annexin V⁺/PI^- (Quadrant 4). B, The effects of CXCR7 upregulation on the angiogenic function of EPCs under ox-LDL treatment condition were determined by tube formation assay. Tube length was normalized to the control Null-EPC group. C, The trans-endothelial migration of EPCs was analyzed by trans-well assay. Images are representatives of 3 independent experiments. Data shown in graphs represents the Mean ± SD. * P<0.05, vs respective control in Null-EPCs or CXCR7-EPCs; # P< 0.05, vs Null-EPCs with the same treatment; & P<0.05, vs CXCR7-EPCs with ox-LDL treatment. CXCR7-EPCs or Null-EPCs: EPCs from WT mice were transduced with CXCR7 recombinant lentiviral vector or control vector.

Figure 4

Upregulating CXCR7 expression attenuates the superoxide level and oxidative damage in EPCs induced by ox-LDL. A, Fluorescent Images and quantitation of superoxide levels in EPCs treated with or without ox-LDL (50 μg/mL) in the presence or absence of SDF-1 (100 ng/mL) for 6 h. Superoxide was determined with the fluorescent indicator DHE, and the fluorescent intensity of DHE was measured by a fluorescent microplate reader. B, Levels of the oxidative damage marker 3-nitroryrosine (3-NT) in EPCs treated with or without ox-LDL (50μg/ml) in the presence or absence of SDF-1 (100 ng/mL) for 12 h was detected by Western blot. Three independent experiments were performed. Data shown in graphs represents the Mean ± SD. * P<0.05, vs respective control in Null-EPCs or CXCR7-EPCs; # P< 0.05, vs Null-EPCs with the same treatment; & P<0.05, vs CXCR7-EPCs with ox-LDL treatment.

Figure 5

Upregulating CXCR7 activates nuclear Nrf2 signaling in EPCs. Null-EPCs or CXCR7-EPCs were exposed to ox-LDL (0 or 50μg/ml) in the presence or absence of SDF-1 (100 ng/mL) for 12 h. A, Protein levels of Nrf2 and its downstream target genes HO-1, NQO-1 and CAT and nuclear expression of Nrf2 (n-Nrf2) were detected by Western blot. B, The mRNA expression of Nrf2 and its downstream target genes HO-1, NQO-1 and CAT were determined by real-time PCR. C, Nrf2 nuclear translocation was determined in fixed cells by immunofluorescent staining. Three independent experiments were performed. Data shown in graphs represents the Means ± SD. * P<0.05, vs respective control in Null-EPCs or CXCR7-EPCs; # P< 0.05, vs Null-EPC with the same treatment; & P<0.05, vs CXCR7-EPC with ox-LDL treatment. NQO-1: NAD(P)H dehydrogenase quinone 1; HO-1: Heme oxygenase-1; CAT: catalase.

Figure 6

Upregulating CXCR7 activates Nrf2 via Akt/GSK-3β/Fyn pathway. Null-EPCs or CXCR7-EPCs were pretreated with or without PI3K inhibitor wortmannin for 30 min, and then exposed to ox-LDL (50μg/ml) for 12 h in the presence or absence of SDF-1 (100 ng/mL). A, The phosphorylation of Akt and GSK-3β, and the nuclear translocation of Fyn (n-Fyn) were evaluated by Western blot. B, C, The expression of nuclear Nrf2 (n-Nrf2) and its downstream target genes (HO-1, NQO-1 and CAT) was evaluated by Western blot (B) and real-time PCR (C). The results were normalized to the control group of Null-EPCs. D, The apoptosis of EPCs was analyzed by flow cytometry using Annexin V/PI staining. E, The angiogenic function of EPCs was determined by tube formation assay, the tube length was normalized to the control group of Null-EPCs. F, The trans-endothelial migration abilities of EPCs was analyzed by trans-well assay. Three independent experiments were performed for each study. Data shown in graphs represents the Means ± SD. * P<0.05 vs respective control in Null-EPCs or CXCR7-EPCs; # P< 0.05 vs Null-EPC with the same treatment; & P<0.05 vs CXCR7-EPC with ox-LDL treatment in presence of SDF-1.

Figure 6

Upregulating CXCR7 activates Nrf2 via Akt/GSK-3β/Fyn pathway. Null-EPCs or CXCR7-EPCs were pretreated with or without PI3K inhibitor wortmannin for 30 min, and then exposed to ox-LDL (50μg/ml) for 12 h in the presence or absence of SDF-1 (100 ng/mL). A, The phosphorylation of Akt and GSK-3β, and the nuclear translocation of Fyn (n-Fyn) were evaluated by Western blot. B, C, The expression of nuclear Nrf2 (n-Nrf2) and its downstream target genes (HO-1, NQO-1 and CAT) was evaluated by Western blot (B) and real-time PCR (C). The results were normalized to the control group of Null-EPCs. D, The apoptosis of EPCs was analyzed by flow cytometry using Annexin V/PI staining. E, The angiogenic function of EPCs was determined by tube formation assay, the tube length was normalized to the control group of Null-EPCs. F, The trans-endothelial migration abilities of EPCs was analyzed by trans-well assay. Three independent experiments were performed for each study. Data shown in graphs represents the Means ± SD. * P<0.05 vs respective control in Null-EPCs or CXCR7-EPCs; # P< 0.05 vs Null-EPC with the same treatment; & P<0.05 vs CXCR7-EPC with ox-LDL treatment in presence of SDF-1.

Figure 7

Knockdown of Nrf2 attenuates the protective effects of CXCR7 upregulation on EPCs. CXCR7-EPCs or Null-EPCs were transfected with lentivirus vector encoding Nrf2 shRNA (CXCR7/sh-Nrf2-EPCs) or control shRNA (CXCR7/sh-Ctrl-EPCs or Null/sh-Ctrl-EPCs). Following shRNA transfection the protective effects of SDF-1/CXCR7 on EPC survival, angiogenesis and trans-endothelial migration were evaluated as described in Figure 6. A, Apoptosis was analyzed by flow cytometry. B, Angiogenic function was determined by tube formation assay. C, Trans-endothelial migration was analyzed by trans-well assay. Three independent experiments were performed for each study. Data shown in graphs represents the Means ± SD. * P<0.05 vs respective control in Null/sh-Ctrl-EPCs, CXCR7/sh-Ctrl-EPCs or CXCR7/sh-Nrf2-EPCs; # P< 0.05 vs Null/sh-Ctrl-EPC with the same treatment; & P<0.05 vs CXCR7/sh-Ctrl-EPC with the same treatment.

Figure 8

Knockdown of Nrf2 attenuates the beneficial effects of CXCR7 upregulation on EPC mediated angiogenesis in ischemic limb of db/db diabetic mice. The effects of Nrf2 knockdown on the beneficial effects of CXCR7 upregulation on EPC mediated angiogenesis and blood perfusion were evaluated in the HLI model in db/db diabetic mice as described in Figure 2 and Online Figure VI. A, Time course of blood perfusion after HLI surgery with or without EPCs transplantation shown in images and quantitative data analysis. Blood perfusion is the ratio of ischemic to non-ischemic limb perfusion measured by PSI. B, Immunofluorescent staining and quantitation of isolectin-positive capillaries (white arrows) in transverse sections of gastrocnemius muscle tissue from ischemic hind limbs 28 days after HLI surgery. Capillary density was expressed as isolectin-positive capillaries per muscle fiber. n=8 mice per group. Data shown in graphs represents the Means ± SD. * P<0.05 vs PBS, # P< 0.05 vs Null/sh-Ctrl-EPC group; & P<0.05 vs CXCR7/sh-Ctrl-EPC group. C, Schematic illustration of the protective effects of SDF-1/CXCR7 on EPCs under diabetic conditions. Diabetes decreases expression of CXCR7 in EPCs and induces oxidative stress, which impairs the survival and angiogenic function of EPCs. Under diabetic conditions upregulation of SDF-1/CXCR7 signaling improves EPC survival and function predominantly by Nrf2 activation mediated by increasing phosphorylation of Akt and GSK-3β and inhibiting Fyn-mediated export and degradation of nuclear Nrf2.

Figure 8

Knockdown of Nrf2 attenuates the beneficial effects of CXCR7 upregulation on EPC mediated angiogenesis in ischemic limb of db/db diabetic mice. The effects of Nrf2 knockdown on the beneficial effects of CXCR7 upregulation on EPC mediated angiogenesis and blood perfusion were evaluated in the HLI model in db/db diabetic mice as described in Figure 2 and Online Figure VI. A, Time course of blood perfusion after HLI surgery with or without EPCs transplantation shown in images and quantitative data analysis. Blood perfusion is the ratio of ischemic to non-ischemic limb perfusion measured by PSI. B, Immunofluorescent staining and quantitation of isolectin-positive capillaries (white arrows) in transverse sections of gastrocnemius muscle tissue from ischemic hind limbs 28 days after HLI surgery. Capillary density was expressed as isolectin-positive capillaries per muscle fiber. n=8 mice per group. Data shown in graphs represents the Means ± SD. * P<0.05 vs PBS, # P< 0.05 vs Null/sh-Ctrl-EPC group; & P<0.05 vs CXCR7/sh-Ctrl-EPC group. C, Schematic illustration of the protective effects of SDF-1/CXCR7 on EPCs under diabetic conditions. Diabetes decreases expression of CXCR7 in EPCs and induces oxidative stress, which impairs the survival and angiogenic function of EPCs. Under diabetic conditions upregulation of SDF-1/CXCR7 signaling improves EPC survival and function predominantly by Nrf2 activation mediated by increasing phosphorylation of Akt and GSK-3β and inhibiting Fyn-mediated export and degradation of nuclear Nrf2.