Rosiglitazone via upregulation of Akt/eNOS pathways attenuates dysfunction of endothelial progenitor cells, induced by advanced glycation end products

Abstract

Background and purpose: Advanced glycation end products (AGEs) and endothelial progenitor cells (EPCs) play key roles in pathogenesis of diabetes-related vascular complications. AGEs can induce dysfunction in EPCs. The peroxisome proliferator-activated receptor-gamma (PPARgamma) agonists are widely used in the treatment of type 2 diabetes, and it remains unknown if they could attenuate EPC dysfunction induced by AGEs.

Experimental approach: EPCs isolated from healthy adults were cultured with various concentrations of AGEs (0, 50, 100 and 200 mg L(-1)) with or without rosiglitazone (10 nM), antibody for the receptors for AGE-human serum albumin (anti-receptor for advanced glycation end products (RAGE); 50 microg mL(-1)), phosphatidylinositol-3-kinase (PI3K) inhibitor (LY294002, 5 microM), nitric oxide (NO) synthase inhibitor (L-N(G)-nitro-arginine methyl ester (L-NAME), 100 microM) or sodium nitroprusside (SNP, 25 microM). Proliferation, apoptosis, cell adhesion, migration and NO production in EPCs were assessed, and expressions of endothelial NO synthase (eNOS) and Akt were determined.

Key results: Number, proliferation/migration capacities, eNOS and Akt phosphorylation as well as NO synthesized by EPCs were increased by rosiglitazone and reduced by AGEs. AGEs promoted while rosiglitazone reduced EPC apoptosis. The AGE-induced effects were significantly ameliorated by pre-incubation with rosiglitazone, RAGE antibody and SNP. The beneficial effects of rosiglitazone could be blocked by pretreatment with L-NAME and LY294002.

Conclusions and implications: The PPARgamma agonist rosiglitazone increased EPC function and attenuated EPC dysfunction induced by AGEs via upregulating the Akt-eNOS signal pathways of EPCs.

Figure 1

Effect of AGE-HSA and rosiglitazone on the proliferation (A), apoptosis (B), adhesion (C) and migration (D) capacities of EPCs. (A) Rosiglitazone (Rosig) increased EPC proliferation and reversed the inhibited proliferation induced by AGE-HSA: *P < 0.05 versus control, HSA (200 µg·mL⁻¹), AGE-HSA (50 µg·mL⁻¹); #P < 0.05 versus AGE-HSA (200 µg·mL⁻¹). (B) Effect of AGE-HSA and rosiglitazone on apoptosis of EPCs: *P < 0.05 versus control and HSA (200 µg·mL⁻¹); #P < 0.05 versus AGE-HSA (200 µg·mL⁻¹). (C) Effect of AGE-HSA and rosiglitazone on adhesion of EPCs. *P < 0.05 versus control and HSA (200 µg·mL⁻¹); #P < 0.05 versus AGE-HSA (200 µg·mL⁻¹). (D) Effect of AGE-HSA and rosiglitazone on migration of EPCs: *P < 0.05 versus control and HSA (200 µg·mL⁻¹); #P < 0.05 versus AGE-HSA (200 µg·mL⁻¹). Data are mean ± SEM for four independent experiments. AGE, advanced glycation end product; EPC, endothelial progenitor cell; HSA, human serum albumin; HPF, high-power (×100) microscope fields.

Figure 2

Effect of AGE-HSA, the anti-RAGE antibody and rosiglitazone on expression of phosphorylated-Akt (p-Akt) in EPCs. *P < 0.05 versus control and HSA (200 µg·mL⁻¹); #P < 0.05 versus AGE-HSA (AGEs; 200 µg·mL⁻¹). †P < 0.05 versus rosiglitazone (Rosig; 10 nM). Data are mean ± SEM for three independent experiments. AGE, advanced glycation end product; EPC, endothelial progenitor cell; HAS, human serum albumin; RAGE, receptor for advanced glycation end products.

Effect of AGE-HSA, the anti-RAGE antibody and rosiglitazone on expression of phosphorylated-eNOS (p-eNOS) in EPCs. *P < 0.05 versus control and HSA (200 µg·mL⁻¹); #P < 0.05 versus AGE-HSA (AGEs; 200 µg·mL⁻¹); †P < 0.05 versus rosiglitazone (Rosig; 10 nM). Data are mean ± SEM for four independent experiments. AGE, advanced glycation end product; EPC, endothelial progenitor cell; HSA, human serum albumin; RAGE, receptor for advanced glycation end products.

Figure 4

Effect of AGE-HSA, the anti-RAGE antibody, Rosiglitazone, SNP, Ly29002 and L-NAME on the proliferation, apoptosis, adhesion, migration capacities, and NO production of EPCs. (A) Rosiglitazone, anti RAGE antibody and SNP significantly reversed the inhibition by AGE-HSA of proliferation of EPCs, which could be blocked by LY29002 and L-NAME: *P < 0.05 versus control; #P < 0.05 versus AGE-HSA (AGEs; 200 µg·mL⁻¹); †P < 0.05 versus rosiglitazone (Rosig; 10 nM). (B) Rosiglitazone, anti RAGE antibody and SNP significantly attenuated AGE-HSA induced EPCs apoptosis, and this effect could be blocked by LY29002 and L-NAME: *P < 0.05 versus control; #P < 0.05 versus AGE-HSA (200 µg·mL⁻¹); †P < 0.05 versus rosiglitazone (10 nM). (C) Rosiglitazone, anti RAGE antibody and SNP significantly attenuated the inhibition effect of AGE-HSA on adhesion capacity of EPCs, which could be blocked by LY29002 and L-NAME: *P < 0.05 versus control; #P < 0.05 versus AGE-HSA (200 µg·mL⁻¹); †P < 0.05 versus rosiglitazone (10 nM). (D) Rosiglitazone, anti RAGE antibody and SNP significantly reversed the inhibition by AGE-HSA of migration of EPCs, addition of LY29002 and L-NAME significantly blocked this effect: *P < 0.05 versus control; #P < 0.05 versus AGE-HSA (200 µg·mL⁻¹); †P < 0.05 versus rosiglitazone (10 nM). E, Rosiglitazone, anti RAGE antibody and SNP significantly reversed the inhibitory effect of AGE-HSA on NO production of EPCs, and LY29002 and L-NAME blocked this effect: *P < 0.05 versus control; #P < 0.05 versus AGE-HSA (200 µg·mL⁻¹); †P < 0.05 versus rosiglitazone (10 nM). Data are mean ± SEM for three independent experiments. AGE, advanced glycation end product; EPC, endothelial progenitor cell; HAS, human serum albumin; HPF, high-power (×100) microscope fields; L-NAME, L-N^G-nitro-arginine methyl ester; RAGE, receptor for advanced glycation end products; SNP, sodium nitroprusside.