Insulinlike Growth Factor-Binding Protein-1 Improves Vascular Endothelial Repair in Male Mice in the Setting of Insulin Resistance

Abstract

Insulin resistance is associated with impaired endothelial regeneration in response to mechanical injury. We recently demonstrated that insulinlike growth factor-binding protein-1 (IGFBP1) ameliorated insulin resistance and increased nitric oxide generation in the endothelium. In this study, we hypothesized that IGFBP1 would improve endothelial regeneration and restore endothelial reparative functions in the setting of insulin resistance. In male mice heterozygous for deletion of insulin receptors, endothelial regeneration after femoral artery wire injury was enhanced by transgenic expression of human IGFBP1 (hIGFBP1). This was not explained by altered abundance of circulating myeloid angiogenic cells. Incubation of human endothelial cells with hIGFBP1 increased integrin expression and enhanced their ability to adhere to and repopulate denuded human saphenous vein ex vivo. In vitro, induction of insulin resistance by tumor necrosis factor α (TNFα) significantly inhibited endothelial cell migration and proliferation. Coincubation with hIGFBP1 restored endothelial migratory and proliferative capacity. At the molecular level, hIGFBP1 induced phosphorylation of focal adhesion kinase, activated RhoA and modulated TNFα-induced actin fiber anisotropy. Collectively, the effects of hIGFBP1 on endothelial cell responses and acceleration of endothelial regeneration in mice indicate that manipulating IGFBP1 could be exploited as a putative strategy to improve endothelial repair in the setting of insulin resistance.

Figure 1.

Endothelial regeneration after wire injury of the femoral artery. IGFBP-1 rescues endothelial regeneration in insulin-resistant mice. (A) Representative in situ Evans blue staining 5 days after vascular injury (blue staining indicates denuded endothelium) in WT, IGFBP1-tg, IR^+/− and IR^+/− IGFBP1-tg mice (magnification ×20). (B) Endothelial regeneration 5 days after vascular injury in WT and IGFBP-1tg mice (n = 7 mice per group). No significant difference was seen between WT and IGFBP-1tg mice. (C) Endothelial regeneration 5 days after vascular wire injury in WT, IR^+/− and IR^+/−IGFBP1-tg mice (n = 5 to 10 per group). Data shown are mean +/− SEM. *P < 0.05; **P < 0.01.

Figure 2.

Progenitor cell abundance and function. (A–C) Enumeration of MACs derived from blood, spleen, and bone marrow by cell culture after 7 days. Numbers of (A) peripheral blood–derived (n = 5 to 6), (B) spleen-derived (n = 6), and (C) bone marrow–derived (n = 6 to 9) cultured MACs from uninjured mice are shown. *P < 0.05. (D) Adhesion capacity of spleen-derived MACs expressed as number of cells adhering to fibronectin-coated plates (n = 5 to 6). No significant difference between groups was observed. (E) Enumeration of circulating Sca-1⁺/Flk-1⁺ cells. Number of Sca-1⁺/Flk-1⁺ cells was quantified in peripheral blood by flow cytometry. *P < 0.05. (n = 6). Data shown are mean +/− SEM.

Figure 3.

hIGFBP1 improves adhesion of human endothelial cells to denuded human saphenous vein and upregulates cell-surface integrins. (A and B) Representative images of cell-tracker–labeled HCAECs adherent to denuded saphenous vein after preincubation with (A) control medium or (B) hIGFBP1 (500 ng/mL) for 60 minutes (magnification ×10). (C) Significantly more cells were adherent to the saphenous vein after preincubation with hIGFBP1 (500 ng/mL; 60 minutes) (n = 5). ***P < 0.001. (D) Adhesion of HCAECs to glass coverslips. HCAECs were incubated in 1% FCS with or without hIGFBP1 (500 ng/mL) for indicated times before cells were fixed with paraformaldehyde and stained with hematoxylin and eosin. Adherent cells were quantified in 10 random fields at ×400 magnification. There were no significant differences between control and hIGFBP1-treated cells at each time point. (E and F) Cell-surface integrin expression. HCAECs were incubated with or without hIGFBP1 (500 ng/mL) for 1 hour before quantification of cell-surface integrins by using an integrin-mediated cell adhesion array kit (Millipore). Expression of α-integrins (E) and β-integrins (F) are indicated (n = 6). Data shown are mean +/− SEM. *P < 0.05; **P < 0.01.

Figure 4.

hIGFBP1 abrogates TNFα-induced inhibition of endothelial cell (EC) migration. (A and B) No significant difference in migration in response to hIGFBP1 (500 ng/mL; 48 hours) was observed in (A) HUVECs or (B) HCAECs in a scratch wound healing assay (n = 3). (C and D) Preincubation with TNFα (10 ng/mL) for the indicated times inhibited insulin-stimulated (100 nmol/L; 15 minutes) Akt phosphorylation in HUVECs. (C) Representative immunoblot and (D) mean data of phospho Akt (pAkt)/Akt ratio are shown. (E and F) hIGFBP1 (500 ng/mL) partially restored endothelial migratory responses after exposure to TNFα (10 mg/mL) in scratch wound assays. (E) HUVECs (n = 9). *P < 0.01. (F) HCAECs (n = 6). *P < 0.05. Data shown are mean +/− SEM.

Figure 5.

IGFBP-1 does not act as chemotactic agent for endothelial cell migration in Boyden chamber assays. (A) Effect of hIGFBP1 (500 ng/mL; 6 hours) on migration in HUVECs (n = 3). No significant difference was seen. (B) Effect of hIGFBP1 (500 ng/mL; 6 hours) on migration in HCAECs (n = 5). P = 0.07. (C) Effects of IGFBP-1 (500 ng/mL; 6 hours) and VEGF (50 ng/mL; 6 hours) on cell migration (HCAECs) (n = 5). Control vs. VEGF: ** P < 0.01; VEGF vs. VEGF + IGFBP-1: no significant difference. Data shown are mean +/− SEM.

Figure 6.

hIGFBP1 improves endothelial cell proliferation in a proinflammatory setting. (A) HUVEC proliferation. Quiesced cells treated with 2.5% FCS supplemented with insulin (100 nmol/L) or hIGFBP1 (500 ng/mL). Cells counted after 5 days with insulin or hIGFBP1 treatment (n=4). *P < 0.05. (B) HCAEC proliferation. Quiesced cells treated with 20% FCS supplemented with vehicle or 500 ng/mL hIGFBP1. Cells counted after 5 days with control or hIGFBP1 treatment (n = 4). (C) Concentration-dependent effect of TNFα on inhibition of proliferation in HCAECs. Cells counted after 5 days following TNFα treatment (0.01 to 10 ng/mL) (n = 3). (D) HCAEC proliferation. Quiesced cells treated with 20% FCS supplemented with TNFα (1 ng/mL), hIGFBP1 (500 ng/mL), or a combination of TNFα (1 ng/mL) and IGFBP1 (500 ng/mL). Cells counted after 5 days. Analysis of variance: P < 0.01. Post hoc: **P < 0.01; *P < 0.05 (n = 6). Data shown are mean +/− SEM. Abbreviation: NS, not significant.

Figure 7.

IGF-independent effects of hIGFBP1 and involvement in its RGD domain and FAK on endothelial cell proliferation. (A) hIGFBP1 (500 ng/L) and IGF-1 (18 nM) both independently stimulated proliferation of HUVECs on an EdU assay. There was no additive effect of IGF-1 and hIGFBP1 on cell proliferation. (B and C) WT hIGFBP1 stimulated proliferation of HUVECs. Proliferation was not significantly increased by hIGFBP1 when the RGD domain was mutated to WGD. (D) The positive effect of hIGFBP1 on proliferation of HUVECs was abrogated by the focal FAK inhibitor (FAK-i) PZ0117 (100 nmol/L) (n = 4). *P < 0.05. Abbreviation: NS, not significant. Data shown are mean +/− SEM.

Figure 8.

hIGFBP1 stimulates phosphorylation of FAK, activates the small GTPase RhoA, and ameliorates TNFα induced cytoskeletal rearrangement in endothelial cells. (A and B) hIGFBP1 (500 ng/mL; 15 minutes) induced rapid ³⁹⁷Tyr phosphorylation of FAK in HUVECs. (A) Representative immunoblot. (B) Mean data for phospho-FAK (pFAK)/FAK ratio (n = 6). *P < 0.05. (C and D) Mutation of the RGD domain of IGFBP1 to WGD (incapable of integrin binding) abrogates phosphorylation of FAK. (C) Representative immunoblot. (D) Mean data for pFAK/FAK ratio (n = 6). (E) hIGFBP1 (500 ng/mL) induced time-dependent activation of RhoA in HCAECs (n = 5). **P < 0.01. (F and G) Effects of TNFα (10 ng/mL) and hIGFBP1 (500 ng/mL) on actin fiber anisotropy in HUVECs. (F) Representative images (scale bar represents 100 µm). (G) Mean data from four repeat experiments with 188 to 287 cells per experiment. *P < 0.05; **P < 0.01.