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Hyperinsulinemia-induced KLF5 mediates endothelial angiogenic dysfunction in diabetic endothelial cells

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Abstract

Reduced expression of endothelial nitric oxide synthase (eNOS) is a hallmark of endothelial dysfunction in diabetes, which predisposes diabetic patients to numerous cardiovascular complications including blunted angiogenesis. The Krüppel-like factor (KLF) five has been implicated as a central regulator of cardiovascular remodeling, but its role in endothelial cells (ECs) remains poorly understood. We show here that expression of endothelial KLF5 was significantly increased in the ECs from mouse diabetes mellitus type 2 (T2DM) model, when compared to non-diabetic or T1DM mouse. KLF5 up-regulation by insulin was dependent on activation of multiple pathways, including mammalian target of rapamycin, oxidative stress and Protein kinase C pathways. Hyperinsulinemia-induced KLF5 inhibited endothelial function and migration, and thereby compromised in vitro and in vivo angiogenesis. Mechanistically, KLF5 acted in concert with the MTA1 coregulator to negatively regulate NOS3 transcription, thereby leading to the diminished eNOS levels in ECs. Conversely, potentiation of cGMP content (the essential downstream effector of eNOS signaling) by pharmacological approaches successfully rescued the endothelial proliferation and in vitro tube formation, in the HUVECs overexpressing the exogenous KLF5. Collectively, the available data suggest that the augmentation of endothelial KLF5 expression by hyperinsulinemia may represent a novel mechanism for negatively regulating eNOS expression, and may thus help to explain for the T2DM-related endothelial dysfunction at the transcriptional level.

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Correspondence to Jian-rong Liu.

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10735_2019_9821_MOESM1_ESM.ppt

Supplementary material 1—Measurement of diabetes induction was carried out through the assessment of whole blood glucose (a) and serum insulin levels (b) in different experimental groups (PPT 149 KB)

10735_2019_9821_MOESM2_ESM.ppt

Supplementary material 2—Isolation and purification of ECs from mouse liver or kidney. (a) Cultures of ECs isolated from mouse liver or kidney exhibited the classical cobblestone morphology. (b) Representative FACS analyses of VECAM1 and CD144 in isolated ECs. (c) Immunofluorescence images shows a cytoplasm expression of the endothelial cell-specific marker CD34 in isolated ECs, further confirming the cell identity. Nuclear are demonstrated using DAPI staining (PPT 256 KB)

10735_2019_9821_MOESM3_ESM.ppt

Supplementary material 3—CD34+ cells were isolated from the peripheral blood samples of T2DM patients and normal volunteers using FACS method (PPT 134 KB)

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Supplementary material 4—Human endothelial colony forming cells (ECFCs), isolated from the mononuclear cell fractions of donors’ cord blood, were identified using RT-qPCR method. UN, undetectable (PPT 124 KB)

10735_2019_9821_MOESM5_ESM.ppt

Supplementary material 5—Effects of KLF5 knockdown on endothelial function in HUVECs. (a) HUVECs were transfected with KLF5 siRNA or Ctrl siRNA (Sigma-Aldrich, Shanghai, China) using FuGENE® HD. 48 h later, cells were harvested, and ablation of endogenous KLF5 was validated using immunoblotting. (b) Effects of KLF5 knockdown on endothelial proliferation was determined using Sulforhodamine-B (SRB) absorption assay, as described in Materials and methods section. (c) Knock down of KLF5 promotes migration of HUVECs in Transwell assays stimulated by 10 ng/ml VEGF. (d) Knock down of KLF5 potentiates the tube-forming activity in HUVECs (PPT 216 KB)

10735_2019_9821_MOESM6_ESM.ppt

Supplementary material 6—Effects of KLF5 knockdown on endothelial function in HUVECs. (a) HUVECs were transfected with KLF5 siRNA or Ctrl siRNA (Sigma-Aldrich, Shanghai, China) using FuGENE® HD. 48 h later, cells were harvested, and ablation of endogenous KLF5 was validated using immunoblotting. (b) Effects of KLF5 knockdown on endothelial proliferation was determined using Sulforhodamine-B (SRB) absorption assay, as described in Materials and methods section. (c) Knock down of KLF5 promotes migration of HUVECs in Transwell assays stimulated by 10 ng/ml VEGF. (d) Knock down of KLF5 potentiates the tube-forming activity in HUVECs (PPT 215 KB)

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Wang, Xh., Yan, Cy. & Liu, Jr. Hyperinsulinemia-induced KLF5 mediates endothelial angiogenic dysfunction in diabetic endothelial cells. J Mol Hist 50, 239–251 (2019). https://doi.org/10.1007/s10735-019-09821-3

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  • DOI: https://doi.org/10.1007/s10735-019-09821-3

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