Nuclear factor-κB signaling negatively regulates high glucose-induced vascular endothelial cell damage downstream of the extracellular signal-regulated kinase/c-Jun N-terminal kinase pathway

Abstract

Diabetes mellitus (DM)-induced high blood sugar severely damages vascular endothelial cells (VECs), which are in direct contact with the blood. Diabetic complications cause difficulties in skin wound healing and VECs are important for this process. Previous studies demonstrated that high blood sugar delayed the repair of wounded VECs, but the underlying mechanism has remained elusive. To explore the effects of diabetic conditions on VEC damage, cells were incubated in a medium with high glucose and then subjected to RNA-sequencing based transcriptome analysis. The results revealed that numerous biological processes were altered by HG stress, including extracellular matrix-receptor interaction, NOD-like receptor signaling and the nuclear factor (NF)-κB pathway. HG treatment increased the levels of phosphorylated inhibitor of NF-κB (IκB-α), the key NF-κB signaling regulator as well as the transcripts of plasminogen activator inhibitor-1 and interleukin-8, two inflammatory response markers. Treatment with extracellular signal-regulated kinase (ERK)- and c-Jun N-terminal kinase (JNK)-specific inhibitors U0126 and sp600125, respectively, led to the activation of IκB-α; however, the inhibitor of IκBα phosphorylation Bay11-7082 did not affect ERK and JNK activity, suggesting that ERK/JNK signaling occurs upstream of NF-κB in VECs. The present study provided useful information regarding the effects of diabetes on VECs, which may provide approaches for therapies of diabetes-associated complications in the future.

Keywords: damage; extracellular signal-regulated kinase/c-Jun N-terminal kinase; high glucose; nuclear factor-κB; vascular endothelial cell.

Figure 1.

HG altered the transcriptome profile in VECs. (A) Heat map diagram of the genes whose expression levels in VECs were altered after HG (35 mM) treatment for 3 h. Gene expression is indicated by a pseudocolor scale with red denoting higher and green denoting lower gene expression levels. (B) Reverse-transcription quantitative polymerase chain reaction analysis was performed to verify the expression levels of IL-8, PAI-1, CCL13 and Caspase-3. GAPDH was used as an internal control. *P<0.05 vs. Low Glc. Low Glc/Con, low glucose (5.5 mM); High Glc/HG, high glucose (35 mM); VECs, vascular endothelial cells; IL, interleukin; CCL, chemokine ligand 13; PAI-1, plasminogen activator inhibitor-1.

Figure 2.

Top-ranking canonical pathways altered in vascular endothelial cells by high glucose stimulation. The top ranking canonical Kyoto Encyclopedia of Genes and Genomes pathways identified are listed according to P-values. The 15 pathways listed were significantly enriched (P<0.05), which included ECM-receptor interaction, NF-κB signaling, and NOD-like receptor signaling pathways. P-value, probability that the association between the more than 1.5-fold changed genes and the canonical pathway can be accounted for by chance only. NF, nuclear factor; ECM, extracellular matrix; NOD, nucleotide-binding oligomerization domain; HTLV, human T-lymphotropic virus.

Figure 3.

Association between IκB-α and JNK/ERK. (A) Western blot analysis was performed to analyze the effect of HG on the levels IκB-α and p-IκB-α. (B) Effect of inhibitor of IκBα phosphorylation Bay117082 on ERK/JNK and p-ERK/p-JNK levels, and (C) effects of ERK inhibitor U0126 and JNK inhibitor sp600125 on IκB-α and p-IκB-α levels. IκB-α, inhibitor of NF-κB; NF, nuclear factor; p-ERK, phosphorylated extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; HG, high glucose; Con, control.