Sitagliptin protects against hypoxia/reoxygenation (H/R)-induced cardiac microvascular endothelial cell injury

Abstract

Inhibition of hypoxia/reoxygenation (H/R)-induced insult in cardiac microvascular endothelial cells (CMECs) has been considered as a promising therapeutic strategy for the treatment of ischemic cardiovascular disease. In the present study, we found that H/R significantly increased the expression of dipeptidyl peptidase (DPP)-4 in CMECs. Treatment with the DPP-4 inhibitor sitagliptin, a licensed drug used for the treatment of type 2 diabetes mellitus (T2DM), ameliorated H/R-induced oxidative stress by decreasing the expression of NOX-4 and restoring the intracellular level of GSH in CMECs. Sitagliptin could also improve H/R-induced mitochondrial dysfunction by increasing intracellular MMP and ATP. Additionally, we found that the presence of sitagliptin prevented H/R-induced reduced cell viability and LDH release. Notably, our findings indicate that sitagliptin possesses an anti-inflammatory effect against H/R-induced expression of IL-6, IL-8, and TNF-α as well as secretion of HMGB1. Mechanistically, we found that sitagliptin suppresses activation of p38/NF-κB signaling. These findings suggest that sitagliptin may have potential as a therapeutic agent for the treatment of cardiovascular diseases.

Keywords: Hypoxia/reoxygenation; NF-κB; cardiovascular diseases; oxidative stress; sitagliptin.

Figure 1

Hypoxia/reoxygenation (H/R) increased the expression of DPP-4 in primary cardiac microvascular endothelial cells (CMECs). Hypoxia/reoxygenation experiments were performed by putting cells in an air-tight chamber with pure N₂ followed by incubation at 37°C for 6 h. Cells were then cultured with normal medium in a normoxic incubator (95% air plus 5% CO₂) for 12 h for reoxygenation. A. Expression of DPP-4 at the mRNA levels was determined by real time PCR analysis; B. Expression of DPP-4 at the protein levels was determined by western blot analysis (*, P<0.01 vs. the control group, n=6).

Figure 2

Sitagliptin pretreatment ameliorated hypoxia/reoxygenation (H/R)-induced oxidative stress in primary cardiac microvascular endothelial cells (CMECs). Cells were pre-incubated with 100, 200 nM sitagliptin or DMSO vehicle for 12 h, followed by exposure to hypoxia/reoxygenation (H/R) conditions. A. Western blot analysis of NOX-4; B. Reduced glutathione (GSH) was determined (*, P<0.01 vs. vehicle group; #, P<0.01 vs. H/R group; $, P<0.01 vs. H/R+100 nM sitagliptin, n=5-6).

Figure 3

Sitagliptin pretreatment ameliorated hypoxia/reoxygenation (H/R)-induced mitochondrial dysfunction in primary cardiac microvascular endothelial cells (CMECs). Cells were preincubated with 100, 200 nM sitagliptin or DMSO vehicle for 12 h, followed by exposure to hypoxia/reoxygenation (H/R) conditions. A. Mitochondrial membrane potential (MMP) was assayed by the TMRM staining; B. Intracellular levels of ATP were determined (*, P<0.01 vs. vehicle group; #, P<0.01 vs. H/R group; $, P<0.01 vs. H/R+100 nM sitagliptin, n=5-6).

Figure 4

Sitagliptin pretreatment ameliorated hypoxia/reoxygenation (H/R)-induced cell death of primary cardiac microvascular endothelial cells (CMECs). Cells were preincubated with 100, 200 nM sitagliptin or DMSO vehicle for 12 h, followed by exposure to hypoxia/reoxygenation (H/R) conditions. A. Cell viability of CMECs was assessed by MTT assay; B. LDH released from the cytoplasm to the medium was assayed (*, P<0.01 vs. vehicle group; #, P<0.01 vs. H/R group; $, P<0.01 vs. H/R+100 nM sitagliptin, n=5-6).

Figure 5

Sitagliptin pretreatment ameliorated hypoxia/reoxygenation (H/R)-induced production of pro-inflammatory cytokines in primary cardiac microvascular endothelial cells (CMECs). Cells were preincubated with 100, 200 nM sitagliptin or DMSO vehicle for 12 h, followed by exposure to hypoxia/reoxygenation (H/R) conditions. A. Expressions of IL-6, IL-8, and TNF-α at the gene levels were measured by real time PCR analysis; B. Secretions of IL-6, IL-8, and TNF-α at the protein levels were determined by ELISA assay (*, P<0.01 vs. vehicle group; #, P<0.01 vs. H/R group; $, P<0.01 vs. H/R+100 nM sitagliptin, n=5-6).

Figure 6

Sitagliptin pretreatment ameliorated hypoxia/reoxygenation (H/R)-induced secretion of high mobility group box 1 (HMGB1) in primary cardiac microvascular endothelial cells (CMECs). Cells were pre-incubated with 100, 200 nM sitagliptin or DMSO vehicle for 12 h, followed by exposure to hypoxia/reoxygenation (H/R) conditions. Secretion of HMGB1 was determined by the ELSA assay (*, P<0.01 vs. vehicle group; #, P<0.01 vs. H/R group; $, P<0.01 vs. H/R+100 nM sitagliptin, n=6).

Figure 7

Sitagliptin pretreatment ameliorated hypoxia/reoxygenation (H/R)-induced activation of p38 in primary cardiac microvascular endothelial cells (CMECs). Cells were pre-incubated with 100, 200 nM sitagliptin or DMSO vehicle for 12 h, followed by exposure to hypoxia/reoxygenation (H/R) conditions. Activation of p38 was determined by western blot analysis (*, P<0.01 vs. vehicle group; #, P<0.01 vs. H/R group; $, P<0.01 vs. H/R+100 nM sitagliptin, n=6).

Figure 8

Sitagliptin pretreatment attenuated hypoxia/reoxygenation (H/R)-induced activation of NF-κB signaling in primary cardiac microvascular endothelial cells (CMECs). Cells were pre-incubated with 100, 200 nM sitagliptin or DMSO vehicle for 12 h, followed by exposure to hypoxia/reoxygenation (H/R) conditions. A. Nuclear translocation of p65 was determined by western blot analysis with Lamin B1 as a positive control; B. Luciferase activity of NF-κB (*, P<0.01 vs. vehicle group; #, P<0.01 vs. H/R group; $, P<0.01 vs. H/R+100 nM sitagliptin, n=5-6).