The Transcription Factor Nrf2 Protects Angiogenic Capacity of Endothelial Colony-Forming Cells in High-Oxygen Radical Stress Conditions

Abstract

Background: Endothelial colony forming cells (ECFCs) have shown a promise in tissue engineering of vascular constructs, where they act as endothelial progenitor cells. After implantation, ECFCs are likely to be subjected to elevated reactive oxygen species (ROS). The transcription factor Nrf2 regulates the expression of antioxidant enzymes in response to ROS.

Methods: Stable knockdown of Nrf2 and Keap1 was achieved by transduction with lentiviral shRNAs; activation of Nrf2 was induced by incubation with sulforaphane (SFN). Expression of Nrf2 target genes was assessed by qPCR, oxidative stress was assessed using CM-DCFDA, and angiogenesis was quantified by scratch-wound and tubule-formation assays Results. Nrf2 knockdown led to a reduction of antioxidant gene expression and increased ROS. Angiogenesis was disturbed after Nrf2 knockdown even in the absence of ROS. Conversely, angiogenesis was preserved in high ROS conditions after knockdown of Keap1. Preincubation of ECFCs with SFN reduced intracellular ROS in the presence of H₂O₂ and preserved scratch-wound closure and tubule-formation.

Results: Nrf2 knockdown led to a reduction of antioxidant gene expression and increased ROS. Angiogenesis was disturbed after Nrf2 knockdown even in the absence of ROS. Conversely, angiogenesis was preserved in high ROS conditions after knockdown of Keap1. Preincubation of ECFCs with SFN reduced intracellular ROS in the presence of H₂O₂ and preserved scratch-wound closure and tubule-formation.

Conclusion: The results of this study indicate that Nrf2 plays an important role in the angiogenic capacity of ECFCs, particularly under conditions of increased oxidative stress. Pretreatment of ECFCs with SFN prior to implantation may be a protective strategy for tissue-engineered constructs or cell therapies.

Figure 1

Nrf2 pathway modulation—effects on gene expression: (a) transduction with shRNAs against Nrf2 resulted in a significant reduction in Nrf2 expression (p < 0.001). No effect on Keap1 was observed. (b) shRNAs against Keap1 reduced its expression, (p = 0.02). (c) Effects of shRNAs on Nrf2/ARE target gene HO-1, shNrf2 transduction induced a reduction in HO-1 expression (p = 0.01) and shKeap1 increased HO-1 expression (p = 0.004), all experiments reflect data from 3 independent replicates.

Figure 2

Nrf2 pathway modulation and oxidative stress: (a) intracellular ROS levels as measured by CM-H₂DCFDA. ECFCs show a dose-dependent increase in CM-H₂DCFDA signal in response to H₂O₂ (p < 0.001). Keap1 knockdown reduces oxidative stress in the presence of higher concentrations of H₂O_2, (p = 0.04), whereas Nrf2 knockdown increases susceptibility to higher concentrations of H₂O₂ (p = 0.01). Graphs represent mean +/− SEM, and data are from 3 independent biological replicates.

Figure 3

Nrf2 pathway modulation and endothelial function. (a) Endothelial scratch-wound closure in Nrf2 and Keap1 knockdown ECFCs in the presence of increasing concentrations of H₂O₂. Graphs represent mean +/− SEM, and data are from 4 independent biological replicates. (b) Tubule formation assay on Matrigel shows that tubule formation is sensitive to ROS. Nrf2 knockdown markedly impairs ECFC ability to form tubules even in the absence of H₂O₂, whereas Keap1 knockdown prevents H₂O₂-mediated inhibition of tubule formation. (c) Quantification of tubule formation by number of junctions. The number of junctions in the endothelial network greatly decreases in increasing concentrations of H₂O₂. Nrf2 knockdown significantly impairs tubule formation compared to control (p < 0.001), and Keap1 knockdown confers some resistance to H₂O₂-mediated impairment in tubule formation (p = 0.06). Graphs represent mean +/− SEM, and data are from 6 independent biological replicates.

Figure 4

Nrf2 activation by SFN pretreatment in ECFCs. (a) Western blot of nuclear extracts of ECFCs exposed to SFN (Nrf2 band at ~72 kD). Data represent two donors. (b) Densitometric quantification of Western blot, showing dose-dependent increase of nuclear Nrf2 translocation (p = 0.02, n = 3) (c). Upregulation of Nrf2 target genes after incubation with sulforaphane (SFN). Glutamate-cysteine ligase expression, catalytic (GCLC) and regulatory subunit, is moderately upregulated in ECFCs treated with SFN. Heme oxygenase 1 (HO-1) shows a strong dose-dependent increase in expression with a plateau at ca. 5 μM. Graphs represent mean +/− SEM, and data are from 3 independent biological replicates.

Figure 5

SFN pretreatment reduces ROS. Intracellular ROS as measured by CM-DCFDA fluorescence increased dose dependently with exposure to H₂O₂. Pretreatment of ECFCs with SFN ameliorated the H₂O₂-induced rise in ROS (p = 0.03). Graphs represent mean +/− SEM, and data are from 3 independent biological replicates.

Figure 6

Pretreatment with SFN protects endothelial function. (a) Scratch-wound closure was reduced in the presence of increasing concentrations of H₂O₂. Pretreatment with 2.5 μM SFN partially prevented the ROS reduction in migration (n = 6, p < 0.001). (b) SFN pretreatment increases numbers of junctions overall (p = 0.002), and no additional effect in the presence H₂O₂ is observed. Graphs represent mean +/− SEM, and 6 independent biological replicates were used for (a) and 4 replicates for (b).