A paradox: Fe2+-containing agents decreased ROS and apoptosis induced by CoNPs in vascular endothelial cells by inhibiting HIF-1α

Abstract

Cobalt nanoparticles (CoNPs) released from hip joint implants are known to have a toxic effect on several organs probably through increasing reactive oxygen species (ROS). Ferrous ion (Fe2+) is well-known to enhance oxidative stress by catalysing the production of ROS. However, in our pilot study, we found that Fe2+ conversely inhibited the ROS production induced by CoNPs. To elucidate the underlying mechanism, the present study treated vascular endothelial HUVEC and HMEC-1 cells with CoNPs alone or in combination with ferrous lactate [Fe(CH3CHOHCOO)2], ferrous succinate [Fe(CH2COO)2], and ferrous chloride (FeCl2). CoNP toxicity was evaluated by measuring cell viability, rate of apoptosis and lactose dehydrogenase (LDH) release, and intracellular ROS levels. Treatment with CoNPs decreased cell viability, LDH release, and ROS production and increased apoptosis. CoNPs increased hypoxia-inducible factor-1α (HIF-1α) protein level and mRNA levels of vascular endothelial growth factor (VEGF) and glucose transporter 1 (GLUT1) downstream of HIF-1α signalling. Silencing HIF-1α attenuated CoNP toxicity, as seen by recovery of cell viability, LDH release, and ROS levels and reduced apoptosis. CoNPs caused a pronounced reduction of Fe2+ in cells, but supplementation with Fe(CH3CHOHCOO)2, Fe(CH2COO)2, and FeCl2 restored Fe2+ levels and inhibited HIF-1α activation. Moreover, all three Fe2+-containing agents conferred protection from CoNPs; Fe(CH3CHOHCOO)2 and Fe(CH2COO)2 more effectively than FeCl2. In summary, the present study revealed that CoNPs exert their toxicity on human vascular endothelial cells by depleting intracellular Fe2+ level, which causes activation of HIF-1α signalling. Supplements of Fe2+, especially in the form of Fe(CH3CHOHCOO)2 and Fe(CH2COO)2, mitigated CoNP toxicity.

Keywords: CoNPs; Fe2+-containing agents; hypoxia-inducible factor; vascular endothelial cells.

Figure 1. The toxic effect of CoNPs on vascular endothelial cells

(A) Electron microscopic observation of HUVEC cells after treatment with CoNPs for 24 h. (B) HUVEC and HMEC-1 cells were exposed to varying concentrations of CoNPs (0, 100, 200, 400, 800, and 15000 μM) for 24 h or these cells were exposed to 800 μM CoNPs for 0, 2, 6, 12, 24, and 48 h. MTT was performed to evaluate cell viability. The viability of HUVEC and HMEC-1 cells were decreased by CoNPs in dose- and time-dependent manner. Apoptosis (C) and LDH assays (D) were performed, after HUVEC and HMEC-1 cells were treated with 800 μM CoNPs for 24 h. CoNPs increased the apoptosis and LDH concentration in culture medium. *P<0.05, **P<0.01 and ***P<0.001 vs. control group.

Figure 2. CoNPs caused the activation of HIF-1α signalling and ROS production but decreased intracellular Fe²⁺ level

After HUVEC and HMEC-1 cells were treated with 800 μM CoNPs for 24 h, protein level of HIF-1α (A) and mRNA levels of VEGF and GLUT1 (B) were measured by Western blot and PCR assays, respectively. CoNPs increased HIF-1α, VEGF and GLUT1 in HUVEC and HMEC-1 cells. Intracellular ROS (C), LPO (D), MDA (E) as well as Fe²⁺ and total iron levels (F) were measured using flow cytometer or detection kits following the CoNPs treatment. CoNPs increased intracellular ROS, LPO and MDA, but decreased Fe²⁺ levels. *P<0.05, **P<0.01 and ***P<0.001 vs. control group.

Figure 3. HIF-1α knockdown attenuated the toxic effect of CoNPs on vascular endothelial cells

HIF-1α was knocked down before treating vascular endothelial cells with CoNPs. (A) PCR and (B) Western blot assays were performed to detect the mRNA level of HIF-1α in HUVEC and HMEC-1 cells. MTT (C), apoptosis (D) and LDH (E) assays were performed to evaluate the cell viability, apoptosis rate and damage in cell membrane, respectively. HIF-1α knockdown attenuated the toxic effect of CoNPs on vascular endothelial cells. **P<0.01 and ***P<0.001 vs. control group; ^#P<0.05 vs. CoNPs group.

Figure 4. The regulatory effects of HIF-1α on intracellular ROS level in vascular endothelial cells treated with CoNPs

Intracellular ROS (A), LPO (B) and MDA (C) were measured using flow cytometer or detection kits following the CoNPs treatment. Expression of NOX1, 2, and 4 as well as NOS3 were tested using PCR assay. Silencing HIF-1α blocked increase in ROS, LPO, and MDA in HUVEC and HMEC-1 cells induced by CoNPs. However, the reduction of Fe²⁺ level did not improve after HIF-1α knockdown. HIF-1α knockdown decreased expression of NOX1, 2, and 4 as well as NOS3 which were increased by CoNPs. *P<0.05, **P<0.01 and ***P<0.001 vs. control group; ^#P<0.05 and ^##P<0.01 vs. CoNPs group.

Figure 5. Ferrous agents conferred protection against the toxicity of CoNPs in vascular endothelial cells

HUVEC and HMEC-1 cells were treated with 800 μM CoNPs in combination with Fe(CH3CHOHCOO)₂, Fe(CH₂COO)₂, or FeCl₂. MTT (A), apoptosis (B) and LDH (C) assays were performed 24 h after the treatments. All three ferrous agents improved cell viability and decreased the apoptosis and the LDH concentrations in culture medium. **P<0.01 vs. control group; ^#P<0.05 vs. CoNPs group. Abbreviations: FC, ferrous chloride, (FeCl₂); FL, ferrous lactate, [Fe(CH₃CHOHCOO)₂]; FS, ferrous succinate, [Fe(CH₂COO)₂].

Figure 6. Hoechst33258 and PI staining

The morphological changes of apoptosis were observed through a dual staining with Hoechst33258 and PI. The morphological changes of apoptosis include chromatin condensation (in early stage), and cell membrane fragmentation (in later stage). After Hoechst33258 staining, the nuclei of normal cells were normal blue, while the nuclei of apoptotic cells were dense stained or fragmented, and some of them turned white from blue colour. The nuclei of normal cells were not stained by PI, because PI can not pass through the complete cell membrane. However, the integrity of cell membrane was broken in the later stage of apoptosis, thus the nuclei of apoptotic cells can be stained. We observed that the cell number was reduced after treatment with CoNPs, with notable increase in PI staining. Silencing HIF-1α blocked the reduction in cell number caused by CoNPs, whereas a portion of cells showed chromatin condensation as indicated by Hoechst33258 staining. Treatments with Fe(CH3CHOHCOO)₂, Fe(CH₂COO)₂, and FeCl₂ attenuated the increase in PI staining induced by CoNPs.

Figure 7. The regulatory effects of ferrous agents on HIF-1α signal and Fe²⁺ levels in vascular endothelial cells treated with CoNPs

HUVEC and HMEC-1 cells were treated with 800 μM CoNPs in combination with Fe(CH3CHOHCOO)₂, Fe(CH₂COO)₂, or FeCl₂. Fe²⁺ and total iron levels were measured following the CoNPs treatment. (A) Expression of HIF-1α, VEGF and GLUT1 were tested using Western blot (B) or PCR assay (C). Treatment with these three agents increased Fe²⁺ and total iron levels in HUVEC and HMEC-1 cells that were exposed to CoNPs. All three ferrous agents inhibited increase in HIF-1α, VEGF, and GLUT1 induced by CoNPs. **P<0.01 and ***P<0.001 vs. CoNPs group. Abbreviations: FC, ferrous chloride, (FeCl₂); FL, ferrous lactate, [Fe(CH₃CHOHCOO)₂]; FS, ferrous succinate, [Fe(CH₂COO)₂].

Figure 8. The regulatory effects of ferrous agents on intracellular ROS level in vascular endothelial cells treated with CoNPs

HUVEC and HMEC-1 cells were treated with 800 μM CoNPs in combination with Fe(CH3CHOHCOO)₂, Fe(CH₂COO)₂, or FeCl₂. Intracellular ROS (A), LPO (B) and MDA (C) were measured using flow cytometer or detection kits following the CoNPs treatment. Expression of NOX1, 2, and 4 as well as NOS3 were tested using PCR assay (D). All three ferrous agents inhibited increase in ROS, LPO, and MDA in HUVEC and HMEC-1 cells induced by CoNPs. Moreover, all three ferrous agents decreased expression of NOX1, 2, and 4 as well as NOS3 which were increased by CoNPs. *P<0.05, and **P<0.01 vs. CoNPs group. Abbreviations: FC, ferrous chloride, (FeCl₂); FL, ferrous lactate, [Fe(CH₃CHOHCOO)₂]; FS, ferrous succinate, [Fe(CH₂COO)₂].