KLF2 regulates eNOS uncoupling via Nrf2/HO-1 in endothelial cells under hypoxia and reoxygenation

Abstract

Kruppel-like factor 2 (KLF2) is known to regulate endothelial functions by modulating the endothelial nitric oxide synthase (eNOS)/nitric oxide (NO) pathway. Tetrahydrobiopterin (BH4) and S-glutathionylation of eNOS are crucial for eNOS uncoupling and activation. However, the exact influence of KLF2 on eNOS uncoupling and the mechanism of eNOS activation are not fully understood.

This study used a hypoxia and reoxygenation (H/R) model of human umbilical vein endothelial cells (HUVECs). Cell viability and the eNOS uncoupling-related oxidative stress index were measured. The Nrf2 inhibitor ML385 and HO-1 siRNA were used to elucidate the activation mechanism.

The results demonstrated that KLF2 overexpression increased cell viability, reduced lactate dehydrogenase leakage, decreased the production of superoxide anions (O₂•⁻) and peroxynitrite (ONOO⁻), and increased NO levels and eNOS activity.

KLF2 overexpression also increased the BH4/BH2 ratio and the GSH/GSSG ratio, significantly improving eNOS uncoupling in the H/R model.

KLF2 did not directly regulate upstream proteins involved in eNOS activation. However, when combined with the Nrf2 inhibitor or HO-1 siRNA, the regulatory effect of KLF2 on eNOS uncoupling was significantly reduced.

These findings suggest that KLF2 can improve eNOS uncoupling via the Nrf2/HO-1 pathway in H/R-induced endothelial injury.

Introduction

Endothelial dysfunction (ED) is an early marker of various vascular diseases and a significant factor in ischemia-reperfusion (I/R) injury. Nitric oxide (NO) is essential for maintaining normal endothelial function, and endothelial NO synthase (eNOS) is the primary enzyme regulating NO production.

Kruppel-like factor 2 (KLF2) is a transcription factor induced by laminar flow, predominantly expressed in endothelial cells, and plays a crucial role in regulating endothelial function. Studies have demonstrated that KLF2 induces eNOS expression and increases NO production in cultured human umbilical vein endothelial cells (HUVECs). It has also been shown to activate eNOS and heme oxygenase-1 (HO-1) in liver I/R injury.

Furthermore, KLF2 expression is mediated by the phosphorylation of extracellular signal-regulated kinase 5 (ERK5), and KLF2 subsequently activates eNOS. However, the precise mechanisms by which KLF2 activates eNOS are not fully understood.

Vascular nitric oxide (NO) is produced by endothelial NO synthase (eNOS) through the conversion of L-arginine to L-citrulline in the presence of molecular oxygen. Tetrahydrobiopterin (BH4) is a crucial eNOS cofactor that facilitates the coupling of L-arginine and the heme group within the eNOS oxygenase domain. In this coupled state, eNOS produces NO, which supports normal blood flow and maintains an anti-inflammatory and anti-thrombotic vascular endothelial surface.

A deficiency of BH4 can lead to eNOS uncoupling. This uncoupling not only results in NO insufficiency but also contributes to oxidative stress under pathological conditions like ischemia-reperfusion (I/R). Additionally, increased oxidized glutathione (GSSG) levels, due to oxidative stress, can induce dose-dependent eNOS S-glutathionylation (eNOS-SG), further exacerbating eNOS uncoupling in I/R-induced endothelial dysfunction (ED).

Phosphorylation of eNOS also plays a role in regulating NO levels. The phosphatidylinositol-3-kinase (PI3K)/Akt/eNOS pathway is involved in the protective effects of ischemic postconditioning. Akt, when phosphorylated by activated PI3K, phosphorylates eNOS at serine 1177 (p-eNOS), activating it to produce NO.

However, the relationship between KLF2, BH4, the PI3K pathway, and their combined effects on eNOS activity is not well-documented. Therefore, the mechanisms by which KLF2 regulates eNOS activity and provides vascular protection remain unclear.

This study aimed to clarify the relationship between KLF2, eNOS uncoupling, and the Akt pathway.

Materials and methods

Hypoxia and reoxygenation model

Human umbilical vein endothelial cells (HUVECs) were washed with phosphate-buffered saline (PBS) and maintained in serum-free Dulbecco’s modified Eagle’s medium (DMEM) within a hypoxic environment. This environment was created by placing confluent cell flasks into a Billups-Rothenberg modular incubator chamber, which was then flushed with a gas mixture consisting of 95% nitrogen (N₂) and 5% carbon dioxide (CO₂).

The oxygen level inside the incubator chamber was monitored using an oxygen electrode connected to an Apollo 4000 control unit. Cells were incubated at 37 °C for 4 hours within the chamber, maintaining a controlled gas mixture of 1% oxygen (O₂), 94% nitrogen (N₂), and 5% carbon dioxide (CO₂).

Following the hypoxic period, cells underwent reoxygenation for 24 hours. This was achieved by replacing the hypoxic medium with normoxic PBS supplemented with calcium, magnesium, 1 g/L D-glucose, and 36 mg/L sodium pyruvate.

Small interfering RNAs (siRNAs) were transfected into HUVECs 48 hours before the hypoxia/reoxygenation (H/R) treatments, while plasmids were transfected 24 hours before the H/R treatments.

Cell transfection

The KLF2 sequence was, respectively, subcloned into the pcDNA3.1 vector (GENECHEM, Shanghai, China) to generate the pcDNA-KLF2 vector for ectopic expression in cells. Empty pcDNA3.1 vector was used as a control [13]. HiPerFect transfection reagent (QIAGEN, Valencia, CA, USA) was used to deliver siRNA against HO-1 into cells. HO-1 FlexiTube GeneSolution siRNA was purchased from QIAGEN [14].

Cell viability assay and LDH leakage rate assay

Cell viability was assessed using the MTT assay. Cultured HUVECs were randomly divided into different groups. Briefly, 100 μL of a suspension of exponentially growing HUVECs (1 × 10⁴ cells) was seeded into each well of a 96-well plate, with six wells per group. After hypoxia/reoxygenation (H/R) treatment, 20 μL of MTT (5 mg/mL final concentration) was added to each well. The plates were then incubated at 37 °C for an additional 4 hours.

Following incubation, the culture medium containing the MTT solution was removed, and the formazan crystals were dissolved in 100 μL of dimethyl sulfoxide (DMSO). The absorbance was measured at 546 nm.

Cell viability was calculated using the following formula: cell viability (%) = (OD sample − OD background) / (OD control − OD background) × 100%, where OD represents optical density.

For lactate dehydrogenase (LDH) detection, after H/R treatment, the cell culture medium from each group was centrifuged, and the supernatant was collected. The supernatant from each sample was transferred to a 96-well plate, LDH detection substrate mix was added to each well, and samples were read at 450 nm using a microplate absorbance reader (Tecan infinite M200 Pro, Switzerland).

Measurement of superoxide by high-performance liquid chromatography (HPLC) using CT02-H probe

Superoxide levels were quantified using high-performance liquid chromatography (HPLC) with the CT02-H probe, as previously described. Upon reacting with superoxide anions (O₂•⁻), the green CT02-H probe undergoes dehydrogenation, yielding a purple diamagnetic quinone methide, which is then detected using a Shimadzu HPLC system via electrochemical oxidation.

CT02-H was added to approximately 3 × 10⁶ cells at a final concentration of 50 μM, in PBS containing calcium, magnesium, 1 g/L D-glucose, and 36 mg/L sodium pyruvate.

After 4 hours of reoxygenation, the supernatant was collected and injected into the HPLC system, followed by isocratic elution at a flow rate of 1.0 mL/min.

Statistical analysis

Statistical analysis was performed with SPSS Version 17 software. Student’s t-test was used for statistical analysis, with P < 0.05 being considered statistically significant.

Results

Overexpression of KLF2 increased cell viability and reduced LDH leakage and superoxide and peroxynitrite generation in a hypoxia/reoxygenation (H/R) human umbilical vein endothelial cell (HUVEC) model. Exposure of HUVECs to H/R resulted in cell damage, characterized by a decrease in cell viability and an increase in extracellular LDH levels.

In HUVECs overexpressing KLF2, cell viability was higher and LDH levels were lower compared to normal HUVECs after H/R exposure. KLF2 overexpression had no effect on the viability and LDH levels of normal cells. KLF2 plays a key role in HUVECs in the protection against H/R injury. In the H/R HUVEC model, superoxide and peroxynitrite generation was increased.

The levels of superoxide and peroxynitrite were also higher in KLF2 overexpressing HUVECs after H/R, but lower than in normal HUVECs after H/R. Under H/R conditions, the expression levels of Nrf2 and HO-1 were increased compared to control cells.

In KLF2 overexpressing HUVECs, the expression levels of Nrf2 and HO-1 were increased significantly. Overexpression of KLF2 could further increase the expression of Nrf2 and HO-1 in cells exposed to H/R compared to the nontransfected H/R model.

Overexpression of KLF2 increased NO levels and eNOS activity in H/R HUVEC model

Nitric oxide (NO) levels decreased after hypoxia/reoxygenation (H/R) in HUVECs. KLF2 overexpression significantly increased NO levels post-H/R. NO is produced from L-arginine by endothelial nitric oxide synthase (eNOS). eNOS activity was reduced in both normal and KLF2-overexpressing cells.

H/R caused a significant reduction in eNOS activity. KLF2 overexpressing cells inhibited this reduction more effectively. eNOS and p-eNOS expression decreased in H/R HUVECs. KLF2 overexpression increased eNOS and p-eNOS levels.

KLF2 had no significant effect on Akt phosphorylation. There was no difference in Akt phosphorylation after H/R. This indicates KLF2 affects eNOS through pathways other than Akt. eNOS uncoupling is a key factor in endothelial dysfunction.

Inhibition of Nrf2 or knockdown of HO-1 significantly reduced KLF2′s regulatory effect on eNOS uncoupling. Treatment with the Nrf2 inhibitor (ML385) or HO-1 siRNA showed no significant change in cell viability or LDH leakage compared to untreated cells after H/R. However, KLF2′s protective effects on cell viability and LDH leakage were almost eliminated.

Compared to KLF2-overexpressing HUVECs after H/R, superoxide and peroxynitrite levels were higher, and NO levels and eNOS activity were lower, when cells were treated with the Nrf2 inhibitor or HO-1 siRNA. There was no significant difference between treated KLF2-overexpressing cells and normal cells under H/R. The changes in the BH4/BH2 and GSH/GSSG ratios caused by KLF2 overexpression were also reversed by the Nrf2 inhibitor or HO-1 siRNA.

KLF2 overexpression, with or without the Nrf2 inhibitor or HO-1 siRNA, had no significant effect on eNOS-SG levels under normal conditions. Under H/R, KLF2 inhibited the increase in eNOS-SG levels, but the Nrf2 inhibitor or HO-1 siRNA increased eNOS-SG levels. HO-1 is a key downstream molecule that regulates oxidative stress by Nrf2. After HO-1 knockdown or Nrf2 inhibition, KLF2′s protective effects on H/R injury were almost completely lost.

Discussion

Ischemia-reperfusion injury (IRI) leads to cardiac problems. Nitric oxide (NO) regulates many essential functions. eNOS activation is key for heart protection. PI3K/Akt/eNOS is a primary signaling pathway.

Akt phosphorylates eNOS, boosting NO production. KLF2 reduces H/R cell damage and oxidative stress. It activates eNOS and increases its phosphorylation. KLF2 does not affect Akt phosphorylation.

KLF2 activates eNOS through a pathway other than Akt. This indicates KLF2 does not use the PI3K/Akt pathway. eNOS uncoupling is a major cause of endothelial dysfunction.

eNOS activation depends on signaling and coupling. BH4 is essential for eNOS coupling. BH2, an oxidized form, causes uncoupling. Oxidative stress affects eNOS coupling.

During H/R, eNOS produces superoxide instead of NO. This increases the BH2/BH4 ratio. This further promotes eNOS uncoupling and oxidative stress. Oxidative stress and eNOS uncoupling accelerate disease.

Our study shows increased oxidative stress during H/R. Superoxide and peroxynitrite levels increased. The BH4/BH2 ratio decreased significantly. This indicates eNOS uncoupling occurred.

eNOS uncoupling leads to NO deficiency. KLF2 overexpression increased the BH4/BH2 ratio. It also reduced superoxide and peroxynitrite levels. KLF2 downregulates molecules that transform BH4 to BH2.

High superoxide and peroxynitrite lower the BH4/BH2 ratio. Oxidative stress depletes glutathione and forms GSSG. This induces eNOS S-glutathionylation, another uncoupling pathway. KLF2 decreased eNOS-SG, increased eNOS activity, and restored the GSH/GSSG ratio.

These results show KLF2 regulates eNOS mainly by controlling uncoupling. The mechanisms of KLF2′s oxidative stress regulation are unclear. Nrf2 and HO-1 regulate oxidative stress. KLF2 can activate Nrf2 and HO-1.

Nrf2 was inhibited or HO-1 knocked down in KLF2-overexpressing HUVECs. KLF2 increased Nrf2 and HO-1 expression. KLF2′s protective effects on H/R injury and eNOS uncoupling were lost. KLF2 protects against H/R injury by regulating eNOS uncoupling via Nrf2/HO-1.