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  • br Materials and methods br Results


    Materials and methods
    Results Overexpression of KLF2 increased the cell viability and reduced the LDH leakage rate and O2•− and ONOO− generation in H/R HUVEC model. To evaluate the effect of KLF2 on H/R HUVEC injury, KLF2 was overexpressed in HUVECs. Cell viability and extracellular LDH levels were detected in the H/R model. As expected, exposure of HUVECs to H/R resulted in cell damage, characterized by a notable decrease in cell viability and a remarkable increase in extracellular LDH levels. However, in HUVECs overexpressing KLF2, cell viability was significantly higher and LDH levels were lower compared to normal HUVECs after H/R exposure. Overexpression of KLF2 has no effect on the viability and LDH levels of normal cells. Our results suggest that KLF2 plays a key role in HUVECs in the protection against H/R injury (Fig. 1A). Oxidative stress is an important cause of H/R injury. In the H/R HUVEC model, O2•− and ONOO− generation was significantly increased. The levels of O2•− and ONOO− were also higher in KLF2 overexpressing HUVECs after H/R, but significantly lower than in normal HUVECs after H/R. Oxidative stress plays an important role in eNOS uncoupling. Nrf2 is a transcription factor that plays an important role in the defense mechanisms against oxidative stress and exogenous toxic substances. 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 (Fig. 1B). Moreover, overexpression of KLF2 could further increase the expression of Nrf2 and HO-1 in AMD-070 exposed to H/R compared to the nontransfected H/R model (Fig. 1B).
    Discussion IR injury (IRI) is one of the main causes of cardiac failure, morbidity, and mortality after cardiac operations or heart infarctions. NO is a critical regulator of numerous processes, such as maintenance of vasomotor tone, metabolism, signal transduction, cell-to-cell interaction, and immune modulation [[17], [18], [19]]. Activation of eNOS is a key step to maintain endothelial NO levels and plays a cardioprotective role by preconditioning tissues to IRI [20]. PI3K/AKt/eNOS is the main signal pathway for the activation of eNOS and the regulation of NO levels. It was reported that Akt phosphorylates eNOS serine 1177, inducing NO production [21,22]. Our study shows that overexpression of KLF2 can effectively reduce cell damage caused by H/R, increase cell viability, reduce LDH release, and decrease the oxidative stress response. At the same time, it can significantly activate eNOS and promote its phosphorylation. eNOS activity and NO levels increased significantly. However, overexpression of KLF2 had no significant effect on the phosphorylation by Akt under H/R conditions. Therefore, although KLF2 can activate eNOS and increase its phosphorylation, the mechanism is not related to the Akt pathway. These results indicate that KLF2 does not regulate eNOS activity through the PI3K/Akt pathway. In vivo, the activation of eNOS is related not only to signal pathways but also to the eNOS coupling state. BH4 is an essential cofactor of eNOS that aids in the coupling between l-arginine and the heme group within the oxygenase domain of eNOS [23,24]. By contrast, BH2, an oxidized form of BH4, can cause uncoupling between l-arginine and eNOS. Therefore, oxidative stress plays a key role in the occurrence of the uncoupling state of eNOS. Under H/R conditions, eNOS utilizes molecular oxygen as the substrate in the absence of l-arginine to generate SO instead of NO, thus increasing the BH2/BH4 ratio and further promoting eNOS uncoupling and aggravating oxidative stress. The interaction between oxidative stress and eNOS uncoupling can then accelerate disease development [25,26]. Our study shows that under H/R conditions, oxidative stress increases significantly (O2•− and ONOO− levels increased significantly), and the BH4/BH2 ratio decreases significantly. In this regard, eNOS uncoupling may induce NO insufficiency. However, overexpression of KLF2 significantly increased the BH4/BH2 ratio and reduced the O2•− and ONOO− levels. It is important to note that overexpression of KLF2 could downregulate the levels of O2•− and ONOO−, which participate in the transformation of BH4 to BH2. High levels of O2•− and ONOO− in the body can lead to a decrease in the BH4/BH2 ratio [27,28]. Oxidative stress can also lead to the depletion of glutathione, promote the formation of GSSG, and induce dose-dependent eNOS S-glutathionylation, which is another possible pathway of eNOS uncoupling in I/R [29,30]. In our study, overexpression of KLF2 significantly decreased eNOS-SG levels, increased eNOS activity, and restored the GSH/GSSG ratio caused by H/R. These results show that the regulatory effect of KLF2 on eNOS is mainly related to the regulation of eNOS uncoupling. However, the mechanisms by which KLF2 regulates oxidative stress remain to be studied.