In this study, we observed that propofol could inhibit TNF-α-modulated occludin expression, more importantly, the HIF-1α/ VEGF/ VEGFR-2/ ERK signaling pathway was involved in this process.
It is known that BBB plays a crucial role in maintaining a stable environment of the central neural system. The BBB is mainly consisted of the microvascular endothelial cells which are linked by intercellular tight junction protein complexes and lack fenestrae, thus restricting paracellular transport between the blood and brain tissues [16]. Tight junctions among endothelial cells form the primary structure of BBB to limit its permeability [17]. The multiple tight junction proteins includes claudins, occludin and zonula occludens-1 (ZO-1) [18]. It has been reported that the imflammation factors could impact the expression of tight junction proteins [19]. It also has been reported that the levels of imflammation factors were raised due to surgical trauma during perioperative period [20]. Thus we inferred that the alterations in the levels of tight junction proteins following imflammation response may contribute to the BBB disruption. In our study, we found that TNF-α could significantly decrease the expression of occludin and ZO-1, while the expression of claudin-5 was not changed in hCMEC/D3 cells.
Propofol is the most commonly used intravenous anesthetic which has been reported to have anti-imflammatory effects. A study revealed that propofol could reverse TNF-α-induced human vascular endothelial cells apoptosis [21]. In this study, we wonder if propofol could attenuate the effect of TNF-α on the expression of occludin and ZO-1. As shown in Fig 2, we found that the effect of TNF-α on occludin expression could be markedly attenuated by propofol in a concentration of 25μM, which is within clinical plasma concentration that ranges from 5 to 50μM during the induction and maintenance of general anesthesia [22]. Moreover, we also found propofol had no effect on TNF-α-modulated ZO-1 expression (data not shown). In an ongoing project of our study, we found that propofol could attenuate hypoxia-modulated occludin and ZO-1 expression in hCMEC/D3 cells. We reasoned that propofol exert different effects under different stimuli.
Hypoxia-inducible factor-1α (HIF-1α) can be activated by many inflammatory mediators such as TNF-α [23]. HIF-1a is an important regulator of the cellular and systemic hypoxia response and is involved in cell survival, apoptosis, cellular metabolic shift and cancer progression [24]. HIF-1a is usually induced by low-oxygen microenvironment. Recently studies have reported that various stimuli, including inflammation, could increase the expression of HIF-1α even under normoxic conditions [25]. More importantly, the role of HIF-1α in tight junction damage has recently become evident [26]. It is reported that hypoxia selectively disrupts brain microvascular endothelial tight junction complexes through a HIF-1α dependent mechanism [26].
In our study, HIF-1α and its target gene vascular endothelial growth factor (VEGF) were shown to be activated by TNF-α. VEGF is a homodimeric 45-kDa glycoprotein and is secreted by a variety of cells including cerebral microvascular endothelial cells [27]. It is recognized that VEGF could affect angiogenesis, endothelial cell survival and function. Studies also have revealed that VEGF was involved in occludin expression [28]. The study revealed that the level of occludin was lowered by VEGF treatment and found that VEGF increased BBB permeability in brain microvascular endothelial cells monolayer cultures [28]. Consistently, we found that TNF-α could induce the expression of HIF-1α and VEGF, and decrease the expression of occludin in hCMEC/D3 cells. Furthermore, the inhibitor of HIF-1α markedly decrease the expression of VEGF also demonstrated the correlate between HIF-1α and VEGF.
VEGF exerts its physiological function via interacting with receptors on vascular endothelial cells [29]. The receptors of VEGF are receptor tyrosine kinases, including VEGFR-1, VEGFR-2, and VEGFR-3 [30]. Although the receptors of VEGF are highly similar in Amino acid compositions, different VEGFRs have disparate functions and play key role in pathological processes and signal transduction [31]. In previous studies, VEGFR-1 is mainly involved in angiogenesis [32] and VEGFR-3 seems to be involved in migration and invasion in cancer cells [33], while VEGFR-2 is closely involved with inflammation response.
It has been reported that VEGF is mainly induced by hypoxia and inflammation, and it activates VEGFR-2 to promote its auto-phosphorylation to generate p-VEGFR-2 [34], which could affect cell proliferation, differentiation, and migration, along with specific effects on regulation of cell permeability [35]. The phosphorylation of VEGFR-2 is induced by the inflammation cytokine TNF-α [36], suggesting that the VEGF/VEGFR-2 pathway is involved in the inflammatory process. Consistently, our data showed that TNF-α could induce the phosphorylation of VEGFR-2 in hCMEC/D3 cells, while the inhibitor of VEGF could depress this effect. Little literature has revealed the effect of propofol on the phosphorylation of VEGFR2. However, we found that propofol could significantly inhibit the phosphorylation of VEGFR2 and we reasoned that this effect of propofol may be relevant to the blocking of the upstream pathways such as VEGF.
Studies have revealed that the breakdown of BBB was involved with the activation of ERK signaling pathway [37]. As one of the downstream molecules of VEGF/ VEGFR-2 signaling pathway [38], we inferred ERK may active by TNF-α and may be involved in the expression of occludin. In our study, we found that TNF-α could induce the phosphorylation of ERK, which was attenuated by propfol. In addition, we demonstrated that the inhibitor of ERK, could significantly alleviate the effect of TNF-α on occludin expression as propofol. It also has been reported that p38MAPK was also activated by TNF-α and was involved in occludin expression [13]. Although we did not examine the phosphorylation and expression of p38MAPK in this study, we found that the inhibitor of ERK could almost completely block TNF-α-modulated occludin expression in hCMEC/D3 cells. Thus, we inferred that compared with p38MAPK, ERK plays a more important role in TNF-α-modulated occludin expression.
We realized the protective effects of propofol and the mechanism found in this in vitro study should be confirmed in animal models and we are planning to exam the effect of propofol protect TNF-α-modulated occludin expression and BBB impairment in the mouse model. In addition, the signaling pathway should be proved repeatedly by different inhibitors and this is going to be one of our next works.
Conclusion: TNF-α could decrease the expression of occludin via inducing HIF-1α/ VEGF/ VEGFR-2/ ERK signaling pathway, which was also involved in the effect of propofol.