High glucose-induced apoptosis in human vascular endothelial cells is mediated through NF-κB and c-Jun NH2-terminal kinase pathway and prevented by PI3K/Akt/eNOS pathway
Introduction
Diabetes mellitus is an important risk factor for cardiovascular diseases, and hyperglycemia is generally regarded as one of the major causes of vascular complications in diabetic patients [1], [2]. Endothelial cell dysfunction plays a major role in the pathogenesis of vascular abnormalities. Evidences suggest that, in endothelial cells, high glucose would induce production of reactive oxygen species (ROS), which can cause cellular dysfunction and even cell death [3], [4]. In our previous studies, we demonstrated that, in human umbilical vein endothelial cells (HUVECs), the activation of ROS-dependent c-Jun N-terminal protein kinase (JNK) and caspase-3 mediates the vascular endothelial cell apoptosis induced by high glucose [5].
It has been shown that the transcriptional factor NF-κB can be induced by a multitude of stimuli, including cytokines and ROS [6], [7]. NF-κB mainly consists of a dimmer, either a homo- or a heterodimer of p50 and p65, and an inhibitor IκB. Degradation of IκB results in NF-κB activation which controls the transcription of several genes involved in inflammatory responses, cell growth, survival and apoptosis [8], [9], [10], [11], [12], [13]. In some recent studies, it has been demonstrated that NF-κB activation can down-regulate pro-apoptotic JNK signaling and thus prevent apoptosis in many cells types [14], [15]. However, in endothelial cells, NF-κB activation has been found to produce cell apoptosis instead of preventing the cells from apoptosis [12], [13], [16]. Du et al. [16] have demonstrated that ROS-dependent activation of NF-κB plays a major role in cell apoptosis induced by high glucose [16]. Yet, the mechanisms underlying the hyperglycemia-induced endothelial cell apoptosis involving the activation of NF-κB is still not well studied. Furthermore, whether NF-κB has a crosstalk with JNK cascade and thereby regulates endothelial cell apoptosis remains unclear.
Through activation of the downstream serine/threonine kinase Akt, phosphatidylinositol 3′-kinase (PI3K) plays an important role in averting cell death produced by many death stimuli [17], [18], [19], [20], [21]. In endothelial cells, Akt activation has been reported to promote cell survival [19], [22]. Akt can activate endothelial nitric oxide synthase (eNOS), which leads to nitric oxide (NO) production [22]. Because NO can avidly scavenge superoxide anion, it can prevent superoxide anion from forming its dismutation product, hydrogen peroxide [23]. Evidences show that PI3K/Akt/NO pathway plays an important role in preventing ROS-induced endothelial cell injury [24]. In our previous studies, we have demonstrated that exposure of HUVECs to high glucose leads to a progressive increase of eNOS expression, reaching a maximum at 6 h, and decreasing gradually after 24 h [25]. We also demonstrated that the NO donor, sodium nitroprusside (SNP), could partially reverse high glucose-induced apoptosis in HUVECs [25]. Yet, the upstream signaling mechanisms involved in the up-regulation of eNOS expression by high glucose, and the contribution of endogenous NO production in preventing high glucose-induced endothelial cell apoptosis are still unclear.
In this study, for the first time, we demonstrated that the sustained activation of NF-κB following exposure to high glucose leads to activation of pro-apoptotic JNK signaling, which is followed by cell apoptosis. Moreover, we also showed that gradual loss of Akt phosphorylation mediated by ROS also contributes to cell death while early Akt-dependent eNOS activation provides protection on the endothelial cells from apoptosis.
Section snippets
Cell culture and cell treatment
HUVECs were cultured as previously described [5]. Cells were seeded at a density of 1 × 105 per 75-cm2-flask in medium 199 (Gibco, Grand Island, New York), supplemented with 20 mM HEPES, 100 μg/ml endothelial cell growth substance (Collaborative Research Inc, Bedford, MA), and 20% fetal calf serum (Gibco). The cultures were maintained at 37 °C with gas of 5% CO2-95% air mixture. All media were supplemented with 5 U/ml heparin, 100 IU/ml penicillin, and 0.1 μg/ml streptomycin. Medium was refreshed
Induction of apoptosis by high glucose
As reported in our previous study [25], we again demonstrated the induction of apoptosis by high glucose (33 mM) in HUVECs in this study. Apoptosis was not evident until 36 h of high glucose treatment. In fact, a paradoxical increase in cell proliferation was observed at 24 h (3.5-fold increase of 3H-thymidine incorporation, data not shown). Thus, similar to that observed in other cell types and in some pathological conditions, high glucose treatment induces a biphasic response in cell growth,
Discussion
In our previous study, we have demonstrated that ROS induced by high glucose treatment can elicit JNK activation, which in turn activates caspase-3 to produce apoptosis in HUVECs [5]. Here, we extended to clarify the role of the transcriptional factor NF-κB in the regulation of JNK signaling induced by high glucose treatment. Furthermore, we investigated the PI3K/Akt and downstream eNOS signals that might prevent apoptosis in the same experimental settings.
The protein kinase Akt, a
Acknowledgement
This work was supported by a grant (NSC 91-2314-B-087-001) from the National Science Council of Taiwan, Republic of China.
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