High glucose induced nuclear factor kappa B mediated inhibition of endothelial cell migration
Introduction
Disrupted integrity of the endothelial cells plays a significant role in the genesis of vascular complications of diabetes such as delayed wound healing and accelerated atherosclerosis [1], [2]. The initiation of cell migration is critical to repair mechanisms that restore the normal integrity of endothelial cell lining of blood vessels and for the process of wound healing [3], [4]. Though the altered environment in vascular cells due to elevated glucose levels has been known to contribute delayed wound healing in diabetic patients, the underlying molecular mechanisms are not clearly understood.
Recent reports indicate that one of the key transcriptional regulator, nuclear factor kappa B (NF-κB), and its downstream signaling pathway may play an important role in diabetes [6], [8]. The Rel/NF-κB family of proteins consists of homo- or hetero-dimers of p49, p50, p52, p65, Rel B and c rel subunits. The heterodimeric complex of p50 and p65 is a predominant NF-κB species in cultured endothelial cells [7]. In its inactive cytosolic state, NF-κB is complexed with an inhibitor subunit called IκB. NF-κB activation requires multiple phosphorylations of IκB followed by ubiquitination and proteolytic degradation [9]. As a consequence, the p50–p65 heterodimeric complex of NF-κB translocates into the nucleus and binds specific DNA sequences, which results in the transcription of target genes [5]. Activation of NF-κB can be triggered by various extracellular stimuli, including TNF-α, IL-1β, bacterial endotoxin, viral infection, calcium ionophore, radiation, reactive oxygen radicals, hypoxia, and hyperglycemia [8], [9], [10].
In the present study, the effect of high glucose induced NF-κB activity on endothelial cell migration was studied. High glucose-induced NF-κB activity could mediate the inhibition of endothelial cell migration by regulating eNOS production and thereby influencing intracellular nitric oxide (NO) levels. These sequential events may subsequently lead to delayed wound healing. Investigation of high glucose induced pathogenic mechanisms involved in early lesion formation may lead to interventions that delay or prevent lesion progression and associated complications in diabetes mellitus.
Section snippets
Cell culture
Human aortic endothelial cells (HAEC; Clonetics, San Diego, CA) of passages 4–8 were used for the following studies. Cells were trypsinized and sub-cultured in the MCDB-131 medium (Sigma, St. Louis, MO), supplemented with 250 ng/ml fibroblast growth factor (Pepro Tech, Rocky Hill, NJ), 1 mg/ml epidermal growth factor (Pepro Tech), 1 mg/ml hydrocortisone (Sigma), and 100 U/ml penicillin/streptomycin (Mediatech, Herndon, VA) with 10% iron supplemented bovine calf serum (BCS, Hyclone, OR) at 37 °C
Statistical Analysis
Data are shown as mean±S.E.M. or S.D. Differences among treatment groups were evaluated by using an unpaired ‘t’ test and Stat View 5.0 software. Each experiment was performed three times independently. In addition, coefficients of variation were calculated for proliferation and migration assays to determine the reproducibility of the results by using one way (for migration studies) and two-way (for proliferation assays) variance analysis.
High glucose inhibits endothelial cell migration
Inhibitory effect of high glucose on migration of HAEC was studied by evaluating the number and characteristics of cells that migrated into denuded area during 24 h incubation. First, to rule out the influence of cell proliferation over migration and to determine, precisely, the number of migrating cells, HAEC were treated with 5 mM hydroxyurea, which arrested cell proliferation. Cells were incubated in the presence or absence of hydroxyurea for 24 h and examined for cell proliferation using
Discussion
Delayed wound healing observed in diabetic patients can be accelerated and the vascular complications can be prevented by tight control of blood glucose level [11]. We, for the first time, show evidence for the involvement of NF-κB mediating high glucose induced inhibition of endothelial cell migration. The in vitro wound model (Fig. 2) used in the present study clearly demonstrates that elevated glucose inhibits endothelial migration. In addition to the number of migrating cells as shown in
Acknowledgements
We thank Dr Thomas Prihoda for his assistance in statistical analysis. This work was supported by Juvenile Diabetes Foundation International research grant (1-2000-203) and funds from NIH-NHLBI (1 R01 HL63032-01A1) to SM.
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