Specific inhibition of NF-κB subunits prevents norepinephrine dependent, athero-thrombotic gene expression in experimental models of psychosocial stress

Aims: Disease-related psychosocial stress is common in patients with diabetes. Stress evoked elevated catecholamine levels have been shown to induce the redox-sensitive, proinflammatory transcription factor NF-κB, which is supposed to contribute to the pathogenesis of the micro and macrovascular late diabetic complications. Since most of the NF-κB inducers also trigger the release of radical oxygen species, antioxidants were supposed to effectively block NF-κB activation. Although certain studies have indicated beneficial effects of NF-κB inhibition, numerous recent reports have shown that long-term systemic inhibition of NF-κB does not lead to the better outcome of disease. This absence of beneficial effect of NF-κB lowering drugs in the prevention and therapy of diabetic vascular complications and atherosclerosis might be due to the parallel inhibition of NF-κB regulated protective genes. Therefore, we hypothesized that differential inhibition of defined NF-κB subunits might inhibit atherothrombotic gene expression, while preserving cellular defense mechanisms.

Materials and methods: THP-1 cells were induced with 10nM norepinephrine (NE) in the presence or absence of pathway inhibitors. Subunit-specific NF-κB activation and expression of NF-κB regulated proatherogenic (ICAM-1, Tissue Factor (TF)) and protective(superoxide dismutase (MnSOD)) genes was studied using EMSA, Chromatin Immunoprecipitation, Western Blot and RT-PCR. Aortic tissue of ApoE-/–mice subjected to repeated restrain-stress were studied for atherosclerotic lesions and gene expression using Red Oil staining and immunohistochemistry.

Results: Induction of THP-1 cells with 10nM NE or repeated restraint stress in ApoE-/-mice both resulted in increased binding of different NF-κB-subunits at the ICAM-1, TF and MnSOD-promoter and subsequent increase in mRNA synthesis and gene expression. While ICAM-1 was controlled by NF-κB subunits p50, p65 and cRel, TF expression was driven by p65/cRel. In contrast, MnSOD expression was dependent on binding of p50/p65 heterodimers. In vitro and in vivo nuclear translocation of NF-κBp50 was dependent on PKC-activation, NF-κBp65 was activated via p38MAPKinase and NF-κBcRel via the PI3/Akt pathway. Analysis of aortic tissue from ApoE-/–mice chronically stressed in the absence or presence of pathway inhibitors demonstrated that only inhibition of cRel resulted in reduction of atherosclerotic lesions. In contrast long time inhibition of neither NF-κBp50 nor NF-κBp65, both reducing MnSOD expression, had effects on vascular damage.

Conclusion: NE induces differential activation of different NF-κB subunits that control proatherogenic, atherothrombotic and cellular defense mechanisms. Targeted and specific inhibition of NF-κB-subunits controlling proatherogenic gene expression, but not affecting cellular defence mechanisms, might therefore provide a future therapeutic option in the prevention and therapy of vascular complications in diabetes.