Abstract
Peripheral artery disease (PAD) is atherosclerotic occlusion of vessel outside the heart and most commonly affects the lower extremities. Diabetes (DM) accelerates the course and severity of PAD. Studies have shown that vascular endothelial cell NF-κB activity is required for post ischemic adaptation in experimental PAD. To better understand how DM contributes to PAD severity, we investigated the role of DM hyperglycemia in the activation of NF-κB under ischemic conditions. Induced ischemia in human vascular endothelial cell (HUVEC) cultures increased components of both canonical and non-canonical NF-κB pathways in the nucleus (p65 1.0 ± 0.1 vs 1.5 ± 0.2, p< 0.05, RelB 1.0 ± 0.1 vs 1.5 ± 0.2, p<0.01). Similarly, HUVEC acutely exposed to high glucose (HG, 25 mM) activated both canonical (IκB-α degradation, normal vs. HG 1.25 ± 0.02 vs 0.9 ± 0.0, p<0.05) and non-canonical NF-κB (p100 degradation, normal vs HG 0.021±0.001 vs 0.016±0.000, p<0.05) pathways. Prolonged exposure (3 days) of HUVEC to high glucose before ischemia resulted in impaired NF-κB activation as evident from decreased IκB phosphorylation (pIκB/IκB in normal glucose and ischemia 1.56 ± 0.22 vs 1.12 ± 0.35, p<0.01). To understand the signaling pathways underlying the ischemic activation of the NF-κB pathway, we used an array of antibodies to phosphoproteins involved in the inflammatory pathway. Compared to the lysates from cells grown in normal glucose, the lysates from cells grown in prolonged high glucose had dramatically increased phosphorylation of PKC-β2 (PKC-β2pSer661, 8-fold increase). To test whether this increase in PKC-β2pSer66 impairs NF-κB activation by ischemia, we treated HUVECS with prolonged high glucose exposure and ruboxystaurin (Rbx) (20 nM), an inhibitor of PKC-β2 phosphorylation, prior to ischemic exposure. Immunoblotting results confirmed that inhibition of PKC-β2 phosphorylation enhanced the ischemia induced NF-κB activation in HUVEC in this condition. We then tested the effect of Rbx on PKC-β2 phosphorylation and NF-κB activation in vivo in Akita mice, a model for type 1 diabetes. Consistent with our in vitro findings, in experimental PAD, NF-κB activity in the ischemic hind limb of Akita mice was significantly lower than those of the wild type (WT) mice as measured by IκB-α degradation (WT ischemic vs Akita ischemic; 0.04 ± 0.03 vs 0.10 ± 0.04 p<0.05). However, treatment of Akita mice with Rbx increased NF-κB activation in the ischemic hind limb (Akita ischemic 0.10 ± 0.04 vs ischemic+ Rbx 0.05 ± 0.02, p<0.05). Moreover, compared to the WT mice, the untreated Akita mice showed an impaired perfusion in the ischemic limbs (% perfusion recovery, WT vs Akita; 80.1 ± 10.3 vs 55.7 ± 10.1, p<0.05, n=5-8) that was improved in Rbx treated Akita mice (96.3 ± 2.3, p<0.01). Thus, hyperglycemic conditions increase PKC-β2pSer66 in endothelial cells attenuating salutary NF-κB activation contributing to poor PAD outcomes in DM.
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