Hemoglobin-induced endothelial cell permeability is controlled, in part, via a myeloid differentiation primary response gene-88-dependent signaling mechanism.

The release of hemoglobin (Hb) with hemolysis causes vascular dysfunction. New evidence implicates Hb-induced NF-κB and hypoxia inducible factor (HIF) activation, which may be under the control of a Toll-like receptor (TLR)-signaling pathway. Nearly all TLR-signaling pathways activate the myeloid differentiation primary response gene-88 (MyD88) that regulates NF-κB. We hypothesized that the differing transition states of Hb influence endothelial cell permeability via NF-κB activation and HIF regulation through a MyD88-dependent pathway. In cultured human dermal microvascular endothelial cells (HMECs-1), we examined the effects of Hb in the ferrous (HbFe(2+)), ferric (HbFe(3+)), and ferryl (HbFe(4+)) transition states on NF-κB and HIF activity, HIF-1α and HIF-2α mRNA up-regulation, and monolayer permeability, in the presence or absence of TLR4, MyD88, NF-κB, or HIF inhibition, as well as superoxide dismutase (SOD) and catalase. Our data showed that cell-free Hb, in each transition state, induced NF-κB and HIF activity, up-regulated HIF-1α and HIF-2α mRNA, and increased HMEC-1 permeability. The blockade of either MyD88 or NF-κB, but not TLR4, attenuated Hb-induced HIF activity, the up-regulation HIF-1 and HIF-2α mRNA, and HMEC-1 permeability. The inhibition of HIF activity exerted less of an effect on Hb-induced monolayer permeability. Moreover, SOD and catalase attenuated NF-κB, HIF activity, and monolayer permeability. Our results demonstrate that Hb-induced NF-κB and HIF are regulated by two mechanisms, either MyD88 activation or Hb transition state-induced ROS formation, that influence HMEC-1 permeability.

Cardiac Per2 functions as novel link between fatty acid metabolism and myocardial inflammation during ischemia and reperfusion injury of the heart.

Disruption of peripheral circadian rhyme pathways dominantly leads to metabolic disorders. Studies on circadian rhythm proteins in the heart indicated a role for Clock or Per2 in cardiac metabolism. In contrast to Clock(-/-), Per2(-/-) mice have larger infarct sizes with deficient lactate production during myocardial ischemia. To test the hypothesis that cardiac Per2 represents an important regulator of cardiac metabolism during myocardial ischemia, we measured lactate during reperfusion in Per1(-/-), Per2(-/-) or wildtype mice. As lactate measurements in whole blood indicated an exclusive role of Per2 in controlling lactate production during myocardial ischemia, we next performed gene array studies using various ischemia-reperfusion protocols comparing wildtype and Per2(-/-) mice. Surprisingly, high-throughput gene array analysis revealed dominantly lipid metabolism as the differentially regulated pathway in wildtype mice when compared to Per2(-/-). In all ischemia-reperfusion protocols used, the enzyme enoyl-CoA hydratase, which is essential in fatty acid beta-oxidation, was regulated in wildtype animals only. Studies using nuclear magnet resonance imaging (NMRI) confirmed altered fatty acid populations with higher mono-unsaturated fatty acid levels in hearts from Per2(-/-) mice. Unexpectedly, studies on gene regulation during reperfusion revealed solely pro inflammatory genes as differentially regulated 'Per2-genes'. Subsequent studies on inflammatory markers showed increasing IL-6 or TNFα levels during reperfusion in Per2(-/-) mice. In summary, these studies reveal an important role of cardiac Per2 for fatty acid metabolism and inflammation during myocardial ischemia and reperfusion, respectively.