FINAL REPORT FP7-MC-ERG-ATHEROHEME
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The central hypothesis of this proposal was that Fe-protoporphyrin IX (heme) that is a potent inducer of lowdensity lipoprotein (LDL) oxidation in vitro is an important pathophysiological mediator of LDL oxidation in vivo and plays an important role in the progression of atherosclerosis.
The major source of heme in the vasculature is hemoglobin (Hb). We hypothesized that upon hemolysis cell free
Hb oxidize and release heme that can target directly endothelial cells and macrophages causing endothelial cell and macrophage activation. Alternatively, heme can intercalate into low-density lipoprotein (LDL) and cause
LDL oxidation, which in turn can also target endothelial cells and macrophages.
Figure 1. Proposed mechanisms of heme-mediated LDL oxidation and proatherogenic effects of oxLDL.
Upon RBC lysis cellfree Hb can be oxidized by reactive oxygen/nitrogen species (ROS/RNS) to MetHb. MetHb readily releases its heme groups that can target LDL and cause its oxidative modification. OxLDL activates endothelial cells and Mφ. The deleterious effects of Hb and heme can be inhibited by Hp and Hx, respectively.
Several defense mechanisms evolved to cope with the deleterious effects of cell-free Hb as well as those of heme itself. Under homeostasis plasma cell-free Hb is bound by haptoglobin (Hp), forming Hb-Hp complexes that are cleared from circulation by hemophagocytic macrophages via the Hp receptor (CD163) that promotes heme catabolism by heme oxygenase-1 (HO-1). When the protective effect of Hp is overwhelmed, cell-free Hb accumulates in plasma and becomes oxidized, releasing its heme prosthetic groups. Hx is an acute-phase protein that binds free heme in plasma with the highest affinity of any protein described so far. The resulting heme-Hx complexes are removed via the Hx receptor (CD91), which promotes endocytosis of the heme-Hx complexes in macrophages.
Our objective was to study the effect of Hb and Hb oxidation products in atherogenesis and to test whether defense mechanisms - rely on the removal of cell free Hb and heme by Hp and Hx respectively - are important in athero-protection.
To test our hypothesis first we assessed whether chronic heme and Hb administration accelerate high-cholesterol diet-induced atherosclerosis in apolipoprotein E deficient (ApoE-/-) mice, a relevant atherogenic mice model.
Then we tested whether chronic hemolysis accelerate progression of high-cholesterol diet-induced atherosclerosis in ApoE-/- mice. To study the role of defense mechanism we tested whether atherosclerosis is exacerbated in ApoE-/- mice deficient for Hp, Hx, or heme oxygenase-1 (Hmox1). We studied the interactions between Hb and plaque lipids in vitro and their effects on vascular endothelial cells. Finally, we determined whether drugs that are able to inhibit heme-mediated LDL oxidation in vitro are able to inhibit progression of high-cholesterol diet-induced atherosclerosis in ApoE-/- mice.

FINAL REPORT FP7-MC-ERG-ATHEROHEME
1.
Chronic administration of Hb accelerate atherosclerosis in apoE deficient mice
2.
Hemolysis accelerates atherosclerosis in apoE deficient mice.
3.
Oxidized hemoglobin species induce oxidative modification of LDL.
4.
Oxidized Hb species trigger EC death.
5.
Oxidized hemoglobin species induce HO-1 and ferritin expression in HUVECs.
6.
OxLDL and reactive lipid mediators derived from complicated atherosclerotic lesions initiate Hb oxidation and globin-globin crosslinking.
7.
Hb oxidation and crosslinking induced by H2O2 and reactive lipid mediators can be inhibited by haptoglobin (Hp) or GSH/GPx.
8.
FerrylHb increases EC monolayer permeability and enhances monocyte adhesion.
9.
Deficiency of haptoglobin and hemopexin provide atheroprotection in ApoE deficient mice.
10.
Chronic hemolysis accelerates atherosclerosis in ApoE-/-Hp-/- and ApoE-/-Hx-/- mice.
In summary, we found that Hb and intravascular hemolysis can accelerate disease progression and that oxidized
Hb species behave in a pro-inflammatory and pro-oxidant manner in the atherosclerotic vessel wall. Our in vitro results highlighted the importance of Hb-lipid interactions in the atherosclerotic plaque in triggering these proinflammatory and pro-oxidant mechanisms. Based on these results one can assume that deficiency of molecules responsible for clearing of these dangerous molecules (Hb and heme) from circulation could facilitate atheroma formation. Our results indicate that in fact, this is not the case. Mice deficient for Hp or Hx are somehow protected in this rodent model of atherosclerosis. The molecular mechanism behind this phenomenon remained to be elucidated, but certainly that is our next question. We assume that physiological level of hemolysis in the absence of Hp or Hx results in continuous low-level of oxidative stress that switch on stress-adaptation mechanisms in these mice.
The project was undertaken at the Vascular Biology Research Laboratory at the Department of Medicine that is a very well equipped and well-prospering laboratory at the University of Debrecen with a strong financial stability. The researcher is the most senior scientist in the group and served as a “group-leader” during these 3 years with the support of Dr. József Balla. The researcher obtained a Hungarian Government Grant (OTKA
2010-2013) and recently applied for a new grant. She also received a 20-month fellowship (Zoltán Magyary
Postdoctoral Fellowship) that aims to support the career perspectives of outstanding young researchers.
Currently she supervises 5 PhD students very successfully: two of them are planning to obtain the PhD degree this year. Many undergraduate students joined her team to learn state of the art research methodology and to write their diploma thesis under her supervision. She obtained a permanent senior research fellow position at the University of Debrecen that provide her long-term stability and integration. She joined to the Vascular
Biology, Thrombosis and Haemostasis Research team of the Hungarian Academy of Sciences that was established in 2011 at the University of Debrecen.
In summary during this reintegration period the researcher has built up her own research group, attracted money for her independent research projects, demonstrated abilities to train PhD students and could achieve a permanent senior research fellow position at the University of Debrecen.