Hemoglobin and Its Scavenger Protein Haptoglobin Associate with ApoA-1-containing Particles and Influence the Inflammatory Properties and Function of High Density Lipoprotein

Hemoglobin (Hb) uniquely associates with proinflammatory HDL in atherogenic mice and coronary heart disease (CHD) patients. In this paper, we report that Hb and its scavenger proteins, haptoglobin (Hp) and hemopexin (Hx) are significantly increased in apoA-1-containing particles of HDL both in mouse models of hyperlipidemia and in CHD patients, when compared with wild type mice and healthy donors, respectively. We further demonstrate that the association of Hb, Hp, and Hx proteins with HDL positively correlates with inflammatory properties of HDL and systemic inflammation in CHD patients. Interestingly, HDL from Hp^?/? mice under atherogenic conditions does not accumulate Hb and is anti-inflammatory, suggesting that (i) Hp is required for the association of Hb with HDL and (ii) Hb·Hp complexes regulate the inflammatory properties of HDL. Moreover, treatment of apoE^?/? mice with an apoA-1 mimetic peptide resulted in significant dissociation of Hb·Hp complexes from HDL and improvement of HDL inflammatory properties. Our data strongly suggest that HDL can become proinflammatory via the Hb·Hp pathway in mice and humans, and dissociation of Hb·Hp·Hx complexes from apoA-1-containing particles of HDL may be a novel target for the treatment of CHD.Atherosclerosis is the leading cause of morbidity and mortality in Western society. The inverse relationship between HDL² cholesterol and the risk of atherosclerosis is well established. Although HDL cholesterol is an epidemiological predictor of risk for coronary heart disease (CHD) (1), a significant number of CHD events occur in patients with normal LDL and HDL cholesterol levels (1, 2). Based on a number of recent studies in both animal models and human samples, it appears that the anti- or proinflammatory nature of HDL may be a more sensitive indicator of the presence or absence of atherosclerosis than HDL cholesterol levels. HDL exerts anti-inflammatory functions by promoting reverse cholesterol transport and preventing the oxidation of LDL (3, 4). We have previously shown that the anti-inflammatory functions of HDL can be impaired in humans (5) rabbits (6), and mice (7) during inflammatory processes. This impaired HDL is proinflammatory in nature, as characterized by (i) decreased levels and activity of anti-inflammatory, antioxidant factors, including apolipoprotein A1 (apoA-1) and PON1 (paraoxonase 1) (8); (ii) gain of proinflammatory proteins, such as serum amyloid A and ceruloplasmin (6); (iii) increased lipid hydroperoxide (LOOH) content (9); (iv) reduced potential to efflux cholesterol (10); and (v) diminished ability to prevent LDL oxidation (11). The molecular changes and mechanisms that promote anti-inflammatory HDL conversion to proinflammatory HDL are currently not well understood.We recently reported the identification and characterization of Hb associated with proinflammatory HDL in atherogenic/hyperlipidemic mice and in human CHD patients (12). We demonstrated that under normal circumstances, a small amount of Hb is always found outside of red blood cells (RBC) in the non-lipoprotein fractions of serum (on the order of 10 μm compared with the >1 m concentration of Hb in RBC). We further demonstrated that under conditions of hyperlipidemia in mice and in CHD patients, the non-RBC Hb moves out of the non-lipoprotein fractions and associates with HDL. This HDL-associated Hb was shown to play an important role in the modulation of HDL function, suggesting that Hb is not only a novel biomarker but may also serve as a therapeutic target for atherosclerosis (12). We therefore sought to determine the molecular mechanisms that play a role in the association of Hb with HDL.Hp and Hx are plasma proteins with the highest binding affinity for Hb (K_d ≈ 1 pm) and heme (K_d < 1 pm), respectively. They are expressed mainly in the liver and belong to the family of acute phase proteins, whose synthesis is induced during inflammatory processes (13, 14). Under conditions of increased hemolysis, Hb becomes highly toxic because of the oxidative properties of heme, which participates in the Fenton reaction to produce reactive oxygen species causing cell injury (15). Under these conditions, Hb is known to be scavenged by Hp·Hx complexes that utilize specific receptor pathways, thus protecting the body against the harmful effects of excess free Hb. We set out to determine whether the Hb·Hp·Hx system (i) also participates in the association of Hb with proinflammatory HDL and (ii) plays a role in the inflammatory properties of HDL.In this paper, we demonstrate that (i) Hb·Hp·Hx complexes associate with HDL in CHD patients and mouse models of hyperlipidemia but not in healthy human donors and wild type mice, and (ii) Hb·Hp·Hx association with HDL positively correlates with proinflammatory properties of HDL. We further show that HDL from Hp^?/? mice on an atherogenic diet is anti-inflammatory and did not contain any Hb, suggesting that (i) Hp is required for the association of Hb with HDL, and (ii) Hp regulates the inflammatory properties of HDL. In contrast to HDL from Hp^?/? mice, HDL from Hx^?/? mice on normal chow was proinflammatory and associated with Hb and Hp, suggesting a novel protective role for Hx in HDL function. When apoE^?/? mice were treated in vivo with an apoA-1 mimetic peptide, 4F, Hb·Hp·Hx dissociated from HDL. Our data strongly suggest that the association of Hb·Hp·Hx with HDL plays an important role in the functional status and inflammatory properties of HDL.