Mechanisms of haptoglobin protection against hemoglobin peroxidation triggered endothelial damage

Abstract

Extracellular hemoglobin (Hb) has been recognized as a disease trigger in hemolytic conditions such as sickle cell disease, malaria, and blood transfusion. In vivo, many of the adverse effects of free Hb can be attenuated by the Hb scavenger acute-phase protein haptoglobin (Hp). The primary physiologic disturbances that can be caused by free Hb are found within the cardiovascular system and Hb-triggered oxidative toxicity toward the endothelium has been promoted as a potential mechanism. The molecular mechanisms of this toxicity as well as of the protective activities of Hp are not yet clear. Within this study, we systematically investigated the structural, biochemical, and cell biologic nature of Hb toxicity in an endothelial cell system under peroxidative stress. We identified two principal mechanisms of oxidative Hb toxicity that are mediated by globin degradation products and by modified lipoprotein species, respectively. The two damage pathways trigger diverse and discriminative inflammatory and cytotoxic responses. Hp provides structural stabilization of Hb and shields Hb's oxidative reactions with lipoproteins, providing dramatic protection against both pathways of toxicity. By these mechanisms, Hp shifts Hb's destructive pseudo-peroxidative reaction to a potential anti-oxidative function during peroxidative stress.

Figures

Figure 1

Hb oxidation, endothelial damage, and Hp protection. (a) Endothelial monolayer electrical resistance during treatment with human Hb (huHb, 2 mg/ml), a low/nontoxic level of GOX (2.5 mU/ml), or the combination of huHb+GOX. Only the combination of huHb+GOX resulted in a significant decline of endothelial resistance over time. ECIS traces represent means±S.D. of four replicate measurements. (b) The decline of HPAEC monolayer resistance was morphologically associated with a redistribution of the adherens junction protein β-catenin (red) in fluorescence light microscopy (blue=DAPI-stained nuclei). Imaging of the same specimen region with a correlative SEM revealed intercellular gap formation (arrow head) with intercellular space spanning filopodia in the regions with decreased β-catenin staining. The arrow indicates a conglomerate of Hb aggregates. No changes were observed when cells were only treated with Hb or GOX alone (data not shown). The incubation time in these experiments was 6 h. (c) β-Catenin (red) redistribution in HPAECs was completely prevented when Hp (2 mg/ml) was present during the combined huHb+GOX treatment in confocal laser scanning microscopy images (original magnification × 630)

Figure 2

Hb oxidative stability, aggregate formation, and endothelial damage. (a) Aggregate formation during the reaction of H2O2 (GOX 2.5 mU/ml) with the different types of Hb (2 mg/ml) was recorded by time-resolved spectrophotometry at 700 nm under identical conditions as in the ECIS experiment with HPAEC shown in (b). At equal H2O2 exposure, endothelial barrier function was compromised in the presence of huHb but not in the presence of the structure-stable αXLHb or bvHb. The addition of Hp (2 mg/ml) to the huHb+GOX reaction (red line) completely prevented aggregate formation and endothelial barrier breakdown (blue line). (c) The aggregates/precipitates can be localized on HPAEC by SEM (images were recorded after 5 h). No aggregates can be visualized in the presence of Hp. (d) Internalization of the Hb aggregates (arrows) by the endothelial cells can be observed on zoom-in cell surface views (insert) and in a 3D reconstruction of serial sections recorded with a FIB-SEM instrument. The white color appearance of the Hb precipitates on the front section of the 3D image is indicative of the high density of these particles that allowed automatic recognition and reconstruction by the image processing software (Imaris Software), which clearly show internalized particles as well as particle aggregates on the surface of the cell

Figure 3

Biochemical characterization of the H2O2 reactions with human Hb, bvHb, α-α-cross-linked Hb, and the Hb/Hp complex. (a) Time-resolved analysis of the reactions of the different types of Hb in complete endothelial cell culture medium at 37 °C. Spectra were recorded at 2-min intervals and deconvoluted to calculate the concentrations of the individual Hb oxidation states. (b) Heme release and transfer to Hpx from ferric Hb. Serial spectra were recorded from a mixture of ferric Hb (12.5 μM) with a 10-fold molar excess of Hpx. The first spectrum is red (ferric Hb) and the last recorded spectrum is blue (heme-Hpx). The insert shows the reference spectra of ferric Hb (red) and heme-Hpx (blue), respectively. (c) Decay of ferric Hb over time. (d) Radical generation during Hb-H2O2 reactions was explored with spin-trapping EPR under the same conditions used for endothelial cell culture (cell culture medium, Hb (2 mg/ml), and GOX (2.5 mU/ml)) in the presence of 50 mM DEPMPO. EPR spectra (left panel) of the different experimental samples were recorded sequentially over time, and the integrated spectral values are plotted in the right panel. The insert shows an anti-DMPO Western blot of Hbs reacted with H2O2 in the presence of DMPO. The signals indicate generation of protein based radicals

Figure 4

Intrinsic and extrinsic reaction pathways of Hb peroxidative endothelial damage. (a) The ‘intrinsic' oxidation pathway (red) involves globin oxidation with the subsequent formation of Hb aggregates that induce direct endothelial damage. The proposed ‘extrinsic' pathway (violet) involves oxidative transformation of bystander molecules (i.e., LDL) into toxic reaction products. (b) HUVECs and HPAECs were incubated with either LDL (0.5 mg/ml)+GOX 2.5 mU/ml, bvHb (2 mg/ml)+GOX, or LDL +bvHb+GOX to explore the extrinsic pathway of peroxidative toxicity. To compare the extrinsic pathway with the intrinsic pathway, some cells were treated with huHb (2 mg/ml)+GOX. Transendothelial resistance was measured with an ECIS instrument. (c) Hb-mediated LDL oxidation and protection by Hp. Left panel: O2 consumption was measured with an O2 sensitive micro-electrode in stirred reaction mixtures of LDL with huHb-Fe3+ (red), αXLHb-Fe3+ (dark violet), bvHb-Fe3+ (black), or huHb-Fe3+/Hp complex (blue). Right panel: the Hb/Hp complex (blue line) delayed the breakdown of endothelial electrical resistance compared with when αXLHb (dark violet) or bvHb (violet) were used in the reaction mixture with LDL+GOX. The control treatment (black line) was LDL+GOX in the absence of any Hb (LDL 0.5 mg/ml and Hbs 0.1 mg/ml). All ECIS traces represent mean±S.D. values of four replicate measurements

Figure 5

Gene expression analysis of endothelial cells exposed to Hb-catalyzed oxidative reactions. (a) Reaction components (= endothelial cell treatments) and the proposed in situ reaction intermediates and end products. (b) HPAECs were exposed to the indicated components for 8 h. The global mRNA expression pattern was analyzed by Agilent gene expression arrays. In the PCA, the global expression profile of each sample is represented as a ball that is color coded for its treatment condition. (c) Hierarchical clustering analysis of all genes that were significantly changed from baseline expression by any treatment condition. Each column represents the color-coded gene expression pattern of a single sample. Treatment conditions are indicated at the top of the graph. The treatment discriminative gene clusters that were used for functional network analysis are indicated at the right border of the graph. (d) The treatment discriminative gene clusters A (LDL peroxidation) and B (Hb degradation) were analyzed with Metacore software for the enrichment of functional gene networks. The 10 most significantly enriched networks are indicated

Figure 6

The Hb/Hp complex is a protective sink for cytotoxic extracellular H2O2. High-dose H2O2-mediated endothelial cell toxicity was mimicked by treating confluent HUVEC endothelial monolayers with 20 mU/ml GOX (high GOX). (a) β-Catenin (red) immunofluorescence of endothelial cells incubated with high GOX, high GOX+huHb, or high GOX+Hb/Hp (blue=DAPI-stained nuclei, original magnification × 630). (b) Global gene expression changes observed after 8 h with the treatments indicated on the top of the graph. The heat-map shows the result of a hierarchical clustering of the most significantly upregulated (red) and downregulated (blue) genes (compared with untreated controls). While there appeared to be almost no significant gene expression changes after high GOX+Hb/Hp treatment, distinct and mutually exclusive clusters of highly regulated genes appeared after treatment with either high GOX alone (→ oxidative stress) or high GOX+huHb (→ inflammation). (c) The breakdown of electrical resistance observed during high GOX treatment (red line) was exaggerated when huHb (2 mg/ml) was added to the system (blue line) and completely halted by the addition of Hb/Hp (2 mg/ml) (grey line). (d) RT-PCR confirmation of the representative signature genes of the oxidative stress response (ATF3), which was induced by high GOX alone, and inflammation (SELE), which was only induced by high GOX+Hb. Hp attenuates both responses to control levels (date are presented as mean±S.D. of three independent experiments)

Figure 7

Schematic summary of Hb-catalyzed oxidative endothelial damage and protection by Hp. Graphical illustration of the principal Hb toxicity and Hp protection pathways described in this study. We propose that Hp protects against peroxidative Hb toxicity by stabilizing the Hb structure (mechanism 1) and by shielding extrinsic molecules against Hb/heme-mediated peroxidative damage (mechanism 2)