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Next we examined the bioenergetic profile of pulmonary endothelium following a prolonged incubation 12 h with various ferryl species of Hb. In a similar experimental setup, ferryl species of both mutant Hb proteins i. Figure 6. Oxidized hemoglobins cause bioenergetic reprogramming in pulmonary endothelial cells. Cell free plasma Hb, and its prosthetic heme, are known to disrupt endothelial function, drive oxidative and inflammatory stress. Heme, the byproduct of Hb oxidation is also recognized as a damage-associated molecular pattern DAMP molecule Gladwin et al.

Studies in a sickle cell mice model showed unequivocally that heme released from plasma oxidized Hb binds to the toll-like receptor-4 TLR-4 on macrophages that can activate the innate immune system Belcher et al. In both diseases, RBC-derived microparticles were shown to promote vascular complications as they carry both highly oxidized Hb and considerable quantities of free heme to the vascular system Chaichompoo et al. Naturally occurring human Hb variants resulting mostly from single point mutations alter Hb structure and biochemical properties with physiological effects ranging from insignificant to severe Thom et al.

These Hb mutants, which are subjected to evolutionary pressures, can provide unique model systems to address the question as to why some Hb variants are more oxidatively stable while others succumb to oxidative stress and develop into a full circulatory disorder Thom et al.

To assess the role of HbS and HbE in promoting endothelial functional impairment, we monitored endothelial permeability, intracellular oxidative stress response parameters and some key metabolic changes involving mitochondrial bioenergetics and changes in glycolytic flux. Vascular endothelium very selectively allows passage of blood or plasma components into the adjacent tissues by acting as a dynamic barrier. However, vascular endothelial integrity is highly susceptible to damage by various stress conditions including production of inflammatory mediators Nooteboom et al. This phenomenon may be attributed to the previously documented higher rates of autoxidation, oxidative instability and heme release from the two mutant Hb proteins compared to common HbA Table 1.

Higher rates of autoxidation of mutant Hbs coupled with the formation of more persistent and damaging ferryl species under oxidative stress conditions can target other biological molecules Kassa et al. We have recently shown that ferric Hb loses heme at rates substantially higher than that of ferryl Hb Kassa et al. This was also supported by a higher expression of heme oxygenase-1 HO-1 when ferric Hb was added to cultured lung alveolar epithelial cells E The mitochondrial dysfunction reported here may therefore be attributed to a combination of accelerated heme loss from the ferric form and protein radical formation associated with the ferryl Hb.

This is consistent with a recent study in which it was shown that ferric metHb causes oxidative damage to myelin basic protein MBP and myelin lipids, partly by transferring its heme moiety to protein and lipid, but mostly as an intact protein possibly via formation of a ferryl radical Bamm et al. Interestingly, we also noticed a high degree of HPAEC membrane leakage mediated by the oxidized Hb molecules; this finding is consistent with a recent observation where a greater loss of endothelial monolayer integrity was evident under ferryl Hb exposure Lisk et al.

Our results further show that mutant Hb proteins, e. Findings from another previous work showing differential reactivity of these mutant Hbs causing lipid peroxidation also strongly supports our results Chiu et al. These events can be attributed to their heme releasing capability in addition to their high redox potential Bonaventura et al. Studies on endothelial energy metabolism have shown the dominance of glycolysis over mitochondrial ATP production, although endothelial mitochondria are considered critical for the maintenance of functional integrity of these special vascular cells Davidson and Duchen, ; Groschner et al.

Endothelial mitochondria can integrate several cell death and survival pathways through transition pore opening, intracellular calcium signaling and redox signaling Davidson and Duchen, Our present study is undoubtedly the first to observe bioenergetic changes in human endothelial cells mediated by the Hb variants. Since, endothelial cells are more dependent on glycolytic ATP production, we also for the first time monitored changes in glycolytic rates under the influence of Hb exposure. Despite some early signs of leakage in endothelial monolayers, neither of the Hb variants in reduced states showed any imbalance in basal respiration or glycolysis in HPAECs.

Effects of phase separating systems on lyophilized hemoglobin - Dimensions

However, an augmentation of uncoupled respiration by both HbS and HbE associated with more mitochondrial oxygen radical production and lipid peroxidation supports a mechanism linked to higher rates of oxidation and greater heme release by the two proteins. Carbon monoxide is one of the major byproduct of heme catabolism by HO-1 and is known to cause inhibition of glycolysis and uncoupling of mitochondrial respiration by opening mitochondria specific ion-channels in endothelium Kaczara et al. Our results clearly show that direct interactions of oxidized Hbs with vascular endothelium can cause severe impairment of selective electron transport chain complexes especially cytochrome c oxidase.

This explains the ferryl Hb mediated loss of mitochondrial function in intact endothelial cells during a short co-incubation. In contrast, all the ferryl Hbs also augmented uncoupled respiration quite significantly after a longer incubation. We have previously shown that HO-1 is strongly expressed by both ferric and ferryl Hbs Chintagari et al. Therefore, a boost in maximal respiration following uncoupling by FCCP can be easily correlated with our finding that Hb proteins promote high expression of stress response proteins in varying degree depending on their oxidative profile and possibly be considered as an outcome of HO-1 mediated CO production.

A concomitant loss of glycolytic reserve in HPAEC by ferryl Hbs after a long incubation also indicates a metabolic reprogramming in the endothelium probably due to an exhaustive loss of glycolytic capacity in HPAEC. Similar observations have been seen in some cancer cells where a prolonged uncoupled state of mitochondrial respiration resulted in metabolic exhaustion and loss of glycolysis Wegiel et al. In summary, we show for the first time a comparative toxicity of different mutant Hbs on a pulmonary endothelial model based on their distinct oxidative profile; and also propose a mechanism that explains Hb-induced metabolic and functional alteration.

Our results elucidating the mechanism of extracellular Hb-induced oxidative damage to the mitochondrial respiratory chain will contribute to better understanding of some of the underlying pathophysiology of pulmonary arterial hypertension caused by cell free Hbs; and, in addition, lend new insight into potential antioxidative therapeutic interventions in hemolytic diseases.

Conception and design: SJ and AA.

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Experiments: SJ and FM. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Abraham, B. Isolated Hb Providence beta82Asn and beta82Asp fractions are more stable than native HbA 0 under oxidative stress conditions. Biochemistry 50, — Aebi, H.

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X-ray Crystallography of Hemoglobins

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Blood Cells Mol. Chintagari, N. Oxidized ferric and ferryl forms of hemoglobin trigger mitochondrial dysfunction and injury in Alveolar Type I Cells. Cell Mol. Chiu, D. Correlation of membrane lipid peroxidation with oxidation of hemoglobin variants: possibly related to the rates of hemin release.