Effects Of Cell-free Hemoglobin Research Articles

Abstract 267: Hemoglobin, Endothelial Cells, and TLRs. Common Connection?

Introduction: Patients suffering from chronic hereditary hemolytic anemic syndromes, such as sickle cell disease (SCD) and thalassemia, are often at risk for systemic and pulmonary vascular disease. It has been suggested that chronic exposure to cell free hemoglobin (CFH) may contribute to some vascular diseases associated with these syndromes such as pulmonary arterial hypertension. To date, the vasculotoxic effects of CFH have mostly been attributed to its pro-oxidant and nitric oxide scavenging characteristics. However, emerging evidence suggests CFH may contribute to inflammation by directly activating a signaling cascade event by binding to a pattern recognition receptor (PRR) or a toll like receptor (TLR) on vascular endothelial cells. Hypothesis: We hypothesized that CFH would increase the activity of transcription factors, NF-κb and HIF-1α, via a MyD88-dependent pathway. Methods: Human microvascular endothelial cells (HMEC) were transfected with either an NF-κB or HIF-1α luciferase reporter gene and treated with CFH (ferrous, ferric, and ferryl forms) in the presence or absence of SOD, catalase, dexamethasome, MyD88 inhibitor, or, the PHD inhibitor, DMOG. Messenger RNA for HIF-1α and HIF-2 were also measured after treatments. Results: All three states of hemoglobin increased NF-κB and HIF-1α activity in a dose response fashion, with ferryl inducing the greatest activity of both NF-κB and HIF-1α. Time course studies showed that NF-κB and HIF-1α activity tracked together. A unique synergy was noted with co-treatment of ferryl and DMOG. Co-treatment with SOD or catalase did not inhibit the CFH-induced NF-κB or HIF-1α response. Dexamthasome and MyD88 inhibition reduced the CFH-induced NF-κB and HIF-1α activity. Conclusion: Our results support the hypothesis, that CFH may activate a TLR or PRR signaling cascade subsequently activating MyD88-NF-κB and HIF-1α. Our data, that showed SOD and/or catalase did not block CFH effects, suggests that this event is not mediated by CFH pro-oxidant characteristics. CFH-induced HIF-1α was blocked by NF-κB inhibition with either, Dexamethasome or MyD88 inhibition emphasizing the importance of NF-κB in the HIF-1α pathway.

Pathophysiological consequences of hemolysis. Role of cell-free hemoglobin

Abundant hemolysis is associated with a number of inherent and acquired diseases including sickle-cell disease (SCD), polycythemia, paroxysmal nocturnal hemoglobinuria (PNH) and drug-induced hemolytic anemia. Despite different etiopathology of hemolytic diseases, many concomitant symptoms are comparable and include e.g. hypertension, hemoglobinuria and hypercoagulation state. Studies in the last years have shown a growing list of mechanisms lying at the basis of those symptoms, in particular irreversible reaction between cell-free hemoglobin (Hb) and nitric oxide (NO) - endogenous vasorelaxant and anti-thrombotic agent. Saturation of protective physiological cell-free Hb-scavenging mechanisms results in accumulation of Hb in plasma and hemoglobinemia. Extensive hemoglobinemia subsequently leads to hemoglobinuria, which may cause kidney damage and development of Fanconi syndrome. A severe problem in patients with SCD and PNH is pulmonary and systemic hypertension. It may lead to circulation failure, including stroke, and it is related to abolition of NO bioavailability for vascular smooth muscle cells. Thrombotic events are the major cause of death in SCD and PNH. It ensues from lack of platelet inhibition evoked by Hb-mediated NO scavenging. A serious complication that affects patients with excessive hemolysis is erectile dysfunction. Also direct cytotoxic, prooxidant and proinflammatory effects of cell-free hemoglobin and heme compose the clinical picture of hemolytic diseases. The pathophysiological role of plasma Hb, mechanisms of its elimination, and direct and indirect (via NO scavenging) deleterious effects of cell-free Hb are presented in detail in this review. Understanding the critical role of hemolysis and cell-free Hb is important in the perspective of treating patients with hemolytic diseases and to design new effective therapies in future.

Haptoglobin Genotypes among Pediatric and Adult Sickle Cell Patients: An Emerging Genetic Modifier?.

The role of hemolysis and its end product, cell free hemoglobin, as a Nitric Oxide (NO) scavenger in the pathogenesis of some complications of sickle cell disease (SCD) such as pulmonary hypertension has recently been recognized. Hemoglobin (Hb) released from red blood cells as a result of hemolysis complexes with haptoglobin (Hp). Hb-Hp complexes are removed from the circulation by tissue macrophages in the liver and spleen through CD163, the newly identified hemoglobin scavenger receptor expressed on the monocytes and macrophages. Hp is a polymorphic protein with two well-known alleles, Hp-1 and Hp-2 with worldwide distribution. Although the worldwide frequencies of these two allelles are approximately equal, there is significant ethnic and geographic variation in the distribution of Hp-1 and Hp-2 among different populations. Hp locus is on chromosome 16q22. each Hp monomer consists of one α-chain and one β-chain. An intragenic duplication event during evolution resulted in an elongated α-chain thus generating the Hp-2 allele. Thus individuals homozygous for the long α-2 chain express large multimeric molecules (Hp 2-2). Over the past 5 years, Hp-2 allele has emerged as a major susceptibility gene for development of vascular complications such as coronary artery restenosis and risk of cardiovascular disease, particularly in diabetic patients. It has been reported that the Hb-Hp-2 complexes have a 10-fold greater affinity for the CD163 receptor compared to Hb-Hp-1, and the interaction of Hb-Hp-2 complexes with CD163 on macrophages generates a more powerful inflammatory response with the release of pro-inflammatory cytokines such as IL-1β, IL-6, and GM-CSF. These observations led us to examine the role of Hp polymorphisms in sickle cell disease. We performed genotyping of the Hp-alleles with PCR in 21 pediatric patients (age 4 mo-10 years) and 41 adults patients (age 16–60). The results of this genotyping are shown in the table below. The Hp-1 allele frequency was 0.24 among pediatric patients and 0.52 among adults; while that of Hp-2 was 0.76 in the pediatric age group and 0.48 in adults. This difference was significant (p=0.0023). The significantly higher allele frequency of Hp-2 in pediatric patients compared to adults may indicate a selection against this genotype and suggests that Hp-1 allele may be associated with a survival advantage. This is in keeping with the deleterious effects of Hp-2 allele reported in the literature and suggests yet another mechanism whereby hemolysis may lead to adverse outcomes associated with Hp polymorphisms, in addition to the NO scavenging effects of cell free hemoglobin. Detailed studies are currently being undertaken to elucidate the mechanism whereby Hp polymorphisms may impact on the pathophysiology of SCD.PERCENTAGE OF HAPTOGLOBIN GENOTYPES BETWEEN ADULT AND PEDIATRIC SICKLE CELL PATIENTSADULTPEDSsHp 1–141.4%9.5%Hp 1–222.5%28.6%Hp 2–236.6%62.0%

Effects of Cell-Free Hemoglobin on Hypoxia-Inducible Factor (HIF-1α) and Heme Oxygenase (HO-1) Expressions in Endothelial Cells Subjected to Hypoxia

Effects of Cell-Free Hemoglobin on Hypoxia-Inducible Factor (HIF-1α) and Heme Oxygenase (HO-1) Expressions in Endothelial Cells Subjected to Hypoxia

Effects of Cell-Free Hemoglobin on Hypoxia-Inducible Factor (HIF-1α) and Heme Oxygenase (HO-1) Expressions in Endothelial Cells Subjected to Hypoxia

We have investigated the impact of diaspirin cross-linked hemoglobin (DBBF-Hb), a blood substitute, on cell signaling pathways that are modulated in part by biological peroxides (i.e., hydrogen peroxide, lipid peroxide, and peroxynitrite). Bovine aortic endothelial cells (BAECs) subjected to hypoxia expressed hypoxia-inducible factor (HIF-1alpha) in a time course that paralleled the expressions of heme oxygenase (HO-1). Co-incubation of the oxy form (HbFe(2+)) with hypoxic BAECs resulted in an increase in the expression of HIF-1alpha in a manner that corresponded linearly with the decay of HbFe(2+) and accumulation of the ferric form (HbFe(3+)). Inclusion of HbFe(3+) with hypoxic BAECs produced twice as much expression in the HIF-1alpha and HO-1 proteins as opposed to HbFe(2+) alone, or HbFe(2+) plus hypoxia. In addition, higher and more persistent levels of the ferryl form (HbFe(4+)), due to the consumption of endogenous peroxides, were found in the hypoxic media containing hemoglobin. Nitric oxide (NO) released from an NO donor reduced the levels of HIF-1alpha in the hypoxic cells treated with either HbFe(2+) or HbFe(3+), but had little or no effect on the levels of HO-1. DBBF-Hb modulates key cell-signaling pathways by competing with peroxides required for the deactivation of HIF-1alpha, which may modulate important physiological mediators.

The Hemodynamic Effects of Cell-Free Hemoglobin During General and Epidural Anesthesia

The Hemodynamic Effects of Cell-Free Hemoglobin During General and Epidural Anesthesia

Evaluation of the Capabilities of a Hemoglobin Vesicle as an Artificial Oxygen Carrier in a Rat Exchange Transfusion Model

Encapsulation of hemoglobin within a liposome is one of the strategies in the development of artificial oxygen carriers. It maintains the oxygen transporting properties of hemoglobin and, at the same time, eliminates the side effects of cell free hemoglobin. Hemoglobin vesicles (HbV) are a type of liposome encapsulated hemoglobin. They have a particle size of approximately 250 nm, a hemoglobin concentration of 10 g/dl, and the oxygen affinity, P50, is regulated to 32 Torr. In this study the authors examined the oxygen transporting capability of HbV in vivo, by performing exchange transfusions in rats. Exchange transfusion (90% of the estimated circulatory volume) with HbV suspended in 5% albumin (containing 160 mEq/L, sodium and 107 mEq/L, chloride) was carried out in male Wistar rats. Mean arterial pressure and heart rate were monitored through the arterial catheter. Arterial blood samples for gas analyses were also obtained from the arterial catheter. Abdominal aortic blood flow was measured by an ultrasonic pulsed Doppler flowmeter as an indicator of cardiac output. The oxygen tension of blood withdrawn from the right atrium was measured as an indicator of mixed venous oxygen tension. These values were employed to calculate oxygen delivery and consumption. Renal cortical and skeletal muscle tissue oxygen tensions were monitored as indicators of tissue perfusion. Five percent albumin and washed rat red blood cells suspended in 5% albumin containing 10 g/dl of hemoglobin; were employed as controls. At the completion of a 90% exchange transfusion, renal cortical and skeletal muscle tissue oxygen tensions, along with oxygen delivery and consumption, were sustained almost equally well with the HbV suspension compared to the washed rat red blood cell suspension, but declined significantly with the albumin suspension. The results indicate that the oxygen transporting capability of HbV was almost equivalent to that of rat red blood cells.

Blood volume and cardiac index in rats after exchange transfusion with hemoglobin-based oxygen carriers.

We have measured plasma volume and cardiac index in rats after 50% isovolemic exchange transfusion with human hemoglobin cross-linked between the alpha-chains with bis(3,5-dibromosalicyl)fumarate (alpha alpha Hb) and with bovine hemoglobin modified with polyethylene glycol (PEGHb). alpha alpha Hb and PEGHb differ in colloid osmotic pressure (23.4 and 118.0 Torr, respectively), oxygen affinity (oxygen half-saturation pressure of hemoglobin = 30.0 and 10.2 Torr, respectively), viscosity (1.00 and 3.39 cP, respectively), and molecular weight (64,400 and 105,000, respectively). Plasma volume was measured by Evans blue dye dilution modified for interference by plasma hemoglobin. Blood volumes in PEGHb-treated animals were significantly elevated (74.0 +/- 3.5 ml/kg) compared with animals treated with alpha alpha Hb (49.0 +/- 1.2 ml/kg) or Ringer lactate (48.0 +/- 2.0 ml/kg) or with controls (58.2 +/- 1.9 ml/kg). Heart rate reduction after alpha alpha Hb exchange is opposite to that expected with blood volume contraction, suggesting that alpha alpha Hb may have a direct myocardial depressant action. The apparently slow elimination of PEGHb during the 2 h after its injection is a consequence of plasma volume expansion: when absolute hemoglobin (concentration x plasma volume) is compared for PEGHb and alpha alpha Hb, no difference in their elimination rates is found. These studies emphasize the need to understand blood volume regulation when the effects of cell-free hemoglobin on hemodynamic measurements are evaluated.