Quantitative assessment of hemoglobin-induced endothelial barrier dysfunction

Dull, Randal O.; DeWitt, Bracken J.; Dinavahi, Ramani; Schwartz, Louis B.; Hubert, Christopher G.; Pace, Nathan L.; Fronticelli, Clara

doi:10.1152/japplphysiol.00102.2004

Cited by 31 publications

(11 citation statements)

References 37 publications

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“…RBC-endothelial interactions, even in the absence of flow, need to be considered, in addition to the extravasation of cell-free Hb into the subendothelial space. This latter process is thought to play a significant role in NO scavenging as indicated in studies in the development of blood substitutes (42)(43)(44)(45). It has been shown that the hypertensive effect of various hemoglobins is dependent on their size (43,44).…”

Section: Discussionmentioning

confidence: 98%

“…At low concentrations of Hb a significant fraction of dimers will form, which can penetrate the endothelial glomerulus, and the remaining Hb tetramers are likely to extravasate through larger pores (44,46). It is possible that extravasation in vessel bioassays plays an especially significant role, because free Hb is known to increase Hb permeability itself due to its interaction with the endothelium (45). Thus, in the absence of flow, extravasation of free Hb may contribute significantly to enhanced NO scavenging by free Hb compared with RBCs in addition to any intrinsic differences.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Nitric Oxide Scavenging by Red Blood Cells as a Function of Hematocrit and Oxygenation

Azarov

Huang

Basu

et al. 2005

Journal of Biological Chemistry

171

199

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The reaction rate between nitric oxide and intraerythrocytic hemoglobin plays a major role in nitric oxide bioavailability and modulates homeostatic vascular function. It has previously been demonstrated that the encapsulation of hemoglobin in red blood cells restricts its ability to scavenge nitric oxide. This effect has been attributed to either factors intrinsic to the red blood cell such as a physical membrane barrier or factors external to the red blood cell such as the formation of an unstirred layer around the cell. We have performed measurements of the uptake rate of nitric oxide by red blood cells under oxygenated and deoxygenated conditions at different hematocrit percentages. Our studies include stopped-flow measurements where both the unstirred layer and physical barrier potentially participate, as well as competition experiments where the potential contribution of the unstirred layer is limited. We find that deoxygenated erythrocytes scavenge nitric oxide faster than oxygenated cells and that the rate of nitric oxide scavenging for oxygenated red blood cells increases as the hematocrit is raised from 15% to 50%. Our results 1) confirm the critical biological phenomenon that hemoglobin compartmentalization within the erythrocyte reduces reaction rates with nitric oxide, 2) show that extraerythocytic diffusional barriers mediate most of this effect, and 3) provide novel evidence that an oxygen-dependent intrinsic property of the red blood cell contributes to this barrier activity, albeit to a lesser extent. These observations may have important physiological implications within the microvasculature and for pathophysiological disruption of nitric oxide homeostasis in diseases. Nitric oxide (NO)3 is an endothelium-derived relaxation factor that is synthesized in endothelial cells (1-4). To elicit its vasodilatory activity, NO must diffuse to the smooth muscle cells and activate soluble guanylate cyclase. In 1994, Lancaster suggested that the proximity of the endothelium to the millimolar concentrations of hemoglobin (Hb), an avid NO scavenger, would severely compromise the efficiency of the NO/soluble guanylate cyclase pathway (5). However, later studies have indicated that the physical compartmentalization of hemoglobin within the red blood cell (RBC) effectively reduces the apparent rate at which NO is consumed by Hb (6 -15). One contributory element to this effect is a RBC-free zone at the blood/endothelium interface that is present during laminar flow (7, 9, 10). In addition, the rate of NO consumption has been reported to occur up to 1000 times more slowly by red blood cells than by an equivalent concentration of cell-free hemoglobin. Two potential mechanisms for this effect involve either the presence of an unstirred layer surrounding the red blood cell that is formed as a result of NO diffusion (6, 13) or a physical barrier to NO diffusion that is integral to the protein-rich RBC submembrane (11). The faster effective reaction of NO with cell-free Hb compared with RBC-encapsulated hemoglobin may ...

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Section: Discussionmentioning

confidence: 98%

Section: Discussionmentioning

confidence: 99%

Nitric Oxide Scavenging by Red Blood Cells as a Function of Hematocrit and Oxygenation

Azarov

Huang

Basu

et al. 2005

Journal of Biological Chemistry

171

199

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“…Consequently, extravasation has been proposed to play a major role in the hypertensive effect of hemoglobin-based blood substitutes. 39,[51][52][53][54][55] The hypertensive effect of various hemoglobins is partially dependent on their size. 52,53 It is possible that in addition to rate limitations caused by diffusion, extravasation of hemoglobin into the interstitial space may have contributed to the 1000-fold difference in scavenging between cell-free and RBC-encapsulated Hb observed by Liao et al in the absence of flow.…”

Section: Hemoglobin Extravasationmentioning

confidence: 99%

Unraveling the Reactions of Nitric Oxide, Nitrite, and Hemoglobin in Physiology and Therapeutics

Kim‐Shapiro

Schechter

Gladwin

2006

ATVB

262

204

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Abstract-The ability of oxyhemoglobin to inhibit nitric oxide (NO)-dependent activation of soluble guanylate cyclase and vasodilation provided some of the earliest experimental evidence that NO was the endothelium-derived relaxing factor (EDRF). The chemical behavior of this dioxygenation reaction, producing nearly diffusion limited and irreversible NO scavenging, presents a major paradox in vascular biology: The proximity of large amounts of oxyhemoglobin (10 mmol/L) to the endothelium should severely limit paracrine NO diffusion from endothelium to smooth muscle. However, several physical factors are now known to mitigate NO scavenging by red blood cell encapsulated hemoglobin. These include diffusional boundaries around the erythrocyte and a red blood cell free zone along the endothelium in laminar flowing blood, which reduce reaction rates between NO and red cell hemoglobin by 100-to 600-fold. Beyond these mechanisms that reduce NO scavenging by hemoglobin within the red cell, 2 additional mechanisms have been proposed suggesting that NO can be stored in the red blood cell either as nitrite or as an S-nitrosothiol (S-nitroso-hemoglobin). The latter controversial hypothesis contends that NO is stabilized, transported, and delivered by intra-molecular NO group transfers between the heme iron and ␤-93 cysteine to form S-nitrosohemoglobin (SNO-Hb), followed by hypoxia-dependent delivery of the S-nitrosothiol in a process that links regional oxygen deficits with S-nitrosothiol-mediated vasodilation. Although this model has generated a field of research examining the potential endocrine properties of intravascular NO molecules, including S-nitrosothiols, nitrite, and nitrated lipids, a number of mechanistic elements of the theory have been challenged. Recent data from several groups suggest that the nitrite anion (NO 2 Ϫ ) may represent the major intravascular NO storage molecule whose transduction to NO is made possible through an allosterically controlled nitrite reductase reaction with the heme moiety of hemoglobin. As subsequently understood, the hypoxic generation of NO from nitrite is likely to prove important in many aspects of physiology, pathophysiology, and therapeutics. Key Words: nitric oxide Ⅲ nitrite Ⅲ hemoglobin Ⅲ vasodilation Ⅲ red blood cell N itric oxide (NO) is the endothelium-derived relaxing factor that modulates vascular tone by activating soluble guanylyl cyclase (sGC) in smooth muscle. [1][2][3][4] It is now appreciated that NO is produced in endothelial cells by the endothelial NO synthase enzyme and participates in many aspects of normal vascular physiology, including tonic vasodilation and inhibition of platelet activation and endothelial adhesion molecule expression. Endothelial dysfunction, characterized by reduced bioavailability of endothelial derived NO, is a central mechanistic feature of coronary artery disease and its risk factors, including diabetes, hypertension, smoking, and obesity. Excessive NO production from inducible NO synthase is a central mechanism of septic shock. Nov...

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“…Three mechanisms contribute to reduced NO scavenging by RBCs [46]. : (1) the rate of the reaction is largely limited by external diffusion of NO through the plasma to the surface of the RBC [44], especially due to the presence of a red cell free zone adjacent to the vessel walls where NO is made [37][38][39]; (2) NO diffusion is partially blocked by a physical barrier across the RBC membrane [40,43,47]; and (3) RBC-encapsulated Hb is efficiently compartmentalized in the lumen; it does not extravasate into the endothelium and interstitium [44,[48][49][50][51][52].…”

Section: Introductionmentioning

confidence: 99%

The potential of Angeli's salt to decrease nitric oxide scavenging by plasma hemoglobin

Azarov

Jeffers

et al. 2008

Free Radical Biology and Medicine

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Release of hemoglobin from the erythrocyte during intravascular hemolysis contributes to the pathology of a variety of diseased states. This effect is partially due to the enhanced ability of cellfree plasma hemoglobin, which is primarily found in the ferrous, oxygenated state, to scavenge nitric oxide. Oxidation of the cell-free hemoglobin to methemoglobin, which does not effectively scavenge nitric oxide, using inhaled nitric oxide has been shown to be effective in limiting pulmonary and systemic vasoconstriction. However, the ferric heme species may be reduced back to ferrous hemoglobin in plasma and has the potential to drive injurious redox chemistry. We propose that compounds that selectively convert cell-free hemoglobin to ferric, and ideally iron-nitrosylated heme species that do not actively scavenge nitric oxide would effectively treat intravascular hemolysis. We show here that nitroxyl, generated by Angeli's salt (Sodium α-oxyhyponitrite, Na 2 N 2 O 3 ), preferentially reacts with cell-free hemoglobin compared to that encapsulated in the red blood cell under physiologically relevant conditions. Nitroxyl oxidizes oxygenated ferrous hemoglobin to methemoglobin and can convert the methemoglobin to a more stable, less toxic species, iron-nitrosyl hemoglobin. These results support the notion that Angeli's salt or a similar compound could be used to effectively treat conditions associated with intravascular hemolysis.

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Quantitative assessment of hemoglobin-induced endothelial barrier dysfunction

Cited by 31 publications

References 37 publications

Nitric Oxide Scavenging by Red Blood Cells as a Function of Hematocrit and Oxygenation

Nitric Oxide Scavenging by Red Blood Cells as a Function of Hematocrit and Oxygenation

Unraveling the Reactions of Nitric Oxide, Nitrite, and Hemoglobin in Physiology and Therapeutics

The potential of Angeli's salt to decrease nitric oxide scavenging by plasma hemoglobin

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