Elsevier

Microvascular Research

Volume 95, September 2014, Pages 15-25
Microvascular Research

Computational analysis of nitric oxide biotransport to red blood cell in the presence of free hemoglobin and NO donor

https://doi.org/10.1016/j.mvr.2014.06.004Get rights and content

Highlights

  • NO availability in the presence of extracellular Hb decreases with increasing hematocrit.

  • Extracellular free Hb affects NO–RBC interactions only at low hematocrit.

  • NO donor increased plasma NO concentrations despite the presence extracellular free Hb.

  • The first model to quantify NO supplementation via NO donors in vascular lumen

Abstract

Red blood cells (RBCs) modulate nitric oxide (NO) bioavailability in the vasculature. Extracellular free hemoglobin (Hb) in the vascular lumen can cause NO bioavailability related complications seen in pathological conditions such as pancreatitis, sickle cell disease and malaria. In addition, the role of extracellular free Hb has been critical to estimate kinetic and transport properties of NO–RBCs interactions in ‘competition experiments’. We recently reported a strong dependence of NO transport on RBC membrane permeability and hematocrit. NO donors combined with anti-inflammatory drugs are an emergent treatment for diseases like cancer, cardiovascular complications and wound healing. However, the role of RBCs in transport NO from NO donors is not clearly understood. To understand the significance of extracellular free Hb in pathophysiology on NO availability and estimation of the NO-RBC interactions, we developed a computational model to simulate NO biotransport to the RBC in the presence of extracellular free Hb. Using this model, we studied the effect of hematocrit, RBC membrane permeability and NO donors on NO–RBC interactions in the presence and absence of extracellular free Hb. The plasma NO concentration gradients and average plasma NO concentrations changed minimally with increase in extracellular free Hb concentrations at the higher hematocrit as compared to those at the lower hematocrit irrespective of the NO delivery method, indicating that the presence of extracellular free Hb affects the NO transport only at a low hematocrit. We also observed that NO concentrations increased with NO donor concentrations in the absence as well as in the presence of extracellular free Hb. In addition, NO donor supplementation may increase NO availability in the plasma in the event of loss of endothelium-derived NO activity.

Introduction

Nitric oxide (NO) is generated in the endothelial cells as a response to shear stress from blood flow in the vasculature and acts a vasodilator (Ignarro et al., 1987, Moncada et al., 1991). The red blood cell (RBC) has emerged as a key player in the modulation of NO bioavailability (Owusu et al., 2012). Questions have been raised on the mechanisms of RBC–NO interaction and the transport of NO in physiologic and pathophysiologic conditions (Deonikar and Kavdia, 2010a). These issues have been raised for a number of reasons including the close proximity of RBCs to the endothelial cells, the high affinity of hemoglobin (Hb) for NO (Eich et al., 1996), and the probable pathway of NO escape from RBCs (Owusu et al., 2012). In addition, NO releasing drugs are being used in treatment of several pathological conditions including cancer, cardiovascular disease and wound healing (Baetta et al., 2013, Blecher et al., 2012, Cheng et al., 2012, Schade et al., 2011).

Endothelium-derived NO diffuses into the smooth muscle cell, activates soluble guanylate cyclase (sGC) that catalyzes conversion of GTP to cGMP and thus initiates vasodilation (Arnold et al., 1977). Majority of the NO enters the vascular lumen and is consumed by Hb in the RBCs (Butler et al., 1998, Gladwin et al., 2000, Joshi et al., 2002). RBC interaction with NO interaction leads to formation of methemoglobin and nitrate in oxygenated conditions and nitrosyl hemoglobin (HbNO) in deoxygenated conditions (Eich et al., 1996, Herold et al., 2001). On the other hand, RBCs can ‘store’ NO bioactivity in the form of S-nitrosohemoglobin (SNOHb) (Stamler et al., 1997) and deoxy-Hb can act as nitrite reductase to form NO (Gladwin et al., 2006, van Faassen et al., 2009). NO availability for vasodilation at the smooth muscle layer is determined by the transport barriers in the vascular lumen for NO transport from endothelium to the RBC–Hb or for transport of NO/active NO species from RBC to the vascular wall (Chen et al., 2007, Chen et al., 2008a, Deonikar and Kavdia, 2010b, Deonikar and Kavdia, 2013). NO consumption by RBC–Hb is much lower than that by extracellular free Hb due to the barriers for NO biotransport that include the extracellular free layer near the vessel wall (Deonikar and Kavdia, 2010a, Liao et al., 1999), extracellular diffusion (Liu et al., 2007), RBC membrane (Deonikar and Kavdia, 2013, Vaughn et al., 2001) and intracellular diffusion (Sakai et al., 2008).

In the absence of some of the transport barriers for free Hb, the presence of extracellular free Hb causes vasoconstriction (Doherty et al., 1998). Extracellular free Hb is also implicated in the hypertensive effects observed in pancreatitis and myocardial infarction (Winslow, 2013) as well as pulmonary hypertension seen in sickle cell disease, malaria and hemolytic anemia (Deonikar and Kavdia, 2012, Gladwin et al., 2010). Additionally, the lysis of blood or packed RBCs during storage is reported to cause post-transfusion toxicity (Donadee et al., 2011, Gladwin and Kim-Shapiro, 2009, Stapley et al., 2012). Theoretical analysis of hemolytic pathologies such as sickle cell disease (Deonikar and Kavdia, 2013) showed that even small amounts of extracellular free Hb led to insufficient NO concentrations at smooth muscle cell layer indicating insufficient vasodilation and subsequent hypertension.

In addition to its role in reducing NO bioavailability in physiological and pathophysiological conditions (Deonikar and Kavdia, 2012, Jeffers et al., 2006); extracellular free Hb is used in analyzing the various NO transport barriers in the vascular lumen and estimation of kinetic parameters for the NO–RBC interactions (Azarov et al., 2005, Liu et al., 2002, Vaughn et al., 2000). One such technique is an integrated experimental and theoretical analysis of competition for NO between extracellular free Hb and RBCs (‘competition experiments’). The ‘competition experiments’ using extracellular free Hb and well dispersed NO donor in the RBC suspension, (Vaughn et al., 2000, Vaughn et al., 2001) reported an ~ 1000–2000 times lower NO uptake by RBCs compared to that of free Hb owing to transport barriers from either the RBC membrane or intracellular diffusion. Moreover in these studies, NO–RBC interactions did not change significantly for hematocrit higher than 5%. Subsequent analyses of competition experiments implicated extracellular diffusion resistance as the main transport barrier for NO transport to RBC (Azarov et al., 2005, Liu et al., 2002) and the RBC membrane limits the NO transport effectively only under oxygenated conditions (Azarov et al., 2005).

Most of the ‘competition experiments’ to analyze NO–RBC reaction kinetics and the role of transport resistance were conducted at low hematocrit (≤~15%) (Liu et al., 1998, Liu et al., 2002, Vaughn et al., 2000, Vaughn et al., 2001). However, in a recent computational analysis, we have shown that the contribution from extracellular diffusion and RBC membrane resistance towards the overall transport resistance depended on the hematocrit and membrane permeability (Deonikar and Kavdia, 2013). These new insights indicate that the transport and kinetic characteristics of NO interactions with RBCs may be different at physiological range of hematocrit than those reported at a lower hematocrit.

To explore this possibility, an understanding of NO–RBC interactions in the presence of extracellular Hb at physiologic hematocrit is needed. For this purpose, we developed a computational model for the NO–RBC interactions for hematocrit ranging from 1 to 60% in the presence and absence of extracellular free Hb. This mathematical model also gives us a reliable platform to understand the NO–RBC interactions and NO distribution in the vascular lumen under pharmacological interventions where NO donors can be used to supplement NO activity.

Towards this end, we used two types of NO sources: 1) a constant NO source at a distance from the center of the RBC to simulate the endothelial NO transport to an RBC; and 2) homogenous NO source to simulate NO donor or NO delivery drug in the plasma layer surrounding each RBC. We quantified the NO availability inside the RBC, at the RBC membrane and at the outer edge of plasma. These results will help understand NO transport to and from RBCs and elucidate the role of RBCs in modulating the NO availability in vasculature in the presence of extracellular free Hb and an NO donor in the plasma layer. In addition, these results help us interpret results from the competition experiments.

Section snippets

Mathematical model

In order to simulate NO biotransport to an RBC, we developed a 1-dimensional model in spherical geometry based on an earlier RBC model (Deonikar and Kavdia, 2013). Briefly, the RBC core, the RBC membrane and the plasma layer surrounding the RBC are treated as concentric spheres as seen in Fig. 1. Extracellular free Hb is assumed to be uniformly dispersed in the plasma layer. We simulated two cases of NO biotransport in the present geometry. For Case 1, NO diffuses from outer boundary of the

Effect of extracellular free Hb and RBC membrane permeability on NO concentration profiles

To assess the effect of RBC membrane permeability in the presence of extracellular free Hb, we analyzed NO transport to RBC for permeability values of 0.0415, 0.4, 1 and 4 cm·s 1. NO concentration at the edge of the plasma layer was 100 nM for these simulations. Figs. 2a and b shows the NO concentration profiles for 10% Hct and 45% Hct, respectively. The NO concentrations inside the RBC core were almost zero for all studied hematocrit, RBC membrane permeability and extracellular free Hb

Discussion

In this investigation, a 1-dimensional computational transport model of NO–RBC interactions is presented. The computational model analyzes the effects of extracellular free Hb on transport of NO to RBC at low and high hematocrit values for fixed distant NO source at the edge of the plasma layer and a uniformly distributed NO donor in the plasma. Understanding the mechanism of RBC interactions with NO in the presence of extracellular free Hb and quantification of NO supplementation in the form

Acknowledgments

This study is supported by NIH grants R01 HL084337.

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