Nitric Oxide Scavenging, Abnormal Vasoregulation and Oxidative Damage in sph/sph Mice with Severe Hereditary Spherocytosis: Possible Consequences of Red Blood Cell Hemolysis. (2005)

Abstract The membrane skeleton, a multiprotein complex located just beneath the plasma membrane, provides the red blood cell (RBC) with the mechanical strength and deformability required to withstand high shear forces generated in the microcapillaries. Spectrin, a tetramer composed of a- and b- subunits, is the backbone of the erythroid membrane skeleton. Previously, we have shown that sph/sph mice have severe hereditary spherocytosis (HS) due to a spontaneous single-base deletion in the murine erythroid a-spectrin gene, Spna1. HS mice have severe hemolytic anemia, compensatory reticulocytosis, altered RBC morphology and increased fragility. Vascular dysfunction in sph/sph mice likely plays an important role in the mechanism by which these mice develop a high incidence of cardiac thrombosis and stroke between 6 and 12 weeks of age. We hypothesize that serum free hemoglobin released from intravascular hemolysis of sph/sph RBCs and xanthine oxidase, released from ischemic tissues, impairs endothelial cell function by scavenging nitric oxide (NO) and increasing oxidative damage. To test this hypothesis, we used helium electroparamagnetic resonance (EPR), to quantify plasma free Hb and NO scavenging capacity in the plasma of the mice; immunohistochemistry to determine tissue and vascular levels of xanthine oxidase and 3-nitrotyrosine; and, facialis arteries to measure changes in acetylcholine, endothelium and eNOS-dependent vasodilation. By EPR we found that the plasma free Hb and NO scavenging capacity in the plasma of sph/sph mice is much greater than that of the normal +/+ mice. Immunohistochemistry (IHC) for XO and NTyr revealed XO staining was decreased in livers of sph/sph mice as compared to livers from normal +/+ mice. XO staining was increased in local patches on the endothelium of lungs isolated from sph/sph mice compared to lungs from +/+ mice. NTyr, a marker of peroxynitrite formation was also increased in a focal manner in lungs of sph/sph mice compared to lungs of +/+ mice. Acetylcholine-induced and eNOS-dependent vasodilation in sph/sph mice was significantly impaired compared to vasodilation in normal +/+ mice. Taken together these data suggest the hemoglobin removal system in sph/sph mice is saturated, leading to increased free Hb and nitric oxide scavenging. IHC studies reveal XO is released from liver in sph/sph mice and once released binds the endothelium of lung, quite distal from the original site of injury. Such changes likely contribute to marked increases in NTyr staining and impaired endothelium and eNOS-dependent vasodilation in facialis arteries isolated from sph/sph mice. Taken together, these data indicate that sph/sph mice with severe HS have increased plasma free Hb and NO scavenging capacity as well as increased release of xanthine oxidase and subsequent binding to vascular endothelial cells to locations that are distal the original site of injury. Such plasma and vascular changes in hemoglobin and oxidative enzymes likely play a critical role in the mechanisms contributing to aberrant vasoregulation and initiating the pathways of oxidative damage found in sph/sph mice.