Vascular Dysfunction in Murine Models of Hemolytic Anemia.
Authors: Wandersee Nancy J. , Frei Anne C. , Holzhauer Sandra L. , Scott J. Paul , Pritchard Kirkwood A. , Fagan Karen A. , Hogg Neil , Hillery Cheryl A. 
Published: October 2019

Pulmonary hypertension affects approximately 30% of young adults with sickle cell disease (SCD) and is a risk factor for early death. There is increasing evidence that intravascular hemolysis contributes to the pathophysiology of pulmonary hypertension in SCD as well as other hemolytic disorders. In this study, we compare measures of vascular dysfunction in Berkeley sickle cell mice (SCD mice) with a murine model of hereditary spherocytosis (sph/sph mice) that exhibit severe hemolytic anemia due to alpha spectrin deficiency, but without HbS-induced RBC sickling. We assessed right ventricular systolic pressure in vivo as a measure of pulmonary arterial function and endothelial-dependent vasodilation of facialis arteries ex vivo as a measure of systemic arterial function. Right ventricular systolic pressures and right ventricle to body weight ratios were increased to similar levels in both SCD and sph/sph mice as compared to control mice (p <0.025), consistent with pulmonary hypertension in both SCD and sph/sph mice. Acetylcholine-induced vasodilation of facialis arteries (a branch of the carotid artery) was severely impaired in both SCD and sph/sph mice compared to control mice (p<0.02). We also found increased plasma levels of soluble VCAM-1, E-selectin, and P-selectin in SCD and sph/sph mice compared to control mice (p<0.005 for all groups), providing further evidence for increased endothelial injury in both murine models. Interestingly, sVCAM-1 and E-selectin were more elevated in SCD mice compared to sph/sph mice (p<0.0001), suggesting hemolysis combined with HbS induces further endothelial injury. We hypothesized that plasma free hemoglobin released by intravascular hemolysis initiates endothelial impairment and injury due to scavenging of nitric oxide (NO) and generation of oxidative damage. Plasma methemoglobin levels were determined by helium electron paramagnetic resonance (EPR) spectroscopy before and after the addition of the NO donor molecule PROLI/NO. The difference between these two signals represents the total NO scavenging capacity of plasma. We found that both the plasma free Hb and NO scavenging capacity in plasma from SCD and sph/sph mice was much greater than that of healthy control mice (p<0.05). In addition, we developed an oxygen electrode assay to assess the oxidizing potential of plasma. The initiation of lipid peroxidation by oxidants present in the plasma results in the consumption of oxygen. We found that plasma from SCD and sph/sph mice has a significantly higher oxidizing potential than plasma from control mice (p<0.05). We obtained similar results (increased plasma free Hb, NO scavenging capacity, and oxidizing potential) comparing plasma from individuals with SCD to plasma from healthy control individuals. Consistent with the higher levels of hemolysis in the sph/sph mice, plasma free Hb, NO scavenging and oxidizing capacity were greater in sph/sph mice compared SCD mice (p<0.05). These results indicate that both pulmonary and systemic vascular function is impaired in these two different murine models of hemolysis, and suggest that nitric oxide-based vasoregulatory mechanisms are particularly affected. These data, combined with previous pathologic reports, suggest that while hemolysis importantly contributes to aberrant vasoregulation and initiating pathways of oxidative damage, sickle hemoglobin may uniquely provoke additional vascular and organ injury.