Endothelial Deficiency Augments and Inhaled Nitric Oxide Prevents the Adverse Hemodynamic Effects of Transfusing Syngeneic Stored Blood in Mice

During storage, red blood cells (RBC) undergo progressive deleterious functional, biochemical and structural changes, producing a “storage lesion”. The storage lesion includes reversible and irreversible changes that begin within hours of storage, progress during storage, release free hemoglobin (Hb) and Hb-containing microvesicles, and impair RBC function and lifespan after transfusion. Our recent studies in mice with endothelial dysfunction revealed an enhanced systemic vasoconstrictor response to infusion of tetrameric Hb or Hb-based oxygen carriers. Therefore, we sought to test the hypothesis that endothelial dysfunction would predispose mice to the vasoconstrictor effects of infusion of fresh and stored murine blood.

Murine leukoreduced RBC from C57BL/6 mice were prepared with 14% CPDA-1 anticoagulant and stored at 4°C for either ≤24 h (fresh red blood cells, FRBC) or 2 weeks (stored red blood cells, SRBC). RBC morphology, as well as ATP levels, 2,3-diphosphoglycerate (2,3-DPG) levels, and P₅₀ were measured in FRBC and SRBC before transfusion. We also prepared murine RBC storage components, i.e. supernatant from FRBC or SRBC, oxidized supernatant from SRBC, and washed SRBC. We studied three groups of mice, awake wild-type mice (WT, C57BL/6) fed a standard diet, WT mice fed a high-fat diet (HFD) for 4–6 weeks (to induce endothelial dysfunction), and diabetic (db/db, C57BL/6 background) mice. Each group was transfused with FRBC, SRBC, and db/db mice also received RBC components (10% of total blood volume). Systolic blood pressure (SBP) was measured every 10 min in awake mice before and 2 h after transfusion with FRBC, SRBC or RBC components. A subgroup of mice transfused with SRBC also breathed air (FiO₂=0.21) supplement with nitric oxide (NO, 80 parts per million (ppm)). Invasive hemodynamic measurements were performed in anesthetized mice in order to obtain mean arterial blood pressure, heart rate, and cardiac output. Blood and tissue samples were collected 2 h after FRBC or SRBC transfusion for determination of plasma Hb and iron levels, and measurement of lung and liver levels of mRNA encoding inflammatory cytokines. In addition, heme oxygenase-1 (HO-1) in lung and liver was measured 2 h after FRBC or SRBC transfusion.

SRBC were characterized by altered RBC morphology, decreased ATP and 2,3-DPG levels, and a reduced P₅₀. Transfusion of SRBC into awake WT mice fed a standard diet or HFD produced no systemic hemodynamic changes. In contrast, transfusion of SRBC or supernatant from SRBC into db/db mice induced systemic hypertension that was prevented by concurrent inhalation of NO. Infusion of washed SRBC or oxidized SRBC supernatant into db/db mice did not induce systemic vasoconstriction or hypertension. Invasive hemodynamic studies confirmed that transfusion of SRBC and SRBC supernatant induced systemic vasoconstriction and hypertension, but transfusion of washed SRBC did not. Plasma Hb levels were greater in all mouse groups at 2 h after transfusion of SRBC but not after FRBC transfusion. Two hours after transfusion of SRBC, plasma interleukin-6 and iron levels, as well as hepatic HO-1 mRNA levels, were increased in all mouse groups.

Syngeneic transfusion of SRBC or only the supernatant from SRBC but not washed SRBC produces systemic hypertension and vasoconstriction in db/db mice, which is prevented by oxidizing the supernatant of SRBC or breathing NO during SRBC transfusion. Infusion of SRBC induced a mild systemic inflammatory response in WT fed a standard diet or HFD, and db/db mice. Transfused cell-free oxyHb in the supernatant released from RBC during storage appears to be responsible for the vasoconstriction produced in db/db mice, since it is prevented by oxidizing the supernatant. Our current data support examining the link between the RBC storage lesion and cardiovascular and immunological perturbations in highly susceptible recipients with endothelial dysfunction.

Yu:Massachusetts General Hospital: patents on inhaled nitric oxide and blood transfusion. Bloch:MGH has received sponsored research grant funding from Ikaria LLC, the maker of nitric oxide gas for inhalation in the US, in support of Dr. Bloch's research program.: Research Funding. Zapol:Dr. Warren Zapol receives royalties from patents on inhaled nitric oxide licensed by Massachusetts General Hospital to Linde Corp, Munich, Germany, and Ikaria Corp, Clinton, New Jersey. Dr. Zapol has applied for patents on inhaled nitric oxide and blood t: Patents & Royalties.