Effects of Nitric Oxide on the Endothelium During Hemolysis.

Endothelial cells regulate tissue perfusion by releasing nitric oxide (NO), a potent
endogenous dilator of vascular smooth muscle cells, which modifies vascular tone. Under
normal physiological conditions, vascular NO is released by endothelial NO synthase (eNOS).
Impairment of the eNOS, as seen in patients with atherosclerosis, peripheral vascular
disease, hypertension, obesity, and diabetes, is a feature of endothelial dysfunction.The
inability to increment eNOS activity is particularly evident in conditions of decreased
vascular NO bioavailability, such as during hemolysis associated with prolonged
cardiopulmonary bypass (CPB>90 min). During hemolysis, ferrous plasma free hemoglobin
(Oxy-Hb) is released into the circulation and can be injurious for the endothelial cells by
exerting an oxidative and proinflammatory effect. Moreover, plasma free Oxy-Hb can scavenge
vascular NO, reducing its bioavailability as ferrous Oxy-Hb is transformed into ferric
methemoglobin (Met-Hb). The clinical results of reduced bioavailability of vascular NO have
been found to be associated with both systemic and pulmonary vasoconstriction, ultimately
leading to reduced tissue perfusion.

The exogenous administration of NO has been shown to prevent the scavenging of endogenous NO
by inactivating the highly oxidative-reactive ferrous plasma Oxy-Hb to ferric Met-Hb. Our
group is conducting a randomized controlled trial at Massachusetts General Hospital (Boston,
USA) in patients with signs and symptoms of endothelial dysfunction, undergoing cardiac
surgery requiring prolonged CPB and randomized to receive NO or placebo. However, the
mechanisms underlying the beneficial systemic effects of NO administration have still to be
determined. This is an ancillary study that aims to (I) assess the effects of hemolysis on
vascular responsiveness and on endothelial function in patients with pre-operative
endothelial dysfunction and (II) to determine the vascular protective effects of NO
administration.

Inclusion Criteria:

- Eligible and randomized in the trial NCT02836899

- Provide written informed consent

- Age ≥ 18 years of age

- Elective cardiac or aortic surgery with CPB >90 minutes

- Clinical evidence of endothelial dysfunction assessed by a specifically designed
questionnaire

Exclusion Criteria:

- Estimated Glomerular Filtration Rate less than 30 ml/min/1.73 m2

- Emergent cardiac surgery

- Life expectancy < 1 year at the time of enrollment

- Hemodynamic instability as defined by a systolic blood pressure <90 mmHg.

- Mean pulmonary artery pressure ≥ 40 mm Hg and PVR > 4 Wood Units.

- Left ventricular ejection fraction < 30% by echocardiography obtained within three
months of enrollment

- Administration of one or more Packed Red Blood Cell (PRBC) transfusions in the week
prior to enrollment

- X-ray contrast infusion less than 48 hours before surgery

- Evidence of hemolysis from any other origin:

a. Intravascular: i. Intrinsic RBC defects leading to hemolytic anemia (eg, enzyme
deficiencies, hemoglobinopathies, membrane defects) ii. Extrinsic: liver disease,
hypersplenism, infections (eg, bartonella, babesia, malaria), treatment with oxidizing
exogenous agents (eg, dapsone, nitrites, aniline dyes), exposure to other hemolytic
agents (eg, lead, snake and spider bites), lymphocyte leukemia, autoimmune hemolytic
disorders b. Extravascular: Infection (eg, clostridial sepsis, severe malaria),
paroxysmal cold hemoglobinuria, cold agglutinin disease, paroxysmal nocturnal
hemoglobinuria, iv infusion of Rho(D) immune globulin, iv infusion of hypotonic
solutions