Table 2.

Common Misunderstandings of Vascular Physiology with Regard to Hypoxic Vasodilation and SNO-βCys93-Hb

Physiology	Misunderstanding	Actuality
Blood pressure (baseline)	C93A mice should have elevated blood pressure (because vasodilation will decrease systemic blood pressure). C93A mice should have elevated pulmonary blood pressure, reflected in histological changes in the lungs.	Blood pressure should not change in C93A mouse. Systemic blood pressure is effectively independent of hypoxic vasodilation, which affects blood flow primarily. Microvascular flow regulating oxygen delivery is not a primary contributor to systemic blood pressure (109). Classic autoregulation as described by Guyton is independent of blood pressure (100). Histological changes are not a useful measure of blood pressure in the lungs or response to hypoxia (31, 123).
Blood pressure (under systemic hypoxia)	Elevated systemic blood pressure in C93A mice will be worsened in systemic hypoxia.	There is no predicted relationship between blood pressure and hypoxic vasodilation governing tissue blood flow (117).
Pulmonary hypertension	C93A mice will have elevated pulmonary artery blood pressure and develop lung disease.	There is no relationship between systemic hypoxic vasodilation and pulmonary pressure (117). Lack of histological change does not inform pulmonary pressure (31, 123). Pulmonary pressure was not measured in C93A mice.
SNO-mediated vasodilation	C93A mouse RBCs will lack SNO-mediated effects on vessels in vitro at 0% O2.	On the contrary, C93A mouse RBCs compensate to contain normal total amounts of SNO, at non-Cys93 sites on HbA and elsewhere (43, 134). These SNOs will cause vasodilation in bioassays at 0% O2 (100% T state) but will lack R/T allosteric control by O2 (96). Also, C93A mouse RBCs contain fetal Hb that carries C93-SNO (97, 98), which is subject to allosteric regulation.
SNO-mediated vasodilation	Artificially SNO-loaded C93 and C93A mouse RBCs are informative for hypoxia-driven SNO-mediated effects on vessels in vitro.	The literature includes two sets of data: physiological loading (NO <1 μM; NO/Hb <1:100) vs. artificial overloading (NO ∼1 mM; NO:Hb >1:10). SNO-overloaded RBCs (and C93A RBCs with SNO at non-C93 sites) will produce nonspecific vasodilation lacking allosteric/O2-dependent control (Table 1). High pharmacological SNO-loading of C93A and C93 RBCs artifactually oxidizes Hb and eliminates all oxygen responsivity even in normal C93 cells (113, 117).
Hb Cys93Ala mutation	Elimination of Cys93 will prevent RBC-mediated SNO-based vasodilatory activity.	Mutation of Cys93 does not change amount of SNO in RBCs due to compensations: SNO binds other sites on Hb and the amounts of SNO on other RBC thiols increase (43, 134). Therefore, total SNO-mediated vasodilation should be comparable in C93- vs. C93A-mutant RBCs under low PO2. What should change is sensitivity to PO2 and/or thiol at intermediate range of Hb oxygen-saturation values (R/T transition points). Also, effects of γC93-SNO should be accounted for in βC93A.
Ischemia–perfusion injury in an isolated mouse heart model system	Normal RBCs injected in coronary artery at the start of an acute ischemic insult will be protective of heart function during later reperfusion due to SNO-mediated effects, but C93A RBCs will be deficient.	Model is intended as a surrogate of reactive hyperemia (which is markedly abnormal in C93A mice), but is not: reperfusion/injury models are critically dependent on duration of hypoxia/reperfusion and species (90); cardiac function after injury is not a surrogate for blood flow. Model is also complicated by ROS (which eliminates SNO) that is generated by ischemia/reperfusion (11).

RBC-mediated vasodilation responses are linked to blood flow responses, not blood pressure. Blood flow responses linked to tissue oxygenation are disrupted by excessive/nonphysiological loading of RBCs, which disrupts physiological allosteric (Hb O₂-saturation-dependent) control of flow responses. Physiological SNO loading of RBCs entails SNO/Hb <10/1000 and met Hb <5%.