Unravelling one more piece of the erythrocytes‐vascular function puzzle in humans: Does the double‐edged sword have NO sharper side?

Subsequent to the paradigm-shifting discovery of Professor Robert Furchgott (Furchgott & Zawadzki, 1980), vascular tone regulation has become a multifaceted puzzle that involves paracrine signalling to smooth muscle cells and a complex communication through the vessel wall. No longer considered an inert monolayer barrier of ‘nucleated cellophane’ cells, the endothelium plays a crucial role in this puzzle by synthesizing and releasing a broad spectrum of vasoactive autacoids in response to neurohumoral factors and mechanical forces imposed by the pulsatile characteristic of blood flow. Shear stress, the frictional force of the flowing blood, acts tangentially to the apical surface of endothelial cells, causing deformation and triggering a piece of mechanotransduction machinery. Endothelial nitric oxide synthase is then activated and catalyses l-arginine to l-citrulline and nitric oxide (NO), a short-lived free-radical that acts as a prototypical regulatory pathway of vascular tone and function. In paracrine cross-talk, endothelium-derived NO diffuses to vascular tunica media, activating specific cGMP-dependent protein kinases and initiating smooth muscle cells relaxation and vasodilatation. Based on this major regulatory pathway of vascular homeostasis, Celermajer et al. (1992) developed a landmark approach in which high-resolution ultrasound imaging tracks the changes in the diameter of a peripheral conduit artery exposed to the transient increase in blood flow (reactive hyperaemia) following a period of distal limb ischaemia. This approach has been termed flow-mediated dilatation (FMD) and is recognized as an index of vascular function as a result of its high dependence on endothelium-derived NO and predictive/prognostic value. FMD has then been applied in integrative human physiology settings to unravel a complex piece of this multifaceted puzzle that has been mostly investigated using basic molecular approaches: the erythrocytes–vascular function interaction. As the principal element suspended in the plasma, the density of erythrocytes [red blood cells (RBCs)] and their capacity to deform play key roles in dictating the blood's rheological behaviour. However, evidence implicates RBCs in the regulation of vascular homeostasis not only as a result of their influence on viscosity/shear stress-related signalling, but also the dichotomy that characterizes haemoglobin-related modulation of NO bioavailability. Haemoglobin is a potent scavenger that rapidly reacts and oxidizes endothelium-derived NO, limiting its paracrine diffusion to smooth muscle cells. Despite the immense content of haemoglobin paradoxically close to endothelial cells, NO still exerts control over vascular tone because scavenging is mitigated by diffusional barriers such as the RBCs membrane and the red-cell–free layer (RCFL), a thin zone between the blood column and the endothelium formed by RBCs axial migration in response to laminar flow. Thus, RBCs act as a double-edged sword in which the sharper side, NO formation or scavenging (and hence the impact on vascular function) would be defined by the extent that changes in haematocrit and haemoglobin affect the balance between their regulatory factors. The study by Hoiland et al. (2020) in this issue of The Journal of Physiology sheds light on how this intricate balance affects vascular function, quantifying brachial artery FMD, arterial blood gases, haematocrit, haemoglobin, viscosity and NO metabolites before and after isovolumic haemodilution, a procedure in which ∼20% of the whole blood was removed and then replaced with 5% human serum albumin. As expected, isovolumic haemodilution did not affect cardiac output and arterial pressure but reduced haematocrit, haemoglobin and whole blood viscosity. Plasma nitrite, S-nitrosothiols and total RBCs NO were also reduced, whereas brachial artery haemodynamics were not affected by isovolumic haemodilution. FMD abruptly increased (∼160%) after haemodilution, a change that persisted even when brachial artery diameter or shear-stress were applied as covariates. By contrast to most evidence exploring the hypothesis that viscosity would proportionally affect shear stress-dependent signalling and FMD, the approach adopted by Hoiland et al. (2020) is distinctive with respect to dealing with the interdependency that NO formation and scavenging have on the rheological behaviour of blood. Although specific findings such as the uncoupling that characterizes reduced resting NO levels and the stability of central and brachial artery haemodynamics deserve further discussion, the robustness of the study's approach permits (if not firm conclusions) elegant educated guesses on the erythrocytes–vascular function interaction in humans. Although the quantification of arterial-venous gradients of NO metabolites (Gladwin et al. 2000) could provide additional insights on this uncoupling mentioned above, FMD increment was not related to blood viscosity-induced changes in shear stress, raising the hypothesis that haematocrit/haemoglobin reductions affected paracrine signalling to smooth muscle cells via modulation of NO scavenging regulatory factors. Because haemodilution is not expected to disturb the membrane properties of RBCs, a change in RCFL width appears as a hypothetical candidate to explain the significative increase in FMD. Determined by the RBC content and the vessel wall–flow velocity gradient, RCFL width proportionally affects NO half-life in microvessels models (Kim-Shapiro et al. 2006). Thus, the assumption of a hypothetical ‘blindness’ of the scavenging side of the ‘double-edged sword’ is tempting and supported by the reduction in haematocrit and the increase in reactive hyperaemia after isovolumic haemodilution. In summary, Hoiland et al. (2020) elegantly emphasize the importance of the simultaneous consideration of NO production and inactivation in the investigation of vascular function in humans. Because RBC content may vary according to age, sex, disease and environmental stress, these variations and the resulting impact on the determinant factors of NO bioavailability are of clinical and experimental relevance and must be considered with respect to further improving the discriminatory power of FMD as an index of vascular function. None. Sole author. None.