The cellular deformability of red blood cells (RBC) is exceptional among mammalian cells and facilitates nutrient delivery throughout the microcirculation; however, this physical property is negatively impacted by oxidative stress. It remains unresolved whether the molecular determinants of cellular deformability – which in the contemporary model of RBC are increasingly recognized – are sensitive to free radicals. Moreover, given cellular deformability has recently been demonstrated to increase following exposure to specific doses of mechanical stimulation, the potential for using shear “conditioning” as a novel method to reverse free-radical induced impairment of cell mechanics is of interest. We thus designed a series of experiments that explored the effects of intracellular superoxide (O2-) generation on the deformability of RBC and also activation of pivotal molecular pathways known to regulate cell mechanics – i.e., PI3K/Akt kinase and RBC nitric oxide synthase (NOS). In addition, RBC exposed to O2- were conditioned with specific shear stresses, prior to evaluation of cellular deformability and activation of PI3K/Akt kinase and RBC-NOS. Intracellular generation of O2- decreased phosphorylation of RBC-NOS at its primary activation site (Ser1177) (p < 0.001), while phosphorylation of Akt kinase at its active residue (Ser473) was also diminished (p < 0.001). Inactivation of these enzymes following O2- exposure occurred in tandem with decreased RBC deformability. Shear conditioning significantly improved cellular deformability, even in RBC previously exposed to O2-. The improvement in cellular deformability may have been the result of enhanced molecular signaling, given RBC-NOS phosphorylation in RBC exposed to O2- was restored following shear conditioning. Impaired RBC deformability induced by intracellular O2- may be due, in part, to impaired activation of PI3K/Akt, and downstream signaling with RBC-NOS. These findings may shed light on improved circulatory health with targeted promotion of blood flow (e.g., exercise training), and may prove fruitful in future development of blood-contacting devices.

It was classically thought that the function of mammalian red blood cells (RBC) was limited to serving as a vehicle for oxygen, given the cell's abundance of cytosolic haemoglobin. Over the past decades, however, accumulating evidence indicates that RBC have the capacity to sense low oxygen tensions in hypoxic tissues, and subsequently release signalling molecules that influence the distribution of blood flow. The precise mechanisms that facilitate RBC modulation of blood flow are still being elucidated, although recent evidence indicates involvement of: i) adenosine triphosphate (ATP) - capable of binding to purinergic receptors located on the vascular wall prior to initiating nitric oxide (NO; a powerful vasodilator) production in endothelial cells, and/or ii) non-vascular NO - which is now known to have several modes of production within RBC, including an enzymatic process via a unique isoform of NO synthase (i.e., RBC-NOS), that has potential effects on the vascular smooth muscle. The physical properties of RBC - including their tendency to form three-dimensional structures in low shear flow (i.e., aggregation) and their capacity to elongate in high shear flow (i.e., deformability) - are only recently being viewed as mechanotransductive processes, with profound effects on vascular reactivity and tissue perfusion. Recent developments in intracellular signalling in RBC, and the subsequent effects on the mechanical properties of blood, and blood flow, thus present a vivid expansion on the classic perspective of these abundant cells.

Red blood cell populations respond differently to mechanical stimuli: older (more dense) cells are highly susceptible to sublethal mechanical trauma, while cell age (density) does not appear to alter the magnitude of improved cell deformability following low-shear conditioning.

Specific shear conditioning results in either temporarily increased cell deformability, or a less reversible decrease of RBC deformability.

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