Sitosterolemia is a metabolic disorder leading to excessive accumulation of phytosterols. Hemolytic stomatocytosis and macrothrombocytopenia are part of the clinical picture. However, the impact of ...phytosterols on red blood cell (RBC) deformability, membrane lipid composition and distribution and the efficiency of the reference treatment, Ezetimibe, are largely unknown. This study addresses these issues using RBCs from three patients with sitosterolemia and healthy RBCs exposed to β-sitosterol. Patients presented an increased proportion of stomatocytes, decreased RBC deformability and increased RBC hydration and osmotic fragility compared to healthy donors. At the membrane level, patient RBCs showed (i) very high content in β-sitosterols, (ii) increased proportions of saturated fatty acids and polyunsaturated fatty acid species with long and unsaturated carbon chains, and (iii) decreased content in phosphatidylethanolamine species. These lipid changes were accompanied by an almost complete abrogation of cholesterol-enriched domains, which could result from: (i) the reduced phosphatidylethanolamine content which positively correlated with domain abundance; and (ii) the fatty acid modifications and increased phytosterol content, both compatible with higher membrane stiffness. The role of β-sitosterol was supported by comparable changes in RBC morphology and cholesterol-enriched domains upon β-sitosterol integration at the healthy RBC membrane. Finally, Ezetimibe treatment combined with a sterol restricted diet lowered phytosterols and improved anemia and RBC deformability and hydration. However, this treatment had no or limited effect on RBC morphology and cholesterol-enriched domain abundance. This study reveals for the first time that phytosterols affect RBC membrane lipid composition and distribution but also RBC morphology, hydration, deformability and fragility.
Bioreactors are increasingly implemented for large scale cultures of various mammalian cells, which requires optimization of culture conditions. Such upscaling is also required to produce red blood ...cells (RBC) for transfusion and therapy purposes. However, the physiological suitability of RBC cultures to be transferred to stirred bioreactors is not well understood. PIEZO1 is the most abundantly expressed known mechanosensor on erythroid cells. It is a cation channel that translates mechanical forces directly into a physiological response. We investigated signaling cascades downstream of PIEZO1 activated upon transitioning stationary cultures to orbital shaking associated with mechanical stress, and compared the results to direct activation of PIEZO1 by the chemical agonist Yoda1. Erythroblasts subjected to orbital shaking displayed decreased proliferation, comparable to incubation in the presence of a low dose of Yoda1. Epo (Erythropoietin)-dependent STAT5 phosphorylation, and Calcineurin-dependent NFAT dephosphorylation was enhanced. Phosphorylation of ERK was also induced by both orbital shaking and Yoda1 treatment. Activation of these pathways was inhibited by intracellular Ca
chelation (BAPTA-AM) in the orbital shaker. Our results suggest that PIEZO1 is functional and could be activated by the mechanical forces in a bioreactor setup, and results in the induction of Ca
-dependent signaling cascades regulating various aspects of erythropoiesis. With this study, we showed that Yoda1 treatment and mechanical stress induced via orbital shaking results in comparable activation of some Ca
-dependent pathways, exhibiting that there are direct physiological outcomes of mechanical stress on erythroblasts.
Red blood cell (RBC) rheology is markedly abnormal in sickle cell disease (SCD) and is associated with clinical complications. Even fully oxygenated, sickle RBC are less deformable than those of HbAA ...or HbAS individuals; upon deoxygenation, deformability further declines. The goal of any cell-based therapy of curative intent should be to normalize SCD RBC rheology. We propose to functionally assess a series of post-allogeneic hematopoietic cell transplant (alloHCT) patients (pts) with SCD using rheological biomarker measurements of deformability and sickling to determine if the rheology has been normalized.
Blood from 6 SCD pts post-alloHCT were analyzed using an ektacytometer with oxygenscan (Lorrca) to measure RBC deformability (elongation index EI), under a range of pO2 (150-0 mmHg). EImax is the deformability of the oxygenated sickle RBC; EImin is the deformability of deoxygenated RBCs. The point of sickling (PoS) is the pO2 at which RBC deformability rapidly declines. Reference ranges were generated using n=45 HbSS samples age 2-21, on hydroxyurea, chronic transfusion, or untreated; n=14 HbAS, and n=43 HbAA.
Figure 1 shows measurements obtained on 5 pts post alloHCT with a range of donor chimerism, as well as typical values for HbAA, HbAS, and HbSS. Pts 1 and 3 exhibit normal rheology; both were transplanted with an HbAA donor, had high chimerism and no detectable HbS. Pt 2 had 40% whole blood chimerism and hbS < 50%, yet their plot resembles that of untransplanted HbSS. Pts 5 and 6 exhibit rheology in the HbAS range; they have intermediate chimerism from an AS donor. Pt 2 is the only subject with values in the HbSS range, and the only subject with SCD symptoms. Clinical details and biomarker values for all 6 analyzed are in Table 1, as well as ranges of rheological values for HbSS and HbAS pts generated in the Sheehan and Shevkoplyas labs.
Current HCT evaluation depends on chimerism and hemoglobin profiles, and do not assess RBC function. Using the Lorrca with oxygenscan, we identified a post-alloHSCT patient with rheological values consistent with an HbSS patient despite donor chimerism >20% and HbS <50% thought sufficient for cure from an HbAA donor. Our results suggest that this functional analysis may help with management post-alloHCT and may be essential in assessing new gene-based therapy approaches to curing pts with SCD.
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