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. 2016 Sep 15;129(18):3511-7.
doi: 10.1242/jcs.189225. Epub 2016 Aug 9.

Visualizing Red Blood Cell Sickling and the Effects of Inhibition of Sphingosine Kinase 1 Using Soft X-ray Tomography

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Free PMC article

Visualizing Red Blood Cell Sickling and the Effects of Inhibition of Sphingosine Kinase 1 Using Soft X-ray Tomography

Michele C Darrow et al. J Cell Sci. .
Free PMC article

Abstract

Sickle cell disease is a destructive genetic disorder characterized by the formation of fibrils of deoxygenated hemoglobin, leading to the red blood cell (RBC) morphology changes that underlie the clinical manifestations of this disease. Using cryogenic soft X-ray tomography (SXT), we characterized the morphology of sickled RBCs in terms of volume and the number of protrusions per cell. We were able to identify statistically a relationship between the number of protrusions and the volume of the cell, which is known to correlate to the severity of sickling. This structural polymorphism allows for the classification of the stages of the sickling process. Recent studies have shown that elevated sphingosine kinase 1 (Sphk1)-mediated sphingosine 1-phosphate production contributes to sickling. Here, we further demonstrate that compound 5C, an inhibitor of Sphk1, has anti-sickling properties. Additionally, the variation in cellular morphology upon treatment suggests that this drug acts to delay the sickling process. SXT is an effective tool that can be used to identify the morphology of the sickling process and assess the effectiveness of potential therapeutics.

Keywords: Cryogenic soft X-ray tomography; Red blood cell; Red cell morphology; Sickle cell disease; Sphingosine kinase inhibitor.

Conflict of interest statement

The authors declare no competing or financial interests.

Figures

Fig. 1.
Fig. 1.
The number of cell protrusions is correlated with volume, and therefore density. (A) Results from sickle RBCs under hypoxia conditions both with and without treatment with compound 5C were combined after statistical tests revealed no significant difference between the two populations (see also Fig. S1). This combined dataset (n=105) represents four individual SXT experiments. The distribution of cells based on number of protrusions for the combined dataset is shown. In subsequent presentations all RBCs with ≥10 protrusions were combined into a 10+ category. (B) Volumetric analysis of the combined sickle RBC dataset revealed a correlation between volume and the number of protrusions (see also Fig. S2). The volume measurement can be used as an inverse proxy for density, indicating the number of protrusions is correlated to density, a hallmark of SCD associated with severity of sickling. Circled data points have been statistically identified as outliers (as described in the Materials and Methods), but not excluded.
Fig. 2.
Fig. 2.
Continuum of the stages of sickling. Following volumetric analysis to correlate protrusion number with density, a hallmark of SCD, data were binned into four categories. The none category is composed of RBCs with zero or one protrusion; the mild category is composed of RBCs with eight, nine, ten or greater protrusions; the moderate category is composed of RBCs with five, six or seven protrusions; and finally, the severe category is composed of RBCs with two, three or four protrusions. Multiple examples of RBCs as imaged by SXT that fall into each of these categories are displayed here (see also Fig. S2).
Fig. 3.
Fig. 3.
Sickle RBCs under hypoxia conditions differ significantly from controls. Only 19±8% of cells from the experimental condition (SCD cells, hypoxia) fall into the none category. The rest of these cells was split unevenly throughout the remaining categories, with the majority falling into the severe category (43±6%), with 24±5% in the moderate category and 14±6% in the mild category. Importantly, these data indicate that up to 80% of the RBCs cultivated under these conditions (4% oxygen, 37°C, 2 h) are sickled to some degree. Note: data are presented as normalized mean±s.e.m. and represent four individual experiments.
Fig. 4.
Fig. 4.
Compound 5C significantly shifts the population of RBCs toward the less severe categories of sickling. One-way analysis of variance indicates that the population shift due to treatment with compound 5C is significant as compared to the control sickle RBC population (*P<0.05; see also Table S1). Tukey's multiple comparison test indicates that this shift is towards the less severe categories of sickling and a pairwise χ2 test indicates the greatest change is between the moderate and severe categories. Taken together, these data indicate the treatment is acting to keep sickle RBCs in the morphological mild and moderate categories. Note: data are presented as normalized mean±s.e.m. and represent four individual experiments.
Fig. 5.
Fig. 5.
Effects of compound 5C treatment on sickling RBCs using light microscopy. Inhibition of Sphk1 reduced sickling of SCD RBCs under hypoxia conditions. The percentage of sickled cells was significantly reduced in the RBCs treated with the Sphk1-specific inhibitor compound 5C. *P<0.05 versus saline treatment control (Student's t-test). Data are presented as mean±s.d. (n=1000).
Fig. 6.
Fig. 6.
Treatment of SCD mice by compound 5C increased the lifespan of RBCs. Specific inhibition of Sphk1 with 5C in SCD mice significantly increased RBC lifespan. *P<0.05 versus saline treatment control (Student's t-test). Data are presented as mean±s.e.m. (n=6).

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