Membrane Lesions and Reduced Life Span of Red Blood Cells in Preeclampsia as Evidenced by Atomic Force Microscopy

Abstract

Preeclampsia (PE) presents with maternal de novo hypertension and significant proteinuria and is one of the leading causes of maternal and perinatal morbidity and mortality with unknown etiology. The disease is associated with inflammatory vascular response and severe red blood cell (RBC) morphology changes. This study examined the nanoscopic morphological changes of RBCs from PE women versus normotensive healthy pregnant controls (PCs) and non-pregnant controls (NPCs) applying atomic force microscopy (AFM) imaging. The results revealed that the membrane of fresh PE RBCs differed significantly from healthy ones by the presence of invaginations and protrusions and an increased roughness value (R_rms) (4.7 ± 0.8 nm for PE vs. 3.8 ± 0.5 nm and 2.9 ± 0.4 nm for PCs and NPCs, respectively). PE-cells aging resulted in more pronounced protrusions and concavities, with exponentially increasing R_rms values, in contrast to the controls, where the R_rms parameter decreased linearly with time. The R_rms, evaluated on a 2 × 2 µm² scanned area, for senescent PE cells (13 ± 2.0 nm) was significantly higher (p < 0.01) than that of PCs (1.5 ± 0.2 nm) and NPCs (1.9 ± 0.2 nm). Furthermore, the RBCs from PE patients appeared fragile, and often only ghosts were observed instead of intact cells at 20-30 days of aging. Oxidative-stress simulation on healthy cells led to RBC membrane features similar to those observed for PE cells. The results demonstrate that the most pronounced effects on RBCs in PE patients are related to impaired membrane homogeneity and strongly altered roughness values, as well as to vesiculation and ghost formation in the course of cell aging.

Keywords: atomic force microscopy; cell senescence; membrane impairment; membrane roughness; preeclampsia; red blood cells.

Figure 1

3D high-resolution images of membrane nanostructure of freshly isolated RBCs from non-pregnant controls (NPCs, (A)) and pregnant controls (PCs, (B)) and from patients with preeclampsia (PE, (C)). The height profiles defined by the dashed lines on panels (A–C) are presented on panels (D–F), respectively.

Figure 2

Histogram of the relative contribution of RBCs assigned to the main morphological classes, along with the senescence of cells derived from healthy non-pregnant controls (NPCs, (A)); healthy pregnant controls (PCs, (B)), and patients with preeclampsia (PE, (C)). Each morphological type is presented as a percentage of the total number of cells.

Figure 3

Selected 3D images of RBCs isolated from non-pregnant women (NPC, (A–C)), healthy pregnant women (PC, (D–F)), and patients with preeclampsia (PE, (G–I)). The images show morphological changes in aging cells over a storage period of 30 days.

Figure 4

Example of 3D images of a whole RBC (A) and RBC membrane patch scanned in an area of 2 × 2 µm² (B) from a patient with preeclampsia, representing the initial formation of vesicles. Panels (C,D) illustrate the disturbances on the membrane surface after vesicle extrusion (denoted with arrows). The scale bar in panel (A) is 13 µm, and in panels (B–D) is 2 µm.

Figure 5

Selected 3D AFM images, representing the ultrastructural alteration of the membrane surface of RBCs isolated from patients with preeclampsia (PE (G–I)), non-pregnant controls (NPCs (A–C)), and pregnant controls (PCs (D–F)) during the cells’ senescence. The scanned area is 2 × 2 µm².

Figure 6

Membrane roughness value (R_rms) determined as a function of storage time for RBCs derived from non-pregnant controls (NPCs), healthy pregnant controls (PCs), and patients with preeclampsia (PE). R_rms values were evaluated from topographic images with an area of either 2 × 2 µm² (A) or 1 × 1 µm² (B).

Figure 7

Representative 3D images of RBC membrane for non-treated, freshly isolated cells from healthy donors (A) and cells treated with 100 mM (B) and 200 mM (C) H₂O₂. The respective cross-sectional profiles defined by the dashed line at each panel are presented in panels (D–F). Arrows in panels B and C indicate the appearance of invaginations after treatment with H₂O₂.