Correspondence

Circulating Endothelial Cells in Sickle Cell Anemia

N Engl J Med 1998; 338:1162-1163April 16, 1998

Article

To the Editor:

Solovey et al. reported an increased number of activated circulating endothelial cells in patients with sickle cell anemia (Nov. 27 issue).1 Although the mechanism that releases endothelial cells into the circulation is unclear, several points suggest that apoptosis may be responsible. An average of 34 percent of these circulating endothelial cells were dead, as assessed by nuclear staining with ethidium homodimer.1 Dead cells stain with ethidium homodimer when the cell membrane is damaged and becomes permeable. A permeable cell membrane is a feature of necrotic cells and of the late stages of apoptosis, whereas in the early stages of apoptotic cell death the cell-membrane integrity is preserved.2 Thus, a proportion of apparently live circulating endothelial cells might already be committed to death through apoptosis. A cell-culture medium rich in survival factors may rescue some of these cells and allow them to remain alive in culture. Other features of apoptosis include cell detachment, which may account for the presence of circulating endothelial cells, blebbing of cell membranes, and cell fragmentation into membrane-bound bodies (apoptotic bodies).2 Indeed, the cell in Figure 1B of the article by Solovey et al. appears to display blebbing. Circulating anucleate P1H12-positive cell fragments were noted by the authors.1 Furthermore, apoptotic endothelial cells become procoagulant and are primed to express tissue factor,3 a feature noted in circulating endothelial cells in sickle cell anemia.

We propose that apoptosis may account for the increased number of circulating endothelial cells in sickle cell anemia. If this is the case, and given the short half-life of apoptotic cells, the number of endothelial cells released into the circulation may be far greater than the counts obtained by Solovey et al. suggest. Various factors may induce endothelial-cell apoptosis. At least one of them, tumor necrosis factor, may be present in the blood of patients with sickle cell disease and induces both the expression of adhesion molecules and apoptosis in cultured endothelial cells.4,5

Alberto Ortiz, M.D.
Fundación Jiménez Díaz, 28040 Madrid, Spain

5 References

1
Solovey A, Lin Y, Browne P, Choong S, Wayner E, Hebbel RP. Circulating activated endothelial cells in sickle cell anemia. N Engl J Med 1997;337:1584-1590
Full Text | Web of Science | Medline

2
Ortiz A, Cuadrado SG, Lorz C, Egido J. Apoptosis in renal diseases. Front Biosci 1996;1:30-47

3
Bombeli T, Karsan A, Tait JF, Harlan JM. Apoptotic vascular endothelial cells become procoagulant. Blood 1997;89:2429-2442
Web of Science | Medline

4
Robaye B, Mosselmans R, Fiers W, Dumont JE, Galand P. Tumor necrosis factor induces apoptosis (programmed cell death) in normal endothelial cells in vitro. Am J Pathol 1991;138:447-453
Web of Science | Medline

5
Hebbel RP, Vercellotti GM. The endothelial biology of sickle cell disease. J Lab Clin Med 1997;129:288-293
CrossRef | Medline

To the Editor:

Dr. Solovey and her colleagues found that the normal number of circulating endothelial cells is about a millionth that of white cells. Though the number rose during a sickle cell crisis, the range of 1 to 66 endothelial cells per milliliter shows that this was not always so, and suggests that there may be an additional factor. Endothelium may be affected physically and chemically by a jet of foreign liquid. It would be interesting to know whether the count was affected by intravenous treatment before sampling.

T.H. Hughes-Davies, F.R.C.P.
Breamore Marsh, Fordingbridge, Hampshire SP6 2EJ, United Kingdom

Author/Editor Response

The authors reply:

To the Editor: Dr. Ortiz proposes that apoptosis causes the appearance of circulating endothelial cells in patients with sickle cell anemia, and he emphasizes that our methods would fail to detect cells that are early in the apoptotic process. We were also concerned about this for the additional reason that endothelial-cell apoptosis is promoted if the cells lose contact with substratum.1 However, in ongoing studies we are finding that only about 30 percent of circulating endothelial cells from patients with sickle cell disease are undergoing apoptosis (as detected by the accumulation of 3'-OH DNA ends), whereas approximately 60 percent of circulating endothelial cells from normal donors show the same changes. It may be that endothelium in sickle cell disease is actually protected from apoptosis, which we hypothesize could be due to elevated levels of antiapoptotic growth factors. Although we have no data on the circulatory life span of circulating endothelial cells, apoptotic or otherwise, these preliminary data argue against apoptosis as the proximate stimulus of the release of circulating endothelial cells.

We share Dr. Ortiz's interest in the possible presence of small vesicular particulates from blebbed or injured endothelial-cell membranes. It may be that plasma from patients with sickle cell disease contains procoagulant endothelial “dust” in addition to its known content of procoagulant vesicles derived from red-cell membranes.2

In terms of possible induction of an endothelial phenotype by the apoptotic process, apoptosis does not actually cause expression of tissue factor but instead enhances the activity of any tissue factor that was expressed for some other reason — e.g., prior stimulation with endotoxin.3 In any case, the inverse relation we are finding between the degree of circulating-endothelial-cell apoptosis (as described above) and the degree of circulating-endothelial-cell activation argues against the hypothesis that the phenotype we observed actually resulted from ongoing apoptosis.

Dr. Hughes-Davies asks whether a fluid jet might cause endothelial release. We found no differences between donors who were receiving intravenous-fluid administration and those who were not. We have not yet applied our methods to analysis of patients with other clinical conditions that might be instructive as to the mechanisms of endothelial release or the nature of their activation state.

Robert P. Hebbel, M.D.
Anna Solovey, M.D., Ph.D.
University of Minnesota, Minneapolis, MN 55455

3 References

1
Re F, Zanetti A, Sironi M, et al. Inhibition of anchorage-dependent cell spreading triggers apoptosis in cultured human endothelial cells. J Cell Biol 1994;127:537-546
CrossRef | Web of Science | Medline

2
Allan D, Limbrick AR, Thomas P, Westerman MP. Release of spectrin-free spicules on reoxygenation of sickled erythrocytes. Nature 1982;295:612-613
CrossRef | Web of Science | Medline