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In vitro monitoring of endothelial complications following hematopoietic allogeneic stem cell transplantation

Bone Marrow Transplantation volume 33, pages 355–357 (2004)Cite this article

Hematopoietic allogeneic stem cell transplantation (SCT) is an established method for the treatment of hematologic malignancies and increasingly used also for older patients as well as other neoplasias. Owing to transplantation across major or, more importantly, minor histocompatibility barriers, severe inflammatory reactions, such as acute and chronic graft-versus-host disease (GvHD), veno-occlusive disease (VOD), endothelial leakage syndrome (ELS), thrombotic thrombocytopenic purpura (TTP) and microangiopathic hemolytic anemia (microangiopathy), can occur.1 Previous work from our group and other investigators strongly argues that almost all of these complications are associated with endothelial damage, which is the primary event of organ dysfunction following SCT,2,3,4 and is initiated even prior to transplant. However, progress in the diagnosis, prediction of and prophylaxis against endothelial cell damage is limited so far. Studies on the presence of serum plasminogen activator inhibitor 1 as being indicative of VOD5 as the major hepatic complication prompted us to examine whether in the course of SCT there are also apoptotic factors present.

We previously established an indicator cell system for the detection of apoptotic factors in SCT patients’ serum.6 Briefly, serum samples from 30 patients were collected at various time points pre and post SCT and used for the treatment of a microvascular endothelial cell line (CDC/EU.-HMEC-1, further referred to as HMEC) as indicator cells. Endothelial damage was assessed by apoptosis assays using computer-guided microscopic analysis of DNA dye (DAPI, 4,6-diamidino-2-phenylindole, Sigma, Deisenhofen, Germany) stained HMEC after 48 h of treatment with 30% serum. Nuclear condensation as revealed by DAPI staining, in the absence of trypan blue uptake, is considered characteristic of apoptosis as opposed to necrosis.7 Results are given as % apoptotic HMEC±s.d. out of n=4 microscopic fields. Spontaneous apoptosis in untreated (medium) HMEC was below 10% in all cases. The relevant results have been confirmed by an alternative apoptosis assay using flow cytometric detection of apoptosis-specific granularity (data not shown). Percent apoptotic HMEC was normalized to untreated control cells (apoptotic index, AI). The AI was set to a cutoff of 1.7 representing the mean AI of serum collected prior to any therapeutic intervention plus one s.d. For Tables 1 and 2, the AI was taken from the maximum of HMEC apoptosis in the first 100 days post SCT. As a further control, the apoptosis-inducing capacity of serum from healthy control individuals was assessed and never reached patient levels (n=11, ranging from 10.5±1.6 to 13.4±3.9% apoptotic HMEC). Statistical analysis of these assays was done by the χ2 test.

Table 1 Endothelial cell apoptosis-inducing capacity in patients' serum correlates with GvHD
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Table 2 Endothelial cell apoptosis-inducing capacity in patients’ serum correlates with severe microangiopathy
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Acknowledgements

We wish to thank Drs Ades and Candal from the Centers for Disease Control and Prevention (Atlanta, GA, USA) for kindly providing the dermal microvascular endothelial cell line CDC/EU.-HMEC-1 as indicator cells.10 This work was supported by Grants No. Ei68/1-1 and Ei68/2-1-3 from the Deutsche Forschungsgemeinschaft (DFG).

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  1. A Ganster and I Brucker: These two authors contributed equally to this work.

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  1. Department of Hematology and Oncology, University of Regensburg, Franz-Josef-Strauss-Allee, D-93053, Regensburg, Germany

    A Ganster, I Brucker, E Holler, J Hahn, H Bremm, R Andreesen & G Eissner

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Ganster, A., Brucker, I., Holler, E. et al. In vitro monitoring of endothelial complications following hematopoietic allogeneic stem cell transplantation. Bone Marrow Transplant 33, 355–357 (2004). https://doi.org/10.1038/sj.bmt.1704354

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