Quantification of circulating endothelial progenitor cells in systemic sclerosis

Jerome Avouac, ,

Other Contributors:

February 21, 2012

Dear Editor,

We read with great interest the manuscript from Kuwana and coworkers related to the external validation of the EUSTAR recommendations on endothelial progenitor cells (EPCs)(1, 2). The side-by-side comparisons of methods for quantifying EPCs performed on 11 SSc patients and 11 age- matched controls showed a statistically significant correlation between EPC counts obtained by the MACS (enrichment of CD34+ cells by a magnetic- activated cell sorter) and rosette methods when combined with fluorosphere calibration (r=0.81, p<0.0001). These findings confirmed the validation of EUSTAR recommendations and support the incorporation of a fluorosphere technique into the EUSTAR recommendations. These data are of importance because standardisation and reproducibility are critical issues in this context.
Regarding the discussion part of the manuscript, it is stated that a direct link between CD34+CD133+VEGFR2+ cell counts and "true EPCs" (also called late outgrowth EPCs or endothelial colony-forming cells) is still missing. In fact, we would like to remind that we already reported that levels of circulating EPCs detected by flow cytometry were associated with i) the formation of true EPC colonies and ii) with the delay before colony appearance in patients with SSc (respectively p=0.02 and 0.006). In addition, the number of true EPC colonies positively correlated with levels of EPCs (R=0.73, p=0.0004) defined as Lin-7AAD-CD34+CD133+VEGFR2+ cells in patients with SSc (3). This correlation was not found with other putative EPC populations (Lin-7AAD-CD133+VEGFR2+ and Lin-7AAD-CD34+VEGFR2+ cells), suggesting that the Lin-7AAD-CD34+CD133%VEGFR2+ should be considered as the most suitable definition for EPC detection by flow cytometry. These findings also support that late outgrowth EPCs are "true EPCs" referring to the population detected by FACS. The authors also state that studies conform to EUSTAR recommendations should assess associations between EPC counts and clinical features. We do agree that translational data are needed to establish the face validity of a biomarker. We would like to highlight that studies assessing this issue have been recently published. Indeed, we previously reported in a cohort of 50 consecutiveSSc patients that low EPC counts (defined by Lin-7AAD- CD34+CD133+VEGFR2+ cells) were associated with the higher Medsger's severity score (p=0.01) and the presence of past and/or current digital ulcers (p=0.03). In addition, we recently reported the results of a 3-year prospective study aiming at the evaluation of the possible merit of endothelial markers for the prediction of cardiac/vascular events in patients with SSc, in particular ischaemic digital ulcers(4). By multivariate Cox proportional analysis, circulating Lin-7AAD- CD34+CD133+VEGFR2+ cells were identified as independent predictors of the occurrence of new ischaemic ulcers (hazard ratio, HR: 2.33, 95% confidence interval, CI 1.44-12.22, p=0.03) in association with increased serum levels of placenta growth factor (HR: 7.95, 95% CI 2.09-30.10). Low EPC levels were also predictive of the occurrence of cardiac and vascular events, defined by an exploratory composite index (HR: 3.18, 95% CI: 1.19- 12.80). We believe that these data obtained through a prospective study support the reliability and value of EPCs to evaluate vascular risk.
Indeed, these results at least suggest that EPC counts quantified by flow cytometry, accordingly to EUSTAR recommendations, can be used to identify SSc patients who are at risk of the development of digital ulcers.

Altogether, Kuwana's results together with data from our groupsupport the use of EUSTAR recommendations for assessing EPCs, which can yet be used as a biomarker for vascular risk and raise the critical importance of angiogenesis and vasculogenesis in the devastating condition that is SSc.

References

1. Kuwana M, Okazaki Y. Quantification of circulating endothelial progenitor cells in systemic sclerosis: a direct comparison of protocols. Ann Rheum Dis 2012, in press.

2. Distler JH, Allanore Y, Avouac J, et al. EULAR Scleroderma Trials and Research group statement and recommendations on endothelial precursor cells. Ann Rheum Dis 2009;68(2):163-8.

3. Avouac J, Juin F, Wipff J, et al. Circulating endothelial progenitor cells in systemic sclerosis: association with disease severity. Ann Rheum Dis 2008;67(10):1455-60.

4. Avouac J, Meune C, Ruiz B, et al. Angiogenic biomarkers predict the occurrence of digital ulcers in systemic sclerosis. Ann Rheum Dis 2012;71(3):394-9.

Conflict of Interest:

None declared

Conflict of Interest

None declared