Increased hypoxic proliferative response in PBMCs‐derived erythroid progenitor cells of Andean highlanders with Chronic Mountain Sickness
Authors: Bermudez Daniela , Anza‐Ramirez Cecilia , Orrego‐Solano Miguel Ángel , Guerra‐Giraldez Cristina , Villafuerte Francisco C. 
Published: June 2021

Rationale

Excessive erythrocytosis (EE; Hb≥21g/dL in men and ≥19g/dL in women) is the main sign of Chronic Mountain Sickness (CMS), a maladaptive clinical syndrome prevalent in Andean and other high‐altitude populations. Although the pathophysiological mechanism for EE is still controversial, chronic hypoxemia is accepted as its underlying stimulus.

Although EE is common to all CMS subjects, most studies have shown similar mean values for serum erythropoietin compared to healthy highlanders (HH). We have previously shown that plasma concentration of the soluble Epo receptor (sEpoR), an endogenous Epo antagonist, is decreased in highlanders with CMS, which would lead to higher Epo circulating availability and contribute to EE. However, it is also possible that erythroid progenitor cells of CMS subjects are more sensitive to Epo, and have a stronger proliferative response than HH cells exposed to similar circulating Epo concentration. Thus, the aim of the present study was to determine the hypoxic proliferative response of erythroid progenitor cells (BFU‐E) of CMS and HH to equal rhEpo concentration.

Methods

Samples

Thirty‐six highlanders (CMS, n=17; HH, n=19) from Cerro de Pasco, Peru, at 4340m, were recruited for the study. A small blood sample was obtained for hematocrit determination and CMS score was assessed. A 10ml blood sample was taken in heparin‐coated BD Vacutainer tubes for peripheral blood mononuclear cells (PBMCs) isolation. Two additional 6ml blood samples were also obtained in clot‐activator‐coated tubes for Epo and iron profile determination. Serum was separated by centrifugation and stored in liquid N2 until analysis.

Culture

PBMCs were isolated using Histopaque 1077 (Sigma) and were cultured in Methocult H‐4534 medium (Stemcell Technologies) at a final density of 2.5×105cells/ml in the presence of 3U/ml of rhEpo (Stemcell Technologies) for 14 days. Cultures were maintained at 37°C in a Biospherix X3 hood set for cellular hypoxia (1%O2, 5% CO2).

BFU‐E identification and count

Colony identification and count were performed at days 7 and 10 using an inverted microscope (LS 560, Lumascope) inside the hypoxic hood at a magnification of 20X. On day 14 cultures were removed from the hood and analyzed using an AxioZoom Stereomicroscope (Carl Zeiss Microscopy) at a magnification of 10X.

Data analysis

Student's t‐test for equal and unequal variances was applied as parametric test of comparison and Wilcoxon as nonparametric to evaluate differences between CMS and HH.

Results

As expected, Hct and CMS score were significantly higher in the CMS group (Table 1). Mean serum Epo concentration was higher in CMS subjects (12.79±2.8 vs 26.8±8.1pg/ml, p<0.05) due to two participants showing very high Epo values (high‐Epo CMS highlanders, as previously described).

Percentage of change in BFU‐E number from day 7 to day 14 was larger in the CMS group (87.84±27.2 vs 258.6±69.12%, Figure 1B), and CMS BFU‐E colonies occupied a larger total area compared to HH (4.31±0.5 vs 6.4±0.5mm2, Figure 1A).

Conclusion

Our findings show that CMS erythroid progenitor cells have a greater proliferative response compared to HH when cultured under hypoxic conditions at the same rhEpo concentration, suggesting a higher Epo‐sensitivity of BFU‐E cells of CMS subjects.

Support or Funding Information

This research was supported by a Wellcome Trust Public Health and Tropical Medicine Fellowship (107544/Z/15/Z) to F. C. Villafuerte.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.