High-Altitude Trekking in the Himalayas Increasesthe Activity of Circulating Endothelial Cells
M.M. Ciulla,1* A. Giorgetti,2 L. Lazzari,2 M. Cortiana,3 I. Silvestris,3 G. Annoni,1 C. De Asmundis,1
A.V. Fiore,2 E. Montelatici,2 R. Paliotti,1 F. Magrini,1 P. Rebulla,2 and A. Cortelezzi3
1 Istituto di Medicina Cardiovascolare, Centro di Fisiologia Clinica e Ipertensione, University of Milan,
IRCCS Ospedale Maggiore di Milano, Milan, Italy2Cell Factory Franco Calori, Centro Trasfusionale e di Immunologia dei Trapianti, IRCCS Ospedale Maggiore di Milano, Milan, Italy
3Department of Hematology, University of Milan, IRCCS Ospedale Maggiore di Milano, Milan, Italy
Circulating endothelial progenitor cells (EPCs) are believed to contribute to vascular
homeostasis; unfortunately, the response of EPCs in physiological conditions remains
largely unknown. Herein we report our observations of a 44-year-old healthy subject after a
trek in the Himalayas that support high-altitude hypoxia and exercise oxygen demands are
strong stimuli for clonogenic endothelial cell activation and activity, as shown by the
increase in the number of mature EPCs and in the endothelial colony-forming unit capa-
city. Both of these effects were completely reverted at sea level, 45 days after the subjects
trek. Am. J. Hematol. 79:7678, 2005. 2005 Wiley-Liss, Inc.
Key words: endothelial cell; exercise; hypobaric hypoxia
There is a growing understanding that circulatingendothelial progenitor cells (EPCs) derived from bonemarrow (BM) play a role in vascular homeostasis,possibly by both inducing and modulating angiogen-esis in areas with reduced oxygen supply or by stimu-lating the re-endothelization of injured vessels . Inphysiological conditions, exercise is known to upre-gulate EPCs and to decrease the rate of EPC apopto-sis . Pathologic tissue ischemia in experimentalanimal models has been demonstrated to increasethe frequency of EPCs, thereby contributing to neo-vascularization . Furthermore, in vitro inducedanoxia has been shown to enhance the differentiationof peripheral blood mononuclear cells from healthysubjects into EPCs . However, no information isavailable on the effects of hypoxia that occurs at highaltitude (hypobaric hypoxia) on the EPCs and on theEPC activity quantified by evaluation of endothelialcolony-forming units (CFU-endo).
Herein we report our observations from a 44-year-oldhealthy, nonsmoking, male, sea-level-native subject(height, 175 cm; body weight, 74 kg; BMI, 24) following
trekking in the Himalayas, an activity that combinesaltitude hypoxia and increased exercise oxygen demand.The subject, who underwent a screening evaluation toexclude any underlying cardiac or pulmonary diseases,was moderately trained (max VO2 45.2 1.9 mL/min/kg) and unacclimatized to high altitudes beforetrekking. Three samples (20 mL each) of peripheralblood were obtained at 11:0012:00 am at sea levelbefore the trek (basal conditions), the day after a20-day stay at a mean of 3,900 400 m, and 45 daysafter the trek, at sea level. The number of CD45()/CD146(+)/CD144(+) circulating cells was assessed by
Contract grant sponsor: Cariplo; FIRB; Programma NazionaleCellule Staminali-Istituto Superiore di Sanita`
*Correspondence to: Michele M. Ciulla, M.D., Ph.D., Istituto diMedicina Cardiovascolare, Centro di Fisiologia Clinica eIpertensione, Universita` di Milano, IRCCS Ospedale Maggioredi Milano, Via F. Sforza 35, 20122 Milano, Italy.E-mail: firstname.lastname@example.org
Received for publication 17 September 2004; Accepted 5November 2004
Published online inWiley InterScience (www.interscience.wiley.com).DOI: 10.1002/ajh.20332
American Journal of Hematology 79:7678 (2005)
2005 Wiley-Liss, Inc.
flow-cytometric analysis of 100,000 events per sample(FACScan, Becton Dickinson, Franklin Lakes, NJ).This phenotype was assumed to be a marker of circulat-ingmatureEPCs.Thenumber ofCFU-endo [CD31(+)/CD105(+)/CD144(+)/CD146low/CD133()/vWf low)]was scored by microscopy in at least 6 wells by twoblinded operators and was assumed to be a marker ofEPC activity according to a previously described proce-dure . At baseline, before the trek, the number of EPCcells was 4.88 cells/mLwhile noCFU-endo colonies wereformed.At 1 day after the trek, about a 5-fold increase inthe EPCs population was found and a mean of 12.5CFU-endo colonies/well were formed (Table I). It isimportant to note that the mean number of CFU-endocolonies in 17 randomly selected, healthy, nonsmokingsubjects utilized as a reference was 2.48 0.5 (SE)colonies/well (unpublished data). Endothelial activationwas confirmed by an increase in CD45() events (from0.41% to 2.41%). At day 45 after the trek, completenormalization of EPCs and CFU-endo was observed(Table I; Fig. 1). No significant changes in RBC countor Hb concentration were documented at any of the
checked timepoints. To establish if the changes observedfrom baseline were associatedwith a concurrent increasein the number of hematopoietic/endothelial precursorcells, the number of [CD45()/CD34(+)/CD133(+)]cells was assessed and an increase from 0 to 31.8 cells/mL was confirmed. Furthermore, both CD45()/KDR(+) and CD45(+)/KDR(+) cells were increased75-fold and 240-fold, respectively. These preliminaryfindings indicate that high-altitude trekking activatesthe endothelial lineage by increasing the number ofmature EPCs. These data are supported by an increasein EPC activity, as shown by the CFU-endo capacity.Both of these effects are normalized 45 days after thetrek. In conclusion, the association of a low oxygensaturation and a higher oxygen demand induced byexercise are strong stimuli for clonogenic endothelialcell activation.
This work was supported by grants from Cariplo,Plasticita` delle cellule staminali da sangue placentaree midollo osseo in senso neurale, muscolare, endote-liale ed epatico; Progetto Ricerca Finalizzata 2002,Criobanca automatizzata di materiale biologico;Ricerca Finalizzata 2003, Differenziamento e trans-differenziamento di cellule progenitrici endoteliali inpatologie ischemiche e pazienti a rischio; FIRB2001, Identificazione, purificazione, transdifferenzia-zione e banking di cellule staminali indirizzate versofenotipi propri e non propri da utilizzare per ripara-zione tissutale; and Programma Nazionale CelluleStaminali-Istituto Superiore di Sanita`, Homing ofperipherally injected bone marrow stem cells in ratafter experimental myocardial injury.
TABLE I. Changes inCirculating and Inducible Endothelial Cells
(no. of cells/mL)CFU-endo
(no. of colonies/well)
Normoxiaa 7.0 1.1 (SE) 2.48 0.5 (SE)
7 days before 4.88 0
1 day after 20.49 12.5
45 days after 8.22 0
aEPC reference values are from 31 randomhealthy subjects (M/F 15:16)aged 41.3 16.7 (SD) years. CFU-endo reference values are from
17 random healthy subjects (M/F 9:8) aged 40.8 15.8 (SD) years.
Fig. 1. Phase-contrast images of CFU-endo differentiated from peripheral blood mononuclear cells obtained after 7 daysof culture. Images (a) and (c) were obtained before and after the trek, respectively, and show no colony-forming unitcapacity. Image (b) was obtained 1 day after the trekking and shows a typical endothelial progenitor cell (EPC) colony(original magnification 200).
Brief Report: High-Altitude Trekking and Circulating Endothelial Cell Activity 77
We acknowledge trekker Fernando Sala for mak-ing these observations possible.
1. Hristov M, Erl W, Weber PC. Endothelial progenitor cells: mobi-
lization, differentiation, and homing. Arterioscler Thromb Vasc
2. Laufs U, Werner N, Link A, et al. Physical training increases
endothelial progenitor cells, inhibits neointima formation, and
enhances angiogenesis. Circulation 2004;109:220226.
3. Takahashi T, Kalka C, Masuda H, et al. Ischemia- and cyto-
kine-induced mobilization of bone marrow-derived endoth-
elial progenitor cells for neovascularization. Nat Med 1999;5:
4. Akita T, Murohara T, Ikeda H, et al. Hypoxic precondi-
tioning augments efficacy of human endothelial progenitor
cells for therapeutic neovascularization. Lab Invest 2003;83:
5. Hill JM, Zalos G, Halcox JP, et al. Circulating endothelial progeni-
tor cells, vascular function, and cardiovascular risk. N Engl J Med
78 Brief Report: Ciulla et al.