May 20, 2017 - The Undersea and Hyperbaric Medical Society has documented the beneficial effects of hyperbaric oxygen (HBO) therapy in at least 14 ...
Acta Medica Mediterranea, 2017, 33: 801
EFFECTS OF HYPERBARIC OXYGEN EXPOSURE ON MOBILIZATION OF ENDOTHELIAL PROGENITOR CELLS IN HEALTHY VOLUNTEERS
GIULIANO VEZZANI1, MANUELA IEZZI2, ALEX RIZZATO1, SILVIA QUARTESAN1, DEVANAND MANGAR3, ENRICO M CAMPORESI3, MATTEO PAGANINI4, GERARDO BOSCO1 1 Master II level in Hyperbaric Medicine and Physiological Lab, Department of Biomedical Sciences, University of Padova, Italy 2 Department of Medicine and Aging Science, CESI-MeT, University G. D’Annunzio, Chieti, Italy - 3Anesthesia, Tampa general Hospital, TEAMHealth Research Institute, TGH, Tampa, Florida, USA - 4Emergency Medicine Residency Program - Azienda Ospedaliera Università di Padova, Italy ABSTRACT Background: Hyperbaric oxygen therapy (HBO) has been successfully utilized for the treatment of a wide variety of diseases, including diabetes mellitus, osteomyelitis and radiation induced necrotic lesions. These diseases are characterized by significant vascular damage and prolonged wound healing process. HBO has been recently shown to mobilize stem/progenitor cells from the bone marrow. Because of its potential to promote neovascularization it is believed that HBO contributes to vascular damage recovery. In this study we questioned whether hyperbaric oxygen therapy might exert pro-angiogenic effects via the modulation of endothelial progenitor cells. Methods: Peripheral blood samples were collected from 15 healthy human volunteers (5 women and 10 men; mean age: 49.4 ± 2.56 years; body weight: 70.3 ± 4.58 kg; height: 1.67 ± 4.19 m) before and after exposure to hyperbaric oxygen (n=8) for 20 days (90 min at 2.4 ATA once a day) and exposure to hyperbaric compressed air (n= 7), for 90 minutes at 2.4 ATA twice per day for 20 days. Flow cytometry immunophenotypic analysis was performed on these samples to identify and enumerate endothelial progenitor cells. Results: Our results show a significant effect of hyperbaric oxygen therapy on surface markers of endothelial progenitor cells in healthy volunteers. The number of CD34/CD133-expressing cells increased in volunteers after both the 1st (p < 0.02) and the 10th HBO exposures (p < 0.05). HBO group shows also significant increase of CD34/CD133/VEGFR-2 – expressing cells only at 1st treatment (p < 0.05), compared to basal values, returning to normal values at wash out. Conclusion: HBO might induce modulation of endothelial progenitor cells biology and this mechanism could initiate clinical benefits exerted by neoangiogenesis and neovascularization. Keywords: hyperbaric oxygen therapy, stem cell expression, endothelial progenitor cells, angiogenesis. DOI: 10.19193/0393-6384_2017_5_118 Received November 30, 2016; Accepted May 20, 2017
Introduction The Undersea and Hyperbaric Medical Society has documented the beneficial effects of hyperbaric oxygen (HBO) therapy in at least 14 different disease categories(1). Although the benefits of HBO have already been observed in chronic-wound healing processes - such as diabetic ulcers, wounds with hypoxic tissue environment and thermal burns - the mechanisms underlying HBO therapeutic effects
are partially understood(2-4). It has been repeatedly demonstrated that “wound healing” relies on formation and maintenance of a functional vascular system. Previous studies in animal models as well as in humans provided evidence that endothelial progenitor cells (EPCs) have notable ability to induce functional improvement in ischemic organs. The EPCsinduced effects appear to be related to the induction and modulation of angiogenesis in tissues with reduced oxygen supply and/or to their ability to
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Giuliano Vezzani, Manuela Iezzi et Al
stimulate re-endothelialization of damaged blood vessels(5). It has been previously demonstrated that EPCs and bone marrow-derived multipotent progenitor cells possess inherent ability to localize to sites of neovascularization and to differentiate into endothelial cells in situ(6). Although no EPC-specific antigen has been identified to date, EPCs are usually characterized by the co-expression of 3 antigens: CD34, CD133, and VEGFR-2 (the Vascular Endothelial Growth Factor Receptor-2)(7). EPCs appear to lose CD133 during their differentiation (8), and VEGFR-2 is known to be important in the process of angiogenesis(9-11). The endothelial progenitor cells (EPC) research could be the key to clarify the distinct process of vasculogenesis and neovessels origin from bone marrow-derived progenitor cells (12). Ischemic limb perfusion and wound healing seems to be related to EPC mobilization into circulation and triggered by hyperoxia through induction of bone marrow nitric oxide(13). The aim of this study is to examine the effects of HBO on the number of EPCs in healthy human volunteers in order to investigate the physiological reaction of EPCs to HBO, without the influence of pre-existing inflammatory elements. We had two goals for this investigation: (i) to measure the various population of circulating EPC in blood; (ii) using fluorescence with markers CD34+, CD133 and VEGFR-2, surface markers related to new vessels formation.
All subjects gave written consent to participate in the research protocol approved by the local Ethics Committee, assuring anonymity of personal data. All the procedures were in accordance with the ethical standards of the responsible committee on human experimentation and with the Helsinki Declaration of 1975, as revised in 2000 and 2008. Subjects were exposed inside a multiplace hyperbaric chamber with either compressed oxygen (FiO2 100%) or compressed air at 2.4 ATA for 90 minutes. Each person was provided with a wellsealed breathing mask from which he or she received either 100% oxygen (HBO) or 100% compressed air (HBA). Oxygen concentration in the mask was measured every 15 minutes to ensure adequacy of the gas supply and the ability of providing a tight seal around the face. Figure 1 demonstrates the O2 levels measured during all treatments of HBO. The mask was applied at the beginning of the session and was kept in place throughout the 90-minute treatment session without removal. All subjects received 20 treatments of HBO or HBA from Monday through Friday for a period of 4 weeks. Subjects were blind to which gas they were breathing.
Material and methods Human subjects and HBO exposure For this study 15 healthy human volunteers (5 women and 10 men; mean age: 49.4 ± 2.56 years; body weight: 70.3 ± 4.58 kg; height: 1.67 ± 4.19 m) were recruited and divided in two groups that underwent hyperbaric oxygen (HBO100%, Oxygen) or compressed air (HBA) exposure (Table 1).
Figure 1: The average concentration of O2 percentage inspired for the 5 males and 3 females receiving HBO (C) in a total of 20 sessions is represented. In order to ensure O2 concentration, a device within the volunteers’ face mask monitored CO2 expired every 15 min during each session of the therapy. These numbers exemplify an average inspired O2 of 87,08%. C: compression, each HBO session.
Sex N° of subjects
Mean age Male
Female
Total
15
10
5
49,4
HBO
8
5
3
49,8
HBA
7
5
2
49
Table 1: Demographic features of volunteers enrolled in this study and treatment subgroups.
Collection of peripheral blood samples and isolation of mononuclear cell Venous blood samples were obtained at various times from the subjects. Lymphomonocytes were separated from venous blood samples before exposure to HBO or HBA (t0), then after the 1st (t1), 10th (t10), and 20th (t20) exposures, respective-
Effects of hyperbaric oxygen exposure on mobilization of endothelial progenitor cells in healthy volunteers
ly. An additional follow up peripheral blood sample was also collected at one month after the conclusion of the last exposures (wash out, two). EPCs characterization was performed by flow cytometry and culture (in endothelial medium). EDTA-anticoagulated blood (16 ml) was diluted in HBSS (Sigma Aldrich, USA) and centrifuged through Histopaque 1077 (Sigma Aldrich, USA) at 800g for 15 min to isolate leukocytes. Leukocyterings (buffy coats) were then repeatedly washed with HBSS 5% FBS (Gibco, Australia), residual erythrocytes were lysed by incubation in 150 mM ammonium chloride, 0.1 M EDTA and 10 mM potassium carbonate (pH 7.2), and finally isolated PBMC were counted and frozen in 1:10 DMSO (Sigma Aldrich, USA) and FBS in liquid nitrogen. Flow - cytometry immuno - phenotypic analysis Flow cytometric analysis of the mononuclear peripheral blood leukocytes was performed using a FACScalibur flow cytometer (Becton Dickinson). Cells were suspended in 250 µl PBS with 0.5% BSA. Cells were then incubated with human serum (Lonza, Belgium) for 15 min at 4°C to block Fc receptors. Thereafter, cells were incubated with fluorescein isothiocyanate (FITC) conjugated mouse anti-human CD34 (Clone AC136, a class III CD34 epitope; Milteny Biotec, Germany), allophycocyanin (APC)-conjugated mouse anti-human vascular endothelial growth factor-2 receptor (VEGFR-2, CD309, clone ES8-20E6; Milteny Biotec, Germany) and R-phycoerythrin (RPE)-conjugated CD133/1 (Clone AC133; Miltenyi Biotec, Germany), for 30 min at 4°C in the dark. Isotypematched mouse immunoglobulin served as control. Cells were then washed with PBS, centrifuged, and resuspended in a 1:100 solution of 7-AAD (Beckman Coulter, Japan) and PBS. The flow analysis was performed on 1.0 - 105 gated cells using forward and side light scatters and the 7AAD negativity EPCs culture For the EPCs culture, thawed peripheral blood mononuclear leukocytes were suspended in EGM®-2 Endothelial Cell Growth Medium-2 (Lonza, Belgium) which contains fetal bovine serum, hydrocortisone, h-FGF-B, VEGF, R3-IGF-1, ascorbic acid, hEGF, GA-1000 and heparin. Cultures were initiated with 1 ml of suspension/well of a six-well Petri plate collagen-1
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coated and incubated at 37°C, air with 5% CO2, in a fully humidified atmosphere. After 24 h of incubation, non-adherent cells were washed and remaining cells were kept in culture in EGM®-2 Endothelial Cell Growth Medium-2 for 21 days, every two days medium was replaced with fresh medium. Statistics All data are expressed as mean ± Standard Deviation (SD) and compared with the statistical GraphPad Prism software (GraphPad Prism 6, Graphpad Software Inc., San Diego, CA). Statistical analysis of EPCs and quantitative changes in expressing protein markers were carried out by Levene’s test for equality of variances followed by two tailed t-test (independent samples). Statistical significance was taken as p
