Hypothermia reduces VEGF-165 expression, but not osteogenic differentiation of human adipose stem cells under hypoxia

• Published in: PloS one Vol. 12; no. 2; p. e0171492
• Main Authors: Leegwater, Nick C, Bakker, Astrid D, Hogervorst, Jolanda M A, Nolte, Peter A, Klein-Nulend, Jenneke
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 06-02-2017; Public Library of Science (PLoS)
• Subjects: Adipose tissue; Adipose Tissue - cytology; Alkaline phosphatase; Anoxia; Apoptosis; BAX protein; Bcl-2 protein; Biocompatibility; Biology and Life Sciences; Biomedical materials; Callus; Cell differentiation; Cell Differentiation - radiation effects; Cell Hypoxia; Cells, Cultured; Cryotherapy; Damage prevention; Deoxyribonucleic acid; Differentiation; DNA; Gene expression; Gene Expression Profiling; Gene Expression Regulation; Health aspects; Humans; Hypothermia; Hypoxia; Kinases; Medicine and Health Sciences; Mesenchymal stem cells; Mesenchymal Stem Cells - cytology; Mesenchymal Stem Cells - metabolism; Mesenchymal Stem Cells - radiation effects; Mesenchyme; Nitric oxide; Osteocalcin; Osteogenesis - genetics; Osteogenesis - radiation effects; Pain; Protein folding; Research and Analysis Methods; Rodents; Sox9 protein; Stem cells; Vascular endothelial growth factor; Vascular Endothelial Growth Factor A - genetics; United States > US; Netherlands
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0171492

Cryotherapy is successfully used in the clinic to reduce pain and inflammation after musculoskeletal damage, and might prevent secondary tissue damage under the prevalent hypoxic conditions. Whether cryotherapy reduces mesenchymal stem cell (MSC) number and differentiation under hypoxic conditions, causing impaired callus formation is unknown. We aimed to determine whether hypothermia modulates proliferation, apoptosis, nitric oxide production, VEGF gene and protein expression, and osteogenic/chondrogenic differentiation of human MSCs under hypoxia. Human adipose MSCs were cultured under hypoxia (37°C, 1% O2), hypothermia and hypoxia (30°C, 1% O2), or control conditions (37°C, 20% O2). Total DNA, protein, nitric oxide production, alkaline phosphatase activity, gene expression, and VEGF protein concentration were measured up to day 8. Hypoxia enhanced KI67 expression at day 4. The combination of hypothermia and hypoxia further enhanced KI67 gene expression compared to hypoxia alone, but was unable to prevent the 1.2-fold reduction in DNA amount caused by hypoxia at day 4. Addition of hypothermia to hypoxic cells did not alter the effect of hypoxia alone on BAX-to-BCL-2 ratio, alkaline phosphatase activity, gene expression of SOX9, COL1, or osteocalcin, or nitric oxide production. Hypothermia decreased the stimulating effect of hypoxia on VEGF-165 gene expression by 6-fold at day 4 and by 2-fold at day 8. Hypothermia also decreased VEGF protein expression under hypoxia by 2.9-fold at day 8. In conclusion, hypothermia decreased VEGF-165 gene and protein expression, but did not affect differentiation, or apoptosis of MSCs cultured under hypoxia. These in vitro results implicate that hypothermia treatment in vivo, applied to alleviate pain and inflammation, is not likely to harm early stages of callus formation.