Effects of Cold Stimulation on Mitochondrial Activity and VEGF Expression in vitro

Abstract

We aimed to clarify the effects of cold stimulation at various temperatures on mitochondrial activity and vascular endothelial growth factor (VEGF) expression in vitro. Human fibroblast, human mesenchymal stem cell, and rat skeletal muscle myoblast cell lines were used. For each cell type, cells were divided into 4 groups and stimulated in various cold temperatures (0, 4, 17 and 25°C) 3 times for 15 min each by placement on crushed ice or floating on cold water set at each temperature. Control cells were subjected to warm water at 37°C. Factors related to mitochondrial activity, mitochondrial DNA copy numbers, and VEGF expression were analyzed 24 h after the last cold stimulation. In all cell types, significant increases of factors related to mitochondrial activity and mitochondrial DNA copy numbers were seen in the 4°C and 17°C-stimulated cells compared with control cells. In rat skeletal muscle cells stimulated at 4°C, VEGF expression significantly increased compared to the control cells. Our data suggest that cold stimulation at certain temperatures promotes mitochondrial activity, biogenesis and VEGF expression.

Key words

cryotherapy - mitochondrial activity - fibroblasts - mesenchymal stem cells - myoblasts

• References

• 1 Abu-Amero KK, Bosley TM. Detection of mitochondrial respiratory dysfunction in circulating lymphocytes using resazurin. Arch Pathol Lab Med 2005; 129: 1295-1298
• 2 Austin S, St-Pierre J. PGC1α and mitochondrial metabolism – emerging concepts and relevance in ageing and neurodegenerative disorders. J Cell Sci 2012; 125: 4963-4971
• 3 Ali SF, LeBel CP, Bondy SC. Reactive oxygen species formation as a biomarker of methylmercury and trimethyltin neurotoxicity. Neurotoxicology 1992; 13: 637-648
• 4 Bleakley C, McDonough S, MacAuley D. The use of ice in the treatment of acute soft-tissue injury: a systematic review of randomized controlled trials. Am J Sports Med 2004; 32: 251-261
• 5 Chen JF, Liu H, Ni HF, Lv LL, Zhang MH, Zhang AH, Tang RN, Chen PS, Liu BC. Improved mitochondrial function underlies the protective effect of pirfenidone against tubulointerstitial fibrosis in 5/6 nephrectomized rats. PLoS One 2013; 8: e83593
• 6 Chiang B, Essick E, Ehringer W, Murphree S, Hauck MA, Li M, Chien S. Enhancing skin wound healing by direct delivery of intracellular adenosine triphosphate. Am J Surg 2007; 193: 213-218
• 7 Coban AY, Uzun M, Akgunes A, Durupinar B. Comparative evaluation of the microplate nitrate reductase assay and the rezasurin microtitre assay for the rapid detection of multidrug resistant Mycobacterium tuberculosis clinical isolates. Mem Inst Oswaldo Cruz 2012; 107: 578-581
• 8 Dikalov S, Griendling KK, Harrison DG. Measurement of reactive oxygen species in cardiovascular studies. Hypertension 2007; 49: 717-727
• 9 Duluc L, Jacques C, Soleti R, Andriantsitohaina R, Simard G. Delphinidin inhibits VEGF induced-mitochondrial biogenesis and Akt activation in endothelial cells. Int J Biochem Cell Biol 2014; 53: 9-14
• 10 Escribano AI, Marcel AM, Tugores YM, Ruiz JJ, Redó VJ, García-Trevijano JA, Barrio AG. Validation of a modified fluorimetric assay for the screening of trichomonacidal drugs. Mem Inst Oswaldo Cruz 2012; 107: 637-643
• 11 Feng YM, Jia YF, Su LY, Wang D, Lv L, Xu L, Yao YG. Decreased mitochondrial DNA copy number in the hippocampus and peripheral blood during opiate addiction is mediated by autophagy and can be salvaged by melatonin. Autophagy 2013; 9: 1395-1406
• 12 Forbes SJ, Rosenthal N. Preparing the ground for tissue regeneration: From mechanism to therapy. Nat Med 2014; 20: 857-869
• 13 Frey SP, Jansen H, Raschke MJ, Meffert RH, Ochman S. VEGF improves skeletal muscle regeneration after acute trauma and reconstruction of the limb in a rabbit model. Clin Orthop Relat Res 2012; 470: 3607-3614
• 14 Gabbiani G, Ryan GB, Majno G. Presence of modified fibroblasts in granulation tissue and their possible role in wound contraction. Experientia 1971; 7: 549-550
• 15 Galiano RD, Tepper OM, Pelo CR, Bhatt KA, Callaghan M, Bastidas N, Bunting S, Steinmetz HG, Gurtner GC. Topical vascular endothelial growth factor accelerates diabetic wound healing through increased angiogenesis and by mobilizing and recruiting bone marrow-derived cells. Am J Pathol 2004; 164: 1935-1947
• 16 Gosain A, DiPietro LA. Aging and wound healing. World J Surg 2004; 28: 321-326
• 17 Harriss DJ, Atkinson G. Ethical standards in sport and exercise science research: 2016 update. Int J Sports Med 2015; 36: 1121-1124
• 18 Howard JD, Sarojini H, Wan R, Chien S. Rapid granulation tissue regeneration by intracellular ATP delivery-A comparison with Regranex. PLoS One 2014; 9: e91787
• 19 Hubbard TJ, Denegar CR. Does cryotherapy improve outcomes with soft tissue injury?. J Athl Train 2004; 39: 278-279
• 20 Ihsan M, Watson G, Choo HC, Lewandowski P, Papazzo A, Cameron-Smith D, Abbiss CR. Postexercise muscle cooling enhances gene expression of PGC1α. Med Sci Sports Exerc 2014; 46: 1900-1907
• 21 Ihsan M, Watson G, Lipski M, Abbiss CR. Influence of postexercise cooling on muscle oxygenation and blood volume changes. Med Sci Sports Exerc 2013; 45: 876-882
• 22 Ijiri D, Kanai Y, Hirabayashi M. Possible roles of myostatin and PGC-1alpha in the increase of skeletal muscle and transformation of fiber type in cold-exposed chicks: expression of myostatin and PGC-1alpha in chicks exposed to cold. Domest Anim Endocrinol 2009; 37: 12-22
• 23 Imray CH, Richards P, Greeves J, Castellani JW. Nonfreezing cold-induced injuries. J R Army Med Corps 2011; 157: 79-84
• 24 Ingram J, Dawson B, Goodman C, Wallman K, Beilby J. Effect of water immersion methods on post-exercise recovery from simulated team sport exercise. J Sci Med Sport 2009; 12: 417-421
• 25 Knight KL. Chapter 2 Historical Perspective. In: Drews C, Roselund D, Wentworth J, Thomas J, Barker D. (eds.). Cryotherapy in Sport Injury Management. Champaign: Human Kinetics; 1995: 13-16
• 26 Liang H, Ward WF. PGC-1alpha: a key regulator of energy metabolism. Adv Physiol Educ 2006; 30: 145-151
• 27 Li R, Stewart DJ, von Schroeder HP, Mackinnon ES, Schemitsch EH. Effect of cell-based VEGF gene therapy on healing of a segmental bone defect. J Orthop Res 2009; 27: 8-14
• 28 Mathieu D, Linke JC, Wattel F. Chapter 2.2.9 Non-healing wounds. In: Mathieu DE. (ed.). Handbook on hyperbaric medicine. Dordrecht: Springer; 2006: 401-427
• 29 Merrick MA, Jutte LS, Smith ME. Cold modalities with different thermodynamic properties produce different surface and intramuscular temperatures. J Athl Train 2003; 38: 28-33
• 30 Merrick MA, Knight KL, Ingersoll CD, Potteiger JA. The effects of ice and compression wraps on intramuscular temperatures at various depths. J Athl Train 1993; 28: 236-245
• 31 Molloy T, Wang Y, Murrell GAC. The roles of growth factors in tendon and ligament healing. Sports Med 2003; 33: 381-394
• 32 Nauta A, Seidel C, Deveza L, Montoro D, Grova M, Ko SH, Hyun J, Gurtner GC, Longaker MT, Yang F. Adipose-derived stromal cells overexpressing vascular endothelial growth factor accelerate mouse excisional wound healing. Mol Ther 2013; 21: 445-455
• 33 Neutelings T, Lambert CA, Nusgens BV, Colige AC. Effects of mild cold shock (25°C) followed by warming up at 37°C on the cellular stress response. PLoS One 2013; 8: e69687
• 34 Nusair YM. Local application of ice bags did not affect postoperative facial swelling after oral surgery in rabbits. Br J Oral Maxillofac 2007; 45: 48-50
• 35 Oryan A, Alidadi S, Moshiri A. Current concerns regarding healing of bone defects. Hard Tissue 2013; 2: 13-25
• 36 Ozturk BY, Inci I, Egri S, Ozturk AM, Yetkin H, Goktas G, Elmas C, Piskin E, Erdogan D. The treatment of segmental bone defects in rabbit tibiae with vascular endothelial growth factor (VEGF)-loaded gelatin/hydroxyapatite “cryogel” scaffold. Eur J Orthop Surg Traumatol 2013; 23: 767-774
• 37 Park CH, Lee MJ, Ahn J, Kim S, Kim HH, Kim KH, Eun HC, Chung JH. Heat shock-induced matrix metalloproteinase (MMP)-1 and MMP-3 are mediated through ERK and JNK activation and via an autocrine interleukin-6 loop. J Invest Dermatol 2004; 123: 1012-1019
• 38 Pivonka P, Dunstan CR. Role of mathematical modeling in bone fracture healing. Bonekey Reports 2012; 1: 221
• 39 Pournot H, Bieuzen F, Duffield R, Lepretre PM, Cozzolino C, Hausswirth C. Short term effects of various water immersions on recovery from exhaustive intermittent exercise. Eur J Appl Physiol 2011; 111: 1287-1295
• 40 Puntel GO, Carvalho NR, Dobrachinski F, Salgueiro AC, Puntel RL, Folmer V, Barbosa NB, Royes LF, Rocha JB, Soares FA. Cryotherapy reduces skeletal muscle damage after ischemia/reperfusion in rats. J Anat 2013; 222: 223-230
• 41 Rampersad SN. Multiple applications of alamar blue as an indicator of metabolic function and cellular health in cell viability bioassays. Sensors (Switzerland) 2012; 12: 12347-12360
• 42 Reers M, Smith TW, Chen LB. J-aggregate formation of a carbocyanine as a quantitative fluorescent indicator of membrane potential. Biochemistry 1991; 30: 4480-4486
• 43 Saaristo A, Tammela T, Farkkilā A, Kärkkäinen M, Suominen E, Yla-Herttuala S, Alitalo K. Vascular endothelial growth factor-C accelerates diabetic wound healing. Am J Pathol 2006; 169: 1080-1087
• 44 Slivka DR, Dumke CL, Tucker TJ, Cuddy JS, Ruby B. Human mRNA response to exercise and temperature. Int J Sports Med 2012; 33: 94-100
• 45 Springer JE, Azbill RD, Carlson SL. A rapid and sensitive assay for measuring mitochondrial metabolic activity in isolated neural tissue. Brain Res Protoc 1998; 2: 259-263
• 46 Sugasawa T, Tanba T, Iwasawa Y, Shibuya T, Hayakawa K, Oshiro S. The effect of icing on fracture healing in rats. Riryo 2013; 43: 59-65
• 47 Sugasawa T, Mukai N, Oshiro S, Handa T, Tanabe I, Kumaki Y, Tamura K, Tanba T, Moriyama N, Takekoshi K. The effects of cold stimulations of various temperatures on cell growth and metabolic activity of human cells in vitro. Gazz Med Ital (in press)
• 48 Tadaishi M, Miura S, Kai Y, Kano Y, Oishi Y, Ezaki O. Skeletal muscle-specific expression of PGC-1α-b, an exercise-responsive isoform, increases exercise capacity and peak oxygen uptake. PLoS One 2011; 6: e28290
• 49 Takagi R, Fujita N, Arakawa T, Kawada S, Ishii N, Miki A. Influence of icing on muscle regeneration after crush injury to skeletal muscles in rats. J Appl Physiol 2011; 110: 382-388
• 50 Thorsson O, Lilja B, Ahlgren L, Hemdal B, Westlin N. The effect of local cold application on intramuscular blood flow at rest and after running. Med Sci Sports Exerc 1985; 17: 710-713
• 51 Wang J, Zhang Q, Wan R, Mo Y, Li M, Tseng MT, Chien S. Intracellular adenosine triphosphate delivery enhanced skin wound healing in rabbits. Ann Plast Surg 2009; 62: 180-186
• 52 Wang J, Wan R, Mo Y, Li M, Zhang Q, Chien S. Intracellular delivery of adenosine triphosphate enhanced healing process in full-thickness skin wounds in diabetic rabbits. Am J Surg 2010; 199: 823-832
• 53 White GE, Wells GD. Cold-water immersion and other forms of cryotherapy: physiological changes potentially affecting recovery from high-intensity exercise. Extrem Physiol Med 2013; 2: 26
• 54 Yan X, Chen B, Lin Y, Li Y, Xiao Z, Hou X, Tan Q, Dai J. Acceleration of diabetic wound healing by collagen-binding vascular endothelial growth factor in diabetic rat model. Diabetes Res Clin Pract 2010; 90: 66-72
• 55 Zhang H, Du G, Zhang J. Assay of mitochondrial functions by resazurin in vitro. Acta Pharmacol Sin 2004; 25: 385-389