Elevated Carbon Dioxide Triggers Angiogenesis
Angiogenesis is the natural process whereby new blood vessels are formed. New blood vessels are needed when the metabolic demand outstrips the delivery of oxygenated blood, as in heavy exercise, and when tissue is stressed or damaged and therefore needs new blood vessels to heal and form fresh new tissue.
This paper describes the biological mechanisms involved in angiogenesis and cites multiple studies showing that the delivery of carbon dioxide to tissue through the skin (transdermal carbon dioxide) can play a critical role in the formation of new blood vessels.
When the use of skeletal muscles increases through, for example, exercise, the body activates multiple mechanisms to make the muscle use oxygen more efficiently. (Skeletal muscles are all muscles except the heart and the muscles that line the walls of several organs.) The most obvious mechanism and the easiest one to measure is the increase in oxygen delivered through the blood that results from a dilating network of blood vessels. Once blood has reached the metabolically active tissue, oxygen release from the hemoglobin is enhanced due to the Bohr effect. If the muscle tissue senses repeated stress on the supply of oxygen, angiogenesis triggers the formation of additional blood vessels that deliver oxygen to the tissue.
Thousands of studies have focused on identifying the mechanisms involved in the formation of new blood vessels through angiogenesis.6,9 One well-known mechanism is the increase of a substance in the tissue called HIF-1 in response to hypoxia, a low level of oxygen in the blood and a potent trigger for angiogenesis. Three other known physiological mechanisms activate angiogenesis in the absence of hypoxia: an increase in blood flow and shear,11 a stretching of the tissue in its microenvironment,24 and metabolic triggers such as adenosine, a compound that is one of the building blocks of RNA.1
Many recent studies now clearly demonstrate that carbon dioxide applied to the skin can also trigger angiogenesis. Given that carbon dioxide is a byproduct of metabolism, and high levels of carbon dioxide suggest that there is a lack of blood flow to carry it away, it makes sense that a high level of carbon dioxide would trigger the formation of new blood vessels. Although the pathways linking angiogenesis to the shearing of blood vessel walls and the stretching of tissue are well described, the link between angiogenesis and metabolic stress remains a mystery.
Multiple studies have also clearly shown that the cascade of biological mechanisms that leads to angiogenesis is directly activated by increases in carbon dioxide in the tissue. Investigators have demonstrated that carbon dioxide induces normoxic angiogenesis in the following: normal skeletal muscle,21 injured skeletal muscle, fractured bone,14,20 tissue in which the nerves have been disabled after an injury in which a limb or other body part has been crushed,19 skin flaps (skin moved from one part of the body to another during surgery),22 hyperglycemic skeletal muscle,16 a hind limb that has been deprived of blood flow,13 and healing wounds.15
How might carbon dioxide activate normoxic angiogenesis? Given that carbon dioxide dilates the blood vessels and forces red blood cells to release their oxygen,5,23,26 angiogenesis occurs in an environment with abundant oxygen, which thereby negates the idea that hypoxia, or HIF, is a controlling factor.
It is also possible that carbon dioxide activates angiogenesis through several other pathways that do not involve hypoxia. One study has suggested that carbon dioxide might cause the release of vascular endothelial growth factor (VEGF), a protein that stimulates the formation of blood vessels.4 Carbon dioxide might also activate angiogenesis by induces the release of nitric oxide,3,10, 18,7. Finally, angiogenesis might be stimulated by the acidity generated by a high level of carbon dioxide.25
Carbon dioxide also increases oxidative metabolism of muscle fibers, which ultimately causes angiogenesis. Carbon dioxide does this by mediating mitochondrial biogenesis. by influencing the creation of mitochondria, tiny structures in every cell that are often called the “powerhouse of the cell” because they convert oxygen into the energy that the body needs to function. One study showed that when carbon dioxide is increased in normal skeletal muscle, the gene expression of PGC-1a, SIRT1, and VEGF increase, as does the number of mitochondria.21 The CO2-induced increase in PGC-1a was confirmed in a study where oxidative capacity was preserved by CO2 in a model of hyperglycemia.17 and again when CO2 was used to abate muscle atrophy.12 These studies clearly demonstrate that carbon dioxide mimics the effects of a muscle during exercise and stimulates the need for oxidative capacity that ultimately causes angiogenesis.
The angiogenic response produced when CO2 is applied through the skin closely matches the response caused by CO2 released naturally during exercise. Haas and Nwadozi (8) reported in a recent review that three stages of angiogenesis are activated during repeated bouts of exercise. It was noted that when exercise is repeated over the long term, VEGF stimulation diminishes and concentrations return to baseline. This normalization of VEGF occurs once adequate remodeling of the vasculature has occurred.
Interestingly, the study of CO2 on exercised muscle mimics this response. After weeks of repeated transdermal CO2 therapy to the muscle, the amount of VEGF generated by the CO2 treatment also returned to baseline (2). This finding offers evidence that CO2 applied to the skin offers a stimulus very similar to that of natively generated CO2 in the tissue.
In summary, recent studies have demonstrated that elevated concentrations of CO2 in the tissue will essentially mimic an increase in metabolic rate and stimulate the tissue to respond accordingly by activating the process for normoxic angiogenesis.
The question is, does the application of carbon dioxide to the skin stimulate angiogenesis in the same way that carbon dioxide does this naturally during exercise? The answer is that, based on two studies, the two processes are very much alike. Both studies focused on the behavior of VEGF, which, as mentioned on page 1 of this paper, is a protein that helps to stimulate angiogenesis.
In one study, the researchers looked at muscles that were routinely exercised over a long period of time.8 They found that the level of VEGF returned to normal as the exercise continued. VEGF does this only once angiogenesis is complete, and carbon dioxide, which also plays a role in the formation of blood vessels, may trigger the release of VEGF.
In the second study,2 the researchers applied carbon dioxide transdermally to the muscle for weeks. After this treatment, they found that the amount of VEGF had returned to normal. The fact that VEGF behaved after this treatment just as it did after repeated sessions of natural exercise shows that high levels of carbon dioxide delivered transdermally to muscle can set the process of angiogenesis in motion.
The evidence from these two studies and from the others described in this paper shows that the transdermal application of carbon dioxide, by triggering the formation of new blood vessels, can create an environment in which fresh, new tissue can form.
References
Adair TAdair T. Growth regulation of the vascular system: An emerging role for adenosine. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology I 289:283-296, 2005.
Akahane S, Sakai Y, Ueha T, Nishimoto H, Inoue M, Niikura T, and Kuroda R. Transcutaneous carbon dioxide application accelerates muscle injury repair in rat models. International Orthopedics 41:1007-1015, 2017.
Baum O, Da Silva-Azevedo L, Willerding G, Wöckel A, Planitzer G, Gossrau R, Pries AR, and Zakrzewicz A. Endothelial NOS is main mediator for shear stress-dependent angiogenesis in skeletal muscle after prazosin administration. American Journal of Physiology – Heart and Circulatory Physiology 287:2300-2308, 2004.
D’Arcangelo D, Facchiano F, Barlucchi L, Melillo G, Illi B, Testolin L, Gaetano C, and Capogrossi M. Acidosis inhibits endothelial cell apoptosis and function and induces basic fibroblast growth factor and vascular endothelial growth factor expression. Circulation Research 86:312-318, 2000.
Duling BR. Changes in microvascular diameter and oxygen tension induced by carbon dioxide. Circulation Research 32:370-376, 1973.
Egginton S. Invited review: Activity-induced angiogenesis. Pflugers Archive - European Journal of Physiology 457:963-977, 2009.
Fathi AR, Yang C, Bakhtian KD, Qi M, Lonser RR, and Pluta RM. Carbon dioxide influence on nitric oxide production in endothelial cells and astrocytes: Cellular mechanisms. Brain Research 1386:50-57, 2011.
Haas TL and Nwadozi E. Regulation of skeletal muscle capillary growth in exercise and disease. Applied Physiology, Nutrition, and Metabolism 40:1221-1232, 2015.
Hoier B and Hellsten Y. Exercise‐induced capillary growth in human skeletal muscle and the dynamics of VEGF. Microcirculation 21:301-314, 2014.
Hudlická O, Brown MD, and Silgram H. Inhibition of capillary growth in chronically stimulated rat muscles by N(G)-nitro-l-arginine, nitric oxide synthase inhibitor. Microvascular Research 59:45, 2000.
Hudlicka O, Wright AJ, and Ziada AM. Angiogenesis in the heart and skeletal muscle. Canadian Journal of Physiology 2:120-123, 1984.
Inoue M, Sakai Y, Oe K, Ueha T, Koga T, Nishimoto H, Akahane S, Harada R, Lee SY, Niikura T, and Kuroda R. Transcutaneous carbon dioxide application inhibits muscle atrophy after fracture in rats. Journal of Orthopaedic Science 25(2):338-343, 2019.
Irie H, Tatsumi T, Takamiya M, Zen K, Takahashi T, Azuma A, Tateishi K, Nomura T, Hayashi H, Nakajima N, Okigaki M, and Matsubara H. Carbon dioxide-rich water bathing enhances collateral blood flow in ischemic hindlimb via mobilization of endothelial progenitor cells and activation of NO-cGMP system. Circulation 111:1523-1529, 2005.
Koga T, Niikura T, Lee SY, Okumachi E, Ueha T, Iwakura T, Sakai Y, Miwa M, Kuroda R, and Kurosaka M. Topical cutaneous CO2 application by means of a novel hydrogel accelerates fracture repair in rats. Journal of Bone and Joint Surgery (American volume) 96:2077-2084, 2014.
Levine D. Scientific Presentation Abstracts: 2019 ACVS Surgery Summit October 16-19, Las Vegas, Nevada. Veterinary Surgery, E1-E69, 2019.
Matsumoto T, Tanaka M, Ikeji T, Maeshige N, Sakai Y, Akisue T, Kondo H, Ishihara A, and Fujino H. Application of transcutaneous carbon dioxide improves capillary regression of skeletal muscle in hyperglycemia. Journal of Physiological Sciences, 69:317-326, 2019.
Matsumoto T, Tanaka M, Nakanish R, Takuwa M, Hirabayashi T, Ono K, Ikeji T, Maeshige N, Sakai Y, Akisue T, Kondo H, Ishihara A, and Fujino H. Transcutaneous carbon dioxide attenuates impaired oxidative capacity in skeletal muscle in hyperglycemia model. General Physiology and Biophysics 38:237-244, 2019.
Milkiewicz M, Hudlicka O, Brown MD, and Silgram H. Nitric oxide, VEGF, and VEGFR-2: Interactions in activity-induced angiogenesis in rat skeletal muscle. American Journal of Physiology – Heart and Circulatory Physiology 289:336-343, 2005.
Nishimoto H, Inui A, Ueha T, Inoue M, Akahane S, Harada R, Mifune Y, Kokubu T, Nishida K, Kuroda R and Sakai Y. Transcutaneous carbon dioxide application with hydrogel prevents muscle atrophy in a rat sciatic nerve crush model. Journal of Orthopaedic Research 36:1653-1658, 2018.
Oda T, Iwakura T, Fukui T, Oe K, Mifune Y, Hayashi S, Matsumoto T, Matsushita T, Kawamoto T, Sakai Y, Akisue T, Kuroda R, and Niikura T. Effects of the duration of transcutaneous CO2 application on the facilitatory effect in rat fracture repair. Journal of Orthopaedic Science 2020.
Oe K, Ueha T, Sakai Y, Nikura T, Lee SY, Koh A, Hasegawa T, Tanaka M, Miwa M, and Kurosaka M. The effect of transcutaneous application of carbon dioxide (CO2) on skeletal muscle. Biochemical and Biophysical Research Communications 407:148-152, 2011.
Saito I, Hasegawa T, Ueha T, Takeda D, Iwata E, Arimoto S, Sakakibara A, Sakakibara S, Akashi M, Sakai Y, Terashi H, and Komori T. Effect of local application of transcutaneous carbon dioxide on survival of random-pattern skin flaps. Journal of Plastic, Reconstructive & Aesthetic Surgery 71:1644-1651, 2018.
Sakai Y, Miwa M, Oe K, Ueha T, Koh A, Niikura T, Iwakura T, Lee SY, Tanaka M, and Kurosaka M. A novel system for transcutaneous application of carbon dioxide causing an "artificial Bohr effect" in the human body. PLOS ONE 6:e24137, 2011.
Tomanek RJ and Torry RJ. Growth of the coronary vascu lature in hypertrophy: Mechanisms and model dependence. Cellular & Molecular Biology Research 40:129, 1994.
Xu L, Fukumura D, and Jain RK. Acidic extracellular pH induces vascular endothelial growth factor (VEGF) in human glioblastoma cells via ERK1/2 MAPK signaling pathway: Mechanism of low pH-induced VEGF. Journal of Biological Chemistry 277:11368-11374, 2002.
Yamada M, Ishikawa T, Yamanaka A, Fujimori A, and Goto K. Local neurogenic regulation of rat hindlimb circulation: CO2‐induced release of calcitonin gene‐related peptide from sensory nerves. British Journal of Pharmacology 122:710-714, 1997.