This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Irie, H. Articles by Matsubara, H. Search for Related Content PubMed PubMed Citation Articles by Irie, H. Articles by Matsubara, H. Related Collections Angiogenesis Endothelium/vascular type/nitric oxide

(Circulation. 2005;111:1523-1529.)
© 2005 American Heart Association, Inc.

Vascular Medicine

Carbon Dioxide–Rich Water Bathing Enhances Collateral Blood Flow in Ischemic Hindlimb via Mobilization of Endothelial Progenitor Cells and Activation of NO-cGMP System

Hidekazu Irie, MD; Tetsuya Tatsumi, MD, PhD; Mitsutaka Takamiya, MD; Kan Zen, MD; Tomosaburo Takahashi, MD, PhD; Akihiro Azuma, MD, PhD; Kento Tateishi, MD; Tetsuya Nomura, MD; Hironori Hayashi, MD; Norio Nakajima, MD; Mitsuhiko Okigaki, MD, PhD; Hiroaki Matsubara, MD, PhD

From the Department of Cardiovascular Medicine, Kyoto Prefectural University School of Medicine, Kyoto, Japan.

Correspondence to Hiroaki Matsubara, MD, Department of Cardiovascular Medicine, Kyoto Prefectural University of Medicine, Kamigyo-Ku, Kyoto, 602-8566, Japan. E-mail matsubah{at}koto.kpu-m.ac.jp

Received June 1, 2004; revision received November 13, 2004; accepted November 19, 2004.

Background— Carbon dioxide–rich water bathing has the effect of vasodilatation, whereas it remains undetermined whether this therapy exerts an angiogenic action associated with new vessel formation.

Methods and Results— Unilateral hindlimb ischemia was induced by resecting the femoral arteries of C57BL/J mice. Lower limbs were immersed in CO₂-enriched water (CO₂ concentration, 1000 to 1200 mg/L) or freshwater (control) at 37°C for 10 minutes once a day. Laser Doppler imaging revealed increased blood perfusion in ischemic limbs of CO₂ bathing (38% increase at day 28, P<0.001), whereas N^G-nitro-L-arginine methyl ester treatment abolished this effect. Angiography or immunohistochemistry revealed that collateral vessel formation and capillary densities were increased (4.1-fold and 3.7-fold, P<0.001, respectively). Plasma vascular endothelial growth factor (VEGF) levels were elevated at day 14 (18%, P<0.05). VEGF mRNA levels, phosphorylation of NO synthase, and cGMP accumulation in the CO₂-bathed hindlimb muscles were increased (2.7-fold, 2.4-fold, and 3.4-fold, respectively) but not in forelimb muscles. The number of circulating Lin–/Flk-1+/CD34– endothelial-lineage progenitor cells was markedly increased by CO₂ bathing (24-fold at day 14, P<0.001). The Lin–/Flk-1+/CD34– cells express other endothelial antigens (endoglin and VE-cadherin) and incorporated acetylated LDL.

Conclusions— Our present study demonstrates that CO₂ bathing of ischemic hindlimb causes the induction of local VEGF synthesis, resulting in an NO-dependent neocapillary formation associated with mobilization of endothelial progenitor cells.

Key Words: carbon dioxide • hypercapnia • angiogenesis • stem cells • endothelium • vasculogenesis