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

Abstract

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. 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 degrees 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. 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.