Carbon dioxide-rich water bathing enhances collateral blood flow in ischemic hindlimb via mobilization of endothelial progenitor cells and activation of NO-cGMP system

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 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. 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.