Repeated phlebotomy augments angiogenesis to improve blood flow in murine ischemic legs

Abstract

Anemia may accelerate angiogenesis in ischemic organs through its ability to augment tissue hypoxia-induced generation of several known angiogenic factors and to increase erythropoietin levels, which are also potently angiogenic. We examined the effect of controlled phlebotomy (bloodletting) on blood flow in a mouse ischemic leg model. We ligated the right femoral artery of BALB/c mice. In the phlebotomy group, 200 microl of blood were drawn from the tail vein once a week. After 4 wk, blood flow in the ischemic leg was significantly better in the phlebotomy group (flow ratio of the ischemic to nonischemic leg, 0.87 + or - 0.04) than the control group (0.59 + or - 0.05, P < 0.05), and capillary density was significantly higher. Repeated phlebotomy increased serum erythropoietin levels as well as the expression of hypoxia-inducible transcription factor-1alpha and vascular endothelial growth factor and both the expression and activity of Akt and endothelial nitric oxide synthase (eNOS) in ischemic legs. Treatment with wortmannin or N(omega)-nitro-l-arginine methyl ester significantly attenuated the phlebotomy-induced improvement of blood flow. In addition, fluorescence-activated cell sorting analysis revealed an increase in circulating peripheral endothelial progenitor cells in the phlebotomy group, and treatment with AMD3100, a specific inhibitor of the chemokine receptor CXCR4, blocked the beneficial effect of phlebotomy. These findings suggest that repeated phlebotomy improves blood flow in ischemic legs through an angiogenic action that involves the Akt/eNOS pathway, endothelial progenitor cell mobilization, and their complicated cross talk. An adequately controlled phlebotomy might be one method by which to induce therapeutic angiogenesis.

LOX-1 Plays an Important Role in Ischemia-Induced Angiogenesis of Limbs

“…Under sufficient anesthesia with sodium pentobarbital (50 mg/kg, intraperitoneally), local fur was removed with depilatory cream. A small incision was made on the right leg to expose the femoral vasculature and dual ligation of the femoral artery was performed at a point just below the inguinal ligament, as previously described [4] .…”

“…We measured the ischemic hindlimb (right)/nonischemic hindlimb (left) blood flow ratio by laser Doppler blood flowmetry (LDBF; Moor LDI, Moor Instruments, Wilmington, DE), which provides a non-invasive measurement of blood flow. Data acquisition was performed by a previously described method [4] . Briefly, the sedated mice were secured on a monochromatic surface and an area of 11×11 cm was scanned from the lower abdomen to the end of the toes.…”

Necrostatin-1 protects C2C12 myotubes from CoCl2-induced hypoxia

“…However, hypoxia is common in the tissues of the majority of individuals. Hypoxia-induced muscle wasting is a condition frequently reported in several environmental and pathological conditions, including exposure to high altitudes, prolonged immobilization, chronic obstructive pulmonary disease, exercise, and anemia ( 1 – 4 ). However, the mechanism underlying the effects of hypoxia in skeletal muscle remains to be elucidated.…”

Effects of Cobalt Chloride, a Hypoxia-Mimetic Agent, on Autophagy and Atrophy in Skeletal C2C12 Myotubes

“…However, hypoxia is common in the tissues of most individuals. Hypoxia-induced muscle wasting is a phenomenon frequently reported in several environmental and pathological conditions, such as exposure to high altitudes, prolonged immobilization, chronic obstructive pulmonary disease, exercise, and anemia [1–4]. However, the mechanism underlying the effects of hypoxia in skeletal muscle is still unknown.…”