Coronary artery bypass is the favored treatment for heart disease and ischemic stroke. It's not the only bypass operation in medical practice. At the far end of the body, remote from the heart, lie the lower limbs - thigh, calf, feet and toes. They also require arterial bypass surgery for atherosclerosis of the extremities.

"Such surgical treatment is not very effective in patients with ischemia of the lower limbs," observed cardiologist Hiroaki Matsubara, at Kansai Medical University in Osaka, Japan. He attributes the etiology of leg ischemia "mainly to diabetes mellitus, with hypercholesterolemia, hyperlipidemia and atherosclerosis." He pointed out that "leg ischemia affects 1 percent of the population in Japan; three percent in the U.S. - and that's too many."

Matsubara said that lower limb ischemia is caused by narrowing of the arteries, which starves the legs of oxygen and nutrients. The condition can lead to unhealing ulcers, gangrene, amputation and sometimes death. Its cardinal symptom is severe leg pain, while resting or walking.

An associate professor of medicine at Kansai University, Matsubara is co-lead author of a paper in The Lancet, dated Aug. 10, 2002. Its title: "Therapeutic angiogenesis for patients with limb ischemia by autologous transplantation of bone-marrow cells: a pilot study and a randomized controlled trial."

"Implantation of bone marrow-mononuclear cells," he told BioWorld Today, "could be a safe and effective strategy for achievement of therapeutic angiogenesis because of the natural ability of marrow cells to supply endothelial progenitor cells and then secrete various angiogenic factors. Current surgical or medical treatments," Matsubara observed, "have no effect on the ischemic conditions. Right now, vascular endothelial growth factor [VEGF] therapy is reported to be effective in patients, but our treatment is more efficacious for removal of pain, in terms of the angiogenic functions."

Matsubara recalled that, "in preclinical studies, implantation of bone marrow mononuclear cells, including endothelial progenitor cells, into ischemic limbs increased collateral blood vessel formation by angiogenesis." Advancing from animal to clinical trials, he and his co-authors began with a pilot study in which 25 patients suffering from unilateral leg ischemia received bone marrow mononuclear cells injected into the calf muscle of the constricted limb.

Favorable Outcomes In All 45 Leg Patients

"We then recruited 22 patients with bilateral leg ischemia," Matsubara recounted. "These we randomly injected with our bone-marrow fraction, and administered peripheral blood mononuclear cells to the other limb as a control. Patients who received the peripheral cells experienced inhibition of ischemic leg pain," Matsubara narrated, "but this treatment did not elevate the transcutaneous oxygen pressure. However, the bone marrow cells not only inhibited leg pain, but also raised that blood pressure in the lower limb. Of 45 patients enrolled, 38 were men, seven women. Ten had unhealing ulcers;18 gangrene. Most were diagnosed with hypertension, hyperlipidemia and diabetes.

"Four weeks after the procedure," Matsubara went on, "37 of 45 patients saw their pain reduced or relieved completely. Fifteen of 20 were able to avoid scheduled amputation of a toe, and leg ulcers improved in six of 10 patients. Results of angiography showed a striking increase in number of visible collateral vessels. Positive benefits of the bone marrow cell transplantation were still evident six months after the intervention.

"The mononuclear portion of our bone marrow cells," he went on, "included progenitor stem cells but not mature neutrophils." He explained, "Neutrophils produce inflammation, which is not good for angiogenesis. The mononuclear cells contained both cytokines and endothelial [blood vessel] progenitors. The immature mononuclears harbor progenitors - stem cells. But the mature ones contain the cells that release angiogenic factors - various VEGFs and cytokines.

"Peripheral blood," Matsubara added, "did not elevate blood pressure and inhibited less pain. The statistical difference between marrow cells and peripherals was significant - three times higher in bone marrow - which is a good therapeutic result."

The yardstick for such results is a physical test called ABI - ankle-brachial index.

"In humans," Matsubara explained, "ABI compares oxygen blood pressure in the legs with the arms. Legs are 20 percent higher, so ABI measures the arm/leg ratio. In patients treated with bone marrow cells, ABI increased around 0.1, but only 0.01 in the peripheral control group. That's the difference.

"Our angiogenesis treatment - restoring blood flow to blocked arteries - involved two mechanisms," Matsubara recounted. "One involved bone marrow cells containing angiogenic factors such as the VEGFs. The CD34 cytokine in the marrow is 3 percent," he noted. "In the second mechanism, the marrow treatment supplied progenitor cells. Since we used bone marrow-mononuclear cells containing CD34-plus and CD34-minus cytokines, we postulated that these cell fractions might release factors to enhance angiogenesis in addition to supplying endothelial progenitor cells. CD34 cells expressed messenger RNAs of fibroblast growth factor much more than VEG, which was expressed more than angiopoietin."

Two-Faced Therapeutic Angiogenesis?

Ongoing in Matsubara's immediate clinical agenda "is moving from leg ischemia to heart disease - coronary artery disease. I have already treated one cardiac patient with this bone marrow therapy six months ago. He had attacks of chest pain - angina pectoris - 15 times a day. Right now this patient's chest pain is gone, and he is very well. No more angina."

This transition required open-heart surgery to inject the bone marrow-mononuclear cell therapeutic. "Our target was so-called ischemic areas of hibernation - sleeping muscle patches of ischemia. Using complicated procedures, we found these areas. First," he related, "we opened the patient's chest under general anesthesia in the OR. Then we located the target areas by means of scintigraphy, echocardiography and other sophisticated scanning machinery. Then we injected the cells directly into the open heart."

Matsubara continued: "Right now I am watching this first patient for any signs of side effects. If, after completion of one year, he shows none, next year we are going to go into many patients with angina. Initially, we are listing three subjects," he concluded, "and planning for 100 patients."

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