ArticlesTherapeutic angiogenesis for patients with limb ischaemia by autologous transplantation of bone-marrow cells: a pilot study and a randomised controlled trial*
Introduction
Results of preclinical studies have shown that angiogenic growth factors promote development of collateral arteries, which is called therapeutic angiogenesis.1 Angiogenesis can be achieved either by use of growth-factors or genes encoding these proteins. Limited clinical data from proteindelivery and gene-delivery trials suggests that both approaches are safe. However, a great deal more clinical experience is needed to resolve safety concerns such as potentiation of pathological angiogenesis (eg, malignancy) and so-called bystander effects of delivered factors (eg, effects on kidney or atheroma).2 In view of the enclosure of formed mature vessels with periendothelial matrix and pericyte, smooth-muscle cells, or both, treatment with various angiogenic growth factors might be preferable in future treatments.3
Endothelial progenitor cells in the CD34+ stem-cell fraction of adult human peripheral blood take part in postnatal neovascularisation after mobilisation from bone marrow.4, 5 Although CD34− cells enhance CD34+ cell-mediated angiogenesis,4 the underlying mechanism remains undefined. We,6 and Kalka and colleagues,7 noted that mononuclear cells from adult human peripheral or cord blood improved capillary density in hindlimb ischaemia. Marrow stromal cells have many of the characteristics of stem cells for mesenchymal tissues, and also secrete many angiogenic cytokines,8, 9, 10 raising the possibility that marrow implantation into ischaemic limbs could enhance angiogenesis by supplying endothelial progenitor cells and angiogenic cytokines or factors.
Consistent with this hypothesis, we have shown in animals that bone marrow-mononuclear cell implantation into ischaemic limbs11 or myocardium12 promotes collateral vessel formation, with incorporation of endothelial progenitor cells into new capillaries, and that local concentrations of angiogenic factors (basic fibroblast growth factor [bFGF], vascular endothelial growth factor [VEGF], and angiopoietin-1) or angiogenic cytokines (interleukin 1β and tumour necrosis factor α) were increased in implanted tissues. Neither tissue injury by inflammatory cytokines released from injected cells nor differentiation into other lineage cells, such as osteoblasts or fibroblasts, was noted in implanted ischaemic tissues.
On the basis of these results in animals, we started a clinical trial to test cell therapy with autologous bone marrow-mononuclear cells in patients with ischaemic limbs.
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Patients
Patients qualified for marrow implantation if they had chronic limb ischaemia, including rest pain, non-healing ischaemic ulcers, or both, and were not candidates for nonsurgical or surgical revascularisation.13 Requisite haemodynamic deficits included resting ankle-brachial pressure index (ABI) less than 0·6 in the affected limb on two consecutive examinations done at least 1 week apart. We excluded patients with poorly controlled diabetes mellitus (HbA1c >6·5% and proliferative retinopathy)
Results
Fluorescence activated cell sorting analyses in all patients showed that 18% (SD 3·8) of CD34+ cells had the characteristic function of endothelial lineage cells (figure 2).
Since we used bone marrow-mononuclear cells containing CD34+ and CD34 cells, we postulated that these cell fractions might release angiogenic factors to enhance angiogenesis in addition to supply of endothelial progenitor cells. CD34 cells expressed mRNAs of bFGF much more than VEGF, which was expressed more than
Discussion
We have shown here that bone-marrow implantation effectively increased blood flow in all 45 legs, as assessed by substantial increases in ABI, TcO2, and pain-free walking time or by formation of new collateral vessels formation on angiogram. Implantation of bone marrow-mononuclear cells strikingly improved rest pain in most patients (complete regression in half), and ischaemic ulcers or gangrene were improved in just under half of all limbs, showing successful limb salvage in these legs.
Chronic
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