through summarize the clinical trials of cell therapy for PAD. Many clinical trials have investigated the safety and efficacy of EPCs in the treatment of PAD. These studies have ranged from case reports to small, randomized, placebo-controlled trials, usually involving Rutherford 4 to 6 (CLI) patients with atherosclerotic or vasculitic disease. Studies that include patients with less severe ischemia (Rutherford class 1–3) run the risk of overestimating stem cell efficacy as applied to its target population of no-option PAD. However, scientific insights gained from such trials remain valuable.
Clinical Trials of Unselected Autologous BM-MNCs Administered Intramuscularly and Intra-Arterially
Clinical Trials of Autologous G-CSF–Mobilized Peripheral Blood-CD34+ Cells Administered Intramuscularly
The majority of trials to date have used either BM-derived or peripheral MNCs, although other cell types have also been tested.108
BM harvests are usually processed by density gradient centrifugation (Ficoll method) or plasmapheresis, both of which require access to certified good clinical practice facilities.109
Newer bedside centrifugation systems have been developed to circumvent this resource-and labor-intensive process.110
Peripheral EPCs are usually mobilized using several doses of subcutaneous G-CSF, and harvests are similarly processed via plasmapheresis to enrich target cell content.
EPCs isolated by these methods can be administered either whole (unselected) or after further selection for cell-surface antigens. Usual routes of delivery are direct intramuscular and intra-arterial injection.
The first major clinical trial of stem cell therapy in peripheral vascular disease was the Therapeutic Angiogenesis using Cell Transplantation study, demonstrating safety and providing evidence for bioactivity of unselected BM-MNCs injected intramuscularly into the ischemic limbs of patients with CLI. BM-MNC therapy resulted in improved rest pain, ABI, transcutaneous oxygen pressure, and pain-free walking distance at 24-week follow-up compared with placebo (saline injection). BM-MNC therapy similarly outperformed intramuscular injection of nonmobilized peripheral MNCs, suggesting EPC number or concentration in the latter may be insufficient for therapeutic effect.111
Improvements in leg pain, walking distance, and ulcer size were maintained at 2-year follow-up.112
Intramuscular BM-MNC implantation has been shown to increase acetylcholine-mediated endothelium-dependent blood flow for up to 4 weeks, suggesting that endothelial dysfunction may be reversible by stem cell therapy in patients with severe atherosclerotic CLI.113
Pathological analysis of amputated tissue suggested functional neocapillarization distal to the site of intramuscular BM-MNC treatment that was not seen in age- and sex-matched controls who required amputation.114
Subsequent trials of unselected BM-MNCs delivered intramuscularly in patients with CLI vasculitis mirrored improvements in rest pain and ulcer size at 24 weeks and in ABI and ulcer size at 2-year follow-up.115,116
Head-to-head comparisons of different cell preparations are scarce and have yielded inconclusive results. One randomized, controlled trial of 150 patients with CLI receiving intramuscular injections of G-CSF–mobilized peripheral MNCs versus BM-MNCs reported greater improvements in ABI and rest pain with peripheral MNCs but no difference in pain-free walking distance, ulcer healing, or amputation rates between the 2 groups.117
A small open-label study in patients with CLI vasculitis reported notable improvement in rest pain with both therapies at 1-month follow-up and greater improvement in blood flow by intra-arterial digital subtraction angiography with BM-MNC treatment in 1 patient.118
Further clinical trials are needed to identify optimal cell source and processing.
Additionally, the benefit of intramuscular versus intra-arterial cell delivery remains unclear. It has been hypothesized that intramuscular delivery results in a transient cell depot within ischemic tissue, allowing local paracrine activity and some degree of cell incorporation into the neovasculature. In contrast, intra-arterial therapy is thought to direct stem cells to viable peri-ischemic zones, with enough oxygen and nutrient content to support cell functions. One clinical trial of unselected BM-MNCs delivered intra-arterially demonstrated a similar degree of improvement in ABI compared with previous trials of intramuscular BM-MNC administration (an ABI increase of ≈0.1 points), as well as 2-fold improvement in capillary density and 2- to 10-fold improvement in pain-free walking distance.119
An intra-arterial study in patients with diabetes mellitus suggested much greater ABI improvements, nearing 0.4 points at 1-year follow-up, as well as marked improvement in wound healing and blood flow by intra-arterial digital subtraction angiography despite the use of lower-dose cell therapy (106
rather than 109
The only multicenter, randomized trial of intra-arterial BM–MNC therapy in patients with CLI to date (the Intraarterial Progenitor Cell Transplantation of Bone Marrow Mononuclear Cells for Induction of Neovascularization in Patients With Peripheral Arterial Occlusive Disease study) showed dose-dependent improvement in wound healing and significant reductions in rest pain compared with placebo, despite a lack of improvement in ABI or limb salvage rates.121
This study used cell concentrates on the order of only 106
cells and demonstrated that the use of multiple cell treatments was associated with greater clinical gains. Finally, on the basis of available data, concurrent intramuscular and intra-arterial administration of unselected BM-MNCs seems to result in a magnitude of benefit similar to that of either therapy alone.122–125
G-CSF–mobilized peripheral MNCs have been also been investigated in PAD. Both intramuscular injection and intra-arterial injection of such unselected mobilized peripheral cells have been shown to result in a >0.1-point improvement in ABI and 2-fold increase in maximum walking distance in small clinical series.126–129
Although an earlier trial showed improvement in soft end points with the use of G-CSF–mobilized selected CD34+
cells administered intramuscularly in CLI, cell yield was lower than expected in this study (as low as 105
Our recent data suggest a dose-dependent improvement in freedom from amputation with G-CSF–mobilized selected CD34 cells administered via intramuscular injection (autologous cell therapy-34 CLI trial).131
In this 28-patient double-blind study, patients were randomized to receive either 105
(n=7) or 106
(n=9) autologous CD34+
cells per 1 kg body weight via intramuscular injection or placebo, consisting of the cell diluent alone (n=12). In the combined cell–treatment groups, the incidence of any amputation trended strongly in favor of cell-treated subjects (P
=0.054); however, a larger sample size is clearly needed in future studies.
One unique approach to G-CSF–mobilized therapy involved the creation of tibial fenestrations to allow unselected BM cells to directly mobilize into ischemic lower extremities. Although administration of G-CSF in this study increased peripheral EPC concentration, efficacy of this method requires further investigation.108
In 1 trial, G-CSF administration following intramuscular BM-MNC transplantation did not result in added clinical benefit despite likely peripheral mobilization.77
Differences have been noted in efficacy of stem cell therapy based on the pathogenesis of PAD. Two-year survival rates and 1-year amputation rates are notably worse in atherosclerotic than vasculitic PAD.132
PROVASA investigators and others have noted lesser overall therapeutic benefit in patients with advanced atherosclerotic CLI compared with patients with vasculitic CLI.121,133
Moreover, these benefits are less likely to be sustained over the long term in patients with atherosclerotic PAD compared with patients with vasculitic PAD.134
Such observations call into question the health of autologous stem cells in the setting of advanced age and chronic cardiovascular disease.
Indeed, traditional cardiovascular risk factors associated with peripheral vascular disease have been associated with decreased circulating progenitor cell number and function. A number of studies report dysfunction of endogenous EPCs in the setting of hypertension, dyslipidemia, smoking, and diabetes mellitus.119,135–141
For example, EPC recruitment in response to tissue hypoxia may be impaired in the setting of diabetes mellitus; even the allogenic injection of healthy EPCs into diabetic mice improved, but did not normalize, ischemic tissue survival to the level seen in nondiabetic mice.142
Additionally, patients with diabetes mellitus can exhibit several-fold-greater intimal plaque neovascularization by histology and microscopy, a process that has previously been associated with greater plaque instability and cardiovascular events.143
Despite these challenges, therapeutic benefit can be derived using autologous strategies.144,145
Reversal of risk factors and modulation of cellular microenvironments, for example, through smoking cessation or use of hyperoxygenation, can overcome intrinsic deficiencies of autologous EPCs.146–148
In 1 study, healthy EPCs injected into diabetic mice resulted in improved allograft function and donor cell recruitment only when transplanted in combination with therapies to promote synergism with local trophic pathways.149
Attention must be given, therefore, to the application of EPCs in advanced cardiovascular states, with special effort to optimize the metabolic and mechanical context of cell therapy.
Finally, a challenge for the field of cell-based therapies and for all therapies that target the microcirculation is the limitation of available surrogate end points for the prediction of major clinical outcomes. Although the traditional end points such as ABI, toe-brachial index, or transcutaneous oxygen-saturation are reliable indicators of prognosis in population studies, they have failed to consistently provide useful metrics for the development of microvascular therapies. Future studies will need to develop new algorithms using existing technologies or identify novel methods for reliably quantifying tissue perfusion that will permit the design of early-phase studies to evaluate dose in modest-sized study populations.
Context of Cell Therapy
Oxidative stress, hormonal milieu, ischemic conditioning, and shear stress have all been shown to affect stem cell efficacy. Adjuvant therapies to gainfully modulate these conditions in vivo are being investigated. For example, although low levels of reactive oxygen species act constructively as signaling molecules, higher levels can lead to increased stem cell senescence.150,151
This identifies a potential target for optimizing EPC function. Preclinical studies in healthy, diabetic, and dyslipidemic mice showed that intravenous and oral antioxidant therapy synergistically improved neovascularization when given in combination with BM-MNC therapy (but not as a stand-alone therapy).141,152,153
A clinical case-control trial of oral L-arginine and antioxidants after intra-arterial BM–MNC transplantation in patients with CLI resulted in significantly improved amputation rates at 1-year follow-up, but this was in comparison with standard medical therapy alone.119
Incremental benefit of antioxidant therapy over stem cell transplant in patients has yet to be quantified.
Similarly, estradiol has been postulated to exert positive effects on endothelial remodeling after injury. Accelerated re-endothelialization and decreased apoptosis/neointimal formation were demonstrated in preclinical models of ovariectomized mice.154,155
Although these mechanisms may also contribute to the protective cardiovascular effects of estrogen observed in premenopausal women, the larger clinical context of hormone therapy must be taken into account before the study or application of this preclinical signal in humans.
Multiscale computational models have predicted that exercise may improve vasculogenesis by increasing angiogenic factor concentrations and gradients toward ischemic tissues.156
Preclinical models of ischemic mice randomized to exercise exhibited greater histological neovascularization and suffered less neointimal hyperplasia than those that were sedentary. This effect was attenuated in endothelial nitric-oxide–deficient states. Patients completing a 4-week standardized exercise program had an increase in circulating EPC numbers and a decrease in EPC apoptosis compared with before exercise.157
The importance of ischemic conditioning through exercise was elegantly demonstrated in a series of 3 prospective, randomized, clinical trials. Nonrevascularized patients with PAD who achieved an increase in serum lactate (a marker of anaerobic metabolism) benefited from >300% increased VEGF concentration and >400% increased EPC mobilization compared with control patients who did not exercise to ischemia.158
Shear stress may have a role in improved neovascularization and may mediate some of the benefits seen with exercise. Vessels exposed to high-shear stress after surgical induction of ischemia in animal models display accelerated collateralization (arteriogenesis).159
The effect of in vivo flow conditions on EPCs has been studied in vitro and is shown to influence EPC alignment and adhesion molecule expression consistent with the endothelial phenotype.160,161
Currently, there is no reliable measure of blood flow at the tissue level in ischemic limbs. This is an impediment to therapeutic development because outcome measures such as limb amputation present major challenges for early-phase trial design. A reliable surrogate end point, capable of assessing improvements in blood flow and predicting physiological and clinical improvement, will greatly enhance the development of novel therapies for PAD.
As can be seen in the trials described above, use of stem cell therapy has vast potential for improved clinical outcomes in PAD. Identifying appropriate end points is vital to the progression of therapeutic neovascularization. To this end, many imaging modalities have been developed in addition to existing pathological analyses and hard end points such as mortality, freedom from amputation, and hemodynamic measures of perfusion. These technologies can be used to aid in tracking EPCs in vivo and in understanding correlations of angiographic outcomes with meaningful clinical end points. The following 5 main imaging modalities exist: ultrasound, computed tomography, magnetic resonance, perfusion scintigraphy, and angiography. Test operating characteristics of each these modalities in CLI are being worked out.162–168
Challenges and Future Directions
In summary, the field of progenitor cell therapy for therapeutic neovascularization in PAD is gaining momentum. Scientific inquiry has yielded many promising tools, and a robust movement toward clinical study has uncovered several questions that remain to be fully answered. Effective cell populations, isolation, and processing methods must continue to be refined to gain a deeper understanding of the feature that defines potency. Optimal delivery method, timing, and dosing regimens will be tailored to the disease state and clinical trajectory. Adjunctive therapies to overcome endogenous impairments in EPC health and vascular responsiveness must also be developed, along with molecular and bioengineering tools to advance therapeutic effects of stem cells in time and space. One area that remains to be explored is the possible synergy of macrovascular revascularization with efforts to restore the microcirculation. Finally, trials incorporating valid surrogate measures of success that predict hard end points must be designed to evaluate cell safety, efficacy, and long-term outcomes. With this broad framework in mind, we are confident that basic, translational, and clinical study of therapeutic neovascularization will move steadily toward safe, improved outcomes in PAD by altering the natural history of this progressive disease via vascular repair and regeneration.