Cell therapy for peripheral arterial disease

NOT FOR CIRCULATION Current Opinion in Molecular Therapeutics 2010 12(5):  Thomson Reuters (Scientific) Ltd ISSN 2040-3445

REVIEW

Cell therapy for peripheral arterial disease Philippe Menasché

Address Assistance Publique-Hôpitaux de Paris, Hôpital Européen Georges Pompidou, Department of Cardiovascular Surgery; Université Paris Descartes; INSERM U 633, Paris, France Email: philippe.menasche@egp.aphp.fr *To whom correspondence should be addressed

Peripheral arterial disease remains an often devastating condition, particularly in patients with diabetes, because of the high rate of functional disability, amputation and death. For those patients for whom conventional endovascular or surgical revascularization procedures have been unsuccessful, new options are eagerly awaited, among which cell therapy has gained increasing interest. Most clinical trials of cell therapy have used multiple intramuscular injections of bone marrow-derived mononuclear cells that have yielded encouraging suggestions of efficacy. The prevailing opinion is that the benefits of cell therapy are not a result of the structural integration of grafted cells within new vessels, but of the paracrine activation of angiogenesis, arteriogenesis and vasculogenesis pathways by the cytokines, chemokines and growth factors released from such cells. An analysis of cell therapy clinical trial outcomes has also identified several key issues that need to be addressed, including the optimal cell type, source and dosing, the most effective route for cell transfer, and methods for enhancing survival of the cellular graft. Finally, because of the strong placebo effect that may confound interpretation of outcome measures, rigorously randomized controlled trials are mandatory in order to assess more thoroughly whether cell therapy will be beneficial for patients with peripheral arterial disease.

Keywords Cell homing, cell therapy, clinical trial, critical limb ischemia, peripheral arterial disease, stem cell

Introduction Peripheral arterial disease is most commonly related to atherosclerosis and affects approximately 8 million individuals in the US [1]. The number of affected individuals is expected to increase in parallel with life expectancy, as the annual incidence of intermittent claudication per 10,000 at-risk individuals increases from 6 in men and 3 in women between the ages of 30 and 44 years to 61 in men and 54 in women between the ages of 65 and 74 years [1]. Among patients with peripheral arterial occlusive disease, it is estimated that approximately 10% will progress toward the stage of chronic limb ischemia (CLI), which is defined by pain at rest or tissue necrosis that manifests as ulceration or gangrene.

The prognosis of patients with CLI remains unfavorable.

Despite improvements in surgical and endovascular procedures [2], these treatments have a limited efficacy when the disease is diffuse and extends below the knee.

Estimates suggest that approximately 40% of patients with CLI are not candidates for revascularization procedures [3]. Such patients often require amputation, and this probability is increased 10-fold in patients with diabetes [3]. Amputation is associated with high mortality (5 to 20%), a poor functional outcome and a low 5-year survival rate [3]. Furthermore, CLI is a marker for systemic atherosclerotic disease and, consequently, patients with peripheral arterial disease have a

3-fold increased risk for all-cause mortality, a 6-fold increased risk for death from cerebrovascular disease complications and an approximately 7-fold increased risk for death from coronary heart disease [1]. Overall, the survival rate of patients with peripheral arterial disease is worse than that for breast cancer or Hodgkin's disease [4].

Among the new treatments designed to improve the outcome for patients with peripheral arterial disease, gene therapy has been the first to enter clinical development. Despite the encouraging results obtained from some of the early phase I clinical trials, more rigorous phase II and phase III trials have failed to establish the efficacy of angiogenic gene therapy conclusively [5]. This failure was attributed initially to the one-shot administration of a single gene (or a single protein) with activity against a single target that, given the complexity of the angiogenic pathways, may be an overly simplistic approach. Unfortunately, this opinion must be revised following the failure of the recent 289-patient, dose-ranging, randomized trial of hypoxiainducible factor-1α This master switch gene controls the downstream expression of multiple transcription factors that should have overcome the limitations of single-gene treatments, but that nevertheless failed to achieve its pre-specified improvement in an exercise treadmill test efficacy endpoint [6]

NOT FOR CIRCULATION

2 Current Opinion in Molecular Therapeutics 2010 Vol 12 No 5

The combination of these observations justify the search for new therapeutic interventions for peripheral arterial disease and, in this setting, cell therapy has emerged as a promising option for influencing the repair of damaged vasculature and subsequently enhancing tissue viability. This review details the objectives of cell therapy in CLI, highlights the most recent clinical results for this treatment and discusses the major issues that need to be addressed for improving the consistently and success of this treatment approach.

Objectives of cell therapy in chronic limb ischemia The landmark studies of Asahara, Isner and colleagues [7,8] pioneered the use of cell therapy in the treatment of limb ischemia. These studies demonstrated that, following ex vivo expansion of a population of cells from the peripheral human blood termed the endothelial progenitor cells (EPCs), transplantation of expanded EPCs into athymic mice with hind limb ischemia increased blood flow and capillary density in the affected limbs. Since these studies, EPCs have been the subject of extensive investigations, although some controversy exists regarding the exact phenotype and origin of this cell type. Currently, there is some agreement that EPCs are heterogeneous, as they comprise a minimally proliferative monocyte-derived population (early-outgrowth EPCs) and a highly proliferative non-myeloid population (late-outgrowth EPCs) [9]. The latter is thought to harbor true endothelial precursors that are defined by the coexpression of hematopoietic stem cell markers, including CD34 and CD133, endothelial VEGFR2, von Willebrand factor and endothelial nitric oxide synthase (eNOS) [9,10]. Circulating levels of EPCs are usually low (in their seminal paper, Kalka et al [8] estimated this number to be 0.05% or 5 x 102 per million PBMCs), but levels can increase in response to pharmacological mobilization or myocardial/peripheral tissue ischemia [10].

Several experimental studies have documented the ability of bone marrow-derived mononuclear cells (MNCs), or of purified fractions of such cells, primarily EPCs or mesenchymal stem cells (MSCs), to relieve ischemia-induced perfusion defects and to increase vessel density in the ischemic tissue [11-13]. These effects were subsequently demonstrated to be conferred by adipose-derived stromal cells [12]. The majority of these studies have used the aforementioned model of hind limb ischemia in rodents, which is convenient for screening therapeutic strategies in large numbers of animals and for yielding proof-of-principle data, but which has several serious limitations including (i) the rapid development of collateral vessels, necessitating the requirement for early assessments to avoid confounding results; (ii) the subsequent difficulty in generating long-term outcome measures because of the development of collateral vessels; and (iii) the lack of risk factors usually present in patients, such as advanced age, diabetes, hypercholesterolemia or hypertension, all of

which can modulate treatment effects [3,13]. Thus, small and preferably large animal models more closely simulating the clinical situation are urgently required.

The mechanisms by which transplanted cells may increase tissue neovascularization should be considered with respect to the three major events that contribute to new vessel development. Thus, while capillary growth (microvessels) occurs through both angiogenesis (ie, sprouting of endothelial cells from pre-existing vasculature) and vasculogenesis (ie, de novo capillary formation), larger arteries (macrovessels) develop through arteriogenesis that is triggered by shear stress subsequent to a stop-flowinitiated pressure gradient [10]. These processes involve the local release of various cytokines, chemokines and growth factors. Therefore, grafted cells might act as 'cytokine factories', and may contribute particularly to collateral artery growth (ie, arteriogenesis), thus boosting endogenous repair processes [13]. The role of EPCs in endogenous vascular repair has been further highlighted by the finding that a reduced number of EPCs in the blood is a predictor of cardiovascular adverse events [14,15], indicating such cells could be used as a potential surrogate biomarker of atherosclerotic disease progression. Conversely, evidence suggests that bone marrow-derived stem cells do not differentiate into endothelial cells [16]. Thus, the initial premise that bone marrow-derived grafted cells would convert into endothelial cells and become physically incorporated into the newly formed vessels, thereby contributing structurally to vessel formation, has shifted considerably toward a paradigm whereby the cells are thought to act in a paracrine manner through the activation of host-associated signaling pathways. These pathways likely involve several outcomes (eg, vessel formation, limitation of apoptosis, extracellular matrix remodeling and recruitment of putative tissue-resident stem cells) that ultimately converge to protect peripheral arteries.

This paracrine hypothesis may account for the frequent observation that a significant discrepancy exists between the protective effects of the transplanted cells and their lack of long-term persistence in the grafted tissue [12].

Clinical data on the use of cell therapy in peripheral arterial disease Several comprehensive reviews of the major clinical trials of cell therapy in patients with peripheral arterial disease, with or without CLI, have recently been published [13,17]. Two cell sources, bone marrow and peripheral blood, after mobilization by G-CSF and apheresis, were investigated in these trials. In most cases, the MNCs used in the trials were unselected. A pooled analysis of the reported data demonstrated a trend toward improvements in both clinical (ie, walking distance and wound healing) and perfusion (ie, ankle-brachial index and transcutaneous oxygen pressure) endpoints. In addition, 2-year results of the seminal Therapeutic Angiogenesis

by Cell Transplantation (TACT) trial, which pioneered the use of bone marrow cells in patients with CLI, suggest a persistent clinical benefit with time [18].

NOT FOR CIRCULATION

Cell therapy for peripheral arterial disease MenaschĂŠ 3

Notably, these studies also established the excellent safety record of this new therapy; the high rate of adverse events reported in a small cohort of patients (n = 8) with thromboangiitis obliterans [19] has remained an isolated finding. Similarly, in contrast to the accelerated atherosclerotic plaque progression observed in apolipoprotein E-knockout mice treated with bone marrow cell transplantation in the setting of hind limb ischemia [20], there has been no similar reports in trials of cell therapy for peripheral vascular diseases.

These results should be interpreted cautiously for several methodological reasons. First, the trials tended to enroll a small number of patients and, consequently, the statistical power of these trials was limited. Second, there was a paucity of controlled studies, including randomization and double-blind evaluations. For example, in the meta-analysis by Fadini et al [17], only 4 trials out of the 37 deemed appropriate for analysis were randomized. The importance of this randomization is illustrated by the observation that, while ankle-brachial index and transcutaneous oxygen pressure were improved significantly when all trials were considered, improvements were downgraded to 'marginal' when the analysis was restricted to the controlled studies. Third, the underlying pathology is heterogeneous, even if atherosclerosis prevails compared with thromboangiitis obliterans. Finally, there was variability in the clinical presentation of patients, number of CD34+ cells, method of cell processing, dosing and endpoints. It is hoped that ongoing trials will clarify most of these issues. A total of 26 trials were listed on the ClinicalTrials.gov website, as of September , 2010, using critical limb ischemia and cell therapy as search terms [21]. These studies are highly heterogeneous with regard to the cell type under investigation (ie, primarily, bone marrow-derived MNCs, PBMCs after cytokine mobilization, bone marrow cell concentrates, allogeneic MSCs, cord blood stem cells, and CD34+ and CD133+ progenitors), the method of cell delivery (ie, subcutaneous, intramuscular, intra-arterial or combinations of such methods) and the clinical presentation of the patients. However, endpoints are more consistent across studies and consist of clinical (ie, death and amputation rates, pain-free walking distance, rest pain, and ulcer healing) and functional (ie, ankle-brachial index and transcutaneous oxygen pressure) parameters. Therefore, despite only 3 out of these 26 trials planning to enroll more than 100 patients and, thus, only 3 trials expected to generate statistically meaningful data, it is hoped that a pooled outcome analysis will yield some useful information. However, from a medical and regulatory standpoint, it is important to note that parameters such as mortality or limb salvage, or even objective anatomic and perfusion data provided by high-resolution imaging and Doppler measurements, are more conclusive than surrogate endpoints focusing on subjective symptomatic feelings. In addition, the strong placebo effect observed in several angiogenic gene therapy trials [22] highlights the critical importance of randomized, placebo-controlled trials for efficacy assessments.

Challenges to the use of cell therapy for peripheral arterial disease Cell phenotype

When selecting the cell type to use for the treatment of peripheral arterial disease, it is unclear whether it is more efficacious to use the bulk of unfractionated bone marrow MNCs or to rely on selected subpopulations.

Because CD34+ and CD133+ cells are endowed with a high angiogenic capacity [23], as demonstrated in a cell therapy clinical trial in patients with refractory myocardial ischemia [24], some investigators have speculated that the efficacy of the cells may be weakened if they are 'diluted' in unpurified MNCs populations, where this cell type represents only a small percentage. The same concern applies to EPCs. In contrast, other investigators [3,25] have suggested that it might be better to use a mixture of cells, with the expectation that crosstalk between the various subpopulations may result in synergistic effects. For example, in the pioneering study

by Tateishi-Yuyama et al [25], the CD34+ and CD34- cell fractions were observed to express angiogenic growth factors and their receptors differentially, highlighting the expectation that a mixture of these cells would optimally promote the development of stable and mature capillary vessels. This hypothesis was consistent with the experimental observations that CD34+-stimulated neovascularization was enhanced greatly by co-culture with CD34- cells [26], and that the delivery of a mixture of early- and late-outgrowth EPCs also had synergistic effects [27], possibly through the release of a more diverse blend of cytokines and growth factors. Similarly, the greater angiogenic capacity of bone marrow cells compared with peripheral blood cells may be the result of a higher number of CD34+ and supportive 'stem' cell populations present in the former cell source [25].

Furthermore, the ex vivo scale-up of select cell populations, such as EPCs, without inducing unwanted phenotypic changes, may be technically challenging, particularly in elderly patients or those with comorbidities that feature a reduced availability and basal dysfunction of their EPCs [15]. In combination, these observations suggest the use of unfractionated MNCs harvested from bone marrow rather than from peripheral blood after cytokine-induced mobilization [2,13].

Cell source Regarding the origin of the cells used in the treatment of peripheral arterial disease, most studies have used autologous cells, which have the advantages of a straightforward procurement and the lack of immunogenicity. However, patient-specific cell products have several disadvantages, the most important of which is interpatient variability in cell functionality, making it challenging to yield consistent, well-defined products; this variability likely accounts for the heterogeneity observed in clinical outcomes. Bone marrow-derived cells harvested from patients of an advanced age [28] or with chronic ischemic heart disease have been reported to have a reduced neovascularization capacity [29], a finding that

NOT FOR CIRCULATION

4 Current Opinion in Molecular Therapeutics 2010 Vol 12 No 5

is consistent with the observation that diabetic rats fail to mobilize EPCs after hind limb ischemia [30]. Moreover, patients who best responded to intracoronary bone marrow cell infusions after myocardial infarction received progenitor cell preparations with a greater CFU capacity than non-responders [31], further highlighting the impact of baseline cell functionality on post-treatment outcomes. Logistical constraints inherent in cell shipping and costs related to quality assessments of personalized batches of cells represent additional limitations to the use of cell therapy, although these challenges could be addressed by the development of point-of-care cell isolation/concentration techniques. Similarly, the defective function of bone marrow cells could be corrected by genetic engineering approaches or the use of small molecules, such as statins or NOS enhancers [32]. However, these procedures are labor-intensive, highlight potential safety issues and may not be pertinent to the treatment of large numbers of patients, particularly if treatment has to be implemented rapidly. In combination, these limitations have generated interest in the use of allogeneic cells, which can be derived from well characterized, virologically secured cell banks, and are available immediately as off-the-shelf products. In this setting, a neural stem line initially developed by ReNeuron Group plc for the treatment of strokes [33] has demonstrated efficacy in animal models of limb ischemia [34]. However, a general issue of allogeneic cell products is the expected induction of an immune response. To overcome this problem, the use of MSCs, which can be retrieved from several sources (ie, primarily the bone marrow and fat tissue, but also from cord blood), offer the dual advantages of a high capacity to secrete trophic factors [35] and immune privilege [36]. Clinical trials are underway to assess MSCs derived from such sources in patients with CLI [21]. Each of the companies responsible for developing MSCs suggeststhat their own proprietary process produces cells with the most suitable properties.

However, it is well known that cells can alter their phenotype during culture to expand the cell population, and it unclear whether these apparently different MSCs have fundamentally different properties that would legitimate profit-targeted IP claims, or whether they simply represent different stages of differentiation of the same cell type that expresses different surface markers at different times during culture. Furthermore, as aging is known to impair MSC function, the use of induced pluripotent cells differentiated into MSCs has been suggested to provide a patient-specific 'rejuvenated' cell product; this approach was demonstrated to be more effective for improving hind limb ischemia than the use of adult MSCs in mice [37]. However, given the complex issues associated with adult somatic cell reprogramming, it is unlikely that such an approach will be used in the clinic in the near future, if at all.

Cell dosing A clear dose-effect relationship for cell therapy is not obvious from published clinical trials [3,13], as similar improvements in outcome have been reported with cell

numbers that differ significantly. By analogy with cell therapy described for the treatment of acute myocardial infarction [38], it is possible that the functionality of the cells, as assessed by their CFU capacity, their migratory properties against a chemotactic gradient and the amount of cytokines that they can release, may be a better predictor of treatment efficacy than the actual number of delivered cells. In turn, cell function must be evaluated when determining which isolation technique is the most appropriate [39]. Interestingly, perspectives on cell processing are provided by the use of automated devices, which allow the fast point-of-care concentration of bone marrow cells; however, these techniques await large-scale clinical validation.

Routes of cell delivery for peripheral arterial disease For the treatment of peripheral arterial disease, cells are usually delivered by multiple intramuscular injections and, more rarely, by intra-arterial infusion, or a combination of both approaches. Regardless of the route of delivery, only low levels of cells are expected to survive the procedure as, at least in the heart, retention rates have been reported to vary from 0.44 to 10% at 4 days post-infusion [13]. Although the effects of cell therapy in peripheral arterial disease are predominantly paracrine and, therefore, the long-term residence of grafted cells in the target tissue is not necessary, a critical pool of cells may be required, at least initially, to maximize the paracrine effects. This assumption is supported by observations of a relationship between engraftment rates and improved functional outcomes in animal models of both heart [40] and limb [41] ischemia. It is therefore important to improve delivery techniques in order to optimize early engraftment rates.

Intramuscular/perivascular delivery The intramuscular delivery of cells, often by injection with a hand-held syringe, may fail to achieve the desired levels of efficiency, accuracy and reproducibility because of the lack of control with this type of procedure. New devices are under development that might overcome these challenges by allowing fast, precisely targeted and controlled cell injections [42]. For example, perivascular injections of matrix-embedded endothelial cells into the space between the perivascular tissue and the vessel wall have been demonstrated to be an effective alternate delivery strategy for fully exploiting the paracrine effects of cells targeted at preventing post-stenting stenosis in a porcine femoral stent model [43].

Intravascular delivery For cells delivered intravascularly, the challenge is to enhance their homing to the target tissue. From a practical and regulatory standpoint, the simplest approach to enhance engraftment is most likely to develop improved delivery catheters, such as the Mercator device (Mercator MedSystems Inc), which features a transendothelial needle to facilitate the perivascular

NOT FOR CIRCULATION

Cell therapy for peripheral arterial disease MenaschĂŠ 5

distribution of cells [44], and/or repeat cell deliveries; this injection device is being evaluated in an ongoing clinical trial (JUVENTAS; ClinicalTrials.gov identifier: NCT00371371). A second strategy to improve cell homing in the target tissue is to use homing signals responsible for cell trafficking to sites of injury. This approach can be achieved by interventions that manipulate the therapeutic cells directly or that modify the recipient tissue.

Cell homing to sites of injury is a complex process whereby the recruitment of cells is orchestrated by a tightly coordinated sequence of events involving 'sensing' of the damaged tissue-emitted signals, selectin-mediated rolling, integrin-mediated firm adhesion to the vascular wall, transendothelial migration and protease-induced degradation of the extracellular matrix to facilitate cell invasion [45]. In the setting of acute ischemia, one of the key receptor is the chemokine CXC receptor (CXCR)4 [46], which is expressed on early hematopoietic stem cells and EPCs, and interacts with its cognate ligand, the stromal cell-derived factor (SDF)-1ι. However, EPCs from patients with coronary artery disease, who frequently also have CLI, demonstrate a decreased responsiveness to SDF-1 because of the dysregulation of CXCR4 signaling [47]. This observation has led to attempts to enhance homing by engineering cells to express one of the components of this receptor-ligand axis. In fact, SDF-1-transfected EPCs improved angiogenesis and vasculogenesis in a nude mouse model of hind limb ischemia; these improvements were associated with the mobilization and partial incorporation of EPCs into neovessels and were mediated through a VEGF/eNOS-related pathway [48]. In addition to these gene-based approaches, drugs such as statins, eNOS enhancers or activators of β2-integrins have also been successful in enhancing cell homing to sites of injury, at least in experimental models [45]. An alternative approach is to modulate the target tissue to increase its capacity to recruit circulating cells. For example, in a rat model of ischemia, intramuscular injection of a collagen matrix containing sialyl Lewis X ligands for L-selectin increased progenitor cell recruitment to the treated limb and, subsequently, improved perfusion [49]. Similarly, in an athymic mouse hind limb ischemia model, the recruitment of transplanted human EPCs in ischemic tissues was augmented with an associated increase in neovascularization when cell transplantation was combined with intramuscular injections of SDF-1 [50].

The rationale for extending this concept to CLI is supported by the finding that chronically ischemic human amputated limbs exhibit SDF-1 downregulation [51].

In parallel, increasing interest has also been directed toward physical interventions, such as low-energy shockwaves, which increased tissue expression of SDF-1 [52], focused ultrasound-mediated destruction of microbubbles, which may enhance transendothelial migration of cells as a result of a series of biochemical responses [53], or magnetic targeting, which has recently been demonstrated to be effective in the homing of iron-loaded EPCs toward the site of vascular [54] or

cardiac [40] injury using an externally applied magnetic device. The lack of head-to-head comparisons between these different approaches precludes a conclusion on which is the most effective approach. However, given the tight constraints of the current regulatory environment, the anticipated hurdles of superimposing gene and cell therapies should be noted, regardless of whether both treatments are combined or whether the cells are genetically modified prior to delivery.

Cell survival following transplantation The high death rate of transplanted cells is another important issue for cell-based therapies. It can be assumed that the longer the grafted cells survive, the more effective their protective effects would be [40,41]. Therefore, enhancing cell survival following transplantation is an important goal to achieve. The development of appropriate strategies first requires an understanding of the causes of cell death. The death of transplanted cells results from an interplay between several factors, including inflammation related to injection-induced tissue injury, ischemia inherent to the hostile environment in which the cells are delivered, apoptosis as a result of the loss of attachments to other cells and to the extracellular matrix and, if allogeneic cells are to be used, eventually an immune response.

To counteract these processes, various strategies have been investigated, in particular the engineering of cells with angiogenic genes [55], the local delivery of growth factors [41] and the incorporation of cells into injectable biomaterials. The latter approach is appealing because the restoration of a 3D environment may facilitate the survival, growth and differentiation of transplanted cells.

In this regard, encouraging data obtained in porcine models demonstrated that embedding allogeneic aortic endothelial cells within collagen-based matrices reduced intimal hyperplasia associated with arteriovenous fistulae [56] and reduced stenosis after stent-induced injury [43].

These effects were attributed to the paracrine effects of the cells, which were transiently protected from an immune response through their spatial interactions with the matrix microarchitecture prior to collagen degradation, which leads to the destruction of the cells by the immune system [57]. This type of approach can be further expanded by the use of an encapsulationbased technology, which may be suitable for cells that are used exclusively for their paracrine effects and are not intended to integrate structurally within the host tissue. Once encased in microparticles, which can be coated with different materials, the cells are expected to release their cytoprotective factors, while an outward-inward flux of oxygen and nutrients maintains the cells. In addition, the pores of the particle membrane are small and prevent host immune cell invasion, which theoretically allows the use of banked allogeneic, or even xenogenic, cells without additional drug immunosuppression. This approach is being tested in patients with diabetes with encapsulated Langerhans islets [58] and, based on the efficacy of nanosphere-

NOT FOR CIRCULATION

6 Current Opinion in Molecular Therapeutics 2010 Vol 12 No 5

mediated gene delivery for improving angiogenesis in mouse ischemic limbs [59], may be a suitable strategy for the treatment of CLI.

Conclusion Compelling evidence suggests that cell therapy might become a useful adjunct to the options available for patients with peripheral arterial disease complicated by CLI. The encouraging results provided by early-phase clinical trials require validation by more rigorous controlled trials involving homogeneous patient populations and focusing on clinically relevant endpoints.

To maximize the likelihood of success, it is likely to be important that previously tested strategies are not simply duplicated in larger numbers of patients, but to learn from the early clinical experience and research that has been conducted in parallel. This approach should thoroughly address the challenges still encountered by cell therapy, such as cell phenotype, processing, dosing, route of delivery and strategies to enhance graft survival. Addressing these issues successfully should provide opportunities to improve the outcome for patients with CLI, while the expected mechanistic data yielded by research conducted in parallel might define new drug targets.

References •• • 1.

of outstanding interest of special interest Rosamond W, Flegal K, Friday G, Furie K, Go A, Greenlund K, Haase N, Ho M, Howard V, Kissela B, Kittner S et al: Heart disease and stroke statistics – 2007 Update: A report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Circulation (2007) 115(5): e69-e171.

2.

ogers JH, Laird JR: Overview of new technologies for lower R extremity revascularization.

Circulation (2007) 116(18): 2072-2085.

3.

prengers RW, Lips DJ, Moll FL, Verhaar MC: Progenitor cell S therapy in patients with critical limb ischemia without surgical options. Ann Surg (2008) 247(3):411-420.

4.

Belch JJ, Topol EJ, Agnelli G, Bertrand M, Califf RM, Clement DL, Creager MA, Easton JD, Gavin JR 3rd, Greenland P, Hankey G et al: Critical issues in peripheral arterial disease detection and management: A call to action. Arch Intern Med (2003) 163(8):884-892.

5.

upta R, Tongers J, Losordo DW: Human studies of angiogenic G gene therapy. Circ Res (2009) 105(8):724-736.

6.

Creager MA, Olin JW, Belch J, Gregory M, Henry TD, Rajagopalan S, Annex BH, Chronos N, Hiatt WR, Walk Investigators: Treatment of intermittent claudication with Hypoxia-Inducible Factor-1α. ACC.09 American College of Cardiology Annual Meeting, Orlando, FL, USA (2009) 58:Abs 404-12

7.

sahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, A Witzenbichler B, Schatteman G, Isner JM: Isolation of putative progenitor endothelial cells for angiogenesis. Science (1997) 275(5302):964-967.

•• A seminal paper that describes EPCs and facilitated the initiation of cell therapy clinical trials in patients with peripheral arterial diseases.

8.

alka C, Masuda H, Takahashi T, Kalka-Moll WM, Silver M, K Kearney M, Li T, Isner JM, Asahara T: Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization. Proc Natl Acad Sci USA (2000) 97(7):3422-3427.

9.

ulati R, Simari RD: Cell therapy for angiogenesis: Embracing G diversity. Circulation (2005) 112(11):1522-1524.

10. T ongers J, Roncalli JG, Losordo DW: Role of endothelial progenitor cells during ischemia-induced vasculogenesis and collateral formation. Microvasc Res (2010) 79(3):200-206.

• An excellent review of the mechanisms involved in the formation of new blood vessels.

11. A l-Khaldi A, Al-Sabti H, Galipeau J, Lachapelle K: Therapeutic angiogenesis using autologous bone marrow stromal cells: Improved blood flow in a chronic limb ischemia model.

Ann Thorac Surg (2003) 75(1):204-209.

12. G ulati R, Simari RD: Defining the potential for cell therapy for vascular disease using animal models. Dis Model Mech (2009) 2(3-4):130-137.

13. L awall H, Bramlage P, Amann B: Stem cell and progenitor cell therapy in peripheral artery disease. A critical appraisal.

Thromb Haemost (2010) 103(4):696-709.

14. S chmidt-Lucke C, Rössig L, Fichtlscherer S, Vasa M, Britten M, Kämper U, Dimmeler S, Zeiher AM: Reduced number of circulating endothelial progenitor cells predicts future cardiovascular events: Proof of concept for the clinical importance of endogenous vascular repair. Circulation (2005) 111(22): 2981-2987.

15. S hantsila E, Watson T, Lip GY: Endothelial progenitor cells in cardiovascular disorders. J Am Coll Cardiol (2007) 49(7): 741-752.

16. P urhonen S, Palm J, Rossi D, Kaskenpää N, Rajantie I, Ylä-Herttuala S, Alitalo K, Weissman IL, Salven P: Bone marrow-derived circulating endothelial precursors do not contribute to vascular endothelium and are not needed for tumor growth. Proc Natl Acad Sci USA (2008) 105(18):6620-6625.

17. F adini GP, Agostini C, Avogaro A: Autologous stem cell therapy for peripheral arterial disease meta-analysis and systematic review of the literature. Atherosclerosis (2010) 209(1):10-17.

• A well-documented update on clinical trials of cell therapy in patients with peripheral arterial diseases.

18. M atoba S, Tatsumi T, Murohara T, Imaizumi T, Katsuda Y, Ito M, Saito Y, Uemura S, Suzuki H, Fukumoto S, Yamamoto Y et al: Long-term clinical outcome after intramuscular implantation of bone marrow mononuclear cells (Therapeutic Angiogenesis

by Cell Transplantation [TACT] trial) in patients with chronic limb ischemia. Am Heart J (2008) 156(5):1010-1018.

19. M iyamoto K, Nishigami K, Nagaya N, Akutsu K, Chiku M, Kamei M, Soma T, Miyata S, Higashi M, Tanaka R, Nakatani T et al: Unblinded pilot study of autologous transplantation of bone marrow mononuclear cells in patients with thromboangiitis obliterans. Circulation (2006) 114(24):2679-2684.

20. S ilvestre JS, Gojova A, Brun V, Potteaux S, Esposito B, Duriez M, Clergue M, Le Ricousse-Roussanne S, Barateau V, Merval R, Groux H et al: Transplantation of bone marrow-derived mononuclear cells in ischemic apolipoprotein E-knockout mice accelerates atherosclerosis without altering plaque composition. Circulation (2003) 108(23):2839-2842.

21. ClinicalTrials.gov: National Institutes of Health, Bethesda, MD, USA (2010). clinicaltrials.gov/ct2/home 22. D e Haro J, Acin F, Lopez-Quintana A, Florez A, Martinez-Aguilar E, Varela C: Meta-analysis of randomized, controlled clinical trials in angiogenesis: gene and cell therapy in peripheral arterial disease. Heart Vessels (2009) 24(5):321-328.

23. B arcelos LS, Duplaa C, Kränkel N, Graiani G, Invernici G, Katare R, Siragusa M, Meloni M, Campesi I, Monica M, Simm A et al: Human CD133+ progenitor cells promote the healing of diabetic ischemic ulcers by paracrine stimulation of angiogenesis and activation of Wnt signaling. Circ Res (2009) 104(9):1095-1102.

24. L osordo DW, Schatz RA, White CJ, Udelson JE, Veereshwarayya V, Durgin M, Poh KK, Weinstein R, Kearney M, Chaudhry M, Burg A et al: Intramyocardial transplantation of autologous CD34+ stem cells for intractable angina: A phase I/IIa double-blind, randomized controlled trial. Circulation (2007) 115(25):3165-3172.

NOT FOR CIRCULATION

Cell therapy for peripheral arterial disease Menasché 7

25. T ateishi-Yuyama E, Matsubara H, Murohara T, Ikeda U, Shintani S, Masaki H, Amano K, Kishimoto Y, Yoshimoto K, Akashi H, Shimada K et al: Therapeutic angiogenesis for patients with limb ischaemia by autologous transplantation of bone-marrow cells: A pilot study and a randomised controlled trial. Lancet (2002) 360(9331):427-435.

•• The first randomized, controlled clinical trial of bone marrow-derived cell therapy in patients with severe peripheral arterial disease.

26. R ookmaaker MB, Verhaar MC, Loomans CJ, Verloop R, Peters E, Westerweel PE, Murohara T, Staal FJ, van Zonneveld AJ, Koolwijk P, Rabelink TJ et al: CD34+ cells home, proliferate, and participate in capillary formation, and in combination with CD34- cells enhance tube formation in a 3-dimensional matrix. Arterioscler Thromb Vasc Biol (2005) 25(9):1843-1850.

27. Y oon CH, Hur J, Park KW, Kim JH, Lee CS, Oh IY, Kim TY, Cho HJ, Kang HJ, Chae IH, Yang HK et al: Synergistic neovascularization by mixed transplantation of early endothelial progenitor cells and late outgrowth endothelial cells: The role of angiogenic cytokines and matrix metalloproteinases. Circulation (2005) 112(11):1618-1627.

28. L i TS, Kubo M, Ueda K, Murakami M, Mikamo A, Hamano K: Impaired angiogenic potency of bone marrow cells from patients with advanced age, anemia, and renal failure.

J Thorac Cardiovasc Surg (2010) 139(2):459-465.

29. H eeschen C, Lehmann R, Honold J, Assmus B, Aicher A, Walter DH, Martin H, Zeiher AM, Dimmeler S: Profoundly reduced neovascularization capacity of bone marrow mononuclear cells derived from patients with chronic ischemic heart disease. Circulation (2004) 109(13):1615-1622.

•• Describes the impaired functionality of bone marrow cells in patients with heart disease, highlighting the issue of the use of autologous cells in individuals with peripheral arterial disease who often also have heart disease.

30. F adini GP, Sartore S, Schiavon M, Albiero M, Baesso I, Cabrelle A, Agostini C, Avogaro A: Diabetes impairs progenitor cell mobilisation after hindlimb ischaemia-reperfusion injury in rats. Diabetologia (2006) 49(12):3075-3084.

31. A ssmus B, Fischer-Rasokat U, Honold J, Seeger FH, Fichtlscherer S, Tonn T, Seifried E, Schächinger V, Dimmeler S, Zeiher AM, TOPCARE-CHD Registry: Transcoronary transplantation of functionally competent BMCs is associated with a decrease in natriuretic peptide serum levels and improved survival of patients with chronic postinfarction heart failure: Results of the TOPCARE-CHD Registry. Circ Res (2007) 100(8): 1234-1241 32. S eeger FH, Zeiher AM, Dimmeler S: Cell-enhancement strategies for the treatment of ischemic heart disease.

Nat Clin Pract Cardiovasc Med (2007) 4(Suppl 1):S110-S113.

33. A lessandri G, Emanueli C, Madeddu P: Genetically engineered stem cell therapy for tissue regeneration. Ann NY Acad Sci (2004) 1015:271-284.

34.

ReNeuron Group plc: ReNeuron to report positive preclinical efficacy data from ReN009: (2010) Press release: March 10. www.reneuron.com/news__events/news/document_239_237.php

35. I wase T, Nagaya N, Fujii T, Itoh T, Murakami S, Matsumoto T, Kangawa K, Kitamura S: Comparison of angiogenic potency between mesenchymal stem cells and mononuclear cells in a rat model of hindlimb ischemia. Cardiovasc Res (2005) 66(3):543-551.

36. N auta AJ, Fibbe WE: Immunomodulatory properties of mesenchymal stromal cells. Blood (2007) 110(10):3499-3506.

• Important review of the possible mechanisms to induce host immune tolerance to allogeneic mesenchymal cells.

37. L ian Q, Zhang Y, Zhang J, Zhang HK, Wu X, Zhang Y, Lam FF, Kang S, Xia JC, Lai WH, Au KW et al: Functional mesenchymal stem cells derived from human induced pluripotent stem cells attenuate limb ischemia in mice. Circulation (2010) 121(9):1113-1123.

38. B ritten MB, Abolmaali ND, Assmus B, Lehmann R, Honold J, Schmitt J, Vogl TJ, Martin H, Schächinger V, Dimmeler S, Zeiher AM: Infarct remodeling after intracoronary progenitor cell treatment in patients with acute myocardial infarction (TOPCARE-AMI): Mechanistic insights from serial contrast-enhanced magnetic resonance imaging. Circulation (2003) 108(18):2212-2218.

39. S eeger FH, Tonn T, Krzossok N, Zeiher AM, Dimmeler S: Cell isolation procedures matter: A comparison of different isolation protocols of bone marrow mononuclear cells used for cell therapy in patients with acute myocardial infarction.

Eur Heart J (2007) 28(6):766-772.

40. C heng K, Li TS, Malliaras K, Davis DR, Zhang Y, Marbán E: Magnetic targeting enhances engraftment and functional benefit of iron-labeled cardiosphere-derived cells in myocardial infarction. Circ Res (2010) 106(10):1570-1581.

41. B hang SH, Cho SW, Lim JM, Kang JM, Lee TJ, Yang HS, Song YS, Park MH, Kim HS, Yoo KJ, Jang Y et al: Locally delivered growth factor enhances the angiogenic efficacy of adiposederived stromal cells transplanted to ischemic limbs.

Stem Cells (2009) 27(8):1976-1986.

42. R ichard PL, Gosselin C, Laliberté T, Paradis M, Goulet M, Tremblay JP, Skuk D: A first semimanual device for clinical intramuscular repetitive cell injections. Cell Transplant (2010) 19(1):67-78.

43. N ugent HM, Ng YS, White D, Groothius A, Kanner G, Edelman ER: Delivery site of perivascular endothelial cell matrices determines control of stenosis in a porcine femoral stent model. J Vasc Interv Radiol (2009) 20(12):1617-1624.

44. W ang X, Jameel MN, Li Q, Mansoor A, Qiang X, Swingen C, Panetta C, Zhang J: Stem cells for myocardial repair with use of a transarterial catheter. Circulation (2009) 120(11 Suppl): S238-S246.

45. C havakis E, Urbich C, Dimmeler S: Homing and engraftment of progenitor cells: A prerequisite for cell therapy. J Mol Cell Cardiol (2008) 45(4):514-522.

• Comprehensive overview of the various treatments that can be implemented for enhancing cell engraftment and survival.

46. S eeger FH, Rasper T, Koyanagi M, Fox S. CXCR4 expression determines bone marrow-derived mononuclear neovascularization in acute ischemia.

Biol (2009) 29(11):1802-1809.

H, Zeiher AM, Dimmeler functional activity of cells for therapeutic Arterioscler Thromb Vasc

47. W alter DH, Haendeler J, Reinhold J, Rochwalsky U, Seeger F, Honold J, Hoffmann J, Urbich C, Lehmann R, Arenzana-Seisdesdos F, Aicher A et al: Impaired CXCR4 signaling contributes to the reduced neovascularization capacity of endothelial progenitor cells from patients with coronary artery disease.

Circ Res (2005) 97(11):1142-1151.

48. H iasa K, Ishibashi M, Ohtani K, Inoue S, Zhao Q, Kitamoto S, Sata M, Ichiki T, Takeshita A, Egashira K: Gene transfer of stromal cell-derived factor-1α enhances ischemic vasculogenesis and angiogenesis via vascular endothelial growth factor/endothelial nitric oxide synthase-related pathway: Next-generation chemokine therapy for therapeutic neovascularization. Circulation (2004) 109(20):2454-2461.

49. S uuronen EJ, Zhang P, Kuraitis D, Cao X, Melhuish A, McKee D, Li F, Mesana TG, Veinot JP, Ruel M: An acellular matrixbound ligand enhances the mobilization, recruitment and therapeutic effects of circulating progenitor cells in a hindlimb ischemia model. FASEB J (2009) 23(5):1447-1458.

50. Y amaguchi J, Kusano KF, Masuo O, Kawamoto A, Silver M, Murasawa S, Bosch-Marce M, Masuda H, Losordo DW, Isner JM, Asahara T: Stromal cell-derived factor-1 effects on ex vivo expanded endothelial progenitor cell recruitment for ischemic neovascularization.

Circulation (2003) 107(9): 1322-1328.

51. v an Weel V, Seghers L, de Vries MR, Kuiper EJ, Schlingemann RO, Bajema IM, Lindeman JH, Delis-van Diemen PM, van Hinsbergh VW, van Bockel JH, Quax PH: Expression of vascular endothelial growth factor, stromal cell-derived factor-1, and CXCR4 in human limb muscle with acute and chronic ischemia.

Arterioscler Thromb Vasc Biol (2007) 27(6):1426-1432.

52. A icher A, Heeschen C, Sasaki K, Urbich C, Zeiher AM, Dimmeler S: Low-energy shock wave for enhancing recruitment of endothelial progenitor cells: A new modality to increase efficacy of cell therapy in chronic hind limb ischemia.

Circulation (2006) 114(25):2823-2830.

•• Describes an innovative method for enhancing the transendothelial migration of intravascularly infused cells and, thus, their subsequent engraftment in the target tissue.

NOT FOR CIRCULATION

8 Current Opinion in Molecular Therapeutics 2010 Vol 12 No 5

53. G hanem A, Steingen C, Brenig F, Funcke F, Bai ZY, Hall C, Chin CT, Nickenig G, Bloch W, Tiemann K: Focused ultrasoundinduced stimulation of microbubbles augments sitetargeted engraftment of mesenchymal stem cells after acute myocardial infarction. J Mol Cell Cardiol (2009) 47(3):411-418.

54. K yrtatos PG, Lehtolainen P, Junemann-Ramirez M, Garcia-Prieto A, Price AN, Martin JF, Gadian DG, Pankhurst QA, Lythgoe MF: JACC Cardiovasc Interv (2009) 2(8):794-802.

55. Y ang F, Cho SW, Son SM, Bogatyrev SR, Singh D, Green JJ, Mei Y, Park S, Bhang SH, Kim BS, Langer R et al: Genetic engineering of human stem cells for enhanced angiogenesis using biodegradable polymeric nanoparticles. Proc Natl Acad Sci USA (2010) 107(8):3317-3322.

56. N ugent HM, Groothuis A, Seifert P, Guerraro JL, Nedelman M, Mohanakumar T, Edelman ER: Perivascular endothelial implants inhibit intimal hyperplasia in a model of arteriovenous fistulae: A safety and efficacy study in the pig. J Vasc Res (2002) 39(6):524-533.

57. M ethe H, Nugent HM, Groothuis A, Seifert P, Sayegh MH, Edelman ER: Matrix embedding alters the immune response against endothelial cells in vitro and in vivo. Circulation (2005) 112(9 Suppl):I89-I95.

•• Important paper describing the incorporation of allogeneic cells into a matrix that allows shedding of their antigenic sites. This approach provides the cells with temporarily protection from the host’s immune system.

58. d e Vos P, Spasojevic M, Faas MM: Treatment of diabetes with encapsulated islets. Adv Exp Med Biol (2010) 670:38-53.

59. K ang SW, Lim HW, Seo SW, Jeon O, Lee M, Kim BS: Nanospheremediated delivery of vascular endothelial growth factor gene for therapeutic angiogenesis in mouse ischemic limbs.

Biomaterials (2008) 29(8):1109-1117.