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Circulation Research. 2004;95:354-363
doi: 10.1161/01.RES.0000137878.26174.66
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(Circulation Research. 2004;95:354.)
© 2004 American Heart Association, Inc.

Reviews

Bone Marrow–Derived Cells for Enhancing Collateral Development

Mechanisms, Animal Data, and Initial Clinical Experiences

Tim Kinnaird, Eugenio Stabile, Mary Susan Burnett, Stephen E. Epstein

From the Cardiovascular Research Institute, MedStar Research Institute, Washington Hospital Center, Washington, DC.

Correspondence to Stephen E. Epstein, MD, Suite 4B-1, Cardiovascular Research Institute, Washington Hospital Center, 110 Irving St NW, Washington, DC 20010. E-mail stephen.epstein{at}medstar.net

This Review is part of a thematic series on Angiogenesis, which includes the following articles:

Endothelial Progenitor Cells: Characterization and Role in Vascular Biology

Bone Marrow–Derived Cells for Enhancing Collateral Development: Mechanisms, Animal Data, and Initial Clinical Experiences

Arteriogenesis

Innate Immunity and Angiogenesis

Syndecans

Growth Factors and Blood Vessels: Differentiation and Maturation
Ralph Kelly Guest Editor


*    Abstract
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 down arrowBone Marrow Cell Therapy:...
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Initial animal studies of single angiogenic agents, such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), generated enthusiasm for the concept that these agents might enhance collateral development and thereby provide alternative therapies for patients with vascular disease not amenable to traditional revascularization. The enthusiasm, apparently justified by the subsequent results of small nonrandomized phase-I clinical trials, was then tempered by the subsequent disappointing results of randomized clinical trials. In light of these disappointing results, investigators have pursued alternative strategies in an attempt to improve tissue perfusion. One such strategy is the utilization of bone marrow-derived cell therapy. This review discusses mechanistic pathways mediating the effects of such cell therapy, summarizes the animal and early clinical experience, and speculates on the potential of genetic manipulation of bone marrow-derived cells in an attempt to further enhance their potency.


Key Words: arteriogenesis • angiogenesis • bone marrow cells • collateral vessels


*    Introduction
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Two distinct postembryogenesis mechanisms are responsible for new blood vessel formation in adult life: (1) angiogenesis, where local capillaries proliferate and thereby increase local distribution of blood flow,4–5 and (2) arteriogenesis, where preexisting high resistance collaterals enlarge, thereby decreasing flow resistance and increasing total flow to an ischemic region.6

Angiogenesis occurs under multiple conditions, including local ischemia. One mechanism by which ischemia leads to angiogenesis is though HIF-1 signaling.7 Cellular hypoxia stabilizes the HIF-1{alpha} transcription factor leading to the expression of many genes involved in the response of the cell to hypoxia including vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), VEGF receptors, nitric oxide, and insulin-like growth factors.8 In response to changes in local concentrations of these and other cytokines, endothelial cells loosen their intracellular connections, sprout, migrate, and form new thin-walled vessels.9–11 Accompanying these cellular events, local increases in vessel permeability and basement membrane composition occur mediated by local changes in proteases such as MMP-9 and plasminogen activator. The extracellular matrix also plays an important role in modulating vessel formation by changing its composition (expressing integrins such as {alpha}vß3, which are proangiogenic) and structure to induce endothelial cell migration. Thus, sprouting of preexisting capillaries forms a new immature capillary network. To complete maturation, these newly formed vessels must associate with pericytes, thus ensuring endothelial cell survival, blood flow, and vascular permeability. Cytokines playing an essential role in neovasculature maturation include Ang-1, platelet-derived growth factor (PDGF), TGF-ß, and PlGF.12–14

An increase in the number of capillaries without a concomitant increase in the number and/or caliber of conducting vessels is insufficient to augment tissue perfusion. It is the second mechanism, arteriogenesis, which involves remodeling of preexisting small arterioles into larger vessels, that is the essential component of a developing functional collateral network.15–16 Under normal circumstances, flow through preexisting collaterals (lying in parallel to the native artery) is low because of their small caliber and resulting high resistance. However, following parent vessel occlusion, a large pressure drop develops across the preexisting collateral, resulting in an acute increase in flow and a concomitant increase in shear stress exerted on the vessel wall.17 This triggers endothelial cell expression of VCAM and ICAM, and the production of several cytokines including monocyte chemoattractant protein-1 (MCP-1), granulocyte-macrophage colony-stimulating factor-10, and tumor necrosis factor-{alpha}.18 In addition, gene array studies in tissue derived from an area of remodeling collaterals demonstrate that the expression of stem cell-derived factor-1 (SDF-1) is significantly upregulated.19 These cytokines recruit circulating monocytes to the activated endothelium, where they adhere, invade the interstitial space, and mature to macrophages.20–21 The macrophages, in addition to other cellular elements, produce abundant cytokines such as VEGF, nitric oxide, more MCP-1, FGF-1, and FGF-2. The new milieu leads to endothelial and smooth muscle cell proliferation, migration, vessel enlargement and maturation, and synthesis of extracellular matrix. Ultimately, the small preexisting arterioles remodel into large functional conducting collaterals.


*    Mechanistic Considerations
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Although an increase in tissue perfusion can only be achieved through efficient arteriogenesis, much of the mechanistic data pertaining to bone marrow-derived cell therapy pertains to angiogenesis. Although there may be considerable mechanistic overlap in the two processes, at this time insufficient information is available to definitively distinguish differences in the molecular processes leading to arteriogenesis and those leading to angiogenesis. Therefore, in the following text, we have considered data from studies addressing vascular remodeling and stem cell therapy in general.

Bone Marrow Cells Incorporate Into the Vessel Wall
Many of the cells required to form neovasculature or remodel existing collaterals derive from local proliferation of endothelial and smooth muscle cells.22–24 However, several studies suggest an additional contribution derived from bone marrow progenitors, which are mobilized in the setting of limb or myocardial ischemia, migrate to ischemic tissue, and actively incorporate into new vessels.25–29 It is proposed that tissue ischemia induces local increases in chemokines such as VEGF, thus promoting migration of VEGFR1- and VEGFR2-expressing progenitor cells to the ischemic territory.30 Thus, one rationale and proposed advantage of cell therapy versus single agent therapy is that cell therapy directly increases the number of stem/progenitor cells in ischemic tissue. The increase in local stem/progenitor cells could be achieved either by systemic delivery (by systemic injection of the cells or inducing cells to exit the marrow microenvironment by chemokine therapy) or by direct injection of cells into or around the ischemic zone. Regardless of the pathway taken, the number of potential candidate cells to incorporate into the vessel wall is substantially increased.

Several cell types within the marrow cavity appear to retain the ability to differentiate into one or more of the cellular components of the vascular bed and thus in theory might incorporate directly into the wall of newly formed or remodeled vessels. Endothelial progenitor cells are one such cell type present in the bone marrow (EPCs) and peripheral circulation (CEPs); the role of these cells in angiogenesis is the subject of a separate review.

One bone-marrow cell considered as a potential candidate for transdifferentiation into vascular wall cells is the hematopoietic stem cell (HSC), which displays multiorgan and multilineage engraftment in animal marrow irradiation studies.31 An enriched CD34–/low, c-kit+, Sca-1+ subpopulation of the HSC, termed the side-population (or SP) cell also appears to exhibit transdifferentiation capabilities with incorporation into infarcted myocardium as new cardiomyocytes and endothelial cells.32–33 The rates of SP incorporation in these studies appears to be relatively low, with only 3% of capillaries in the peri-infarct territory displaying donor cell phenotype. Another HSC derivative, the Lin– c-kitPOS cell, may also possess multilineage capabilities. In murine acute infarction models, Lin– c-kitPOS cells directly injected into infarcted myocardium incorporated as cardiomyocytes, and also as endothelial and smooth muscle cells.34–35 These studies demonstrated higher rates of cell incorporation with up to 40% of new endothelial cells and smooth muscle cells in the peri-infarct region being derived from donor cells. It appears from these studies that the milieu in which cells find themselves is crucial in directing their ultimate differentiation and commitment to a particular lineage.

The marrow stromal cell (MSC) is another bone-marrow cell type with the potential to incorporate into the developing vascular wall. Marrow stromal cells are also termed mesenchymal stem cells, mesenchymal stromal cells, and mesenchymal progenitor cells, and their nomenclature is the subject of on-going discussion. However, cells isolated by diverse protocols as used by different groups appear to produce cells that are phenotypically and functionally indistinguishable.36 MSCs do not express cell surface markers typical of hematopoietic progenitors, such as CD31, CD34, CD45, CD117, or CD133, but are uniformly positive for CD90, CD105, and CD166.37 MSCs retain the ability to differentiate into several mesenchymal lineages including osteoblasts, chondroblasts, and adipocytes.38–39 However, MicroSAGE analysis also confirms that MSCs express RNA’s characteristic of smooth muscle and endothelial cells,40 and in vitro studies reveal that after several weeks of culture, MSCs acquire a phenotype that closely resembles smooth muscle cells.41,42 Although most cardiovascular studies of MSC differentiation have focused on their ability to form cardiomyocytes,43 some evidence of MSC incorporation into neovasculature has been observed.44 One study directly addressing MSC therapy for angiogenesis suggested that locally delivered MSCs were able to incorporate into newly formed vessels and displayed endothelial and smooth muscle cell phenotypes.45

The MSC population consists of a heterogeneous population of cell types. MSC progenitors, termed multipotent adult progenitor cells (MAPCs), copurify with MSC cultures.46–47 Until the isolation of MAPCs, MSCs or their derivatives had not been conclusively demonstrated in vitro to differentiate into endothelial cell lineages. However, when cultured with VEGF, MAPCs, as well as retaining the typical MSC lineages potential, differentiate into CD34+, VE-cadherin+, Flk1+ cells, a phenotype that would be expected for angioblasts. Subsequently, these cells could be induced to express endothelial markers and function as endothelial cells in vitro.48 The crucial phenotypic difference between MSCs and MAPCs is the expression of CD44. This cell marker is expressed when cultured in greater than 2% serum, and appears to signal MSC commitment away from endothelial cell lineages.

Given the large amount of in vitro data regarding the plasticity of various bone marrow-derived cell populations, it is tempting to conclude that cell therapy exerts it dominant effects through incorporation into vessels as endothelial or smooth muscle cells. However, the relative importance of direct incorporation into new or remodeling collaterals is the subject of on-going debate, with the magnitude of actual incorporation of donated cells into vascular structures varying substantially between studies.

Although some studies report more than half of counted capillaries containing transplanted cells, other studies have reported only occasional positive vessels despite impressive improvements in perfusion.49–51 Whether any cells actually incorporate was examined by inducing hind-limb ischemia in a mouse previously undergoing marrow irradiation followed by marrow reconstitution by cells derived from a green fluorescent protein (GFP) transgenic mouse. Colocalization of GFP signals with endothelial or smooth muscle cell markers in collateral arteries developing in the ischemic hindlimb was not observed. In contrast, what was observed was an accumulation of GFP+ fibroblasts, pericytes, and leukocytes adjacent to growing collateral arteries. These results suggest that bone marrow cells do not promote vascular growth by incorporating directly into the vessel wall but play an important supportive role.52 The importance of confocal microscopy was emphasized in this study, as false colocalization of GPF and endothelial signals was observed with normal microscopy.

The issue of adult stem cell plasticity itself has also been questioned, and is currently the subject of intense debate. Single transplanted HSCs were able to reconstitute all peripheral blood components after marrow irradiation, but were not observed in nonhematopoietic tissues, suggesting that transdifferentiation of circulating HSCs and/or their progeny is an extremely rare event.53 Two very recent studies raised further questions regarding stem cell plasticity in vivo. In one of these studies, cardiomyocyte-restricted and ubiquitously expressed reporter transgenes were used to track the fate of hematopoietic stem cells after 145 transplants into normal and injured adult mouse hearts. Although donor HSCs were observed within myocardial scar tissue, the cells had not transdifferentiated into cardiomyocytes.54 In the second study, donor cells again were not found to transdifferentiate into cardiomyocytes, but persisted as HSCs within the myocardium and continued to differentiate into blood cells.55 These studies used genetically tagged HSCs, thus avoiding the potential error introduced by antibody labeling—one mechanism that may explain the conflicting results of these trials with earlier studies.56

Even accepting that donor stem cells are found in non-hematopoietic tissues as differentiated cells, the mechanisms underlying this observation have also been called into question. Coculture of embryonic stem (ES) cells and murine brain cells expressing a transgenic marker, followed by selection of cells expressing the transgenic marker, recovered a population of undifferentiated cells demonstrating an ES cell phenotype. These cells could be differentiated into multiple cell lineages. However, the demonstration that the undifferentiated cells contained a tetraploid complement confirmed that fusion had occurred.57 In a similar study, bone marrow mononuclear cells from transgenic mice expressing both the gene for GFP and puromycin resistance were cocultured with ES cells.58 After the addition of puromycin, multiple GFP+ clones were identified with ES cell morphology. Although these cells were capable of multilineage differentiation, they also expressed a haploid karyotype, again confirming that fusion had occurred.

To examine the fusion phenomenon in an animal model, the Cre/lox reporter system (in which activation of Lac-Z expression only occurs after cell fusion) has been used in several studies.59 Injection of flox+/LacZ+ myoblasts into Cre(+) murine hearts resulted in LacZ-positive cells. In contrast, no LacZ-positive cells were observed when flox+/LacZ+ myoblasts were injected into Cre(–) murine hearts. A further study harnessing the Cre/lox reporter model demonstrated bone marrow-derived cells fused spontaneously with cardiomyocytes, resulting in the formation of multinucleated cells. No evidence of transdifferentiation without fusion was observed in this study.60 Thus, these data reveal the first insights into the potential importance of progenitor cell/recipient cell fusion. Clearly, any future studies of stem cell therapy will have to convincingly rule out cell fusion before any substantive claims as to plasticity can be made.

Bone Marrow Cells Function as Supportive Cells
Despite evidence that bone marrow-derived stem or progenitor cells incorporate into vascular structures, as discussed earlier, several studies suggest that only a small number of vessels contain donated cells. In addition, heterogeneous bone marrow cell populations (ie, mononuclear cells or unfractionated cells) contain very small numbers of stem cells (<0.01% of total cells); but despite this, injection of these cells significantly enhances collateral development. Thus, other mechanisms are likely to contribute to the benefits seen after bone marrow cell therapy.

An alternative or perhaps complementary hypothesis to the concept of bone marrow cell incorporation is that the donated cells act in a supportive role, optimizing the milieu for host vasculature to respond to tissue ischemia. Many bone marrow subpopulations are a source of growth factors previously demonstrated to be central in the initiation and coordination of angiogenesis. An early study of bone marrow cell therapy demonstrated that mononuclear marrow cells secrete angiogenic factors such as VEGF and MCP-1 in culture.61 Other groups confirmed these findings, and observed that after direct myocardial delivery of bone marrow mononuclear cells, increases in cardiac mRNA expression of VEGF, FGF, Ang-1, interleukin-1ß, and tumor-necrosis factor-{alpha} were observed, suggesting localized in vivo secretion of angiogenic growth factors by the injected cells.62

Several stem/progenitor cells are also a source of cytokines, and among this group, MSCs are of particular relevance. These cells play a vital supportive role within the marrow microenvironment; their effects are mediated partly through cell-to-cell contact, but also through paracrine signaling. In gene array studies, MSCs express mRNAs for a wide spectrum of angiogenic/arteriogenic cytokines including VEGF, FGF, monocyte chemoattractant protein 1 (MCP-1), placental growth factor, interleukins 1 and 6, insulin-like growth factor, SDF-1, MMP-9, and plasminogen activator.63 In addition to these cytokines other studies demonstrate that hepatocyte growth factor (HGF) and several insulin growth factors are also released by MSCs.64–67 Media collected from MSC cultures promoted in vitro proliferation and migration of endothelial cells and smooth muscle cells, and enhanced collateral flow recovery and remodeling when directly injected into a mouse ischemic hindlimb.63

Several other cell populations are potential sources of chemokines. Hematopoietic stem cells express mRNA for VEGF and Ang-1,32 whereas EPCs, at least in vitro, secrete VEGF, HGF, and the potent progenitor cell mitogens granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor.68 Interestingly, although another progenitor cell, the angioblast, has not been directly shown to exert paracrine effects, injection of human-derived angioblasts into infarcted rat myocardium stimulated host endothelial cells to proliferate, suggesting that the angioblast may also be a source of proangiogenic factors.69

Other more mature cellular components of the marrow population also demonstrate potential to exert effects through paracrine mechanisms.70–71 Several groups have observed that T-lymphocytes release VEGF and play a central role in coordinating the cellular response to arterial occlusion, in part by recruiting monocytes/macrophages to the site of active collateral artery formation.72 T-lymphocytes also stimulate endothelial cells to produce VEGF through CD40 signaling, thus providing another pathway via which these cells modulate collateral artery growth.73 In addition, monocytes are important mediators of the inflammatory response during arteriogenesis, and are a source of a wide array of chemokines including VEGF, nitric oxide, MCP-1, FGF-1, and FGF-2.74

As discussed earlier, angiogenesis and arteriogenesis are complex processes involving many cellular components and multiple cytokines. These cytokines act not only in a coordinated time- and concentration-dependent manner, but one cytokine may potentiate (or inhibit) the effect of another. For example, synergistic relationships between VEGF and bFGF, placental growth factor and VEGF, PDGF and FGF-2, and angiopoietin-1 and VEGF have all been reported.75–79 In addition, any change in the relative concentrations of one or more of the important cytokines can have catastrophic consequences.80 FGF-1 when delivered chronically to ischemic myocardium of dogs, led to hemangioma formation.81 Similarly, injection of VEGF-overexpressing myoblasts caused myocardial hemangiomas in close proximity to the injected cells.82 It was also demonstrated that microenvironmental concentrations of VEGF were crucial in determining the neovascular response. Below a certain threshold, increased VEGF levels increased the number of morphologically normal capillaries. However, once this threshold was exceeded, increased VEGF resulted in the formation of vascular tumors in every animal treated.83 In contrast, even a small reduction in VEGF levels may have a profound effect on vascular development.84

Thus, it is clear from these data that significant barriers exist in optimizing local cytokine concentrations therapeutically in an attempt to enhance innate responses to tissue ischemia. The theoretical advantage of bone marrow cell therapy over single agent therapy is that as well as delivering vascular cell precursors, such therapy delivers cells that can supply many of the necessary cytokines required to support angiogenesis and arteriogenesis. Although it is tempting to speculate that cell therapy also delivers these cytokines at physiological concentrations in a time-appropriate manner, the large number of cytokines involved and complexity of the processes precludes proof of this concept.

Conclusions Regarding Potential Mechanisms by Which Bone Marrow Cell Therapy May Enhance Collateral Development
Controversy continues regarding the exact mechanisms through which bone marrow cells enhance collateral development. Although initial studies suggested that stem cell plasticity and resultant incorporation into the vascular wall was an important phenomenon, more recent data has raised serious concerns regarding its true significance (or indeed occurrence). Furthermore, several groups have independently demonstrated the potential role of supportive mechanisms in mediating the effect of bone marrow therapy for tissue ischemia. However, it is important to bear in mind that the relative balance of incorporation versus paracrine signaling may well vary depending on which cell type is harnessed and in what setting the cells are delivered (see Figure 1 for a summary of the potential mechanisms mediating bone marrow cell effects). Furthermore, uncertainty exists as to which cell is most efficacious, as there is little published data comparing one cell type versus another. Regardless of the gaps in understanding the mechanistic pathways and optimal cell type, the potential of bone marrow-derived cells to affect local arteriogenic processes via possible transdifferentiation and coordinated secretion of arteriogenic cytokines, make them attractive for interventions aimed at enhancing collateral tissue perfusion.



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  		Figure 1. Mechanisms by which progenitor cells could enhance collateral development. (1) Supportive: Progenitor cells secrete multiple cytokines, growth factors, and chemokines that could facilitate arteriogenesis by (a) influencing the matrix in a way that would be conducive for collateral development, (b) inhibit endothelial and smooth muscle cell apoptosis and stimulate their migration and proliferation, and (c) recruit proarteriogenic inflammatory and progenitor cells. (2) Incorporation: Progenitor cells could directly incorporate into the developing collateral and thereby physically contribute to collateral formation; the biological importance of this mechanism is presently a source of controversy. (3) Fusion: Fusion of progenitor cells with tissue specific cells has been demonstrated, but no data are available suggesting this functionally contributes to collaterogenesis. This schematic diagram demonstrates two parallel conductance vessels and an interconnecting collateral vessel. Although in vivo these vessels are lined by endothelial cells, smooth muscle cells, and adventitia, for the purposes of the figure only the endothelial cell layer is illustrated.


*    Bone Marrow Cell Therapy: Preclinical Studies
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Several differing approaches to bone marrow cell therapy have been undertaken, with some groups administering heterogeneous populations of cells and other groups selecting specific cell subgroups (See Figure 2 for a summary of the cell types delivered).



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  		Figure 2. Different subpopulations of progenitor cells that have been found to enhance collateral development.

Many small animal studies demonstrate the therapeutic potential of heterogeneous cell delivery for improving collateral flow. For example, transepicardial injection of cultured marrow mononuclear cells (no cell marker data supplied) in a rat cardiac cryoinjury model although designed to study myocardial regeneration, increased capillary counts in the myocardial scar.50 In a rat model of myocardial ischemia, freshly isolated mononuclear cells increased the number of CD31-positive vessels compared with saline.85 After femoral artery ligation, intramuscular implantation of marrow mononuclear cells increased the number of visible capillaries measured by microangiography, increased hindlimb blood flow estimated by microsphere injection, and improved exercise tolerance by 50% when compared with controls.86 Mononuclear cell therapy also appeared to augment small and larger vessel remodeling in rabbit hindlimb ischemia, with an increase in capillary count, and enhanced collateral development as assessed by angiographic score and Laser Doppler perfusion imaging.87 Of particular interest is the demonstration that ex vivo exposure of mononuclear cells (isolated by density gradient) to hypoxic stress for 24 hours significantly increased capillary count and microsphere-calculated flow recovery when compared with cells cultured under normoxia.88

The capacity of mononuclear cell injection to enhance vascular remodeling has also been demonstrated in large animal models. Transendocardial injection of filtered whole bone marrow aspirate in a pig ameroid model of chronic ischemia improved collateral flow as assessed by microspheres.61 In a porcine acute infarction model, injection of mononuclear cells (isolated by density gradient) resulted in a 3-fold increase in the capillary count. This therapy also augmented larger vessel remodeling with a 5-fold increase in the number of angiographically visible collaterals, and a reduction in myocardial contrast echo perfusion defect, ultimately leading to a 48% improvement in ejection fraction compared with controls.62 In a canine chronic coronary occlusion model, transepicardial injection of mononuclear cells after coronary occlusion led to a 50% increase in the number of microvessels observed and a significant improvement in LV wall systolic thickening.89

Other groups have examined the role of more selected subpopulations of bone marrow-derived cells. Injection of harvested human CD34+ precursors induced by G-CSF injection (most of this subset were also CD117+ and contained a high proportion of HSCs and EPCs) led to a 5-fold increase in capillary count in a nude rat myocardial infarction model. Echocardiographic myocardial function improved by a mean of 22% compared with controls, with a reduction in the severity of ventricular remodeling observed. These effects persisted to at least 15-weeks after injection.69 As discussed, SP cells isolated by Hoechst dye staining (CD34–/low, c-kit+, Sca-1+ phenotype) also contribute to infarct healing, incorporating as endothelial and smooth muscle cells, although the effect of this on end-points such as ventricular function and remodeling are less certain.32,33 HSC derivatives such as the lin– c-kitPOS cells have also been used in murine infarct models. Although these cells (isolated from fresh marrow cells by monoclonal antibodies) were observed to incorporate into vascular structures, myocardial regeneration was the most striking finding of this study.34 Subsequent studies in mice using granulocyte colony-stimulating factor to mobilize lin– c-kitPOS HSCs from the marrow cavity before the induction of myocardial infarction resulted in significant increase in capillaries, arterioles (with several layers of smooth muscle cells), and myocytes within the scar. The combined effect of this was a 68% reduction in mortality, a 40% reduction in echocardiographic infarct size, and significant improvements in postmortem myocardial hemodynamics.35

MSCs also are effective in augmenting the vascular response to arterial occlusion, increasing capillary counts and hindlimb collateral flow.45,90 In a mechanistic study of cellular arteriogenic potential, MSC (CD34–, CD45– CD90+, and CD105+ cells selected by magnetic bead sorting from cultured murine marrow) injection increased limb perfusion when measured by laser Doppler, and increased conductance vessel number and total cross-sectional area.63,91 MAPCs, as might be expected given their clear multilineage potential, also contribute to new vessel formation, although this potential has only been exploited in wound healing and tumor angiogenesis models.48

In the animal studies conducted thus far, abnormal tissue development subsequent to injection (eg, bone, cartilage, or teratoma formation), or increased cardiac fibrosis, have not been demonstrated. Accordingly, the safety and suggestive potential efficacy results of these preclinical studies served as the basis for several on-going clinical trials.


*    Bone Marrow Cell Therapy: Preliminary Clinical Experience
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In the first human study, mononuclear cells were injected into ungraftable myocardial territories as an adjunct to coronary artery bypass grafting.92 Postoperative evaluation revealed no adverse effects and suggested improvement in perfusion in injected territories in 3 of the 5 patients. In a similar pilot study, AC133+ cells were isolated from mononuclear cells and injected intramyocardially during CABG.93 During follow-up, LV function improved in 4/6 patients and perfusion in 5/6. Clearly, as cell therapy was delivered as an adjunct to CABG, and in the absence of control groups, no conclusions can be drawn from these studies as to the effectiveness of the cells themselves. Sole therapy using transendocardial injection of whole filtered marrow aspirate has been used as part of several pilot and phase I safety and feasibility studies.94–96 These small nonrandomized studies suggested improvements in angina scores and a reduction stress-induced ischemia.

Intracoronary injection of bone marrow cells is an alternative strategy to deliver cells directly to the myocardium. Autologous mononuclear cells were injected into the target vessel approximately 8 days after primary angioplasty for acute infarction (n=10). Although designed to study the role of cells in myogenesis, some improvements in myocardial perfusion were observed, suggesting a contribution of these cells to collateral vessel formation.97 In a second similar study, investigators assessed the safety and feasibility of intracoronary injection of bone marrow-derived mononuclear cells and peripheral blood-derived mononuclear cells 4 days after successful angioplasty for acute myocardial infarction (n=20). When patients with restenosis were excluded, flow reserve in the infarct vessels appeared to improve substantially.98 However, in the absence of randomized control groups in each of these studies, the significance of any conclusions relating to efficacy are uncertain. Finally, in the only randomized human trial of bone marrow cell therapy performed to date, mononuclear cells were injected into the gastrocnemius muscle of patients with peripheral vascular disease, resulting in significant improvements in the ankle-brachial index of the bone marrow-treated leg when compared with the control leg treated with peripheral blood-derived cells.99

Although encouraging, the data emerging from these preliminary trials should be cautiously interpreted. It is well recognized that patients enrolled in such trials experience a powerful placebo effect. Additionally, no trial thus far has used an adequately powered, randomized, double-blinded design, and therefore the results of such studies must be cautiously interpreted.


*    Bone Marrow Cell Therapy: Future Directions
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As discussed earlier, there are significant theoretical explanations for the disappointing results of trials using a single angiogenic agent.1–3 Cell therapy, mainly by virtue of the potential of this strategy to supply stem/progenitor cells and multiple angiogenic-related cytokines to the region of developing collaterals, may overcome some of these problems. However, there are data to suggest that cell therapy itself may also have inherent limitations.

One example of such a potential limitation is that transplanted cells may have low survival rates, significantly impacting on their beneficial effects.100,101 Genetically engineering cells to overexpress prosurvival genes such as Akt may be one approach to overcome this limitation. Transplantation of MSCs overexpressing Akt resulted in a 4-fold greater myocardial volume than equal numbers of MSCs transduced with a reporter gene.102 Another potential limitation to cell therapy is the suggestion that cardiovascular risk factors such as aging may also impair the angiogenic effectiveness of cells derived from the older patient in whom autologous cells are to be injected.103 Furthermore, even if bone marrow cell therapy is more efficacious than single agent therapy, it may still be insufficient to overcome the inhibitory effects of cardiovascular risk factors on collateral development. One approach to this problem may be to genetically engineer the cells to further enhance their therapeutic potential.

Although little is currently known about genetically engineering bone marrow cells to enhance angiogenesis, extrapolation from studies exploring the potential of genetically engineering other types of cells suggests that this direction might be worth further investigation. For example, transduction of skeletal myoblasts with VEGF165 appeared to increase capillary density in the vicinity of the transplanted cells compared with nontransduced cells.104 Whether this conclusion can be extrapolated to the potential of this approach to enhance collateral vessel development is, of course, entirely speculative. Similarly, in a study without a concurrent control group (results were compared with previously performed studies), transduction of endothelial progenitor cells with VEGF164 appeared to reduce the number of cells required to improve ischemic limb salvage when injected into the ischemic hindlimbs of athymic nude mice.105

In an attempt to develop an optimal angiogenic strategy, using an adenoviral vector, a stable HIF-1{alpha} analogue (HIF-1{alpha}/VP16, see Vincent et al106 for a description of this construct) was overexpressed in MSCs resulting in a 4-fold increase in MSC VEGF release. Subsequently, in a murine model of hindlimb ischemia local injection of MSCs transduced with HIF-1{alpha}/VP16 into the ischemic mouse hindlimb significantly increased collateral perfusion compared with nontransduced cells.107 Whether such interventions will ultimately translate into clinical benefits, however, remains to be seen.


*    Conclusions
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Administration of autologous bone marrow cells is a novel therapeutic strategy derived from the concept that a more optimal arteriogenic effect can be achieved by delivering multiple angiogenic cytokines and cells to regions of tissue ischemia. Experimental studies suggest that such cells may retain the ability to differentiate into vascular cells. However, in what may be the predominant mechanism, these studies also confirm that many of these cells express multiple angiogenic cytokines that support vascular remodeling. Regardless of precise mechanism, delivery of these cells appears to increase tissue perfusion. Whether cell therapy can overcome several potential limitations, or whether more novel approaches need to be explored, such as genetic modification of these cells, is unknown. The clinical need for arteriogenic interventions, the encouraging early experimental results, and the existence of advanced molecular technologies, will undoubtedly further stimulate investigational efforts to optimize cell-based approaches to tissue ischemia.


*    Acknowledgments
 
The authors receive research support from Medstar Research Institute, Berlex Pharmaceuticals, Genzyme Corporation, and Myocardial Therapeutics Incorporated.


*    Footnotes
 
Original received February 6, 2004; resubmission received April 2, 2004; revised resubmission received May 28, 2004; accepted May 28, 2004.


*    References
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