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Perivascular Delivery of Encapsulated Mesenchymal Stem Cells Improves Postischemic Angiogenesis Via Paracrine Activation of VEGF-A

Originally publishedhttps://doi.org/10.1161/ATVBAHA.113.301217Arteriosclerosis, Thrombosis, and Vascular Biology. 2013;33:1872–1880

Abstract

Objective—

To test the therapeutic activity of perivascular transplantation of encapsulated human mesenchymal stem cells (MSCs) in an immunocompetent mouse model of limb ischemia.

Approach and Results—

CD1 mice underwent unilateral limb ischemia, followed by randomized treatment with vehicle, alginate microbeads (MBs), MB-encapsulated MSCs (MB-MSCs), or MB-MSCs engineered with glucagon-like peptide-1. Treatments were applied directly in the perivascular space around the femoral artery. Laser Doppler and fluorescent microsphere assessment of blood flow showed a marked improvement of perfusion in the MB-MSCs and MB-MSCs engineered with glucagon-like peptide-1 groups, which was associated with increased foot salvage particularly in MB-MSCs engineered with glucagon-like peptide-1–treated mice. Histological analysis revealed increased capillary and arteriole density in limb muscles of the 2 MSC groups. Furthermore, MB-MSCs engineered with glucagon-like peptide-1 and, to a lesser extent, MB-MSC treatment increased functional arterial collaterals alongside the femoral artery occlusion. Analysis of expressional changes in ischemic muscles showed that MB-MSC transplantation activates a proangiogenic signaling pathway centered on vascular endothelial growth factor A. In contrast, intramuscular MB-MSCs caused inflammatory reaction, but no improvement of reparative vascularization. Importantly, nonencapsulated MSCs were ineffective either by intramuscular or perivascular route.

Conclusions—

Perivascular delivery of encapsulated MSCs helps postischemic reperfusion. This novel biological bypass method might be useful in patients not amenable to conventional revascularization approaches.

Introduction

Peripheral artery disease (PAD) affects up to 15% of people >55 years.1 Critical limb ischemia (CLI) is the end stage of lower extremity PAD in which severe obstruction of blood flow results in ischemic rest pain, ulcers, and high risk for limb loss. Surgical bypass surgery or percutaneous revascularization, the gold standard for the treatment of PAD, produces long-term benefit with a 5-year limb salvage rate of >80%.2 However, ≈30% of patients with CLI cannot be revascularized because of multivascular disease or occlusions of small-caliber blood vessels, which are common in patients with diabetes mellitus and hypertension.3

Gene and stem cell therapies have been accredited to provide a possible alternative to interventional angioplasty.4,5 Clinical trials using bone marrow–derived mononuclear cells showed significant benefit, including improvement of ankle brachial index, transcutaneous partial pressure of oxygen, reduction of pain, and decreased need for amputation.69 Intramuscular, intra-arterial injection, or a combination of both represents the preferred route of cell therapy in clinical trials. Both the methods have limitations depending on the disease pattern. Intra-arterial infusion is not ideal for patients with occluded femoral artery because the stem cells will not reach the affected site by blood flow, whereas intramuscular delivery requires multiple injections to maximize the extension of therapeutic benefit. In BONe Marrow Outcomes Trial 1&2 (BONMOT-1&2), injections were placed instead along the occluded arteries with the intention to increase the formation of collaterals bypassing the occlusion.10

A common drawback of the above methods is that cell retention is generally low because of massive apoptosis in the first few days after implantation. Microencapsulation strategies, surrounding the cells with a semipermeable polymeric membrane, have been proposed for enhancing cell viability.1114 Here, the aim is to use the cells as local drug delivery factories, to secrete a cocktail of beneficial factors to elicit a therapeutic paracrine effect. Recently, we have developed a novel miniaturized encapsulation procedure where stem cell–containing corebeads are surrounded with a permeable shell of biocompatible alginate, which provides a robust immunoisolating barrier, whereas allowing for diffusion of inherent paracrine factors, such as vascular endothelial growth factor (VEGF).15 Furthermore, the optimized shell-to-core ratio ensures full cell viability. This new formulation (CellBeads) contains human mesenchymal stem cells (MSCs) genetically modified to express glucagon-like peptide-1 (GLP-1), an incretin hormone that has antiapoptotic, proangiogenic, and cardioprotective effects.1619 Intracoronary injection of CellBeads passed feasibility and safety tests and showed therapeutic benefit in a porcine model of acute myocardial infarction.20,21 In addition, CellBeads have been evaluated in a clinical study to treat patients with posthemorrhagic stroke to limit the ensuing apoptotic damage.22 Based on these encouraging data, we propose that CellBeads might be used as an off-the-shelf cell product for wide-scale treatment of CLI.

Therefore, in view of clinical application, the present study investigates the feasibility of CellBead delivery into the perivascular space surrounding the occluded femoral artery and the efficiency of CellBead transplantation in improving reparative angiogenesis and collateralization in an immunocompetent mouse model of limb ischemia.

Results

Feasibility of Perivascular Cell Delivery

To assess the retention of microbeads (MB), cryosections from en bloc muscle samples were collected at day 7 after ischemia induction. Hematoxylin and eosin staining of these samples demonstrated the presence of MB-MSCs engineered with GLP-1 (MB-MSC-GLP) surrounding the femoral artery and vein, thus, confirming the feasibility of the procedure (Figure 1A). Furthermore, cell-transplanted muscles showed increased capillary (P<0.02 versus vehicle) and arteriole density (P<0.05 versus vehicle) at the site of implantation (Figure 1B–1D). Arterioles were oriented coaxially with the femoral artery.

Figure 1.

Figure 1. A, Representative hematoxylin-eosin staining showing the persistence of microbeads encapsulated with mesenchymal stem cells transfected with glucagon-like peptide-1 (MB-MSC-GLP) along the femoral artery and vein at 7 days postischemia. B, Representative microphotographs from fluorescence microscopy showing MB-MSC-GLP, isolectin-B4 positive capillaries (box, blue arrowhead) and α-smooth muscle actin positive arterioles (box, pink arrowhead) around the femoral artery and vein. C, Bar graphs showing the quantitative analysis for the number of capillaries and arterioles around femoral artery and vein. D, Bar graph showing arteriole density. Scale bars, 100 µm. Data represented as mean±SE, n=4 per group. *P<0.05 and **P<0.01 vs Vehicle.

Improvement of Clinical Outcomes

Limb salvage (no necrotic toe) occurred in 33% and 42% of mice given vehicle or MB, respectively. This outcome was improved by MSC transplantation, with 66% salvage in MB-MSC and 75% in MB-MSC-GLP–treated groups. Global clinical outcome considering the number of mice with ≥1 necrotic toes or foot necrosis/amputation is illustrated in Figure 2A. Analysis of contingency by the χ2 test was not applicable because the data set comprised several values <1, and <20% of the values were >5. Therefore, an arbitrary score from 0 (no necrosis) to 6 (foot necrosis/amputation) was computed and used to compare the experimental groups. ANOVA detected a difference among groups pertaining to the severity score (P<0.01). MB-MSC-GLP–treated mice showed the lowest severity score (0.4±0.3; P<0.01 versus vehicle and P<0.05 versus MB), followed by MB-MSC (1.2±0.6), MB (2.4±0.7) and vehicle (3.0±0.7). Computation of Cohen d coefficient indicates a large biological effect of MB-MSC-GLP as compared with vehicle (1.3) and MB (1.1).

Figure 2.

Figure 2. A, Stacked bars showing % of necrotic toes at 2 days postischemia. B, Representative Doppler images at 21 days postischemia and line graph showing blood flow recovery. Green and yellow squares delimit the ischemic and contralateral foot, respectively; n=12 per group. C, Bar graph showing adductor muscle blood flow at 21 days, as assessed by fluorescent microspheres; n=5 per group. D, Bar graph showing the level of Po2 at 21 days; n=9 to 12 mice per group. Data are mean±SE.*P<0.05, **P<0.01, and ***P<0.001 vs Vehicle; #P<0.05 and ###P<0.001 vs microbeads (MB). MB-MSCs indicate MB encapsulated with mesenchymal stem cells; and MB-MSC-GLP, MB encapsulated with mesenchymal stem cells transfected with glucagon-like peptide-1.

Improvement of Perfusion

Laser Doppler blood flow recovery was different among groups (2-way ANOVA, group effect: P<0.0001). Bonferroni multiple comparison analysis indicates an improvement of blood flow in MB-MSC-GLP– and MB-MSC–treated groups (P<0.001 versus vehicle or MB from day 7 to day 21; Figure 2B). Cohen d coefficient indicates that the biological effect of MB-MSC-GLP and MB-MSC treatments is large (2.8 and 2.4 versus vehicle and 2.0 and 1.9 versus MB, respectively). In contrast, no difference was observed between MB-MSC and MB-MSC-GLP groups.

Analysis of blood flow to the adductor muscle at 21 days postischemia using fluorescent microspheres indicates a significant difference among groups (ANOVA, group effect: P=0.02), with the MB-MSC-GLP group showing a large improvement compared with vehicle (P<0.01; Cohen d coefficient, 2.6) or MB (P<0.05; Cohen d coefficient, 1.9; Figure 2C). Moreover, the MB-MSC group showed higher blood flow levels compared with vehicle (P<0.05; Cohen d coefficient, 1.9). No difference was detected between MB-MSC and MB-MSC-GLP treatments. Moreover, no group difference was observed in perfusion of contralateral adductor.

Similarly, Po2 levels in the ischemic adductor were markedly improved in the MB-MSC-GLP– or MB-MSC–treated groups compared with vehicle or MB (ANOVA, P<0.0001; Figure 2D). The effects in both treatment groups can be considered very large (Cohen d coefficient, 3.7 and 3.4, respectively).

Promotion of Reparative Angiogenesis

Immunofluorescence microscopy showed a significant increase in the number of capillaries in ischemic adductor muscles of MB-MSC–transplanted mice (879±16 capillaries/mm2) compared with vehicle (673±13 capillaries/mm2; P<0.001; Figure 3A). Similarly increased were small arterioles (15.3±0.5 versus 9.1±0.3 arterioles/mm2 in vehicle; P<0.001; Figure 3B). Analogous improvements were observed in the MB-MSC-GLP–treated group (891±20 capillaries/mm2 and 14.6±0.8 arterioles/mm2; P<0.001 versus vehicle for both comparisons; Figure 3A and 3B). The effect of MB-MSC and MB-MSC-GLP treatments on neovascularization was very large (Cohen d coefficient, 5.5 and 5.0 for capillaries and 6.5 and 3.6 for arterioles, respectively). Use of MB alone did not produce any improvement (736±13 capillaries/mm2 and 10±0.5 arterioles/mm2; P=NS versus vehicle; Figure 3A and 3B).

Figure 3.

Figure 3. A and B, Representative microphotographs and bar graphs showing capillary (A) and arteriole (B) density in the ischemic adductor muscle at 21 days postischemia. Scale bars are 100 µm. C and D, Bar graphs showing capillary (C) and arteriole (D) density around femoral artery and vein (at the site of delivery of cells or vehicle). Data are mean±SE, n ≥5 in each group. ***P<0.001 vs Vehicle; ###P<0.001 vs microbeads (MB); ΦP<0.05 vs MB encapsulated with mesenchymal stem cells (MB-MSCs). MB-MSC-GLP indicates MB encapsulated with mesenchymal stem cells transfected with glucagon-like peptide-1.

Collateral Formation Along the Occluded Artery

Cell therapy approaches mainly enhance the microvasculature downstream to the vascular occlusion. However, unless the artery blockage is removed or bypassed, it is unlikely that the increased microvascular bed will restore optimal perfusion. We verified whether the increased muscular blood flow and oxygenation observed in our study was associated with an increased collateralization along the femoral artery, and whether these collaterals were functionally operative. We found that MB-MSC-GLP– and MB-MSC–treated mice have an increased number of capillaries (460±12 and 389±23 versus 177±10 capillaries/mm2 in vehicle; P<0.001; Figure 3C), small arterioles (10.3±0.3 and 9.0±0.4 versus 5.7±0.2 arterioles/mm2 in vehicle; P<0.01; Figure 3D), and large arterioles (4.0±0.2 and 3.6±0.2 versus 2.2±0.2 arterioles/mm2 in vehicle; P<0.01; Figure 3D) in the implant site around the arterial occlusion. The effects induced by MB-MSC-GLP and MB-MSC treatments were very large (Cohen d coefficient: 10.8 and 4.8 for capillaries, 7.6 and 4.7 for small arterioles, and 4.1 and 3.2 for large arterioles, respectively). Interestingly, pairwise comparison indicates that MB-MSC-GLP is superior to MB-MSC treatment in increasing capillary and small arteriole density (P<0.05 for both comparisons; Cohen d coefficient, 1.5 and 1.6 for capillaries and arterioles, respectively). Treatment with MB did not affect local vascularization (218±4 capillaries/mm2, 5.3±0.6 small arterioles/mm2 and 2.3±0.3 large arterioles/mm2; P=NS versus vehicle).

To verify the functional status of periocclusional vascularization, whole-mount preparations of the implantation site were analyzed after intravenous injection of the endothelial marker isolectin. Three-dimensional reconstruction of confocal microscopy images showed numerous functional capillaries and arterioles bridging the space between implanted MB-MSC-GLP and MB-MSC and the ischemic muscles (Figure 4; Movie I in the online-only Data Supplement).

Figure 4.

Figure 4. Representative confocal images showing numerous functional capillaries (stained green with isolectin) and arterioles (stained with both isolectin and α-smooth muscle actin and pointed by red arrowheads) bridging the gap between microbeads encapsulated with mesenchymal stem cells (MB-MSCs) and ischemic muscle at 21 days postischemia. The dotted line delimits the beads implant. Scale bars, 100 µm.

Activation of Angiogenic Factors

To verify the mechanisms for improved angiogenesis, we measured gene and protein expression in limb muscles at 7 days after ischemia.

Results of cytometric bead arrays show the expression of human angiogenic proteins in ischemic muscles of MB-MSC– and MB-MSC-GLP–treated mice (Figure 5), indicating persistence of paracrinally active human cells. Furthermore, RT-profiler polymerase chain reaction array showed a marked activation of murine genes associated with angiogenesis, such as endoglin, VEGF-A, sphingosine kinase 1, angiopoietin 4, interleukin-8, and heparanase, in ischemic muscles of MB-MSC– and MB-MSC-GLP–treated mice compared with vehicle (Table). Notably, the 2 treatments markedly reduced the expression of β2-microglobulin, which is a biomarker of peripheral vascular disease,23 as well as the expression of several antiangiogenic genes, such as tissue inhibitor of metalloproteinase 2, tissue inhibitor of metalloproteinase 3, and thrombospondin 2, and proinflammatory genes associated with T lymphocytes induction of MSC apoptosis, including interferon-γ and tumor necrosis factor-α. Moreover, quantitative polymerase chain reaction confirmed the expression of the GLP transgene in limb muscles injected with MB-MSC-GLP (Figure III in the online-only Data Supplement).

Figure 5.

Figure 5. Bar graphs showing the level of human proangiogenic proteins among study groups. Data represented as median and 5 to 95 percentile; n=5 in each group. MB indicates microbeads; MB-MSCs, MB encapsulated with mesenchymal stem cells; and MB-MSC-GLP, MB encapsulated with mesenchymal stem cells transfected with glucagon-like peptide-1.

Table. Modulation of Pro- and Antiangiogenic Genes in MB-MSC– and MB-MSC-GLP–Treated Groups Compared With the Vehicle as Measured by the RT-Profiler PCR Angiogenesis Array

GenesDescriptionFold Changes in MB-MSCs vs VehicleFold Changes in MB-MSC-GLP vs Vehicle
ANGPTL4Angiopoietin 42.112.30Upregulated genes
CCL2Chemokine (C-C motif) ligand 21.802.14
CXCL3Chemokine(C-X-C motif) ligand 33.342.73
ENGEndoglin6.274.66
HPSEHeparanase3.142.89
IL8Interleukin-82.753.01
SPHK1Sphingosine kinase 14.222
THBS1Thrombospondin 13.173.76
VEGF-AVascular endothelial growth factor A4.122.49
B2Mβ2-microglobulin−2.85−4.66Downregulated genes
TIMP2Tissue inhibitor of metalloproteinase 2−18.74−10.27
TIMP3Tissue inhibitor of metalloproteinase 3−8.07−9.06
TNFTumor necrosis factor−6.44−7.78
THBS2Thrombospondin 2−5.94−5.43
PLGPlasminogen−9.27−15.67
IL6Interleukin-6−6.71−2.36
IFNGInterferon-γ−4.55−3.84

MB-MSCs indicates microbeads encapsulated with mesenchymal stem cells; MB-MSC-GLP, microbeads encapsulated with mesenchymal stem cells transfected with glucagon-like peptide-1; and PCR, polymerase chain reaction.

The interaction of differentially regulated genes was then investigated using the STRING database (Figure 6). Results indicate that transplantation of MB-MSCs interferes with a molecular network centered on VEGF-A, interleukin-6, interferon-γ, and tumor necrosis factor-α, leading to changes in the balance between angiogenic mediators (the chemokines CXCL3 and CCL2/MCP1, the membrane glycoprotein endoglin, and the S1P activator sphingosine kinase 1), stabilizers of vascular growth (angiopoietin 4 and thrombospondin 1), and enzymes implicated in activation (heparanase) or inhibition (tissue inhibitor of metalloproteinases) of extracellular matrix degradation.

Figure 6.

Figure 6. Molecular network induced by microbeads encapsulated with mesenchymal stem cells (MB-MSCs) in murine ischemic muscle. Upregulated (A) and downregulated (B) murine genes are shown separately. In addition, the full network is shown (C) with indication of the nature of interaction. The size of each circle is proportional to the difference in expression as compared with vehicle. ANGPTL4 indicates angiopoietin 4; B2M, β2-microglobulin; CCL2, chemokine (C-C motif) ligand 2; CXCL3, chemokine(C-X-C motif) ligand 3; ENG, endoglin; HPSE, heparanase; IL6, interleukin-6; IL8, interleukin-8; IFNG, interferon-γ; PLG, plasminogen; SPHK1, sphingosine kinase 1; TIMP2, tissue inhibitor of metalloproteinase 2; THBS1, thrombospondin 1; THBS2, thrombospondin 2; TIMP3, tissue inhibitor of metalloproteinase 3; TNF, tumor necrosis factor; and VEGF-A, vascular endothelial growth factor A.

Importance of Cell Delivery Route and Encapsulation

Next, we evaluated the benefit of direct transplantation of encapsulated MSC in ischemic muscles. Intramuscular delivery of MB-MSCs was ineffective in improving the blood flow recovery or muscular capillary density in immunocompetent mice with limb ischemia (Figure IVA and IVB in the online-only Data Supplement). Furthermore, bead injection caused distortion of the muscle structure (Figure IVCi and IVCii in the online-only Data Supplement) and marked infiltration of inflammatory cells expressing isolectin and the macrophage/monocyte marker F4/80 (Figure IVCiii–IVCvi in the online-only Data Supplement). Therefore, perivascular delivery of the current CellBead formulation seems to be superior to intramuscular injection.

In separate experiments, we evaluated the therapeutic activity of perivascular or intramuscular transplantation of nonencapsulated MSCs. In both cases, allogeneic cell therapy was unable to produce improvement of blood flow recovery in immunocompetent mice with limb ischemia (Figure VA and VE in the online-only Data Supplement). Furthermore, no difference was observed in the capillary and arteriole density of muscles directly injected with nonencapsulated MSCs or vehicle (Figure VB–VD in the online-only Data Supplement). Perivascular delivery of nonencapsulated MSCs caused a mild increase in capillary density of the ischemic muscle (P<0.04 versus vehicle), but no change in arteriole density (Figure VF–VH in the online-only Data Supplement). To verify whether the lack of therapeutic activity is related to the rapid clearance of cells that are not protected by an immunoisolating shell, we next measured the expression of human angiogenic proteins in mice given nonencapsulated MSCs (either intramuscularly or perivascularly) or vehicle. Human proteins were undetectable in ischemic murine muscles from all studied groups. These data suggest functional inactivation of nonencapsulated MSCs at 7 days from transplantation in perivascular or interstitial muscular space.

Discussion

This study is the first to show the therapeutic activity of a cell product consisting of encapsulated, genetically modified MSCs, which were delivered perivascularly in a mouse model of limb ischemia. On stabilization at the implantation site, the cell product induces microvascular angiogenesis in the ischemic muscle as well as collateralization alongside the occluded femoral artery, leading to restoration of perfusion, oxygenation, and remarkable improvement of limb salvage. The therapeutic effect is mainly attributable to paracrine modulation of a molecular network in which VEGF-A represents the central hub.

The originality in the design of CellBeads consists of a miniaturized 2-step encapsulation, which is critical for the product performance because it allows for improved bioavailability and tolerability (Figure I in the online-only Data Supplement). This technology will only work with nontumorigenic cell lines because proliferating cells would burst the specifically designed capsule and result in the loss of function. Importantly, independent investigation on toxicity and tumorigenicity on CellBeads is reassuring (unpublished data). Furthermore, the novel approach described here combines several implementations of cell therapy in 1 single cell product. The CellBeads formulation enables long-lasting cell retention in the transplanted site and integrates the native paracrine activity of MSCs with a genetic modification conferring encapsulated cells with the ability to produce and secrete GLP-1, an incretin hormone that has both antiapoptotic and cytoprotective effects.24 Delivery of MB-MSC-GLP alongside the occluded femoral artery allows for authentic stimulation of collateralization together with potentiation of muscular vascularization.

Individual elements of this approach proved to benefit postischemic angiogenesis. For instance, cell therapy with bone marrow–derived MSCs reportedly improves limb function, reduces the incidence of autoamputation, and attenuates muscle atrophy in a mouse model of limb ischemia.25 Moreover, MSCs promote lower limb perfusion and foot ulcer healing in patients with CLI, either given alone26 or in combination with endothelial progenitor cells.27 MSCs engineered with growth factors or antiapoptotic agents, including Akt,28 adrenomedullin,29 and angiopoietin,30 showed incremental enhancements of therapeutic activity in models of ischemia. Likewise, intracoronary delivery of GLP-1–overexpressing MSCs induces substantial cardiac recovery in an acute myocardial infarction model.20 Furthermore, the BONMOT-1&2 trials showed the feasibility and advantage of perivascular delivery of dispersed stem cells in CLI.10 Rapid inactivation of transplanted cells occurs, however, in an allogeneic setting, as documented by the present study. Encapsulation of human MSCs prevents this phenomenon, thus providing a potential means of treating patients with severely debilitating CLI with a single off-the-shelf cell product.

All the above concepts were integrated in the strategy of delivering immunoprotected MSCs to the vascular occlusion site. Our study shows for the first time that the procedure is feasible and therapeutically useful. Data highlight the additional benefit from 1 particular manifestation of the CellBead technology, whereby MSCs have been genetically modified to secrete a GLP-1 fusion protein. The GLP-1–enriched cell product showed superior foot-salvaging and collateral-forming capacities as compared with MB-MSCs. After arterial occlusion, preexisting vessels start enlarging through an arteriogenic process that is mainly triggered by increased shear stress, but also involves soluble factors, inflammatory cells, cell proliferation, and the remodeling of the extracellular matrix. We found that the arteriogenic process is enhanced by MB-MSC-GLP and, to a lesser extent, by MB-MSCs, most likely through secreted factors that are released into the surrounding environment in the immediate vicinity of the occluded artery. The method used in this study advantageously direct GLP-1 and other native growth factors to the anatomic region where collateralization is maximally desirable, resulting in a robust neovascularization of arterioles coaxially oriented with the femoral artery. This represents a significant improvement over conventional intramuscular cell therapy, which disperses cells and therapeutic mediators in an unpredictable manner.24 In addition, perivascular delivery of MB-MSC-GLP enhanced the tributary microvascular bed in hindlimb muscles, as documented by increased counts of capillaries and small arterioles in the ischemic adductor. Measurement of human protein expression confirms that CellBeads are secreting proangiogenic factors at day 7 postimplantation, and this is accompanied by an upregulation of murine proangiogenic genes and corresponding downregulation of antiangiogenic genes. The functional improvements in the model are, therefore, likely to arise from a combination of direct effects of the beneficial secretion of proteins, such as VEGF-A from the CellBeads (which is highly conserved and, therefore, active in mouse), together with additional modulation of gene expression in the tissues local to the implant site by the secreted paracrine factors. These factors might reach the skeletal muscle by diffusion through the interstitium or via the expanded collaterals. Therefore, the method allows for additively advantageous implementation of proximal collateralization and distal microvascular angiogenesis.

The present CellBead formulation does not seem to be suitable for intramuscular delivery as benefits of cell therapy are overwhelmed by spatial restriction and excessive inflammatory reaction in the mouse muscle in this model. Studies in large animal models are necessary to compare feasibility and therapeutic activity of direct intramuscular injection versus delivery around the neurovascular femoral bundle.

In summary, the described methodology using microCellBead technology may offer potential as a treatment for peripheral vascular disease, whereby administration of multiple microCellBead depots within the vicinity of diseased vessels could promote revascularization and re-established blood flow to the ischemic limb. The approach is particularly attractive for use in patients in whom interventional revascularization is not amenable because of multiple or distal obstructions. Moreover, perivascular cell therapy might be used as an adjuvant treatment in conjunction with or preparation to operative revascularization.

Conclusions

After femoral arterial ligation in a mouse, application of CellBeads in the perivascular space enhances collateralization and neoangiogenesis through secretion of a variety of paracrine factors that may act directly as well as indirectly by inducing the upregulation of proangiogenic chemokines and downregulation of antiangiogenic genes. Functional neovascularization made by capillaries and small-medium size arterioles is significantly enhanced around the site of administration. This leads to significantly improved blood flow, increased tissue oxygenation, and reduced toe necrosis. These results demonstrate the potential for CellBead technology in the treatment of peripheral vascular disease.

Footnotes

*These authors contributed equally.

The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.113.301217/-/DC1.

Correspondence to Paolo Madeddu, MD, CS FAHA, Chair of Experimental Cardiovascular Medicine, Head of Regenerative Medicine, Section Bristol Heart Institute, School of Clinical Sciences, University of Bristol, Level 7, Bristol Royal Infirmary, Upper Maudlin St, Bristol BS2 8HW, United Kingdom. E-mail

References

  • 1. Hiatt WR. Medical treatment of peripheral arterial disease and claudication.N Engl J Med. 2001; 344:1608–1621.CrossrefMedlineGoogle Scholar
  • 2. Conte MS. Bypass versus Angioplasty in Severe Ischaemia of the Leg (BASIL) and the (hoped for) dawn of evidence-based treatment for advanced limb ischemia.J Vasc Surg. 2010; 51(5 suppl):69S–75S.CrossrefMedlineGoogle Scholar
  • 3. Faglia E, Clerici G, Losa S, Tavano D, Caminiti M, Miramonti M, Somalvico F, Airoldi F. Limb revascularization feasibility in diabetic patients with critical limb ischemia: results from a cohort of 344 consecutive unselected diabetic patients evaluated in 2009.Diabetes Res Clin Pract. 2012; 95:364–371.CrossrefMedlineGoogle Scholar
  • 4. Blum A, Balkan W, Hare JM. Advances in cell-based therapy for peripheral vascular disease.Atherosclerosis. 2012; 223:269–277.CrossrefMedlineGoogle Scholar
  • 5. Tongers J, Roncalli JG, Losordo DW. Therapeutic angiogenesis for critical limb ischemia: microvascular therapies coming of age.Circulation. 2008; 118:9–16.LinkGoogle Scholar
  • 6. Matoba S, Tatsumi T, Murohara T, Imaizumi T, Katsuda Y, Ito M, Saito Y, Uemura S, Suzuki H, Fukumoto S, Yamamoto Y, Onodera R, Teramukai S, Fukushima M, Matsubara H; TACT Follow-up Study Investigators. 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; 156:1010–1018.CrossrefMedlineGoogle Scholar
  • 7. Burdon TJ, Paul A, Noiseux N, Prakash S, Shum-Tim D. Bone marrow stem cell derived paracrine factors for regenerative medicine: current perspectives and therapeutic potential.Bone Marrow Res. 2011; 2011:207326.CrossrefMedlineGoogle Scholar
  • 8. Ruiz-Salmeron R, de la Cuesta-Diaz A, Constantino-Bermejo M, Pérez-Camacho I, Marcos-Sánchez F, Hmadcha A, Soria B. Angiographic demonstration of neoangiogenesis after intra-arterial infusion of autologous bone marrow mononuclear cells in diabetic patients with critical limb ischemia.Cell Transplant. 2011; 20:1629–1639.CrossrefMedlineGoogle Scholar
  • 9. Powell RJ. Update on clinical trials evaluating the effect of biologic therapy in patients with critical limb ischemia.J Vasc Surg. 2012; 56:264–266.CrossrefMedlineGoogle Scholar
  • 10. Amann B, Lüdemann C, Rückert R, Lawall H, Liesenfeld B, Schneider M, Schmidt-Lucke J. Design and rationale of a randomized, double-blind, placebo-controlled phase III study for autologous bone marrow cell transplantation in critical limb ischemia: the BONe Marrow Outcomes Trial in Critical Limb Ischemia (BONMOT-CLI).Vasa. 2008; 37:319–325.CrossrefMedlineGoogle Scholar
  • 11. Weber C, Pohl S, Poertner R, Pino-Grace P, Freimark D, Wallrapp C, Geigle P, Czermak P. Production process for stem cell based therapeutic implants: expansion of the production cell line and cultivation of encapsulated cells.Adv Biochem Eng Biotechnol. 2010; 123:143–162.MedlineGoogle Scholar
  • 12. Goren A, Dahan N, Goren E, Baruch L, Machluf M. Encapsulated human mesenchymal stem cells: a unique hypoimmunogenic platform for long-term cellular therapy.FASEB J. 2010; 24:22–31.CrossrefMedlineGoogle Scholar
  • 13. Montanucci P, Pennoni I, Pescara T, Blasi P, Bistoni G, Basta G, Calafiore R. The functional performance of microencapsulated human pancreatic islet-derived precursor cells.Biomaterials. 2011; 32:9254–9262.CrossrefMedlineGoogle Scholar
  • 14. Hernández RM, Orive G, Murua A, Pedraz JL. Microcapsules and microcarriers for in situ cell delivery.Adv Drug Deliv Rev. 2010; 62:711–730.CrossrefMedlineGoogle Scholar
  • 15. Wallrapp C, Thoenes E, Thürmer F, Jork A, Kassem M, Geigle P. Cell-based delivery of glucagon-like peptide-1 using encapsulated mesenchymal stem cells.J Microencapsul. 2013; 30:315–324.CrossrefMedlineGoogle Scholar
  • 16. Bose AK, Mocanu MM, Carr RD, Brand CL, Yellon DM. Glucagon-like peptide 1 can directly protect the heart against ischemia/reperfusion injury.Diabetes. 2005; 54:146–151.CrossrefMedlineGoogle Scholar
  • 17. Sokos GG, Nikolaidis LA, Mankad S, Elahi D, Shannon RP. Glucagon-like peptide-1 infusion improves left ventricular ejection fraction and functional status in patients with chronic heart failure.J Card Fail. 2006; 12:694–699.CrossrefMedlineGoogle Scholar
  • 18. Liu Z, Stanojevic V, Brindamour LJ, Habener JF. GLP1-derived nonapeptide GLP1(28-36)amide protects pancreatic β-cells from glucolipotoxicity.J Endocrinol. 2012; 213:143–154.CrossrefMedlineGoogle Scholar
  • 19. Huang WC, Newby GB, Lewis AL, Stratford PW, Rogers CA, Newby AC, Murphy GJ. Periadventitial human stem cell treatment reduces vein graft intimal thickening in pig vein-into-artery interposition grafts.J Surg Res. 2013;183:33–39.CrossrefGoogle Scholar
  • 20. Houtgraaf JH, de Jong R, Monkhorst K, Tempel D, Dendekker WK, Kazemi K, Hoefer I, Pasterkamp G, Lewis AL, Stratford PW, Wallrapp C, Zijlstra F, Duckers HJ. Feasibility of intracoronary glp-1 eluting cellbead infusion in acute myocardial infarction.Cell Transplant. 2013;22:535–543.CrossrefGoogle Scholar
  • 21. Wright EJ, Farrell KA, Malik N, Kassem M, Lewis AL, Wallrapp C, Holt CM. Encapsulated glucagon-like peptide-1-producing mesenchymal stem cells have a beneficial effect on failing pig hearts.Stem Cells Transl Med. 2012; 1:759–769.CrossrefMedlineGoogle Scholar
  • 22. Coppen D. Company profile: Biocompatibles International plc: local drug delivery for targeted therapies.Regen Med. 2010; 5:189–195.CrossrefMedlineGoogle Scholar
  • 23. Wilson AM, Kimura E, Harada RK, Nair N, Narasimhan B, Meng XY, Zhang F, Beck KR, Olin JW, Fung ET, Cooke JP. Beta2-microglobulin as a biomarker in peripheral arterial disease: proteomic profiling and clinical studies.Circulation. 2007; 116:1396–1403.LinkGoogle Scholar
  • 24. Gnecchi M, Zhang Z, Ni A, Dzau VJ. Paracrine mechanisms in adult stem cell signaling and therapy.Circ Res. 2008; 103:1204–1219.LinkGoogle Scholar
  • 25. Kinnaird T, Stabile E, Burnett MS, Shou M, Lee CW, Barr S, Fuchs S, Epstein SE. Local delivery of marrow-derived stromal cells augments collateral perfusion through paracrine mechanisms.Circulation. 2004; 109:1543–1549.LinkGoogle Scholar
  • 26. Lu D, Chen B, Liang Z, Deng W, Jiang Y, Li S, Xu J, Wu Q, Zhang Z, Xie B, Chen S. Comparison of bone marrow mesenchymal stem cells with bone marrow-derived mononuclear cells for treatment of diabetic critical limb ischemia and foot ulcer: a double-blind, randomized, controlled trial.Diabetes Res Clin Pract. 2011; 92:26–36.CrossrefMedlineGoogle Scholar
  • 27. Lasala GP, Silva JA, Gardner PA, Minguell JJ. Combination stem cell therapy for the treatment of severe limb ischemia: safety and efficacy analysis.Angiology. 2010; 61:551–556.CrossrefMedlineGoogle Scholar
  • 28. Gnecchi M, He H, Noiseux N, Liang OD, Zhang L, Morello F, Mu H, Melo LG, Pratt RE, Ingwall JS, Dzau VJ. Evidence supporting paracrine hypothesis for Akt-modified mesenchymal stem cell-mediated cardiac protection and functional improvement.FASEB J. 2006; 20:661–669.CrossrefMedlineGoogle Scholar
  • 29. Hanabusa K, Nagaya N, Iwase T, Itoh T, Murakami S, Shimizu Y, Taki W, Miyatake K, Kangawa K. Adrenomedullin enhances therapeutic potency of mesenchymal stem cells after experimental stroke in rats.Stroke. 2005; 36:853–858.LinkGoogle Scholar
  • 30. Piao W, Wang H, Inoue M, Hasegawa M, Hamada H, Huang J. Transplantation of Sendai viral angiopoietin-1-modified mesenchymal stem cells for ischemic limb disease.Angiogenesis. 2010; 13:203–210.CrossrefMedlineGoogle Scholar

Significance

Surgical bypass surgery or percutaneous revascularization is the gold standard for the treatment of peripheral vascular disease, producing long-term benefit, with a 5-year limb salvage rate of >80%. Gene or stem cell therapies have accredited to provide a possible alternative to surgical bypass surgery in patients with multivascular disease or occlusions of small-caliber blood vessels, which are commonly seen in those with diabetes mellitus or hypertension. However, the mode of cell delivery for optimal revascularization is highly debatable. The new methodology of administration of multiple microCellBead depots within the vicinity of the diseased vessels as described in this study could be attractive for use in these patients to promote revascularization and re-establish the blood flow to the ischemic limb. Moreover, perivascular cell therapy might be used as an adjuvant treatment in conjunction with or preparation to operative revascularization.