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Comparison of treatments of peripheral arterial disease with mesenchymal stromal cells and mesenchymal stromal cells modified with granulocyte and macrophage colony-stimulating factor
Cytotherapy, 2013; 15: 820e829
Comparison of treatments of peripheral arterial disease with mesenchymal stromal cells and mesenchymal stromal cells modified with granulocyte and macrophage colony-stimulating factor
FLAVIA FRANCO DA CUNHA*, LEONARDO MARTINS*, PRISCILA KEIKO MATSUMOTO MARTIN, ROBERTA SESSA STILHANO, EDGAR JULIAN PAREDES GAMERO & SANG WON HAN Department of Biophysics, Universidade Federal de São Paulo, São Paulo-SP, Brazil Abstract Background aims. Granulocyte macrophage-colony stimulating factor (GM-CSF) promotes vessel formation through several molecular signaling pathways. Mesenchymal stromal cells (MSCs) have an important role in neovasculogenesis during ischemia because they release pro-angiogenic paracrine factors, pro-survival and immunomodulatory substances and can differentiate into endothelial cells. The objective of this study was to evaluate whether there is synergy between GM-CSF and MSCs in recovering ischemic limbs. Methods. MSCs from mouse bone marrow were transduced with a lentiviral vector expressing GM-CSF and injected into animals with surgically induced limb ischemia, with unmodified MCSs used as control. The evolution of limb necrosis was evaluated for 1 month. Muscle strength was assessed on the 30th day, and the animals were euthanized to determine the muscle mass and to perform histological analyses to determine the degree of cellular infiltration, capillary and microvessel densities, fibrosis, necrosis and tissue regeneration. Results. Both treatments were able to ameliorate ischemia, decrease the areas of fibrosis, necrosis, adipocytes and leukocyte infiltrates and increase the number of capillaries. The addition of GM-CSF promoted the formation of larger vessels, but it also resulted in more fibrosis and less muscle mass without affecting muscle force. Conclusions. Both treatments resulted in a remarkable amelioration of ischemia. More fibrosis and less muscle mass produced by the overexpression of GM-CSF did not affect muscle functionality significantly. Importantly, MSCs overexpressing GM-CSF produced larger vessels, which is an important long-term advantage because larger vessels are more efficient in the reperfusion of ischemic tissues physiologically. Key Words: cell therapy, gene therapy, GM-CSF, ischemia, stromal cells
Introduction Peripheral arterial disease is caused by the obstruction of arteries, leading to decreased blood flow. It is a chronic disease that affects 3e10% of the world population (1). With disease progression, approximately 30% of patients are faced with the possibility of limb amputation within 1 year (1). These patients depend on the adaptation of preexisting collateral vessels (arteriogenesis) or the formation of new vessels through vasculogenesis or angiogenesis to recover tissue oxygenation (2,3). Thus, therapeutic neovascularization by means of angiogenic factors or stem cells, aiming at rapid revascularization of the ischemic area, represents a potential treatment option for regenerating the damaged tissue and preventing amputations (4).
Mesenchymal stromal cells (MSCs) are cells that can adhere to plastic, self-renew and differentiate into osteoblasts, chondrocytes and adipocytes (5). Therapies that use MSCs have been widely used in cardiovascular medicine. Several studies indicate that MSCs are an important means for neovasculogenesis, especially during ischemia (6e9), because the reduction of oxygen levels (hypoxia) induces MSC to form capillary-like structures in vitro (10). When implanted, MSCs can differentiate into and acquire the characteristics of mature endothelial cells (7,11), vascular smooth muscle cells and cardiomyocytes (12,13). However, the greatest potential of MSCs for neovascularization is related to its trophic effect, producing cytokines with paracrine
*These authors contributed equally to this work. Correspondence: Sang Won Han, PhD, Research Center for Gene Therapy, Department of Biophysics, Universidade Federal de São Paulo, Rua Mirassol 207, São Paulo-SP, CEP 04044-010, Brazil. E-mail: [email protected] (Received 26 June 2012; accepted 25 February 2013) ISSN 1465-3249 Copyright Ó 2013, International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jcyt.2013.02.014
Treatment of PAD with GM-CSF-modified MSCs effects, preventing fibrosis and apoptosis, promoting angiogenesis and arteriogenesis and stimulating the proliferation and differentiation of tissue-specific progenitors, thereby contributing to tissue repair (14). Several studies have shown that the injection of MSCs into ischemic limbs increased blood flow and vascular density (7,9,15). Granulocyte macrophage-colony stimulating factor (GM-CSF) promotes the proliferation, differentiation and survival of hematopoietic cells as neutrophils, monocytes, macrophages and dendritic cells (16,17), in addition to inducing effector functions of monocytes and macrophages, thereby contributing to the defense against pathogenic microbial agents (the inflammatory response) (18). GM-CSF also plays an important role in neovascularization (19), primarily because of its ability to prolong the life of monocytes by inhibiting apoptosis (20), thereby giving monocytes more time in circulation (21,22). Monocytes are involved in tissue remodeling during ischemia by playing important roles in arteriogenesis and angiogenesis through the secretion of several growth factors (23e26). A specific population of monocytes has promoted a new concept of vasculogenesis, which is a process of vessel formation by progenitor cells, specifically endothelial progenitor cells (27). Additionally, GM-CSF is the main chemotactic agent for neutrophils (28), in addition to its ability to inhibit the apoptosis of the same (29,30). A recent study from our laboratory (31) showed that the administration of a plasmid vector expressing GM-CSF into an ischemic mouse hind limb caused a strong therapeutic effect by mobilizing bone marrow stem cells and neutrophils, which increased the blood vessel density and muscle mass and strength. On the basis of these observations, the goal of this study was to evaluate the therapeutic effect of MSCs modified to express GM-CSF in the treatment of mouse ischemic limbs, to compare this treatment with mice treated with only MSCs, and to look for a synergistic effect of MSCs and GM-CSF.
Methods Construction of a lentiviral vector with GM-CSF GM-CSF was isolated from the pVAX-GM-CSF (31) with Bam HI and inserted into the pIRESe2GFP (internal ribosome entry site-green fluorescent protein; addgene, Cambridge, MA, USA), which had been previously digested with the same enzyme. The DNA fragment containing GM-CSF and IRES-GFP was isolated from this vector with NheI and HpaI and inserted into the lentiviral vector M107 (kindly provided by Yvonne Heidemarie Fisher, University of Lund, Stem Cell Center, Lund,
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Sweden), which was previously digested with XbaI and HincII. The final vector was named 107-GMGFP (Figure 1B). Plasmid vectors were amplified and purified with the use of a Qiagen mega-prep kit (Sao Paulo, Brazil). Viral vector production, concentration and titration were performed in a biosafety level 2 (NB2) laboratory, following a protocol established by Naldini et al. (32), after approval of the project by the National Biosecurity Council (CTNBIO) (approval No.: UNIFESP CQB 028/97, 18.07.97) and the Internal Biosecurity Council of the UNIFESP (approval No.: CIBIO No. 13/2010). A set of packaging vectors containing Gag-Pol (10 mg), Rev (5 mg) and VSV-G (6 mg) and the transfer vector (20 mg) containing the gene of interest were used to transfect human endothelial kidney cell (HEK)293T cells (2 106 cells in 75 cm2 plate) with the use of co-precipitation with calcium phosphate. The culture supernatant was collected after 24 and 48 h and filtered through a 0.45-mm syringe filter (Millipore, New York, NY, USA). For each 26 mL of supernatant, 4 mL of a 20% sucrose solution was added, and this mix was centrifuged in the a Sorvall centrifuge with a SS34 rotor at 16,000 rpm for 2 h. After centrifugation, the supernatant was discarded, and the pellet was resuspended in Dulbecco’s modified Eagle’s medium (DMEM) without serum, aliquoted and stored at 80 C. To determine viral titer, HEK293T cells were transduced with different concentrations of lentiviral vectors in the presence of 8 mg/mL Polybrene (Sigma, St Louis, MO, USA), and after 3 days, the GFP-positive cells were counted with the use of an inverted microscope (IX70; Olympus America Inc, Center Valley, PA, USA). The titer was calculated according to the following formula: Titer ¼ % GFP-positive cells total number of cells on the day of transduction/volume of virus. Isolation, characterization and modification of MSCs from mouse bone marrow Eight-week-old BALB/c mice were killed by cervical dislocation, following the procedure recommended and approved by the ethics and research committee of the Federal University of São Paulo (UNIFESP) (approval No.: CEP 0327/10). The tibia and femur were dissected, and DMEM with low concentrations of glucose, supplemented with 10% fetal bovine serum (FBS), 2 mmol/L L-glutamine, 200 U/mL penicillin and 200 mg/mL streptomycin (this supplemented DMEM was named DMEMc), was injected into the dissected bone to collect the bone marrow cells, which were maintained in six-well plates. The
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Figure 1. Characterization and genetic modification of MSC and GM-CSF gene expression analysis. (A) MSC differentiation. Bone marrow MSC (I) were differentiated into adipocytes (II) or osteoblasts (III) and stained with oil red O and alizarin red, respectively. (B) Schematic representation of human immunodeficiency virusebased lentiviral vector. DU5 LTR indicates self-inactivating long-terminal repeat with deletion at the U5 region; J, packaging signal; RRE, Rev responsive element; CMV, cytomegalovirus promoter; iRES, internal ribosome entry site; GFP, green fluorescent protein; WPRE, woodchuck hepatitis virus post-transcriptional regulatory element; LTR, long terminal repeat. (C) Transduction of MSC. Phase-contrast micrographs (I, IV), fluorescent image of GFP expression (II, V) and overlay of phase-contrast and fluorescent images (III, VI) are shown. Images were acquired 3 (I, II, III) and 30 (IV, V, VI) days after transduction. (D) GM-CSF gene expression analysis of MSCs and MSCs transduced with GM-CSF after 3 days by quantitative RT-PCR. *P < 0.05.
Treatment of PAD with GM-CSF-modified MSCs medium was regularly replaced with fresh DMEMc (33). Adherent cells were treated with a 0.25% trypsin solution, centrifuged, resuspended and plated in culture bottles (25 or 75 cm2) with DMEMc at 37 C with 5% CO2 using cell culture incubators (Model 3158; Thermo Fisher Scientific Inc, Waltham, MA, USA). All reagents used in this step were purchased from Invitrogen (Grand Island, NY, USA). The capacity of these cells to differentiate into adipocytes and osteoblasts was evaluated on the basis of an established protocol (33). For osteogenic differentiation, 2 105 cells were plated per well in a six-well plate. DMEM, supplemented with 108 mol/L dexamethasone, 5 mg/mL ascorbic acid 2-phosphate, 10 mmol/L b-glycerophosphate and 10% FBS, was used, with continuous medium exchange every 3 days for 3e4 weeks. The osteoblasts were stained with 2% alizarin red S, pH 4.1 (SigmaAldrich, St Louis, MO, USA). For adipogenic differentiation, 2 105 cells were plated per well in six-well plates containing DMEM supplemented with 10% FBS, 108 mol/L dexamethasone, 2.5 mg/mL insulin and 5 mmol/L rosiglitazone. The cells were maintained with continuous medium exchange every 3 days for 3e4 weeks. The adipocytes were stained with oil red O (3.75% in 60% isopropyl alcohol) (Sigma-Aldrich) For staining, the medium was aspirated, cells were fixed with 4% paraformaldehyde, washed with phosphate-buffered saline (PBS) and incubated with the solution of oil red O or alizarin red S for 5 minutes. For transduction, 1 105 MSC per well were initially seeded in six-well-plates with DMEMc. Eighteen hours later, the medium was replaced with 1 mL of fresh DMEMc, and 20 mL of the concentrated viral solution was added in the presence of Polybrene (Sigma) to a final concentration of 8 mg/mL. After 24 h, the medium was replaced with 3 mL of fresh DMEMc and the cells were maintained in culture at 37 C and 5% CO2 for 3 days. The transduction efficiency was determined after this period by counting GFP-positive cells with an inverted fluorescence microscope. To verify the maintenance of transgene expression in the transduced MSCs, GFP expression was monitored weekly for 1 month with a fluorescence microscope. Analysis of GM-CSF expression by quantitative realtime polymerase chain reaction The total RNA of MSCs was extracted with the use of an RNAeasy kit (Qiagen) and treated with DNAse I (Sigma-Aldrich). The cDNA was synthesized with the use of a High Capacity Reverse Transcription kit (Life Technologies, Carlsbad, CA, USA), and a
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quantitative real-time polymerase chain reaction (RTPCR) assay was performed with the use of the SYBR Green QuantiFast RT-PCR Kit (Qiagen, Hilden, Germany) in the Rotor Gene-Q (Qiagen). The following primers were used for the quantitation of murine GM-CSF: GM-CSF_F, CCC AAG AAG TGA TCC ACC TG and GM-CSF_R, GGG CAC TCT GCA ATG TTA GCT T. The relative gene expression was calculated by 2DDCT. The murine b-actin gene was used as a normalizer with the following primers: b-actin_F: GCT CCT CCT GAG CGC AAG, and b-actin_R: CAT CTG CTG GAA GGT GGA CA. Each reaction was performed in duplicate, and each experiment was performed at least three times. Hind limb ischemia induction and cell therapy Induction of ischemia was performed surgically in 10- to 12-week-old BALB/c mice. After anesthesia with ketamine (40 mg/kg) and xylazine (10 mg/kg), ischemia was induced in the right leg by the removal of the entire femoral artery and the closure of its branches (deep femoral, epigastric, saphenous and popliteal arteries), on the basis of the procedure already established in our laboratory (31,34). Five days after ischemic induction, mice were anesthetized again, the quadriceps were exposed and 5 105 cells in 50 mL DMEM were injected into the middle of the quadriceps with the use of a 21-gauge needle. MSCs between 7 and 10 passages were used in the in vivo experiments. The mice were divided into five groups (n ¼ 6 per group) as follows: nonischemic mice (N-IS), sham-operated mice (S), ischemic mice (IS), ischemic mice treated with MSCs (MSC) and ischemic mice treated with MSCs modified with GM-CSF and GFP (MSC-GM). The animals were followed for 35 days, and the visual assessment of limbs was performed weekly on the basis of the following scale: I, no change; II, nailblackening; III, necrosis on toes; IV, necrosis below the heel. On the 35th day, before the euthanasia of the animals, the isometric muscle force was determined according to the method standardized in our laboratory (31,35). Briefly, a mouse was anesthetized and set on a table; the gastrocnemius muscle was isolated, to maintain the vascular connections and the origin of the muscle, and the tendinous insertion of the muscle was isolated and connected to the force transducer (iWorx/CB Science, Inc, Dover, NH, USA). The distal portion of the sciatic nerve was exposed and connected to bipolar electrodes, which were connected to an electrostimulator (Grass S88; Grass Instruments, Quincy, MA, USA). The muscle function was evaluated by the isometric contraction
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response, with an adjustment of the rest tension, to obtain the maximum muscle strength (tetanus), with the use of the peak voltage curve caused by the electrostimulator. Muscle strength was recorded and analyzed by Powerlab 8/30 software (AD Instruments Pty Ltd, Colorado Springs, CO, USA). Histological analysis The animals were euthanized and perfused with intravascular injection of PBS, and manipulated muscles were removed and washed with PBS to eliminate blood. The mass of the quadriceps and gastrocnemius muscle was determined on an analytical balance. The muscles were fixed in 4% paraformaldehyde for 48 h, dehydrated and embedded in paraffin. Sections of 4-mm thickness were obtained and stained with hematoxylin and eosin (HE) to determine the degree of muscle regeneration and the presence of adipocytes and infiltrated cells or with picrosirius red to quantify the degree of fibrosis. Other sections were collected on glass slides coated with poly-L-lysine and subjected to immunohistochemistry with the use of antibody anti-aeactin (1:50) (Dako Denmark A/S, Glostrup, Denmark, clone 1A4) to mark smooth muscle cells or stained with lectin Griffonia (bandeiraea) simplicifolia I biotinylated (1:100) (Vector Laboratories, Peterborough, UK) to mark endothelium, followed by incubation with streptavidinperoxidase (1:100) (Sigma-Aldrich) and detection with chromogen diaminobenzidine (Dako). The images were obtained with the use of an optical microscope (Olympus BX60) and analyzed digitally. Morphometric analyses of skeletal muscle tissue were performed on each slide. Images were captured in at least 10 fields of the wounded area per slide to evaluate necrosis, muscle regeneration, and degeneration, fibrosis and angiogenesis, with the use of Image Pro Plus software (Media Cybernetics, Bethesda, MD, USA). Statistical analysis GraphPad Prism software (version 5.01) was used for comparison between groups. Student’s t and MannWhitney tests were applied for parametric and nonparametric analyses. Values of P < 0.05 were considered statistically significant. Results To evaluate whether an ex vivo gene therapy with the use of MSCs that constitutively secrete GM-CSF is more effective in treating limb ischemia, MSCs were first obtained from the bone marrow of BALB/c mice for characterization. Most of the MSCs used were
spindle-shaped cells after approximately 2 weeks of extraction. The cells that were cultivated in predetermined media to differentiate into adipocytes or osteocytes showed the expected phenotypes and staining (Figure 1A); this is one of the most important characterizations of MSCs (5). A lentiviral vector was chosen for MSC modification because of its high ability to transduce quiescent or low-growth-rate cells; it also produces long-term gene expression (36). Several authors have demonstrated that MSCs are difficult to transduce because of the low expression of viral membrane receptors, and lentiviral vectors have been more efficient than other viral vectors to transduce MSCs (37e39). To verify the functionality of the viral vector preparations, concentrated viral stocks were used to transduce HEK293T cells, and the number of GFPpositive cells was determined by means of an inverted fluorescence microscope: in most cases, a 6 105 transducing units/mL viral titer was obtained. To determine the best conditions for in vivo transduction, MSCs were plated (1 105 cells per well in six-well plates) and transduced with different volumes of the above vector (5, 10 and 20 mL), and the efficiency of transduction was evaluated after 72 h. The best outcome was obtained with 20 mL, resulting in 40% transduction (Figure 1C). In addition, during 1 month of observation, there was no significant variation in the percentage of GFPpositive cells, which confirmed stable integration and gene expression (Figure 1C). GM-CSF gene expression as analyzed by RT-PCR also confirmed much higher expression in transduced cells compared with nontransduced cells (Figure 1D). These results demonstrated that the MSCs that were genetically modified with GM-CSF were of high enough quality to perform the in vivo experiments. The limb ischemia model used to test our hypothesis should represent human peripheral arterial disease, with stable, uniform and relatively severe ischemia. To reach this condition, the femoral artery was removed completely and its branches (deep femoral, epigastric, saphenous and popliteal arteries) were closed surgically (40). This procedure severely affects limb circulation, and within 1 week, the limbs become darkened. However, it is very important to note that this is not a chronic ischemia model, a mouse model of which is not yet available. Because of their different physiology and the size of the animals, it is difficult to establish a chronic ischemia model in small rodents. MSCs were injected only 5 days after ischemia because, in our previous studies, we found that injecting MSCs in the first few days after injury worsened the limbs, with highly affected necrotic areas and increased rates of amputation (results not
Treatment of PAD with GM-CSF-modified MSCs shown). It is likely that the growth factors and proteases, which are released by acute inflammatory cells after ischemic surgery and by the injected MSCs, exacerbated the inflammatory reaction, leading to greater tissue damage. During 30 days of follow-up, a visual evaluation of the necrotic tissue was performed. Seventy percent of the ischemic animals proceeded to advanced necrosis (grade IV), affecting the functionality of the limbs, and the other 30% proceeded to grade III necrosis (Figure 2A,B). In the MSC-treated group, 87.5% had no visual sign of necrosis (grade I), and 12.5% of animals had only blackened nails (grade II). In the group treated with MSC-GM, 83% of the mice were at grade I, whereas 17% were at grade III (Figure 2A), a slightly worse result than the groups treated with MSCs only. To better understand this result, functional and histological analyses were performed. To assess muscle function, a parameter that assesses the whole tissue repair process, including angiogenesis, myogenesis and fibrosis, the strength of the gastrocnemius muscle was measured after 35 days of ischemic induction. The gastrocnemius muscle was chosen for assessment because ischemia affects distant tissues, and this muscle was not manipulated during ischemic surgery. The shamoperated animals had muscle strength of 1.01 N, but ischemic surgery produced almost total loss of strength, with an average muscle strength of 0.14 N. The animals treated with MSCs or MSC-GM showed great recovery, with muscle strength
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reaching 0.79 N and 0.67 N, respectively (Figure 2C). There was a similar profile of muscle recovery for gastrocnemius muscle mass. MSCtreated and MSC-GMetreated groups had a muscle mass of 79% (157 mg) and 63% (124 mg), respectively, of the sham-operated group (197 mg), whereas the untreated ischemic mice had a muscle mass of only 95 mg (Figure 2D). Because inflammation is an important process for muscle cell degeneration and regeneration, muscle tissues were histologically analyzed to evaluate cell infiltrates. HE staining of the gastrocnemius muscle showed a large area that was occupied by adipocytes in the untreated ischemic group (62.8%), which was reduced to 30% and 26% in the groups treated with MSCs or MSC-GM, respectively (Figures 3 and 4A). The infiltration of leukocytes was also greatly reduced in the treated animals compared with ischemic animals: from 12.8% to 0.55% and 1.1% in the groups treated with MSC-GM and MSC, respectively. The cellular degeneration was similar in all groups (Figure 4A); however, a great number of regenerating muscle cells were observed in the MSC and MSC-GM groups, at 57% and 51.8%, respectively, whereas the sham-operated group and the untreated group both presented only 5% of regenerating muscle cells (Figure 4A). These results indicate that the treatment of limb ischemia with MSCs, with or without GM-CSF, was able to promote a marked regeneration of muscle tissue and confirmed the previous visual assessment and strength and muscle mass measurements.
Figure 2. Therapeutic assessments of the MSCs-treated ischemic limbs. (A, B) Limb quality was evaluated visually according to the degree of necrosis. Gastrocnemius muscle force (C) and mass (D) were determined after 30 days of cell therapy. I indicates no change; II, nailblackening; III, necrosis on toes; IV, necrosis below the heel. *P < 0.05.
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Figure 3. Histological analysis of limb muscles. Gastrocnemius muscles were collected after 4 weeks of cell therapy. Tissue samples were stained with (A) HE to assess regenerative (*), necrotic (**), endomysial fibrosis (***) and normal areas, (arrow down) adipocytes, (B) picrosirius red to fibrotic area (stained in red), (C) lectin Griffonia and (D) a-actin antibody to capillary (arrowheads) density and mature vessels (arrow), respectively. High concentration of leucocytes can be found in necrotic area (**). Macrophages are marked with “M” and vessels are marked in brown. Scale bar ¼ 50 mm (A, B) and 20 mm (C, D).
One of the more negative consequences of tissue healing is fibrosis, which leaves the recovered tissue fragile and predisposes it to develop new lesions. The primary cause of fibrosis is the excessive deposition of collagen, which can be stained with picrosirius red. All treated groups had a decrease in fibrosis, reaching 2.59% (MSC) and 3.56% (MSCGM), whereas the ischemic group had 6.77% (Figures 3 and 4B), clearly demonstrating the effects of MSCs in the reduction of fibrosis with or without GM-CSF. The anti-fibrotic role of MSCs has previously been established (41,42), and we previously saw a similar effect with mice treated with GM-CSF (data not shown). However, no synergistic effect was observed in the GM-MSC group; on the contrary, the overexpression of GM-CSF partially affected the anti-fibrotic role of MSCs. This small variation between the two groups should not have
significantly affected the recovery of muscle functionality because the muscle strength did not vary significantly between them (Figure 2C). Finally, we evaluated the neovascularization promoted by mesenchymal cells (6e9) and GMCSF (31,43). Blood vessels were stained with Griffonia, which marks endothelium and activated macrophages, cells that can be easily identified by microscopy, and with the antibody anti-a actin, which marks mature vessels. By staining with Griffonia, we noted that both treatments led to a significant increase in the number of capillaries when compared with the untreated group (MSCs: 619 capillaries/mm2, MSC-GM: 564 capillaries/mm2, IS: 497 capillaries/mm2). Despite a small difference between the treated groups, there was no significance difference between the MSC-treated and MSC-GMetreated groups (Figures 3 and 4C).
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Figure 4. Morphometric analyses of limb muscles. From Figure 3, (A) necrotic, regenerative and normal areas, (B) fibrotic area, (C) capillary density and (D) matured vessels were determined. More than 50 fields of lesions were counted for each group. AI, adipocyte infiltration; LI, leukocyte infiltration; MD, muscle degeneration; MR, muscle regeneration. *P < 0.05.
The immunohistochemistry analysis also showed an increase in the number of mature vessels in both treated groups compared with the ischemic untreated group (40 vessels/mm2), but it was more prominent in the MSC-GM group (61.6 vessels/mm2) than in the MSC group (51.5 vessels/mm2) (Figures 3 and 4D). This difference is probably caused by the arteriogenic property of GM-CSF (20), which we have also previously demonstrated (31). In terms of physiologic functionality, arteriogenesis plays a more important role than angiogenesis because the collateral vessels remodeled by arteriogenesis are larger than those formed by angiogenesis (2,3). This study confirmed the important role of MSCs in the treatment of limb ischemia. All treated groups had a remarkable improvement compared with the untreated ischemic group, decreasing the adipocytes and inflammatory infiltrates, increasing the muscle mass and strength and increasing the number of capillaries and larger vessels. However, the combination of MSCs with GM-CSF did not markedly improve recovery compared with animals treated with only MSCs, except for an increase in larger vessels. The formation of larger vessels is the best way to increase tissue reperfusion after ischemia, especially in larger animals that circulate a greater blood volume. Therefore, ex vivo gene therapy with MSC modified with GM-CSF will be more beneficial for human patients with ischemic
limbs. On the other side, the overexpression of GM-CSF by MSCs also resulted in more fibrosis and less muscle mass. Even these variations did not affect muscle functionality in our ischemic model; in larger animals with chronic limb ischemia, these parameters must be monitored carefully because they contribute greatly in the evolution of physiopathology. Acknowledgments F.F.C. was recipient of a CNPq scholarship, and L.M., R.S.S. and P.K.M.M. were recipients of FAPESP scholarships. This work was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP: Processo No. 2011/00859-6). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. Disclosure of interest: The authors have no commercial, proprietary, or financial interest in the products or companies described in this article. References 1. Norgren L, Hiatt WR, Dormandy JA, Nehler MR, Harris KA, Fowkes FG, et al. Inter-Society Consensus for the Management of Peripheral Arterial Disease (TASC II). Eur J Vasc Endovasc Surg. 2007;33(Suppl 1):S1e75.
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20. Buschmann IR, Hoefer IE, van Royen N, Katzer E, BraunDulleaus R, Heil M, et al. GM-CSF: a strong arteriogenic factor acting by amplification of monocyte function. Atherosclerosis. 2001;159:343e56. 21. Hashimoto SI, Komuro I, Yamada M, Akagawa KS. IL-10 inhibits granulocyte-macrophage colony-stimulating factordependent human monocyte survival at the early stage of the culture and inhibits the generation of macrophages. J Immunol. 2001;167:3619e25. 22. Zbinden R, Vogel R, Meier B, Seiler C. Coronary collateral flow and peripheral blood monocyte concentration in patients treated with granulocyte-macrophage colony stimulating factor. Heart. 2004;90:945e6. 23. Arras M, Ito WD, Scholz D, Winkler B, Schaper J, Schaper W. Monocyte activation in angiogenesis and collateral growth in the rabbit hindlimb. J Clin Invest. 1998;101:40e50. 24. Bergmann CE, Hoefer IE, Meder B, Roth H, van Royen N, Breit SM, et al. Arteriogenesis depends on circulating monocytes and macrophage accumulation and is severely depressed in op/op mice. J Leukoc Biol. 2006;80:59e65. 25. Heil M, Ziegelhoeffer T, Pipp F, Kostin S, Martin S, Clauss M, et al. Blood monocyte concentration is critical for enhancement of collateral artery growth. Am J Physiol Heart Circ Physiol. 2002;283:H2411e9. 26. Buschmann IR, Busch HJ, Mies G, Hossmann KA. Therapeutic induction of arteriogenesis in hypoperfused rat brain via granulocyte-macrophage colony-stimulating factor. Circulation. 2003;108:610e5. 27. Takahashi T, Kalka C, Masuda H, Chen D, Silver M, Kearney M, et al. Ischemia- and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization. Nat Med. 1999;5:434e8. 28. Weisbart RH, Golde DW, Clark SC, Wong GG, Gasson JC. Human granulocyte-macrophage colony-stimulating factor is a neutrophil activator. Nature. 1985;314:361e3. 29. Brach MA, deVos S, Gruss HJ, Herrmann F. Prolongation of survival of human polymorphonuclear neutrophils by granulocyte-macrophage colony-stimulating factor is caused by inhibition of programmed cell death. Blood. 1992;80: 2920e4. 30. Kobayashi SD, Voyich JM, Whitney AR, DeLeo FR. Spontaneous neutrophil apoptosis and regulation of cell survival by granulocyte macrophage-colony stimulating factor. J Leukoc Biol. 2005;78:1408e18. 31. Sacramento CB, Cantagalli VD, Grings M, Carvalho LP, Baptista-Silva JC, Beutel A, et al. Granulocyte-macrophage colony-stimulating factor gene based therapy for acute limb ischemia in a mouse model. J Gene Med. 2009;11:345e53. 32. Naldini L, Blomer U, Gallay P, Ory D, Mulligan R, Gage FH, et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science. 1996;272:263e7. 33. Meirelles Lda S, Nardi NB. Murine marrow-derived mesenchymal stem cell: isolation, in vitro expansion, and characterization. Br J Haematol. 2003;123:702e11. 34. Sacramento CB, da Silva FH, Nardi NB, Yasumura EG, Baptista-Silva JC, Beutel A, et al. Synergistic effect of vascular endothelial growth factor and granulocyte colony-stimulating factor double gene therapy in mouse limb ischemia. J Gene Med. 2010;12:310e9. 35. Yasumura EG, Stilhano RS, Samoto VY, Matsumoto PK, de Carvalho LP, Valero Lapchik VB, et al. Treatment of mouse limb ischemia with an integrative hypoxia-responsive vector expressing the vascular endothelial growth factor gene. PloS One. 2012;7:e33944. 36. Kay MA, Glorioso JC, Naldini L. Viral vectors for gene therapy: the art of turning infectious agents into vehicles of therapeutics. Nat Med. 2001;7:33e40.
Treatment of PAD with GM-CSF-modified MSCs 37. McGinley L, McMahon J, Strappe P, Barry F, Murphy M, O’Toole D, et al. Lentiviral vector mediated modification of mesenchymal stem cells & enhanced survival in an in vitro model of ischaemia. Stem Cell Res Ther. 2011;2:12. 38. McMahon JM, Conroy S, Lyons M, Greiser U, O’Shea C, Strappe P, et al. Gene transfer into rat mesenchymal stem cells: a comparative study of viral and nonviral vectors. Stem Cells Dev. 2006;15:87e96. 39. Van Damme A, Thorrez L, Ma L, Vandenburgh H, Eyckmans J, Dell’Accio F, et al. Efficient lentiviral transduction and improved engraftment of human bone marrow mesenchymal cells. Stem Cells. 2006;24:896e907. 40. Goto T, Fukuyama N, Aki A, Kanabuchi K, Kimura K, Taira H, et al. Search for appropriate experimental methods to create stable hind-limb ischemia in mouse. Tokai J Exp Clin Med. 2006;31:128e32.
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41. Ortiz LA, Gambelli F, McBride C, Gaupp D, Baddoo M, Kaminski N, et al. Mesenchymal stem cell engraftment in lung is enhanced in response to bleomycin exposure and ameliorates its fibrotic effects. Proc Natl Acad Sci U S A. 2003;100:8407e11. 42. Suga H, Eto H, Shigeura T, Inoue K, Aoi N, Kato H, et al. IFATS collection: fibroblast growth factor-2-induced hepatocyte growth factor secretion by adipose-derived stromal cells inhibits postinjury fibrogenesis through a c-Jun Nterminal kinase-dependent mechanism. Stem Cells. 2009;27: 238e49. 43. Seiler C, Pohl T, Wustmann K, Hutter D, Nicolet PA, Windecker S, et al. Promotion of collateral growth by granulocyte-macrophage colony-stimulating factor in patients with coronary artery disease: a randomized, double-blind, placebo-controlled study. Circulation. 2001;104:2012e7.
Comparison of treatments of peripheral arterial disease with mesenchymal stromal cells and mesenchymal stromal cells modified with granulocyte and macrophage colony-stimulating factor
FLAVIA FRANCO DA CUNHA*, LEONARDO MARTINS*, PRISCILA KEIKO MATSUMOTO MARTIN, ROBERTA SESSA STILHANO, EDGAR JULIAN PAREDES GAMERO & SANG WON HAN Department of Biophysics, Universidade Federal de São Paulo, São Paulo-SP, Brazil Abstract Background aims. Granulocyte macrophage-colony stimulating factor (GM-CSF) promotes vessel formation through several molecular signaling pathways. Mesenchymal stromal cells (MSCs) have an important role in neovasculogenesis during ischemia because they release pro-angiogenic paracrine factors, pro-survival and immunomodulatory substances and can differentiate into endothelial cells. The objective of this study was to evaluate whether there is synergy between GM-CSF and MSCs in recovering ischemic limbs. Methods. MSCs from mouse bone marrow were transduced with a lentiviral vector expressing GM-CSF and injected into animals with surgically induced limb ischemia, with unmodified MCSs used as control. The evolution of limb necrosis was evaluated for 1 month. Muscle strength was assessed on the 30th day, and the animals were euthanized to determine the muscle mass and to perform histological analyses to determine the degree of cellular infiltration, capillary and microvessel densities, fibrosis, necrosis and tissue regeneration. Results. Both treatments were able to ameliorate ischemia, decrease the areas of fibrosis, necrosis, adipocytes and leukocyte infiltrates and increase the number of capillaries. The addition of GM-CSF promoted the formation of larger vessels, but it also resulted in more fibrosis and less muscle mass without affecting muscle force. Conclusions. Both treatments resulted in a remarkable amelioration of ischemia. More fibrosis and less muscle mass produced by the overexpression of GM-CSF did not affect muscle functionality significantly. Importantly, MSCs overexpressing GM-CSF produced larger vessels, which is an important long-term advantage because larger vessels are more efficient in the reperfusion of ischemic tissues physiologically. Key Words: cell therapy, gene therapy, GM-CSF, ischemia, stromal cells
Introduction Peripheral arterial disease is caused by the obstruction of arteries, leading to decreased blood flow. It is a chronic disease that affects 3e10% of the world population (1). With disease progression, approximately 30% of patients are faced with the possibility of limb amputation within 1 year (1). These patients depend on the adaptation of preexisting collateral vessels (arteriogenesis) or the formation of new vessels through vasculogenesis or angiogenesis to recover tissue oxygenation (2,3). Thus, therapeutic neovascularization by means of angiogenic factors or stem cells, aiming at rapid revascularization of the ischemic area, represents a potential treatment option for regenerating the damaged tissue and preventing amputations (4).
Mesenchymal stromal cells (MSCs) are cells that can adhere to plastic, self-renew and differentiate into osteoblasts, chondrocytes and adipocytes (5). Therapies that use MSCs have been widely used in cardiovascular medicine. Several studies indicate that MSCs are an important means for neovasculogenesis, especially during ischemia (6e9), because the reduction of oxygen levels (hypoxia) induces MSC to form capillary-like structures in vitro (10). When implanted, MSCs can differentiate into and acquire the characteristics of mature endothelial cells (7,11), vascular smooth muscle cells and cardiomyocytes (12,13). However, the greatest potential of MSCs for neovascularization is related to its trophic effect, producing cytokines with paracrine
*These authors contributed equally to this work. Correspondence: Sang Won Han, PhD, Research Center for Gene Therapy, Department of Biophysics, Universidade Federal de São Paulo, Rua Mirassol 207, São Paulo-SP, CEP 04044-010, Brazil. E-mail: [email protected] (Received 26 June 2012; accepted 25 February 2013) ISSN 1465-3249 Copyright Ó 2013, International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jcyt.2013.02.014
Treatment of PAD with GM-CSF-modified MSCs effects, preventing fibrosis and apoptosis, promoting angiogenesis and arteriogenesis and stimulating the proliferation and differentiation of tissue-specific progenitors, thereby contributing to tissue repair (14). Several studies have shown that the injection of MSCs into ischemic limbs increased blood flow and vascular density (7,9,15). Granulocyte macrophage-colony stimulating factor (GM-CSF) promotes the proliferation, differentiation and survival of hematopoietic cells as neutrophils, monocytes, macrophages and dendritic cells (16,17), in addition to inducing effector functions of monocytes and macrophages, thereby contributing to the defense against pathogenic microbial agents (the inflammatory response) (18). GM-CSF also plays an important role in neovascularization (19), primarily because of its ability to prolong the life of monocytes by inhibiting apoptosis (20), thereby giving monocytes more time in circulation (21,22). Monocytes are involved in tissue remodeling during ischemia by playing important roles in arteriogenesis and angiogenesis through the secretion of several growth factors (23e26). A specific population of monocytes has promoted a new concept of vasculogenesis, which is a process of vessel formation by progenitor cells, specifically endothelial progenitor cells (27). Additionally, GM-CSF is the main chemotactic agent for neutrophils (28), in addition to its ability to inhibit the apoptosis of the same (29,30). A recent study from our laboratory (31) showed that the administration of a plasmid vector expressing GM-CSF into an ischemic mouse hind limb caused a strong therapeutic effect by mobilizing bone marrow stem cells and neutrophils, which increased the blood vessel density and muscle mass and strength. On the basis of these observations, the goal of this study was to evaluate the therapeutic effect of MSCs modified to express GM-CSF in the treatment of mouse ischemic limbs, to compare this treatment with mice treated with only MSCs, and to look for a synergistic effect of MSCs and GM-CSF.
Methods Construction of a lentiviral vector with GM-CSF GM-CSF was isolated from the pVAX-GM-CSF (31) with Bam HI and inserted into the pIRESe2GFP (internal ribosome entry site-green fluorescent protein; addgene, Cambridge, MA, USA), which had been previously digested with the same enzyme. The DNA fragment containing GM-CSF and IRES-GFP was isolated from this vector with NheI and HpaI and inserted into the lentiviral vector M107 (kindly provided by Yvonne Heidemarie Fisher, University of Lund, Stem Cell Center, Lund,
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Sweden), which was previously digested with XbaI and HincII. The final vector was named 107-GMGFP (Figure 1B). Plasmid vectors were amplified and purified with the use of a Qiagen mega-prep kit (Sao Paulo, Brazil). Viral vector production, concentration and titration were performed in a biosafety level 2 (NB2) laboratory, following a protocol established by Naldini et al. (32), after approval of the project by the National Biosecurity Council (CTNBIO) (approval No.: UNIFESP CQB 028/97, 18.07.97) and the Internal Biosecurity Council of the UNIFESP (approval No.: CIBIO No. 13/2010). A set of packaging vectors containing Gag-Pol (10 mg), Rev (5 mg) and VSV-G (6 mg) and the transfer vector (20 mg) containing the gene of interest were used to transfect human endothelial kidney cell (HEK)293T cells (2 106 cells in 75 cm2 plate) with the use of co-precipitation with calcium phosphate. The culture supernatant was collected after 24 and 48 h and filtered through a 0.45-mm syringe filter (Millipore, New York, NY, USA). For each 26 mL of supernatant, 4 mL of a 20% sucrose solution was added, and this mix was centrifuged in the a Sorvall centrifuge with a SS34 rotor at 16,000 rpm for 2 h. After centrifugation, the supernatant was discarded, and the pellet was resuspended in Dulbecco’s modified Eagle’s medium (DMEM) without serum, aliquoted and stored at 80 C. To determine viral titer, HEK293T cells were transduced with different concentrations of lentiviral vectors in the presence of 8 mg/mL Polybrene (Sigma, St Louis, MO, USA), and after 3 days, the GFP-positive cells were counted with the use of an inverted microscope (IX70; Olympus America Inc, Center Valley, PA, USA). The titer was calculated according to the following formula: Titer ¼ % GFP-positive cells total number of cells on the day of transduction/volume of virus. Isolation, characterization and modification of MSCs from mouse bone marrow Eight-week-old BALB/c mice were killed by cervical dislocation, following the procedure recommended and approved by the ethics and research committee of the Federal University of São Paulo (UNIFESP) (approval No.: CEP 0327/10). The tibia and femur were dissected, and DMEM with low concentrations of glucose, supplemented with 10% fetal bovine serum (FBS), 2 mmol/L L-glutamine, 200 U/mL penicillin and 200 mg/mL streptomycin (this supplemented DMEM was named DMEMc), was injected into the dissected bone to collect the bone marrow cells, which were maintained in six-well plates. The
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Figure 1. Characterization and genetic modification of MSC and GM-CSF gene expression analysis. (A) MSC differentiation. Bone marrow MSC (I) were differentiated into adipocytes (II) or osteoblasts (III) and stained with oil red O and alizarin red, respectively. (B) Schematic representation of human immunodeficiency virusebased lentiviral vector. DU5 LTR indicates self-inactivating long-terminal repeat with deletion at the U5 region; J, packaging signal; RRE, Rev responsive element; CMV, cytomegalovirus promoter; iRES, internal ribosome entry site; GFP, green fluorescent protein; WPRE, woodchuck hepatitis virus post-transcriptional regulatory element; LTR, long terminal repeat. (C) Transduction of MSC. Phase-contrast micrographs (I, IV), fluorescent image of GFP expression (II, V) and overlay of phase-contrast and fluorescent images (III, VI) are shown. Images were acquired 3 (I, II, III) and 30 (IV, V, VI) days after transduction. (D) GM-CSF gene expression analysis of MSCs and MSCs transduced with GM-CSF after 3 days by quantitative RT-PCR. *P < 0.05.
Treatment of PAD with GM-CSF-modified MSCs medium was regularly replaced with fresh DMEMc (33). Adherent cells were treated with a 0.25% trypsin solution, centrifuged, resuspended and plated in culture bottles (25 or 75 cm2) with DMEMc at 37 C with 5% CO2 using cell culture incubators (Model 3158; Thermo Fisher Scientific Inc, Waltham, MA, USA). All reagents used in this step were purchased from Invitrogen (Grand Island, NY, USA). The capacity of these cells to differentiate into adipocytes and osteoblasts was evaluated on the basis of an established protocol (33). For osteogenic differentiation, 2 105 cells were plated per well in a six-well plate. DMEM, supplemented with 108 mol/L dexamethasone, 5 mg/mL ascorbic acid 2-phosphate, 10 mmol/L b-glycerophosphate and 10% FBS, was used, with continuous medium exchange every 3 days for 3e4 weeks. The osteoblasts were stained with 2% alizarin red S, pH 4.1 (SigmaAldrich, St Louis, MO, USA). For adipogenic differentiation, 2 105 cells were plated per well in six-well plates containing DMEM supplemented with 10% FBS, 108 mol/L dexamethasone, 2.5 mg/mL insulin and 5 mmol/L rosiglitazone. The cells were maintained with continuous medium exchange every 3 days for 3e4 weeks. The adipocytes were stained with oil red O (3.75% in 60% isopropyl alcohol) (Sigma-Aldrich) For staining, the medium was aspirated, cells were fixed with 4% paraformaldehyde, washed with phosphate-buffered saline (PBS) and incubated with the solution of oil red O or alizarin red S for 5 minutes. For transduction, 1 105 MSC per well were initially seeded in six-well-plates with DMEMc. Eighteen hours later, the medium was replaced with 1 mL of fresh DMEMc, and 20 mL of the concentrated viral solution was added in the presence of Polybrene (Sigma) to a final concentration of 8 mg/mL. After 24 h, the medium was replaced with 3 mL of fresh DMEMc and the cells were maintained in culture at 37 C and 5% CO2 for 3 days. The transduction efficiency was determined after this period by counting GFP-positive cells with an inverted fluorescence microscope. To verify the maintenance of transgene expression in the transduced MSCs, GFP expression was monitored weekly for 1 month with a fluorescence microscope. Analysis of GM-CSF expression by quantitative realtime polymerase chain reaction The total RNA of MSCs was extracted with the use of an RNAeasy kit (Qiagen) and treated with DNAse I (Sigma-Aldrich). The cDNA was synthesized with the use of a High Capacity Reverse Transcription kit (Life Technologies, Carlsbad, CA, USA), and a
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quantitative real-time polymerase chain reaction (RTPCR) assay was performed with the use of the SYBR Green QuantiFast RT-PCR Kit (Qiagen, Hilden, Germany) in the Rotor Gene-Q (Qiagen). The following primers were used for the quantitation of murine GM-CSF: GM-CSF_F, CCC AAG AAG TGA TCC ACC TG and GM-CSF_R, GGG CAC TCT GCA ATG TTA GCT T. The relative gene expression was calculated by 2DDCT. The murine b-actin gene was used as a normalizer with the following primers: b-actin_F: GCT CCT CCT GAG CGC AAG, and b-actin_R: CAT CTG CTG GAA GGT GGA CA. Each reaction was performed in duplicate, and each experiment was performed at least three times. Hind limb ischemia induction and cell therapy Induction of ischemia was performed surgically in 10- to 12-week-old BALB/c mice. After anesthesia with ketamine (40 mg/kg) and xylazine (10 mg/kg), ischemia was induced in the right leg by the removal of the entire femoral artery and the closure of its branches (deep femoral, epigastric, saphenous and popliteal arteries), on the basis of the procedure already established in our laboratory (31,34). Five days after ischemic induction, mice were anesthetized again, the quadriceps were exposed and 5 105 cells in 50 mL DMEM were injected into the middle of the quadriceps with the use of a 21-gauge needle. MSCs between 7 and 10 passages were used in the in vivo experiments. The mice were divided into five groups (n ¼ 6 per group) as follows: nonischemic mice (N-IS), sham-operated mice (S), ischemic mice (IS), ischemic mice treated with MSCs (MSC) and ischemic mice treated with MSCs modified with GM-CSF and GFP (MSC-GM). The animals were followed for 35 days, and the visual assessment of limbs was performed weekly on the basis of the following scale: I, no change; II, nailblackening; III, necrosis on toes; IV, necrosis below the heel. On the 35th day, before the euthanasia of the animals, the isometric muscle force was determined according to the method standardized in our laboratory (31,35). Briefly, a mouse was anesthetized and set on a table; the gastrocnemius muscle was isolated, to maintain the vascular connections and the origin of the muscle, and the tendinous insertion of the muscle was isolated and connected to the force transducer (iWorx/CB Science, Inc, Dover, NH, USA). The distal portion of the sciatic nerve was exposed and connected to bipolar electrodes, which were connected to an electrostimulator (Grass S88; Grass Instruments, Quincy, MA, USA). The muscle function was evaluated by the isometric contraction
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response, with an adjustment of the rest tension, to obtain the maximum muscle strength (tetanus), with the use of the peak voltage curve caused by the electrostimulator. Muscle strength was recorded and analyzed by Powerlab 8/30 software (AD Instruments Pty Ltd, Colorado Springs, CO, USA). Histological analysis The animals were euthanized and perfused with intravascular injection of PBS, and manipulated muscles were removed and washed with PBS to eliminate blood. The mass of the quadriceps and gastrocnemius muscle was determined on an analytical balance. The muscles were fixed in 4% paraformaldehyde for 48 h, dehydrated and embedded in paraffin. Sections of 4-mm thickness were obtained and stained with hematoxylin and eosin (HE) to determine the degree of muscle regeneration and the presence of adipocytes and infiltrated cells or with picrosirius red to quantify the degree of fibrosis. Other sections were collected on glass slides coated with poly-L-lysine and subjected to immunohistochemistry with the use of antibody anti-aeactin (1:50) (Dako Denmark A/S, Glostrup, Denmark, clone 1A4) to mark smooth muscle cells or stained with lectin Griffonia (bandeiraea) simplicifolia I biotinylated (1:100) (Vector Laboratories, Peterborough, UK) to mark endothelium, followed by incubation with streptavidinperoxidase (1:100) (Sigma-Aldrich) and detection with chromogen diaminobenzidine (Dako). The images were obtained with the use of an optical microscope (Olympus BX60) and analyzed digitally. Morphometric analyses of skeletal muscle tissue were performed on each slide. Images were captured in at least 10 fields of the wounded area per slide to evaluate necrosis, muscle regeneration, and degeneration, fibrosis and angiogenesis, with the use of Image Pro Plus software (Media Cybernetics, Bethesda, MD, USA). Statistical analysis GraphPad Prism software (version 5.01) was used for comparison between groups. Student’s t and MannWhitney tests were applied for parametric and nonparametric analyses. Values of P < 0.05 were considered statistically significant. Results To evaluate whether an ex vivo gene therapy with the use of MSCs that constitutively secrete GM-CSF is more effective in treating limb ischemia, MSCs were first obtained from the bone marrow of BALB/c mice for characterization. Most of the MSCs used were
spindle-shaped cells after approximately 2 weeks of extraction. The cells that were cultivated in predetermined media to differentiate into adipocytes or osteocytes showed the expected phenotypes and staining (Figure 1A); this is one of the most important characterizations of MSCs (5). A lentiviral vector was chosen for MSC modification because of its high ability to transduce quiescent or low-growth-rate cells; it also produces long-term gene expression (36). Several authors have demonstrated that MSCs are difficult to transduce because of the low expression of viral membrane receptors, and lentiviral vectors have been more efficient than other viral vectors to transduce MSCs (37e39). To verify the functionality of the viral vector preparations, concentrated viral stocks were used to transduce HEK293T cells, and the number of GFPpositive cells was determined by means of an inverted fluorescence microscope: in most cases, a 6 105 transducing units/mL viral titer was obtained. To determine the best conditions for in vivo transduction, MSCs were plated (1 105 cells per well in six-well plates) and transduced with different volumes of the above vector (5, 10 and 20 mL), and the efficiency of transduction was evaluated after 72 h. The best outcome was obtained with 20 mL, resulting in 40% transduction (Figure 1C). In addition, during 1 month of observation, there was no significant variation in the percentage of GFPpositive cells, which confirmed stable integration and gene expression (Figure 1C). GM-CSF gene expression as analyzed by RT-PCR also confirmed much higher expression in transduced cells compared with nontransduced cells (Figure 1D). These results demonstrated that the MSCs that were genetically modified with GM-CSF were of high enough quality to perform the in vivo experiments. The limb ischemia model used to test our hypothesis should represent human peripheral arterial disease, with stable, uniform and relatively severe ischemia. To reach this condition, the femoral artery was removed completely and its branches (deep femoral, epigastric, saphenous and popliteal arteries) were closed surgically (40). This procedure severely affects limb circulation, and within 1 week, the limbs become darkened. However, it is very important to note that this is not a chronic ischemia model, a mouse model of which is not yet available. Because of their different physiology and the size of the animals, it is difficult to establish a chronic ischemia model in small rodents. MSCs were injected only 5 days after ischemia because, in our previous studies, we found that injecting MSCs in the first few days after injury worsened the limbs, with highly affected necrotic areas and increased rates of amputation (results not
Treatment of PAD with GM-CSF-modified MSCs shown). It is likely that the growth factors and proteases, which are released by acute inflammatory cells after ischemic surgery and by the injected MSCs, exacerbated the inflammatory reaction, leading to greater tissue damage. During 30 days of follow-up, a visual evaluation of the necrotic tissue was performed. Seventy percent of the ischemic animals proceeded to advanced necrosis (grade IV), affecting the functionality of the limbs, and the other 30% proceeded to grade III necrosis (Figure 2A,B). In the MSC-treated group, 87.5% had no visual sign of necrosis (grade I), and 12.5% of animals had only blackened nails (grade II). In the group treated with MSC-GM, 83% of the mice were at grade I, whereas 17% were at grade III (Figure 2A), a slightly worse result than the groups treated with MSCs only. To better understand this result, functional and histological analyses were performed. To assess muscle function, a parameter that assesses the whole tissue repair process, including angiogenesis, myogenesis and fibrosis, the strength of the gastrocnemius muscle was measured after 35 days of ischemic induction. The gastrocnemius muscle was chosen for assessment because ischemia affects distant tissues, and this muscle was not manipulated during ischemic surgery. The shamoperated animals had muscle strength of 1.01 N, but ischemic surgery produced almost total loss of strength, with an average muscle strength of 0.14 N. The animals treated with MSCs or MSC-GM showed great recovery, with muscle strength
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reaching 0.79 N and 0.67 N, respectively (Figure 2C). There was a similar profile of muscle recovery for gastrocnemius muscle mass. MSCtreated and MSC-GMetreated groups had a muscle mass of 79% (157 mg) and 63% (124 mg), respectively, of the sham-operated group (197 mg), whereas the untreated ischemic mice had a muscle mass of only 95 mg (Figure 2D). Because inflammation is an important process for muscle cell degeneration and regeneration, muscle tissues were histologically analyzed to evaluate cell infiltrates. HE staining of the gastrocnemius muscle showed a large area that was occupied by adipocytes in the untreated ischemic group (62.8%), which was reduced to 30% and 26% in the groups treated with MSCs or MSC-GM, respectively (Figures 3 and 4A). The infiltration of leukocytes was also greatly reduced in the treated animals compared with ischemic animals: from 12.8% to 0.55% and 1.1% in the groups treated with MSC-GM and MSC, respectively. The cellular degeneration was similar in all groups (Figure 4A); however, a great number of regenerating muscle cells were observed in the MSC and MSC-GM groups, at 57% and 51.8%, respectively, whereas the sham-operated group and the untreated group both presented only 5% of regenerating muscle cells (Figure 4A). These results indicate that the treatment of limb ischemia with MSCs, with or without GM-CSF, was able to promote a marked regeneration of muscle tissue and confirmed the previous visual assessment and strength and muscle mass measurements.
Figure 2. Therapeutic assessments of the MSCs-treated ischemic limbs. (A, B) Limb quality was evaluated visually according to the degree of necrosis. Gastrocnemius muscle force (C) and mass (D) were determined after 30 days of cell therapy. I indicates no change; II, nailblackening; III, necrosis on toes; IV, necrosis below the heel. *P < 0.05.
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Figure 3. Histological analysis of limb muscles. Gastrocnemius muscles were collected after 4 weeks of cell therapy. Tissue samples were stained with (A) HE to assess regenerative (*), necrotic (**), endomysial fibrosis (***) and normal areas, (arrow down) adipocytes, (B) picrosirius red to fibrotic area (stained in red), (C) lectin Griffonia and (D) a-actin antibody to capillary (arrowheads) density and mature vessels (arrow), respectively. High concentration of leucocytes can be found in necrotic area (**). Macrophages are marked with “M” and vessels are marked in brown. Scale bar ¼ 50 mm (A, B) and 20 mm (C, D).
One of the more negative consequences of tissue healing is fibrosis, which leaves the recovered tissue fragile and predisposes it to develop new lesions. The primary cause of fibrosis is the excessive deposition of collagen, which can be stained with picrosirius red. All treated groups had a decrease in fibrosis, reaching 2.59% (MSC) and 3.56% (MSCGM), whereas the ischemic group had 6.77% (Figures 3 and 4B), clearly demonstrating the effects of MSCs in the reduction of fibrosis with or without GM-CSF. The anti-fibrotic role of MSCs has previously been established (41,42), and we previously saw a similar effect with mice treated with GM-CSF (data not shown). However, no synergistic effect was observed in the GM-MSC group; on the contrary, the overexpression of GM-CSF partially affected the anti-fibrotic role of MSCs. This small variation between the two groups should not have
significantly affected the recovery of muscle functionality because the muscle strength did not vary significantly between them (Figure 2C). Finally, we evaluated the neovascularization promoted by mesenchymal cells (6e9) and GMCSF (31,43). Blood vessels were stained with Griffonia, which marks endothelium and activated macrophages, cells that can be easily identified by microscopy, and with the antibody anti-a actin, which marks mature vessels. By staining with Griffonia, we noted that both treatments led to a significant increase in the number of capillaries when compared with the untreated group (MSCs: 619 capillaries/mm2, MSC-GM: 564 capillaries/mm2, IS: 497 capillaries/mm2). Despite a small difference between the treated groups, there was no significance difference between the MSC-treated and MSC-GMetreated groups (Figures 3 and 4C).
Treatment of PAD with GM-CSF-modified MSCs
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Figure 4. Morphometric analyses of limb muscles. From Figure 3, (A) necrotic, regenerative and normal areas, (B) fibrotic area, (C) capillary density and (D) matured vessels were determined. More than 50 fields of lesions were counted for each group. AI, adipocyte infiltration; LI, leukocyte infiltration; MD, muscle degeneration; MR, muscle regeneration. *P < 0.05.
The immunohistochemistry analysis also showed an increase in the number of mature vessels in both treated groups compared with the ischemic untreated group (40 vessels/mm2), but it was more prominent in the MSC-GM group (61.6 vessels/mm2) than in the MSC group (51.5 vessels/mm2) (Figures 3 and 4D). This difference is probably caused by the arteriogenic property of GM-CSF (20), which we have also previously demonstrated (31). In terms of physiologic functionality, arteriogenesis plays a more important role than angiogenesis because the collateral vessels remodeled by arteriogenesis are larger than those formed by angiogenesis (2,3). This study confirmed the important role of MSCs in the treatment of limb ischemia. All treated groups had a remarkable improvement compared with the untreated ischemic group, decreasing the adipocytes and inflammatory infiltrates, increasing the muscle mass and strength and increasing the number of capillaries and larger vessels. However, the combination of MSCs with GM-CSF did not markedly improve recovery compared with animals treated with only MSCs, except for an increase in larger vessels. The formation of larger vessels is the best way to increase tissue reperfusion after ischemia, especially in larger animals that circulate a greater blood volume. Therefore, ex vivo gene therapy with MSC modified with GM-CSF will be more beneficial for human patients with ischemic
limbs. On the other side, the overexpression of GM-CSF by MSCs also resulted in more fibrosis and less muscle mass. Even these variations did not affect muscle functionality in our ischemic model; in larger animals with chronic limb ischemia, these parameters must be monitored carefully because they contribute greatly in the evolution of physiopathology. Acknowledgments F.F.C. was recipient of a CNPq scholarship, and L.M., R.S.S. and P.K.M.M. were recipients of FAPESP scholarships. This work was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP: Processo No. 2011/00859-6). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. Disclosure of interest: The authors have no commercial, proprietary, or financial interest in the products or companies described in this article. References 1. Norgren L, Hiatt WR, Dormandy JA, Nehler MR, Harris KA, Fowkes FG, et al. Inter-Society Consensus for the Management of Peripheral Arterial Disease (TASC II). Eur J Vasc Endovasc Surg. 2007;33(Suppl 1):S1e75.
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