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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2004;24:e192.)
© 2004 American Heart Association, Inc.

Vascular Biology

Molecular Evaluation of Endothelial Progenitor Cells in Patients With Ischemic Limbs

Therapeutic Effect by Stem Cell Transplantation

Koji Yamamoto; Takahisa Kondo; Satoshi Suzuki; Hideo Izawa; Masayoshi Kobayashi; Nobuhiko Emi; Kimihiro Komori; Tomoki Naoe; Junki Takamatsu; Toyoaki Murohara

From the Departments of Transfusion Medicine (K.Y., S.S., J.T.), Cardiology (T.K., H.I., T.M.), Vascular Surgery (M.K., K.K.), and Hematology (N.E., T.N.), Nagoya University Hospital, Nagoya, Japan.

Correspondence to Dr K. Yamamoto, Department of Transfusion Medicine, Nagoya University Hospital, 65 Tsurumai, Showa, Nagoya 466-8550, Japan. E-mail kojiy{at}med.nagoya-u.ac.jp

	Abstract

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Objective— Although some patients with limb ischemia have recently undergone therapeutic angiogenesis by cell transplantation, their angiogenic potential has not been well characterized. It is also important to evaluate endothelial progenitor cell (EPC) contents in different stem cell sources to choose the best material for therapeutic angiogenesis.

Methods and Results— We quantitated the mRNA expression of EPC-specific molecules (eg, Flk-1, Flt-1, CD133, VE-cadherin, etc) in bone marrow-derived or peripheral blood-derived mononuclear cells obtained from patients with ischemic limbs, using real-time reverse-transcription polymerase chain reaction technique. The mRNA expression level of EPC markers was significantly lower in the patients than in healthy controls, which was consistent with results of flow cytometric analysis. However, the implantation of autologous bone marrow mononuclear cells increased the circulating EPCs in the peripheral blood of patients. We furthermore revealed the different expression pattern of EPC markers in possible sources for stem cell transplantation, including normal bone marrow, peripheral blood obtained from recombinant granulocyte colony–stimulating factor-treated donor, and umbilical cord blood.

Conclusions— Patients with peripheral obstructive arterial diseases may have lower angiogenic potential because of decreased expression of EPC specific molecules in their marrow and blood. Therapeutic angiogenesis by transplantation of autologous marrow mononuclear cells increased circulating EPCs in the patients and improved ischemic symptoms. (Arterioscler Thromb Vasc Biol. 2004;24:e192–e196.)

The gene expression of EPC-specific molecules in bone marrow-derived and peripheral blood-derived mononuclear cells, analyzed by real-time RT-PCR, was lower in patients with ischemic limbs than in healthy subjects. Therapeutic angiogenesis by autologous stem cell transplantation was effective for the patients and increased circulating EPCs.

Key Words: angiogenesis • stem cell • endothelial progenitor cells • transplantation • ischemia

	Introduction

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Until recently, it was thought that blood vessel formation in postnatal life was mediated by sprouting of endothelial cells (ECs) from existing vessels. However, recent studies have suggested that endothelial stem cells may persist into adult life, when they contribute to the formation of new blood cells.^1–3 Neovascular formation in adults has been considered to result exclusively from the proliferation, migration, and remodeling of preexisting mature ECs, a process referred to as angiogenesis.⁴ Endothelial progenitor cells (EPCs) in the CD34-positive stem cell fraction of adult peripheral blood take part in postnatal neovascularization, defined as vasculogenesis, after mobilization from bone marrow.^3,5,6 In this context, therapeutic neovascularization is an important strategy to salvage tissue from critical ischemia.^7–9 Previous studies using animals have shown that bone marrow–mononuclear cell implantation into ischemic limbs promotes collateral vessel formation, with incorporation of EPCs into new capillaries.¹⁰ Based on these studies, Japanese clinical research groups, including us, have developed therapeutic angiogenesis by cell transplantation for patients with limb ischemia.¹¹ However, the angiogenic potential for therapeutic angiogenesis by cell transplantation in the patients has not been evaluated at the molecular level before and after stem cell transplantation. Moreover, it is difficult to decide the best cell source for therapeutic angiogenesis, not only because it is necessary to take stem cells most safely and effectively but also because it is difficult to evaluate EPCs existing in different cell sources.

In the present study, we established a highly sensitive method to evaluate the gene expression of several molecules specific for EPC and endothelial lineage in bone marrow-derived and peripheral blood-derived mononuclear cells. The mRNA expression of vascular endothelial growth factor (VEGF) receptors (eg, Flk-1 and Flt-1), CD133, VE-cadherin, PECAM-1, and a universal marker for EC, von Willebrand factor (vWF), in mononuclear cells obtained from patients with ischemic limbs was quantitated before and after receiving stem cell transplantation using real-time reverse-transcription polymerase chain reaction (RT-PCR) technique. We further analyzed the gene expression of each molecule in different stem cell sources, including normal bone marrow, peripheral blood of recombinant granulocyte colony-stimulating factor (G-CSF)-treated donors, and umbilical cord blood, revealing the different expression pattern of EPC/EC-specific molecules between them.

	Methods

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Patients
Four patients were qualified for bone marrow implantation because they had chronic limb ischemia, including rest pain, nonhealing ischemic ulcers, and they were not candidates for nonsurgical or surgical revascularization. The profile of patients is as follows: patient 1, a 38-year-old woman with Buerger disease who had a painful ulcer in her left foot; patient 2, a 57-year-old man with arteriosclerosis obliterans (ASO) who had an intractable ulcer in his left foot; patient 3, a 67-year-old man with ASO who had severe ischemia in his right foot; and patient 4, a 51-year-old man with Buerger disease who had a ischemic lesion in his right foot. Patient 2 and 4 have diabetes and have been treated with sulfonylureas. Both ASO patients have undergone hemodialysis because of chronic renal failure for several years and have taken prostaglandin E₁ for their ischemic ulcers. The study protocol for therapeutic angiogenesis by cell transplantation was approved by the Committees on the Ethics of Human Research of Nagoya University Hospital, and written informed consent was obtained from each participant. The patients have undergone the transplantation of autologous bone marrow mononuclear cells.

Bone Marrow Aspiration and Implantation
We aspirated 400 to 500 mL of bone marrow from the posterior iliac crest under general systemic anesthesia, and a part of the marrow (500 µL) was used for RT-PCR analysis. Autologous bone marrow mononuclear cells were isolated by centrifugation using AS104-Plus blood-cell separator (Baxter, Deerfield, Ill) and concentrated to a final volume of 30 to 40 mL containing 2.8 to 4.4x10⁹ mononuclear cells. A small fraction was assessed morphologically and tested for viability with trypan blue exclusion, absence of clots, bone spicules, and gross bacterial contamination. Bone marrow cell population was analyzed by flow cytometry using anti-CD34 antibody and contained 3.0x10⁷ cells positive for CD34. Then, each 0.8 mL of concentrated mononuclear cells was intramuscularly injected into 40 sites of the ischemic limb where grafting is not possible, as described previously.¹¹ Isolated red blood cells from bone marrow were returned to the patients.

Samples and Real-Time RT-PCR Assay
Different stem cell sources (ie, bone marrow or peripheral blood) were obtained from patients with ischemic limbs, age-matched healthy (disease-free) volunteers, or recombinant G-CSF–treated donors after obtaining written informed consent. Each 5 mL of umbilical cord blood was also obtained from delivered mothers based on the informed consent. Mononuclear cells were separated with Ficoll-Plaque Plus (Amersham Biosciences) from each sample as follows: bone marrow, 4.2±1.1x10⁶ cells/mL; peripheral blood, 1.3±0.4x10⁶ cells/mL; and cord blood, 3.9±0.9x10⁶ cells/mL. Total cellular RNA was isolated from mononuclear cells using STAT-60 total RNA isolation reagent (Stratagene, La Jolla, Calif). One hundred nanograms of each cellular RNA was reverse-transcribed, and then the expression levels of mRNA for Flk-1, Flt-1, CD133, VE-cadherin, PECAM-1, vWF, and ß-actin were determined by real-time quantitative RT-PCR with the ABI Prisms 7700 Sequence Detection (Perkin-Elmer Biosystems, Foster City, Calif) and SYBR Green PCR Kit (Perkin-Elmer Biosystems), according to the manufacturers’ recommendations. A synthetic DNA template containing the sequences for the upstream and downstream primers for each gene was used as a standard. The sequences of primer pairs used to quantitate mRNAs of the aforementioned genes were described in the NCBI Sequence Viewer. Various concentrations of the standard DNA template (eg, 1x10⁵ to 1x10¹¹ molecules/µL) were used for the calibration curve for each primer set.¹² After 30 cycles of PCR reaction (94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 30 seconds), the amount of gene transcripts was calibrated by the comparison with the standard curve. All the RT-PCR experiments were performed in duplicate.

Flow Cytometry
We further examined circulating EPCs using cell surface antigen, as previously described.¹³ Circulating mononuclear cells with CD34⁺CD133⁺ or CD34⁺CD133⁺VEGFR2⁺ were defined as tentative progenitor cells or EPCs, respectively.¹⁴ Samples were subjected to a 2-dimensional side-scatter fluorescence dot plot analysis (FACScan; Becton-Dickinson). After appropriate gating with low-cytoplasmic granularity and with low expression of CD45, the numbers of CD34⁺, CD133⁺, CD34⁺CD133⁺, and CD34⁺CD133⁺VEGFR2⁺ cells were quantified and expressed as number of cells per 10⁶ total events. Then, the number of CD45^lowCD34⁺CD133⁺ or CD45^lowCD34⁺ CD133⁺VEGFR2⁺ cells was counted.

	Results

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First, we examined the mRNA expression level of several molecules specific for EPC and/or EC in bone marrow-derived and peripheral blood-derived mononuclear cells from patients with ischemic limbs and healthy volunteers (Table 1). In general, the mRNA content of all molecules examined was lower (50%, except vWF) in mononuclear cells from the patients’ bone marrow compared with the marrow obtained from healthy volunteers. In peripheral blood-derived mononuclear cells of the patients, the mRNA expression of Flk-1, CD133, and VE-cadherin, all of which were specifically expressed in EPCs, was not detected, even by sensitive real-time RT-PCR method. Although Flk-1 mRNA expression in peripheral mononuclear cells from healthy volunteers was also below the detection limit of our assay, we detected their steady-state levels of mRNA expression for CD133 and VE-cadherin. Again, the expression levels of other molecules (eg, Flt-1, PECAM-1, vWF) in peripheral mononuclear cells were relatively lower in patients with Buerger disease or ASO than those of healthy subjects. We performed conventional flow cytometric analysis and confirmed that circulating and marrow progenitor cells/EPCs also decreased in patients with ischemic limbs (Table 1).

View this table: [in this window] [in a new window]   TABLE 1. Quantitative Evaluation of EPC/EC Specific Molecules and PC/EPC Numbers in Patients With Ischemic Limbs and in Healthy (disease-free) Volunteers

Changes in the mRNA expression of EPC/EC-specific molecules were examined in peripheral mononuclear cells from the patients with Buerger disease (patient 1) or ASO (patient 3), who received autologous marrow mononuclear cell transplantation (Figure A). Both patients underwent transplantation with 3.0x10⁹ marrow mononuclear cells, containing 1% of cells positive for CD34. Basal mRNA expression level of each gene was quite low in both patients, especially in the ASO patient. However, marrow mononuclear cell transplantation significantly increased the mRNA expression of all genes examined with the maximum increase in VE-cadherin mRNA in peripheral blood of the patient with Buerger disease. In contrast, only slight increases in the mRNA expression of these genes were observed in peripheral blood of the ASO patient after marrow mononuclear cell transplantation. Flk-1 mRNA was not detected at all in peripheral mononuclear cells from both patients, even after stem cell transplantation (not shown). Flow cytometric analysis revealed that the number of circulating progenitor cells (CD45^lowCD34⁺CD133⁺) in patient 1 increased to 174 per 1x10⁸ CD45⁺ cells after cell therapy (before therapy: 96), whereas progenitor cells in patient 3 increased to 115 (before therapy: 97). Ischemic status (eg, rest pain, transcutaneous oxygen pressure, regional blood flow evaluated by thermography, ulcer size) was dramatically improved in the patient with Buerger disease (patient 1; Figure B), but not in the patient with ASO (patient 3; not shown).

View larger version (41K): [in this window] [in a new window]   The expression of EPC/EC-specific molecules and the therapeutic effect of stem cell transplantation in patients with ischemic limbs. A, The mRNA expression of EPC/EC-specific molecules in peripheral mononuclear cells from the patients with Buerger disease (patient 1) and ASO (patient 3) was quantitated before (hatched bars) and the day (closed bars) after transplantation of autologous marrow mononuclear cells (3.0x109 cells). B, Healing process of foot ulcer observed in patient 1 (Buerger disease) after transplantation of autologous marrow mononuclear cells. VE-cad indicates VE-cadherin; ND, not detected before marrow implantation; wk, weeks; mo, months.

Finally, we compared the mRNA expression of EPC/EC-specific molecules between normal bone marrow, peripheral blood of G-CSF–treated donors, and umbilical cord blood, all of which are possible source for therapeutic angiogenesis by cell transplantation (Table 2). In general, higher expression of most molecules was detected in bone marrow-derived mononuclear cells. However, the expression of VE-cadherin mRNA was relatively higher in peripheral blood obtained from G-CSF–treated donors as compared with bone marrow. Umbilical cord blood showed relatively less expression of EPC/EC-specific molecules, except PECAM-1, in comparison with bone marrow or G-CSF–treated blood.

View this table: [in this window] [in a new window]   TABLE 2. Quantitative Evaluation of EPC/EC Specific Molecules in Different Stem Cell Sources

	Discussion

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Therapeutic angiogenesis by cell transplantation has recently started as effective therapy for patients with ischemic limbs who have no graftable lesions.¹¹ Marrow mononuclear cells have many characteristics of stem cells for mesenchymal tissues and also secrete many angiogenic cytokines,^15,16 raising the possibility that marrow implantation into ischemic limbs could enhance angiogenesis by supplying EPCs and angiogenic factors or cytokines. Subjects with risk factors for coronary artery disease (eg, hyperlipidemia, diabetes, hypertension, smoking, aging, etc) have low numbers and migratory activity of circulating EPCs.^17,18 A few medicines and hormones (eg, statins, estrogen, erythropoietin) could enhance the proliferation of EPC and/or the mobilization of EPCs into the circulation,^19–21 thus increasing the angiogenic potential. Previous studies noted that mononuclear cells from adult human peripheral blood improved capillary density in limb ischemia.^22,23 In the present study, we evaluated the angiogenic potential in patients with arterial obstructive disease by quantitating the mRNA expression of EPC/EC-specific molecules, including Flk-1, Flt-1, CD133, VE-cadherin, PECAM-1, and vWF, in their marrow and peripheral blood as compared with healthy donors. In general, less expression of EPC/EC marker molecules was detected in mononuclear cells from patients’ marrow and blood than that of healthy controls (Table 1). Of course, the mRNA expression level is not the same as the cell number of EPCs. We quantified progenitor cell/EPC numbers by flow cytometric analysis and the obtained data were consistent with the analysis of the molecular markers. We cultured mononuclear cells obtained from peripheral blood of patients, but they did not proliferate and differentiate to endothelial-like cells. Thus, we could evaluate the potential to differentiate endothelial-like cells based on the RT-PCR and flow cytometric analysis. Taken together, these results suggest that patients with ischemic limbs may have lower potential for neovascularization, which possibly results in the formation of intractable ulcer and in the progression of ischemic lesion.

The molecular mechanism and process of angiogenic response in the lesion have not been fully elucidated, and how implanted stem cells are incorporated into new vessels has not clarified. We have observed that autologous bone marrow implantation dramatically improved ischemic status in patients with Buerger disease, but not in the patient with ASO (Figure B). The mRNA expression of EPC-specific molecules in mononuclear cells from peripheral blood was increased by stem cell transplantation (Figure A), suggesting that EPCs implanted into the ischemic lesions came into the circulation of the patients and that the evaluation of the expression of EPC markers may predict the outcome of patients who received autologous marrow implantation. Less elevation of the expression level of EPC markers in blood of the ASO patient after marrow implantation may be because of the deprivation of transplanted EPCs from the circulation by hemodialysis. It is also speculated that ASO patients have impaired EPC/EC function, low responses to angiogenic cytokines secreted by transplanted stem cells, and decreased potential of stem cells to be incorporated to endothelial lineage in comparison with patients with Buerger disease. Thus, we hypothesize that the efficacy of implantation of bone marrow mononuclear cells depends on supply of EPCs and multiple angiogenic factors. To increase regional angiogenic potential by implantation of autologous marrow mononuclear cells may be a promising therapeutic strategy for the patients with arterial obstructive diseases.

Although hematopoietic stem cells obtained from recombinant G-CSF–treated donors and umbilical cord blood have been used for therapeutic transplantation in patients with hematologic malignancies, whether both materials can be sources for isolating EPCs has not yet been resolved. Previous studies showed that mononuclear cells obtained from human umbilical cord blood contained significant amounts of EPCs and that transplantation of cord blood-derived EPCs augmented neovascularization in the ischemic limb of immunodeficient nude rats.¹⁷ We detected less expression of EPC-specific molecules in mononuclear cells from umbilical cord blood as compared with bone marrow and G-CSF–treated blood (Table 2), suggesting that umbilical cord blood may not have enough angiogenic potential for clinical use. Meanwhile, the peripheral blood from G-CSF–treated donors showed similar expression level of EPC/EC markers with bone marrow. This indicates that peripheral mononuclear cells isolated from patients after G-CSF treatment may be an alternative source for therapeutic angiogenesis instead of bone marrow.

In summary, we revealed that angiogenic potential as evaluated by the expression of EPC-specific molecules in marrow and blood was relatively low in patients with arterial obstructive diseases, and that therapeutic implantation with autologous marrow mononuclear cells increased EPCs in the circulation. Our comparative data on the expression of EPC/EC markers in different stem cell materials may be helpful in choosing cell source for therapeutic angiogenesis in a variety of clinical settings.

	Acknowledgments

 
The authors thank Dr A. Itakura (Department of Gynecology, Nagoya University Hospital) for consultation to obtain umbilical cord blood. The authors also thank M. Aoki, K. Kinoshita, and T. Nashida for their technical assistance.

Received June 16, 2004; accepted September 27, 2004.

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This Article Abstract Full Text (PDF) All Versions of this Article: 24/12/e192    most recent 01.ATV.0000147730.78953.60v1 Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Yamamoto, K. Articles by Murohara, T. Search for Related Content PubMed PubMed Citation Articles by Yamamoto, K. Articles by Murohara, T.