iMedPub Journals

TRANSLATIONAL BIOMEDICINE

2010Vol.1No. 3:2doi: 10:3823/415

Introduction

Buerger’s disease or thromboangiitis obliterans (TAO) is a non-atherosclerotic, imammatory, vasooclusive disease. Clinicalndings show occlusive segmental and more often multipleinammatory lesions of medium and small blood vessels andsupercial vein thrombosis [1,2, 3]. The occlusion of blood ves-sels in the aected limbs and hands restricts the availability of blood supply to the aected tissues, causing severe pain, andeventually leading to tissue destruction. The disease progressesto ulceration and gangrene and necrosis of aected limb whichmay lead to amputation [4,5]. The etiology of TAO remains unknown, but previous studieshave suggested a strong link with cigarette smoking. Almostall the aected patients are smokers. In spite of strong associa-tion indicating that tobacco smoking (amongst other factors)has a role in the pathogenesis of the disease, no sucient datais available for underlying cellular and molecular mechanism of the disease. Therapeutic options for TAO patients are limited. Their conditions are often refractory to conservative measuresand are typically unresponsive to drug therapy [6-11].In 2002, Tateishi-yayuma et. al [12] published impressive resultsin patients with critical ischemia treated by the autologoustransplantation of bone marrow cells. This study showed somesuccess in pain relief with Buerger’s disease. Subsequent experi-ments by several workers have shown that autologous wholebone-marrow stem cells or bone-marrow derived stem cellsfrom peripheral blood following mobilization with granulocytecolony-stimulating factor have the potential to stimulate angio-genesis and thereby modulate hemodynamic decit in ischemiclimbs in vivo and improve the life quality of the aected patients[13-21].In the current work, stem cells in the bone marrow were mo-bilized into the peripheral blood by administration of G-CSF,followed by collection of peripheral blood stem cells using anapheresis technique. The collected cells were then injected intoaected limbs.

Bone Marrow-derived Mononuclear Stem Cell Implantationin Patients with Buerger’s Disease

1Tissue Engineering and Nanomedicine Research Center, Taleghani Hospital,Shahid Beheshti University of Medical Sciences, Tehran, Iran.*Correspondent Address: Dr. S Shahghasempour, Tissue Engineering and Nanomedicine Research Center, Taleghani Hospital,Shahid Beheshti University of Medical Sciences, Velenjak, Tehran, Iran. Tel: +98(21) 22439847, Fax +98(21) 22439848, E-mail:sgasempour@yahoo.com.

Shapour Shahgasempour, Ph.D

*, Habibullah Peirovi, M.D

and Afshin Fathi, M.D

Abstract

Background:

Patients suering from Thromboangiitis obliterans (TAO) have endothelial cell dysfunction and the severityof the disease lies in the need for amputation in more than a quarter of all suerers.

Methods and Findings:

We report the safety and feasibility of autologous implantation of circulating mononuclear cellsfor patients suering from Buerger’s disease following bone-marrow mobilization with granulocyte colony stimulatingfactor (5

g/kg/day for 5-7 days). Six patients participated in this study. Mononuclear cells were separated by Cobas Spectracell separator. MNCs, CD34+ and CD133+ cells were enumerated prior to intramuscular injection into the aected foot/limb muscles at multiple sites on the collection day. Stem cell injection prevented disease progression in all six patients.In this small cohort of patients with critical limb ischemia, quality of life improved signicantly over a two year period.Also, pain-free walking distance in all patients showed signicant improvement.

Conclusions:

Autologous mononuclear cell containing CD34+/CD133+ stem cells collected from peripheral bloodfollowing G-CSF mobilization is eective, safe and results in sustained clinical results for patients with severe peripheralocclusive arterial disease.

Keywords:

Buerger’s disease, Autologous, Bone-Marrow, Stem cell, Endothelial Progenitor, Angiogenesis.

iMedPub Journals

TRANSLATIONAL BIOMEDICINE

2010Vol.1No. 3:2doi: 10:3823/415

Patients and Methods

Seven patients with Buerger’s disease were enrolled for autolo-gous peripheral blood stem cell therapy. Patient’s eligibility wasapproved by the vascular surgery department bioethics groupat the Shahid Beheshti University of Medical Sciences. One pa-tient was excluded from the study due to lower percentagesof circulating CD4+ T helper lymphocytes. All patients had se-vere claudication, rest pain with nonhealing ischemic wounds.Patients had already received currently available medical treat-ment and surgical interventions such as sympathectomy. Theprole of six patients (with mean age 39.5

6.50

)

was as follows:Patient 1, a 36 year-old man with a severe gangrene on his toeand nonhealing wound in his left foot who had undergonemedical treatments and lumbar sympathectomy. Patient 2 wasa 42 year-old man with intractable pain in his ngers and an ul-cer on his second digit and for whom a lumbar sympathectomyand medical treatments had failed. Patient 3 was a 35 year-oldman with severe rest pain with an ulcer on his foot who hadexperienced a variety of medical treatments and interventions.Patients 4 and 5 had ulcers in fourth toe of the left lower extre-mity. Patient 6, a 42 year-old man had ulcer at the tip of his rsttoe along with deep ulcer on aected left foot. Pulse oxymetry,O2 Saturation , rest pain, pain-free walking, skin temperature,the size of skin ulceration, color dopler sonography and angi-ography were done pre- and post-implantation. Patients werescreened for HIV, HCV and HBV. Cardiac, hematological, infec-tious, renal, hepatic, metabolic and clinical parameters were alsodetermined prior to stem cell transplantation. Also, ow cyto-metric analysis (BD FACSCalibur CA, USA) were carried out forthe following marker CD19, CD3, CD4, CD8, CD34, CD13, CD14,and CD45 pre- and post-operatively. Circulating mononuclearcells with CD34+ and CD133+ markers were enumerated on theday of stem cell administration, using ow cytometry. Follow uphas been over 24 months.

Granulocyte Colony-Stimulating FactorAdministration and Stem Cell Collection

The patients received subcutaneous injections of recombinanthuman G-CSF (Filgrastim, Pooyesh Daru, Iran) daily at 5

g/kg/day for 5-7 days prior to apheresis. Blood samples were collectedand leukocytes were measured daily. Once the WBCs reachedapproximately to 25000-30000/ml, then, approximately 400-500ml suspensions of circulating peripheral blood mononucle-ar cells were collected using Cobe Spectra instrument (GambroBCT, CO, USA). The cells were then concentrated to a nal vol-ume of 60-70ml, followed by injection of the cell suspension intothe gastrocnemius muscle of aected limb under general and/or spinal anesthesia (60-70 sites, 0.5-1ml per site).

Statistical Analysis

Data were analyzed by using Wilcoxon non-parametric test.P<0.05 was considered signicant.

Results

In all six patients (table 1), normalization of limb temperaturein the aected area showed signicant improvement after 2-3weeks post-transplantation as observed by thermograph (an av-erage of 1

C). Rest pain and pain-free walking distances weresignicantly improved 1 to 2 months post-transplantation. O

saturation also improved gradually after the treatment in allsix patients. Table 2 shows percentage MNCs and CD34+ andCD133+ cells before stem cell therapy. In all patients signicanthealing of wound was noted. Color Doppler sonographic assess-ment indicates an increase in blood ow in the aected areapossibly of improved collateral circulation, Table 3.

Case#Age/YearGender/MaleSmokingHistory(Years)Pain Free WalkingDistance (m)Before-After(p=0.043)Prior TherapiesSkin Temperature (0C)Before-After(p<0.05)Ulcerin LowextremitiesBefore-After136>20150±30RILS35.036.5yesWH242>22280±50RILS36.037.0yesWH335>15250±50RILS/AP35.536.0yesWH432>17156±10RILS34.535.5yesWH550>3045±2RINone34.536.0yesWH642>2535±2RILS35.036.0yesWH

TABLE 1. Characteristics and clinical evaluation of six patients before andafter stem cell therapy transplantation. TABLE 2. Total number of MNC and percentages of CD34+/CD133+Cells Implanted in aected Limb/Foot of Patients(Double Positive Cells).

RI = Remarkably Improved, WH = Wound Healed, LS = Lumbar Sympathectomy,AP = AngioplastyCase#MNCs CountX 108/KgPercentages of CD34+and (CD133+/CD34+ImplantedInjection DayImplantation Day18.497.8 (2.13)27.988.5 (2.19)37.454.9 (1.22)45.414.8 (1.16)57.565.6 (1.61)66.674.8 (0.93)

iMedPub Journals

TRANSLATIONAL BIOMEDICINE

2010Vol.1No. 3:2doi: 10:3823/415

Discussion

Management of obstructive vascular diseases involving theextremities poses great challenges for physicians and patients.Peripheral arterial disease is often a consequence of obstructiveatherosclerosis aecting the ilefemoral circulation but is also aresult of arteriosclerotic conditions such as thromboangiitis ob-literans. Consequences range from the presence of asymptom-atic obstruction to intermittent claudication, development of rest pain, ulceration, gangrene, and amputation [10,13,14,16,17].A relatively new and promising approach using stem cell hasin recent years been developed to treat intractable symptomsrelated to ischemia in subjects with peripheral arterial diseasein whom conventional treatments has failed [1]. It has been pos-tulated that marrow stem cell implantation into ischemic limbscould enhance angiogenesis by supplying endothelial progeni-tor cells (EPCs) and angiogenic factors and/or cytokines [7].In the present study, we demonstrated that the transplanta-tion of bone-marrow derived peripheral blood mononuclearcells containing CD34+/CD133+ into lower ischemic limbs of sixBuerger’s patients was associated with a dramatic improvementin ischemic signs and symptoms possibly of distal circulationimprovement as shown in table 1and g 1. Improvement of restpain, walking distance limitation, healing of skin ulcer and anincrease in distal limb temperature was observed in all six cas-es. After stem cell injection, limb amputation in all patients hasbeen avoided for 24 months of follow-up. The formation of newcollateral vessels was observed in three patients as determinedby angiogram and color Doppler sonography. In this regard, thecurrent work supports previous observations by establishingthe potential benets of G-CSF mobilized bone-marrow stemcell transplantation for the treatment of arterial diseases suchas TAO. No adverse reaction was observed with G-CSF adminis-tration, although some reports suggest that that G-CSF mightpose harmful eect. There was no abnormality in the laboratoryndings of hematology, immunology, kidney, and liver functiontests as well as the level of serum proteins in all patients afterthe treatment, indicating that the injection of EPCs were safe forpatients with Buerger’s disease.It should be noted that the mo-lecular mechanism and process of angiogenesis in the aectedlesions have not been fully understood, and how transplantedCD34+ and/or CD34+/CD133+ stem cells are incorporated intonew vessels has not fully been elucidated. Further studies wouldbe required to illustrate the relationship between the numbersof CD34+/CD133+ stem cells injected, angiogenic promotingfactors such as cytokines and growth factors. We are currentlyworking on the molecules that are responsible for the homingand the recruitment of endothelial progenitor cells (EPC) intoischemic area.In conclusion, despite the small number of patients and techni-cal limitations on the assessment of microvascular blood ow,the eectiveness of therapeutic angiogenesis with bone-mar-row derived mononuclear cells in patients with Buerger’s dis-ease has been established. Therefore, BM-MNC transplantationmay provide a promising therapy in such patients in whom con-ventional medical therapy has been exhausted.

CasenumberPeak systolic volume(cm/s) in Posterior Tibial artery (aected limb)(P<0.05)Resistance index(%) in Posterior Tibial artery (aected limb)(P<0.05)BeforeAfterBeforeAfterCase 131.344.27462Case 24248.88660Case 326.327.55952Case 436.846.27264Case 528.8366455Case 633.441.27158

TABLE 3. Color Doppler Sonographic indicespre- and post-stem cell transplantation.Fig.1. Improvement of wound healing following stem cell transplantationin selected patients