Journal of Vascular Surgery
Volume 48, Issue 6, Supplement , Pages 53S-60S, December 2008

Therapeutic angiogenesis in Buerger's disease (thromboangiitis obliterans) patients with critical limb ischemia by autologous transplantation of bone marrow mononuclear cells

Jain Institute of Vascular Sciences, Bhagawan Mahaveer Jain Hospital, Bangalore, India

Received 11 July 2008; accepted 4 September 2008.

Article Outline

Background

Peripheral arterial disease is a significant problem worldwide. In developing countries such as India, the increased incidence of smoking and other forms of nicotine intake has resulted in a large proportion of young individuals with Buerger's disease. The results of surgical and endovascular treatment for this condition have not been very rewarding. Hence, we focused on providing alternative therapies. Neovascularization by autologous bone marrow mononuclear cell transplantation is being tried as an alternative therapeutic option. We have reviewed our series of patients who underwent autologous bone marrow mononuclear cell transplantation during the last 2 years.

Methods

We enrolled 38 patients who were chosen to undergo autologous bone marrow mononuclear cell transplantation for nonreconstructible Buerger's disease. We injected the bone marrow mononuclear cells into the calf muscles of the affected limbs in 36 patients. We monitored ulcer healing, ankle-brachial index (ABI), and transcutaneous oximetry (TcPo2) level.

Results

No procedurally related complications occurred, although one injected sample of bone marrow aspirate later revealed infestation with Strongyloides stercoralis. Two patients were seropositive on the Venereal Disease Research Laboratory test and were not injected with the bone marrow mononuclear cells. Three patients (12%) underwent major amputations ≤6 months. The others had improvements in their ulcer healing, an increase in the mean ABI of 0.14 (range, 0.1-0.19; P < .01), and improvement in the mean TcPo2 of 52 mm Hg (range, 40-68 mm Hg, P < .01), with resultant limb salvage in all at 6 months. All patients discontinued smoking during the study period.

Conclusions

Use of bone marrow–derived progenitor cell transplantation into ischemic limbs is a relatively safe procedure with no demonstrable side effects at 6 months. These study data support conducting controlled and multicenter trials to evaluate the efficacy of this therapy in preventing amputation in selected patients with Buerger's disease who have critical limb ischemia.

 

Therapeutic angiogenesis represents an attempt to relieve inadequate blood flow by the directed growth and proliferation of blood vessels. Neovascularization is a complex process involving multiple growth factors, receptors, extracellular signaling pathways, and local and bone marrow–derived cells. New blood vessel formation can occur in adult life as a result of two distinct mechanisms. The first is angiogenesis,1, 2 which involves the proliferation of capillaries and hence an increase in the local blood flow. The second is arteriogenesis, wherein the already existing high-resistance collateral vessels increase in size, resulting in a decrease in resistance and an increase in blood flow to the ischemic region.3

Angiogenesis occurs under various conditions, including local ischemia. Local cellular ischemia is said to affect the transcription factor hypoxia-inducible factor 1,4 which in turn leads to expression of vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), VEGF receptors, nitric oxide, and insulin-like growth factors.5 These local changes result in migration of the endothelial progenitor cells and formation of new thin walled blood vessels.6, 7, 8 The extracellular matrix also plays a role in this process. The sprouting of capillaries forms a new immature capillary network. The maturation of these immature capillaries involves the incorporation of pericytes under the influence of various cytokines and growth factors.9, 10, 11

Therapeutic angiogenesis can be induced by the implantation of bone marrow mononuclear cells. Kamihata et al12 and Shintani et al13 have shown that bone marrow mononuclear cells contain not only endothelial progenitor cells but also angiogenic factors and cytokines and that implantation of bone marrow mononuclear cells into ischemic tissues augments collateral vessel formation. Although CD34 is the most popular marker for stem cell selection for inducing therapeutic angiogenesis, others such as CD133 stem cells14, 15, 16 and CD117 stem cells17 also are important in therapeutic angiogenesis.

India is experiencing an exponential increase in peripheral arterial disease due to a large population of diabetic patients (42 million in 2007) and unabated smoking. Although the exact data are unavailable, 20% to 40% of these patients are estimated to have Buerger's disease, also known as thromboangiitis obliterans, especially in those who smoke a crude form of rolled tobacco leaves called beedies or have other forms of tobacco intake, including chewing of gutka, a highly concentrated form of tobacco leaves.

A significant social problem in patients with thromboangiitis obliterans is that they are young, most are from lower socioeconomic strata, and many are single breadwinners for their families and are generally unskilled manual workers. This socioeconomic problem, coupled with the fact that present medical, surgical, and endovascular treatment modalities have not been very successful, has resulted in a dire need for alternative therapies. Thus, investigations have been initiated of the role of bone marrow–derived progenitor cell transplantation into ischemic limbs in these patients, in whom loss of a limb could have serious repercussions on the entire family. Various studies have concluded that transplantation of bone marrow mononuclear cells could contribute to a safe and effective strategy for achievement of therapeutic angiogenesis and hence prevention of amputation.18, 19

Back to Article Outline

Materials and methods 

Patients 

Thromboangiitis obliterans was diagnosed by the modified Olin20 criteria and included smoking history, age of onset <45 years, infrapopliteal arterial occlusive disease, angiographic evidence of medium and small vessel disease, upper limb involvement or phlebitis migrans, absence of atherosclerotic risk factors other than smoking/tobacco use at the time of initial diagnosis, and exclusion of autoimmune diseases, diabetes mellitus, proximal source of emboli, and hypercoagulable states.

Inclusion criteria 

Our inclusion criteria for patients qualifying to undergo bone marrow progenitor cells transplantation, as approved by Ethics Committee for Human Research of the hospital and after providing informed consent, were age <45 years, no history of diabetes mellitus, no evidence of malignancy, patients with Buerger's disease, has limb-threatening ischemia, and established methods of treatment were not possible or were tried and were not successful.

Exclusion criteria 

Patients with diabetes mellitus, atherosclerosis, thrombotic disease, proximal embolic source, malignancy, or hypercoagulable states were excluded. Those who would require amputation proximal to transmetatarsal level were also excluded.

Procedures 

The study enrolled 38 men (38 limbs) with thromboangiitis obliterans. Patients' mean age was 34 years. All 38 patients had nonhealing ischemic wounds of a minimum of 2 months' duration that showed no improvement with conventional therapy. All patients also had rest pain of >2 months' duration.

The baseline evaluation for all patients included a complete blood count, erythrocyte sedimentation rate, blood glucose level, renal and liver function tests, an electrocardiogram, and a chest radiograph. An echocardiogram and tests for connective tissue disorders and hypercoagulable states were not done routinely due to financial constraints in these patients.

An epidural catheter was placed on admission in all patients (except one with upper limb ischemia) for pain control. All were counseled against smoking. Appropriate wound care was commenced and they were prescribed systemic antibiotics. Intra-arterial digital substraction angiography was done for the indexed extremity before the patient was diagnosed as having nonreconstructible disease.

The primary outcome was limb salvage at 6 months. We looked for decrease in rest pain, healing of ulcers, improvements in the ankle-brachial index (ABI) scores, and rise in transcutaneous oxygen pressure (TcPo2) levels in the patients undergoing bone marrow progenitor cell transplantation.

Regional anesthesia was used for the procedure. To increase the yield, bone marrow was aspirated from the posterior iliac crests in different directions using a bone marrow aspiration needle and 20-mL syringes.The average volume of bone marrow aspirated was 400 mL, with minimum mixing of sinusoidal blood from each patient. The collection was done in a sterile container.

The marrow was taken to the sterile stem cell processing laboratory where stem cell enrichment took place in cyclic guanosine monophosphate conditions. Mainly, the procedure consisted of red blood cell (RBC) sedimentation by 6% hydroxyethyl starch (HAES) at unit gravity. This resulted in RBC depletion of >90%. The nucleated cells present in the supernatant were concentrated by centrifugation at 1500 rpm. The nucleated cell pellet was reconstituted in sterile 6% HAES, and 0.5 mL of cell suspension was taken for cell count viability and CD34 count by flow cytometry.

After RBC depletion and volume reduction, the nucleated cell count was 5.8 × 107 ± 4 × 107/mL. The final volume of the RBC-depleted cell suspension was 45 to 60 mL, which was used for injections into the patient. The total number of CD34 in the cell suspension was 9.8 ± 9.91 × 106. Viability was 99%. CD34 count was done using BD Procount (Becton Dickinson, Franklin Lakes, NJ) using International Society of Hematotherapy and Graft Engineering protocol.

The cell suspension was cultured and found to be free of contamination in 36 patients. This was then injected into multiple sites in the gastrocnemius muscle of the ischemic lower limb. The sites marked on the gastrocnemius muscle were 1 cm apart. A 2-mL syringe and a 20-gauge needle were used to inject 0.75 mL into each site intramuscularly (Fig 1). A baseline ABI and TcPo2 were performed preoperatively.

We followed up the patients weekly for 4 weeks and monthly thereafter. Monitoring of ulcer healing, decrease in rest pain, ABI and TcPo2 was done at the end of 1 month and 6 months. We judged an increase in ABI of >0.1 as an improvement according to the standard assessment of interventional therapy for peripheral arterial diseases.21 TcPo2 was measured on the dorsum of the foot using a TCM400 TcPo2 monitoring system (Radiometer Medical APS, Denmark). This report includes 38 patients recruited between November 2005 and 2007. Two bone marrow aspirates were seropositive on the Venereal Disease Research Laboratory test, although the initial test results were negative, and hence were not processed. Three patients required amputation by the 6-month follow-up, which left 33 patients in the study. In addition, one patient was found to have Strongyloides stercoralis (Fig 2) in the collection, which we came to know a day after the therapy.

Statistical analysis 

The study data were analyzed using SPSS 16.0 software (SPSS Inc, Chicago, Ill). Statistical analysis has been provided for baseline, 1 month, and 6 months for ABI (Table I, Table I), TcPo2 (Table II, Table II), and visual analog scale scores (Table III, Table III). The difference in the baseline and end of study has been compared using the paired-samples t-test. The level of significance was fixed at P = .05.

Table I. A, Descriptive statistics for ankle-brachial index
VariableNo.MinimumMaximumMeanSDSE
Baseline380.120.470.26370.068040.0111
1 mon360.270.600.36810.066020.01100
6 mon330.300.650.41730.069830.01216
Table I. B, Paired samples statistics for ankle-brachial index
VariableMeanSDSEMP
Baseline-1 mon.09861.03735.00623<.001
Baseline-6 mon.14636.02608.00454<.001
1-6 mon.05030.02675.00466<.001

SD, Standard deviation; SE, standard error; SEM, standard error of the mean.

Table II. A, Descriptive statistics transcutaneous oximetry
VariableNMinimumMaximumMeanSDSE
Baseline38205128.185.526.896
1 mon36346048.176.1341.022
6 mon33406852.125.9041.028
Table II. B, Paired sample test analysis of transcutaneous oximetry data
VariableMeanSDSEMP
Baseline-1 mon19.8335.940.990<.001
Baseline-6 mon23.6976.3071.098<.001
1-6 mon3.4243.666.638<.001

SD, Standard deviation; SE, standard error; SEM, standard error of the mean.

Table III. A, Descriptive statistics for the visual analog scale
VariableNMinimumMaximumMeanSDSE
Baseline388108.74.724.117
1 mon36153.361.046.174
6 mon33042.451.660.289
Table III. B, Paired samples test analysis of the visual analog scale results
VariableMeanSDSEMP
Baseline-1 mon5.3611.222.204<0.001
Baseline-6 mon6.2421.696.295<0.001
1-6 mon.8791.139.198<0.001

SD, Standard deviation; SE, standard error; SEM, standard error of the mean.

Back to Article Outline

Results 

We tested the ABI in all study patients before the procedure and also obtained a follow-up ABI measurement in all the patients at 4 weeks and 6 months after the procedure. The ABI improved in all 33 patients (Fig 5). The mean preprocedural ABI was 0.26 (range, 0.12-0.47). An increase in ABI of >0.1 at the end of 1 month and 6 months was noted in 33 patients, with a mean increase of 0.14.

Similarly, TcPo2 was measured before the procedure and again at the end of 4 weeks and 6 months. Compared with baseline values, the 33 patients showed a significant increase in TcPo2 levels from the mean baseline TcPo2 of 28 mm Hg (range, 20-51 mm Hg). The mean TcPo2 level was 48 mm Hg (range, 34-60 mm Hg) at 4 weeks after the procedure and 52 mm Hg (range, 40-68 mm Hg) at the end of 6 months (Fig 7).

Marrow implantation induced no local inflammatory reaction in the form of redness, pain, erythema, or swelling in any of the study participants. No major adverse events were reported in any of the patients during their hospital stay or in the 6-month follow-up period.

We used bone marrow mononuclear cells containing CD34 cells, and it is postulated that these cell fractions release multiple angiogenic factors to enhance angiogenesis in addition to supplying endothelial progenitor cells. The mean (SD) CD34 count was 9.88 ± 9.91 million (range, 1.62-33.6 million), the total mean mononuclear cell count (×108) was 26.23 ± 17.98 (range, 1.80-64.93), and the mononuclear cell count per mL (×107) was 0.3 to 14.4.

Clinical improvement was shown by decrease in rest pain on a visual analog scale score in 25 patients, with complete regression in eight (Fig 6). Also, ulcer healing was noted in 23 patients, and seven patients had complete healing of their ulcer in a time frame of 1 to 6 months. In three patients the size of the ulcer did not change during the study period. Improvement in rest pain, ulcer healing, ABI, and TcPo2 levels were maintained in all patients during their 6-month follow-up, except in three patients (12%) in whom an amputation was necessary due to severe rest pain with progression of foot gangrene.

Back to Article Outline

Discussion 

We have shown that implantation of bone marrow–derived progenitor cells into the ischemic limbs resulted in an increase in blood flow in the ischemic limbs in 33 patients. This was assessed by documenting an increase in the ABI and in the TcPo2 measurements. Rest pain also decreased in most patients. Similarly on follow-up, we also noted an improved ulcer healing and that seven of the ulcers completely healed (Fig 3, Fig 4).

  • View full-size image.
  • Fig 7. 

    A, Representation of mean transcutaneous oximetry data and the standard error (bars). B, Representation of individual subject data for transcutaneous oximetry levels.

Chronic critical limb ischemia is defined as persistent recurring rest pain of >2 weeks' duration not responding to regular, adequate analgesia and an ankle pressure of <50 mm Hg or a toe pressure of <30 mm Hg. The study inclusion criteria were that we enroll patients with Buerger's disease who did not have diabetes and had undergone or were unable to undergo the standard treatment options for their disease. All of the 38 patients in our study had critical limb ischemia despite having undergone earlier surgical treatment in the form of lumbar sympathectomy before they were included in the study.

Angiogenesis1, 22, 23 that occurs can result in growth of new capillaries or growth of pre-existing collateral vessels. For newly formed vessels to survive, they must undergo remodeling and acquire a layer of smooth muscle cells. In view of the complex process of vessel formation, treatments that use only one angiogenic factor might result in suboptimal endothelial channels.

The CD34 fraction in bone marrow mononuclear cells reportedly synthesized not only angiogenic growth factors like VEGF and basic FGF but also angiopoietin I,9 which is important in the maturation and maintenance of the vascular system; hence, resulting in blood vessels with an adequate composition of smooth muscle cells, periendothelial matrix, and pericytes.24

Buerger's disease, also known as thromboangiitis obliterans, is characterized by the occurrence of segmental thrombotic occlusions of the medium- and small-sized arteries. This entity is seen in young smokers with no other risk factors and is more common in Asia than in the West. The incidence of major amputation in this patient population according to multiple studies is 12% to 31% and has included both patients with claudication and those with critical limb ischemia.25

Because this is probably the first study of this kind in India in patients with ischemic limbs, and also owing to the small number of patients included in the study, the interpretation of the results, which appear promising, are limited. Absence of a control, the small number of patients enrolled, and absence of a postprocedural angiogram are the limitations of this study. A point to be remembered in this discussion is that most of the patients belong to lower socioeconomic strata and are single breadwinners for their families. This procedure, which was done in 36, revealed no adverse effects on any of them and more important, the approximate cost of the total procedure and stay in the hospital was US $200 to $300 compared with peripheral vascular bypass, which would cost approximately US $1000 to $1300. Because these patients belonged to a low-income group, with out any medical insurance, their care was completely funded.

Another factor to be kept in mind is that although all the patients in the study said they did not restart smoking during their 6 month follow-up, no testing for urine cotinine by radioimmunoassay was done. Hence, the contribution of persistent smoking to the disease outcome could not be assessed. We would also like to state in conclusion that this study is a preliminary one to show safety, but that efficacy requires a placebo-controlled study, because other associated treatment, namely smoking cessation, could have substantially influenced the outcome.

Back to Article Outline

Author contributions 


Conception and design: VM

Analysis and interpretation: KR

Data collection: GI

Writing the article: VM

Critical revision of the article: VI

Final approval of the article: KR

Statistical analysis: SU

Obtained funding: SU

Overall responsibility: VM

Back to Article Outline

 

We acknowledge Ms Rohini Nilekeni of Arghyam Trust and Dr Majid for funding these patients, and Dr Anand Rao of SR Biotechnology for bone marrow processing and technical assistance.

Back to Article Outline

References 

  1. Risau W. Mechanisms of angiogenesis. Nature. 1997;86:671–674
  2. Carmeliet P. Angiogenesis in health and disease. Nat Med. 2003;9:653–660
  3. Schaper W, Buschmann I. Collateral circulation and diabetes. Circulation. 1999;99:2224–2226
  4. Wang GL, Jiang BH, Rue EA, Semenza GL. Hypoxia-inducible factor 1 is a basic–helix loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc Natl Acad Sci U S A. 1995;92:5510–5514
  5. Jiang BH, Rue E, Wang GL, Roe R, Semenza GL. Dimerization, DNA binding and transactivation properties of hypoxia inducible factor 1. J Biol Chem. 1996;271:17771–17778
  6. Gale NW, Yancopoulos GD. Growth factors acting via endothelial cell specific receptor tyrosine kinases: VEGFs, angiopoietins, and ephrins in vascular development. Genes Dev. 1999;13:1055–1066
  7. Dvorak HF, Brown LF, Detmar M, Dvorak AM. Vascular permeability factor/vascular endothelial growth factor, microvascular permeability and angiogenesis. Am J Pathol. 1995;146:1029–1039
  8. Nabel EG, Yang Zy, Plautz G, Forough R, Zhanx , Haudenschild CC, et al. Recombinant fibroblast growth factor-1 promotes intimal hyperplasia and angiogenesis in arteries in vivo. Nature. 1993;362:844–846
  9. Thurston G, Rudge JS, Ioffe E, Zhou H, Ross L, Croll SD, et al. Angiopoietin–1 protects the adult vasculature against plasma leakage. Nat Med. 2000;6:460–463
  10. Hirschi K, Rohovsky S, D'Amore P. PDGF, TGF-b and heterotypic cell-cell interactions mediate endothelial cell induced recruitment of 10T1/2 cells and their differentiation to a smooth muscle fate. J Cell Biol. 1998;141:805–814
  11. Luttun A, Tjwa M, Moons L, Wu Y, Angelillo-Scherrer A, Liao F, et al. Revascularization of ischemic tissues by PlGF treatment, and inhibition of tumor angiogenesis, arthritis and atherosclerosis by anti-Flt1. Nat Med. 2002;8:831–840
  12. Kamihata H, Matsubara H, Nishiue T, Fujiyama S, Tsutsumi Y, Ozono R, et al. Implantation of bone marrow mononuclear cells into ischemic myocardium enhances collateral perfusion and regional function via side supply of angioblasts, angiogenic ligands and cytokines. Circulation. 2001;104:1046–1052
  13. Shintani S, Murohara T, Ikeda H, Veno T, Sasaki K, Dvan J. Augmentation of postnatal neovascularization with autologous bone marrow transplantation. Circulation. 2001;103:897-5
  14. Qvirici N, Soligo D, Caneva L, Servida F, Bossolasco T, Lambertenghi G. Differentiation and expansion of endothelial cells from human bone marrow CD 133+ cells. Br J Haematol. 2001;115:186–194
  15. Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T. Isolation of putative progenitor endothelial cells for angiogenesis. Science. 1997;275:964–967
  16. Peichev M, Naiyer AJ, Pereira D, Zhu Z, Lane UJ, Williams M. Expression of VEGFR-2 and AC 133 by circulating human CD 34+ cells identifies a population of functional endothelial precursors. Blood. 2000;95:952–958
  17. Li TS, Hamano K, Nishida M, Hayashi M, Ito H, Mikamo A, et al. CD117+ stem cells play a key role in therapeutic angiogenesis induced by bone marrow cell implantation. Am J Physiol Heart Care Physiol. 2003;285:H931–H937
  18. Higashi Y, Kimura M, Hara K, Noma K, Jitsuiki D, Nakagawa K. Autologous bone marrow mononuclear cell implantation improves endothelium dependant vasodilation in patients with limb ischemia. Circulation. 2004;109:1215–1218
  19. Saigawa T, Kato K, Ozawa T, Toba K, Makiyama Y, Mingawa S. Clinical application of bone marrow implantation in patients with arteriosclerosis obliterans and the association between efficacy and the number of implanted bone marrow cells. Circ J. 2004;68:1189–1193
  20. Olin JW. Thromboangiitis obliterans (Buerger's disease). N Engl J Med. 2000;343:864–869
  21. Tateishi-Yuyama E, Matsubara H, Murohara T, Ikeda U, Shintani S, Masaki H, et al. Therapeutic angiogenesis for patients with limb ischemia by autologous transplantation of bone marrow cells: a pilot study and a randomized controlled trial. Lancet. 2002;360:427–435
  22. Asahara T, Kawamoto A. Endothelial progenitor cells for postnatal vasculogenesis. Am J Physiol Cell Physiol. 2004;287:C572–C579
  23. Isner JM, Asahara T. Angiogenesis and vasculogenesis as therapeutic strategies for postnatal neovascularization. J Clin Invest. 1999;103:1231–1236
  24. Kinnaird T, Stabile E, Burnett MS, Epstein SE. Bone marrow derived cells for enhancing collateral development. Circulation. 2004;95:354–363
  25. Borner C, Heidrich H. long-term follow-up of thromboangiitis obliterans. Vasa. 1998;27:80–86

 STATEMENT OF CONFLICT OF INTEREST: These authors report that they have no conflicts of interest with the sponsor of this supplement article or products discussed in this article.

PII: S0741-5214(08)01519-X

doi:10.1016/j.jvs.2008.09.005

Journal of Vascular Surgery
Volume 48, Issue 6, Supplement , Pages 53S-60S, December 2008

Article Tools

* Image Usage & Resolution