Bone Marrow Stem Cell Therapy (SCT) for Peripheral Arterial Disease (PAD): an Initial Experience

Introduction: 30% of patients with critical limb ischaemia (CLI) are not suitable for conventional treatment. Use of stem cell therapy (SCT) is relatively new. This study shares an initial experience using SCT in 4 patients. Methods: Approval from Institutional Medical Board Ethics Committee was obtained prior to commencement and informed consent was sought. Patients included had extensive CLI history that was no longer amenable to standard treatment. Bone marrow aspiration from the iliac crest was carried out under regional anaesthesia. This was later centrifuged and injected intramuscularly and adjacent to affected vessels. Wound surveillance was then performed. Results: SCT was well tolerated in all 4 patients and 2 had favourable results. None developed related complications. Patients 1 and 2 showed improvement of rest pain, claudication symptoms and healing of ulcers. Angiogenesis and neovascularization can be seen in follow up angiography for patient 1. Wound healing was not noted in the other 2 patients, with both requiring amputations eventually. Discussion: The experience while early has been invaluable. The varied response suggests that factors determining treatment success remained unknown. Likewise, most other trials have consisted of small uncontrolled patient series, with few randomized studies. Haemodialysis, diabetes mellitus and coronary arterial disease factors seemed to negatively affect angiogenesis. Severity of rest pain and number of repeated interventions, in particular bypass procedures, may negatively intervene with neo-capillary formation. SCT may eventually provide hope to patients and physicians. More research can help determine a specific group of patients that will benefit most. Journal of Blood & Lymph Jo rn al o f Blood Lym ph


Introduction
Critical limb ischaemia (CLI) is the most severe form of peripheral arterial disease where there is inadequate blood flow to maintain metabolic requirements of the tissues at rest [1]. Treatment is often challenging, with surgical or endovascular revascularisation considered to be the gold standard. Up to 30% of patients however, are not suitable for treatment and require limb amputation, while 25% suffer mortality from the disease [2]. This is due to the high operative risks conferred by concomitant chronic co-morbidities such as ischemic heart disease, chronic renal impairment, as well as the often adverse extent of vasculopathy with diffuse involvement of multiple levels and distal stenosis. Locally, 8.9% of the population between 18 and 69 years are diabetic, and the risk of CLI is increased 4 fold in these patients [3]. CLI in diabetes preferentially involve infra-popliteal distal calf vessels with diffuse, multi-level distal stenosis compounding the difficulties faced during revascularization. Patients require repeated procedures due to a combination of premature advanced atherosclerosis, peripheral neuropathy, impaired cellular immunity and impaired wound healing. Up to 45% of all amputees are diabetic, and a diabetic patient with CLI is 10 times more likely to require an amputation despite new technology and innovation in revascularization [4].

Bone Marrow Stem Cell Therapy (SCT) in clinical practice
Embryonic and adult stem cells have the capacity to renew and generate differentiated cells. Embryonic stem cells are pluripotent, while adult stem cells are partially lineage committed giving rise to specialized cells of the germ layer. (i.e., they are multipotent rather than pluripotent). Bone marrow stem cells include progenitor cells such as the multipotent adult progenitor cells, mesenchymal and haematopoietic stem cells. Endothelial progenitor cells are adult haemangioblast-derived cells. In addition to having all functional properties of endothelial cells, they secrete paracrine mediators, interleukins, growth factors (angiopoietins Ang1 and Ang2, vascular endothelial growth factor (VEGF)) and chemokines to encourage migration of endothelial and support cells and the further proliferation and differentiation of the progenitor cells. Likewise, mesenchymal stem cells stimulate endothelial cell migration and vessel tube formation. They too release factors, migrate to injured sites, differentiate and secrete further trophic factors for paracrine signalling, activating endothelial cells [5]. SCT in the treatment of PAD is relatively new. In 1997, Asahara et al. identified a class of bone marrow-derived, circulating endothelial progenitor cells that contribute to angiogenesis in ischemic tissues [6]. Thereafter, the first clinical trial of cell therapy for PAD was performed by Tateishi-Yuyama et al. in 2002 known as the  Therapeutic Angiogenesis using Cell Transplantation Study (TACT), where bone marrow derived mononuclear cells were injected into the gastrocnemius muscle, and subsequent improvement in ankle-brachial index (ABI), transcutaneous oxygen tension, pain-free walking time, and rest pain was documented [7]. Since then, several studies have reported variations of stem cells usage, including embryonic, adult and induced pluripotent stem cells, examining the efficacy and safety of cell therapy while obtaining varying results [8,9]. The procedure has also been generally well tolerated, with the most frequent adverse reaction being local pain or anaemia. In this study, an initial experience with 4 patients who underwent SCT as an alternative therapy when standard revascularisation was no longer an option for them is described. The aim of the study is to see if these patients can avoid amputation through improved wound healing brought about by angiogenesis and vascular collateral formation.

Materials and Methodology
Approval from the Institutional Medical Board Ethics Committee was obtained prior to commencement of this study. Informed consent was sought from all patients undergoing this treatment. Patients selected had no evidence of underlying malignancy, with life expectancy of more than 6 months, not pregnant and were without known haematological disease. All patients selected had an extensive CLI history, multiple revascularisation procedures, failed revascularisation therapy or recalcitrant, recurrent, persistent disease not amenable to treatment for reasons including high surgical risk, multi-level distal disease or both as summarized in Table 1.

SCT procedure
Bone marrow aspiration from the iliac crest was carried out under regional anaesthesia in the operating theatre. The aspirate is then centrifuged with the Terumo SmartPRep2 system (Figures 1A-E) to allow concentration of Bone Marrow-Mononuclear cells (BM-MNC). This was then injected intramuscularly, adjacent to the affected vessels, 1 mL at 2 cm intervals under ultrasound guidance with the remaining around ulcers and wounds (peri-lesional). Wound surveillance was performed at both inpatient and outpatient settings post procedure.
Clinical improvement in terms of reduced or resolution of symptoms was also noted ( Figure 1).

Results
SCT was well tolerated in all 4 patients and patients 1 and 2 were discharged well. None of them developed direct SCT-related complications such as anaemia or severe injection site pain. Patients 1 and 2 benefitted from SCT with improvement of rest pain, claudication symptoms and healing of ulcers. We achieved the best results in Patient 1 who had excellent clinical improvement and was well enough to go abroad half a year after treatment. Angiogenesis and neovascularization can be seen in her follow up angiography 3 months post SCT. In patients 3 and 4, results of the treatment were not as encouraging. Wound healing was not noted in either. Patient 3, whose wound deteriorated further, underwent further multiple amputations to cope with the sepsis. Patient 4 was readmitted for exacerbation of cardiac failure brought about by NSTEMI about a month after treatment. Prior to his demise, the patient had returned to smoking, neglected foot care and declined further intervention or amputation. Table 2 summarizes the results of the patients undergoing SCT.

Discussion
The experience with injection of BM-MNCs for CLI in these 4 patients, while early, has been invaluable. Healing of ischemic ulcers demands an improvement in blood supply, good glycaemic control as well as tissue that is infection free. The treatment of CLI is often fraught with despair and the disease process is known to take a toll both physically and psychologically. Many patients develop fatalistic attitudes as they lose their limbs, give up on good glycaemic control      Left mid shin and dorsal foot non-infected ulcers with exposed tendons, absent pedal pulses Brisk flow to profunda via collaterals

US Duplex and Angiography (Figure 2)
Bilateral EIA, CFA, SFA severely calcified and occluded to popliteal, peroneal and L DP occluded.
<1mm trickle to R PTA DEB to L CIA and EIA performed

CT Peripheral Angiography
Failed SFA plasty-with balloon rupture secondary to calcified occlusion.
Extensive atherosclerosis involving aorta, stenosis of common, internal, external iliac arteries and CFA Considered Aorto-illiac bypass

Months Later
Left lower limb cellulitis and distal ulcers with wet gangrene and exposed mid-foot tendon             mononuclear cells and/or peripheral blood-derived mononuclear cells harvested with or without granulocyte colony-stimulating factor mobilization. Cells were delivered by direct intramuscular injection at multiple sites of the affected limb or by intra-arterial injection via the femoral artery. Reported endpoints of these studies have included ABI, transcutaneous oxygen tension, and angiography examined at baseline and following cell therapy, with an average follow-up period of approximately 6 months to 1 year. Subjective outcomes have also been reported, including patient-perceived rest pain and pain-free walking time or distance. Collectively, results were promising and the procedures were well tolerated by the patients, with few adverse events reported (Figures 2-4). Haemodialysis, diabetes mellitus and complications with coronary arterial disease are all known factors affecting amputation-free survival in patients as these risk factors can negatively affect angiogenesis [10][11][12]. Matoba et al. postulate that severity of rest pain and number of repeated vascular interventions, in particular bypass procedures may negatively intervene with neocapillary formation sprouting from collateral vessels retarding angiogenesis [13]. While severe rest pain from ischemic limbs may lead to an active production of cytokines promoting angiogenesis, multiple attempts at revascularisation may retard this and render the patient unable to achieve any healing of ischemic wounds. SCT might thus work best through delivery of these angiogenic factors.

Conclusion
This study, likely the first of its kind in Singapore and Southeast Asia, has proven no different from others that SCT may eventually provide hope to patients and surgeons that have exhausted conventional treatment options save for amputation(s). The science is not perfect but it remains a potential treatment for chronic limb ischaemia recalcitrant to standard of care techniques. Perhaps more widespread use of SCT can help determine a specific group of patients that will benefit most from it.