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Adult Stem Cells and the Clinical Arena: Are we Able to Widely Use this Therapy in
Patients with Chronic Limbs Arteriopathy and Ischemic Ulcers without Possibility of
Revasculariza...

Article  in  Cardiovascular & hematological agents in medicinal chemistry · February 2012

DOI: 10.2174/187152512800388920 · Source: PubMed

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 Cardiovascular & Hematological Agents in Medicinal Chemistry, 2012, 10, 99-108 99

Adult Stem Cells and the Clinical Arena: Are we Able to Widely Use this
Therapy in Patients with Chronic Limbs Arteriopathy and Ischemic Ulcers
without Possibility of Revascularization?

Amelia Casamassimi1,#, Vincenzo Grimaldi1,#,*, Teresa Infante2,#, Mohammed Al-Omran3,

Valeria Crudele1 and Claudio Napoli1,2

1
Department of General Pathology, Chair of Clinical Pathology, U.O.C. Immunohematology, and Excellence Research
Centre on Cardiovascular Disease, 1st School of Medicine, Second University of Naples, 80138 Naples, Italy (AC, VG,
VC, C.N.) 2Fondazione Studio Diagnostica Nucleare (SDN), IRCCS, 80134 Naples, Italy (T.I., C.N.); 3Peripheral
Vascular Disease Research Chair, College of Medicine, King Saud University, Riyadh, Saudi Arabia (M.AO.)

Abstract: The following paper is an overview on stem cells therapy in patients with peripheral vascular diseases. Recent
research shows the ability of stem cells to develop and strengthen the collateral network in ischemic legs. Here,
we discuss this clinical and therapeutic approach. To date, research has been mainly focused on patients with ischemic
ulcers without possibility of revascularization. Non-invasive stem cell therapy has been proposed as an alternative to the
amputation of such patients, but when the ulcers appear it is sometime too late. In our point of view, the selection of
patients is a very important issue and we believe that the best candidate for this treatment is the patient with intermittent
claudication before the development of ulcers. This choice could allow the optimization of results by the type of treated
patients and not only by the type of infused cells. Indeed, several variables still remain to be elucidated for stem cell
therapy, including the type of cells to be used, the infusion route, and more importantly, the stage of patients to be treated.
Keywords: Angiogenesis, autologous transplantation, bone marrow cells, circulating endothelial progenitor cells, clinical trials,
Fontaine’s stage, gangrene, intermittent claudication, limb ischemia, peripheral arterial disease, revascularization.

INTRODUCTION
The peripheral arterial disease (PAD) is caused by an
inadequate supply of oxygen to the limbs. PAD has two
main stages: initially, the lack of oxygen occurs after
muscular exertion, causing the Intermittent Claudication
(IC); then, necrosis and gangrene are the result of chronic
oxygenation absence [1]. Unfortunately, many patients cannot
be helped with surgery or endovascular revascularization.
Moreover, this type of intervention is not conclusive,
because after a few years occlusion and thrombosis can
occur. To date, we have not yet found a way to prevent
this complication; antiplatelet therapy has only prolonged
the time of thrombosis occurrence, as well as, monitoring
with echocolordoppler and endovascular technique (PTA).
Stem cell therapy has been proposed as an alternative to
amputation of limbs [2]. Some clinical studies have shown
that injection of bone marrow cells (BMC) in ischemic limbs
is an effective therapy and can improve tissue vascularization
[3].
But our question is: which patients may actually benefit
from progenitor cells therapy? During the past years the
main end-point of most studies has been the improvement of
*Address correspondence to this author at the Department of General
Pathology, U.O.C. Immunohematology, Second University of Naples, Piazza
Miraglia, 2, 80138, Naples, Italy; Tel: +390815667567; Fax: +390819700943;
E-mail: vincenzo.grimaldi@policliniconapoli.it
#
Contributed equally to this study
ulcers lesions and leg savage. Only few studies were
concentrated on pain free walking distance without lesions.
We think that this point is very important in the research of
this therapeutic approach, since, of course, not all patients
can get the same outcome after cell therapy. Thus, proper
patient selection is necessary to change this therapy
assessment and to provide new opportunities of positive
outcomes.
In this review, we focus on clinical trials that use stem
cells to treat patients suffering from limb ischemia.
THE PREVALENCE OF PERIPHERAL VASCULAR
DISEASE
The recent prevalence of the disease has been derived
from numerous studies. In the Limburg study on 3654
individuals, the prevalence of IC was 0.6% in aged between
45 and 54 years, 2.5% between 55 and 65 years and 8.8%
between 67 and 74 years [4]. The Edinburgh Artery Study
(1592 patients) showed a prevalence of 4.5% between 55 and
74 years [5]. In the Scotland study, performed on 10,042
patients, the prevalence of IC between 40 and 59 years
turned out to be 1,1% [6]. In the Palermo study on 1558
patients, a prevalence of 4.7% IC was observed in the range
between 40 and 49 years and 9.2% between 50 and 59 years
[7]. Finally, in the Rotterdam Study evaluating 7715 subjects,
the presence of PAD and IC was determined through the
measurement of ankle-arm index and PAD was considered
present with an index was below a 0.9 value. In these
subjects, the prevalence of PAD was 19.1%, whereas IC was

1875-6182/12 $58.00+.00 © 2012 Bentham Science Publishers

100 Cardiovascular & Hematological Agents in Medicinal Chemistry, 2012, Vol. 10, No. 2
1,6% [8]. In most of these studies there is a reasonable
correlation of results with an average prevalence of PAD
varying between 3% and 6% around the age of 60 years.
CLINICAL TREATMENT
Usually PAD patients are subdivided according to the
Fontaine’s classification (Table 1). The risk factors associated
with PAD are old age, diabetes, smoking, hypertension, and
dyslipidemia (TASC II) [1]. Interestingly, the presence of
these risks is associated with a lower number and reduced
functionality of circulating vascular progenitor cells [9, 10].
The patient with critical limb ischemia has a poor prognosis
and is at high risk for limb amputation [1].
Table 1. Leriche Fontaine’s classification.
Fontaine’s Stages Symptomatology
Ist stage Asymptomatic
IInd stage Claudicatio intermittens
rd
III stage Chronic pain
IVth stage Trophic lesion-gangrene
The first step of PAD treatment is deleting the risk
factors. In the Ist and IInd stages medical procedures are used
(antiplatelet, muscular work, carnitin, Pentoxifylline, etc).
The aim of therapy is to increase the free pain walking
distance as well as to obtain a normal relationship life. In
patients without response to conservative and pharma-
cological therapies, the revascularization intervention and
control of pain can save the limbs.
In patients at IIIrd and IVth stages, revascularization
intervention is directly used because of the promptness
of disease progression rapidly leading to amputation
for massive gangrene in a few months. Two types of
revascularization procedures can be used: surgical by-pass or
endovascular techniques. The choice depends on the
localization of the atherosclerotic obstruction and on the
lesion type. A significant proportion of patients are not
eligible for these revascularization procedures, due to the
distal localization of the obstruction or its length or the
absence of receiving vessels [9]. Indeed, the aim of these
interventional procedures is to maintain the blood on feet.
To this purpose, an artery “giver” and an artery “receiving”
(run off) are necessary; but, unfortunately, they are not
always present. In the last patients, to date, therapy is based
on pain control (either pharmacological or with spinal cord
stimulation).
In the patient at the IVth stage with important gangrene,
sometimes, the amputation is the only option to save life.
In the TASC II manuscript [1], there are only few words
about the implantation of borrow marrow cells, simply
indicated as a promising research. Particularly, it mentioned
the ability to stimulate vascular growth by bone marrow cells
or treatment with vascular endothelial growth factor (VEGF)
and basic fibroblast growth factor (bFGF). At this time,
therapy for critical ischemia of lower limbs is based on
Casamassimi et al.
surgical intervention or prostanoids usage. However, most
patients, after failure of surgery, have to undergo to major
amputation after few years or months. Moreover, as stated
above, not all patients can have surgery revascularization [9].
Indeed, the assessment of run-off is the prerequisite to
achieve a by-pass. Then, a lot of randomized studies show
the no effect of surgical treatment on amputation tax [1]. In
this context, therapy with progenitor cells can be introduced.
In most preclinical and clinical studies on progenitor cells
the end-point used has been the amputation of limbs or the
regression of necrosis. But, how long is needed to obtain a
collateral network in the stem cell therapy? Is the gangrene
faster than stem cell implantation?
ADULT STEM-LIKE CELL THERAPY
The main goal of cell therapy is to improve peripheral
vascularization by inducing therapeutic angiogenesis. Two
distinct post-embryogenesis mechanisms are essentially
responsible for new blood vessel formation in adult life:
angiogenesis, where local capillaries proliferate and thereby
increase local distribution of blood flow [10]; arteriogenesis,
where pre-existing high resistance collaterals enlarge,
thereby decreasing flow resistance and increasing perfusion
in the ischemic region [11]. Angiogenesis occurs in response
to local hypoxia and it is mediated by hypoxia-inducible
factor 1-alpha (HIF-1
) release of chemokines and
cytokines, particularly VEGF and related signalling growth
factors, FGF and nitric oxide; then, endothelial cells loosen
their intracellular connections, sprout, migrate and form new
thin-walled vessels [12]. An increase in the number of
capillaries without a concomitant increase in the number and
caliber of conducting vessels is insufficient to augment tissue
perfusion.
It is the second mechanism, arteriogenesis, involving
remodelling of pre-existing small arterioles into larger
vessels, that is the essential component of a developing
functional collateral network [13, 14]. Under normal
circumstances, flow through pre-existing collaterals (lying in
parallel to the native artery) is low because of their small
caliber and resulting high resistance. However, following
parent vessel occlusion, the consequent high pressure
leads to an increase of blood flow exerted on the wall of
arterioles connecting the vessels proximal and distal to the
occlusion [15]. This pressure gradient induces a concomitant
shear stress, which promotes proliferation, differentiation
and tube formation of endothelial cells, other than a higher
mitotic activity of both endothelial cells and smooth-muscle
cells required to form the neovasculature or to remodel
existing collaterals [16, 17]. At molecular level, shear stress
induces a cascade triggered by nitric oxide production; then,
endothelial cell activation leads to the infiltration and
adhesion of monocytes that secrete proteases, growth factors
and chemokines. Finally, the whole process promotes the
enlargement of arterioles, which can form the collateral
vasculature turning blood flow beyond the occlusion [14-18].
Bone Marrow Cells and Circulating Endothelial
Progenitor Cells
Several studies suggest an additional contribution derived
from bone marrow progenitors, which are mobilized in the
Adult Stem Cells and the Clinical Arena Cardiovascular & Hematological Agents in Medicinal Chemistry, 2012, Vol. 10, No. 2
limb setting, migrate to ischemic tissues and incorporate into
new vessels [18]. It is proposed that tissue ischemia induces
a local increase of chemokines, such as VEGF, thus
promoting migration of progenitor cells, expressing vascular
endothelial growth factor receptor 1- and 2- (VEGFR1 and
VEGFR2), to the ischemic territory [19]. Thus, one rationale
and proposed advantage of cell therapy versus conventional
therapies is that cell therapy can directly increase the number
of stem/progenitor cells in the ischemic tissue.
The increase of local stem/progenitor cells could be
achieved either by systemic delivery (i.e. systemic injection
of progenitor cells or inducing them to mobilize from the
bone marrow microenvironment by chemokine therapy) or
by direct cell injection into or around the ischemic zone.
Regardless of the pathway taken, the number of potential
candidate cells to incorporate into the vessel wall is
substantially increased [17, 20]. Many clinical studies have
used adult stem cells derived from autologous bone marrow.
This choice is due to the easy availability, security and
lack of ethical controversies associated with the use of
embryo derived stem cells. The bone marrow contains
several subpopulations of cells, which can be isolated by
direct extraction or by mobilization into peripheral blood
by using cytokines (such as G-CSF) [17, 20]. The two
main groups of BMCs are hematopoietic stem cells
and mesenchymal stem cells, which can be further divided
into different subpopulations according to the expression
of specific cell surface receptors. A subpopulation of
heterogeneous cells includes endothelial progenitor cells
(EPC), which are also found, although in less amount, in
other districts like peripheral blood, fetal liver or umbilical
cord blood [21, 22].
In 1997, the existence of bone marrow-derived EPCs in
the peripheral circulation was reported for the first time [21,
23]. These cells contribute to the re-endothelization of
injured vessels as well as to neovascularization of ischemic
lesions, suggesting that EPCs play a major role in the
pathogenesis of atherosclerosis and cardiovascular diseases.
The number and function of EPCs are regulated both by
several angiogenic growth factors, cytokines and chemokines
as well as by cardiovascular risk factors or lifestyle
modifications. An increase in the number of EPCs induced
by ischemic stimuli contributes to reduction in ischemic
damage through neovascularization. EPCs number and
function directly influence the maintenance and development
of cardiovascular diseases [24].
Although a precise definition of EPCs is still lacking,
these cells are identified through the expression of a
combination of hematopoietic stem cell markers, such as
CD34, CD45 and CD133, and endothelial-cell markers,
including VEGFR2, von Willebrandfactor, endothelial nitric
oxide synthase among others [17, 21, 22]. However, there
is no a gold standard marker, resulting in confusion
regarding their definition. Moreover, EPCs have also been
characterized by other methods like the uptake of
diacetylated low-density lipoprotein and binding of
fluorescently labeled Ulex europaeus agglutinin 1 lectin or
by several in-vitro and in-vivo functional assays, such as
colony formation, tube formation, and vascular integration
[17, 21]. However, all these identification methods lack
101
specificity. For instance, in EPCs functional assays, different
culture conditions produce different EPC phenotypes: early
EPCs (CD133+CD34+ cells) and late outgrowth EPCs
(CD133
CD34+ cells) or mixed ones. More importantly,
these cell populations (early and late EPCs) seem to show a
different contribution to neovascularization of ischemic
tissues and re-endothelization of injured vessels [17, 21, 25].
Paracrine Mechanisms in Adult Stem Cell Therapy
Despite some evidence that bone marrow-derived stem or
progenitor cells incorporate into vascular structures, several
studies suggest that only a small number of vessels contain
donor cells. A recent study shows that EPCs are not
frequently observed in the femoral muscle after 8 weeks and
they preferentially home to the sciatic nerve. The authors
hypothesize that the role of EPCs on muscle angiogenesis is
mediated by indirect mechanism with cytokines on resident
progenitor cells [26].
In addition, despite heterogeneous bone marrow cell
populations (i.e., mononuclear cells or unfractionated cells)
contain very small numbers of stem cells (0.01% of total
cells), injection of these cells significantly enhances
collateral development. Thus, other mechanisms are likely to
contribute to the overall improvement of vascular function
and benefits observed after bone marrow cell therapy [27].
An alternative or perhaps complementary hypothesis to
the concept of BMC and EPC incorporation is that the
injected cells act in a supportive role, optimizing the milieu
for host vasculature to respond to tissue ischemia. Many
bone marrow subpopulations (for example EPCs) are a
source of growth factors previously demonstrated to be
central in the initiation and coordination of angiogenesis
[28]. Thus, a paracrine hypothesis has been recently
advanced establishing that the injected stem cells release into
the surrounding tissue soluble factors, such as cytokines,
chemokines and growth factors, that contribute to vascular
repair and regeneration by paracrine mechanisms [29].
Indeed, these released factors are able to induce angiogenesis
and neovascularization also through the recruitment and
activation of resident stem cells. The whole understanding of
these novel mechanisms can have many future clinical
implications in the field of cell therapy for vascular diseases.
Since the fate of cells injected into peripheral muscle is
unknown, even the rationale of intramuscular injection is
based on the paracrine activity provided by injected cells in
the ischaemic area [29].
CLINICAL STUDIES AND CRITICISMS
The first pilot study on the treatment of patients with
limb ischemia by autologous transplantation of BMCs was a
randomized controlled trial published in 2002 [30] (Table 2).
Since then, considering the whole published studies, more
than 900 patients have been enrolled. Most of these studies
have analysed patients in the IVth stage of Fontaine’s
classification, without any possibility of revascularization
[30-64]. Only two studies [42, 44] made a clear distinction of
the patients in subgroups by differentiating the clinical stages
and trophic lesions of patients with diabetes, haemodialysis,
vasculitis etc. Noteworthy, the most favourable results were
102 Cardiovascular & Hematological Agents in Medicinal Chemistry, 2012, Vol. 10, No. 2 Casamassimi et al.

Table 2. Published studies of cell therapy in peripheral vascular diseases.

Published Studies Delivery Route Condition Subjects Cell Type Follow-UP Time End-Point and Clinical Indexes

2002 Tateishi-Yuyama BMCs or ABI and TcO2 improvement; rest

IM PAD, DM 45 4 and 24 weeks
et al., [30] PBMNCs pain; pain-free walking time

2002 Esato et al., [31] IA PAD, TAO 8 BMCs N/D Ulceration healing

2004 Saigawa et al., [32] IM PAD, DM 8 BMCs 4 weeks ABI and TcO2 improvement

2004 Higashi et al., BMCs or BM- ABI and TcO2 improvement; pain-
IM PAD 8 4 and 24 weeks
[33] MNCs free walking time

2004 Miyamoto ABI improvement; pain-free

IM PAD, CLI 12 BMCs - EPCs N/D
et al., [34] walking time

ABI, blood flow, laser Doppler blood

2004 Huang et al., [35] IM PAD 5 PBMNCs 3 months perfusion and angio-graphic scores
improvement

Normalization of limb temperature

2005 Kawamura
IM PAD, CLI 30 PBMNCs N/D was observed by thermograph, and
et al., [36]
symptoms also improved

ABI and TcO2 improvement; rest

2005 Lenk et al., [37] IA CLI 7 PBMNCs 20 weeks pain; pain-free walking time; flow-
dependent vasodilation

ABI, limb pain and ulcers, were

2005 Huang et al., [38] IM CLI, DM 28 M-PBMNCs 3 months
significantly improved

2005 Ishida et al., [39] IM TAO 6 M-PBMNCs 4 and 24 weeks ABI and ischemic ulcers improvement

ABI, rest pain scores and peak

2006 Durdu et al., [40] IM TAO 28 BM-MNCs 3 and 6 months
walking time improvement

2006 Miyamoto 4 weeks, 4 and 7 Rest pain and ischemic ulcers

IM TAO, CLI 8 BM-MNCs
et al., [41] months, 1 year improvement

2006 Kawamura
IM CLI 92 PBMNCs 1,5 month Capillary formation
et al., [42]

2007 Bartsch et al., ABI and pain-free walking distance

IM and IA PAD, CLI 13 BMCs 2 and 13 months
[43] improvement

BM-MNCs or
2007 Huang et al., [44] IM PAD 150 12 weeks ABI and rest pain improvement
M-PBMNCs

2007 Kajiiguchi et al., BM-MNCs or

IM CLI, TAO 7 1 month ABI improvement (for 3 patients)
[45] PBMNCs

2007 Hernandez ABI, SaO2, pain-free walking time

IM CLI, DM 12 BM-MNCs 24 months
et al., [46] and rest pain improvement

2007 Saito et al., [47] IM TAO 7 BM-MNCs N/D TcO2 improvement

2008 Matoba Leg pain scale, ulcer size and pain-

IM PAD, TAO 115 BM-MNCs 3 years
et al., [48] free walking distance improvement

3,6,12 and 18 ABI, ischemic ulcers and pain-free

2008 Napoli et al., [3] IA PAD 18 BM-MNCs
months walking distance improvement

ABI and TcO2 improvement and

2008 Gu et al., [49] IM and IA PAD, DM 16 BM-MNCs 4 weeks
limb salvage

2008 Chochola Collateral vessel development and

IA PAD, DM 28 BM-MNCs 1 year
et al., [50] indexes improvement

2008 Wester et al., [51] IM PAD/CLI 8 BMCs 4 and 8 months Pain relief and indexes improvement
Adult Stem Cells and the Clinical Arena Cardiovascular & Hematological Agents in Medicinal Chemistry, 2012, Vol. 10, No. 2 103

Table 2. contd….

Published Studies Delivery Route Condition Subjects Cell Type Follow-UP Time End-Point and Clinical Indexes

Pain-free walking distance, ABI

2008 Van Tongeren
IM and IA CLI, PAD 27 BMCs 6 and 12 months improvement and pain scores
et al., [52]
reduction

Modest improvement of resting pain,

2008 De Vriese
IM CLI 16 BM-MNCs 12 weeks TcO2 improvement, no ABI
et al., [53]
significant improvement

2008 Cobellis et al., ABI and pain-free walking distance

IA PAD 10 BM-MNCs 12 months
[54] improvement

No clinical or perfusion improvement.

2009 Amann et al., 3 months Limb salvage, ABI and TcO2 and
IM PAD 51 BM-MNCs
[55,56] 6 months pain-free walking distance
improvement

BM-MNCs or
2009 Capiod et al., [57] IM CLI 24 N/D Therapeutic angiogenesis
PBMNCs

Improvement in symptom severity.

2009-2011 Franz et al., 3 and 8 months ABI improvement; absence of rest
IM and IA PAD 18 BM-MNCs
[58,59] 3 months pain; absence of ulcers; and absence
of major limb amputations

2010 Zafarghandi et al., BM-MNCs ABI and pain-free walking distance

IM CLI 50 4 and 24 weeks
[60] and G-CSF improvement

2010 Prohazka et al., ABI improvement and absence of

IM PAD 42 BM-MSCs 4 months
[61] major limb amputations

2010 Lara-Hernandez 14, 18 months

IM CLI 28 mobilized EPCs ABI improvement, limb savage
et al., [62] and 1 year

Not significantly increase ankle-

2011 Walter et al., [63] IA CLI 40 BM-MNCs 3 months brachial index, ulcer healing and
improvement of resting pain

First toe pressure and toe-brachial

2011 Murphy et al., index increase, perfusion index by
IM CLI 29 BM-MNCs 1 year
[64] PET-CT increase and rest pain
improvement

ABI: ankle-brachial pressure index; BMCs: bone marrow cells; BM-MNCs, bone marrow-derived mononuclear cells; BM-MSCs: bone marrow-mesenchymal stem cells; CLI: critical
limb ischemia; DF: diabetic foot; DM, diabetes mellitus; EPCs: endothelial progenitor cells; G-CSF: granulocyte colony-stimulating factor; IA: intra-arterial injection; IM:
intramuscular injection; IV: intravenous injection; LASO: lower limb arteriosclerosis obliterans; LEI: lower extremity ischemia; M-PBMNCs: G-CSF-mobilized peripheral blood
mononuclear cells; PAD: peripheral artery disease; PET-CT: positron emission tomography-computed tomography; PB-MNCs: peripheral blood derived mononuclear cells; SaO2:
arterial oxygen saturation; SLI: severe leg ischemia; TAO, thromboangiitis obliterans; TcO2: transcutaneous oxygen pressure.

obtained in patients with diabetes or haemodialysis or
vasculitis lesions with leg arteries in discrete function (good
run-in). In these studies the best results were achieved in
patients with stages II and III, whereas unsatisfactory
outcomes were obtained in patients with stage IV and
diabetic ulcers. This result can be rationally explained by the
progression of the disease. Indeed, a long period is needed to
detect the development of a collateral circulation stimulated
by BMCs (at least 6 months to have an angiographic result).
However, patients with ulcers at stage IV (with the exception
of patients with Burger’s disease or diabetic patients with
arterial permeability) generally do not have such a period
before the disease progression into gangrene. In our opinion,
the main target of stem cell therapy is the patient with IC
(stage II) or night pain (stage III). Thus, the outcome of
patients should be the increase of the pain-free walking
distance, the ulcers prevention and the rest pain loss, before
the skin lesions occur.
Most of the studies enrolled a limited number of patients
and did not include a placebo arm, with no double-blinded
randomized design and a very short follow-up period. Thus,
these studies do not provide definitive proof of the positive
therapeutic effect of cell therapy.
In most of the trials the preferred administration route
was the injection into the ischemic muscle [30, 32-36, 38-42,
44-48, 49, 51-53, 55-62, 64] whereas only fewer studies with
a little number of treated patients utilized the intra-arterial
injection [3, 31, 37, 43, 49, 50, 52, 54, 58, 59, 63] (Table 2).
Thus, to date we still do not know which is the better
104 Cardiovascular & Hematological Agents in Medicinal Chemistry, 2012, Vol. 10, No. 2 Casamassimi et al.

infusion way. In addition, a combination of both methods
has been tested in some small studies showing no significant
differences in the clinical characteristics between patients
treated with intra-arterial or intra-arterial plus intramuscular
cell administrations [43, 49, 52, 58, 59]. However, there are
no direct comparisons of these two different routes of
administration of cell therapy in patients with limb ischemia,
although the intra-arterial administration offered similar
levels of angiogenic activity as intramuscular injection in a
rat model study [65].
Most of the published studies are small studies and show
the results of short or medium term follow-up. To date, only
one study reported a long-term follow-up (36 months),
suggesting that intramuscular implantation of bone marrow
mononuclear cells may lead to extension of amputation-
free interval and improvement of ischemic pain, ulcer size
and walk without pain [56]. In addition, the safety and
efficacy of this therapy was not lower than conventional
revascularization therapies. Several phase III studies
are currently undergoing to evaluate the efficacy of
intramuscular injection of autologous BMC in patients with
CLI, such as the NCT00904501 and NCT01245335 trials
(Table 3).
Interestingly, in the study of Kawamura et al., [42],
where 92 patients were treated, the authors made a precise
classification of patients in Fontaine’s stages with diabetes
or haemodialysis treatment. We agree with the conclusions
of this study that non diabetic, non dialyzed patients
with ischemic limbs are strongly indicated for stem cell
transplantation regardless of Fontaine classification.
Therapeutic angiogenesis is effective for critically ischemic
limbs resulting from hemodialysis and diabetes until
Fontaine stage III, but it is of limited effectiveness for stage
IV cases. In the 92 patients only the stage I, II, III had a good
response, whereas it was of limited effectiveness for stage IV
cases.

Table 3. NIH ongoing interventional trials with a number of patients
30.

Patients
NIH Registration Number Status Cell Type Condition Delivery Route Phases
Treated

NCT00904501 Unknown 110 BMCs CLI - PAD IM III

NCT00987363 Active, not recruiting 60 BMCs CLI - PAD IA I and II

NCT00539266 Recruiting 108 BMCs CLI - PAD IM II and III

NCT00411840 Unknown 100 BMCs CLI - PAD IM and IA I

NCT00468000 Active, not recruiting 150 BMCs CLI - PAD IM II

NCT01245335 Recruiting 210 BMCs CLI - PAD IM III

NCT00721006 Completed 35 BMCs CLI - PAD - SCI - PVD IM II

NCT01065337 Completed 30 BMCs CLI - DF IM and IA II

NCT01232673 Active, not recruiting 40 BMCs CLI - DF IM II

Local
NCT00292357 Unknown 40 BMCs DF I
application

NCT00595257 Active, not recruiting 60 MSCs CLI - PAD IM II

NCT01079403 Recruiting 36 MSCs CLI IA I and II

NCT01351610 Recruiting 30 MSCs CLI - PAD IV I and II

Umbilical
NCT01216865 Not yet recruiting 50 CLI IM I and II
cord MSCs

CD133+
NCT00753025 Completed 42 CLI - LEI IM II
from BM

NCT00922389 Unknown 36 M-PBMNCs CLI - DF IM I and II

GM-CSF
NCT01408901 Not yet recruiting 267 M-PBMNCs PAD ?
administration

GM-CSF
NCT01041417 Recruiting 180 M-PBMNCs PAD I
administration

BMCs: bone marrow cells; CLI: critical limb ischemia; DF: diabetic foot; G-CSF: granulocyte colony-stimulating factor; IA: intra-arterial injection; IM: intramuscular injection; IV:
intravenous injection; LEI: lower extremity ischemia; M-PBMNCs: G-CSF-mobilized peripheral blood mononuclear cells; MSCs: Mesenchymal Stem Cells; PAD: peripheral artery
disease; PB-MNCs: peripheral blood-derived mononuclear cells; SLI: severe leg ischemia.
Detailed information about the NIH registered clinical trials are reachable visiting the web site http://clinicaltrials.gov/.
Adult Stem Cells and the Clinical Arena Cardiovascular & Hematological Agents in Medicinal Chemistry, 2012, Vol. 10, No. 2
Good impact
Fig. (1). Selection of PAD patient and impact of cell therapy. Patients with peripheral artery disease have been subdivided into three major
subgroups based on the disease stage. Patients with Intermittent claudication are considered at high impact for cell therapy, whereas patients
with pain or ulcers are at middle and bad impact respectively.
The same results have been confirmed by another study
where a comparison of G-CSF-mobilized peripheral blood
mononuclear cells (M-PBMNCs) versus bone marrow-
mononuclear cells (BM-MNCs) for the treatment of patients
with lower limb arteriosclerosis obliterans was made [44];
also in this case a minor response in patients with diabetes
was observed. This result was associated with the
impairment of diabetic M-PBMNCs capacity to differentiate
into EPCs, to incorporate into vessel-like tubules in vitro,
to participate in vascular-like structure formation in a
subcutaneous matrigel plug and to stimulate the recruitment
of pericytes/smooth muscle cells. In addition, there was an
impairment of vasculogenesis, which was related to the
reduced adhesion ability of EPCs from diabetic M-PBMNCs
[44].
Another question to be addressed is the technique to
obtain stem cells. Of course, the borrow marrow cells
aspirated from the iliac crest is the best technique and the
most tested. By contrast, this method requires a local or
general anaesthesia and a collection of a large amount of
marrow by aspiration, which includes the risk of anaesthetic
accident and excludes patients with contraindications to
anaesthesia [44]. About 250-500 ml bone marrow (5.8 ±
2.3
108 mononuclear cells) can be usually aspirated from
the ileum, which is enough for only one implantation.
To aspirate or collect more bone marrow cells the patient
risk increases to an unacceptable degree. In this study,
comparative analysis indicated that M-PBMNCs should
be more practical in comparison with BM-MNCs for
treatment [44]. However, to obtain M-PBMNCs, the patients
received recombinant human G-CSF, 300 μg twice a day
subcutaneously for five consecutive days. In the study of
Huang [44] no collateral effects were found, but because of
the possible dangerous consequences of G-CSF, a long time
observation would be necessary to validate the study. This
key point has been also discussed in a more recent study
where mobilized EPCs have been administered in a small
105
Middle impact Bad impact
number of treated patients [62]. Thus, further research is
needed to decide which cells should be used for therapy and
we have to wait for the results from the currently ongoing
clinical trials (Table 3).
Another important consideration is about the patient at
IVth stage. It is not correct to evaluate the patient at this stage
only for the presence of ulcers. We think it is necessary to
divide this stage into subgroups for the presence or absence
of best run-in for lesions of big artery in the legs. It is not
possible to compare a Burger’s disease with pure chronic
arteriopathy or a diabetic foot with only a micro-angiopathy
with diabetic patient that have also macro-angiopathy
(obstruction of big artery). Indeed, these diseases have
different physiopathology and different evolution, especially
for the duration life of leg.
CONCLUSIONS
At the examination of the literature, there are no
randomized, double-blinded trials, as well as only few
studies have reported the results obtained on more than 50
patients [42, 44, 48, 56]. Obviously, these data are not
enough to achieve significant conclusions about the
effectiveness of this therapy. The limit of most publications
is the selection of patients. Probably, not all the patients in
the IVth Fontaine stage are suitable for the infusion of stem
cells. In the few studies where patients were divided into
clinical sub-groups, the best results were achieved in patients
with stages II and III, which can be explained by the
progression of the disease. In our opinion, these patients
should be the main targets of stem cell therapy. Obviously,
our considerations require further clinical studies to confirm
the effectiveness of therapy in patients with first disease
stages. It would be interesting to assess the impact on
pain-free walking distance not only in patients with
severe claudication in the absence of run-off (without
revascularization chance), but also in those with good run-
106 Cardiovascular & Hematological Agents in Medicinal Chemistry, 2012, Vol. 10, No. 2 Casamassimi et al.

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Accepted: January 23, 2012