General Review

An Update on Therapeutic Angiogenesis for

Peripheral Vascular Disease
Annalisa Pacilli,
1
Gianluca Faggioli,
1
Andrea Stella,
1
and Gianandrea Pasquinelli,
2
Bologna, Italy
Background: We reviewed the issue of stem cells and therapeutic angiogenesis in the treatment
of peripheral vascular disease.
Methods: MEDLINE (1997e2008) with the following search terms: stem cell therapy, endo-
thelial progenitor cells, peripheral blood mononuclear cells, and peripheral vascular
disease. Relevant published papers involving the above search terms, preclinical studies,
and clinical trials using stem cells and progenitors for the treatment of peripheral occlusive
vascular disease were included.
Results: Transplantation of bone marrowederived progenitor cells or peripheral blood mononu-
clear cells promotes tissue angiogenesis, as has already been explored in preclinical studies;
angiogenesis can also be sustained using genetic, protein therapeutic approaches. Engineered
scaffolding with stem cells is a further strategy, which is still in its infancy. The treatment of
patients with severe peripheral arterial disease is generally reported as a series of case reports;
all studies generally show an improvement in clinical symptoms, e.g., rest pain and pain-free
walking time, as well as transcutaneous oxygen pressure, without any important adverse reac-
tions. The few clinical trials also report similar encouraging results. All the studies have their
shortcomings, including absence of control groups and objective evaluation of the results of
treatments as well as short-term follow-up.
Conclusion: Promoting angiogenesis using genetic, protein, stem cellebased therapies is
a promising option for the treatment of peripheral vascular disease when unresponsive to
medical and surgical therapy.
INTRODUCTION
Replacement of dead cells exclusively by proliferation
of terminally differentiated neighboring endothelial
cells (ECs) in case of massive loss of endothelium,
e.g., hypertension, dyslipidemia, ischemia, angio-
plasty, or stenting, is not sufcient to recover the
structural and functional integrity of the endothelial
lining. Arterial healing following tubular graft
implantation cannot in its turn be fully explained by
spreading and phenotypic modulation of smooth
muscle cells.
In such conditions the support and crucial contri-
butionof stemcells andendothelial or smoothmuscle
progenitors is necessary. Progenitors can reside in the
vascular wall itself or can be mobilized fromthe bone
marrow and delivered in the blood; in any case,
progenitors concentrate in the area of tissue damage
responding to local microenvironmental signals;
tissue repair is then achieved by several still debated
mechanisms, which include extracellular matrix
remodeling, angiogenesis, and differentiation of
progenitors into mature and functional cells.
Accumulating evidence shows the ability of mobi-
lized endothelial progenitor cells (EPCs) to repair
1
Department of Specialistic Surgery and Anesthesiological Sciences,
University of Bologna, Bologna, Italy.
2
Department of Radiological and Histocytopathological Sciences,
Clinical Pathology Section, University of Bologna, Bologna, Italy.
Correspondence to: Annalisa Pacilli, Department of Specialistic
Surgery and Anesthesiological Sciences, Chair of Vascular Surgery,
Policlinico S. Orsola-Malpighi, via Massarenti 9, Bld. 18, 40138,
Bologna, Italy, E-mail: annalisa.pacilli@gmail.com
Ann Vasc Surg 2010; 24: 258-268
DOI: 10.1016/j.avsg.2008.12.003
Annals of Vascular Surgery Inc.
Published online: May 21, 2009
258
injured vessels in animal models;
1-7
however, one
potential limitationtothe use of autologous stemcells
in clinical application is their low number and the
documented decline in the number and function
with aging. These shortcomings can be overcome
by increasing the number of circulating EPCs through
pharmacological administration of growth factors,
such as granulocyte colony-stimulating factor
(G-CSF) or vascular endothelial growth factor
(VEGF), which activate progenitor cellereleasing
factors that are able to promote the mobilization of
stem cells from the bone marrow. Mononuclear cells
from peripheral blood (PB-MNCs) can be harvested
for in vitro expansion and differentiation and then
transfused back into the patient. Thus, stem cell
therapy is a realistic option for the treatment of
vascular diseases.
Transfusion of ex vivo expanded EPCs
8
or endog-
enous mobilization of them
4,9-11
enhances reendo-
thelialization in different models of endothelial
denudation. Moreover, EPCs may also contribute to
angiogenesis in wound healing, tissue ischemia, or
myocardial infarction. Delivery of bone marrowe
derived Lin-/c-kit
+
cells onto the border of an infarct
promotes angiogenesis in situ,
12
and intramuscular
(IM) injection of bone marrow-mononuclear cells
(BM-MNCs) intothe ischemic limbs of rat andmouse
induces collateral vessel formation.
13,14
Some in vivo functional approaches have already
been applied in clinical trials, mainly involving
patients with myocardial infarction; these trials are
extensively reviewed elsewhere.
15,16
In this review we focus on the less covered eld of
promoting angiogenesis in peripheral vascular disease
through the use of stem cell, protein, and gene thera-
pies; innovative attempts at using stem celleengi-
neered biomaterials are also dealt with. From the
literature survey, patients with peripheral arterial
disease (PAD) who underwent these pioneering
approaches were unresponsive to conventional
medical andsurgical therapy, hadareducedfunctional
number of EPCs, and lower amounts of angiogenic
growthfactors. Thus, thetherapeutic options available
are limited in these cases, and only novel therapeutic
strategies based on induced angiogenesis may be
expected to check an otherwise irreparable destiny.
Protein and Gene Therapy
The easiest therapeutic strategy is to overcome the
angiogenic factor (i.e., VEGF or broblast growth
factor, FGF) decit by exogenous administration of
them in the form of protein or gene transfer
approaches. Both procedures have their advantages
and disadvantages and have been explored in
preclinical and clinical studies. The results of phase I
clinical studies are encouraging: Intramuscular injec-
tion of recombinant FGF-2 (rFGF-2)
17
in PADpatients
is well toleratedas well as injectionof adenoviral vector
encoding VEGF 121
18
and plasmid encoding VEGF
165
19-21
or FGF-1;
22
this last method was also found
safe and effective in patients with chronic limb
ischemia, although its clinical efcacy is reported
sometimes to be minimal
18
and side effects may
arise.
19e21
Different approaches have beenusedincompleted
phase II randomized trials, ranging from exogenous
administrationof vasculogenic protein to gene trans-
fer. The Therapeutic Angiogenesis withRecombinant
Fibroblast GrowthFactor 2for Intermittent Claudica-
tion (TRAFFIC) study demonstrated that rFGF-2
provides signicant improvement in peak walking
time (PWT) and ankle-brachial index (ABI) with
minimal side effects,
23
while the Regional Angiogen-
esis with Vascular Endothelial Growth Factor in
Peripheral Arterial Disease (RAVE) study did not
report any differences in symptom reduction
between placebo and treated groups.
24
In both trials
peripheral edema occurred at the injectionsite, prob-
ably due to the augmented vascular permeability
induced by growth factors, and the benets were
not dose-dependent. Furthermore, a randomized,
placebo-controlled, double-blinded phase II study
demonstrated that VEGF gene transfer (adenovirus-
or plasmid-mediated) also increases vascularity in
patients with critical lower limb ischemia.
25
Very
recentlytwoadditional double-blinded, randomized,
placebo-controlled clinical trials have been pub-
lished: the Study to Assess the Safety of Intramus-
cular Injection of Hepatocyte Growth Factor
Plasmid to Improve Limb Perfusion in Patients with
Critical Limb Ischemia also known as the HGF-
STAT trial.
26
and the Therapeutic Angiogenesis Leg
Ischemia Study for the Management of Arteriopathy
and Nonhealing Ulcer also known as the TALISMAN
201 trial.
27
The rst trial determined the effect of
hepatocyte growth factor (HGF) plasmid on the
safetyandlimbtissueperfusionas measuredbytrans-
cutaneous oxygen tension (TcPO
2
) in patients with
critical limb ischemia (CLI) and the second aimed to
evaluate the efcacy and safety of IMadministration
of NV1FGF, a plasmid-based angiogenic gene
delivery system for local expression of FGF-1, versus
placebo in patients with CLI. Both trials provide
encouraging results: HGF-STAT reports an improved
TcPO
2
after IM injection of plasmid cDNA encoding
humanHFG(phHGF) andTALISMAN201, areduced
risk of major amputation and death following
NV1FGF administration. To date, some disadvan-
tages, e.g., short half-life of protein and long-term
Vol. 24, No. 2, February 2010 Therapeutic angiogenesis for peripheral vascular disease 259
Table I. Clinical studies using a protein and/or gene therapy approach for PAD
Reference Vascular disease Study type Therapeutic strategies Results
Isner et al.
19
Ischemic limb Case report Injection of phVEGF
165
applied to hydrogel
polymer coating of
angioplastic balloon
Increase in collateral vessel formation
and in resting and maximum ows,
no signicant adverse events
occurred
Baumgartner et al.
20
PAD Pilot study with
9 patients
IM injection of
phVEGF
165
Improvement in ABI and distal ow,
new vessel formation, ulcer
healing; development of
transient edema
Lazarous et al.
17
Intermittent
claudication
Double-blind,
placebo-controlled,
dose-escalation phase I
trial with 19 patients
IA injection of bFGF Procedure well tolerated; greater
improvement in blood perfusion
than placebo group
Comerota et al.
22
PAD Phase I trial with 51
patients
IM injection in different
doses of phFGF
(NV1FGF)
No adverse events related to study
treatment; reduction of pain and
ulcer size; improvement of ABI and
TcPO
2
; dose response not evident
Shyu et al.
21
CLI Phase I trial with 21
patients
IM injection of
phVEGF
165
Improvement in ABI, distal ow, and
rest pain; ulcer healing
Ma kinen et al.
25
CLI Randomized, phase II,
double-blind, placebo-
controlled study with 54
patients
Catheter-mediated VEGF
gene therapy (VEGF-
Ad, VEGF-P/L)
Improvement in ABI and vascularity
Rajagopalan et al.
18
PAD Controlled phase I trial
with 18 patients
IM injection of Ad
VEGF
121
Improvement in ABI and PWT, well-
tolerated procedure
Lederman et al.
23
Intermittent
claudication
TRAFFIC study: phase II,
double-blind, placebo-
controlled study with
190 patients
IA injection of single or
double doses of rFGF-
2
Increase in PWT, signicant
differences between single-dose
and placebo-treated groups, dose-
independent efcacy
Rajagopalan et al.
24
PAD RAVE study: phase II,
double-blind, placebo-
controlled study with
105 patients
IM injection of different
doses of Ad VEGF
121
No difference in PWT, ABI,
claudication onset time, and
quality of life between different
groups
Powell et al.
26
CLI HGF-STAT: 104 patients IM injection of phHGF Improvement in TcPO
2
between
high-dose and other groups, no
differences in secondary end points
Nikol et al.
27
CLI TALISMAN 201: double-
blind, randomized,
controlled trial with 125
patients
IM injection of phFGF-1
(NV1FGF)
Reduced risk of all and major
amputations and death
2
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safety, related to these therapeutic strategies have
been overcome; but more comprehensive clinical
experience is needed to conrm their possible ef-
cacyinpromotingangiogenesis. Highlights frompub-
lished clinical studies using protein and/or gene
therapies are reported in Table I.
Cell Therapy
In the meantime, besides gene therapy, cell therapy
has been emerging. Asahara et al.
28
were the rst to
demonstrate that bone marrowederived EPCs and
peripheral bloodederived angioblasts are key
factors in postnatal vasculogenesis; these ndings
were subsequently supported by a great number of
preclinical studies. The main characteristics and
functions of the stem cells and progenitors used in
clinical studies are listed in Table II.
Therapeutic angiogenesis with stem cell trans-
plantation has been undertaken in humans as well.
The rst clinical trial was published by Tateishi-
Yuyama in 2002.
29
The initial pilot study was
a randomized controlled trial involving 25 patients
with unilateral limb ischemia; the patients were
considered unsuitable for surgical revascularization
and therefore treated with autologous BM-MNC
injections in the gastrocnemius of the ischemic leg;
after 24 weeks a signicant increase in TcPO
2
, relief
from rest pain, and pain-free walking time without
any important adverse reaction were recorded in
all patients. The second part of the study was
a randomized controlled trial in 22 patients with
bilateral limb ischemia who randomly received
implantation of BM-MNCs in one leg and PB-
MNCs as a control in the other. The symptoms
improved signicantly in the BM-MNC-injected
leg; a slight clinical improvement was also seen in
the control leg.
29
The same efcacy and safety were
reported in several uncontrolled studies of limited
size where autologous BM-MNCs were injected IM
in patients with a spectrum of PAD ranging from
CLI
30-37
to thromboangiitis obliterans (TAO).
35,37-39
Miyamoto and colleagues
39
also evaluated the
efcacy in a long-term follow-up, but the results
are not encouraging because of adverse events,
including death and unfavorable angiogenesis.
The clinical efcacy of combined intra-arterial
(IA) and IM transplantation of autologous BM-
MNCs has also been investigated; in such patients,
perfusion indices improved signicantly compared
withbaseline values inthe short and long terms.
33,34
Particularly, van Tongeren et al.
34
compared the IM
and combined IM+ IA delivery options of autolo-
gous BM-MNCs in patients who were unsuited to
surgical or endovascular treatment. This study
conrmed that bothIMalone and IM+ IAcombined
administrations are feasible, safe, and effective in
terms of limb salvage, pain-free walking distance,
ABI, and pain score results. Furthermore, Higashi
et al.
30
demonstrated that BM-MNC implantation
improved not only limb ischemic symptoms but
also endothelium-dependent vasodilation. A case
report documents complete wound healing of
chronic foot ulcers due to CLI 20 weeks after treat-
ment.
40
There seems to be a relationship between
the number of bone marrowederived CD34
+
cells
implanted and the clinical efcacy of the treatment,
particularly if one considers the ABI improvement;
however, this observation is limited by the low
number of patients included in the study and needs
further conrmation.
36
Although several studies have investigated the
efcacy of cell therapy in peripheral vascular
diseases, none has focused on the angiogenesis
induced by transplanted cells. The rst report of an
active angiogenesis process in four patients treated
with BM-MNCs for CLI comes from the Optimiza-
tion of Progenitor Endothelial Cells inthe Treatment
of Critical Leg Ischemia (OPTIPEC) clinical trial. This
study shows that therapeutically injected cells
induce new vessel formation in the ischemic and
distal parts of the treated limb, although this may
not prevent amputation.
41
One of the major drawbacks of the studies
reported above is that BM-MNC collection requires
local or general anesthesia as well as collection of
a large amount of marrow aspirate (up to 500 mL);
this procedure may raise the risk of anesthetic acci-
dents and excludes patients with contraindications
to anesthesia. The use of PB-MNCs could overcome
these disadvantages. Circulating EPCs are only
a small fraction of total circulating cells, but their
percentage can be increased by various methods,
e.g., intravenous administration of cytokines (VEGF
and G-CSF), treatments with statins, estrogens, and
exercise. However, one should bear in mind that
patients who may benet from cell therapy usually
have not only PAD but also a diffuse, systemic
vascular disease. This is a condition in which endo-
thelial progenitors are altered in number and func-
tionality; hence, attempts to increase the number of
circulating EPCs might fail in such patients.
A more effective approach could be endogenous
mobilization of EPCs from bone marrow to the
peripheral blood, through harvesting and IM injec-
tion of ex vivo expanded cells.
42
By this strategy
a heterogeneous cell population, including EPCs
and angiogenic factors delivering PB-MNCs, is
directly delivered into the ischemic area. Experi-
mental studies have demonstrated that the two
Vol. 24, No. 2, February 2010 Therapeutic angiogenesis for peripheral vascular disease 261
Table II. Characteristics and properties of stem cells/progenitors used in the treatment of peripheral arterial disease.
Cell type characteristics Origin Main hypothetical role
Endothelial
progenitor cells
(EPCs)
Heterogeneous group of circulating cells
characterized by the expression of
surface markers,i.e., CD34, CD133,
KDR, uptake of the acetylated low
density lipoprotein and by the
binding of the Ulex Europeus lectin
Bone marrow; a contribution from
peripheral tissues and vascular wall
cannot be excluded
Differentiation into mature
endothelium
Peripheral blood
mononuclear cells
Heterogeneous group of circulating
mononuclear cells which include
many distinct angiogenic cytotypes,
e.g., early and late EPCs, CD146+
circulating endothelial cells, CD14+
monocytes, CD117+ mast cells and
other cells.
Mainly bone marrow; peripheral tissues
and vascular wall also involved
Angiogenic factor delivery
CD133+ cells Homogeneous group of circulating cells
expressing the CD133 surface
molecule
Bone marrow Largely unknown; these cells have
hemangioblastic potentiality, i.e., the
ability to differentiate in vitro into
hematopoietic and endothelial
lineages.
CD34+ cells Homogeneous group of circulating cells
expressing the CD34 surface
molecule; most of these cells belong
to the EPC category
Bone marrow; a contribution from
peripheral tissues and vascular wall
cannot be excluded
These cells have angioblastic
potentiality, i.e., the ability to
differentiate in vitro into endothelial
lineages
2
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cell types cooperate in sustaining angiogenesis and
activating resident repairing cells.
Huang et al.
43
demonstrated for the rst time that
autologous transplantation of mobilized peripheral
blood stem cells (PBSCs) is a practical, safe, and
effective method of lower limb occlusive disease
treatment in ve patients.
In 2004 Kawamura et al.
44
published the rst
large report on the application of regenerative medi-
cine to peripheral ischemic limbs. Thirty patients
who were candidates for imminent limb amputation
were treated by autologous PBSC implantation after
subcutaneous administration of G-CSF (5 mg/kg/
day) for 4 days. Limb salvage was achieved in 73%
of them. In 2006 the study was extended to a greater
number of patients: 92 cases withcritical ischemia or
intermittent claudication not improved by medical
therapy were enrolled; 68/92 were hemodialysis
patients; cases with necrotic limbs or foot infections
were left out; according to the Fontaine classica-
tion, 75/92 patients scored classes III and IV. On
a clinical basis the authors reported an improvement
in symptoms at 1 month in 86% of limbs; by three-
dimensional computerized tomography (CT)
arteries which were not visible at morphological
analysis became visible after treatment in 82%; the
authors reported 91% limbs free from amputation,
with an amputation rate of 59% in Fontaine stage
IV at 1 month. The authors concluded that thera-
peutic angiogenesis is effective for preventing limb
amputation resulting from diabetes and hemodial-
ysis until Fontaine stage III. Concerns about the
study are that no control group was included, the
outcome of the treated patients was similar to the
natural history of the disease, and results were
analyzed on clinical grounds exclusively.
45
In a later randomized controlled trial involving
28 diabetic patients with CLI, Huang et al.
46
demon-
strated an improvement in clinical symptoms in the
G-CSF-mobilized PB-MNC-transplanted group
compared with the control group, who received
conventional care. An improvement in ABI,
TcPO
2
, pain-free walking time, limb blood perfu-
sion, ischemic ulcers, and newly formed collateral
vessels in patients without surgical options was
also reported in this small trial.
42,47,48
Implantation of cells selected on the basis of
specic surface antigenic markersdi.e., CD34
+49
or CD133
+50
dhas been also attempted after G-CSF
mobilization. A series of case reports show that
treatments are safe and effective as well; however,
results coming from another study
48
with unse-
lected PBSCs showno difference among treatments;
thus, additional handling of these cells seems to be
unnecessary.
All in all, the use of PB-MNCs is more practical
and easier than using the bone marrow approach,
even though it should be taken into consideration
that ex vivo expansion is required to obtain a thera-
peutic amount of mobilized EPCs. This raises the
issue of cell handling, which in most European
countries is subject to specic regulations and proce-
dures requiring authorization.
To date, there is no clear evidence as to the best
therapeutic cells to use in vascular diseases. In
a randomized treatment of patients with lower
limb arteriosclerosis obliterans, injection of G-CSF-
mobilized PB-MNCs or BM-MNCs led to similar
results after 12 weeks.
51
Furthermore, the best
method of application (IM, IA, or both) and the
optimal cell dose are still unknown.
It should particularly be underlined that such
clinical studies are small, with no double-blinded
controls or short-termfollow-up. Application proto-
cols and outcome measures in the future should be
standardized if we are to make comparisons.
In the future, combined therapy (cell therapy and
gene therapy or conventional care) also needs to be
investigated, as well as the use of different sources
of stem cells such as umbilical cord bloodederived
multipotent stem cells or adipose tissue progenitor
cells, whose therapeutic potential in vascular
diseases has alreadybeenexploredinanimal models.
Results from published clinical studies using
BM-MNCs and PB-MNCs are summarized in Tables
III and IV, respectively.
ALOOKAT THE TISSUE-ENGINEERING
FUTURE
Given the encouraging clinical evidence and
because of their self-renewal and differentiating
property, stemcells and progenitors can be of poten-
tial use in treating many vascular diseases.
Regenerative medicine is a newmode of therapy,
still in its infancy, which could radically change the
management of some diseases in the future.
Improved knowledge of the biology of adult resident
stem cells and progenitors, development of novel
cell culture techniques aimed at expanding thera-
peutic cells rapidly, and the designing and
manufacturing of new classes of bioresorbable and
biomimetic polymers are emerging as the most
advanced research elds in regenerative medicine.
Tissue engineering is a science aimed at replacing
or repairing damaged tissues with biocompatible
synthetic structures endowed with the patients bio-
logical and biochemical characteristics. The latest
form encompasses the use of cells and their
Vol. 24, No. 2, February 2010 Therapeutic angiogenesis for peripheral vascular disease 263
Table III. Clinical studies using cell therapy with BM-MNCs for PAD
Reference Vascular disease Study type Therapeutic strategies Results
Tateishi-Yuyama
et al.
29
Unilateral/bilateral
leg ischemia
Pilot study/randomized
controlled trial
IM injection of
BM-MNCs/PB-MNCs
and BM-MNCs
Improvement in ABI, TcPO
2
, rest pain,
PWT; smaller increase in collateral vessel
formation in PB-MNC-injected leg
Esato et al.
35
PAD (4 TAO,
4 ASO)
Experimental
observations on 8
patients
IM injection of
BM-MNCs
Improvement in symptoms and rest pain,
ulcer healing
Higashi et al.
30
CLI Experimental
observations on 7
patients
IM injection of
BM-MNCs
Improvement in ABI, TcPO
2
, rest pain,
PWT, and endothelium-dependent
vasodilation
Miyamoto et al.
31
Severe chronic
PAD (limbs
and hands)
Experimental
observations on 12
patients
IM injection of
BM-MNCs
Signicant improvement in PWT, ABI,
TcPO
2
, rest pain, and distal perfusion
Saigawa et al.
36
ASO Experimental
observations on 8
patients
IM injection of BM-MNCs Improvement in symptoms, relief of rest
pain, ulcer healing, increase of small
blood vessels, strong correlation between
CD34
+
cells and the clinical efcacy of
BMI
Durdu et al.
38
CLI (Buerger disease) Experimental
observations on 28
patients
IM injection of BM-MNCs Ulcer healing; relief of rest pain;
improvement in ABI, PWT, quality of
life; de novo collateral vessel formation
Miyamoto et al.
39
TAO Unblinded and
uncontrolled study
on 8 patients
IM injection of BM-MNCs Short-term improvement in symptoms and
ulcer healing, adverse events during
long-term follow-up
Bartsch et al.
33
CLI Controlled open label
trial on 13 patients
Combined IM+ IA injection
of BM-MNCs
Improvement in pain-free walking
distance, ABI, oxygen saturation, venous
occlusion plethysmography at short- and
long-term examination
Kajiguchi et al.
37
TAO and ASO Unblinded and
uncontrolled study
on 7 patients
IM injection of BM-MNCs or
PB-MNCs (only 1 patient)
Improvement in symptoms, ABI, TcPO
2
,
extent of ulcer but only for TAO patients
Kirana et al.
40
PAOD Case report IM injection of BM-MNCs Complete wound healing, angiogenesis of
the forefoot, improvement in
microcirculation
Van Tongeren et al.
34
PAD Experimental
observations on 27
patients
IM injection of BM-MNCs
compared with IM+ IA one
Safety and feasibility of both methods;
improvement in PWT, ABI, rest pain at
short- and long-term follow-up; no
adverse reactions
PAOD, peripheral arterial occlusive disease; ASO, arteriosclerosis obliterans; BMI, body mass index
2
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Table IV. Clinical studies using cell therapy with PB-MNCs for PAD
Reference Vascular disease Study type Therapeutic strategies Results
Kudo et al.
49
CLI Case report IM injection of G-CSF-mobilized
CD34
+
cells
Increase in TcPO
2
, improvement in
symptoms; collateral vessel formation
Huang et al.
43
LASO Pilot study on 5 patients IM injection of G-CSF-mobilized
PB-MNCs
Improvement in clinical manifestations,
ABI, blood ow, angiographic scores
Kawamura et al.
44
CLI Pilot study on 30 patients IM injection of G-CSF-mobilized
PB-MNCs
Improvement in symptoms; prevention of
limb amputation in 73% of treated
patients
Lenk et al.
42
PAOD Experimental observations
in 7 patients
IA injection of G-CSF-mobilized
PB-MNCs after ex vivo culturing
Increase in walking distance, ABI, TcPO
2
,
ow, and endothelium-dependent
vasodilation
Huang et al.
46
Diabetic patients
with CLI
Randomized controlled
trial with 28 patients
IM injection of G-CSF-mobilized
PB-MNCs
Increase in limb blood perfusion, ABI,
ulcer healing; reduction in limb
amputation in treated group
Ishida et al.
47
TAO and ASO Pilot study on 6 patients IM injection of G-CSF-mobilized
PB-MNCs
Improvement in ABI, ulcer healing,
walking distance, and physiological
functioning subscale (SF-36)
Kawamura et al.
45
CLI Pilot study on 92 patients IM injection of G-CSF-mobilized
PB-MNCs
Improvement in symptoms, new vessel
formation; prevention of limb
amputation
Huang et al.
51
LASO Randomized trial with 150
patients
IM injection of G-CSF-mobilized
PB-MNCs or BM-MNCs
Treatments safe and effective;
improvement of the main clinical index
in both groups; no signicant difference
from comparative analysis regarding
walking distance, TcPO
2
, ulcer healing,
and lower limb amputation
Can izo et al.
50
ASO Case report IM injection of G-CSF-mobilized
CD133
+
cells
Limb salvage after 17 months,
improvement in symptoms and blood
ow
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molecules in articial constructs that compensate
for lost or impaired body functions. It is based
upon scaffold-guided tissue regeneration and
involves seeding porous, biodegradable scaffolds
with donor cells, which differentiate and mimic
occurring tissues.
52
Thus, vascular progenitor cells
could be used to develop tissue-engineered vessels.
This is a very important issue because many patients
do not have healthy vessels suitable for surgical or
endovascular revascularization.
53,54
Stem cells are
highly suited to this application. Early articial
conduits did not show much clinical applicability,
but recently articial vessels have been grown
from bone marrow cells in the peritoneal cavity of
the same animal in which they were grafted,
ensuring no rejection and perfect integration in
preexistent vessels.
53
Recently, a Japanese group
provided an effective method for the revasculariza-
tion of ischemic tissues in athymic rats, resulting
in transplantation of tissue-engineered human
smooth muscle cell sheets. Therapeutic strategies
aim either at inducing stable localized growth factor
delivery so as to recruit ECs for the ischemic site or
directly transplanting smooth muscle cells that can
actively participate in new vessel development.
55
It should be noted, lastly, that articial vessels
produced without the need for any exogenous
plastic scaffolds have already been used successfully
as arteriovenous stulas for hemodialysis access.
56,57
CONCLUSION
It is clear fromthe literature that a great deal of work
has already been performed in the eld of thera-
peutic angiogenesis for the treatment of peripheral
vascular disease. The results obtained so far may
help in nding a way to successfully manage threat-
ened ischemic limbs. However, most studies seem
inadequately categorized in their clinical presenta-
tion and, therefore, are of little practical signicance
for the expert vascular surgeon. In order to obtain
clinically useful information and to promote mean-
ingful research in this promising eld, more strict
interaction between pure researchers and special-
ized vascular surgeons should be encouraged.
Candidates for therapeutic angiogenesis should be
chosen from the really threatened ischemic limb
category in order to avoid possible misinterpretation
with a benign course of a mild arteriopathy. Such
patients should be unfavorable candidates for other
kinds of known vascular procedures in order to
avoid ethical conicts in treating them. In this sense,
therapeutic angiogenesis treatment may be tested
on patients undergoing surgical or endovascular
revascularization and compared with patients
treated similarly without the adjunct of this novel
therapy, thus comparing possible differences in the
healing of ischemic lesions. To this end, vascular
centers with high turnover and expertise in treating
peripheral vascular disease should be involved in
designing and carrying out the clinical arm of
research into these innovative strategies.
We thank Ralph Nisbet for his help in reviewing the
manuscript. G. P. and A. S. are supported by grants
from the Italian Ministry of University and Research,
RFO, Fondazione Luisa Fanti Melloni, Programma di
Ricerca Regione Emilia RomagnaeUniversit a di Bologna,
and Fondazione del Monte di Bologna e Ravenna.
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