Journal of Diabetes and Its Complications

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Therapeutical potential of autologous peripheral blood mononuclear cell

transplantation in patients with type 2 diabetic critical limb ischemia

Ahmet Ozturk

a

, Yasar Kucukardali

b,

⁎

, Fatih Tangi

c

, Alev Erikci

a

, Gunalp Uzun

d

, C

_ı

nar Bashekim

e

,

Huseyin Sen

f

, Hakan Terekeci

c

, Yavuz Narin

g

, Mustafa Ozyurt

h

, Sezai Ozkan

f

, Ozkan Sayan

a

,

Osman Rodop

i

, Selim Nalbant

c

, Onur S

_ı

ld

_ı

roglu

e

, Fevzi Firat Yaln

_ı

z

b

,

_İ

brahim Volkan Senkal

b

,

Hilmi Sabuncu

a

, Cagatay Oktenli

c

a

Gulhane Military Medical Academy Hematology Section, Istanbul, Turkey

b

Yeditepe University Medical School Department of Internal Medicine, Istanbul, Turkey

c

GMMA Department of Internal Medicine, Istanbul, Turkey

d_{GMMA Sualt}_ı_Hekimli_ğ_{i Servisi, Istanbul, Turkey} e_{GMMA Department of Radiology, Istanbul, Turkey} f

GMMA Department of Anesthesiology, Istanbul, Turkey

g

GMMA Department of Nuclear Medicine, Istanbul, Turkey

h

GMMA Department of Microbiology, Istanbul, Turkey

i

GMMA Department of Orthopedic Medicine, Istanbul, Turkey

j

Yeditepe University Department of Biostatistics, Istanbul, Turkey

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 15 May 2010

Received in revised form 20 November 2011 Accepted 30 November 2011

Available online 11 January 2012

Keywords:

Diabetes

Critical limb ischemia Angiogenesis

Peripheral blood mononuclear cells Transplantation

Aim:The aim was to evaluate the therapeutic effectiveness of granulocyte colony-stimulating factor (G-CSF) mobilized peripheral blood mononuclear cells (PBMNCs) in critical limb ischemia (CLI) of type 2 diabetic patients.

Method:Forty diabetic patients with CLI were enrolled and randomized to treatment and control groups. In the treatment group, the patients received subcutaneous injections of recombinant human G-CSF (30 MU/day) for 5 days to mobilize stem cells. PBMNCs were collected and transplanted by multiple intramuscular injections of 1 ml in 1–1.5-cm depth into ischemic limbs.

Results:At the end of 12 weeks of follow-up, the baseline and end point results in transplant group were as follows: Fontaine score improved from 3.8±03 to 3±0.5 (P=.0001), ankle brachial pressure index increased from 0.68±0.24 to 0.87±024 (P=.001), transcutaneous oxygen increased from 33±14 mmHg to 44±10 mmHg (P=.0001), and 6-min walking distance improved from 280±82 m to 338±98 m (P=.0001). Pain score decreased from 8.2±1.3 to 5.63±1.6 (P=.001), and the number of patients with limb ulcers was reduced from 9/20 (45%) to 3/20 (15%) (P=.031). In the control group, Fontaine score, 6-min walking distance, and pain score were improved; ankle brachial pressure index and transcutaneous oxygen pressure were not improved. The number of patients with limb ulcers did not change in the control group. There are improvement in amputation rates, collateral vessel development, and number of limb ulcers healed. Conclusions:These results indicate that the autologous transplantation of G-CSF that mobilized PBMNCs in CLI diabetic patients is safe and effective in patient compliant reduction and improved perfusion.

1. Introduction

Despite conventional treatment options, a substantial number of patients with thromboangiitis obliterans and diabetic limb ulcer experience limb loss. Physical disability, psychiatric problems, and high reamputation rates force investigators to identify novel therapeutic approaches in this area of medicine (Trautner, Haastert,

Spraul, Giani, & Berger, 2001). Treatment with vasodilators, antico-agulants, and/or vascular bypass is indicated. However, despite these treatment options, a substantial number of patients undergo limb amputations. Recent studies in thisﬁeld target cell-derived treatment strategies (Chen, Lee, Chiu, Shyu, Lee, Huang, & Li, 2006). Cell-derived treatment strategies aimed at revascularization and/or neovascular-ization for ischemic damage prevention.

Stem cell collection is made through either the bone marrow or peripheral route. Bone marrow aspiration carries the risk of anemia and also requires anesthetic medication. Peripheral stem cell collection is more risk free, and cells are collected by centrifuge. ⁎Corresponding author.

Contents lists available atSciVerse ScienceDirect

Journal of Diabetes and Its Complications

j o u r n a l h o m e p a g e : W W W. J D C J O U R N A L . C O M

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A recent study compared therapeutic angiogenesis, ankle brachial pressure index (ABI), pain at rest, and transcutaneous oxygen pressure (TcPO2) in patients with bilateral diabetic critical limb

ischemia (CLI). One extremity was given bone-marrow-derived stem cells, and the other one was given peripheral blood derived mononuclear cells (PBMNCs). Bone-marrow-derived mononuclear cells showed better results in that extremity than the other one (Tateishi-Yuyama et al., 2002). However, recent studies established the potential beneﬁt of autologous transplantation of PBMNCs in CLI diabetic patients (Huang, Li, Han, Xiao, Yang, & Han, 2005). After granulocyte colony-stimulating factor (G-CSF) mobilization, the number of CD34 antigen-positive expressing hematopoietic proge-nitor cells increased, and these cells act as endothelial progeproge-nitor or angioblasts (Asahara, Murohara, Sullivan, Silver, van Der Zee, & Li, 1997). Recent and also ongoing studies showed positive effects of circulating progenitor cells on vascular healing.Rehman, Li, Orschell, and March (2003) suggest that angiogenesis-promoting effect of endothelial progenitor cells comes from endothelial cell activation by angiogenesis-stimulating factor secretion (Rehman et al., 2003). Endothelial progenitor cells collected from type 1 diabetics has impaired angiogenetic capacity; the ones derived from type 2 diabetics had impaired proliferation and adhesion functions ( Loo-mans et al., 2004; Tepper et al., 2002). The problems discussed explain insufﬁcient vascular development in PBMNCs transplanted in diabetic patients. But in this aspect, future studies are needed. Studies also report the potential retinopathy side effect of endothelial progenitor cells (Sata, 2006).

The aim of this study is to evaluate the efﬁcacy of the application of autologous transplantation of G-CSF mobilized PBMNCs in the treatment of CLI of diabetic patients.

2. Materials and methods

2.1. Study design

This was a prospective, randomized, open-label, controlled intervention study.

2.2. Study location

This clinical study was approved by the ethical committee board of the GMMA Hospital (1491-299-06, 30 November 2006) and Republic of Turkey Ministry of Health (14362/05.07.2007). Patients admitted to GMMA Internal Medicine, Yeditepe University Internal Medicine, GMM hematology section, from December 2008 to December 2009 were enrolled in this prospective controlled clinical trial. All participants gave written informed consent.

2.3. Population

The Fontaine classiﬁcation stratiﬁed patients as class III–IV, type 2 diabetic patients with proven CLI.

2.4. Inclusion criteria

Diabetic patients with proven CLI but without hypercoagulable states or gangrene above the ankle and/or severe coronary, cerebral, and renal vascular diseases were eligible for participation in this trial. Causes of hypercoagulable states in patients' history and medical records include medications (female hormones, estrogens, and birth control pills), postsurgery phospholipid antibodies in blood (anti-cardiolipin antibodies, lupus anticoagulant), cancer, elevated blood homocysteine levels, and inherited protein deﬁciencies (antithrom-bin III, factor V Leiden, protein S, protein C). History and evidence in

medical records for acute or chronic coronary event (coronary angiography, myocardial perfusion scintigraphy) were determined.

2.5. Exclusion criteria

Patients with proliferative retinopathy, malignant neoplasm, myocardial infarction, angina pectoris, cerebrovascular accident (b6 weeks), terminal renal and hepatic failure, anemia, leukopenia, and thrombocytopenia were excluded.

2.6. Initial patient visit

Patients with CLI symptoms were staged according to their Fontaine scores. Demographic characteristics, duration of diabetes, glycemia levels, hepatic and renal functions, inﬂammatory markers, and medications of stage III–IV patients were noted. Initial pain score, ABI, TcPO2, 6-min walking distance (in patients without

ulcer or superﬁcial ulcer), and Doppler ultrasonography/digital substraction angiography (DSA) /magnetic resonance angiography of leg ulcer were determined.

Forty diabetic patients with CLI were enrolled and randomized as 20 patients in the treatment group and 20 patients in the control group. Randomization was made via an internet-based system. Main clinical manifestations were observed 12 weeks after treatment. Mean age for treatment group and control group is as follows: 71±9 years and 70±8 years (P=.691), with a male/female ratio of 16/4 for the treatment group and 13/7 for the control group (P=.288). The baseline features (Fontaine score, ABI, TcPO2, 6-min walking distance,

number of limb ulcers, pain score, duration of diabetes, HbA1c levels) and clinical characteristics of the two groups were similar before the trial (Table 1).

2.7. Fontaine classiﬁcation

The Fontaine classiﬁcation is as follows: stage I: no symptoms, stage II: intermittent claudication subdivided into II-a (without pain on resting, but with claudication at a distance of greater than 200 m) and II-b (without pain on resting, but with a claudication distance of less than 200 m), stage III: nocturnal and/or resting pain, stage IV: necrosis and/or gangrene in the limb (Fontaine, Kim, & Kieny, 1954).

Table 1

Baseline features and clinical characteristics of the patients enrolled Treatment group Control group P

N 20 20 Age (mean ±S.D.) 71.9±9.2 70.8±8.8 (62-79) .691 Sex (M/F) 16/4 13/7 .288 Mean duration of diabetes (years) 13.5±8.5 (5-22) 12.8±9.8 (4-21) 1.30 Mean Hba1c (%) 7.4±1.5 7.8±1.7 1.80 Fontaine score 3.84±0.37 3.75±0.44 .442 ABI 0.68±0.24 0.70±0.26 .858 TcPO2(mmHg) 33.81±14.73 29.53±13.11 .261 6-min walking distance (m) 280±82 292±57 .577 Pain score 8.2±1.3 8.5±0.7 .313

No. of patients with limb ulcer (n/%)

9/ 45% 8/ 40% .749

No. of patients applied HBO (n/%)

9/ 45% 8/ 40% .749

There were no differences in baseline features between the two groups. Between-group comparisons were performed using Mann–Whitney U test for continuous variables andχ2

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2.8. Pain score

We used numeric pain scale in this study. The pain score scale is from 0 to 10, 0 beingno painand 10 beingmaximum pain(Williamson & Hoggart, 2005).

2.9. ABI

The sensitivity and speciﬁcity of ABI for peripheral artery disease prediction were 99% and 99% (Bebe, 2001). The severity of occlusion is inversely proportional with result (Iwagura et al., 2002). An ABIN1.3 incompressibility of calciﬁed arteries, 1.0–1.3: normal, 0.9–1.0: acceptable, 0.7–0.89: mild disease (intermittent claudication fre-quent), 0.5–0.7: moderate arterial disease (intermittent claudication), b0.5: severe peripheral vascular disease (pain at rest),b0.2: tissue loss (severe limb-threatening peripheral arterial disease is probably present) (Potier, Abi Khalil, Mohammedi, & Roussel, 2011).

2.10. Angiographic analysis

The patients were subjected to analysis of magnetic resonance angiography before and 12 weeks after treatment. The angiographic scores for the formation of new collateral vessels were assessed as 0 (no collateral development) and 1 (collateral development) (Graziani et al., 2007).

2.11. TcPO2

TcPO2 monitor consists of a combined platinum and silver

electrode covered by an oxygen-permeable hydrophobic membrane, with a reservoir of phosphate buffer and potassium chloride trapped inside the electrode. TcPO2 is a safe, noninvasive, and accurate

method for evaluation of ischemic extremity perfusion and oxygen-ation (Ruangsetakit, Chinsakchai, Mahawongkajit, Wongwanit, & Mutirangura, 2010).

Hyperbaric oxygen therapy

Galeazzi brand twin lock chamber pressure rooms were used for his study. Patients received 100% oxygen at 2.5 atm chamber pressure for 120 min. All of the patients enrolled in this study received approved medication (vasodiladator medication, anticoagulation, antiaggregation, if needed antibiotics, glycemia regulation, statin, wound care). In addition to these, all patients who have leg ulcer received hyperbaric oxygen (HBO) therapy. Baseline measurements were obtained 2 weeks later after HBO therapy (Niinikoski, 2003).

In the treatment group, the patients received subcutaneous injections of recombinant human G-CSF (30 MU/day) for 5 days. Their mean leukocyte counts were 25 000± 6000/ml on the day of apheresis (day 6).

2.12. PBMNC collection

The Haemonetics MCS3p cell separator with 871 coded apheresis kit was used.

2.13. Analysis of CD34+ cells

A Becton Dickinson FACSCalibur ﬂow cytometer was used for the study. BD FACSFlow solution, BD Trucount Tubes kit, FACS Lysing Solution, Calibrite 3 solution, and BD Falcon 5-ml polysty-rene tubes were also used. Cell suspension of 40–70 ml was obtained by apheresis. The number of cell CD 34+ cells retrieved was 24.8×106_/ml.

2.14. PBMNC transplantation

Three hours later, each ischemic lower limb was injected intramus-cularly at 40–50 sites, nearly 3×3-cm distance, 1–1.5 cm deep, with 1 ml CD34+ cell suspension. Five patients received bilateral and 15 patients received unilateral injections. Low-molecular-weight heparin was used for 5 days for thromboembolic prophylaxis.

2.15. Twelve-week follow-up period

Fontaine score, pain score, ABI, DSA/magnetic resonance angio-graphy, TcPO2, number of patients with ulcer, and number of patients

with amputation were measured. Healing of the ulcer was accepted when it showed a reduction in size in a 1-month treatment period; these patients were classiﬁed as responders.

2.16. Statistical analysis

Data analysis was performed using Statistical Package for Social Sciences for Windows (version 15.0). Data are expressed as number, percentage, mean, median, and standard deviation. Between-group comparisons were performed using Mann-Whitney

U test for continuous variables and χ2 _{for categorical variables.}

Within-group comparisons were performed using Wilcoxon signed-rank test. AP value less than .05 was considered an indication of statistical signiﬁcance.

3. Results

Twelve weeks after PBMNC administration, the scale of pain score decreased, 6-min walking distance increased, Fontaine score dec-reased, and ABI score and TcPO2improved. In addition to these, the

number of patients with ulcers was reduced from nine to three. The results for the control group, respectively, were as follows: Fontaine score decreased, ABI and TcPO2were not changed, 6-min walking

Table 2

Comparison of the parameters in groups at baseline and at the end of 12 weeks

Treatment group Control group

Baseline After 12 weeks P Baseline After 12 weeks P

Fontaine score 3,84±0.37 3.05±0.52 .001 3.75±0.44 3.50±0.60 .021

ABI 0.68±0.24 0.87±0.24 .001 0.70±0.26 0.73±0.28 .331

TcPO2(mmHg) 33.81±14.73 44.31±10.03 .010 29.53±13.11 31.35±14.70 .080

6-min walking distance (m) 14 patients 280±82 11 patients 338±98 .001 15 patients 292±57 10 patients 306±63 .005

Pain score 8.2±1.3 5.6±1.6 .001 8.5±0.7 7.7±0.8 .001

No. of patients with ulcer (n/%) 9/ 45% 3/15% .031 8/40% 8/40% 1.00

No. of patients with collateral vessel (n/%) 9/ 45% 5/ 25% .138

No. of amputations 3/15% 5/25% .429

All parameters showed signiﬁcant improvement in the treatment group. Within-group comparisons were performed using Wilcoxon signed-rank test; between-group comparisons were performed using Mann–WhitneyUtest for continuous variables andχ2_{for categorical variables.}

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distance increased, pain score decreased, but there were no differences in ulcer numbers (Table 2).

In this clinical trial, we observed that all clinical manifestations in the treatment group were signiﬁcantly improved after autologous transplantation of PBMNCs, including outcome of limb pain, 6-min walking distance, limb ulcers, ABI, Fontaine score, and TcPO2

(P=.001–.003)(Graphics 1, 2).

After 12-week follow-up, three (15%) patients from the treatment group andﬁve (25%) patients from the control group underwent limb amputation (P=.429). The number of ischemic limbs with new collateral vessel development in the treatment group was higher than in control patients (45% vs.25%,P=.138) (Table 2). No signiﬁcant statistical difference was found between the control and treatment populations in amputation rates and collateral vessel development.

4. Discussion

Our present study is the second randomized controlled study with a larger number of patient group that investigates the therapeutic effectiveness of G-CSF mobilized PBMNC in CLI of diabetic patients.

At the end of the study, the main manifestations including clinic limb ischemia score, ABI, transcutaneous oxygen pressure, 6-min walking distance, pain score, and number of ulcer healed improved

with signiﬁcant statistical difference in the patients of the treatment group. However, no signiﬁcant statistical difference was found between the control and treatment populations in amputation rates and collateral vessel development.

Interventional and surgical revascularization is the treatment of choice in critical limb ischemic patients. Despite the technical advances in revascularization procedures, a substantial number of patients with widespread and distal vascular disease in whom revascularization is not possible with these techniques remain. The uses of pharmacologic drugs also give limited beneﬁt to these patients.

One such opinion is therapeutic angiogenesis. Recent studies demonstrated the beneﬁcial effects of peripheral progenitor cells in CLI even in totally occluded patients. At present, there is a great deal of interest in cell-based treatment strategies for the repair of ischemic damage. (Kalka et al., 2000; Iwagura et al., 2002).

Huang, Li, Han, Xiao, Yang, and Han (2004) reported that transplantation of G-CSF mobilized autologous PBMNCs improves limb ischemia in patients with arteriosclerosis obliterans of lower extremities (Huang et al., 2004). Following these, they designed a new study and investigated potential beneﬁts of G-CSF mobilized PBMNCs in CLI of a diabetic patient group (Huang et al., 2005). In this pilot clinical trial, they showed that many clinical manifestations in the transplanted patients were signi_ﬁcantly improved after autolo-gous transplantation of PBMNCs, including outcome of lower limb pain, pain-free walking distance, diabetic foot ulcers, and angio-graphic scores. They reported minor amputation rates and better collateral vascular development than our study. The likely basis for this result is that they retransplant critical patients on day 40.

Cardiac, infectious, renal, hepatic, metabolic, and clinical para-meters were measured before and after cell transplantation, and both were noted to be in normal limits. We did not detect infection or any abnormality in vital signs both during and after the trial.

The autologous transplantation of bone marrow mononuclear cells was also found to be safe and effective for achievement of therapeutic angiogenesis (Tateishi-Yuyama et al., 2002); however, because it is a noninvasive procedure and there is a lack of anesthesia need, peripheral stem cell collection is the choice for most of the investigators.

G-CSF used for peripheral stem cell mobilization increases the cost. But it has been suggested that G-CSF by itself is possible to improve neovascularization (Seiler et al., 2001) and wound healing in ischemic diseases because of its ability to mobilize endothelial progenitor cells into peripheral blood (deLalla et al., 2001). In the first study, angiographic collateral degree increased significantly after treatment with GM-CSF, and it was statistically unchanged in the placebo group after treatment in the GM-CSF but not in the placebo group. But in this study, GM-CSF had been applied in an intracoronary manner. In our study, application was perivascular. There was no evidence that show beneficial effect of G-CSF itself applied perivascularly. Because of the potential risks of G-CSF, patients who have proliferative retinopathy were excluded, and low-molecular-weight heparin was used for thromboembolic prophylaxis.

The mainﬁndings in recent studies reported that implantation of PBMNC into ischemic limbs shows its beneﬁt by two mechanisms. First, it promotes angiogenesis and endothelial progenitor cell migration to the ischemic region; second, it secretes several angiogenetic factors that promote neovascularization and vascular cell repair (Iba et al., 2002). Several investigators reported that angiogenetic factors such as vascular endothelial growth factor (Isner, Pieezek, Schainfeld, Blair, Haley, & Asahara, 1996), basic

ﬁbroblast growth factor (Wanataba, Smith, Sun, Smart, Delcarpio, & Roberts, 1998),and hepatocyte growth factor (Taniyama, Morishita, Aoki, Nakagami Yamatoko, & Yamazaki, 2001) promote collateral vascular development in limb and myocardial ischemia models

0,5 -0,5 -1,5 -2 -3 -2,5 -1 0 Diff erences

treatment group control group P:.003

P:.001

Fontaine score Ankle brachial Pain score index

Graphic 1.Comparison of the differences between groups for ABI, Fontaine, and Pain score. 60 50 40 30 20 10 0 Diff erence s

Treatment group Control group

P:.001

TcPO2(mmHg) 6 minute walking distance (m)

Graphic 2.Comparison of the differences between groups for TcPO2 and 6-min

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experimentally. Recent studies demonstrated a limited number of bone-marrow-derived endothelial progenitor cells in peripheral circulation (Asahara et al., 1997). Endothelial progenitor CD34+ cells need to be mobilized before peripheral collection.

This is theﬁrst randomized controlled study following Huang's pilot study. We thought that improvement in limb perfusion is responsible for clinic and laboratory healing. Collateral vascular development around occluded vessels also remains an important factor. In our study, we detected more collateral development in the treatment group. Future studies are needed in a large number of patients to determine the mechanism of action of angiogenetic factors in collateral vessel development. Also, patients should be followed for longer (couple of years) to detect possible side effects of cell-based therapies.

In conclusion, angiogenesis is an important process during pathologic conditions such as wound healing in diabetic patients with CLI. In our data, there was a clear trend for many clinical manifestations in the transplanted patients to be signiﬁcantly improved after autologous transplantation including outcome of limb pain, walking distance, tissue oxygen content, ankle brachial index, and ulcers. We also observed more new collateral vessels, but there was no signiﬁcant statistical difference.

Acknowledgment

This study was supported by Research Grants of GATA Haydarpasa Training Hospital. We thank Professor Mustafa Gultepe, Chief of the Epidemiology Study Group, for support and assistance.

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