treatment for pad: can we turn humans into · pdf file« every 30 seconds a lower limb...

Inserm

Joseph EMMERICH

Dept of Vascular Medicine

University Paris Descartes - INSERM U765

Treatment for PAD: can we turn humans

into salamanders ?

Prevalence of intermittent claudication (IC). Edinburgh Artery Study. Int J Epidemiol 1991;20:384-92

Prevalence of IC was 4.5% and incidence 15.5 per 1000 person-years.

In individuals who were initially symptomatic :

- 28.8% continued to have IC after 5 years

- 8.2% underwent revascularization or amputation

- 1.4% developed ischemic ulcers

Mean prevalence of IC in large population studies (From the TASC consensus)

0

1

2

3

4

5

6

7

30-34 35-39 40-44 45-49 50-54 55-59 60-64 65-69 70-74

Age group

Critical leg ischemia

a) persistently recurring ischemic rest pain requiring

regular adequate analgesia for more than 2 weeks, with

an ankle systolic pressure of < 50 mmHg and/or a toe

systolic pressure of < 30 mmHg, or

b) ulceration or gangrene of the foot or toes, with an

ankle systolic pressure of < 50 mmHg or a toe systolic

systolic pressure of < 30 mmHg.

CLI, in both diabetic and nondiabetic patients, is

defined by either of the following two criteria :

Incidence of CLI is around 500 per million per year

Inserm

Hiatt WR. N Engl J Med 2001;344:1608

Ankle Brachial Index (ABI)

Higher ankle pressure

Higher humeral pressure

Below 0.4 severe PAD

« Every 30 seconds a lower limb

is lost somewhere in the world

as a consequence of diabetes »

Boulton, Vol 366 November 12, 2005

Stage I Stage IIa Stage IIb Stage III or IV

Asymptomatic Mild claudication Moderate claudication Critical leg ischemia

(rest pain, ulcer or gangrene)

Bypass

Percutaneous angioplasty

Treatment strategy of Critical Limb Ischemia

To control pain and infection

Rapid evaluation of the arterial lesions (arteriography)

and rapid revascularization (surgery or angioplasty). In some cases (gangrene or septicemia), poor general condition,

a major amputtion is initially preferred.

Revascularization No possibility of

revascularization

Success

• Medical treatment

• Amputation Failure

Inserm

Tibioperoneal (outflow lesion) angioplasty can be used as primary

treatment in 235 patients with CLI (5-year follow-up)

Dorros G et al. Circulation 2001;104:2057-62.

Actuarial analysis of CLI patients

Fontaine

Inserm

Recommendation 100 : Spinal cord stimulation in CLI.

On current evidence, spinal cord stimulation cannot be

recommended in the treatment of CLI.

Recommendation 101 : Chelation therapy in CLI.

There is no scientific basis for the use of chelation therapy

in the treatment of peripheral arterial disease.

Recommendation 102 : Lumbar sympathectomy in CLI.

There is currently insufficient scientific evidence for the

selection of patients likely to benefit from lumbar

sympathectomy for the treatment of CLI.

Useless treatment

TASC. J Vasc Surg 2000;31(Suppl).

Inserm

ICAI study : Ann Intern Med 1999;130:412-21

Total patients

(n = 1334)

12.6%

11.2%

30.4%

0.9%

55.1%

Alprostadil

(n = 661)

10.9%

11.0%

29.3%

1.2%

52.6%

Controls

(n = 673)

14.3%

11.3%

31.4%

0.6%

57.5%

0.064

> 0.2

> 0.2

> 0.2

0.074

Death

Amputation

Persistant CLI

MI, stroke

Combined events

Events at 6 months

Inserm

Superficial femoral occlusion - Collateral vessels

Arteriogenesis: remodeling of newly formed

or preexisting vascular vessels

Angiogenesis: sprouting of new capillaries from preexisting vessels

Vasculogenesis: formation of blood vessels from endothelial progenitor cells

Ramsauer M. JCI 2002;110:1615

Angiogenesis

Vascular lesion

Tumor

Ischemic tissue Inserm Moore M.A.S.

JCI 2002.

Vasculogenesis

Drawing by M. Malphighi (1661), showing the vascular network of arteries, capillaries

and veins in a developing chicken embryo.

From Carmeliet P, Nature 2005

Inserm

Isolation of putative progenitor endothelial cells for angiogenesis Asahara et al, Science, 1997, 275, 964

20 ml of human peripheral blood

Culture

Cell sorting: CD34+ et VERFR-2+

EPC

Functional and metabolic

Characteristic of endothelial cells

Incorporation

Witin angiogenesis sites

Endothelial progenitor cells

(EPC)

Inserm

CLI

Pro-angiogenic GF

Anti-angiogenic GF

of PEC

VEGF

PlGF

FGF1,2

Angiopoietin 1,2

HGF

IGF

EPO

G-CSF,GM-CSF

MCP-1

HIF-1

TSP-1

Inserm

Circulating Endothelial Progenitor Cells, Vascular Function and CV risk.

Hill JM et al. N Engl J Med 2003;348:593-600

Circulating Endothelial Progenitor Cells and Cardiovascular Outcome.

Werner N et al. N Engl J Med 2005;353:999-1007

Inserm

Critical overexpression of thrombospondin 1 in chronic leg ischaemia.

J. Favier et al. J Pathol. 2005;207:358-66.

mRNA

Western-blot Overexpression of TSP1 in macrophages

and endothelial cells of ischemic tissues Inserm

EPCs C-EPCs

ischemia

VEGF-A

PLGF

Mobilisation

CD 34 +

CD 133+

VEGFR2+

VE-cadherin+

CD 146+

CXCR4 +

vWF +

CD 31 +

GM-CSF

EPO

Statins

Inserm

From Carmeliet P, Nature Med 2003

Participate directly

or indirectly

What are able to do the salamanders ?

The stages of newt limb regeneration. One day: a wound epithelium covers the

amputation site. One to four weeks: dedifferentiation, migration, and proliferation of cells

leads to the formation of a regeneration blastema. Three to six weeks: the limb flattens out

and cartilage begins to form. Four to ten weeks: the new digits develop and regeneration

of the limb is completed (From Odeberg SJ, 2005).

From Whited & Tabin, 2009

and Odelberg, 2005

Msx1, BMP, Notch 1, MMPs pathways

GENE

and / or

CELL THERAPY

Gene therapy of PAD

From : Losordo DW, Dimmeler S. Circulation 2004;109:2487-91

Clinical evidence of angiogenesis after arterial

gene transfer of phVEGF165 in patient with

ischaemic limb.

Isner JM. Lancet 1996;348:370.

Constitutive expression of phVEGF165 after

intramuscular gene transfer promotes collateral

vessel development in patients with critical limb

ischemia.

Baumgartner I. Circulation 1998;97:1114-23.

Inserm

Baumgartner I.

Circulation 1998;97:1114-23.

Intramuscular vascular endothelial growth factor gene therapy in patients with

chronic critical leg ischemia. Shyu KG. Am J Med 2003;114:85-92

21 patients with CLI – 24 legs treated (5 Buerger).

Naked plasmid coding for VEGF (same plasmid as J. Isner)

(dose 400 to 2000 mg – 2 injections IM/calf after 1 month)

Increase circulating VEGF from 26 to 63 pg/mL (p<0.005) 2 weeks

after the injection.

Transient edema in 25% of the patients.

Follow-up : 6 months.

- Disappearance of rest pain : 83%

- Improvement or healing of ischemic ulcer : 75%

ABI : 0.58±0.24 -> 0.72±0.28 (p<0.001) 4 weeks after second injection

Perfusion scoring of the leg (ARM) : 0.37±0.10 -> 0.47±0.11 (p<0.01)

Naked plasmid DNA encoding fibroblast growth factor type 1 for the treatment

of end-stage unreconstructible lower extremity ischemia : preliminary results of

a phase I trial. Comerota AJ et al. J Vasc Surg 2002;35:930-6

51 patients.

Tolerance and dose finding (dose max. 2x8000 mg)

Biodistribution of plasmid was limited and transient in plasma ;

absent in urine.

No increase of FGF-1 was detected.

Significant reduction of pain and ulcer size was seen, as well

as increase in ABI (P<0.01).

Absence of control group.

Inserm

Increased vascularity detected by digital subtraction angiography after VEGF

gene transfer to human lower limb artery: a randomized, placebo-controlled,

Double-blinded phase II study.Mäkinen K, et al. Mol Therapy 2002;6:127-133

3 groups – 19 Controls - 17 VEGF Plasmid/liposome – 18 VEGF adenovirus

Intra-arterial administration after peripheral angioplasty.

40 IC and 14 CLI patients.

Digital subtraction angiography revealed increased vascularity in the

VEGF-treated groups distally to the gene transfer site .

Mean Rutherford class and ABI showed statistically significant

improvements in the VEGF-Ad and VEGF-P/L groups, but similar

improvements were also seen in the control patients.

Anti-adenovirus antibodies increased in 61% of the patients treated

with VEGF-Ad.

Inserm

Phase 2 study

Regional angiogenesis with vascular endothelial growth factor in peripheral arterial

disease. A phase II randomized, double-blind, controlled study of adenoviral

delivery of VEGF 121 in patients with disabling intermittent claudication.

Rajagopolan S, et al. Circulation 2003;108:1933-38

101 patients, randomized in 3 groups.

Patients were randomized to low-dose (4x109 PU) AdVEGF121, high-dose

(4x1010 PU) AdVEGF121, or placebo, administered as 20 intramuscular

injections to the index leg in a single session.

A single unilateral intramuscular administration of AdVEGF121

was not associated with improved exercise performance or quality

of life in this study.

This study does not support local delivery of single-dose VEGF121

as a treatment strategy in patients with unilateral PAD and IC.

Inserm

Phase 2 study

Permanent ischemia as seen

in CLI, but not in IC, is probably

required to induce significant

angiogenesis.

Treatment with intramuscular vascular endothelial growth factor gene

compared with placebo for patients with diabetes mellitus and critical limb

ischemia: a double-blind randomized trial. . Kusumanto YH, et al. Human Gene Therapy 2006;17:683-91.

A double-blind, placebo-controlled study was performed in 54 adult

diabetic patients with CLI.

Patients were randomized to receive phVEGF165 or placebo (0.9% NaCl).

A single unilateral intramuscular administration of AdVEGF121

was not associated with improved exercise performance or quality

of life in this study.

This study failed to meet the primary objective of significant amputation

reduction (3 amputations vs 6; NS).

Overall, 3 versus 14 responding patients (p=0.003). Mainly decreased in

pain and ABO improvement.

Inserm

Inserm

Treatment with Intramuscular VEGF Gene Compared with Placebo for

Patients with Diabetes Mellitus and Critical Limb Ischemia:

A Double-Blind Randomized Trial. Kusumanto YH et al. Hum Gene Ther, 2006

In this small randomized study gene therapy failed to meet the primary

objective of significant amputation reduction.

However, significant and meaningful improvement was found in patients

treated with a VEGF165-containing plasmid.

There were no substantial adverse events.

125 patients. Multicenter study (37 sites).

Randomized, double blind study : FGF-1 vs Placebo.

CLI with non healing ulcers were randomized to receive

eight intramuscular injections of placebo or 2.5 ml of

NV1FGF at 0.2 mg/ml on days 1, 15, 30, and 45 (total

16 mg: 4 × 4 mg).

The primary end point was occurrence of complete

healing of at least one ulcer in the treated limb at week 25.

Secondary end points included ankle brachial index (ABI),

amputation, and death.

125 patients included – 107 eligible for evaluation Inserm

Therapeutic angiogenesis with intramuscular NV1FGF improves

amputation-free survival in patients with CLI. TALISMAN study.

Nikol S et al. Molecular Therapy 2008;16:972-8.




Amputation rate (week 52)

All

Major

Death rate (week 52)

Combined major

Amputation + deaths

Complete healing of > 1 ulcer

Mean change

ABI

TBI

TcPO2

Placebo

(N=56)

31 (55.4%)

19 (33.9%)

13 (23.2%)

29 (51.8%)

8 (14.3%)

0.01 ± 0.04

0.03 ± 0.02

9.81 ± 3.15

NV1FGF

(N=51)

19 (37.3%)

8 (15.7%)

6 (11.8%)

14 (27.4%)

10 (19.6%)

0.05 ± 0.04

0.04 ± 0.02

8.55 ± 3.41

p value

0.013

0.019

0.111

0.011

0.514

NS

NS

NS

Endpoints at week 25

Intramuscular Fibroblast Growth Factor-1 gene transfer in patients with CLI :

A double-blind, placebo-controlled study. Etude TALISMAN (Mol Ther 2008)

Major amputations Deaths

All

Serious

Severe

Possibly related to study drug

Leading to discontinuation

Neoplasm

Cardiovascular

Retinopathy

Death

Placebo

(N=61)

91.8%

80.3%

68.9%

21.3%

16.4%

8.2%

11.5%

0

24.6%

NV1FGF

(N=57)

93%

70.2%

47.4%

26.3%

7.0%

8.8%

24.6%

1.8%

17.5%

Adverse events at week 25

Total

(N=118)

92.4%

75.4%

58.5%

23.7%

11.9%

8.5%

17.8%

0.8%

21.2%




Inserm

Interestingly, the benefit relating to the amputation rate

observed with angiogenic therapy in the present study

was not associated with a benefit in respect of

hemodynamic end points (ABI, TBI), and no significant

differences in these were observed between the two

groups.

These data indicate that ABI and TBI, as well as ulcer

healing and ulcer size, may not be useful markers

of NV1FGF efficacy in patients with CLI,

Inserm

Isner

(1996)

Baumgartner

(1998)

Makinen

(2002)

Comerota

(2002)

Shyu

(2003)

Rajagopalan

(2003)

Motyas L

(2005)

Kusumanto

(2006)

Rajagopalan

(2007)

Nikol

(2008)

Powell

(2008)

IA VEGF165 plasmid

IM VEGF165 plasmid

IA VEGF adenovirus

IA VEGF plasmid/lipo

IM FGF1 plasmid

IM VEGF 165 plasmid

IM VEGF121 adenovirus

IM FGF-4 adenovirus

IM VEGF 165 plasmid

IM HIF-1a adenovirus

IM FGF1 plasmid

IM HGF plasmid

1 -

9 -

18 19

17 19

51 -

24 -

72 33

10 3

27 27

34 7

51 56

78 26

12 wks

6 mths

2 yrs

6 mths

6 mths

6 mths

6 mths

3 mths

1 yr

6 mths

6 mths

Increased collaterals (angio). Spider angioma.

Transient oedema.

Increased collaterals (angio) and increased ABI.

Increased collaterals. No change in ABI.

Increased collaterals. No change in ABI.

Reduction in pain and ulcer size.

Increased ABI and TcPO2.

Increased collaterals and improved ABI.

Reduced rest pain and ulcer healing.

Patients without CLI, but IC.

No improvement of peak walking time, ABI or IC.

Increased collaterals (angiography)

Reduction in pain and ulcer healing

No change in amputatioin rate

Safety of the treatment. No correlation between

ABI and outcomes

Decrease in minor and major amputations

Similar improvement in ulcer healing

No difference between treated and placebo groups

Author

(Year)

Treatment N° of patients

Active Control

FU Results

Summary of gene therapy trials in PAD

Total number of treated patients > 500

TAMARIS study Sanofi-Aventis

NV1-FGF

Confirmation of theTalisman study

Inserm

• METHODE:

TAMARIS est donc une étude internationale de phase III multicentrique.

Elle a inclus 525 malades présentant une ischémie critique des membres inférieurs avec ulcères

cutanés, non accessible à la revascularisation. Ils ont été randomisés, en double aveugle, pour être soit

traités par transfert de gène sous la forme de 4 injections intramusculaires multiples réalisées à 2

semaines d’intervalle, soit par injection de placebo.

• RESULTATS:

Pas d’impact sur les amputations et les décès

A 12 mois de la première injection de la thérapie à visée angiogénique, le taux d’amputation majeure et

de décès qui constituait le critère principal de l’étude a été identique dans les 2 groupes avec 37% dans

le groupe actif contre 33% dans le groupe placebo (RR = 1,11 ; p = 0,48).

Le taux d’amputation majeure seul a été de 25,9% contre 21% dans le groupe placebo

(RR = 1,2 ; p = 0,31).

• TOLERANCES

un cancer ou une rétinopathie proliférative avait été exclus de l’étude, aucun effet secondaire de ce type

n’a été observé à 1 an. Le taux de cancer, de rétinopathie, d’infarctus et d’insuffisance rénale a été

identique dans les 2 groupes. Une surveillance prolongée jusqu’à 3 ans sur ces critères est prévue.

Etude TAMARIS

AHA NOVEMBRE 2010,CHICAGO

HIATT W et al. AHA 2010

Amputation et décès à 1 an dans TAMARIS

Hiatt WR. NV1FGF gene therapy on amputation-free survival in

critical limb ischemia: phase 3 randomised, double-blind placebo-

controlled trial (TAMARIS). AHA 2010

Placebo (n = 259)

Th. Gèn. (n = 266)

RR p

Amputation majeure ou décès

86 (33,2 %) 96 (37,07 %) 1,11 0,48

Amputation majeure

55 (21,24 %) 67 (25,87 %) 1,20 0,31

Décès 39 (15,06 %) 46 (17,76 %) 1,15 0,53

Angiogenic synergism by the

combination of PDGF-BB and

FGF-2. Cao R. et al.

Nature Med 2003;9:604.

Use of a constituvely active HIF-1a transgene as a therapeutic strategy

in no-option critical limb ischemia patients.

Rajagopolan S, et al. Circulation 2007;115:1234-43

34 patients.

Escalation dose from 1x108 to 2x1011 viral particles.

HIF-1a therapy in patients with CLI was well tolerated, supporting further,

larger, randomized efficacy trials.

Inserm

Cell therapy of PAD

Therapeutic angiogenesis for patients

with limb ischaemia by autologous

transplantation of bone-marrow cells :

a pilot study and a randomised

controlled trial. Tateishi-Yuyama E et al.

Lancet 2002;360:427-35

General anesthesia.

Bone marrow cells (500 ml)

Mononuclear cells (95% purity)

Concentrated in 30 ml.

Cells implanted within 3 hours, by IM

injection in gastrocnemius (40 sites,

1.5 cm deep).

ABI 0.34 --> 0.47

Increase in ABI in 17 of 25

No change in the leg treated

with saline

ABI 0.37 --> 0.46

Increase in ABI in 13 of 20

No change in the leg treated

with PBMNCs

TcPO2 28.8 --> 46.3

Pain resolved in 16 of 20 legs

treated with mononuclear cells

but in only 3 of 20 legs treated

by peripheral blood monon. cells

Group B

Group A Groupe A

Groupe B

Therapeutic angiogenesis for patients

with limb ischaemia by autologous

transplantation of bone-marrow cells :

a pilot study and a randomised

controlled trial. Tateishi-Yuyama E et al.

Lancet 2002;360:427-35

Autologous transplantation of peripheral blood stem cells as an effective

therapeutic approach for severe arteriosclerosis obliterans of lower extremities.

Huang PP, et al. Thromb Haemost 2004;91:606-9

5 patients with CLI.

CD34+ mobilsation by G-CSF (600 mg/JD) injection during 5 days.

Cell therapy product defined as « peripheral blood stem cells »

containing 0,35% of CD34+ (concentration 1 x108 cell / mL).

Injection of 30 mL of this cell therapy product (40 injections

of 0.75 mL).

Inserm

Autologous transplantation of GCS-F mobilized peripheral blood mononuclear cells

improves critical limb ischemia in diabetes. Huang P et al. Diab Care 2005;28:2155

28 patients randomized : PBMNCs after G-CSF (600 mg/D) during 5 days

or usual treatment.

40 injections in leg and thigh (3x109 PBMNCs / 7.5x108 each shot).

40 days later, identical treatment using frozen cells.

Results after 3 months

Treated group(14)

14/18 (77.8%)

0

0.50 -> 0.63

76.9%

Control group (14)

7/8 (38.9%)

5

0.49 -> 0.51

18.2%

Ulcer healing

Amputation

ABI

Collateral vessels

(angiography)

Inserm

Author

(year)

Tateishi-Yuyama

(2003)

Higashi

(2004)

Huang

(2004)

Saigawa

(2004)

Huang

(2005)

Lenk K

(2005)

Yang

(2005)

…..

Treatment

(N of cells)

BMMNCs

(0.7 ± 2.9x109)

BMMNCs

(1.6 ± 0.3x109)

PBMNCs

(3x109)

BMMNCs

(6.04 ± 1.58x107/kg)

PBMNCs

(3x109)

PBMNCs

(39x106)

PBMNCs

…..

Injection

IM, calf

40 x 0.75 ml

IM, calf

40 x 0.75 ml

IM, thigh, leg

and foot

IM

IM, calf, thigh

40 x 0.75 ml

Intra-arterial

4 days of cell cult

IM

calf and foot

…..

N of

patients

45

7

5

8

14 treated

14 controls

(randomized)

7

62

……

FU

(weeks)

24

24

12

4

12

Results

Collaterals, ABI, TcPO2

Collaterals, ABI, TcPO2

Endothel-dependent VD

ABI and laser-Doppler

Correlation between CD34+

and in ABI

ABI and laser-Doppler

78% of ulcers healed vs

39% in controls ABI, TcPO2

Endothel-dependent VD

Decrease pain

ABI, TcPO2, ulcer healing

…..

Review of the trials of cell therapy in CLI

Since 2002, a steady rise in the number of clinçical reports, currently accumulating to at least

39 reports, including more than 750 patients.

Autologous stem cell therapy for PAD. Meta-analysis and systematic

review of the literature. Fadini et al. Atherosclerosis 2010;209:10-17.

Walter D H et al. Circ Cardiovasc Interv 2011;4:26-37

Study flow chart of the PROVASA study.

ABI during the randomized-start phase and until 6 months follow-up (open-label phase).

Walter D H et al. Circ Cardiovasc Interv 2011;4:26-37

Currently ongoing randomized, placebo controlled trials investigating

the effects of stem cell therapy in CLI patients (from Sprengers RW et al. 2010).

Year started

2006

2007

2007

2007

2007

2007

2008

2009

Name

Juventas

BONMOT

RESTORE-CLI

Harvest Technologies

ABC Trial

ACT34-CLI Trial

MESENDO Trial

BALI Trial

NCT

00371371

00434616

00468000

00498069

00539266

00616980

00721006

00904501

Nber of pts

110-160

90

150

48

108

75

30

110

Country

NL

Germany

USA

USA

NL

USA

USA

France

Intervention

IA BMMNC

IM BMMNC

IM « Aastrom TRC » BMC

IM aspirate concentrate

IM BMMNC

IM CD34+ cells

IM «stem cell mixture »

IM BMMNC

OPTIPEC trial

« Optimisation d'un produit de thérapie

cellulaire autologue par cellules mononucléées

médullaires dans l’ischémie critique des membres

inférieurs liée à l’athérosclérose. »

Promoter : AP-HP (PHRC national 2003 - AOM 03 034)

CCPPRB de Paris-Broussais-HEGP : 3/12/2004

Inserm

No side effects within 30 days after the treatment.

No side effects in the follow-up due to the teatment :

- 1 sudden death

- 1 GI K

No retinal side effect

50% clinical improvement ; ulcer healing or minor amputation

healing.

4 major amputations in 15 treated patients.

All the amputation specimens were submitted to a standardized

dissection protocol.

When the limb was amputated below the knee, a large sample of gastrocnemius

was sampled, around the site of cell injection and along the tibial arteries.

The forefoot tissues and arteries (plantar and toe arteries) were also sampled in

each case.

Between 15 and 20 paraffin blocks were prepared in each case and were stained

with hematoxylin and eosin (H&E).

Immunihistochemistry :

Tissue sampling

- anti-CD31, anti-CD34, and anti-von Willebrand factor : endothelial cell markers

- anti-podoplanin : lymphatic endothelial cell marker

- anti-a-smooth muscle actin : vascular smooth muscle cell marker

- anti-Ki67 antibody : proliferation cell marker

- anti-CD117 (c-kit) : precursor endothelial cell marker

- anti-Glut-1 : endothelial cells of some angiomas and angiosarcomas.

Modern Pathology, 2008

Inserm

Modern Pathology, 2008

Magnitude of incorporation of bone-marrow derived cells into

vasculature varies : 50% to occasional cells.

Other mechanisms apart from cell incorporation contribute to

collateral remodeling observed after BM cell therapy in various

models of ischemia.

Numerous arteriogenic cytokines are released by marrow stromal

cells.

Local delivery of marrow-derived stromal cells augments collateral perfusion

through paracrine mechanisms. Kinnaird T, et al. Circulation, 2004.

Marrow-derived stromal cells express genes encoding a broad spectrum of

arteriogenic cytokines and promote in vitro and in vivo arteriogenesis through

paracrine mechanisms. Kinnaird T, et al. Circ Res 2004

Bone marrow-derived progenitor cells can improve tissue ischemia

at least in part through paracrine mechanisms.

Perivascular

incorporation

Differentiation

to an endothelial

phenotype

Production of

paracrine factors

Improvement of neovascularization

Inserm

Progenitor endothelial cells

endothelial cell

Inserm

From Aranguren et al. J Mol Med 2009

Late EPC culture from cord blood

Day 0

Cord blood

mononuclear cells

adhesion

colonies

Day 1 Day 7/14

CD34+ isolation

Day 18 Day 25 Day 48 …

Expansion

In EGM-2 medium during

5 weeks

EPC characterisation after expansion

Weibel-Palade bodies

-- IGg1

-- KDR --

Iie-2

-- CD31

Surface markers Gene expression

KDR, CXCR4….

VEGF, SDF1….

Integrins

0.0

2.5

5.0 EPDC

HUVEC

[*H3] Thymidine incorporation

VEGF

SDF-1

Migration Pseudo-tube

formation

In vitro In vivo

Laser Doppler

in hind limb ischemia

Early EPC Late EPC

• Circulating angiogenic cells (CAC)

• CFU-Hill

• Culture modified mononuclear

cells (CMMC)

• Endothelial colonies forming

cells (ECFC)

• Endothelial progenitor derived

cells (EPDC)

• Outgrowth endothelial cells (OECs)

• Blood outgrowth endothelial cells (BOECs)

• LPP-HPP endothelial cells

• Appear in culture after 4-7 days

• Monocytic and endothelial cell markers

• High levels of growth factors secretion

• Appear in culture after 7-20 days

• Cobblestone appearance

• Endothelial cell markers

• High proliferative potential

Early EPC Late EPC

KDR, VE-Cadherin,

VWF, Flt-1, CD14+, CD45+

Positive

No

VEGF, SDF-1, IL-8, HGF

Low

No

Cathepsin L

Good

KDR, VE-Cadherin,

VWF, Flt-1, CD14-, CD45-

Positive

Yes

Low

High

Yes

u-PA

Good

Inserm

Surface Ag expression

AcLDL and lectin binding

Tube formation

Cytokines secretion

NO production

BMP 2/4

Proteolytic capacity

Angiogenic potential

Inserm

The 2 types of EPCs showed comparable angiogenic potentials when each

type was transplanted alone.

Early EPCs may contribute to neovascularization by secretion of cytokines

and MMP-9,

whereas OECs participate by providing building blocks and secreting MMP-2.

Improved perfusion to the ischemic limb and subsequent improved limb salvage

in mice receiving mixed EPC transplantation through cross-talk between

2 types of EPCs, with a synergistic effect.

Synergistic neovascularization by mixed transplantation of early

endothelial progenitor cells and late outgrowth endothelial cells.

The role of angiogenic cytokines and matrix metalloproteinases.

Yoon CH, et al. Circulation 2005;112:1618-27.

Inserm

Inserm

Pleiotropic role of PAR-1 stimulation on EPCs

SFLLRN SFLLRN SFLLRN

Pseudotube

formation Migration Proliferation Inflammatory cells

recruitment

Smadja et al. J Thromb Haemost 2006.

Smadja et al. J Cell Mol Med 2007.



Stimulation of EPCs by PAR-1 activation does not influence significantly

VEGF/VEGFR2 pathway.

BMPs increase late EPC proangiogenic potential

Hindlimb ischemia (nude mice)

Late EPC Late EPC-

pretreated with BMP4

Late EPC-

pretreated with BMP2

Smadja et al, 2008, ATVB

Expression of BMP 2/4 characterized late EPC compared to early EPC

Newly Formed vessels after BM-MNC injection in CLI

are positive for BMP

CD34 vWF

CD45 a-SMA

Ki-67 BMP2/4

Smadja et al, 2008, ATVB

Newly Formed

vessels

originate from

LATE EPC

LINEAGE

CD34

Tie-2

CD34 Commitment Expansion

VEGF, Angiopoïétin-1 BMP 2/4

VEGF, Angiopoïétin-2 BMP 2/4, SDF-1, Thrombin, E-selectin, Fc-ephrin B2…

VEGFR2

mouse embryonic cell line

10T1/2

served as the perivascular

component of the vascular

networks

Vessel density with adult PB-EPC

Vessel density with adult PB-EPC

Autologuous cells

Expansion Transfection (Oct3/4, Sox2, Klf4, c-Myc)

iPS

Embryonic stem cells

Late EPC

ECFC

Ethical and immunological

problems

?

Endo-iPS Endo-

ES

Very rare cells in

peripheral adult

blood of pts with CLI

and low angiogenic

property ?

Mobilisation of progenitor cells in-vivo

GM-CSF

G-CSF

VEGF

SDF-1

EPO

Angiopoietin-1

Statins

Exercise

Estrogen

(PPARg agonists)

Eff

icie

ncy

Infl

am

mati

on

From Aicher A. et al. Hypertension 2005;45:1-5 Inserm

START Trial. A pilot study on Stimulation of ARTeriogenesis

susingsubcutaneousapplication of GM-CSF as a new treatment of PAD

Van Royen N et al. Circulation 2005;112:1040-46

A double-blinded, randomized, placebo-controlled study was performed in 40 patients with

moderate or severe intermittent claudication

Placebo or subcutaneously applied GM-CSF (10 mg/kg) for a period of 14 days (total of 7 injections)

PWT day 14 PWT day 90

START Trial. A pilot study on Stimulation of ARTeriogenesis

susingsubcutaneousapplication of GM-CSF as a new treatment of PAD

Van Royen N et al. Circulation 2005;112:1040-46

Stem cell mobilization induced by granulocyte colony-stimulating factor to

Improve cardiac regeneration after acute ST-elevation MI.

STEMMI trial

Ripa RS et al. Circulation 2006;113:1983-92

Stem cell mobilization by G-CSF therapy in patients with acute

myocardial infarction and successful mechanical reperfusion has no

influence on infarct size, left ventricular function, or coronary restenosis.

Stem cell mobilization by granulocyte colony-stimulating factor in

patients with acute myocardial infarction: a randomized controlled trial.

Zohlnhofer D et al. JAMA 2006;295:1058-60 (REVIVAL-2)

Inserm

Iwaguro H, Yamaguchi J, Kalka C, et al. Endothelial progenitor cell

vascular endothelial growth factor gene transfer for vascular regeneration.

Circulation. 2002;105:732–738.

VEGF

Murasawa S, Llevadot J, Silver M, et al. Constitutive human telomerase

reverse transcriptase expression enhances regenerative properties of

Endothelial progenitor cells. Circulation. 2002;106:1133–1139.

Telomerase

Kong D, et al. Enhanced inhibition of neointimal hyperplasia by genetically

Engineered EPCs. Circulation 2004;109:1769-75.

eNOS.

Combination of gene and cell therapy ?

Inserm

Potential side effects of the stimulation of angiogenesis

Heistad, D. D. N Engl J Med 2003;349:2285-2287

Intraplaque hemorrhage and progression of coronary atherosclerosis. Kolodgie FD et al. N Engl J Med 2003;349:2316-25

Increase risk of cancer on the long term

by promoting the angiogenic switch

Cancer without disease. Do inhibitors of blood-vessel growth found naturally in our bodies defend most

of us against progression of cancer to a lethal stage ?

Judah Folkman and Raghu Kalluri. Nature 2004;427:787.

Systematic autopies after traumatic deaths:

- Women 40-50 y.o.: 30% in situ breast K, but only 1%

of breast cancer diagnosed at this age.

- Similar pattern for men concerning prostate K.

- Between 50 and 70 years , in situ thyroid K in almost 100%

of subjects, but incidence of clinical cancer in only 0.1%.

Two critical steps in cancerogenesis:

1. Cell mutations due to genetic instability that will trigger cell

transformation (stable if cell divisions compensated by cell deaths)

2. Angiogenic switch, converts in-situ tumors that are non-fatal to

grow up and generates metastasis.

(In trisomia 21, few solid tumors due to elevated levels of endostatin

and conversely increase rate of K prostate associated with endostatin SNPs)

Another approach of

cell therapy in CLI

Fibroblast cells, which form the

structural backbone of blood

vessels, are extracted from a

small dermal biopsy (the size of

a postage stamp) and

expanded in culture. Part of

Cytograft’s core technology

includes techniques to grow

these cells into robust sheets

that can be manipulated into

three-dimensional structures.

Cytograft is currently applying

this technology to create the

world's first clinically available

completely autologous tissue

engineered vascular grafts:

Lifeline™

Tissue-Engineered Blood Vessel for Adult Arterial Revascularization.

L’Heureux N, Mac Allister T. N Engl J Med 2007;356:1451-2

From Kumar & Caplice, ATVB 2010

Emerging and future clinical trials of adult vascular progenitor therapy.

Need for new treatment of PAD (CLI)

Clinical need High

Comorbidity High

Patients homogenicity Low

Endpoints Pain

Ulcer healing

Gangrene

Limb loss

Death

Effective therapy Revascularisation (if possible)

Inserm

1) Which patient population should be considered for stem cell

or gene-therapy ?

2) What type of stem cell / gene therapy should be used ?

3) What is the best route for cell / gene delivery ?

4) What is the optimum number of cells / plasmid that should be

used to achieve functional effects ?

5) Is stem cell / gene therapy safer and more effective than

conventional therapies ?

Inserm

INSERM U765 : David Smadja

Pascale Gaussem

Anne-Marie Fischer

Catherine Boisson-Vidal

Blandine Dizier

Gabrielle Sarlon

Isabelle Martinez

Saul Azouelos

Laetitia Mauge

Clément d’Audigier

Centre de Thérapie Cellulaire

de Necker : M. Cavazzana-Calvo

L. Dal Cortivo

F. Lefrère

Inserm

« Nutrisco et extinguo »

François 1er’ motto

What is the relationship between the war

in Iraq and regenerative limb medicine ?

Do you believe in miracles ?

Durand (1997) Fra Angelico (1440)

Deacon Justinian’s recovery.

by transplantation of a black’s leg by St Côme and St Damien)

Clinical Application Cell and Source

Therapeutic application and source of adult vascular progenitor cells

used in clinical trials.

From Kumar & Caplice, ATVB 2010

http://www.cbsnews.com/stories/2008/03/22/sunday/main3960219.shtml

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