There is an increasing interest in cellular therapy for cardiovascular diseases. The first well-designed clinical trials have shown tantalizing hints that bone marrow cells may improve cardiac function post-myocardial infarction (post-MI)1,2 and enhance perfusion in patients with critical limb ischaemia.3 This cautious optimism comes on the heels of the great disappointment with pro-angiogenic gene therapy, which failed to live up to the promise based on encouraging pre-clinical studies and early, largely anecdotal clinical experience.4 However, even accepting that cell therapy may have a significant beneficial effect, the magnitude of this benefit would seem to be rather modest.5 Although there are many reasons for this, related both to the host (age, cardiovascular risk factors, and co-morbidities) as well as the cells themselves (type, ‘dose’, delivery route, and activity), it seems clear that strategies to enhance the regenerative capacity of stem or progenitor cells will be needed to effect robust clinical improvements.

Bone marrow-derived mesenchymal stromal cells (MSCs) offer exciting potential in therapy of cardiovascular diseases and have been studied extensively. They have been reported to induce angiogenesis in models of myocardial6,7 and hindlimb ischaemia.8 They have also been shown to inhibit cardiomyocyte apoptosis, reduce remodelling, and improve myocardial function in the post-infarct myocardium.9 Their functional benefit in the heart was originally attributed to their ability to differentiate into cardiomyocytes,10 although evidence of engraftment and differentiation is often lacking. Greater attention has recently been placed on their ability to secrete paracrine growth factors,11 which act to reduce cardiomyocyte apoptosis and immune cell proliferation,12 together reducing inflammation and fibrosis. MSCs have been shown to secrete several mitogenic and angiogenic growth factors, including VEGF, IGF-1, EGF, angiopoietin-1, and SDF-1.12 In addition, they secrete adrenomedullin, which was shown to have direct anti-fibrotic effects in the myocardium following MSC delivery.13 MSCs have also been shown to modulate the immune system14 and to be immune-privileged in allograft models,15 which, if truly the case, would make them attractive candidates for use in the clinic. Indeed, allogeneic MSCs are currently being studied in clinical trials for a number of indications, including cardiac disease.16,17

Despite generally positive outcomes following MSC delivery in pre-clinical infarct models, cells do not persist long in the myocardium, and this is largely due to low cell survival.18 Lack of engraftment precludes any possibility of trans-differentiation and limits the opportunities for paracrine and local immune modulatory effects. Therefore, genetic engineering strategies to improve the survival of MSCs or increase their paracrine activity may enhance their regenerative function. The combination of gene and cell therapy may be a ‘happy marriage’ for a number of reasons. First, the manufacture and processing of cells ex vivo provides an ideal opportunity for effective gene transfer using a variety of approaches, including simple non-viral strategies. This obviates one of the main barriers to effective in vivo application of gene therapy for cardiovascular disease, namely transfection efficiency. Second, one can select a therapeutic gene whose product will enhance the paracrine activity of a progenitor or stem cell. For example, overexpression of VEGF165, a pro-angiogenic cytokine already produced by MSCs, has been shown to improve the efficacy of cell delivery post-MI,19–21 which was attributed to the paracrine effect of VEGF. Third, one can use a trans-gene, or combination of genes, that will act on the cell itself in an autocrine manner. An example of this third strategy is the overexpression of Akt,22 a potent pro-survival protein that enhanced MSC survival, resulting in reduced inflammation and cardiac remodelling, and improved myocardial function in the post-MI model.

Erythropoetin (Epo) is a glycoprotein hormone produced in the kidney and liver that act primarily as a cytokine for erythroid precursors in the bone marrow. However, the receptor for Epo (EpoR) has been found in various cardiovascular cell types (cardiomyocytes, smooth muscle cells, and endothelial cells) and is thought to activate downstream signalling through Erk1/2, PI3K, and Akt in cardiomyocytes.23–25 In models of myocardial ischaemia or infarction, Epo produces cardioprotection that is independent of haematocrit26 through inhibition of apoptosis and inflammation as well as induction of neovascularization.27 Apart from its direct effects on the vasculature and the myocardium, Epo is thought to improve myocardial function through the mobilization of regenerative cells from the bone marrow, such as endothelial progenitor cells (EPCs).28 Recently, Epo was used in combination with MSC delivery and shown to enhance the angiogenic effect of MSC therapy,29 suggesting a synergistic effect.

In this issue of Cardiovascular Research, Copland et al. have elegantly demonstrated that retroviral transduction of mouse MSCs with erythropoietin (Epo-MSCs)30 improves their regenerative function via autocrine and paracrine effects. They showed that wild-type MSCs do not typically produce Epo but do express the receptor (EpoR), which activates signalling cascades (Jak2 and Erk1/2) in response to augmenting doses of Epo in vitro. They suggest that the overexpression of Epo creates an ‘autocrine loop’ in the transduced cells, although increased pro-survival signalling was not confirmed. Nevertheless, compared to sham-transduced MSCs (WT-MSCs), Epo-MSCs were more resistant to apoptosis and produced a greater in vivo angiogenic response in a Matrigel plug assay. In a mouse MI model, injection of Epo-MSCs into the infarct border zone led to improved left ventricular (LV) remodelling, with increased fractional shortening and contractility (dP/dT) compared to WT-MSCs and saline controls. The Epo-MSCs also improved vascularization of the myocardium and reduced neutrophilic infiltration, which may explain some of the LV functional benefits observed. This study is the first to overexpress a gene not normally expressed by MSCs and show benefit in clinically relevant models.

The dual role of Epo in this study as an autocrine and paracrine agent makes this approach very compelling. Delivering Epo locally using cells as a form of ‘mini-pump’ reduces the potential side effects of systemic delivery, although the haematocrit was still increased in the present study for over a week and may have had a role in the observed therapeutic effect (i.e. improved oxygen delivery to peri-infarct zone). Epo may also be mobilizing pro-angiogenic EPCs from the bone marrow, which could contribute to the improved vascularization seen in the Matrigel plug assay and in the myocardium following delivery. It would be interesting to compare this approach to systemic delivery of low-dose Epo to further validate the benefits of local delivery. The autocrine anti-apoptotic effects of Epo may help MSCs survive oxidative stress within the infarct or ischaemic zone, or reverse functional deficit secondary to advanced age or cardiovascular risk factors.31,32 Apart from inhibiting apoptosis, Epo overexpression may generate further autocrine effects, such as an improvement in MSC chemotaxis or the upregulation of angiogenic growth factors, which would further improve their paracrine role in the myocardium. In this way, the autocrine and paracrine roles of Epo may not be mechanistically distinct and, rather, may act to amplify the potential therapeutic functions of MSCs. This represents a novel method of improving persistence of the cells, and possibly improving their ability to stimulate regeneration of the injured tissue.

Genetic manipulation of regenerative cells (i.e. MSCs, EPCs, and multipotent adult progenitor cells) prior to delivery is a powerful technique that could modulate and potentially improve the efficacy of using cells alone. Rescue of cell death secondary to deleterious host factors such as advanced age31 or diabetes32 may be achieved by the overexpression of human telomerase reverse transcriptase in EPCs33 and MSCs.34 Targeting cells to tissues by overexpression of specific adhesion molecules or integrins may also increase their persistence and engraftment, while potentially reducing the number of cells required for therapeutic benefit. Furthermore, overexpression of stem-cell transcription factors in relatively differentiated progenitor cells may represent a method of ‘programming’ to induce a more ‘stem-like’ phenotype, or to unmask multipotent characteristics. Recent reports have demonstrated that even somatic skin fibroblasts can be induced to a pluripotent, embryonic-like, stem-cell phenotype by overexpressing a cocktail of transcriptional and differentiation regulators.35,36 Also, upregulation of Oct4, Rex-1, and Gata-4 expression in human MSCs induced an undifferentiated status with an increased degree of differentiation capacity.37 However, such reprogramming may be a double-edged sword with increased growth and differentiation potential on the one hand and enhanced possibility of malignant transformation on the other.38,39 Thus, identifying ways of more subtly manipulating cells to enhance their regenerative ability, as demonstrated by Copland et al.30 in this issue of Cardiovascular Research, may have more immediate clinical application.

References

1
Abdel-Latif
A
Bolli
R
Tleyjeh
IM
Montori
VM
Perin
EC
Hornung
CA
, et al.  . 
Adult bone marrow-derived cells for cardiac repair: a systematic review and meta-analysis
Arch Intern Med
 , 
2007
, vol. 
167
 (pg. 
989
-
997
)
2
Lipinski
MJ
Biondi-Zoccai
GG
Abbate
A
Khianey
R
Sheiban
I
Bartunek
J
, et al.  . 
Impact of intracoronary cell therapy on left ventricular function in the setting of acute myocardial infarction: a collaborative systematic review and meta-analysis of controlled clinical trials
J Am Coll Cardiol
 , 
2007
, vol. 
50
 (pg. 
1761
-
1767
)
3
Bartsch
T
Brehm
M
Zeus
T
Kogler
G
Wernet
P
Strauer
BE
Transplantation of autologous mononuclear bone marrow stem cells in patients with peripheral arterial disease (the TAM-PAD study)
Clin Res Cardiol
 , 
2007
, vol. 
96
 (pg. 
891
-
899
)
4
Rissanen
TT
Yla-Herttuala
S
Current status of cardiovascular gene therapy
Mol Ther
 , 
2007
, vol. 
15
 (pg. 
1233
-
1247
)
5
Schachinger
V
Erbs
S
Elsasser
A
Haberbosch
W
Hambrecht
R
Holschermann
H
, et al.  . 
Intracoronary bone marrow-derived progenitor cells in acute myocardial infarction
N Engl J Med
 , 
2006
, vol. 
355
 (pg. 
1210
-
1221
)
6
Tang
YL
Zhao
Q
Zhang
YC
Cheng
L
Liu
M
Shi
J
, et al.  . 
Autologous mesenchymal stem cell transplantation induce VEGF and neovascularization in ischemic myocardium
Regul Pept
 , 
2004
, vol. 
117
 (pg. 
3
-
10
)
7
Silva
GV
Litovsky
S
Assad
JA
Sousa
AL
Martin
BJ
Vela
D
, et al.  . 
Mesenchymal stem cells differentiate into an endothelial phenotype, enhance vascular density, and improve heart function in a canine chronic ischemia model
Circulation
 , 
2005
, vol. 
111
 (pg. 
150
-
156
)
8
Iwase
T
Nagaya
N
Fujii
T
Itoh
T
Murakami
S
Matsumoto
T
, et al.  . 
Comparison of angiogenic potency between mesenchymal stem cells and mononuclear cells in a rat model of hindlimb ischemia
Cardiovasc Res
 , 
2005
, vol. 
66
 (pg. 
543
-
551
)
9
Schafer
R
Northoff
H
Cardioprotection and cardiac regeneration by mesenchymal stem cells
Panminerva Med
 , 
2008
, vol. 
50
 (pg. 
31
-
39
)
10
Toma
C
Pittenger
MF
Cahill
KS
Byrne
BJ
Kessler
PD
Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart
Circulation
 , 
2002
, vol. 
105
 (pg. 
93
-
98
)
11
Kinnaird
T
Stabile
E
Burnett
MS
Lee
CW
Barr
S
Fuchs
S
, et al.  . 
Marrow-derived stromal cells express genes encoding a broad spectrum of arteriogenic cytokines and promote in vitro and in vivo arteriogenesis through paracrine mechanisms
Circ Res
 , 
2004
, vol. 
94
 (pg. 
678
-
685
)
12
Iyer
SS
Rojas
M
Anti-inflammatory effects of mesenchymal stem cells: novel concept for future therapies
Expert Opin Biol Ther
 , 
2008
, vol. 
8
 (pg. 
569
-
581
)
13
Li
L
Zhang
S
Zhang
Y
Yu
B
Xu
Y
Guan
Z
Paracrine action mediate the antifibrotic effect of transplanted mesenchymal stem cells in a rat model of global heart failure
Mol Biol Rep
 , 
2008
 
March 27 [Epub ahead of print]
14
Zhang
W
Qin
C
Zhou
ZM
Mesenchymal stem cells modulate immune responses combined with cyclosporine in a rat renal transplantation model
Transplant Proc
 , 
2007
, vol. 
39
 (pg. 
3404
-
3408
)
15
De Kok
IJ
Peter
SJ
Archambault
M
van den Bos
C
Kadiyala
S
Aukhil
I
, et al.  . 
Investigation of allogeneic mesenchymal stem cell-based alveolar bone formation: preliminary findings
Clin Oral Implants Res
 , 
2003
, vol. 
14
 (pg. 
481
-
489
)
16
Taupin
P
OTI-010 Osiris therapeutics/JCR pharmaceuticals
Curr Opin Invest Drugs
 , 
2006
, vol. 
7
 (pg. 
473
-
481
)
17
Schuleri
KH
Boyle
AJ
Hare
JM
Mesenchymal stem cells for cardiac regenerative therapy
Handb Exp Pharmacol
 , 
2007
, vol. 
180
 (pg. 
195
-
218
)
18
Muller-Ehmsen
J
Krausgrill
B
Burst
V
Schenk
K
Neisen
UC
Fries
JW
, et al.  . 
Effective engraftment but poor mid-term persistence of mononuclear and mesenchymal bone marrow cells in acute and chronic rat myocardial infarction
J Mol Cell Cardiol
 , 
2006
, vol. 
41
 (pg. 
876
-
884
)
19
Gao
F
He
T
Wang
H
Yu
S
Yi
D
Liu
W
, et al.  . 
A promising strategy for the treatment of ischemic heart disease: mesenchymal stem cell-mediated vascular endothelial growth factor gene transfer in rats
Can J Cardiol
 , 
2007
, vol. 
23
 (pg. 
891
-
898
)
20
Zhou
WW
Hu
JG
Yang
JF
Lin
L
Zhou
XM
Tang
T
Angiogenic effect of bone marrow mesenchymal stem cells transfected with human VEGF gene on myocardial infarcts in rats
Zhong Nan Da Xue Xue Bao Yi Xue Ban
 , 
2006
, vol. 
31
 (pg. 
763
-
766
771
21
Wang
Y
Haider
HK
Ahmad
N
Xu
M
Ge
R
Ashraf
M
Combining pharmacological mobilization with intramyocardial delivery of bone marrow cells over-expressing VEGF is more effective for cardiac repair
J Mol Cell Cardiol
 , 
2006
, vol. 
40
 (pg. 
736
-
745
)
22
Mangi
AA
Noiseux
N
Kong
D
He
H
Rezvani
M
Ingwall
JS
, et al.  . 
Mesenchymal stem cells modified with Akt prevent remodeling and restore performance of infarcted hearts
Nat Med
 , 
2003
, vol. 
9
 (pg. 
1195
-
1201
)
23
Burger
D
Lei
M
Geoghegan-Morphet
N
Lu
X
Xenocostas
A
Feng
Q
Erythropoietin protects cardiomyocytes from apoptosis via up-regulation of endothelial nitric oxide synthase
Cardiovasc Res
 , 
2006
, vol. 
72
 (pg. 
51
-
59
)
24
von Lindern
M
Parren-van Amelsvoort
M
van Dijk
T
Deiner
E
van den Akker
E
van Emst-de Vries
S
, et al.  . 
Protein kinase C alpha controls erythropoietin receptor signaling
J Biol Chem
 , 
2000
, vol. 
275
 (pg. 
34719
-
34727
)
25
Mudalagiri
NR
Mocanu
MM
Di Salvo
C
Kolvekar
S
Hayward
M
Yap
J
, et al.  . 
Erythropoietin protects the human myocardium against hypoxia/reoxygenation injury via phosphatidylinositol-3 kinase and ERK1/2 activation
Br J Pharmacol
 , 
2008
, vol. 
153
 (pg. 
50
-
56
)
26
Lipsic
E
Westenbrink
BD
van der Meer
P
van der Harst
P
Voors
AA
van Veldhuisen
DJ
, et al.  . 
Low-dose erythropoietin improves cardiac function in experimental heart failure without increasing haematocrit
Eur J Heart Fail
 , 
2008
, vol. 
10
 (pg. 
22
-
29
)
27
Westenbrink
BD
Voors
AA
Ruifrok
WP
van Gilst
WH
van Veldhuisen
DJ
Therapeutic potential of erythropoietin in cardiovascular disease: erythropoiesis and beyond
Curr Heart Fail Rep
 , 
2007
, vol. 
4
 (pg. 
127
-
133
)
28
Westenbrink
BD
Lipsic
E
van der Meer
P
van der Harst
P
Oeseburg
H
Du Marchie Sarvaas
GJ
, et al.  . 
Erythropoietin improves cardiac function through endothelial progenitor cell and vascular endothelial growth factor mediated neovascularization
Eur Heart J
 , 
2007
, vol. 
28
 (pg. 
2018
-
2027
)
29
Zhang
D
Zhang
F
Zhang
Y
Gao
X
Li
C
Ma
W
, et al.  . 
Erythropoietin enhances the angiogenic potency of autologous bone marrow stromal cells in a rat model of myocardial infarction
Cardiology
 , 
2007
, vol. 
108
 (pg. 
228
-
236
)
30
Copland
IB
Jolicoeur
EM
Gillis
M-A
Cuerquis
J
Eliopoulos
N
Annabi
B
, et al.  . 
Coupling erythropoietin secretion to mesenchymal stromal cells enhances their regenerative properties
Cardiovasc Res
 , 
2008
, vol. 
79
 (pg. 
405
-
415
)
31
Roobrouck
VD
Ulloa-Montoya
F
Verfaillie
CM
Self-renewal and differentiation capacity of young and aged stem cells
Exp Cell Res
 , 
2008
, vol. 
314
 (pg. 
1937
-
1944
)
32
Li
YM
Schilling
T
Benisch
P
Zeck
S
Meissner-Weigl
J
Schneider
D
, et al.  . 
Effects of high glucose on mesenchymal stem cell proliferation and differentiation
Biochem Biophys Res Commun
 , 
2007
, vol. 
363
 (pg. 
209
-
215
)
33
Murasawa
S
Llevadot
J
Silver
M
Isner
JM
Losordo
DW
Asahara
T
Constitutive human telomerase reverse transcriptase expression enhances regenerative properties of endothelial progenitor cells
Circulation
 , 
2002
, vol. 
106
 (pg. 
1133
-
1139
)
34
Kobune
M
Kawano
Y
Ito
Y
Chiba
H
Nakamura
K
Tsuda
H
, et al.  . 
Telomerized human multipotent mesenchymal cells can differentiate into hematopoietic and cobblestone area-supporting cells
Exp Hematol
 , 
2003
, vol. 
31
 (pg. 
715
-
722
)
35
Rajasingh
J
Lambers
E
Hamada
H
Bord
E
Thorne
T
Goukassian
I
, et al.  . 
Cell-free embryonic stem cell extract-mediated derivation of multipotent stem cells from NIH3T3 fibroblasts for functional and anatomical ischemic tissue repair
Circ Res
 , 
2008
36
Stadtfeld
M
Maherali
N
Breault
DT
Hochedlinger
K
Defining molecular cornerstones during fibroblast to iPS cell reprogramming in mouse
Cell Stem Cell
 , 
2008
, vol. 
2
 (pg. 
230
-
240
)
37
Roche
S
Richard
MJ
Favrot
MC
Oct-4, Rex-1, and Gata-4 expression in human MSC increase the differentiation efficiency but not hTERT expression
J Cell Biochem
 , 
2007
, vol. 
101
 (pg. 
271
-
280
)
38
Shima
Y
Okamoto
T
Aoyama
T
Yasura
K
Ishibe
T
Nishijo
K
, et al.  . 
In vitro transformation of mesenchymal stem cells by oncogenic H-rasVal12
Biochem Biophys Res Commun
 , 
2007
, vol. 
353
 (pg. 
60
-
66
)
39
Rubio
D
Garcia
S
De la Cueva
T
Paz
MF
Lloyd
AC
Bernad
A
, et al.  . 
Human mesenchymal stem cell transformation is associated with a mesenchymal–epithelial transition
Exp Cell Res
 , 
2008
, vol. 
314
 (pg. 
691
-
698
)

Author notes

The opinions expressed in this article are not necessarily those of the Editors of Cardiovascular Research or of the European Society of Cardiology.