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.