Coronary (CAD) and peripheral (PAD) artery disease are major causes of morbidity and mortality, requiring bypass surgery or angioplasty in approximately one million patients/year in the world (MERIT-HF Study Group, 1999). While collateral vessel formation as an alternative pathway for blood supply occurs in some of these patients, many do not form vascular networks adequate to compensate for the loss of the original blood supply (Hirsch et al., 2006). These patients might therefore benefit from stem cell transplantation therapies that would accelerate natural processes of postnatal collateral vessel formation, an approach referred to as therapeutic angiogenesis. On the other hand, recent seminal reports have indicated that the adult heart is self-healing and self-renewing. Specifically, these studies have demonstrated that there is a pool of resident cardiac stem cells (CSCs) that are clonogenic and multipotent and are capable of differentiating into new blood vessels or into new myocytes, and of cardiac progenitor cells (CPCs) (Marban, 2007). This suggests the possibility of using a therapeutic angiogenesis approach to complement other treatments (e.g., stem cell therapy) that facilitate myocardial repair. Such combined modalities may facilitate myocardial regeneration by inducing endogenous cardiac cells to migrate, differentiate, and proliferate
2. Stem cell basics and selection
Stem cells are a population of immature tissue precursor cells capable of self-renewal as well as of differentiation into a spectrum of different cell types in appropriate conditions. In general, they share the following characteristics: (1) a high capacity for self-renewal; (2) the potential for differentiation in multiple cell types; (3) the ability to be cultured
Selection of a suitable type of stem cells is a key issue for the success of stem cell therapy. Stem/progenitor cells used for transplantation should have the following characteristics: (1) high rates of survival and proliferation; (2) high capability of differentiation; and (3) the potential for engraftment and integration with native or host cardiac cells. Currently, both embryonic and adult stem cells are used in experimental cardiac cell transplantation studies, while only adult stem cells (e.g., bone marrow-derived mesenchymal cells, skeletal myoblasts, endothelial progenitor cells) are used in clinical trials. Each stem cell type has unique biological properties that offer both advantages and limitations to their use. Therefore, selection of the most suitable stem cells for use in ischemic patients is still a major focus of current research. The skeletal myoblasts or satellite cells are precursor cells of human skeletal muscle that originate from muscle stem cells (Angelis et al., 1999). They normally lie in a quiescent state under the basal membrane of muscular fibers, and have the potential for reentry into the cell cycle in response to injury, where they can divide and differentiate into functional muscle cells. Skeletal myoblasts can be obtained from individual patients themselves. Other theoretical advantages of using autologous skeletal myoblast are their rapid expansion in culture and their lower likelihood of tumor formation after transplantation. In addition, these cells have a chance of engrafting with native cardiomyocytes and surviving in infarcted regions of the heart since they are relatively resistant to ischemia. Animal studies have demonstrated that skeletal myoblasts can successfully accommodate themselves in the infarcted region of the heart, forming striated muscle fibers with intercalated discs in the host myocardium under the influence of factors in the cardiac environment (Murry et al., 1996). Improvement in systolic function has been noted after skeletal myoblast transplantation in ischemic and non-ischemic heart failure models (Atkins et al., 1999; Hagegeet al., 2001; Siminiak & Kurpisz, 2003). Controversy still exists regarding the capability of stem cells to engraft and connect with native cardiomyocytes, despite promising results from preclinical studies (Suzuki et al., 2001; Menasche et al., 2003; Pagani et al., 2003; Smits et al., 2003). Bone marrow-derived stem cells are currently the most commonly used cells in cell transplantation therapy. The ideal stem cells from bone marrow for cardiac regeneration remain to be identified and many details remain to be elucidated. Yet, the clinical results from recent trials show the capability of these cells to ameliorate systo-diastolic heart function and decrease the size of the infarct region after intracoronary injection (Assmus et al., 2002; Strauer et al., 2002; Britten et al., 2003). Other studies (Perin et al., 2002; Perin et al., 2003) have demonstrated the safety and efficacy of transendocardial injection of autologous bone marrow mononuclear cells in patients with end-stage ischemic heart disease. Fetal cardiomyocytes still can enter the cell cycle and be expanded in culture. Successful cell transplantation using fetal cardiomyocytes was initially demonstrated in mice (Soonpaa et al., 1994; Li et al., 1995; Li et al., 1996), with findings in improvement of heart function and formation of new blood vessels in and around the cell graft area (Watanabe et al., 1998). These experiments show that fetal cardiomyocyte transplantation is feasible and potentially clinically relevant. Besides the ethical questions concerning the use of human fetal tissue, however, one limitation of using fetal cardiomyocytes is that lifelong immunosuppressive therapy may be necessary to prevent rejection. The optimal regimen and dose of immunosuppressive agents for cell transplantation would be here still unknown. A large number of studies have examined the differentiation of embryonic stem cells into cardiomyocytes, aiming at clarifying the mechanism of differentiation, identifying cell markers, and developing techniques for purifying embryonic stem cell-derived cardiomyocytes. The pluripotency of embryonic stem cells gives rise to their differentiation into more than 200 hundred kinds of cell lines. Protocols for the
Current experiments suggest that EPCs play an important role in vasculogenesis by differentiating into vascular endothelial cells, inhibiting ventricular remodeling through improvement in myocardial blood supply (Kamihata et al., 2001). Adipose tissue-derived stem cells may overcome major limitations in the use of adult stem cells harvested from essential organs such as muscle, skin, brain. liver and bone marrow. It has been shown that these cells can be induced to differentiate into multiple cell lineages, including adipose, cartilage and bone, muscle cells, neurons and endothelial cells (Zuk et al., 2001; Madonna et al., 2009). Injection of adipose tissue-derived stromal cells (ADSCs) has been recently shown to improve neovascularisation in the ischemic hind limb and the infarcted heart (see Madonna et al., 2009 for a general review of preclinical studies in the heart and hind limb ischemia models). Furthermore, recent studies (Puissant et al., 2005) have reported on the
3. Growth factor selection and vehicle-based delivery approach
Vehicle-based delivery systems for growth factors, derived from diverse biomaterials, are used to increase their retention at treatment sites for a sufficient period of time to allow tissue regenerating cells to migrate into the area of injury, to proliferate and to differentiate, as well as to reduce the loss of bioactivity. They are also used to control toxicity induced by high concentrations of growth factors (Vasita & Katti, 2006; Chou & Leong, 2007). A major challenge inherent in these strategies is to identify growth factors and signaling pathways that selectively promote proliferation, migration, engraftment, and differentiation of resident CSCs or exogenous multipotent stem cells. This challenge relates also to the understanding of cellular uptake mechanisms, cell response to the mechanochemical microenvironment, the potential therapeutic utility of delivered biomolecules, and the exact requirements for multiple signals to drive the ischemic cardiovascular tissue regeneration process to completion. Current knowledge suggests that ‘‘cocktails’’ of biomolecules, or even cocktails of different types of stem cells, should be delivered locally, with specific and distinct pharmacokinetics/pharmacodynamics (e.g., the capacity of each single component of the cocktail to act distinctly in differentiating or in homing different endogenous stem cell/progenitor population), in order to mimic, as far as possible, the different requirements of the ischemic tissue during the various regeneration phases. Engineering natural and/or synthetic scaffolds to release several growth factors (or growth factor-encoding genes) in a sequential manner might affect more than one phase of the ischemic tissue healing process, for example, neovascularization and myogenesis, ultimately leading to cardiovascular tissue regeneration rather than repair. The growth factors that have been most intensively investigated in the regeneration of ischemic cardiovascular tissues include vascular endothelial growth factor (VEGF), transforming growth factor (TGF)-β, platelet-derived growth factors (PDGF, including PDGF-BB and PDGF-AA), insulin-like growth factor (IGF)-1, basic fibroblast growth factor (b-FGF) (Beohar et al., 2010). Overall, results indicate that these growth factors elicit significant proliferative and angiogenic effects. It is now well established that the utilization of multiple growth factors, rather than one, capable to act in a concentration- and time-dependent manner, is essential in the processes involved in the regeneration of ischemic cardiovascular tissues (Chen et al., 2010). An example is given by the need of having VEGF and PDGF together in order to promote angiogenesis. Indeed, VEGF is an initiator of angiogenesis, while PDGF promotes blood vessel maturation. As an example for this, a polymer scaffold constructed from Poly (Lactide-co Glycolide) (PLG), capable of delivering VEGF and PDGF together, with better results in terms of formation of mature vessels compared with the delivery of VEGF or PDGF singularly, has been produced (Richardson et al., 2001). In the selection of growth factors potentially capable to boost the regeneration of ischemic cardiovascular tissue, we and others have investigated on hepatocyte growth factor (HGF) and its receptor receptor mesenchymal-epithelial transition factor (Met). Ligand-receptor systems such HGF and its receptor, the tyrosine kinase Met, are potential candidates for therapeutic angiogenesis and for boosting migration, engraftment and commitment of CSCs because they promote the translocation of CSCs into the injured area, activate their growth and differentiation, and stimulate endothelial cell migration (Rappolee et al., 1996; Forte et al., 2006; Madonna et al., 2010). The strategy of combining stem cells, either native or gene-engineered to overexpress growth factors, with biopolymers that are functionalized with growth factors such as HGF, would facilitate myocardial regeneration: a) by supplying exogenous stem cells or GFs that stimulate resident CSC migration, engraftment and commitment to cardiomyocytes, and that induce and modulate arterial responses to ischemia; b) by supporting the maintenance of GFs and transplanted stem cells in the damaged tissues through the use of biocompatible and biodegradable polymers for a period of time sufficient to allow histological and anatomical restoration of the damaged tissue. These polymers can provide vehicles to deliver bioactive factors and stem cells into the infarcted heart or ischemic cardiovascular tissues. Finally, this approach would promote the ability of resident CSCs or of exogenous multipotent stem cells, such as adipose tissue-derived mesenchymal stem cells (AT-MSCs), to induce the healing of damaged tissue, by recruiting and directing these cells into the damaged area, by improving angiogenesis and, finally, by promoting the reperfusion of ischemic tissues.
4. Growth factor-delivery systems and devices in the treatment of tissue ischemia
Many limitations of stem cell therapies could be resolved by stimulating specific cellular functions for cell populations that normally are quiescent in the adult heart or that are not capable of replacing the dying cells. Compounds (cytokines and growth factors) that are simply injected into the lesions quickly disappear from the site of injection because they are removed by the blood flow and degraded by specific enzymes located in the extracellular microenvironment. To overcome this drawback, a possible strategy is to install a polymer functionalized with growth factors and stem cells into the damaged heart to stimulate the natural process of cardiac repair. Polymers tested in the past for their ability to support transplanted cells, without any conjugation with functional molecules, have been marginally effective. More novel polymers are conjugated with functional molecules (growth factors, chemotactic factors, cytokines), and are capable of stimulating specific normally quiescent cellular functions (Tatard et al., 2005). They are known as “smart” polymers because they “persuade” and guide the regenerative process, and are “biomimetics” because they use strategies occurring during the physiological regenerative process. Unlike the previous ones, novel polymers are “smart”, in that they can acquire several biological functions depending on the bioactive factor or stem cell type to which they are conjugated. These polymers can prolong and amplify specific stem cell functions. They can stimulate the recruitment of circulating and resident stem cells and subsequently promote their adhesion to the damaged area, and can enhance survival, proliferation, and differentiation of stem cells into cardiac and vascular cells (Madonna & De Caterina, 2009). Several of these features are critical to tissue regeneration, including restoration of the delivery of factors, nutrients, oxygen, and blood to necrotic tissues. To accomplish this, polymers must be functionalized by conjugation with bioactive factors. To date, delivery systems of growth factors are basically classified as: 1) reservoir systems; 2) environmentally responsive systems. Reservoir systems are one of the oldest methods used successfully to deliver drugs. The primary drug release mechanism from reservoir systems is diffusion-controlled release, characterized by an initial ‘‘burst release’’ phase followed by a phase of slower drug release from the carrier (Langer, 1983). Examples are hydrophilic matrices, that degrade when water enters and in this way release the drug (Franssen et al., 1999). Several reservoir systems have been developed to more closely control the release kinetics and avoid ‘‘burst type’’ release of the encapsulated factor(s). For example, the
Environmentally responsive systems are able to match a patient’s physiological needs at the appropriate time and/or the correct site. They are able to deliver a certain amount of growth factor(s) in response to a biological state (Qiu & Park, 2001). They are constituted by sensitive hydrogels that can control the release of drugs by changing the gel structure according to environmental stimulation, such as temperature, pH, and/or ion concentration. Temperature-sensitive hydrogels are able to swell or shrink as the temperature of the surrounding fluid varies (Ramanan et al., 2006). The poly-N-isopropylacrylamide (PNIPAAm) hydrogel is a typical example of temperature-sensitive hydrogel, featuring sol-to-gel transition at a critical solution temperature of about 35 °C. This polymer releases the drug with the transition from gel to sol (Zhang et al., 2004), and is of particular interest in those clinical situations, such as tissue ischemia, characterized by low tissue temperature. Similarly, pH-sensitive polymers contain pendant acidic (e.g., carboxylic and sulphonic acids) or basic (e.g., ammonium salts) groups that either accept or release protons in response to changes in environmental pH (Qiu & Park, 2001). Such polymers can release a drug when the environmental pH decrease. These acidic pH-sensitive polymers may be useful for the treatment of tissue ischemia and inflammation (Matsusaki & Akashi, 2005). A very recent work has also shown the capability of new magnetic particles embedded in polymer gels, termed ferrogels, to release drugs in response to magnetic fields (Zhao et al., 2011). Here the authors created alginate-based porous scaffolds containing the arginine-glycine-aspartic acid amino acid sequence, covalently coupled with the alginate and embedded with 10 nm iron oxide particles. Under applied magnetic fields this superparamagnetic gel undergoes prompt deformation, causing water flow through the interconnected pores, thus triggering the release of biological agents. The authors here showed the capability of these ferrogels to promptly release several drugs, including mitoxantrone, plasmid DNA, chemokines, as well as cells under the control of external magnetic fields
5. The gene delivery approach
Beside the incorporation into a carrier vehicle, different approaches and strategies for direct delivery of growth factors have been employed, including the delivery of growth factor genes. In order to mimic the natural healing process of the tissue successfully, this strategy mostly requires a localized application of multiple, rather than one, genes that encode for and activate the synthesis of sequential multiple growth factors with synergistic effects on tissue regeneration (De Laporte et al., 2009; Donofrio et al., 2010; Fujii et al., 2011; Liu et al., 2011). However, low transfection efficiencies, inefficient gene targeting, low gene expression levels, and undesired gene integration into host DNA are all challenges that may undermine growth factor gene delivery as a better approach instead of the growth factor protein delivery (Juillerat-Jeanneret & Schmitt, 2007). Gene delivery can be performed either by directly introducing the delivery vector into the anatomical site (
Although traditional delivery of cells associated with growth factors is still a candidate strategy in laboratory-based trials, the most frequently investigated cell transplantation in tissue engineering to date is cell-based gene therapy. This therapy typically relies on transplanting cells, such as stem cells, lymphocytes, fibroblasts, or – alternatively – the cells of interest, that are removed from the body and injected after therapeutic transgene modifications (Fischer et al., 2009; Cho & Marban, 2010; Madonna et al., 2010). This
6. Lentiviral and non-lentiviral vectors for gene delivery into stem cells
The introduction of growth factor genes in stem cells can be performed by using viral or non-viral vectors. In the choice of using viral vectors, important experimental variables for a successful gene therapy include the multiplicity of infection (MOI), time length for viral incubation and medium used for viral incubation. An optimal combination of such experimental conditions would increase gene transfer efficiency and possibly obviate the need for selective antibiotic-based enrichment and long-term culture, which may contribute to senescence or compromise the long-term engraftment efficiency and/or multipotency of grafted cells (Rombouts & Ploemacher, 2003). In addition, by increasing gene transfer efficiency, fewer cells may be required to achieve a therapeutic effect. This justifies the use of lentiviral vectors for transducing adult stem cells, by virtue of their ability to transduce both dividing and non-dividing cells and their relative ease of use and comparable nature to adeno-associated viral (AAV) vectors, which are clinically preferred. For the transduction of adult stem cells, lentivirus-based systems are virtually ideal, since they overcome most problems, including the short duration of gene expression and the occurrence of significant inflammatory responses, which plague other types of gene vectors (such as adenoviruses). Lentiviruses are a subgroup of retroviruses that include the human type 1 immunodeficiency virus (HIV). While retroviral systems are inefficient in transducing non-dividing or slowly dividing cells, lentivirus-based vectors, after being pseudotyped with vesicular stomatitis virus glycoprotein G (VSV-G) (i.e., using the glycoprotein envelope from the vesicular stomatitis virus to package recombinant retroviruses) (Emi et al., 1991), can mediate genome integration into both non-dividing and dividing cells (Fig. 2). There is evidence that lentiviral vectors can also transduce more primitive, quiescent progenitors with stable transgene integration (Case et al., 1999). In comparison with other retroviral vectors, lentiviral systems allow the immediate transduction without prior expansion, or with growth factor stimulation for only short exposure times. Compared with adenoviral vectors, lentiviral vectors also offer the major advantages of causing little or no disruption of the target cells and of not promoting any inflammatory response (Lever, 1996). AAV vectors represent an alternate type of vector that may also be used for long-term transgene expression in the heart through cell-based therapy (Svensson et al., 1999). Like lentiviruses, AAV can stably integrate into the host genome providing long-term transgene expression, with a minimum inflammatory response. However, AAV can cause insertional mutagenesis and can only carry genes which are less than 5 kb (Donsante et al., 2007). A possible drawback of the use of lentiviral and AAV vectors for delivering genes that encode for growth factors might be that they can cause a chronic overexpression of the protein, with an uncertain therapeutic effect. Short-term gene expression of the growth factor gene would be desirable if the goal is to deliver a secreted protein, such as insulin-like growth factor-1 (IGF-1), vascular endothelial growth factor (VEGF) and HGF, while long-term expression would be preferable if the goal is to express membrane proteins such as receptors for growth factors that require stable expression. Possible strategies to induce short-term gene expression of the transgene include plasmid transfection or the use of adenoviral vectors (Rabbany et al., 2009). Limitations of these strategies are the low transfection efficiency with plasmids and the immunogenic response of the host with adenoviruses.
Growth factor delivery and tissue engineering have emerged as new concepts that focus on tissue regeneration from cells with the support of biomaterials and growth factors (Ikada, 2006). Various delivery methods are available to administer growth factors to ischemic tissues. Among these strategies, the cell-based delivery of growth factor genes is of great potential interest, since the
This work was supported by grants from the Italian Ministry of University and Scientific Research and from the Istituto Nazionale Ricerche Cardiovascolari (INRC).
The Authors declare no conflict of interest as related to the topics here discussed.