Angiogenesis and myogenesis using human skeletal myoblasts for cardiac repair 3

The present study demonstrated the safety and effectiveness of human skeletal myoblast transplantation carrying hVEGF165 and Ang-1 alone or together in xenograft heart models of chronic

Chapter 4 Discussion

The present study demonstrated the safety and effectiveness of human skeletal myoblast transplantation carrying hVEGF165 and Ang-1 alone or together in xenograft heart models of chronic infarction Genetically modulated skeletal myoblasts successfully survived in rat and pig hearts to achieve concurrent myogenesis and angiogenesis for cardiac repair Skeletal myoblasts served as a vehicle for localized delivery and transient expression of hVEGF165 and Ang-1 in the infarcted myocardium

4.1 High purity of human skeletal myoblasts

The purity of donor cells is imperative for successful engraftment of the cells Contaminant populations of cells present in the donor cell preparation could initiate an immunological response from the host and thus jeopardize the survival of the donor cells

at the site of graft Both desmin and CD56 expression have been extensively used as reliable markers for identification of skeletal myoblasts (Menasche et al., 2003; Smits et al., 2003; Haider et al., 2004b) Desmin, a muscle specific intermediate filament protein,

is expressed in all muscle types (Lazarides et al., 1982) It is one of the earliest known myogenic markers (Choi et al., 1990) and one of the first muscle specific proteins to appear during mammalian embryonic development (Hill et al., 1986; Furst et al., 1989; Schaart et al., 1989; Mayo et al., 1992) In contrast to all known muscle-specific genes, desmin is expressed in satellite cells (Allen et al., 1991) and in replicating myoblasts (Kaufman et al., 1988)

CD56 is expressed in approximately 15% of peripheral blood lymphocytes that are mainly composed of natural killer cells and CD3+ T lymphocytes and certain neurons, but not fibroblast (Lanier et al., 1986) It is also an isoform of neural cell adhesion molecules that is constitutively expressed in satellite cells and proliferating

myoblasts However, it is absent in mature muscle (Illa et al., 1992; Belles-Isles et al., 1993)

High purity skeletal myoblast cultures were used in the present study The purity

of the donor myoblasts was demonstrated by fluorescent immunostaining and cytofluorimetry for desmin and CD56 expression (Figure 2 & 3) More than 95% of skeletal myoblasts were positive for desmin and 92% of them expressed CD56 as revealed by cytofluorimetric analysis (Figure 3) About 91% of skeletal myoblasts co-expressed desmin and CD56 (Figure 3) This high purity of the donor myoblasts helped

to exclude the influence of undesired populations of cells and provided an opportunity to explore the fate of skeletal myoblast in the heart A mixture of skeletal myoblasts with other cell types may prove inaccurate in assessing the fate of differentiated skeletal myoblasts in heart after transplantation

Conflicting data regarding the differentiation potential of skeletal myoblasts after transplantation have been published and may be related to the purity of the skeletal myoblast population It has been shown that muscle derived side population of stem cells can develop into hematopoietic progenitor cells (CD45+) instead of myocytes in vitro (Asakura et al., 2002), while the hematopoietic progenitor cells have the potential to differentiate into cardiomyocytes (Jackson et al., 2001, Eisenberg et al., 2003)

We further demonstrated that retroviral and adenoviral vector transduction did not change the phenotype of skeletal myoblasts based on desmin and CD 56 expression (Figure 4 & 5) These results suggest that multiple transduction procedure is well tolerated by skeletal myoblasts The viral vectors at optimum transduction concentration have no ill effects on skeletal myoblasts

4.2 Transduction of skeletal myoblasts with angiogenic genes

Using the Ad-vector as a carrier of angiogenic genes, high transduction and expression efficiencies were achieved with skeletal myoblasts Under optimum transduction conditions, transduced myoblasts expressed biologically active trans-angiogenic proteins at least 30 days in vitro

Considering the capacity for high-titer production, large insert capacity and relatively low pathogenicity as well as the ability to efficiently infect non-dividing cells (Benihoud et al., 1999; Wang et al., 2000a), Ad-vector was chosen to transfer angiogenic genes for therapeutic angiogenesis in this study The replication incompetent Ad-vectors efficiently transduced skeletal myoblasts The maximal transduction efficiency was achieved at a ratio of 1000 PFU viral vector particles: one skeletal myoblast when transduction was carried out for 8 hours exposure in triplicate ELISA and RT-PCR analysis showed that transduced myoblasts were able to express angiogenic protein at least 30 days in vitro The expression of the exogenous angiogenic genes was switched off in the animal heart at around 4 weeks and 6 weeks after cell transplantation in rat heart (Figure 26) and pig heart (Figure 51 & 52) as revealed by heart tissue RT-PCR for hVEGF165 and/or Ang-1 expression The time duration of the transient expression of the angiogenic genes from the donor cells was found sufficient enough to trigger the neovascularization cascade to set in at the site of the cell graft Such an approach of angiogenic growth factor delivery avoided the possible side effects of angiogenic gene therapy associated with the systemic administration of angiogenic growth factors (Hariawala et al., 1996; Lazarous et al., 1996) Previous studies have also reported

expression of the exogenous gene ranging from 1-2 weeks in immuno-competent hosts (Yang et al., 1994) for up to 30 days using hemagglutinating virus of Japan-liposome (Suzuki et al., 2001a)

Although Ad-vectors cause inflammatory reactions, formation of anti-adenoviral antibody, transient fever and increase of liver transaminase in human trials, they have not been linked to any human malignancies so far (Hehir et al., 1996; Kay et al., 2001; Yla-Herttuala et al., 2003) Other viral vectors including retroviral and adeno-associated vectors have also been used for therapeutic angiogenesis (Lee et al, 2000b; Su et al., 2000; Arsic et al., 2003) However, the angiogenic genes transferred by these vectors get integrated into the host genome thus causing long-term transgenic expression (Yla-Herttuala et al., 2000; McTaggart et al., 2002) The long-lasting expression of potent angiogenic growth factors may cause deleterious effects including tumor formation (Lee

et al., 2000b; Schwarz et al., 2000; Masaki et al., 2002; Lutsenko et al., 2003) The use of regulatable promoter system in designing the gene delivery vectors and the use of a non-viral system may help to overcome these problems of unnecessary over-expression of the donor genes (Pachori et al., 2004; Tang et al., 2004) Ozawa et al., (2004) demonstrated that VEGF at 64- 70 ng/ ml (1x106 cell/ day) concentration was the threshold to avoid hemangioma development The safe level of hVEGF165 in this study prevented the hemangioma formation as no hemangioma was observed in any experimental animal

4.3 Biological activity of growth factors secreted from transduced myoblasts

Immunostaining, RT-PCR and Western blot analysis demonstrated that genetically modified skeletal myoblasts efficiently expressed and secreted hVEGF165 and Ang-1 The molecular weights of hVEGF165 and Ang-1 were 42 kDa and 70 kDa

respectively hVEGF165 and Ang-1 were not only actively secreted from transduced myoblasts, they also had biological activity as demonstrated by HUVEC proliferation and thymidine [H3] incorporation assays (Figure 11 & 20)

hVEGF165 secreted from hVEGF165-myoblasts stimulated HUVEC to propagate significantly faster as compared with the control supernatant collected from the non-transduced myoblasts (Figure 11a) The thymidine [H3] incorporation assay further confirmed this (Figure 11b) This effect of hVEGF165 was antagonized by presence of anti-VEGF165 antibody

When hVEGF165 was combined with Ang-1 using Ad-Bic transduced myoblasts, highest rate of proliferation of HUVEC was observed (Figure 20) This was mainly due

to the biological effect of hVEGF165 as the proliferation of HUVEC was totally blocked when anti-VEGF165 antibody alone was added (Figure 20), while the proliferation of HUVEC was still observed when anti-Ang-1 antibody alone was added

The HUVEC proliferation rate was higher with Bic-myoblast supernatant as compared with supernatant containing hVEGF165 alone (when Bic-myoblast supernatant was supplemented with anti-Ang-1 antibody) A synergistic effect of hVEGF165 with Ang-1 on HUVEC proliferation was observed (Figure 20) It is well known that VEGF165 can stimulate the proliferation of endothelial cells, while Ang-1 does not have this biological function in vitro (Koh et al., 2002) The unexpected faster proliferation rate responding to simultaneous hVEGF165 and Ang-1 activity is noteworthy It has been documented that the endothelial cell proliferate stimulated by VEGF is resulted from NO production (Gerber et al., 1998) Increased NO production has been shown to directly result in endothelial cell proliferation (Ziche et al., 1993) More recently, it has been

demonstrated that angiogenic actions of Ang-1 requires endothelium derived NO (Babaei

et al., 2003) Thus, it is possible that Ang-1, through NO production, influences the VEGF mediated signal pathway and leads to faster endothelial cell proliferation The actual mechanism, however, needs further investigation

As is apparent from our data, the transduced skeletal myoblasts efficiently secreted biologically active angiogenic factors in vitro To assess the efficiency of these genetically modified skeletal myoblasts for cardiac repair, they were labeled with the

nLac-z reporter gene or BrdU, and transplanted into rat and pig heart models

4.4 Skeletal myoblast labeled with nLac-z reporter gene or BrdU

Skeletal myoblasts successfully survived in rat and pig heart models for up to 6

and 12 weeks respectively as demonstrated by histochemical staining for nLac-z

expression in the recipient hearts The retroviral vector transduced skeletal myoblasts

expressed nLac-z for up to 7 months in pig heart (Haider et al., 2003) The longer term expression of nLac-z reporter gene allowed us to identify the fate of donor skeletal

myoblasts after transplantation The survival of skeletal myoblasts was further confirmed

by immunostaining for BrdU These suggested successful xeno-transplantation of human skeletal myoblasts in rat and pig heart

4.5 Survival of human skeletal myoblasts in rat and pig heart

The present study has shown successful survival of the donor xenomyoblasts in

both rat and pig heart models as revealed by nLac-z and BrdU expression in the recipient

heart (Figure 24 & 34) This was further confirmed by the presence of the human chromosome in rat and pig myocardium (Figure 26 & 37)

Y-Rapid and extensive death of donor skeletal myoblasts after transplantation is one

of the limiting factors in the success of this approach in the clinical setting The problem

is further accentuated when the donor myoblasts are taken from sources other than autologous More than 99% of the donor cells have been reported to die within 48 hours post transplantation (Fan et al., 1996) The rapid and massive initial death phase is multi-factorial and has been attributed to mechanical injury, tissue ischemia at the site of the graft, contaminant population of the donor myoblast preparation, host inflammatory and immune response together with host complement mediated lysis of the donor cells (Guerrette et al., 1995; Guerrette et al., 1997; Merly et al., 1998) Thus, skeletal myoblasts were transplanted at 10 days (rat) and 3 weeks (pig) after creating animal model This may help transplanted myoblasts escape the attacks from infiltrating inflammatory cells that response to the cryoinjury or infarction

Various strategies have been adopted to overcome this problem, including the use

of immunosuppression (Cosenza et al., 1993; Wu et al., 2000) One of the unique features

of this study is the use of transient immunosuppression for the successful survival of human skeletal myoblasts as xenografts in rat and pig models of myocardial infarction (Haider et al., 2003) The animals were maintained on a minimal dose of 5 mg/ kg Cyc-

A, starting 5 days prior to and until 6 weeks post myoblast transplantation The introduction of Cyc-A alone or in combination with other agents has resulted in excellent donor cell survival (Cosenza et al., 1993; Wu et al., 2000) Our results suggested that transient immunosuppression achieved long-term survival of donor myoblast for up to 3 months of observation In another study from our laboratory, we have observed the survival of xenomyoblasts for until 7 months using transient immunosuppression (Haider

et al., 2003) Cyc-A, a cyclic undecapeptide immunosuppressive agent, is widely used for the prevention of graft-versus-host disease, treatment of allograft rejection and certain auto-immune disorders (Faulds et al., 1993; Opelz et al., 2001) It suppresses T cell mediated immune responses by inhibiting cytokine expression in activated T cells, especially IL-1, IL-2, IL-4, and CD40 (Yoshimura et al., 1994; Rao et al., 1997) It can moderately induce apoptosis in cycling and proliferating lymphoid cells (Strauss et al., 2002) Thus, through the suppression of activated T cell and T cell mediated immune responses, Cyc-A helped human skeletal myoblasts escape immune rejection by the host animals

We observed no infiltration of CD4+ or CD8+ T lymphocytes, monocytes, and granulocytes at the site of the cell graft in the host myocardium (Figure 46 & 47) The lack of host immune cell infiltration suggested that Cyc-A efficiently suppressed the host inflammatory and immune response The successful transient suppression of the host immune system before cell transplantation provided a grace period of grace for the donor myoblasts to acclimatize to the host tissue environment Cytofluorimetric analysis further demonstrated that low level anti-human skeletal myoblast antibodies were produced at 1 week after myoblast transplantation (Figure 48) However, the level was very low and totally disappeared at 2 weeks after myoblast transplantation The low-level antibody concentration might have resulted from the non-specific inflammatory reaction towards the dead skeletal myoblast in the early phase due to anoxic conditions or to a contaminant population of in the donor cell preparation The anti-human skeletal myoblast antibodies completely disappeared at 2 weeks after myoblast transplantation In animals, without the

immunosuppressive treatment, the humoral response has been reported to continue and lead to rejection of the graft within 2 weeks (Vilquin et al., 1994)

The second contributory factor is the down-regulation of human MHC I and II on skeletal myoblasts after differentiation into myotubes Unlike primary myoblasts, mature muscle fibers do not express MHC I and II (Karpati et al., 1988) For the use of immunosuppression to prevent rejection of the donor myoblasts, we hypothesized that suppression of the host immune system during the early phase of cell transplantation may provide opportunity for the MHC expressing myoblast to acclimatize and escape the host immune response Once they have entered in to differentiation pathway, they would start

to down-regulate the expression of MHC and hence the withdrawal of immunosuppression would not effect their survival Based on this hypothesis, we maintained the animals on transient immunosuppression using a daily dose of 5mg/ kg Cyc-A, as described earlier Immunohistochemical studies revealed no expression of human MHC I and II at 6 weeks after myoblast transplantation in pig heart (Figures 44 & 45) and by this time, human skeletal myoblasts were taken as “self” by the host immune system

Histochemical studies for nLac-z expression revealed that skeletal myoblasts survived in the pig heart (Figure 34) Most of the nLac-z positive myofibers had the

typical characteristics of cardiac muscle fibers with branching pattern (anastomosis) One possible mechanism was that myoblasts might integrate into host cardiomyocytes and form hybrid myofibers (Haider et al., 2002) as discussed in section 4.6

Other factors that possibly made contribution to the survival of human skeletal myoblasts included: the effect of angiogenic factors and the high purity of skeletal

myoblasts VEGF could enhance skeletal myoblasts migration and survival and prevent myoblast apoptosis (Germani et al., 2003) Furthermore, hVEGF165 expressed by hVEGF165-myoblasts induced the synthesis of NO and cytosolic phospholipase A2 which would dilate the blood vessels in infarcted area to ameliorate the ischemia and improve the survival of skeletal myoblasts in the early phase (Zachary et al., 2001) The increased neovascularization induced by hVEGF165 or Ang-1 would provide a normalenvironment for skeletal myoblasts in late phase Other growth factors, such as fibroblast growth factor-β and insulin-like growth factor had been shown to stimulate proliferation and enhance survival of the myoblast (Kinoshita et al., 1995; Mitchell et al., 2002) Similarly, desmin contributes towards the survival and differentiation potential of myoblasts (Qu et al., 1998) Skeletal myoblasts expressing high levels of desmin showed better survival after transplantation

Counting of the donor cells surviving in the pig heart post transplantation was

carried out based on nLac-z positive nuclei As shown by our results, the cell number

showed no significant decline at the site of the graft at 6 and 12 weeks after myoblast transplantation (Figure 35) The results were further confirmed by PCR for human Y- chromosome DNA in the pig heart (Figure 37) To identify the implanted cells in vivo, magnetic resonance imaging may be employed (Hill et al., 2003; Dick et al., 2003)

4.6 Differentiation of skeletal myoblast in pig heart

Invariably it has been shown that skeletal myoblast transplantation improves injured heart function, however, the fate of skeletal myoblasts, after transplantation into

heart remains uncertain During the present study, the expression of nLac-z in pig heart

strongly suggested the possibility of human skeletal myoblasts integrated into pig

cardiomyocytes to form heterokaryons which expressed slow and fast isoforms of skeletal myosin heavy chain rather than human cardiac connexin-43 and troponin I

Earlier studies have suggested that myoblasts under milieu dependent differentiation into cardiac-like cells in the rat (Murry et al., 1996b; Dorfman et al., 1998), cryoinjured rabbit (Atkins et al., 1999; Taylor et al., 1998) and the cryoinjured dog hearts (Marelli et al., 1992; Chiu et al., 1995; Yoon et al., 1995) Gap junctions were described during the early stages of muscle development with concurrent expression of N-cadherin and connexin-43, which were down-regulated in the mature stage (Balogh et al., 1993; Reinecke et al., 2002) An interesting in vitro study documented by Iijima et al.,

trans-(2003) has shown that skeletal muscle-derived cells trans-differentiated into

cardiomyocytes upon co-culture with contracting cardiomyocytes Skeletal derived cells expressed cardiac troponin T and atrial natriuretic peptide, cardiac transcription factors Nkx2E and GATA4, andadhesion and gap junction proteins, such as cadherin and connexin-43 These cells even showed cardiomyocyte-like action potential However, it is postulated that they could be hematopoietic progenitor cells in peripheral blood circulation and isolated by muscle biopsy

muscle-Recent reports have shown that the donor skeletal myoblasts failed to differentiate into cardiac-like cells Rather they differentiated into mature skeletal muscle fibers that served as a scaffold to support the infarcted heart muscle wall (Ghostine 2002; Hagege et al., 2003; Pagani et al., 2003) These muscle fibers expressed slow or fast isoform of skeletal myoblast heavy chain or co-expressed both isoforms We have

trans-observed that the nLac-z positive myofibers had the typical structure of the

cardiomyocytes that connected each other through anastomosis (Figure 34f & g) These myofibers either expressed fast or slow isoform of skeletal myosin heavy chain (Figure

hybrid myofiber did not express cardiac connexin-43 and troponin I (Figure 41 & 42) thus suggesting that skeletal myoblasts did not trans-differentiate into cardiomyocyte (Reinecke et al., 2002; Hagege et al., 2003)

Counter-staining of nLac-z expressing myofibers with specific anti-pig Ig-FITC

suggested that they were pig cardiac tissue (Figure 43) Thus, these findings suggested the possibility of fusion between the donor myoblasts and pig cardiomyocytes to form hybrid or mosaic muscle fibers These are interesting findings as regards the fact that both skeletal muscle nuclei and the host cardiomyocyte nuclei co-inhabit the same muscle fiber (Haider et al., 2002; Reinicke et al., 2004) The donor nucleus thus may be in a position to supplement the genes which have been lost from the host cardiomyocyte nuclei during the ischemic injury Another possibility explored from our data is that donor and host nuclei may undergo fusion to form a hybrid nucleus which share characterisitics of both skeletal muscle and cardiac muscle (Table 4.1) These observations get support from our FISH data (Figure 38) Our findings have gained further support from the recently published back to back research papers published by Reinecke et al., (2004) showing that fusion between donor myoblasts and the host cardiac muscle fibers is the possible mechanism of donor cell making a contribution towards improved cardiac function