Our results demonstrate that antibody targeting of ECM-derived peptides to ischemically injured myocardium can effectively help to produce an ECM favorable for angiogenesis. A critical barrier to tissue regeneration is the lack of an adequate vascular network. The creation of a vascular bed in the infracted myocardium should allow for greater cell engraftment and survival 
. Therefore, this strategy of specifically targeting ECM-derived peptides to the ischemic myocardium may provide a more favorable microenvironment for cell transplantation and myocardial regeneration. The use of the ECM-derived peptides alone, without growth factors or cells, was sufficient to promote an angiogenic response in infarcted rat hearts. The induction of new vessel formation suggests that targeting active components of the ECM can influence the microenvironment and allow the body to act as its own bioreactor to regenerate vital structures of the myocardium.
The MHC antibody specifically targets the ECM peptides to the MI region in the heart (Figure S2
). Nuclear imaging studies using I125
-radiolabeled MHC-Ab showed that the majority of the MHC-Ab was concentrated within the MI of the heart and was still detectable within the MI 1 week post-injection. Unconjugated I125
-radiolabeled peptides injected into the rats 1 day post-MI were detectable within the heart 3 hours post-injection, but only at trace levels 24 hours post-injection. Additionally, biodistribution analysis showed that the unconjugated peptides were predominantly in other organs—e.g. liver, intestines—instead of the heart (Figure S3
). Not only did the MHC-Ab concentrate the peptides within the MI, but it also allowed the peptides to remain within the MI for longer periods of time. Hence, in order to expect any benefit from peptide treatment after acute MI, it was necessary for us to target them with an antibody. However, even though most of the antibody was targeted to the infarct region of the heart, there were still trace levels in other organs, which could result in neoplastic angiogenesis within these organs. Future studies will need to be conducted to fully assess the effect of these trace levels in other organ systems in producing angiogenesis.
Our in vitro data found three peptides—HepI, HepIII, RGD—that exhibited similar properties, although to a lesser extent, as their source proteins, particularly in terms of promoting endothelial cell adhesion, proliferation and haptotactic migration. Nanogram amounts of either HepIII or RGD were sufficient to promote significant movement of endothelial cells. This is the same order of magnitude of peptides that we injected into our rats. Using fluorescently labeled peptides, we had determined that our conjugation resulted in crosslinking ~3 moles of peptide per mole of antibody. The presence of the ECM-derived peptides could promote the migration of endothelial cells to the infarct site.
Cells interact with the ECM via receptors, including integrins. Yet, these receptors only interact with certain regions of an ECM protein. Our Western blot analysis showed activation of Erk1/2 by HepI, HepIII, and RGD. Activation of the Erk1/2 signal transduction pathway in ECs is critical for EC proliferation and angiogenesis 
. HepIII has been shown to interact with α2
integrins, thereby promoting cell adhesion to the peptide 
. There is some evidence that α2
, and β1
integrin subunits can also interact with HepI 
. RGD has been shown to interact with the integrin αv
. Endothelial cells migrate via αv
integrins. The interaction of the cells via these integrins can trigger a cascade of signal transduction pathways, some of which could be involved in initiating angiogenesis and/or arteriogenesis. For instance, αv
is involved in the signaling of fibroblast growth factor 2 (FGF2), which is involved in various signaling pathways, including the activation of Erk1/2, which in turn activates the signaling pathways for angiogenesis and/or arteriogenesis 
. Integrin α2
has been shown to support VEGF-stimulated signal transduction 
, which also includes Erk1/2 activation. Integrin α3
-mediated adhesion has been shown to activate focal adhesion kinase (FAK) as well as Erk in keratinocytes 
. Even though HepI, HepIII, and RGD did not promote activation of Erk1/2 to the same extent, their induced angiogenic responses in terms of capillary and arteriole formation were similar. This suggests that in addition to Erk1/2 activation, the ability to induce cell migration and cell proliferation are characteristics that are important if not more so in the promotion of the observed arteriogenesis and angiogenesis.
Furthermore, the ECM peptides may be influencing new vessel formation by interacting with other ECM proteins. Interestingly, HepIII peptides can interact with one another to form a polymer-like matrix (Figure S4
). It is possible that a comparable situation is occurring in rats treated with HepIII and may explain why HepIII was the only peptide that was able to prevent further negative remodeling as indicated by the echocardiography data. The peptides do not necessarily have to be interacting with each other. They may be able to interact with surrounding ECM proteins in a similar manner to form a matrix, thus altering the material properties of the LV and preventing the negative remodeling associated with a MI 
. Also, earlier studies have shown that HepI can interact with whole Col IV 
. The low dissociation equilibrium constant Kd
1.66 nM, indicates that the binding affinity of HepI for Col IV is remarkably high. HepI's ability to interact strongly with Col IV may in some way help to contribute to the formation of neovessels in HepI-treated rats, as Col IV is known to be a major factor in the induction of new vessel formation and in the stabilization of these new vessels 
Despite previous reports 
showing that FC/HV could promote cell adhesion and migration, we did not observe such behavior in our studies with FC/HV. Based on our results, the peptide might promote transient adhesion, as evidenced by our observation of spread cells 1 day after incubation on FC/HV-treated plates. Although FC/HV did induce Erk1/2 activation, we observed no statistically significant increase in angiogenesis or arteriogenesis in the FC/HV-treated rats. It is possible that FC/HV is inducing Erk1/2 activation in the cells already present within the MI region, but the inability of the peptide to promote significant endothelial cell migration and cell proliferation as compared to the other 3 peptides studied may limit its ability to induce any dramatic neovascular formation.
In conclusion, Ab-targeted ECM-derived peptides can be used to alter the myocardial microenvironment and promote the induction of angiogenesis in the injury site after a MI. The exact mechanisms by which the ECM peptides induced the observed in vivo angiogenic response, however, warrant further study. Furthermore, from our echocardiography data only Hep III prevented negative remodeling of the LV following a MI, indicating that neovascularization alone is insufficient to get full recovery of LV function. Perhaps by combining this ECM peptide therapy with cell therapy, we may be able to get full restoration of cardiac function and tissue. Nevertheless, our results present a new non-invasive strategy for regenerative therapies and a tool for investigating tissue repair and regeneration.