The present study identifies a novel matricryptic peptide with in vitro chemotactic activity for several types of progenitor cells and differentiated cells. This peptide is also associated with the increased presence of Sox2+ and Sca1+,Lin− cells at the site of experimentally induced injury in a mouse model. As a short 12 amino acid oligopeptide derived from the C-terminal telopeptide region of the collagen IIIα molecule, the sequence of this molecule is highly conserved amongst at least eight mammalian species.
The C-terminal telopeptide region of fibrillar collagen is known to be a site of interchain cross-linking of cysteine residues that ultimately stabilize the triple helix structure of collagen.38
Thus, in the absence of injury and protease-mediated degradation, it is unlikely that such a sequence would actively interact with cells due to extensive cross-linking. However, protease-mediated matrix degradation at a site of injury would not only destabilize and release peptides from the triple helical domain of collagen, but also expose and cleave the telopeptide regions of collagen to release cryptic peptides similar in sequence to the isolated peptide in the present study. Previous studies have shown that telopeptide sequences can be isolated in the circulating blood after turnover of collagen and soft tissue remodeling in a clinical setting.39–41
Thus, while the cryptic peptide in the present study was isolated by nonphysiologic methods of degradation, it is likely that a similar peptide can and would be released in vivo
at a site of injury.
The concept of cryptic fragments of parent matrix molecules having biologically relevant properties is not new. Antimicrobial activity of matricryptic peptides in the form of defensins,22
has been identified by many groups and is thought to represent an evolutionary survival advantage in response to injury. Angiogenic and anti-angiogenic cryptic peptides such as endostatin,42
have been described and have been used therapeutically for a variety of conditions. Such cryptic peptides can be released from ECM by proteases secreted by immune cells at a site of injury, and thus logically represent a desirable aspect of the host response to tissue injury.
The recruitment of various cell types such as stem and progenitor cells, endothelial cells, and muscle precursor cells to sites of tissue injury represents a logical and plausible host response to support tissue reconstruction. The mechanisms underlying such a recruitment process are largely unknown, but it is feasible that cryptic peptides represent one such strategy. The manner in which the oligopeptide described herein was generated was nonphysiologic, but a previous study has shown naturally occurring degradation products after ECM-mediated tissue reconstruction have similar properties.45
In fact, degradation products of ECM have been shown to regulate the site-directed recruitment of differentiated26–28
and progenitor cells29,31,32 in vivo
Site-specific recruitment of multipotent progenitor cells in response to limb amputation is a prerequisite for blastemal based epimorphic regeneration in species such as newts and axolotls.46
Soluble factors are present that can recruit selected cell types and selected genetic programs are activated to participate in the regeneration process that results in a perfect phenocopy of the missing tissue structure.46–48
While blastema formation does not occur after injury in adult mammalian species, recruitment to and/or directed differentiation of tissue specific progenitor cells at the site of injury has the potential to alter the default scar tissue wound healing response toward a more constructive tissue remodeling response.49,50
In organs such as the liver, bone marrow, and intestinal lining that are capable of mounting a regenerative response to injury, activation and recruitment of progenitor cell compartments is an important prerequisite to site-appropriate tissue regeneration.51–53
Thus, recruitment of multipotent progenitor cells to a site of injury in response to placement of a chemotactic peptide may be considered as a form of endogenous stem or progenitor cell therapy. The clinical efficacy of such therapies, however, remains to be determined.
The present study identified a single peptide derived from a mixture of matricryptic peptides that can recruit stem, progenitor, and differentiated cells in vitro and is associated with an increased accumulation of such cells at sites of injury in vivo. However, the findings of the present study do not preclude the existence of other pro- and anti-chemotactic matricryptic peptides. It is likely that degradation of ECM leads to release of a large mixture of anti- and pro-chemotactic peptides that yields an overall net effect in vivo. Indeed, certain fractions of the ammonium sulfate precipitated peptides in the present study showed inhibited migration of progenitor cells, a finding suggesting that degradation products of ECM such as UBM contain chemotactically positive, negative, and likely also neutral peptides. Isolation of these peptides was not pursued. Although an active peptide described in the present study was isolated from a mix of mammalian matricryptic peptides, the naturally occurring contribution of this particular peptide to the host response to injury was not determined. The present study also showed the efficacy of the isolated oligopeptide in an adult mammalian model of digit amputation via local accumulation of progenitor cells at the site of injury. While the present study cannot determine whether the efficacy of the isolated peptide is dependent on the type of injury, future studies will further investigate the potential role of the peptide in progenitor cell recruitment in various other types of injury.
Additionally, the present study focused on identifying a bioactive cryptic fragment of one source of ECM. ECM derived from the porcine urinary bladder has been used in multiple pre-clinical applications for site-specific remodeling of a variety of soft tissues.54–63
However, commercially available biologic scaffolds composed of ECM are derived from a number of species and organs.64
It is possible, and in fact likely, that a similar cryptic peptide would be found in ECMs from other sources. Collagen III is a common constituent of many soft tissues from which ECM scaffolds are made, and its sequence is highly conserved from species to species. The present study found that the isolated cryptic peptide's sequence is also highly conserved among over eight species, many of which are already used as sources of ECM for commercial applications. Thus, it is likely and expected that the isolated cryptic peptide, or a similar derivative of such a peptide, would qualitatively possess the same bioactive properties in multiple mammalian species and commercially available ECM scaffolds.
In summary, the present study identifies a chemotactic matricryptic peptide capable of site-directed accumulation of selected progenitor cells and differentiated cells in vitro and in vivo. Furthermore, treatment of an injury site in a murine model of digit amputation with this peptide is associated with the accumulation of endogenous Sox2+ and Sca1+,Lin− cells. In the present study, the recruited cells clearly did not spontaneously differentiate and form functional tissue. Nevertheless, the findings of the present study provide a potential strategy for endogenous stem cell recruitment to a site of injury. The next logical step in promoting tissue regeneration of more complex tissues will involve identifying strategies by which to direct the spatiotemporally appropriate proliferation and differentiation of the multipotent progenitor cells.