Investigation into the cyto-protective and wound healing properties of cryptic peptides from bovine Achilles tendon collagen
Introduction
The extracellular matrix (ECM) provides mechanical support and is also involved in biochemical signalling through membrane receptors with the cells that are in contact with it. These signals can have a significant influence on cellular cytoskeletal structure, gene transcription and migration. In turn, the cells remodel the ECM enzymatically and play an active role in sculpting a microenvironment suitable for their phenotypes [1]. This dynamic and reciprocal communication between the cells and ECM is now considered essential for regular functioning and suitably responding to stress. A key component of such communications involves the recognition of a vital ECM component, generally, a short stretch of amino acids, present at a strategic location in the larger protein, by transmembrane receptors such as integrins [2]. The interactions generally require either conformational changes in the ECM protein leading to unmasking of the active region or direct proteolytic action resulting in the excision of the bioactive region as a peptide. Such concealed segments, which upon enzymatic cleavage or conformational change can exhibit physiological activities, are termed as cryptic bioactive peptides [3], [4]. During ECM remodelling and/or wound healing, protease-generated specific fragments of constituent proteins can exhibit a wide array of bioactivities including antioxidative, chemotactic, wound healing, anti tumorogenic and angiotensin I-converting enzyme inhibition [5], [6], [7].
Reactive oxygen species (ROS) are required by living systems to maintain a number of functions including signal transduction, cell adhesion and wound healing [8]. Oxidative stress ensues when the delicate balance between ROS creation and depletion is hampered due to tissue injury or faulty detoxification mechanisms. In case of persistent wounds or the presence of foreign substances in the proximity of a wound site, the resultant uncontrolled stress due to ROS or otherwise, leads to delayed healing [9]. Excess ROS acts through multiple mechanisms to generate stress including oxidation of cellular macromolecules leading to membrane damage, enzyme dysfunction and hampering of metabolic flux along with impaired DNA repair, resulting in mutagenesis or cell death [10]. Oxidative stress generally has an inverse relation with cell adhesion and wound healing; although very low levels of free radicals are required for integrin attachment to ECM, higher levels result in cell death. Excess ROS can, along with other effects, alter the expression of integrin sub-unit genes, consequently leading to improper connectivity with the ECM and eventually causing cell death [11]. Oxidative stress is also involved in reorganization of actin and vimentin arrangements, leading to changes in the focal adhesion site resulting in loss of cell-matrix adhesion. This further leads to the activation of apoptotic pathways, cell loss, compromised tissue integrity and pathologic consequences [12].
In the present study, it is hypothesized that the cell loss due to ROS stress can be minimized in the presence of cryptic peptides. In other words, components of the matrix with prominent cell adhesive and antioxidative activities can provide some form of protection to stress-exposed cells. The increased cell survival, in turn, can also have a substantial effect on cell migration during in vivo stress-generating events like tissue remodelling and wound closure.
The physiological effects of oxidative stress in vitro can be mimicked by the use of exogenously administered H2O2 and heavy metal ions. For the latter scenario, ROS stress can be generated through two distinct mechanisms. With redox-active metal ions like Fe2+ and Cr6+, a Fenton-like reaction increases ROS while redox-inactive toxic metal ions such as Hg2+ and Cd2+ act through depletion of major antioxidant components leading to unquenched free radicals [13]. In this study, oxidative stress has been generated by three methods; subjecting cells to H2O2, a combination of Fe2+-H2O2 and heavy metal ions.
The peptides C2 (sequence: GPOGPOGKNGDDGEAGKPGRPG) and E1 (sequence: GETGPAGPAGPIGPVGARGPAGPQGPRGDKGETGEQ) with molecular weights 2.8 and 3.2 kDa respectively have been isolated from type I collagen in our earlier work [14], [15]. The peptide C2 exhibits cell adhesion properties [14] and the RGD-containing peptide E1 exhibits both cell adhesive and antioxidative properties [15]. The present study was carried out to determine the role of these peptides in countering ROS stress and the ability to aid in vitro wound healing properties.
Section snippets
Materials and methods
Bovine Achilles tendons were collected from a slaughterhouse at Vellore, Tamil Nadu, India and type I collagen was purified from the tendons through acid extraction/salt precipitation technique [16].
T-flasks (Nunclon surface) were procured from Nunc, Roskilde, Denmark and disposable culture dishes (35 × 10 mm) were obtained from Fischer Scientific, Hanover park, IL, USA. Powdered Dulbecco’s modified Eagle’s Medium (DMEM), supplemented with 2 mM glutamine and 10× antibiotic–antimycotic solution were
Hydroxyl radical scavenging assay
Since ROS stress mostly generates 
OH radicals, it was necessary to check for the ability of the peptides to scavenge the same. The activities of BHT, the two peptides and collagen were significantly different (p < 0.004) at a confidence level of 95% based on ANOVA. As depicted in Fig. 1, at 100 nmol, E1 exhibited a scavenging activity of 58.7%, whereas C2 achieved only 15%. BHT, used as the positive control, exhibited highest activity at 100 nmol. To summarize, the results obtained confirmed E1’s
Conclusion
The hypothesis that cryptic peptide fragments could impart cyto-protection to cells under stress was proved through this study. E1 in the dissolved form was capable of enhancing cell survival even at high stressor concentrations by virtue of its antioxidative properties. C2 in the coated form and in combination with E1 in the dissolved form also displayed cyto-protective abilities and rapid wound closure, particularly when subjected to ROS stress. Wound healing is a complex hierarchy of
Conflict of interest
The authors declare no conflict of interest.
Acknowledgements
The authors wish to thank the Department of Science and Technology, Government of India for funding this study. The authors are also grateful to Vellore Institute of Technology University, where the work was carried out.
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