We show here that a homing peptide, named CAR, originally discovered as a wound-homing peptide and used as a delivery vehicle for therapeutics2–4, also possesses an inherent ability to accelerate wound healing. We further show that CAR peptide acts by activating SDC4-dependent mechanisms to drive epithelial cell migration.
The CAR peptide is an example of the propensity of in vivo phage display screening for homing peptides to yield peptides that are bioactive, in addition to being able to home to a particular target in the body. Earlier examples of such peptides include the tumour-penetrating peptides iRGD and LyP-1; iRGD has an inherent anti-metastatic activity25 and LyP-1 elicits apoptosis in tumour cells and tumour macrophages26,27. The reason for this inherent bioactivity is that peptides interact with binding pockets in proteins, and those pockets are generally important active sites (for discussion see28). CAR peptide is different from the existing bioactive peptides that bind to proteins in that its target molecule is a carbohydrate, the heparan sulfate glycosaminoglycan (GAG) chains of HSPGs3,4. CAR contains a classical heparin-binding domain (HBD) that has high homology with the HBD of bone morphogenetic protein-4, and we have previously shown that CAR binds to heparan sulfate and heparin3,4. HBD-GAG interactions are thought to be mostly charge mediated, i.e. dependent on attraction between basic residues in the HBD and sulfated sugar residues in the GAG6,7,29,30. HSPGs are ubiquitous, however the selective homing of CAR to angiogenic vessels and wound tissue suggests additional elements to the CAR specificity, such as a GAG sulfation pattern that creates a molecular signature characteristic of regenerating tissues. This scenario is supported by the recent demonstration that SDC4 expression is very low or absent from normal quiescent blood vessels, but it is the SDC-family member exhibits enhanced expression in angiogenesis31. Furthermore, it was also shown that owing to differences in the heparan sulfate chains of SDC2 and SDC4 (defined by a unique protein sequence in SDC2 ectodomain), the heparin-binding growth factor, vascular endothelial growth factor-A (VEGFA), binds only to SDC2 and not to SDC432. Thus, two factors, distinct GAG chains on specific HSPGs and the selective overexpression of SDC4 in migrating epithelia and angiogenic vessels may contribute to the selectivity of CAR to wounds.
The targeted, organ- and cell-specific mode of action of CAR makes it possible to use systemic administration of the peptide to accelerate wound healing, which circumvents limitations of local treatments, such as difficulty in maintaining the activity of local agents in the wound environment. Systemic treatment is also advantageous when the injured site cannot be accessed by topical application or multiple tissues are injured simultaneously. Our results only address the treatment of skin wounds, so it will now be important to determine whether the beneficial activities of CAR extend to other tissues. It is known that CAR homes to injuries in tendon3 as well as to diseased tissue in pulmonary arterial hypertension, bronchopulmonary dysplasia, cancer (human tumour xenografts), aortic aneurysms, retinopathies, muscular dystrophies and myocardial infarction14–16,33−38. In line with CAR peptide homing to tissue injuries and these diseases, the upregulation of SDC4 expression has been described in all of these instances7,20,31,36,39,40.
Several lines of evidence from our study indicate that CAR promotes wound healing through selective engagement of SDC4. First, wounds in SDC4 knockout mice were impervious to the CAR-mediated effect. Second, our mechanistic data shows that SDC4 is required for internalisation of CAR peptide and that CAR modulates SDC4-dependent ARF6 activity to drive epithelial cell migration. Third, CAR peptide induced SDC4- and ARF6-dependent redistribution of α5β1 integrin, indicating that CAR regulates cell migration by orchestrating integrin engagement. Finally, SDC4 is expressed de novo on keratinocytes in the migrating epidermis (epidermal tongues), and re-epithelialisation is the main aspect of wound healing that is affected by CAR.
We also found that although the size of the granulation tissue was similar in the CAR-treated and control wounds, there was a striking difference in the number of myofibroblasts in the two treatment groups; the CAR-treated wounds almost completely lacked myofibroblasts, whereas myofibroblasts were abundant in the control-treated wounds. Myofibroblast-driven contraction of loose granulation tissue into a scar completes wound healing, but this process comes at a price, with formation of a permanent scar41. As there are fewer myofibroblasts in CAR-treated wounds, CAR may also reduce scarring. This scenario is supported by recent findings that SDC4 presence suppresses the development of fibrosis in fibrotic disease models42–44 .However, this remains to be studied because the 10-day observation period used assess re-epithelialisation in this study was too short to assess permanent scar formation.
The SDC4-dependent cell migration pathway that underpins the mechanistic basis of CAR-accelerated wound healing, has been characterised in detail9–11, 18,45. While many ECM proteins contain HBDs, fibronectin is the main endogenous SDC4 ECM ligand that activates these pro-migratory pathways5,9. Plasma fibronectin is a major component of the blood clot that forms immediately after wounding and fibronectin is also abundantly expressed by fibroblasts in granulation tissue46. Although plasma fibronectin is not essential for wound healing47, cellular fibronectin is48. Lack of fibronectin extra domain A (EDA) leads to selective defects in wound re-epithelialization48. Thus, it is thought that fibronectin provides a bed/scaffold for migrating epidermis to close the wound48. Yet our analysis of wound tissue indicates that very few migrating keratinocytes are in contact with fibronectin during wound repair. Thus, it is probable that a large proportion of keratinocytes express unligated SDC4 that can receive an additional migration-promoting stimulus from CAR. Thus, we propose a model whereby, in the early stages following wounding, elevated SDC4 expression promotes CAR targeting to the site of injury, but also provides a reservoir of unengaged receptors that are available to be bound by the peptide to accelerate the endogenous wound healing response selectively on the epidermis.
Mechanistically, our data indicate that ARF6 is the downstream effector of SDC4 signalling triggered by CAR to drive epithelial cell migration. Accumulating evidence suggests that integrin recycling plays a key role in cell migration49,50. As SDC4 regulates ARF6 to co-ordinate heterodimer-specific recycling of integrins to the plasma membrane11 it is likely that CAR peptide regulates cell migration and wound healing by driving recycling of integrins. Indeed, the SDC4-mediated activation of ARF6 induced by CAR, and the SDC4- and ARF6-dependent redistribution of α5β1 in response to CAR stimulation, are consistent with CAR regulating cell migration and wound healing by orchestrating α5β1 engagement. However, as SDC4 is also a growth factor co-receptor5, CAR-dependent ARF6 activation may also regulate growth factor receptor trafficking to co-ordinate cell migration and wound healing22. A significant level of crosstalk exists between adhesion receptors and growth factor receptors and this interplay has critical roles in cell migration51,52. Reciprocal, and mutually regulatory, trafficking mechanisms are one of the major mechanisms by which adhesion receptor and growth factor receptor signals are integrated49,51,52. Thus, as syndecans and ARF6 regulate trafficking of both integrins and growth factor receptors9,11,22, it is possible that CAR may co-ordinate ligand engagement and signalling of multiple pro-migratory receptors.
Recent papers report the use of fibrin-conjugated peptides as a locally administered biomaterial to enhance wound healing53. The authors attributed the effects of these biomaterials on wound healing to growth factor retention afforded by the growth factor binding to the HBD of these peptides in the wounds53. Since these peptides contain HBD and bind to syndecans53,54, there is some similarity to our work. However, we use soluble peptide, that accumulates in the wounds by homing and cell penetration and is not covalently anchored to ECM, therefore it is unlikely that growth factor retention is a significant part of its mode of action. Moreover, our treatment is systemic which offers considerable advantages over local administration. Furthermore, these topically administered, syndecan-binding peptides are also functionally different from CAR in that their activities include, for example, stimulation of proteolytic activity by matrix metalloproteinases55.
Finally, the classical view of the role of heparan sulfate in the biology of heparin-binding growth factors is that their binding to HSPGs merely provides a way to concentrate and present growth factors to a signalling receptor, such as a receptor tyrosine kinase29,30. As the CAR peptide stimulates wound healing through SDC4-/HSPG-dependent signalling, it may be that heparin-binding growth factor signalling is more complex than currently thought, and that HSPGs, in addition to concentrating and presenting growth factors, also act as signalling receptors in concert with conventional growth factor receptors.
Defective wound healing has been reported in SDC4 knockout mice as well as in mice with a conditional, keratinocyte-specific ARF6 deletion6,7,56,57. These studies, when considered alongside our data, highlight the key roles that both SDC4 and ARF6 play in wound healing and suggest that pharmaceutical manipulation of these pathways to promote wound healing may be therapeutically tractable. Thus, CAR peptide may provide a new way of enhancing wound healing by systemic treatment, and perhaps tissue regeneration in general. Indeed, it has been shown that SDC4 also plays key roles in skeletal muscle regeneration58,59, epithelial regeneration in experimental colitis models60 and fracture and cardiac repair36,61. We have not seen any obvious toxicities in any of the many mice we have treated with CAR over extended periods of time. Moreover, CAR is active in human cells and tissues: the human keratinocyte cell line we used in this study responded to CAR with increased migration, and previous studies have shown that CAR binds to and penetrates human endothelial cells and homes to human tumour xenografts in vivo14. These features bode well for the translational prospects of CAR.