Wound Healing Goes Viral

During cutaneous wounding, many different cell types are mobilized to reinstate homeostasis, including immune cells, keratinocytes, dermal fibroblasts, and fascia fibroblasts. These responses are essential to stop bleeding, clear microbes, and close the wound to re-establish the skin barrier. Humans, similar to most mammals, are incapable of regenerating fully functional skin after cutaneous wound healing. Instead, scar tissue develops, which is inferior to native skin owing to its lack of skin appendages, unsightly appearance, and compromised mechanical properties ().

Patients who suffer from excessive or prolonged scarring typically experience protracted tissue dysfunction, pain, and psychological distress. Treatments that aim to repair chronic scarring are limited to surgical interventions. Small scars may be treated through surgical excision, followed by postsurgical interventions that aim to limit the formation of new scars. Usually, this involves sutures to bring the two sides of the wound into close contact, dressings to alleviate tensile forces on the wound, and corticosteroid injections to limit inflammation. In many cases, these interventions do not prevent the development of pathological scarring ().

The location and extent of an injury are key determinants of scar size and severity. Recently, it was discovered that dermal fibroblasts that have expressed Engrailed-1 (En1) (cells known as En1 past fibroblasts [EPF]) generate scar tissue (). Furthermore, deep wounds have the ability to mobilize fibroblasts from the fascia, a smooth connective tissue that envelops organs. These fibroblasts, also EPF cells, drag along with them the extracellular matrix (ECM) in which they are embedded, in a mechanism that depends on N-cadherin‒based adherens junctions and connexin 43‒based gap junctions (Figure 1a) (). In fact, scars in which the fascia is not mobilized, such as superficial wounds, wounds in the oral cavity, or wounds in which the fascia has been physically impeded, were shown to have smaller scars (). In their recent article in the Journal of Investigative Dermatology, seek to improve wound repair outcomes and reduce scar formation by targeting the collective migration of fascia fibroblasts during wound repair. These proteins represent novel intervention targets that may be able to prevent or reduce fibrotic ECM assembly at wound sites.

Transient regulation of cellular functions in a spatially and temporally specific manner is starting to become a reality with some recent advances in viral delivery technology. Adeno-associated virus (AAV) vectors are appealing candidates because they have an excellent safety record, the genetic material does not integrate into the host DNA, and the genetic material is stable long term in mitotically quiescent cells (). In fact, AAV delivery of genetic material is United States Food and Drug Administratio.n approved for three therapies with many more in clinical trials (). Wound healing is ideally suited for transient gene therapy because the cellular response is spatially and temporally acute, and if the appropriate cell types are reached, the specific wound program can be modified.

attempted to specifically target the wound fibroblasts and, in particular, to reach the fascia fibroblasts. They tested six different AAV serotypes in the delivery and the expression of GFP in cultured fascia explants from mice aged 3 days and found that AAV8 has a higher tropism for fascia fibroblasts than for other skin cell types. They then tested the delivery of GFP in mice aged 3 days by subcutaneous injection upon wounding. Quantification of the cell types that were transduced revealed that 80% of the GFP+ cells were fibroblasts (PDGFRα+) and that of these cells, over half expressed markers of fascia fibroblasts: N-cadherin and SCA1. Next, the authors tested their genetic delivery system to deliver Cre recombinase in the Rosa26-mTmG model in which cells express tdTomato until Cre-mediated recombination permits enhanced GFP expression. Mice were wounded at age 3 days, and analysis conducted 7 days after wounding showed that GFP+ cells were abundant within the wound bed and expressed myofibroblast marker α-smooth mucle actin, thus indicating the feasibility and efficacy of their AAV8-mediated genetic material delivery to the wound fascia fibroblasts (Figure 1b).

Previous work by the authors revealed that N-cadherin is upregulated by the fibroblasts in the wound and resolves upon ECM remodeling (). Intracellularly, cadherin-mediated adherens junctions are mechanically tethered to the actin cytoskeleton through linker proteins such as p120 catenin (). The authors find that wounding induces p120 catenin expression in the wound fibroblasts, similar to upregulation of N-cadherin (). Thus, the authors hypothesized that p120 catenin could be modulated to reduce scar formation. They then took the approach to transiently reduce p120 expression through short hairpin RNA (shRNA). In vitro experiments revealed that this method abrogated mRNA and protein expression. The authors then tested this in vivo, delivering the p120 catenin-targeting shRNA in AAV8 through subcutaneous injection in postnatal day-3 mice upon wounding; control, nontargeting shRNA was delivered to the bilateral wound. Seven days after wounding revealed downregulation of p120 catenin protein as well as expression of N-cadherin in the wound. Ultimately, upon epithelialization, the scar tissue appeared smaller. To dissect the cellular mechanism, the authors excised wound tissue 5 days after wounding to carry out live imaging. The fibroblasts in the control wounds reveal a pattern of directed, collective fibroblast migration with extended protrusions, whereas upon reduction of p120 catenin, the fibroblasts were less motile and exhibited fewer cellular protrusions. Critically, transient abrogation of p120 catenin in fascia fibroblasts showed reduced collagen deposition.

This work lays the promising groundwork for localized delivery of transient genetic modifications, which is a highly relevant method to modify wound healing. Future investigation to detail the long-term outcome of scar mitigation through reduced migration of fascia fibroblasts will be critical. In addition, it will be particularly important to address whether this intervention results in increased tissue strength compared with that of normal scars and further whether altered scarring improves the ability of the skin to regenerate the appendages. Ultimately, this work is highly relevant to keloid scars as well as long term, nonhealing wounds; thus, it would be clinically significant to understand whether AAV8 has the same tropism for fibrotic tissue as for newly wounded fascia. The translational benefit of this work is of great interest; thus, great care will need to be taken to precisely define the duration of the AAV infection as well as lifetime of the exogenous genetic material.