Wound healing requires orchestrated repopulation and regeneration of the lost tissue, a process that often results in scarring in adults especially f the wound is deep and/or extensive. In many tissues, both mesenchymal and epithelial compartments must be regenerated to accomplish proper healing; a deficit in one results in poor to absent function. The pre-eminent model for this is an excisional skin wound in which fibroblasts are recruited from surrounding tissue to regenerate the dermal layer, and keratinocytes from the margins to re-epithelialize the initial clot. Orchestration of repair is conducted by signals from successive waves of growth factors, chemokines, and matrix fragments that occur during the inflammatory, regenerative and resolving phases. A major question is how cells in the different compartments communicate or sense the status of wound repair -- how does a dermal fibroblast 'know' that re-epithelialization is complete and thus must switch to the resolving phase functions of contraction and collagen remodeling? Failure to recognize such signals results in excessively scarred or hyperemic tissue that limits function. Our overarching programmatic hypothesis is that specific and targetable extracellular signals communicate across compartments to synchronize the phenotypes of the key cells in the wound repair process. We have found in the current cycle of the grant that disrupting the transition from the regenerative phase to the resolving phase results in a chronic inflammatory phenotype and a hypertrophic scar. Exciting new data suggest that the changing matrix and signaling milieu directs the function of the skin cells to transition from the synthetic regenerative phase towards a more quiescent resolving phase and thus avoid scarring. By disrupting the 'stop' signaling, the skin matrix remained in an active state with a recrudescence of inflammation after more than three months, resulting in a hypertrophic scar by six months. An open question remains as to whether the matrix is a result of rather than the initiator of the chronic inflammatory situation tat leads to scarring. This is convoluted in that the matrix is derived from the cells, but also signal back to these same cells, and that this directs specific factors to be secreted by these cells that further alters the cell function and matrix generation. This can be reduced to the question of whether changes in matrix composition could alter wound outcomes. The implications of this for cellular therapies are immense. If short term (days to two weeks) alterations in matrix can have prolonged effects, then transient, or even allogeneic cell transplantation can re-educate the resident cells to limit scarring without the need for long term survival of the transplanted cells. Thus, we hypothesize that during wound resolution, the matrix transition from an immature phase directs the resident cells to differentiate, and change their secreted communications, and thereby limits scarring. This will be tested in tissue culture, skin organ cultures and animal models of wound healing. While the matrix contains components that function redundantly, negating the strategy of molecular deletion, the presence of dominant actors can be used experimentally or therapeutically as in the following Aims:
Aim I. That immature matrix proteins prolong the regenerative phase and promote inefficient healing. The onco-fetal-wound ECM component Tenascin C confers both pro-migratory and -proliferative properties as well as protects endothelial and stromal cells from death. Thus, persistence of this multifunctional signaler would be predicted to prolong the hyperplasia and hypervascularity that results in scarring. This will be determined at both the tissue level and the molecular signals that drive both cell activation and survival and modulates the inflammatory response.
Aim II. That suppressive signals of the mature matrix can limit hypertrophic scarring. During the transition to resolution, mature ECM components shift formed elements towards differentiation and even heightened sensitivity to death signals from chemokines, CXCR3 ligands included. These signals come from both structural elements such as fibrillar Collagen I and growth factor interfering molecules such as the small leucine rich proteoglycan Decorin. These in turn are postulated to alter the milieu towards one of regeneration and cellular quiescence. This will be assessed in both ex vivo organotypic and in vivo animal models. These studies investigate a novel hypothesis using innovative organotypic models and a unique animal model of skin hypertrophic scar. This will yield a fuller understanding of the role of the matrix in wound maturation and scarring, suggesting new approaches to limit excessive repair or assist stalled healing. Further, by targeting cell transplantation and matrix composition, this would open underutilized avenues for directed therapies.

Public Health Relevance

Regenerative wound repair requires resolution of the robust cellular responses to injury to restore a functional tissue; persistence of the proliferative and migratory phase of healing leads to scarring that results in a weakened and debilitated tissue. Testing the proposed foundational model of matrix-derived cues as determining cellular behaviors and responses and healing fate would provide novel insights in the basic tissue biology of wound repair and allow for future development of smart matrices and cell transplantation strategies that could harness this information to direct wound healing towards a more regenerative outcome with improved tissue function.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM063569-12
Application #
9388367
Study Section
Surgery, Anesthesiology and Trauma Study Section (SAT)
Program Officer
Somers, Scott D
Project Start
2003-07-01
Project End
2018-11-30
Budget Start
2017-12-01
Budget End
2018-11-30
Support Year
12
Fiscal Year
2018
Total Cost
Indirect Cost
Name
University of Pittsburgh
Department
Pathology
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Shao, Hanshuang; Lauffenburger, Douglas; Wells, Alan (2017) Tyro3 carboxyl terminal region confers stability and contains the autophosphorylation sites. Biochem Biophys Res Commun 490:1074-1079
Yates, Cecelia C; Nuschke, Austin; Rodrigues, Melanie et al. (2017) Improved Transplanted Stem Cell Survival in a Polymer Gel Supplemented With Tenascin C Accelerates Healing and Reduces Scarring of Murine Skin Wounds. Cell Transplant 26:103-113
Shao, Hanshuang; Wang, Anna; Lauffenburger, Douglas et al. (2017) Tyro3-mediated phosphorylation of ACTN4 at tyrosines is FAK-dependent and decreases susceptibility to cleavage by m-Calpain. Int J Biochem Cell Biol 95:73-84
Yates, Cecelia C; Rodrigues, Melanie; Nuschke, Austin et al. (2017) Multipotent stromal cells/mesenchymal stem cells and fibroblasts combine to minimize skin hypertrophic scarring. Stem Cell Res Ther 8:193
Nuschke, Austin; Rodrigues, Melanie; Wells, Albin W et al. (2016) Mesenchymal stem cells/multipotent stromal cells (MSCs) are glycolytic and thus glucose is a limiting factor of in vitro models of MSC starvation. Stem Cell Res Ther 7:179
Nuschke, Austin; Rodrigues, Melanie; Rivera, Jaime et al. (2016) Epidermal Growth Factor Tethered to ?-Tricalcium Phosphate Bone Scaffolds via a High-Affinity Binding Peptide Enhances Survival of Human Mesenchymal Stem Cells/Multipotent Stromal Cells in an Immune-Competent Parafascial Implantation Assay in Mice. Stem Cells Transl Med 5:1580-1586
Bodnar, Richard J; Satish, Latha; Yates, Cecelia C et al. (2016) Pericytes: A newly recognized player in wound healing. Wound Repair Regen 24:204-14
Wells, Alan; Nuschke, Austin; Yates, Cecelia C (2016) Skin tissue repair: Matrix microenvironmental influences. Matrix Biol 49:25-36
Ma, Bo; Khazali, Ahmad; Wells, Alan (2015) CXCR3 in carcinoma progression. Histol Histopathol 30:781-92
Bodnar, Richard J; Wells, Alan (2015) Differential regulation of pericyte function by the CXC receptor 3. Wound Repair Regen 23:785-96

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