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Cutaneous wound healing is an intricate orchestration of three overlapping phases of repair that encompass numerous cell types, signalling cascades, and microenvironment modifications to reach a successful resolution. Disruption of any of these steps will create an abnormal healing response resulting in either ulceration or excessive scarring. It has become evident that the extracellular matrix and its associated components are key orchestrators during this process. One of these essential matrix proteins is decorin, a small leucine-rich proteoglycan (SLRP) that acts as a regulator of collagen fibrillogenesis and a non-competitive inhibitor of multiple growth factors signalling cascades. Decorin is a necessary shut-off switch for the pro-reparative mechanism of the tissue replacement phase and limits the occurrence of hypertrophic scarring by preventing excessive repair. We investigated the use of decorin as a therapeutic by administering the matrix protein anchored in a slow-release coacervate in a hypertrophic scarring mouse model. The results show that early wound healing phase measurements exhibit little difference in performance compared to our coacervate-only baseline or HB-EGF-treated control mice. However, during the resolution phase of wound healing, the decorin-treatment significantly reduces cutaneous thickness, enhances collagen alignment, and improves overall wound scoring in the mice. Thus, mice treated with decorin display better healing outcomes and could limit the hypertrophic scarring phenotype in the coacervate only, and HB-EGF controls. These results suggest that decorin may be a promising tool and alternative therapy for patients who suffer from over-exuberant matrix deposition during wound healing.

Pericytes have generally been considered in the context of stabilizing vessels, ensuring the blood barriers, and regulating the flow through capillaries. However, new reports suggest that pericytes may function at critical times to either drive healing with minimal scarring or, perversely, contribute to fibrosis and ongoing scar formation. Beneficially, pericytes probably drive much of the vascular involution that occurs during the transition from the regenerative to the resolution phases of healing. Pathologically, pericytes can assume a fibrotic phenotype and promote scarring. This perspective will discuss pericyte involvement in wound repair and the relationship pericytes form with the parenchymal cells of the skin. We will further evaluate the role pericytes may have in disease progression in relation to chronic wounds and fibrosis.

Pericytes are mural cell that have been found to play important roles in promoting blood vessel development and regulating blood flow. The signals that attract pericytes to maturing vessels during the resolution phase of wound healing are unknown. In this study, we examine the role of the chemokine receptor CXC receptor 3 (CXCR3) ligands, as they are produced by maturing endothelial cells. Pericytes isolated from muscle and retina were found to by and large only express the B-isoform of CXCR3 (CXCR3B), with expression being independent of the mitotic state of the cells. Pericyte stimulation with the CXCR3 ligands Mig (CXCL9), IP-9/I-TAC (CXCL11), or IP-10 (CXCL10) resulted in the activation of ERK but not AKT. Treatment with Mig or IP-9, but not IP-10, enhanced p38(MAPK) phosphorylation. Interestingly, while cyclic adenosine monophosphate is generated downstream of CXCR3B in other cells, protein kinase A activation was not observed in these pericytes when treated with these three CXCR3 ligands. The increase in ERK activity resulted in a slight increase in cell transmigration, with the inhibition of ERK leading to a decrease in CXCR3B mediated migration and inhibition of p38(MAPK) reducing transmigration through small pores. These ligands did not affect proliferation. These data are the first to characterize CXCR3B as the predominant isoform expressed on pericytes, and was found on these diverse cells isolated from both muscle and eye. We also show that CXCR3B signaling stimulates transmigration of barrier pores in pericytes as opposed to its inhibitory affects on endothelial cells and fibroblasts. These findings characterize a novel role for the CXCR3B in regulating cellular function. Taken together these data show a role for CXCR3B in regulating pericyte function.

Wound healing is a complex, orchestrated series of biological events that is controlled by extracellular components that communicate between cell types to re-establish lost tissue. We have found that signaling by ELR-negative CXC chemokines through their common CXCR3 receptor is critical for dermal maturation during the resolving phase. In addition there needs to be complete maturation of the epidermis and regeneration of a delineating basement membrane for proper functioning. The role of this ligand-receptor system appears confounding as one ligand, CXCL4/(PF4), is present during the initial dissolution and two others, CXCL10/(IP-10) and CXCL11/(IP-9/I-TAC), are expressed by keratinocytes in the later regenerative and resolving phases during which the basement membrane is re-established. We examined CXCR3 signaling role in healing using a mouse lacking this receptor, as all three ligands act solely via the common receptor. Reepithelialization was delayed in CXCR3-deficient mice in both full and partial-thickness excisional wounds. Even at 90 days postwounding, the epidermis of these mice appeared less mature with lower levels of E-cadherin and cytokeratin 18. The underlying basement membrane, a product of both dermal fibroblasts and epidermal keratinocytes, was not fully established with persistent diffuse expression of the matrix components laminin 5, collagen IV, and collagen VII throughout the wound bed. These results suggest that CXCR3 and its ligands play an important role in the re-establishment of the basement membrane and epidermis. These studies further establish the emerging signaling network that involves the CXCR3 chemokine receptor and its ligands as a key regulator of wound repair.

Angiogenesis plays a critical role in wound healing. In wound healing new blood vessels are generated, then later in scar-resolving stages of the repair process, these newly formed blood vessels undergo destabilization and significant regression. ELR-negative chemokines have been shown to have angiostatic properties and are produced in high concentration late in the granulation tissue and resolving stage of wound repair. The receptor for the ELR-negative chemokines, CXCR3, is expressed on endothelial cells but its function is not known. To understand the physiologic role CXCR3 plays in regulating endothelial function, we analyzed the ability of the CXCR3 ligand, IP-10, to inhibit endothelial cell tube formation. IP-10 was able to inhibit endothelial cell tube formation alone and prevent VEGF-induced tube formation on Matrigel®. Treatment of endothelial cells with IP-10 significantly inhibited endothelial motility. Thus, the ELR-negative chemokines inhibit angiogenesis by inhibiting endothelial motility. To identify the signaling pathway(s) mediated by CXCR3 that inhibit endothelial cell motility, we looked at the regulation of key processes. As CXCR3 is a receptor known to generate cAMP, as we show in these cells, we examined PKA-mediated inhibition of calpain. Treatment of endothelial cells with VEGF induced the activation of m-calpain but costimulation with IP-10 significantly decreased m-calpain activity. Using cAMP analogs, 8-Br-cAMP, a PKA activator, is able to inhibit VEGF- induced motility and Rp-8-Br-cAMP, a PKA inhibitor, is able to reverse the inhibitory effects of IP-10 on endothelial migration and calpain activation. To further demonstrate that m-calpain is important in endothelial motility we show that siRNA knockdown of m-calpain inhibited VEGF induced migration. These data indicate that activation of CXCR3 inhibits endothelial migration through the PKA-mediated inhibition of m-calpain. These findings provide new targets to modulate the resolution phase of wound repair.

We find that the ELR-negative CXC chemokines, CXCL10 (IP-10) and CXCL11(IP-9), expressed during wound repair, with IP-9 being produced by redifferentiated keratinocyte. Immunohistochemical analysis for IP-9 on human full thickness wound tissues collected from day 5 confirmed that IP-9 is a wound response protein. We have shown in in vitro coculture systems that IP-9 can serve as an autocrine factor promoting motility in keratinocyte while limiting motility in dermal fibroblasts. These effects occur via modulation of the intracellular proteases calpain isoforms that regulate cell adhesion during motility. Thus, the function of these chemokines in vivo is not obvious. To probe this, we have explored wound repair in mice lacking the receptor for these ligands, CXCR3. Quantitative analysis of IP-9 expression in wounds of these mice collected from day 2 to day 20 showed a significant increase in IP-9 levels in CXCR3-/- mice compared to wild type, which is expected if receptor-mediated feedback attenuation is lost. Full and partial thickness wound healing experiments on these mice showed a significant delay in CXCR3-/- mice when compared to wild type. Histochemical analysis of tissues collected from both full and partial thickness wounds demonstrated differences in epithelialization, granulation tissue formation, and angiogenesis in CXCR3-/- mice when compared to wild type. Collagen, the major extracellular matrix protein organization were analyzed by trichrome and picrosirus red staining methods. We are also examining the effect of this lost receptor on promotility signaling from EGF receptors. These in vivo and in vitro studies confirm the pathophysiologic role of the chemokines IP-9 and IP-10 and their cognate receptor CXCR3 during wound repair.

Recently, extracellular matrix components have been shown to contain domains that can interact with and activate receptors with intrinsic tyrosine kinase activity. These receptor tyrosine kinases are strong mediators of the cell responses of proliferation, migration, differentiation, and dedifferentiation. However, an interesting question is raised as to why cells would present growth factor receptor ligands in such a manner, as the majority of growth factors are small, soluble, or only transiently tethered ligands. With the exception of the discoidin domain receptors that bind collagen, the other described domains interact with a receptor that binds ubiquitous soluble peptide growth factors, the epidermal growth factor receptor. Unlike traditional growth factors, these individual "matrikine" domains within tenascin-C, laminin, collagen, and decorin possess relatively low binding affinity (high nanomolar or micromolar) and are often presented in multiple valency. The presentation of ligands within the extracellular matrix in this fashion might allow for unique biochemical and physiological outcomes. This new class of "matrikine" ligand may be critical for wound healing, as the majority of known extracellular matrix components possessing matrikines play a strong role, or are presented uniquely, during skin repair. Tenascin-C expression, for instance, is uniquely regulated spatially and has been proposed to present pro-migratory tracks during skin repair through its epidermal growth factor-like repeats. The epidermal growth factor-like repeats of laminin-5 act as cryptic ligands revealed upon matrix metalloproteinase-2 degradation of the surrounding extracellular matrix. The deletion of the discoidin domain receptors 1 and 2 for collagen have negative consequences on the role of fibroblasts and epithelial cells for matrix metalloproteinase production, migration, proliferation, and extracellular matrix turnover. Finally, decorin can bind to, inhibit, and down-regulate epidermal growth factor receptor levels and signaling, suggesting a tonic role of the epidermal growth factor binding domain of decorin in the resolution of wound healing. We provide a model framework for further studies into this emerging class of signals.

Keloids, which overgrow the boundaries of the original injury, represent aberrations in the fundamental process of wound healing that include over-abundant cell in-migration, cell proliferation, and inflammation, as well as increased extracellular matrix synthesis and defective remodeling. To understand the key events that result in the formation of these abnormal scars would open new avenues for better understanding of excessive repair, and might provide new therapeutic options. We examined epidermal growth factor receptor (EGFR)-induced cell motility in keloid fibroblasts, as this receptor initiates cell migration during normal wound repair. We show that keloid fibroblasts respond to EGF-induced cell migration but the response is somewhat diminished compared to normal adult fibroblasts (approximately 30% reduced); the mitogenic response was similarly blunted (approximately 5% reduced). Keloid fibroblasts express near normal levels of EGFR (82%), but show a much more attenuated activation of EGFR itself and the motility-associated phospholipase C-gamma. This was reflected in part by rapid loss of EGFR upon exposure to EGF. Interestingly, while extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK-MAPK) activation was relatively robust in keloid fibroblasts, the downstream triggering of the motility-associated calpain activity was blunted. This was reflected by high cell-substratum adhesiveness in the keloid fibroblasts. Thus, the blunted migratory response to EGF noted in keloid fibroblasts appears due to limited activation of two important biochemical switches for cell motility.

Keloids are scars that overgrow the original boundaries of the injury, involving aberrant functioning of the fibroblasts. We have found that during normal wound healing the ELR-negative CXC chemokines IP-9 (CXCL11) and IP-10 (CXCL10) act to limit fibroblast immigration. Our initial goal was to determine if keloid fibroblasts failed to respond to this ‘stop’ signal. Immunohistochemical and immunocytochemical studies on keloid fibroblasts show that these lesions express excessive amounts of IP-9 and CXCR3 receptor. The protein levels of CXCR3 receptor was also not dissimilar between normal and keloid fibroblasts. Though, the expression of the protein and the receptor seems to be normal, the fibroblasts derived from these abnormal lesions did not respond to IP-9 during EGF induced cell migration. We have extended these findings to determine whether other genes are differentially expressed by keloid fibroblasts. The immediate first gene that interest us was the mRNA levels of CXCR3 receptor (GPR9) wherein, we did not find any significant difference between normal and keloid fibroblasts. Further, we also found interestingly, that genes related to tumor progression are up regulated in keloid fibroblasts than the normal fibroblasts. The marker genes like calgranulin, BCL-2 associated anthogen 4 (BAG4), and dual specificity phosphatase 1 (DUSP1). Certain transcription factors like GATA-6 and SpiB are also increased. Apart, the classical growth factor and growth factor receptor related genes which includes, bone morphogenetic protein BMP-1, heparin binding EGF like growth factor, EGF like repeats discoidin1, IGFBP-5 and 3, EphB1&B2, tumor necrosis factor alpha induced protein, protein tyrosine phosphatase receptor, serine threonine kinase 6 along with chondroitin sulfate proteoglycan brevican and versican and vitronectin are also up regulated. The newl identified genes will open new avenues in targeting keloid lesions by understanding the basic signaling mechanism of these specific proteins. This work was supported by grants from NIH/NIGMS and PPRTP.