Cecelia C. Yates

IP-10 Blocks Vascular Endothelial Growth Factor-Induced Endothelial Cell Motility and Tube Formation via Inhibition of Calpain

Angiogenesis plays a critical role in wound repair. Endothelial cells present CXC receptor 3 (CXCR3) for chemokines expressed late in wound regeneration. To understand the physiological role CXCR3 plays in regulating endothelial function, we analyzed the ability of a CXCR3 ligand, IP-10 (CXCL10), to influence endothelial cell tube formation. Treatment of endothelial cells with IP-10 in the presence of vascular endothelial growth factor (VEGF) inhibited tube formation on growth factor-reduced Matrigel and in a subcutaneous Matrigel plug. Furthermore, IP-10 significantly inhibited VEGF-induced endothelial motility, a response critical for angiogenesis. Previous work showed that CXCR3 ligandation initiates protein kinase A (PKA) phosphorylation-dependent inhibition of m-calpain, required for induced cell motility, in fibroblasts but not epithelial cells. Here we show that CXCR3 activation in endothelial cells induces an increase in cAMP and PKA activation. Treatment of endothelial cells with Rp-8-Br-cAMP, an inhibitor of PKA, or small interference RNA to PKA was able to reverse the inhibitory effects of IP-10 on VEGF-mediated tube formation and motility. Importantly, treatment of endothelial cells with VEGF induced the activation of m-calpain, but costimulation with IP-10 significantly decreased this activity. Using Rp-8-Br-cAMP, we show blocking PKA reversed the IP-10 inhibition of VEGF-induced m-calpain activity. These data indicate that the activation of CXCR3 inhibits endothelial tube formation through a PKA mediated inhibition of m-calpain. This provides a means by which late wound repair signals limit the angiogenesis driven early in the wound response process.

Skin Wound Healing and Scarring: Fetal Wounds and Regenerative Restitution

The adverse physiological and psychological effects of scars formation after healing of wounds are broad and a major medical problem for patients. In utero, fetal wounds heal in a regenerative manner, though the mechanisms are unknown. Differences in fetal scarless regeneration and adult repair can provide key insight into reduction of scarring therapy. Understanding the cellular and extracellular matrix alterations in excessive adult scarring in comparison to fetal scarless healing may have important implications. Herein, we propose that matrix can be controlled via cellular therapy to resemble a fetal-like matrix that will result in reduced scarring.

Delayed reepithelialization and basement membrane regeneration after wounding in mice lacking CXCR3.

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.

ELR-Negative CXC Chemokine CXCL11 (IP-9/I-TAC) Facilitates Dermal and Epidermal Maturation during Wound Repair

In skin wounds, the chemokine CXCR3 receptor appears to play a key role in coordinating the switch from regeneration of the ontogenically distinct mesenchymal and epithelial compartments toward maturation. However, because CXCR3 equivalently binds four different ELR-devoid CXC chemokines (ie, PF4/CXCL4, IP-10/CXCL10, MIG/CXCL9, and IP-9/CXCL11), we sought to identify the ligand that coordinates epidermal coverage with the maturation of the underlying superficial dermis. Because CXCL11 (IP-9 or I-TAC) is produced by redifferentiating keratinocytes late in the regenerative phase when re-epithelialization is completed and matrix maturation ensues, we generated mice in which an antisense construct (IP-9AS) eliminated IP-9 expression during the wound-healing process. Both full and partial thickness excisional wounds were created and analyzed histologically throughout a 2-month period. Wound healing was impaired in the IP-9AS mice, with a hypercellular and immature dermis noted even after 60 days. Re-epithelialization was delayed with a deficient delineating basement membrane persisting in mice expressing the IP-9AS construct. Provisional matrix components persisted in the dermis, and the mature basement membrane components laminin V and collagen IV were severely diminished. Interestingly, the inflammatory response was not diminished despite IP-9/I-TAC being chemotactic for such cells. We conclude that IP-9 is a key ligand in the CXCR3 signaling system for wound repair, promoting re-epithelialization and modulating the maturation of the superficial dermis.

Skin tissue repair: Matrix microenvironmental influences.

The process of repair of wounded skin involves intricate orchestration not only between the epidermal and dermal compartments but also between the resident and immigrant cells and the local microenvironment. Only now are we beginning to appreciate the complex roles played by the matrix in directing the outcome of the repair processes, and how this impacts the signals from the various cells. Recent findings speak of dynamic and reciprocal interactions that occurs among the matrix, growth factors, and cells that underlies this integrated process. Further confounding this integration are the physiologic and pathologic situations that directly alter the matrix to impart at least part of the dysrepair that occurs. These topics will be discussed with a call for innovative model systems of direct relevance to the human situation.

Matrix control of scarring.

Repair of wounds usually results in restoration of organ function, even if suboptimal. However, in a minority of situations, the healing process leads to significant scarring that hampers homeostasis and leaves the tissue compromised. This scar is characterized by an excess of matrix deposition that remains poorly organized and weakened. While we know much of the early stages of the repair process, the transition to wound resolution that limits scar formation is poorly understood. This is particularly true of the inducers of scar formation. Here, we present a hypothesis that it is the matrix itself that is a primary driver of scar, rather than being simply the result of other cellular dysregulations.

Shifts in macrophage phenotype at the biomaterial interface via IL-4 eluting coatings are associated with improved implant integration.

The present study tests the hypothesis that transient, early-stage shifts in macrophage polarization at the tissue-implant interface from a pro-inflammatory (M1) to an anti-inflammatory/regulatory (M2) phenotype mitigates the host inflammatory reaction against a non-degradable polypropylene mesh material and improves implant integration downstream. To address this hypothesis, a nanometer-thickness coating capable of releasing IL-4 (an M2 polarizing cytokine) from an implant surface at early stages of the host response has been developed. Results of XPS, ATR-FTIR and Alcian blue staining confirmed the presence of a uniform, conformal coating consisting of chitosan and dermatan sulfate. Immunolabeling showed uniform loading of IL-4 throughout the surface of the implant. ELISA assays revealed that the amount and release time of IL-4 from coated implants were tunable based upon the number of coating bilayers and that release followed a power law dependence profile. In-vitro macrophage culture assays showed that implants coated with IL-4 promoted polarization to an M2 phenotype, demonstrating maintenance of IL-4 bioactivity following processing and sterilization. Finally, in-vivo studies showed that mice with IL-4 coated implants had increased percentages of M2 macrophages and decreased percentages of M1 macrophages at the tissue-implant interface during early stages of the host response. These changes were correlated with diminished formation of fibrotic capsule surrounding the implant and improved tissue integration downstream. The results of this study demonstrate a versatile cytokine delivery system for shifting early-stage macrophage polarization at the tissue-implant interface of a non-degradable material and suggest that modulation of the innate immune reaction at early stages of the host response may represent a preferred strategy for promoting biomaterial integration and success.

Pericytes: A newly recognized player in 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.

MMI-0100 inhibits cardiac fibrosis in myocardial infarction by direct actions on cardiomyocytes and fibroblasts via MK2 inhibition

The cell-permeant peptide inhibitor of MAPKAP kinase 2 (MK2), MMI-0100, inhibits MK2 and downstream fibrosis and inflammation. Recent studies have demonstrated that MMI-0100 reduces intimal hyperplasia in a mouse vein graft model, pulmonary fibrosis in a murine bleomycin-induced model and development of adhesions in conjunction with abdominal surgery. MK2 is critical to the pathogenesis of ischemic heart injury as MK2(-/-) mice are resistant to ischemic remodeling. Therefore, we tested the hypothesis that inhibiting MK2 with MMI-0100 would protect the heart after acute myocardial infarction (AMI) in vivo. AMI was induced by placing a permanent LAD coronary ligation. When MMI-0100 peptide was given 30 min after permanent LAD coronary artery ligation, the resulting fibrosis was reduced/prevented ~50% at a 2 week time point, with a corresponding improvement in cardiac function and decrease in left ventricular dilation. In cultured cardiomyocytes and fibroblasts, MMI-0100 inhibited MK2 to reduce cardiomyocyte caspase 3/7 activity, while enhancing primary cardiac fibroblast caspase 3/7 activity, which may explain MMI-0100s salvage of cardiac function and anti-fibrotic effects in vivo. These findings suggest that therapeutic inhibition of MK2 after acute MI, using rationally-designed cell-permeant peptides, inhibits cardiac fibrosis and maintains cardiac function by mechanisms that involve inhibiting cardiomyocyte apoptosis, while enhancing primary cardiac fibroblast cell death.

Transplanted fibroblasts prevents dysfunctional repair in a murine CXCR3-deficient scarring model.

In skin, the regeneration of the ontogenically distinct mesenchymal and epithelial compartments must proceed in a coordinated manner orchestrated by extracellular signaling networks. We have recently found that the switch from regeneration to remodeling during repair is modulated by chemokines that bind CXCR3 receptor. If this signaling is disrupted wounds continue to be active, resulting in a chronic hypercellular and hypertrophic state characterized by an immature matrix composition. As healing is masterminded in large part by fibroblasts and their synthesis of the extracellular matrix, the question arose as to whether this ongoing scarring can be modulated by transplanted fibroblasts. We examined wounds in the CXCR3-/-mouse scarring model. These wounds exhibited a significant delay in healing in all areas compared to young and aged wild-type mice. Full-thickness wounds were transplanted with fibroblasts derived from newborn CXCR3-/- or wild-type mice. The transplanted fibroblasts were labeled with fluorescent dye (CM-DiI) and suspended in hyaluronic acid gel; by 30 days, these transplanted cells comprised some 30% of the dermal stromal cells regardless of the host or source of transplanted cells. Wild-type fibroblasts transplanted into CXCR3-/- mice wounds reversed the delay and dysfunction previously seen in CXCR3-/- wounds; this correction was not noted with transplanted CXCR3-/- fibroblasts. Additionally, transplant of CXCR3-/-cells into wounds in wild-type animals did not adversely affect those wounds. The transplanted fibroblasts exhibited strong survival and migration patterns and led to an increase in tensile strength. Expression of matrix proteins and collagen in CXCR3-/- wounds transplanted with wild-type fibroblasts resembled normal wild-type healing, and the wound matrix in wild-type mice transplanted with CXCR3-/- cells also presented a mature matrix. These suggest that the major determinant of healing versus scarring lies with the nature of the matrix. These findings have intriguing implications for rational cellular interventions aimed at promoting wound healing via cell therapy.