Patricia A. Hebda

A Patient-Specific in silico Model of Inflammation and Healing Tested in Acute Vocal Fold Injury

The development of personalized medicine is a primary objective of the medical community and increasingly also of funding and registration agencies. Modeling is generally perceived as a key enabling tool to target this goal. Agent-Based Models (ABMs) have previously been used to simulate inflammation at various scales up to the whole-organism level. We extended this approach to the case of a novel, patient-specific ABM that we generated for vocal fold inflammation, with the ultimate goal of identifying individually optimized treatments. ABM simulations reproduced trajectories of inflammatory mediators in laryngeal secretions of individuals subjected to experimental phonotrauma up to 4 hrs post-injury, and predicted the levels of inflammatory mediators 24 hrs post-injury. Subject-specific simulations also predicted different outcomes from behavioral treatment regimens to which subjects had not been exposed. We propose that this translational application of computational modeling could be used to design patient-specific therapies for the larynx, and will serve as a paradigm for future extension to other clinical domains.

Prostaglandin E2 inhibition of keloid fibroblast migration, contraction, and transforming growth factor (TGF)-β1–induced collagen synthesis

Keloid formation has been linked to aberrant fibroblast activity, exacerbated by growth factors and inflammatory mediators. Prostaglandin E2 (PGE2), synthesized from arachidonic acid by cyclooxygenases (COX) and synthases (PGES), acts as both an inflammatory mediator and fibroblast modulator. Although PGE2 has known antifibrotic effects in the lower airway, its role in dermal fibrosis in general, and keloid formation in particular, remains unclear. This study focused on: (1) the effects of PGE2 on keloid fibroblast migration, contraction, and collagen synthesis and (2) endogenous PGE2 synthesis in response interleukin‐1β. PGE2 decreased keloid fibroblast migration and contraction via an EP2/EP4–cAMP mechanism that disrupted actin cytoskeletal dynamics and reversed transforming growth factor‐β1–induced collagen I and III synthesis. Impaired fibroblast PGE2 production has been linked to lower airway fibrosis and recently to keloid formation. Here, we showed that interleukin‐1β stimulation leads to nuclear factor‐κB translocation to the nucleus, resulting in up‐regulation of COX‐2 and microsomal PGE2 synthase 1. Up‐regulation of COX‐2 in, and secretion of PGE2 by keloid fibroblasts are diminished compared with their normal fibroblast counterparts. We suggest that the antifibrotic effects of PGE2 during keloid formation are potentially diminished due to aberrant paracrine fibroblast signaling. Exogenous PGE2 may supplement decreased endogenous levels and inhibit keloid formation or progression.

Mucosal Biofilm Formation on Middle-Ear Mucosa in a Nonhuman Primate Model of Chronic Suppurative Otitis Media†

Background: An increased awareness of bacterial biofilms and their formation has led to a better understanding of bacterial infections that occur in the middle ear. Perhaps the best studied pathogen for its propensity toward biofilm formation is Pseudomonas aeruginosa, also the primary pathogen in chronic suppurative otitis media (CSOM).

Acute Vocal Fold Wound Healing in a Rabbit Model

Several authors have eloquently described the characteristics of vocal fold scar, a long-term consequence of vocal fold injury. However, events in the acute stage of mucosal injury, which lead up to fibrosis, have been largely overlooked. The current study describes acute events with regard to mucosal re-formation in a rabbit model. Vocal fold injury was induced surgically. A fibrinous clot was present 1 day after injury. Massive cellular infiltration was noted on day 3, and complete epithelial coverage was achieved by day 5. Also, neo-matrix deposition was noted as early as 5 days after injury, and more mature collagen was seen by day 7. The general timetable described in the current study can contribute to the experimental foundation for the development of regenerative models of healing in the vocal folds. Most notably, the proliferation phase of wound healing appears to occur approximately 3 days after injury, indicating a critical time for intervention. Manipulation and/or alteration of naturally occurring neo-matrix deposition and organization may yield improved biophysical function of the injured vocal fold.

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.

Repair of the tympanic membrane with urinary bladder matrix

To test urinary bladder matrix (UBM) as a potential treatment for tympanic membrane (TM) healing and regeneration.

Gene expression patterns in isolated keloid fibroblasts

Keloid scars after skin trauma are a significant clinical problem, especially in black populations, in which the incidence of keloids has been estimated at 4–16%. Keloids are abnormal dermal proliferative scars secondary to dysregulated wound healing. Despite several biochemical studies on the role of extracellular matrix proteins and growth factors during keloid formation, we still do not know what molecules and signals induce this change. Fibroblasts are thought to be the major inductive cell for keloid scar formation. The aim of this study was to identify gene expression patterns that characterize keloid fibroblasts; identifying such genetic disequilibrium may shed light on the molecular signaling events responsible for keloid formation. In this study, we performed gene expression analysis of fibroblasts isolated from keloid lesions from three individuals in comparison with the fibroblasts isolated from normal skin using the Affymetrix U133a chip (22,284 genes and expression sequence tags). We found through J5 test score expression analysis that among 22,284 genes, there were 43 genes that were overexpressed and five genes were underexpressed in keloid fibroblasts when compared with dermal fibroblasts from persons without keloids. The overexpression of three genes not previously reported as being up‐regulated in keloids (annexin A2, Transgelin, and RPS18) was confirmed by real‐time polymerase chain reaction. Certain overexpressed genes were similar to previous biochemical observations on the protein levels of these overexpressed genes during keloid formation. We also report for the first time that a few tumor‐related genes are overexpressed in keloid fibroblasts.

Prostaglandin E2 differentially modulates human fetal and adult dermal fibroblast migration and contraction: implication for wound healing.

Cyclooxygenase‐2 is up‐regulated shortly after dermal injury and it has been shown to have important activity during the repair process. Its main product in the skin, prostaglandin E2 (PGE2), modulates both inflammatory and fibrotic processes during wound healing and partially dictates the overall outcome of wound healing. PGE2 signaling has been shown to be altered during fetal wound healing. This study was designed to examine the mechanism(s) by which PGE2 regulates fibroblast migration and contraction and to determine whether these mechanisms are conserved in fetal‐derived dermal fibroblasts. Fetal and adult dermal fibroblasts express all four PGE2 receptors. PGE2 inhibits fetal and adult fibroblast migration in a dose‐dependent manner through the EP2/EP4–cAMP–protein kinase A pathway. However, fetal fibroblasts appear to be refractory to this effect, requiring a 10‐fold higher concentration of PGE2 to achieve a similar degree of inhibition as adult fibroblasts. Inhibition of adult fibroblast migration correlated with disruption of the actin cytoskeleton. In contrast, PGE2 or a cAMP analog did not disrupt the actin cytoskeleton of fetal dermal fibroblasts. These findings were extended using a modified free‐floating, fibroblast‐populated collagen lattice (FPCL) contraction assay designed to measure fibroblast contraction. PGE2‐inhibited FPCL contraction by adult fibroblasts, but fetal fibroblasts exhibited higher rates of FPCL contraction and a blunted response to exogenous modulation by PGE2 or a cyclase activator (forskolin). These findings indicate that fetal dermal fibroblasts are partially refractory to the effects of PGE2, a major inflammatory mediator associated with dermal wound healing. This effect may have significant and specific relevance to the scarless fetal wound‐healing phenotype.

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.