Howard Levinson

Discoidin domain receptors and their ligand, collagen, are temporally regulated in fetal rat fibroblasts in vitro.

The biochemical regulation of collagen deposition during adult cutaneous wound repair is poorly understood. Likewise, how collagen is perceived and modulated in fetal scarless healing remains unknown. Recently, discoidin domain receptors-1 and 2 (DDR1 and DDR2) with tyrosine kinase activity have been identified as novel receptors for collagen. In light of these findings, it was speculated that the production of collagen receptors DDR1 and DDR2 by fetal fibroblasts may be temporally regulated to correlate with the ontogeny of embryonic scar formation. More specifically, because DDRs directly bind collagen and transmit the signals intracellularly, it was hypothesized that they may play an important role in fetal scarless healing by ultimately regulating and modulating collagen production and organization. As part of a fundamental assessment to elucidate the role of DDRs in scarless fetal wound repair, the endogenous expression of DDR1, DDR2, collagen I, and total collagen, as a function of fetal Sprague-Dawley rat skin fibroblasts of different gestational ages, representing scar-free (E16.5) periods was determined. Using explanted dermal fibroblasts of gestational days E13.5, E16.5, E18.5, and E21.5 (term gestation = 21.5 days) fetuses (n = 92), [3H]proline incorporation assay and Northern and Western blotting analysis were performed to compare the expressions of these molecules with scar-free and scar-forming stages of embryonic development. These results revealed a pattern of increasing collagen production with increasing gestational ages, whereas DDR1 expression decreased with increasing gestational age. This observation suggests that elevated levels of DDR1 may play an important role in scarless tissue regeneration by early gestation fetal fibroblasts. In contrast, DDR2 was expressed by fetal rat fibroblasts at a similar level throughout gestation. These data demonstrate for the first time the temporal expression of collagen and DDR tyrosine kinases in fetal rat fibroblasts as a function of gestational ages. Overall, these data suggest that differential temporal expression of the above-mentioned molecules during fetal skin development may play an important role in the ontogeny of scar formation. Future studies will involve the characterization of the biomolecular functions of these receptor kinases during fetal wound repair.

Differential expression of receptor tyrosine kinases and Shc in fetal and adult rat fibroblasts: toward defining scarless versus scarring fibroblast phenotypes.

The remarkable ability of the fetus to heal early gestation skin wounds without scarring remains poorly understood. Taking advantage of recent advances in signal transduction, the tyrosine phosphorylation patterns of fetal rat fibroblasts, representing the scarless cutaneous repair phenotype, and adult rat fibroblasts, representing scar-forming phenotype, were examined whether there were inherent differences in cellular signaling. Specifically, correlation of the phosphorylation patterns with the expression levels of the signaling molecules that transmit information from the plasma membrane receptor to the nucleus was sought. By using three different cell lines of explanted fibroblasts from gestational day 13 fetal rat skin (n = 24) and 1-month-old postnatal adult rat skin (n = 3), immunoblotting was performed to compare tyrosine phosphorylation patterns. The results revealed five major protein bands of interest in fetal rat fibroblasts, but not in the adult rat fibroblasts. These phosphorylated protein bands are of interest because of their possible role in wound repair and may have the potential to regulate cellular responses to the extracellular matrix and their secondary signaling molecules. It was hypothesized that these bands represented receptor tyrosine kinases, epidermal growth factor receptor, and discoidin domain receptor 1, and their downstream adaptor protein Shc that binds receptor tyrosine kinases to transduce signals intracellularly. Furthermore, elevated expression of platelet-derived growth factor receptor-beta in adult compared with fetal fibroblasts was demonstrated, suggesting that decreased expression of certain growth factors may also be important for the scarless phenomenon to occur.

Implant healing in experimental animal models of diabetes.

Diabetes mellitus is becoming increasingly prevalent worldwide. Additionally, there is an increasing number of patients receiving implantable devices such as glucose sensors and orthopedic implants. Thus, it is likely that the number of diabetic patients receiving these devices will also increase. Even though implantable medical devices are considered biocompatible by the Food and Drug Administration, the adverse tissue healing that occurs adjacent to these foreign objects is a leading cause of their failure. This foreign body response leads to fibrosis, encapsulation of the device, and a reduction or cessation of device performance. A second adverse event is microbial infection of implanted devices, which can lead to persistent local and systemic infections and also exacerbates the fibrotic response. Nearly half of all nosocomial infections are associated with the presence of an indwelling medical device. Events associated with both the foreign body response and implant infection can necessitate device removal and may lead to amputation, which is associated with significant morbidity and cost. Diabetes mellitus is generally indicated as a risk factor for the infection of a variety of implants such as prosthetic joints, pacemakers, implantable cardioverter defibrillators, penile implants, and urinary catheters. Implant infection rates in diabetic patients vary depending upon the implant and the microorganism, however, for example, diabetes was found to be a significant variable associated with a nearly 7.2% infection rate for implantable cardioverter defibrillators by the microorganism Candida albicans. While research has elucidated many of the altered mechanisms of diabetic cutaneous wound healing, the internal healing adjacent to indwelling medical devices in a diabetic model has rarely been studied. Understanding this healing process is crucial to facilitating improved device design. The purpose of this article is to summarize the physiologic factors that Influence wound healing and infection in diabetic patients, to review research concerning diabetes and biomedical implants and device infection, and to critically analyze which diabetic animal model might be advantageous for assessing internal healing adjacent to implanted devices.

Functional optical coherence tomography: principles and progress

In the past decade, several functional extensions of optical coherence tomography (OCT) have emerged, and this review highlights key advances in instrumentation, theoretical analysis, signal processing and clinical application of these extensions. We review five principal extensions: Doppler OCT (DOCT), polarization-sensitive OCT (PS-OCT), optical coherence elastography (OCE), spectroscopic OCT (SOCT), and molecular imaging OCT. The former three have been further developed with studies in both ex vivo and in vivo human tissues. This review emphasizes the newer techniques of SOCT and molecular imaging OCT, which show excellent potential for clinical application but have yet to be well reviewed in the literature. SOCT elucidates tissue characteristics, such as oxygenation and carcinogenesis, by detecting wavelength-dependent absorption and scattering of light in tissues. While SOCT measures endogenous biochemical distributions, molecular imaging OCT detects exogenous molecular contrast agents. These newer advances in functional OCT broaden the potential clinical application of OCT by providing novel ways to understand tissue activity that cannot be accomplished by other current imaging methodologies.

Calmodulin‐myosin light chain kinase inhibition changes fibroblast‐populated collagen lattice contraction, cell migration, focal adhesion formation, and wound contraction

Wound healing requires fibroblast migration, synthesis of new extracellular matrix, and organization of that matrix, all of which depend upon myosin ATPase activation and subsequent cytoplasmic actin‐myosin contraction. Myosin ATPase activity is optimized by phosphorylation of myosin light chain at serine 19. Several different signaling pathways can perform that phosphorylation, the focus here is calcium saturated calmodulin dependent ‐myosin light chain kinase (CaM‐MLCK). It is proposed that CaM‐MLCK phosphorylation of myosin light chain and subsequent myosin ATPase activation affects granulation tissue fibroblast behavior and contributes to wound contraction. Myosin ATPase activity generates actin‐myosin contraction within fibroblasts. Myosin ATPase activity is involved in ATP‐induced cell contraction, the generation of focal adhesions, fibroblast migration, fibroblast populated collagen lattice (FPCL) contraction, and wound contraction. The MLCK inhibitors ML‐9 and ML‐7 inhibited ATP‐induced cell contraction, fibroblast migration, FA formation, and FPCL contraction. The calmodulin inhibitors W7 and fluphenazine blocked rat open wound contraction. In addition, fluphenazine delayed re‐epithelialization. These findings support the idea that fibroblast CaM‐MLCK activity is essential for tissue repair. We speculate that inhibition of CaM‐MLCK may reduce or prevent detrimental fibrotic contracture.

Cellular signaling by tyrosine phosphorylation in keloid and normal human dermal fibroblasts.

Keloids represent a dysregulated response to cutaneous wounding that results in disfiguring scars. Unique to humans, keloids are characterized by an accumulation of extracellular matrix components. The underlying molecular mechanisms of keloid pathogenesis, however, remain largely uncharacterized. Similarly, cellular signaling mechanisms, which may indicate inherent differences in the way keloid fibroblasts and normal human dermal fibroblasts interact with extracellular matrix or other cells, have not been investigated. As part of a fundamental assessment of cellular response to injury in keloid fibroblasts, phosphorylation studies were performed using three different keloid (n = 3) and normal human dermal (n = 3) fibroblast cell lines. These studies were undertaken to elucidate whether keloid and normal human dermal fibroblasts exhibit different tyrosine kinase activity. Initially, distinct tyrosine phosphorylation patterns of keloid and normal human dermal fibroblasts were demonstrated. Next, the phosphorylation patterns were correlated with known molecules that may be important to keloid pathogenesis. On the basis of molecular weight, it was hypothesized that the highly phosphorylated bands seen in keloid fibroblasts represented epidermal growth factor receptor (EGFR); discoidin domain receptor 1 (DDR1); and Shc, an adaptor protein known to bind many tyrosine kinases, including EGFR and DDR1. Individual immunoblotting using EGFR, DDR1, and Shc antibodies revealed greater expression in keloid fibroblasts compared with normal human dermal fibroblasts. These data substantiate for the first time the finding of greater phosphorylation by the above-mentioned molecules, which may be important in keloid pathogenesis.

Complex abdominal wall hernias: a new classification system and approach to management based on review of 133 consecutive patients.

Background:Plastic surgeons are increasingly involved in the repair of complex ventral hernias. Although this typically involves recurrent incisional hernias, operative strategies can be applied to most abdominal wall defects, including chronic wounds with or without exposed mesh, enterocutaneous fistulas, or hernias associated with significant pannus formation. Methods:This is a retrospective review of a single institution/single surgeon experience of complex ventral hernia repair performed over a 5-year period. Patients were classified into different hernia types based on their characteristics and underwent hernia repair according to the presented algorithm. Results:A total of 133 patients underwent a complex ventral hernia repair between January 2005 and September 2009. The separation of components technique was used in the majority of cases. Permanent or biologic mesh was added in select patients. Adjunctive procedures were performed as indicated. The majority of short-term (less than 1 year) recurrences occurred in patients expected to have impaired wound healing due to comorbid conditions. In these patients, the recurrence rate was reduced when autologous repair was reinforced with mesh. Conclusion:Autologous tissue is the preferred method for reconstruction of complex ventral hernias. In certain instances, such as contamination, use of an acellular dermal matrix mesh is added as a temporizing measure. A subset of patients who will be prone to recurrence remains. Long-term follow-up is needed to confirm reliable and reproducible results.

Wound contraction is attenuated by fasudil inhibition of Rho-associated kinase.

Background: Dermal scarring and scar contracture result in restriction of movement. There are no effective drugs to prevent scarring. RhoA and Rho-associated kinase have emerged as regulators of fibrosis and contracture. Fasudil, a Rho-associated kinase inhibitor, has been demonstrated to have antifibrotic effects in models of liver, renal, and cardiac fibrosis. The role of fasudil in preventing dermal scarring and contractures has not been studied. The authors used a rat model of dermal wound healing to assess the effects of fasudil with regard to the prevention of scarring. Methods: Human scar tissue and surrounding normal skin were immunostained for RhoA and Rho-associated kinase. Full-thickness wounds were created on Wistar-Han rats, and fasudil (30 mg/kg/day) or saline was continuously delivered subcutaneously. Wound contraction was measured by gravitational planimetry. After 21 days, tissue was harvested for Massons trichrome, hematoxylin and eosin, Ki-67, and CD31 staining. Fibroblast-populated collagen lattices were used to assess the mechanistic effects of fasudil on contractility. Myofibroblast formation was assessed in the presence of fasudil. Results: Human scar tissue in the remodeling phase of repair showed increased expression of RhoA and Rho-associated kinase in scar tissue compared with surrounding normal tissue. Fasudil inhibited wound contraction as compared with controls. Hematoxylin and eosin and Massons trichrome were similar between groups. Fasudil did not alter angiogenesis or proliferation. Fasudil inhibited fibroblast contractility and myofibroblast formation in vitro. Conclusions: There is growing evidence that the RhoA/Rho-associated kinase pathway plays an important role in wound healing and scar contracture. The authors present data showing that inhibition of Rho-associated kinase hinders fibroblast contractility and may be beneficial in preventing scar contracture.

Temporal spatial expression and function of non-muscle myosin II isoforms IIA and IIB in scar remodeling

Scar contracture is believed to be caused by the cell contractility during the remodeling phase of wound healing. Cell contractility is mediated by non-muscle myosin II (NMMII) and actin, but the temporal-spatial expression profile of NMMII isoforms A and B (IIA and IIB) during the remodeling phase and the role of NMMII in scar fibroblast tissue remodeling are unknown. Human scar tissue immunostained for IIA and IIB showed that both isoforms were highly expressed in scar tissue throughout the remodeling phase of repair and expression levels returned to normal after the remodeling phase. Human scar tissue immunostained for β-, γ- and α–smooth muscle actin showed that all isoforms were consistently expressed throughout the remodeling phase of repair. The β- and γ-smooth muscle actin were widely expressed throughout the dermis, but α-smooth muscle actin was only locally expressed within the dermis. In vitro, fibroblasts explanted from scar tissue were shown to express more IIA than fibroblasts explanted from normal tissue and scar fibroblasts contracted collagen lattices to a greater extent than normal fibroblasts. Blebbistatin was used to demonstrate the function of NMMII in collagen lattice contraction. In normal tissue, fibroblasts are stress-shielded from external tensile stress by the extracellular matrix. After dermal injury and during remodeling, fibroblasts are exposed to a matrix of increased stiffness. The effect of matrix stiffness on IIA and IIB expression was examined. IIA expression was greater in fibroblasts cultured in collagen lattices with increasing stiffness, and in fibroblasts cultured on glass slides compared with polyacrylamide gels with stiffness of 1 kPa. In conclusion, NMMII and actin isoform expression changes coordinately with the remodeling phase of repair, and NMMII is increased as matrix stiffness increases. As NMMII expression increases, so does the fibroblast contractility.

The Modified Patient and Observer Scar Assessment Scale: A Novel Approach to Defining Pathologic and Nonpathologic Scarring?

Background: Scarring is a highly prevalent and multifactorial process, yet no studies to date have attempted to distinguish pathologic from nonpathologic scarring. Methods: This article defines and proposes methods of classifying pathologic scarring as it pertains to clinical presentation. Results: The authors propose a new scar scale that incorporates pain and functional impairment. Conclusions: The modified Patient and Observer Scar Assessment Scale is the first of its kind to factor in the functional deficits pain and pruritus of scarring into measurements of associated morbidity. This scale has great potential in evaluating patient response to treatment and analyzing clinical outcomes.