Kemal Levi

Focal adhesion kinase links mechanical force to skin fibrosis via inflammatory signaling

Exuberant fibroproliferation is a common complication after injury for reasons that are not well understood. One key component of wound repair that is often overlooked is mechanical force, which regulates cell-matrix interactions through intracellular focal adhesion components, including focal adhesion kinase (FAK). Here we report that FAK is activated after cutaneous injury and that this process is potentiated by mechanical loading. Fibroblast-specific FAK knockout mice have substantially less inflammation and fibrosis than control mice in a model of hypertrophic scar formation. We show that FAK acts through extracellular-related kinase (ERK) to mechanically trigger the secretion of monocyte chemoattractant protein-1 (MCP-1, also known as CCL2), a potent chemokine that is linked to human fibrotic disorders. Similarly, MCP-1 knockout mice form minimal scars, indicating that inflammatory chemokine pathways are a major mechanism by which FAK mechanotransduction induces fibrosis. Small-molecule inhibition of FAK blocks these effects in human cells and reduces scar formation in vivo through attenuated MCP-1 signaling and inflammatory cell recruitment. These findings collectively indicate that physical force regulates fibrosis through inflammatory FAK–ERK–MCP-1 pathways and that molecular strategies targeting FAK can effectively uncouple mechanical force from pathologic scar formation.

Solar UV radiation reduces the barrier function of human skin

The ubiquitous presence of solar UV radiation in human life is essential for vitamin D production but also leads to skin photoaging, damage, and malignancies. Photoaging and skin cancer have been extensively studied, but the effects of UV on the critical mechanical barrier function of the outermost layer of the epidermis, the stratum corneum (SC), are not understood. The SC is the first line of defense against environmental exposures like solar UV radiation, and its effects on UV targets within the SC and subsequent alterations in the mechanical properties and related barrier function are unclear. Alteration of the SC’s mechanical properties can lead to severe macroscopic skin damage such as chapping and cracking and associated inflammation, infection, scarring, and abnormal desquamation. Here, we show that UV exposure has dramatic effects on cell cohesion and mechanical integrity that are related to its effects on the SC’s intercellular components, including intercellular lipids and corneodesmosomes. We found that, although the keratin-controlled stiffness remained surprisingly constant with UV exposure, the intercellular strength, strain, and cohesion decreased markedly. We further show that solar UV radiation poses a double threat to skin by both increasing the biomechanical driving force for damage while simultaneously decreasing the skin’s natural ability to resist, compromising the critical barrier function of the skin.

Mechanical force prolongs acute inflammation via T-cell-dependent pathways during scar formation

Mechanical force significantly modulates both inflammation and fibrosis, yet the fundamental mechanisms that regulate these interactions remain poorly understood. Here we performed microarray analysis to compare gene expression in mechanically loaded wounds vs. unloaded control wounds in an established murine hypertrophic scar (HTS) model. We identified 853 mechanically regulated genes (false discovery rate <2) at d 14 postinjury, a subset of which were enriched for T‐cell‐regulated pathways. To substantiate the role of T cells in scar mechanotransduction, we applied the HTS model to T‐cell‐deficient mice and wild‐type mice. We found that scar formation in T‐cell‐deficient mice was reduced by almost 9‐fold (P < 0.001) with attenuated epidermal (by 2.6‐fold, P < 0.01) and dermal (3.9‐fold, P < 0.05) proliferation. Mechanical stimulation was highly associated with sustained T‐cell‐dependent Th2 cytokine (IL‐4 and IL‐13) and chemokine (MCP‐1) signaling. Further, T‐cell‐deficient mice failed to recruit systemic inflammatory cells such as macrophages or monocytic fibroblast precursors in response to mechanical loading. These findings indicate that T‐cell‐regulated fibrogenic pathways are highly mechanoresponsive and suggest that mechanical forces induce a chronic‐like inflammatory state through immune‐dependent activation of both local and systemic cell populations.—Wong, V. W., Paterno, J., Sorkin, M., Glotzbach, J. P., Levi, K., Januszyk, M., Rustad, K. C., Longaker, M. T., Gurtner, G. C. Mechanical force prolongs acute inflammation via T‐cell‐dependent pathways during scar formation. FASEB J. 25, 4498–4510 (2011). www.fasebj.org

Drying stress and damage processes in human stratum corneum.

The drying stresses that develop in stratum corneum (SC) are crucial for its mechanical and biophysical function, its cosmetic feel and appearance, and play a central role in processes of dry skin damage. However, quantitative methods to characterize these stresses are lacking and little understanding exists regarding the effects of drying environment, chemical exposures and moisturizing treatments. We describe the application of a substrate curvature technique adapted for biological tissue to accurately characterize SC drying stresses as a function of time following environmental pre‐conditioning and chemical treatment in a range of drying environments. SC stresses were observed to increase to stress levels of up to ∼ 3 MPa over periods of 8 h depending on pretreatment and drying environment. A unique relationship between the SC stress and water in the drying environment was established. The effect of glycerol on lowering SC stresses and damaging surfactants on elevating SC stresses were quantified. Extensions of the method to continuous monitoring of SC stresses in response to changes in environmental moisture content and temperature are reported. Finally, a biomechanics framework to account for the SC drying stress as a mechanical driving force for dry skin damage is presented.

Emollient molecule effects on the drying stresses in human stratum corneum

Background Emollient molecules are widely used in skin care formulations to improve skin sensory properties and to alleviate dry skin but little is understood regarding their effects on skin biomechanical properties.

Scar zones: region-specific differences in skin tension may determine incisional scar formation.

Surgeons have observed for centuries that wounds under tension are prone to exuberant fibrosis.1,2 Accordingly, topographic maps have been described that orient incisions to minimize wound tension and subsequent scar formation.2 Scar width has also been shown to positively correlate with the amount of force required to surgically close excisional wounds.3 Although the mechanisms underlying scar mechanotransduction (the conversion of mechanical signals into biochemical responses) remain unclear, the sensing of physical force is largely determined by the biomechanical properties of skin itself.4–8 These attributes vary considerably across different body regions6,9,10 and can change dramatically throughout life.11–13 In humans, excessive scarring often occurs in locations characterized by greater skin stresses such as the chest and shoulder.14–16 Pathologic scar growth may also be driven by regional variations in mechanical force.17 Although these clinical observations indicate that differences in skin material properties may underlie the propensity to form scar, this concept has not been examined previously. In this study, we use finite element analysis and digital image speckle correlation to substantiate region-specific differences in skin biomechanical properties in the red Duroc pig (a robust large-animal model of human scarring18) and humans. Our analyses suggest the presence of anatomical “scar zones” with distinct tensional states that may define how to optimally minimize scarring in different regions of the body.

Effect of Corneodesmosome Degradation on the Intercellular Delamination of Human Stratum Corneum

Bell EL, Klimova TA, Eisenbart J, Moraes CT, Murphy MP, Budinger GR et al. (2007) The Qo site of the mitochondrial complex III is required for the transduction of hypoxic signaling via reactive oxygen species production. J Cell Biol 177:1029–36 Bodemer C, Rotig A, Rustin P, Cormier V, Niaudet P, Saudubray JM et al. (1999) Hair and skin disorders as signs of mitochondrial disease. Pediatrics 103:428–33

Loss of keratinocyte focal adhesion kinase stimulates dermal proteolysis through upregulation of MMP9 in wound healing.

Objective:To investigate how epithelial mechanotransduction pathways impact wound repair. Background:Mechanical forces are increasingly recognized to influence tissue repair, but their role in chronic wound pathophysiology remains unknown. Studies have shown that chronic wounds exhibit high levels of matrix metalloproteinase 9 (MMP9), a key proteolytic enzyme that regulates wound remodeling. We hypothesized that epithelial mechanosensory pathways regulated by keratinocyte-specific focal adhesion kinase (FAK) control dermal remodeling via MMP9. Methods:A standard wound model was applied to keratinocyte-specific FAK knockout (KO) and control mice. Rates of wound healing were measured and tissue was obtained for histologic and molecular analyses. Transcriptional and immunoblot assays were used to assess the activation of FAK, intracellular kinases, and MMP9 in vitro. A cell suspension model was designed to validate the importance of FAK mechanosensing, p38, and MMP9 secretion in human cells. Biomechanical testing was utilized to evaluate matrix tensile properties in FAK KO and control wounds. Results:Wound healing in FAK KO mice was significantly delayed compared with controls (closure at 15 days compared with 20 days, P = 0.0003). FAK KO wounds demonstrated decreased dermal thickness and collagen density. FAK KO keratinocytes exhibited overactive p38 and MMP9 signaling in vitro, findings recapitulated in human keratinocytes via the deactivation of FAK in the cell suspension model. Functionally, FAK KO wounds were significantly weaker and more brittle than control wounds, results consistent with the histologic and molecular analyses. Conclusions:Keratinocyte FAK is highly responsive to mechanical cues and may play a critical role in matrix remodeling via regulation of p38 and MMP9. These findings suggest that aberrant epithelial mechanosensory pathways may contribute to pathologic dermal proteolysis and wound chronicity.

Application of substrate curvature method to differentiate drying stresses in topical coatings and human stratum corneum.

Despite the extensive use of topical coatings in cosmetics, their effect on the mechanical properties of human skin and the perception of skin tightness in the form of drying stresses is not well understood. We describe the application of a recently developed substrate curvature technique to characterize stresses in drying and non‐drying occlusive topical coatings. We then extend the technique to measure the combined effects of the coating applied to human stratum corneum (SC) where the overall drying stresses may have contributions from the coating, the SC and the interaction of the coating with the SC. We show how these separate contributions in the coating and SC layers can be differentiated.

A histological and mechanical analysis of the cardiac lead-tissue interface: implications for lead extraction.

The major risks of pacemaker and implantable cardioverter defibrillator extraction are attributable to the fibrotic tissue that encases them in situ, yet little is known about the cellular and functional properties of this response. In the present research, we performed a histological and mechanical analysis of human tissue collected from the lead-tissue interface to better understand this process and provide insights for the improvement of lead design and extraction. The lead-tissue interface consisted of a thin cellular layer underlying a smooth, acellular surface, followed by a circumferentially organized collagen-rich matrix. 51.8±4.9% of cells were myofibroblasts via immunohistochemistry, with these cells displaying a similar circumferential organization. Upon mechanical testing, samples exhibited a triphasic force-displacement response consisting of a toe region during initial tensioning, a linear elastic region and a yield and failure region. Mean fracture load was 5.6±2.1N, and mean circumferential stress at failure was 9.5±4.1MPa. While the low cellularity and fibrotic composition of tissue observed herein is consistent with a foreign body reaction to an implanted material, the significant myofibroblast response provides a mechanical explanation for the contractile forces complicating extractions. Moreover, the tensile properties of this tissue suggest the feasibility of circumferential mechanical tissue disruption, similar to balloon angioplasty devices, as a novel approach to assist with lead extraction.