Josemaria Paterno

Mechanical load initiates hypertrophic scar formation through decreased cellular apoptosis

Hypertrophic scars occur following cutaneous wounding and result in severe functional and esthetic defects. The pathophysiology of this process remains unknown. Here, we demonstrate for the first time that mechanical stress applied to a healing wound is sufficient to produce hypertrophic scars in mice. The resulting scars are histopathologically identical to human hypertrophic scars and persist for more than six months following a brief (one‐week) period of augmented mechanical stress during the proliferative phase of wound healing. Resulting scars are structurally identical to human hypertrophic scars and showed dramatic increases in volume (20‐fold) and cellular density (20‐fold). The increased cellularity is accompanied by a four‐fold decrease in cellular apoptosis and increased activation of the prosurvival marker Akt. To clarify the importance of apoptosis in hypertrophic scar formation, we examine the effects of mechanical loading on cutaneous wounds of animals with altered pathways of cellular apoptosis. In p53‐null mice, with down‐regulated cellular apoptosis, we observe significantly greater scar hypertrophy and cellular density. Conversely, scar hypertrophy and cellular density are significantly reduced in proapoptotic BclII‐null mice. We conclude that mechanical loading early in the prolifer‐ative phase of wound healing produces hypertrophic scars by inhibiting cellular apoptosis through an Akt‐dependent mechanism.—Aarabi S., Bhatt, K. A., Shi, Y., Paterno, J., Chang, E. I., Loh, S. A., Holmes, J. W., Longaker, M. T., Yee, H., Gurtner G. C. Mechanical load initiates hypertrophic scar formation through decreased cellular apoptosis. FASEB J. 21, 3250–3261 (2007)

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.

IFATS Collection: Adipose Stromal Cells Adopt a Proangiogenic Phenotype Under the Influence of Hypoxia

Evolving evidence suggests a possible role for adipose stromal cells (ASCs) in adult neovascularization, although the specific cues that stimulate their angiogenic behavior are poorly understood. We evaluated the effect of hypoxia, a central mediator of new blood vessel development within ischemic tissue, on proneovascular ASC functions. Murine ASCs were exposed to normoxia (21% oxygen) or hypoxia (5%, 1% oxygen) for varying lengths of time. Vascular endothelial growth factor (VEGF) secretion by ASCs increased as an inverse function of oxygen tension, with progressively higher VEGF expression at 21%, 5%, and 1% oxygen, respectively. Greater VEGF levels were also associated with longer periods in culture. ASCs were able to migrate towards stromal cell‐derived factor (SDF)‐1, a chemokine expressed by ischemic tissue, with hypoxia augmenting ASC expression of the SDF‐1 receptor (CXCR4) and potentiating ASC migration. In vivo, ASCs demonstrated the capacity to proliferate in response to a hypoxic insult remote from their resident niche, and this was supported by in vitro studies showing increasing ASC proliferation with greater degrees of hypoxia. Hypoxia did not significantly alter the expression of endothelial surface markers by ASCs. However, these cells did assume an endothelial phenotype as evidenced by their ability to tubularize when seeded with differentiated endothelial cells on Matrigel. Taken together, these data suggest that ASCs upregulate their proneovascular activity in response to hypoxia, and may harbor the capacity to home to ischemic tissue and function cooperatively with existing vasculature to promote angiogenesis. STEM CELLS 2009;27:266–274

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

Akt-mediated mechanotransduction in murine fibroblasts during hypertrophic scar formation.

Although numerous factors are implicated in skin fibrosis, the exact pathophysiology of hypertrophic scarring remains unknown. We recently demonstrated that mechanical force initiates hypertrophic scar formation in a murine model, potentially enhancing cellular survival through Akt. Here, we specifically examined Akt‐mediated mechanotransduction in fibroblasts using both strain culture systems and our murine scar model. In vitro, static strain increased fibroblast motility, an effect blocked by wortmannin (a phosphoinositide‐3‐kinase/Akt inhibitor). We also demonstrated that high‐frequency cyclic strain was more effective at inducing Akt phosphorylation than low frequency or static strain. In vivo, Akt phosphorylation was induced by mechanical loading of dermal fibroblasts in both unwounded and wounded murine skin. Mechanically loaded scars also exhibited strong expression of α‐smooth muscle actin, a putative marker of pathologic scar formation. In vivo inhibition of Akt increased apoptosis but did not significantly abrogate hypertrophic scar development. These data suggest that although Akt signaling is activated in fibroblasts during mechanical loading of skin, this is not the critical pathway in hypertrophic scar formation. Future studies are needed to fully elucidate the critical mechanotransduction components and pathways which activate skin fibrosis.

Exercise induces stromal cell-derived factor-1α-mediated release of endothelial progenitor cells with increased vasculogenic function.

Background: Endothelial progenitor cells have been shown to traffic to and incorporate into ischemic tissues, where they participate in new blood vessel formation, a process termed vasculogenesis. Previous investigation has demonstrated that endothelial progenitor cells appear to mobilize from bone marrow to the peripheral circulation after exercise. In this study, the authors investigate potential etiologic factors driving this mobilization and investigate whether the mobilized endothelial progenitor cells are the same as those present at baseline. Methods: Healthy volunteers (n = 5) performed a monitored 30-minute run to maintain a heart rate greater than 140 beats/min. Venous blood samples were collected before, 10 minutes after, and 24 hours after exercise. Endothelial progenitor cells were isolated and evaluated. Results: Plasma levels of stromal cell–derived factor-1&agr; significantly increased nearly two-fold immediately after exercise, with a nearly four-fold increase in circulating endothelial progenitor cells 24 hours later. The endothelial progenitor cells isolated following exercise demonstrated increased colony formation, proliferation, differentiation, and secretion of angiogenic cytokines. Postexercise endothelial progenitor cells also exhibited a more robust response to hypoxic stimulation. Conclusions: Exercise appears to mobilize endothelial progenitor cells and augment their function by means of stromal cell–derived factor 1&agr;–dependent signaling. The population of endothelial progenitor cells mobilized following exercise is primed for vasculogenesis with increased capacity for proliferation, differentiation, secretion of cytokines, and responsiveness to hypoxia. Given the evidence demonstrating positive regenerative effects of exercise, this may be one possible mechanism for its benefits.

Mechanical offloading of incisional wounds is associated with transcriptional downregulation of inflammatory pathways in a large animal model

Cutaneous scarring is a major source of morbidity and current therapies to mitigate scar formation remain ineffective. Although wound fibrosis and inflammation are highly linked, only recently have mechanical forces been implicated in these pathways. Our group has developed a topical polymer device that significantly reduces post-injury scar formation via the manipulation of mechanical forces. Here we extend these studies to examine the genomewide transcriptional effects of mechanomodulation during scar formation using a validated large animal model, the red Duroc pig. We demonstrate that mechanical loading of incisional wounds upregulates expression of genes associated with inflammatory and fibrotic pathways, and that device-mediated offloading of these wounds reverses these effects. Validation studies are needed to clarify the clinical significanceof these findings.

Erratum: Mechanical load initiates hypertrophic scar formation through decreased cellular apoptosis (FASEB Journal (2007) DOI: 10.1096/fj.07-8218com)

In the article titled, “Mechanical load initiates hypertrophic scar formation through decreased cellular apoptosis,” by Shahram Aarabi, Kirit A. Bhatt, Yubin Shi, Josemaria Paterno, Edward I. Chang, Shang A. Loh, Jeffrey W. Holmes, Michael T. Longaker, Herman Yee, and Geoffrey C. Gurtner, which appeared in the October 2007 issue of The FASEB Journal (doi:10.1096/fj.07-8218com), there is an error in the y axis units for Figure 1B. The y axis values for Figure 1B should be in major units of 1, increasing from “0” to “6,” not “0” to “60.” In addition, the y axis for Figures 1A and 1B should be labeled (N/mm2), not (N/mm)2. The authors regret any inconvenience caused by these errors.