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There is a clinical need for new, more effective treatments for chronic wounds in diabetic patients. Lack of epithelial cell migration is a hallmark of nonhealing wounds, and diabetes often involves endothelial dysfunction. Therefore, targeting re-epithelialization, which mainly involves keratinocytes, may improve therapeutic outcomes of current treatments. In this study, we present an integrin-binding prosurvival peptide derived from angiopoietin-1, QHREDGS (glutamine- histidine-arginine-glutamic acid-aspartic acid-glycine-serine), as a therapeutic candidate for diabetic wound treatments by demonstrating its efficacy in promoting the attachment, survival, and collective migration of human primary keratinocytes and the activation of protein kinase B Akt and MAPKp42/44. The QHREDGS peptide, both as a soluble supplement and when immobilized in a substrate, protected keratinocytes against hydrogen peroxide stress in a dose-dependent manner. Collective migration of both normal and diabetic human keratinocytes was promoted on chitosan-collagen films with the immobilized QHREDGS peptide. The clinical relevance was demonstrated further by assessing the chitosan-collagen hydrogel with immobilized QHREDGS in full-thickness excisional wounds in a db/db diabetic mouse model; QHREDGS showed significantly accelerated and enhanced wound closure compared with a clinically approved collagen wound dressing, peptide-free hydrogel, or blank wound controls. The acceleratedwound closure resulted primarily from faster re-epithelialization and increased formation of granulation tissue. There were no observable differences in blood vessel density or size within the wound; however, the total number of blood vessels was greater in the peptide-hydrogel- treated wounds. Together, these findings indicate that QHREDGS is a promising candidate for wound-healing interventions that enhance reepithelialization and the formation of granulation tissue. [ABSTRACT FROM AUTHOR]

In pressure or volume overload, hypertrophic growth of the myocardium is associated with myofibroblast differentiation, a process in which cardiac fibroblasts express smooth muscle α-actin (SMA). The signaling mechanisms that mediate force-induced myofibroblast differentiation and SMA expression are not defined. We examined the role of the Rho-Rho-kinase pathway in force-induced SMA expression in fibroblasts using an in vitro model system that applies static tensile forces (0.65 pN/µm²) to integrins via collagen-coated magnetite beads. Force maximally induced RhoA activation at 10 minutes that was localized to force application sites and required intact actin filaments. Force application induced phosphorylation of LIM kinase (5-10 minutes) and an early dephosphorylation of cofilin (5 minutes) that was followed by prolonged cofilin phosphorylation. These responses were blocked by Y27632, an inhibitor of Rho kinase. Force promoted actin filament assembly at force application sites (10-20 minutes), a process that required Rho kinase and cofilin. Force application induced nuclear translocation of the transcriptional co-activator MRTF-A but not MRTF-B. Nuclear translocation of MRTF-A required Rho kinase and intact actin filaments. Force caused 3.5-fold increases of SMA promoter activity that were completely blocked by transfection of cells with dominant-negative MRTF-A or by inhibition of Rho kinase or by actin filament disassembly. These data indicate that mechanical forces mediate actin assembly through the Rho-Rho-kinase-LIMK cofilin pathway. Force-mediated actin filament assembly promotes nuclear translocation of MRTF and subsequent activation of the SMA promoter to enhance SMA expression. [ABSTRACT FROM AUTHOR]

In pressure or volume overload, hypertrophic growth of the myocardium is associated with myofibroblast differentiation, a process in which cardiac fibroblasts express smooth muscle alpha-actin (SMA). The signaling mechanisms that mediate force-induced myofibroblast differentiation and SMA expression are not defined. We examined the role of the Rho-Rho-kinase pathway in force-induced SMA expression in fibroblasts using an in vitro model system that applies static tensile forces (0.65 pN/microm(2)) to integrins via collagen-coated magnetite beads. Force maximally induced RhoA activation at 10 minutes that was localized to force application sites and required intact actin filaments. Force application induced phosphorylation of LIM kinase (5-10 minutes) and an early dephosphorylation of cofilin (5 minutes) that was followed by prolonged cofilin phosphorylation. These responses were blocked by Y27632, an inhibitor of Rho kinase. Force promoted actin filament assembly at force application sites (10-20 minutes), a process that required Rho kinase and cofilin. Force application induced nuclear translocation of the transcriptional co-activator MRTF-A but not MRTF-B. Nuclear translocation of MRTF-A required Rho kinase and intact actin filaments. Force caused 3.5-fold increases of SMA promoter activity that were completely blocked by transfection of cells with dominant-negative MRTF-A or by inhibition of Rho kinase or by actin filament disassembly. These data indicate that mechanical forces mediate actin assembly through the Rho-Rho-kinase-LIMK cofilin pathway. Force-mediated actin filament assembly promotes nuclear translocation of MRTF and subsequent activation of the SMA promoter to enhance SMA expression.

Cortactin regulates the strength of nascent N-cadherin-mediated intercellular adhesions through a tyrosine phosphorylation-dependent mechanism. Currently, the functional significance of cortactin phosphorylation and the kinases responsible for the regulation of adhesion strength are not defined. We show that the nonreceptor tyrosine kinase Fer phosphorylates cadherin-associated cortactin and that this process is involved in mediating intercellular adhesion strength. In wild-type fibroblasts N-cadherin ligation-induced transient phosphorylation of Fer, indicating that junction formation activates Fer kinase. Tyrosine phosphorylation of cortactin after N-cadherin ligation was strongly reduced in fibroblasts expressing only catalytically inactive Fer (D743R), compared with wild-type cells. In wild-type cells, N-cadherin-coated bead pull-off assays induced fourfold greater endogenous N-cadherin association than in D743R cells. Fluorescence recovery after photobleaching showed that GFP-N-cadherin mobility at nascent contacts was 50% faster in wild-type than D743R cells. In shear wash-off assays, nascent intercellular adhesion strength was twofold higher in wild-type than D743R cells. Cortactin recruitment to adhesions was independent of Fer kinase activity, but was impacted by N-cadherin ligation-provoked Rac activation. We conclude that N-cadherin ligation induces Rac-dependent cortactin recruitment and Fer-dependent cortactin phosphorylation, which in turn promotes enhanced mobilization and interaction of surface expressed N-cadherin in contacting cells.