New insights in wound response and repair of epithelium

Department of Ophthalmology, Boston University School of Medicine, Boston, MA 02118.
Journal of Cellular Physiology (Impact Factor: 3.84). 05/2013; 228(5). DOI: 10.1002/jcp.24268
Source: PubMed


Epithelial wounds usually heal relatively quickly, but repair may be impaired by environmental stressors, such as hypoxic or diabetic states, rendering patients vulnerable to a number of corneal pathologies. Though this response appears simple, at first, years of research have uncovered the complicated biochemical pathways coordinating the wound healing response. Here, we investigate signaling cascades and individual proteins involved in the corneal epithelium's self-repair. We will explore how an epithelial cell migrates across the wound bed and attaches itself to its new post-injury surroundings, including its neighboring cells and the basement membrane, through focal adhesions and hemidesmosomes. We will also discuss how the cell coordinates this motion physiologically, through calcium signaling and protein phosphorylation, focusing on the communication through purinergic, glutamatergic, and growth factor receptors. Many of these aspects reflect and can be extended to similar epithelial surfaces, and can be used to facilitate wound healing in patients with various underlying pathologies. The collective library of laboratory and clinical research done around the world has demonstrated how important precise regulation of these processes is in order for the injured corneal epithelium to properly heal. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

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    • "Directed cell migration of epithelial cells is a critical process in wound healing. This involves interaction of epithelial cells with the BM promoting cell adhesion and migration into the wound [1] as well as coordinated responses to a multitude of soluble biochemical cues that create chemotactic gradients [2] [3]. Matrix metalloproteinases (MMPs) also participate in coordinated movement of cells and matrix dynamics essential to wound repair processes. "
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    ABSTRACT: Directed migration of corneal epithelial cells (CECs) is critical for maintenance of corneal homeostasis as well as wound healing. Soluble cytoactive factors and the intrinsic chemical attributes of the underlying extracellularmatrix (ECM) participate in stimulating and directing migration. Additionally, numerous publications document the central importance of the intrinsic biophysical attributes of the microenvironment of the cell in modulating an array of fundamental epithelial behaviors including migration. Among the best studies of these attributes are the intrinsic topography and stiffness of the ECM and electric fields (EF). How cells integrate these multiple simultaneous inputs is not well understood. Here, we present a method that combines the use of 1. topographically patterned substrates (mean pore diameter of 800 nm) possessing features that approximate those found in the native corneal basement membrane and 2. EF (0-150 mV/mm) mimicking those at corneal epithelial wounds that the cells experience in vivo. We found that topographic cues and EFs synergistically regulated directional migration of human CECs and that this was associated with upregulation of MMP-3. MMP3 expression and activity were significantly elevated with 150 mV/mm applied-EF while MMP2/9 remained unaltered. MMP3 expression was elevated in cells cultured on patterned-surfaces against planar-surfaces. Maximum single cell migration rate was observed with 150 mV/mm applied EF on patterned and planar surfaces. When cultured as a confluent sheet, EFs induced collective cell migration on stochastically patterned surfaces compared with dissociated single cell migration on planar surfaces. These results suggest significant interaction of biophysical cues in regulating cell behaviors and will help define design parameters for corneal prosthetics and help to better understand corneal woundhealing.
    Acta Biomaterialia 10/2014; 12(1). DOI:10.1016/j.actbio.2014.10.007 · 6.03 Impact Factor
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    • "Regeneration of the epithelium requires the participation of several entities, including extracellular matrix proteins and growth factors that collectively promote cell adhesion, migration, and proliferation processes [1-5]. To facilitate healing, several intracellular signaling cascades activated in varying degrees by growth factors coordinate cell migration, adhesion, and proliferation processes [6]. In response to injury, several growth factors are released from the stroma and lacrimal gland [7-13]. "
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    ABSTRACT: Hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) are secreted in the cornea in response to injury. In this study, we investigated the HGF- and KGF-mediated effect on the expression of cell cycle and apoptosis controlling proteins, cell survival, and growth in the corneal epithelium to better understand the possible role of their signaling mechanisms in repairing epithelial injuries. The cell survival capability of HGF and KGF in epithelial primary cultures was evaluated by using a staurosporine-induced apoptosis model. Apoptosis was quantified with image analysis following nuclear staining with Hoechst fluorescent dye and DNA laddering. Western immunoblotting was used to study the effect of growth factors on the expression of cell cycle- and apoptosis-regulating proteins. HGF and KGF protected cells from apoptosis for a short duration (10 h), but only KGF exhibited cell survival capability and maintained cell growth for a longer period (24 h). The onset of apoptosis was accompanied by a significant increase in cell cycle inhibitor p27(kip). HGF and KGF suppressed p27(kip) levels in the apoptosis environment; however, KGF- but not HGF-dependent downregulation in p27(kip) expression was sustained for a longer duration. Inhibition of phosphatidylinositol 3-kinase/Akt activation blocked HGF- and KGF-mediated control of p27(kip) expression. Further, when compared to HGF, the presence of KGF produced significant downregulation of p53 and poly(adenosine diphosphate-ribose) polymerase, the key proteins involved in apoptosis and blocked the degradation of G1/S cell cycle progression checkpoint protein retinoblastoma. HGF and KGF upregulated the levels of p21(cip), cyclins A, D, and E and cyclin-dependent kinases (CDK2 and CDK4) as well, but the KGF-mediated effect on the expression of these molecules lasted longer. Sustained effect of KGF on cell survival and proliferation could be attributed to its ability to inhibit p53, retinoblastoma, caspases, and p27(kip) functions in apoptosis and cell cycle arrest and promote the expression of cell cycle progressing molecules for longer duration. Designing therapeutic strategies targeting cell cycle control through KGF may be beneficial for repairing difficult-to-heal corneal epithelial injuries that require sustained growth and cell survival promoting signals.
    Molecular vision 01/2014; 20:24-xx. · 1.99 Impact Factor
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    ABSTRACT: Cdc42, a member of Rho GTPases participates in cytokine and growth factor controlled biological functions in mammalian tissues. Here, we examined Cdc42 role in corneal epithelial wound healing and the influence of HGF-, KGF- and EGF-mediated signaling on Cdc42. Epithelial wounds were created on the corneas of live rabbits by complete debridement and in rabbit corneal epithelial primary cultures through scratch injury. Cdc42 expression in cultures was suppressed using Cdc42 siRNA. Cdc42 activation was determined by pull down assays with PAK-agarose beads. Cdc42 expression was analyzed by immunoblotting and immunofluorescence. Association of Cdc42 with cell cycle proteins was identified by immunoprecipiatation. Results: In rabbit corneas significant increase in Cdc42 expression that occurred 2-4 days after the injury was coincided with wound closure and by 8 days the expression reached near basal levels. Silencing of Cdc42 expression in cultures caused inhibition of wound closure as a result of 60-75% decrease in epithelial migration and growth. HGF, KGF and EGF increased Cdc2 expression, activation and its phosphorylation on ser71. Inhibition of growth factor mediated-PI-3K signaling resulted in the down regulation of Cdc42 expression and its phosphorylation. Increased association of cell cycle proteins p27kip and CDK4 with Cdc42; and phosphorylated Cdc42 with plasma membrane leading edges was also observed in the presence of growth factors. Conclusions: Cdc42 is required for corneal epithelial wound repair. To promote healing Cdc42 may interact with growth factor receptor tyrosine kinase activated signaling cascades that participate in cell migration and cell cycle progression.
    Investigative ophthalmology & visual science 07/2013; 54(8). DOI:10.1167/iovs.13-11955 · 3.40 Impact Factor
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