Contributions of Ocular Surface Components to Matrix-Metalloproteinases (MMP)-2 and MMP-9 in Feline Tears following Corneal Epithelial Wounding

Purpose

This study investigated ocular surface components that contribute to matrix-metalloproteinase (MMP)-2 and MMP-9 found in tears following corneal epithelial wounding.

Methods

Laboratory short-haired cats underwent corneal epithelial debridement in one randomly chosen eye (n = 18). Eye-flush tears were collected at baseline and during various healing stages. Procedural control eyes (identical experimental protocol as wounded eyes except for wounding, n = 5) served as controls for tear analysis. MMP activity was analyzed in tears using gelatin zymography. MMP staining patterns were evaluated in ocular tissues using immunohistochemistry and used to determine MMP expression sites responsible for tear-derived MMPs.

Results

The proMMP-2 and proMMP-9 activity in tears was highest in wounded and procedural control eyes during epithelial migration (8 to 36 hours post-wounding). Wounded eyes showed significantly higher proMMP-9 in tears only during and after epithelial restratification (day 3 to 4 and day 7 to 28 post-wounding, respectively) as compared to procedural controls (p<0.05). Tears from wounded and procedural control eyes showed no statistical differences for pro-MMP-2 and MMP-9 (p>0.05). Immunohistochemistry showed increased MMP-2 and MMP-9 expression in the cornea during epithelial migration and wound closure. The conjunctival epithelium exhibited highest levels of both MMPs during wound closure, while MMP-9 expression was reduced in conjunctival goblet cells during corneal epithelial migration followed by complete absence of the cells during wound closure. The immunostaining for both MMPs was elevated in the lacrimal gland during corneal healing, with little/no change in the meibomian glands. Conjunctival-associated lymphoid tissue (CALT) showed weak MMP-2 and intense MMP-9 staining.

Conclusions

Following wounding, migrating corneal epithelium contributed little to the observed MMP levels in tears. The major sources assessed in the present study for tear-derived MMP-2 and MMP-9 following corneal wounding are the lacrimal gland and CALT. Other sources included stromal keratocytes and conjunctiva with goblet cells.     

Thymosin beta4 promotes matrix metalloproteinase expression during wound repair

Immobilized patients, diabetics, and the elderly suffer from impaired wound healing. The 43-amino acid angiogenic peptide thymosin beta4 (Tbeta4) has previously been found to accelerate dermal wound repair in rats, aged mice, and db/db diabetic mice. It also promotes corneal repair in both normal rats and mice. Because proteinases are important in wound repair, we hypothesized that Tbeta4 may regulate matrix metalloproteinase (MMP) expression in cells that are involved in wound repair. Analysis by RT-PCR of whole excised mouse dermal wounds on days 1, 2, and 3 after wounding showed that Tbeta4 increased several metalloproteinases, including MMP-2 and -9 expression by several-fold over control on day 2 after wounding. We further analyzed the metalloproteinases secreted in response to exogenous Tbeta4 by cells normally present in the wound. Western blot analysis of cultured keratinocytes, endothelial cells, and fibroblasts that were treated with increasing concentrations of Tbeta4 showed increases in the levels of MMP-1, -2, and -9 in a cell-specific manner. Tbeta4 also enhanced the secretion of MMP-1 and MMP-9 by activated monocytes. The central actin-binding domain, amino acids 17-23, had all of the activity for metalloproteinase induction. We conclude that part of the wound healing activity of Tbeta4 resides in its ability to increase proteinase activity via its central actin-binding domain. Thus, Tbeta4 may play a pivotal role in extracellular matrix remodeling during wound repair.     

Matrix metalloproteinase 2 (gelatinase A) is related to migration of keratinocytes.

The role of matrix metalloproteinases (MMPs) in cell migration was studied by measuring cell growth, migration, and production of MMP-2 and -9 in oral mucosal and skin keratinocytes cultured in the presence of synthetic MMP inhibitors. MMP-2 was the major gelatinolytic MMP produced by these cells while MMP-9 was produced at a low basal level. Inhibitor effects on MMP-9 production were therefore studied in keratinocytes stimulated by tumor necrosis factor alpha (TNFalpha). Tetracycline analogues at concentrations that inhibited the production of MMP-2 but not MMP-9 were able to drastically inhibit migration of both mucosal and skin keratinocytes. Tetracycline analogues also inhibited keratinocyte growth, an effect not found for the other inhibitors tested. Heterocyclic carbonate-derived compounds (LWs) that inhibited MMP-9 but not MMP-2 production had no effect on cell migration. Batimastat, a potent MMP inhibitor, did not have any effect on MMP production or cell growth but did inhibit keratinocyte migration. Tumor growth factor beta (TGFbeta) increased keratinocyte migration as well as both cell-associated and secreted MMP-2 production in wounded cell cultures. The secreted enzyme was partially converted into an active form. In this model batimastat totally blocked TGFbeta-promoted keratinocyte migration. Immunostaining of keratinocytes advancing into the wound revealed that MMP-2 was localized in extracellular matrix contactlike structures against the endogenously produced laminin-5-rich matrix. MMP-9 was localized diffusely along the cell membranes. Using in situ hybridization we observed that in chronically inflamed human gingiva MMP-2 is expressed in epithelium extending into subepithelial connective tissue. These results suggest that MMP-2 plays a specific role in epithelial migration, possibly by detaching the advancing cells from the pericellular matrix or by activating other MMPs.     

Matrix metalloproteinase (MMP) and TGF-β1-stimulated cell migration in skin and cornea wound healing

Cell migration during wound healing is a complex process that involves the expression of a number of growth factors and cytokines. One of these factors, transforming growth factor-beta (TGF-β) controls many aspects of normal and pathological cell behavior. It induces migration of keratinocytes in wounded skin and of epithelial cells in damaged cornea. Furthermore, this TGF-β-induced cell migration is correlated with the production of components of the extracellular matrix (ECM) proteins, and expression of integrins and matrix metalloproteinases (MMPs). MMP digests ECMs and integrins during cell migration, but the mechanisms regulating their expression and the consequences of their induction remain unclear. It has been suggested that MMP-14 activates cellular signaling processes involved in the expression of MMPs and other molecules associated with cell migration. Because of the manifold effects of MMP-14, it is important to understand the roles of MMP-14 not only the cleavage of ECM but also in the activation of signaling pathways.     

Osteopontin, a novel substrate for matrix metalloproteinase-3 (stromelysin-1) and matrix metalloproteinase-7 (matrilysin)

Osteopontin (OPN) is a secreted phosphoprotein shown to function in wound healing, inflammation, and tumor progression. Expression of OPN is often co-localized with members of the matrix metalloproteinase (MMP) family. We report that OPN is a novel substrate for two MMPs, MMP-3 (stromelysin-1) and MMP-7 (matrilysin). Three cleavage sites were identified for MMP-3 in human OPN, and two of those sites were also cleaved by MMP-7. These include hydrolysis of the human Gly166-Leu167, Ala201-Tyr202 (MMP-3 only), and Asp210-Leu211 peptide bonds. Only the N-terminal Gly-Leu cleavage site is conserved in rat OPN (Gly151-Leu152). These sites are distinct from previously reported cleavage sites in OPN for the proteases thrombin or enterokinase. We found evidence for the predicted MMP cleavage fragments of OPN in vitro in tumor cell lines, and in vivo in remodeling tissues such as the postpartum uterus, where OPN and MMPs are co-expressed. Furthermore, cleavage of OPN by MMP-3 or MMP-7 potentiated the function of OPN as an adhesive and migratory stimulus in vitro through cell surface integrins. We predict that interaction of MMPs with OPN at tumor and wound healing sites in vivo may be a mechanism of regulation of OPN bioactivity.     

Rho kinases regulate corneal epithelial wound healing

We have previously shown that Rho small GTPase is required for modulating both cell migration and proliferation through cytoskeleton reorganization and focal adhesion formation in response to wounding. In the present study, we investigated the role of Rho kinases (ROCKs), major effectors of Rho GTPase, in mediating corneal epithelial wound healing. Both ROCK 1 and 2 were expressed and activated in THCE cells, an SV40-immortalized human corneal epithelial cell (HCEC) line, in response to wounding, lysophosphatidic acid, and heparin-binding EGF-like growth factor (HB-EGF) stimulations. The ROCK inhibitor Y-27632 efficiently antagonized ROCK activities without affecting Rho activation in wounded HCECs. Y-27632 promoted basal and HB-EGF-enhanced scratch wound healing and enhanced cell migration and adhesion to matrices, while retarded HB-EGF induced cell proliferation. E-cadherin- and beta-catenin-mediated cell-cell junction and actin cytoskeleton organization were disrupted by Y-27632. Y-27632 impaired the formation and maintenance of tight junction barriers indicated by decreased trans-epithelial resistance and disrupted occludin staining. We conclude that ROCK activities enhance cell proliferation, promote epithelial differentiation, but negatively modulate cell migration and cell adhesion and therefore play a role in regulating corneal epithelial wound healing.     

Transforming growth factor-beta1 increases airway wound repair via MMP-2 upregulation: a new pathway for epithelial wound repair?

In vivo, transforming growth factor (TGF)-beta1 and matrix metalloproteinases (MMPs) present at the site of airway injury are thought to contribute to epithelial wound repair. As TGF-beta1 can modulate MMP expression and MMPs play an important role in wound repair, we hypothesized that TGF-beta1 may enhance airway epithelial repair via MMPs secreted by epithelial cells. We evaluated the in vitro influence of TGF-beta1 on wound repair in human airway epithelial cells cultured under conditions allowing differentiation. The results showed that TGF-beta1 accelerated in vitro airway wound repair, whereas MMP inhibitors prevented this acceleration. In parallel, we examined the effect of TGF-beta1 on the expression of MMP-2 and MMP-9. TGF-beta1 induced a dramatic increase of MMP-2 expression with an increased steady-state level of MMP-2 mRNA, contrasting with a slight increase in MMP-9 expression. To confirm the role of MMP-2, we subsequently evaluated the effect of MMP-2 on in vitro airway wound repair and demonstrated that the addition of MMP-2 reproduced the acceleration of wound repair induced by TGF-beta1. These results strongly suggest that TGF-beta1 increases in vitro airway wound repair via MMP-2 upregulation. It also raises the issue of a different in vivo biological role of MMP-2 and MMP-9 depending on the cytokine microenvironment.     

The immunoreactivity of a chimeric multi-epitope DNA vaccine against IBV in chickens

Lumican is a major proteoglycans of the human cornea. Lumican knock-out mice have been shown to lose corneal transparency and to display delayed wound healing. The purpose of this study was to define the role of lumican in corneal epithelial cell migration. Over-expression of lumican in human corneal epithelial (HCE-T) cells increased both cell migration and proliferation, and increased levels of integrins α2 and β1. ERK 1/2 was also activated in lumican over-expressed cells. When we treated HCE-T cells with the ERK-specific inhibitor U0126, cell migration and the expression of integrin β1 were completely blocked. These data provide evidence that lumican stimulates cell migration in the corneal epithelium by activating ERK 1/2, and point to a novel signaling pathway implicated in corneal epithelial cell migration.     

Activation of MMP-9 by human lung epithelial cells in response to the cystic fibrosis-associated pathogen Burkholderia cenocepacia reduced wound healing in vitro

Burkholderia cepacia complex is a group of bacterial pathogens that cause opportunistic infections in cystic fibrosis (CF). The most virulent of these is Burkholderia cenocepacia. Matrix metalloproteinases (MMPs) are upregulated in CF patients. The aim of this work was to examine the role of MMPs in the pathogenesis of B. cepacia complex, which has not been explored to date. Real-time PCR analysis showed that B. cenocepacia infection upregulated MMP-2 and MMP-9 genes in the CF lung cell line CFBE41o- within 1 h, whereas MMP-2, -7, and -9 genes were upregulated in the non-CF lung cell line 16HBE14o-. Conditioned media from both cell lines showed increased MMP-9 activation following B. cenocepacia infection. Conditioned media from B. cenocepacia-infected cells significantly reduced the rate of wound healing in confluent lung epithelia (P < 0.05), in contrast to conditioned media from Pseudomonas aeruginosa-infected cells, which showed predominant MMP-2 activation. Treatment of control conditioned media from both cell lines with the MMP activator 4-aminophenylmercuric acetate (APMA) also resulted in clear activation of MMP-9 and to a much lesser extent MMP-2. APMA treatment of control media also delayed the repair of wound healing in confluent epithelial cells. Furthermore, specific inhibition of MMP-9 in medium from cells exposed to B. cenocepacia completely reversed the delay in wound repair. These data suggest that MMP-9 plays a role in the reduced epithelial repair observed in response to B. cenocepacia infection and that its activation following B. cenocepacia infection contributes to the pathogenesis of this virulent pathogen.     

Roles of MMP/TIMP in regulating matrix swelling and cell migration during chick corneal development

Tissue remodeling is central to embryonic development. Here, we used immunohistochemistry, Western blotting, and RT-PCR analysis to investigate the roles of matrix metalloproteinases (MMPs) and the related "a disintegrin and metalloproteinase" (ADAM) family proteinases in chick corneal development. While MMP-13 was expressed in developing chick corneas from embryonic day (ED) 5 to ED 10, its inhibitor, tissue inhibitors of metalloproteinase-1 (TIMP-1), was expressed from ED 18 to 2 days post-hatching (P2). Early MMP-13 activity may be associated with degradation of type IX collagen from the primary stroma, which loosens the collagen fibrils and facilitates neural crest (NC) cell migration. The membrane-bound and secreted forms of ADAM10 were both detected throughout corneal development, and active ADAM10 formed a cleavage complex with CD44v6, a CD44 splice variant that is a major cell surface adhesion molecule for hyaluronic acid (HA) and has been implicated in cell migration. Both CD44v6 and its ectodomain cleavage products were detected from ED 5 to ED 14, and a broad-spectrum MMP inhibitor blocked ectodomain cleavage in cultured stromal cells. These findings suggest that ADAM10 mediates CD44v6 cleavage in the developing cornea, facilitating NC cell-derived mesenchymal cell migration. Finally, we identified high levels of active membrane-type 3-MMP (MT3-MMP) in developing corneas at ED 7, ED 14, and ED 18. MT3-MMP takes part in MMP-2 activation and possibly also CD44v6 shedding, suggesting that this pathway may be involved in cell migration. These findings collectively show for the first time that multiple MMPs, ADAMs, and TIMPs appear to functionally interact during corneal development.     

Temporal study of the activity of matrix metalloproteinases and their endogenous inhibitors during wound healing

The restoration of functional connective tissue is a major goal of the wound healing process. This regenerative event requires the deposition and accumulation of collagenous and noncollagenous matrix molecules as well as the remodelling of extracellular matrix (ECM) by matrix metalloproteinases (MMPs). In this study, we have utilized substrate gel electrophoresis, radiometric enzyme assays, and Western blot analyses to determine the temporal pattern of appearance and activity of active and latent MMPs and their inhibitors during the entire healing process in a partial thickness wound model. Through the use of substrate gel electrophoresis, we studied the appearance of proteolytic bands whose molecular weight was consistent with their being members of the MMP family of enzymes. Proteolytic bands whose molecular weight is consistent with both the active and latent forms of MMP-2 (72 kDa, Type IV gelatinase) were detected in wound fluid of days 1–7 after wounding. The number of active MMP-2 species detectable in wound fluid was greatest during days 4–6 after wounding. The most prominent proteolytic band detected each day migrated with a molecular weight consistent with it being the latent form of MMP-9 (92 kDa, Type V pro-collagenase). In contrast to MMP-2, the active form of this enzyme was never detected. The presence of MMP-1 (interstitial collagenase) was detected by immunoblot in the wound fluid from days 1–6 post-injury. Using a radiometric enzyme assay for collagenase inhibitory activity we have also determined the time course of activity of endogenous matrix metalloproteinase inhibitors. We have correlated these data to the known cellular events occurring in the wound during this time period as well. This study establishes a prototypical pattern of MMP appearance in normal wound healing. It may also provide potential intervention sites for the therapeutic use of inhibitors of aberrant MMP activities which characterize chronic wounds. © 1996 Wiley-Liss, Inc.     

Protection of thymosin beta-4 on corneal endothelial cells from UVB-induced apoptosis

Cornea absorbs most of daily ultraviolet (UV) light. An excess of UV damages results in not only keratopathy and cataract but also maculopathy. It has been reported that thymosin beta-4 (Tbeta4) promotes wound healing, decreases inflammatory response and prevents apoptosis of corneal epithelial cells. However, it is not clear whether Tbeta4 protects UVB-induced corneal injury, particularly in corneal endothelial cells because of its non-proliferation in nature. The purpose of this study is to compare the protective effects of Tbeta4 on bovine corneal endothelial (BCE) cells from low- and high-dose UVB damage. In this study, 1 microg/ml of Tbeta4 was added to BCE cells 2 h before low (12.5 mj/cm2) or high dosage (100 mj/cm2) UVB exposure. Using a fluorogenic substrate cleavage assay, we found that Tbeta4 diminished the reactive oxygen species level in BCE cells elicited by UVB. However, the protection of viability by Tbeta4 could only be detected under low-dose UVB exposure. Moreover, both caspase-9 activity and annexin V/propidium iodine staining demonstrated that Tbeta4 only protected BCE cells from low-dose UVB-induced apoptosis but not high-dose UVB-induced necrosis. Together, Tbeta4 protected corneal endothelial cells from UVB-induced oxidative stress and apoptosis after low-dose UVB exposure. The results support further investigation towards topical use or anterior chamber injection of this small hydrophilic peptide in treating and preventing UVB-induced corneal endothelial damage.     

Matrix metalloproteinases process the laminin-5 gamma 2-chain and regulate epithelial cell migration

Matrix metalloproteinase (MMP)-2 and membrane type 1-MMP can process the laminin-5 (Ln-5) gamma2-chain, revealing a cryptic site inducing epithelial cell migration. We investigated whether other MMPs process the Ln-5 gamma2-chain and related their ability to induce epithelial cell migration. The N-terminal sequences of the MMP-3, -12, -13, and -20 processed 80kDa Ln-5 gamma2x-chains were identical whereas the N-terminus of the 80kDa(MMP-8) Ln-5 gamma2x-chain was not. MMP-3, -13, -14, and -20 induced MCF-7 cell migration over Ln-5 while MMP-8 was a poor inducer of MCF-7 cell migration. In conclusion, several MMPs can process the Ln-5 gamma2-chain and induce epithelial cell migration.     

Matrix metalloproteinase (MMP) and TGFβ1-stimulated cell migration in skin and cornea wound healing

Cell migration during wound healing is a complex process that involves the expression of a number of growth factors and cytokines. One of these factors, transforming growth factor-beta (TGFβ) controls many aspects of normal and pathological cell behavior. It induces migration of keratinocytes in wounded skin and of epithelial cells in damaged cornea. Furthermore, this TGFβ-induced cell migration is correlated with the production of components of the extracellular matrix (ECM) proteins and expression of integrins and matrix metalloproteinases (MMPs). MMP digests ECMs and integrins during cell migration, but the mechanisms regulating their expression and the consequences of their induction remain unclear. It has been suggested that MMP-14 activates cellular signaling processes involved in the expression of MMPs and other molecules associated with cell migration. Because of the manifold effects of MMP-14, it is important to understand the roles of MMP-14 not only the cleavage of ECM but also in the activation of signaling pathways.Key words: wound healing, migration, matrix metalloproteinase, transforming growth factor, skin, corneaWound healing is a well-ordered but complex process involving many cellular activities including inflammation, growth factor or cytokine secretion, cell migration and proliferation. Migration of skin keratinocytes and corneal epithelial cells requires the coordinated expression of various growth factors such as platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), transforming growth factor (TGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), epidermal growth factor (EGF), small GTPases, and macrophage stimulating protein (reviewed in refs. ¹ and ²). The epithelial cells in turn regulate the expression of matrix metalloproteinases (MMPs), extracellular matrix (ECM) proteins and integrins during cell migration.^1,3,4 TGF-β is a well-known cytokine involved in processes such as cell growth inhibition, embryogenesis, morphogenesis, tumorigenesis, differentiation, wound healing, senescence and apoptosis (reviewed in refs. ⁵ and ⁶). It is also one of the most important cytokines responsible for promoting the migration of skin keratinocytes and corneal epithelial cells.^3,6,7TGFβ has two quite different effects on skin keratinocytes: it suppresses their multiplication and promotes their migration. The TGFβ-induced cell growth inhibition is usually mediated by Smad signaling, which upregulates expression of the cell cycle inhibitor p21^WAF1/Cip1 or p12^CDK2-AP1 in HaCaT skin keratinocyte cells and human primary foreskin keratinocytes.^8,9 Keratinocyte migration in wounded skin is associated with strong expression of TGFβ and MMPs,¹ and TGFβ stimulates the migration of manually scratched wounded HaCaT cells.¹⁰ TGFβ also induces cell migration and inhibits proliferation of injured corneal epithelial cells, whereas it stimulates proliferation of normal corneal epithelial cells via effects on the MAPK family and Smad signaling.^2,7 Indeed, skin keratinocytes and corneal epithelial cells display the same two physiological responses to TGFβ during wound healing; cell migration and growth inhibition. However as mentioned above, TGFβ has a different effect on normal cells. For example, it induces the epithelial to mesenchymal transition (EMT) of normal mammary cells and lens epithelial cells.^11,12 It also promotes the differentiation of corneal epithelial cells, and induces the fibrosis of various tissues.^2,6The MMPs are a family of structurally related zinc-dependent endopeptidases that are secreted into the extracellular environment.¹³ Members of the MMP family have been classified into gelatinases, stromelysins, collagenases and membrane type-MMPs (MT-MMPs) depending on their substrate specificity and structural properties. Like TGFβ, MMPs influence normal physiological processes including wound healing, tissue remodeling, angiogenesis and embryonic development, as well as pathological conditions such as rheumatoid arthritis, atherosclerosis and tumor invasion.^13,14The expression patterns of MMPs during skin and cornea wound healing are well studied. In rats, MMP-2, -3, -9, -11, -13 and -14 are expressed,¹⁵ and in mice, MMP-1, -2, -3, -9, -10 and -14 are expressed during skin wound healing.¹ MMP-1, -3, -7 and -12 are increased in corneal epithelial cells during Wnt 7a-induced rat cornea wound healing.¹⁶ Wound repair after excimer laser keratectomy is characterized by increased expression of MMP-1, -2, -3 and -9 in the rabbit cornea, and MMP-2, -9 in the rat cornea.^17,18 The expression of MMP-2 and -9 during skin keratinocyte and corneal epithelial cell migration has been the most thoroughly investigated, and it has been shown that their expression generally depends on the activity of MMP-14. MMP-14 (MT1-MMP) is constitutively anchored to the cell membrane; it activates other MMPs such as MMP-2, and also cleaves various types of ECM molecules including collagens, laminins, fibronectin as well as its ligands, the integrins.¹³ The latent forms of some cytokines are also cleaved and activated by MMP-14.¹⁹ Overexpression of MMP-14 protein was found to stimulate HT1080 human fibrosarcoma cell migration.²⁰ In contrast, the attenuation of MMP-14 expression using siRNA method decreased fibroblast invasiveness,²¹ angiogenesis of human microvascular endothelial cells,²² and human skin keratinocyte migration.¹⁰ The latter effect was shown to result from lowering MMP-9 expression. Other studies have shown that EGF has a critical role in MMP-9 expression during keratinocyte tumorigenesis and migration.^23,24 On the other hand, TGFβ modulates MMP-9 production through the Ras/MAPK pathway in transformed mouse keratinocytes and NFκB induces cell migration by binding to the MMP-9 promoter in human skin primary cultures.^25,26 Enhanced levels of pro-MMP-9 and active MMP-9 have also been noted in scratched corneal epithelia of diabetic rats.²⁷There is evidence that MMP-14 activates a number of intracellular signaling pathways including the MAPK family pathway, focal adhesion kinase (FAK), Src family, Rac and CD44, during cell migration and tumor invasion.^19,20,28 In COS-7 cells, ERK activation is stimulated by overexpression of MMP-14 and is essential for cell migration.²⁹ These observations all indicate that MMP-14 plays an important role in cell migration, not only by regulating the activity or expression of downstream MMPs but also by processing and activating migration-associated molecules such as integrins, ECMs and a variety of intracellular signaling pathays.³⁰Cell migration during wound healing is a remarkably complex phenomenon. TGFβ is just one small component of the overall process of wound healing and yet it triggers a multitude of reactions needed for cell migration. It is important to know what kinds of molecules are expressed when cell migration is initiated, but it is equally important to investigate the roles of these molecules and how their expression is regulated. Despite the availability of some information about how MMPs and signaling molecules can influence each other, much remains to be discovered in this area. It will be especially important to clarify how MMP-14 influences other signaling pathways since its role in cell migration is not restricted to digesting ECM molecules but also includes direct or indirect activation of cellular signaling pathways.     

The role of electrical signals in murine corneal wound re-epithelialization

Ion flow from intact tissue into epithelial wound sites results in lateral electric currents that may represent a major driver of wound healing cell migration. Use of applied electric fields (EF) to promote wound healing is the basis of Medicare-approved electric stimulation therapy. This study investigated the roles for EFs in wound re-epithelialization, using the Pax6(+/-) mouse model of the human ocular surface abnormality aniridic keratopathy (in which wound healing and corneal epithelial cell migration are disrupted). Both wild-type (WT) and Pax6(+/-) corneal epithelial cells showed increased migration speeds in response to applied EFs in vitro. However, only Pax6(+/+) cells demonstrated consistent directional galvanotaxis towards the cathode, with activation of pSrc signaling, polarized to the leading edges of cells. In vivo, the epithelial wound site normally represents a cathode, but 43% of Pax6(+/-) corneas exhibited reversed endogenous wound-induced currents (the wound was an anode). These corneas healed at the same rate as WT. Surprisingly, epithelial migration did not correlate with direction or magnitude of endogenous currents for WT or mutant corneas. Furthermore, during healing in vivo, no polarization of pSrc was observed. We found little evidence that Src-dependent mechanisms of cell migration, observed in response to applied EFs in vitro, normally exist in vivo. It is concluded that endogenous EFs do not drive long-term directionality of sustained healing migration in this mouse corneal epithelial model. Ion flow from wounds may nevertheless represent an important component of wound signaling initiation.