Petr Walczysko

Electrical signals control wound healing through phosphatidylinositol-3-OH kinase-γ and PTEN

Wound healing is essential for maintaining the integrity of multicellular organisms. In every species studied, disruption of an epithelial layer instantaneously generates endogenous electric fields, which have been proposed to be important in wound healing. The identity of signalling pathways that guide both cell migration to electric cues and electric-field-induced wound healing have not been elucidated at a genetic level. Here we show that electric fields, of a strength equal to those detected endogenously, direct cell migration during wound healing as a prime directional cue. Manipulation of endogenous wound electric fields affects wound healing in vivo. Electric stimulation triggers activation of Src and inositol–phospholipid signalling, which polarizes in the direction of cell migration. Notably, genetic disruption of phosphatidylinositol-3-OH kinase-γ (PI(3)Kγ) decreases electric-field-induced signalling and abolishes directed movements of healing epithelium in response to electric signals. Deletion of the tumour suppressor phosphatase and tensin homolog (PTEN) enhances signalling and electrotactic responses. These data identify genes essential for electrical-signal-induced wound healing and show that PI(3)Kγ and PTEN control electrotaxis.

The roles of calcium signaling and ERK1/2 phosphorylation in a Pax6+/- mouse model of epithelial wound-healing delay

BackgroundCongenital aniridia caused by heterozygousity at the PAX6 locus is associated with ocular surface disease including keratopathy. It is not clear whether the keratopathy is a direct result of reduced PAX6 gene dosage in the cornea itself, or due to recurrent corneal trauma secondary to defects such as dry eye caused by loss of PAX6 in other tissues. We investigated the hypothesis that reducing Pax6 gene dosage leads to corneal wound-healing defects. and assayed the immediate molecular responses to wounding in wild-type and mutant corneal epithelial cells.ResultsPax6+/- mouse corneal epithelia exhibited a 2-hour delay in their response to wounding, but subsequently the cells migrated normally to repair the wound. Both Pax6+/+ and Pax6+/- epithelia activated immediate wound-induced waves of intracellular calcium signaling. However, the intensity and speed of propagation of the calcium wave, mediated by release from intracellular stores, was reduced in Pax6+/- cells. Initiation and propagation of the calcium wave could be largely decoupled, and both phases of the calcium wave responses were required for wound healing. Wounded cells phosphorylated the extracellular signal-related kinases 1/2 (phospho-ERK1/2). ERK1/2 activation was shown to be required for rapid initiation of wound healing, but had only a minor effect on the rate of cell migration in a healing epithelial sheet. Addition of exogenous epidermal growth factor (EGF) to wounded Pax6+/- cells restored the calcium wave, increased ERK1/2 activation and restored the immediate healing response to wild-type levels.ConclusionThe study links Pax6 deficiency to a previously overlooked wound-healing delay. It demonstrates that defective calcium signaling in Pax6+/- cells underlies this delay, and shows that it can be pharmacologically corrected. ERK1/2 phosphorylation is required for the rapid initiation of wound healing. A model is presented whereby minor abrasions, which are quickly healed in normal corneas, transiently persist in aniridic patients, compromising the corneal stroma.

Influx of extracellular Ca2+ is necessary for electrotaxis in Dictyostelium

Intracellular free Ca2+ ([Ca2+]i) is a pivotal signalling element in cell migration and is thought to be required for chemotaxis of Dictyostelium. Ca2+ signalling may also be important for electrotaxis. However this suggestion has been controversial. We show that electric fields direct Dictyostelium cells to migrate cathodally and increase [Ca2+]i in Dictyostelium cells, as determined by Fluo-3 AM imaging and 45Ca2+ uptake. Omission of extracellular Ca2+([Ca2+]e) and incubation with EGTA abolished the electric-field-stimulated [Ca2+]i rise and directional cell migration. This suggests a requirement for [Ca2+]e in the electrotactic response. Deletion of iplA, a gene responsible for chemoattractant-induced [Ca2+]i increase, had only a minor effect on the electric-field-induced [Ca2+]i rise. Moreover, iplA-null Dictyostelium cells showed the same electrotactic response as wild-type cells. Therefore, iplA-independent Ca2+ influx is necessary for electrotactic cell migration. These results suggest that the [Ca2+]i regulatory mechanisms induced by electric fields are different from those induced by cAMP and folic acid in Dictyostelium cells. Different roles of the iplA gene in chemoattractant-induced and electrically induced Ca2+ signalling, and different effects of [Ca2+]i elevation on chemotaxis and electrotaxis indicate that the chemoattractant and electric cues activate distinctive initial signalling elements.

Control of Patterns of Corneal Innervation by Pax6

PURPOSE Corneal nerves play essential roles in maintaining the ocular surface through provision of neurotrophic support, but genetic control of corneal innervation is poorly understood. The possibility of a neurotrophic failure in ocular surface disease associated with heterozygosity at the Pax6 locus (aniridia-related keratopathy [ARK]) was investigated. METHODS Patterns of corneal innervation were studied during development and aging in mice with different Pax6 dosages and in chimeras. Immunohistochemistry and ELISA-based assays were used to determine the molecular basis of defects seen in Pax6 mutants, and wound healing assays were performed. RESULTS In adults, the Pax6(+/-) epithelium was less densely innervated than the wild-type epithelium, and radial projection of epithelial nerves was disrupted. Neurotrophic support of the corneal epithelium appeared normal. Directed nerve projection correlated with patterns of epithelial cell migration in adult wild-types, but innervation defects observed in Pax6(+/-) mice were not fully corrected in wound healing or chimeric models where directed epithelial migration was restored. CONCLUSIONS Pax6 dosage nonautonomously controls robust directed radial projection of corneal neurons, and the guidance cues for growth cone guidance are not solely dependent on directed epithelial migration. There is little evidence that ARK represents neurotrophic keratitis.

Chronic wound state exacerbated by oxidative stress in Pax6+/-aniridia-related keratopathy

Heterozygosity for the transcription factor PAX6 causes eye disease in humans, characterized by corneal opacity. The molecular aetiology of such disease was investigated using a Pax6+/− mouse model. We found that the barrier function of uninjured Pax6+/− corneas was compromised and that Ca2+–PKC/PLC–ERK/p38 signalling pathways were abnormally activated, mimicking a ‘wounded’ epithelial state. Using proteomic analysis and direct assay for oxidized proteins, Pax6+/− corneas were found to be susceptible to oxidative stress and they exhibited a wound‐healing delay which could be rescued by providing reducing agents such as glutathione. Pax6 protein was oxidized and excluded from the nucleus of stressed corneal epithelial cells, with concomitant loss of corneal epithelial markers and expression of fibroblast/myofibroblast markers. We suggest a chronic wound model for Pax6‐related corneal diseases, in which oxidative stress underlies a positive feedback mechanism by depleting nuclear Pax6, delaying wound healing, and activating cell signalling pathways that lead to metaplasia of the corneal epithelium. The study mechanistically links a relatively minor dosage deficiency of a transcription factor with potentially catastrophic degenerative corneal disease. Copyright

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. J. Cell. Physiol. 226: 1544–1553, 2011.

Intracellular Ca2+ stores are essential for injury induced Ca2+ signaling and re-endothelialization

Endothelialization repairs the lining of damaged vasculature and is a key process in preventing thrombosis and restenosis. It has been demonstrated that extracellular calcium ([Ca2+]o) influx is important for subsequent endothelialization. The role of intracellular Ca2+ stores in mechanical denudation induced intracellular calcium ([Ca2+]i) rise and endothelialization remains to be demonstrated. Using monolayer culture of a human endothelial cell line (human umbilical vein endothelial cell, HUVEC), we investigated [Ca2+]i wave propagation and re‐endothelialization following mechanical denudation. Consistent with previous reports for other types of cells, mechanical denudation induces calcium influx, which is essential for [Ca2+]i rise and endothelialization. Moreover, we found that intracellular Ca2+ stores are also essential for denudation induced [Ca2+]i wave initiation and propagation, and the subsequent endothelialization. Thapsigargin which depletes intracellular Ca2+ stores completely abolished [Ca2+]i wave generation and endothelialization. Xestospongin C (XeC), which prevents Ca2+ release from intracellular Ca2+ stores by inhibition of inositol 1,4,5‐trisphosphate (IP3) receptor, inhibited intercellular Ca2+ wave generation and endothelialization following denudation. Purinergic signaling through a suramin sensitive mechanism and gap junction communication also contribute to in intercellular Ca2+ wave propagation and re‐endothelialization. We conclude that intracellular Ca2+ stores, in addition to extracellular Ca2+, are essential for intracellular Ca2+ signaling and subsequent endothelialization following mechanical denudation. J. Cell. Physiol. 214: 595–603, 2008.

The core planar cell polarity gene, Vangl2 , directs adult corneal epithelial cell alignment and migration

This study shows that the core planar cell polarity (PCP) genes direct the aligned cell migration in the adult corneal epithelium, a stratified squamous epithelium on the outer surface of the vertebrate eye. Expression of multiple core PCP genes was demonstrated in the adult corneal epithelium. PCP components were manipulated genetically and pharmacologically in human and mouse corneal epithelial cells in vivo and in vitro. Knockdown of VANGL2 reduced the directional component of migration of human corneal epithelial (HCE) cells without affecting speed. It was shown that signalling through PCP mediators, dishevelled, dishevelled-associated activator of morphogenesis and Rho-associated protein kinase directs the alignment of HCE cells by affecting cytoskeletal reorganization. Cells in which VANGL2 was disrupted tended to misalign on grooved surfaces and migrate across, rather than parallel to the grooves. Adult corneal epithelial cells in which Vangl2 had been conditionally deleted showed a reduced rate of wound-healing migration. Conditional deletion of Vangl2 in the mouse corneal epithelium ablated the normal highly stereotyped patterns of centripetal cell migration in vivo from the periphery (limbus) to the centre of the cornea. Corneal opacity owing to chronic wounding is a major cause of degenerative blindness across the world, and this study shows that Vangl2 activity is required for directional corneal epithelial migration.