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.     

Motogenic effect of recombinant HGF on airway epithelial cells during the in vitro wound repair of the respiratory epithelium

Cell migration is the earliest mechanism involved in the wound repair process of the respiratory epithelium and could be potentially enhanced by growth factors. In the present work, we investigated the localisation of the hepatocyte growth factor (HGF) receptor (c-Met) during wound repair and evaluated the effect of recombinant HGF (rHGF) on cell migration by using an in vitro model of airway epithelial wound repair. By using immunohistochemical methods, we observed that the immunoreactivity of the c-Met proto-oncogene was increased in epithelial cells engaged in the process of tissue repair. The incubation of wounded cultures with increasing concentrations of rHGF (0.2, 2, 20, and 200 ng/ml) induced a significant (P < 0.02) dose-dependent effect on the wound repair index, with a maximum effect produced at 20 ng/ml (+31.3%). The cell migration speed reached 50.2 micrometer/h at this concentration, compared to 20.4 micrometer/h in the absence of rHGF. No significant effect on cell proliferation was observed in the repairing area in the presence of rHGF. These results suggest that rHGF is able to improve the wound repair process of the airway epithelium by increasing cell migration.     

Human endogenous antibiotic LL-37 stimulates airway epithelial cell proliferation and wound closure

Antimicrobial peptides are endogenous antibiotics that directly inactivate microorganisms and in addition have a variety of receptor-mediated functions. LL-37/hCAP-18 is the only cathelicidin found in humans and is involved in angiogenesis and regulation of the innate immune system. The aim of the present study was to characterize the role of the peptide LL-37 in the regulation of wound closure of the airway epithelium in the cell line NCI-H292 and primary airway epithelial cells. LL-37 stimulated healing of mechanically induced wounds in monolayers of the cell line and in differentiated primary airway epithelium. This effect was detectable at concentrations of 5 mug/ml in NCI-H292 and 1 mug/ml in primary cells. The effect of LL-37 on wound healing was dependent on the presence of serum. LL-37 induced cell proliferation and migration of NCI-H292 cells. Inhibitor studies in the wound closure and proliferation assays indicated that the effects caused by LL-37 are mediated through epidermal growth factor receptor, a G protein-coupled receptor, and MAP/extracellular regulated kinase. In conclusion, LL-37 induces wound healing, proliferation, and migration of airway epithelial cells. The peptide is likely involved in the regulation of tissue homeostasis in the airways.     

EGF and K+ channel activity control normal and cystic fibrosis bronchial epithelia repair

Severe lesions of airway epithelia are observed in cystic fibrosis (CF) patients. The regulatory mechanisms of cell migration and proliferation processes, involved in the repair of injured epithelia, then need to be better understood. A model of mechanical wounding of non-CF (NuLi) and CF (CuFi) bronchial monolayers was employed to study the repair mechanisms. We first observed that wound repair, under paracrine and autocrine EGF control, was slower (up to 33%) in CuFi than in NuLi. Furthermore, EGF receptor (EGFR) activation, following wounding, was lower in CuFi than in NuLi monolayers. Cell proliferation and migration assays indicated a similar rate of proliferation in both cell lines but with reduced (by 25%) CuFi cell migration. In addition, cell migration experiments performed in the presence of conditioned medium, collected from NuLi and CuFi wounded bronchial monolayers, suggested a defect in EGF/EGFR signaling in CF cells. We (49) recently demonstrated coupling between the EGF response and K(+) channel function, which is crucial for EGF-stimulated alveolar repair. In CuFi cells, lower EGF/EGFR signaling was accompanied by a 40-70% reduction in K(+) currents and KvLQT1, ATP-sensitive potassium (K(ATP)), and Ca(2+)-activated K(+) (KCa3.1) channel expression. In addition, EGF-stimulated bronchial wound healing, cell migration, and proliferation were severely decreased by K(+) channel inhibitors. Finally, acute CFTR inhibition failed to reduce wound healing, EGF secretion, and K(+) channel expression in NuLi. In summary, the delay in CuFi wound healing could be due to diminished EGFR signaling coupled with lower K(+) channel function, which play a crucial role in bronchial repair.     

Repair and regeneration of the airway epithelium

Despite an efficient defence system, the airway surface epithelium, in permanent contact with the external milieu, is frequently injured by inhaled pollutants, microorganisms and viruses. The response of the airway surface epithelium to an acute injury includes a succession of cellular events varying from the loss of the surface epithelium integrity to partial shedding of the epithelium or even to complete denudation of the basement membrane. The epithelium has then to repair and regenerate to restore its functions, through several mechanisms including basal cell spreading and migration, followed by proliferation and differentiation of epithelial cells. The cellular and molecular factors involved in wound repair and epithelial regeneration are closely interacting and imply extracellular matrix proteins, matrix metalloproteinases (MMPs) and their inhibitors as well as cytokines and growth factors secreted by airway epithelial and mesenchymal cells. The development of in vitro and in vivo models of airway epithelium wound repair allowed the study of the spatio-temporal modulation of these factors during the different steps of epithelial repair and regeneration. In this context, several studies have demonstrated that the matrix and secretory environment are markedly involved in these mechanisms and that their dysregulation may induce remodelling of the airway mucosa. A better knowledge of the mechanisms involved in airway epithelium regeneration may pave the way to regenerative therapeutics allowing the reconstitution of a functional airway epithelium in numerous respiratory diseases such as asthma, chronic obstructive pulmonary diseases, cystic fibrosis and bronchiolitis.     

Activation of protein kinase A accelerates bovine bronchial epithelial cell migration

Bronchial epithelial cell migration is required for the repair of damaged airway epithelium. We hypothesized that bronchial epithelial cell migration during wound repair is influenced by cAMP and the activity of its cyclic nucleotide-dependent protein kinase, protein kinase A (PKA). We found that, when confluent monolayers of bronchial epithelial cells are wounded, an increase in PKA activity occurs. Augmentation of PKA activity with a cell-permeable analog of cAMP, dibutyryl adenosine 3',5'-cyclic monophosphate, isoproterenol, or a phosphodiesterase inhibitor accelerated migration of normal bronchial epithelial cells in in vitro wound closure assays and Boyden chamber migration assays. A role for PKA activity was also confirmed with a PKA inhibitor, KT-5720, which reduced stimulated migration. Augmentation of PKA activity reduced the levels of active Rho and the formation of focal adhesions. These studies suggest that PKA activation modulates Rho activity, migration mechanisms, and thus bronchial epithelial repair mechanisms.     

RhoA and Rac1 are both required for efficient wound closure of airway epithelial cells

Repair of the airway epithelium after injury is critical for restoring normal lung. The reepithelialization process involves spreading and migration followed later by cell proliferation. Rho-GTPases are key components of the wound healing process in many different types of tissues, but the specific roles for RhoA and Rac1 vary and have not been identified in lung epithelial cells. We investigated whether RhoA and Rac1 regulate wound closure of bronchial epithelial cells. RhoA and Rac1 proteins were efficiently expressed in a cell line of human bronchial epithelial cells (16HBE) by adenovirus-based gene transfer. We found that both constitutively active RhoA and dominant negative RhoA inhibited wound healing, suggesting that both activation and inhibition of RhoA interfere with normal wound healing. Overexpression of wild-type Rac1 induced upregulation of RhoA, disrupted intercellular junctions, and inhibited wound closure. Dominant negative Rac1 also inhibited wound closure. Inhibition of the downstream effector of RhoA, Rho-kinase, with Y-27632 suppressed actin stress fibers and focal adhesion formation, increased Rac1 activity, and stimulated wound closure. The activity of both RhoA and Rac1 are influenced by the polymerization state of microtubules, and cell migration involves coordinated action of actin and microtubules. Microtubule depolymerization upon nocodazole treatment led to an increase in focal adhesions and decreased wound closure. We conclude that coordination of both RhoA and Rac1 activity contributes to bronchial epithelial wound repair mechanisms in vitro, that inhibition of Rho-kinase accelerates wound closure, and that efficient repair involves intact microtubules.     

Changes in expression of ion channels and aquaporins mRNA during differentiation in normal human nasal epithelial cells

Integrity of the airway epithelium is important for pulmonary defense mechanisms to infection. The lining of the airway contains a diverse population of cell types. Understanding about progenitor-progeny relationships during renewal of airway epithelium is important for elucidating mechanisms of injury repair or oncogenesis. Primary culture of airway epithelia is a good model for studying differentiation process of epithelial cells. Ion channels and aquaporins(AQPs) play a critical role on ion and fluid transport across airway epithelia. However, changes in their expression during differentiation of airway epithelial cells have not been reported yet. This study was undertaken to identify isoforms of aquaporins in cultured normal human nasal epithelial cells (NHNE) and effects of various culture conditions on expression of differentiation markers and channels. 1. Degenerative RT-PCR revealed that AQP3 and AQP4 are expressed in cultured NHNE cells. 2. Culture of NHNE cells on permeable filters induced expression of mucin, aquaporins and CFTR. 3. Retinoic acid induced morphological changes in NHNE cells and inhibited their proliferation. The treatment of retinoic acid induced expression of mucin and CFTR, whereas it inhibited expression of cornifin. The effect of retinoic acid was enhanced by culture of cells on permeable filters. 4. Dexamethasone induced ENaC expression in NHNE cells grown on permeable supports only, but did not affect expression of mucin, aquaporins and CFTR. These results indicate that cultured NHNE cells express aquaporins (AQP3 and 4), CFTR and ENaC, and culture of NHNE cells on permeable filters induces differentiation in to mucosecretory and surface epithelial cells, and that effects of retinoic acid and dexamethasone on gene expression are affected by culture conditions.     

Beta-adrenergic receptor agonists delay while antagonists accelerate epithelial wound healing: evidence of an endogenous adrenergic network within the corneal epithelium

Wound healing is a complex and well-orchestrated biological process. Corneal epithelial cells (CECs) must respond quickly to trauma to rapidly restore barrier function and protect the eye from noxious agents. They express a high level of beta2-adrenergic receptors but their function is unknown. Here, we report the novel finding that they form part of a regulatory network in the corneal epithelium, capable of modulating corneal epithelial wound repair. Beta-adrenergic receptor agonists delay CEC migration via a protein phosphatase 2A-mediated mechanism and decrease both electric field-directed migration and corneal wound healing. Conversely, beta-adrenergic receptor antagonists accelerate CEC migration, enhance electric field-mediated directional migration, and promote corneal wound repair. We demonstrate that CECs express key enzymes required for epinephrine (beta-adrenergic receptor agonist) synthesis in the cytoplasm and can detect epinephrine in cell extracts. We propose that the mechanism for the pro-motogenic effect of the beta-adrenergic antagonist is blockade of the beta2-adrenergic receptor preventing autocrine catecholamine binding. Further investigation of this network will improve our understanding of one of the most frequently prescribed class of drugs.     

p38 and ERK1/2 coordinate cellular migration and proliferation in epithelial wound healing: evidence of cross-talk activation between MAP kinase cascades

One important action of growth factors is their participation in tissue repair; however, the signaling pathways involved are poorly understood. In a model of corneal wound healing, we found that two paracrine growth factors, hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF), induced rapid and marked activation and prompt nuclear accumulation of phospho-p38 (p-p38) and -ERK1/2 (p-ERK1/2), but not of JNK (p-JNK1/2), in corneal epithelial cells. Interruption of p38 and ERK1/2 signaling pathways by pretreatment with inhibitors SB203580 and PD98059 and subsequent stimulation with HGF or KGF abolished the activation and nuclear localization. Inhibition of either one of these mitogen-activated protein kinases, p38 or ERK1/2, induced a robust cross-activation of the other. In immunofluorescence studies of wounded cornea, p-p38, unlike p-ERK1/2, was immediately detectable in epithelium after injury. Inhibition of p38 by SB203580 blocked migration of epithelial cells almost completely. In contrast, PD98059 seemed to slightly increase the migration, through concomitant activation of p38. Unlike ERK1/2, p38 did not significantly contribute to proliferation of epithelial cells. Inhibition of either the ERK1/2 or p38 pathway resulted in delayed corneal epithelial wound healing. Interruption of both signaling cascades additively inhibited the wound-healing process. These findings demonstrate that both p38 and ERK1/2 coordinate the dynamics of wound healing: while growth factor-stimulated p38 induces epithelial migration, ERK1/2 activation induces proliferation. The cross-talk between these two signal cascades and the selective action of p38 in migration appear to be important to corneal wound healing, and possibly wound healing in general, and may offer novel drug targets for tissue repair.     

Keratinocyte growth factor accelerates wound closure in airway epithelium during cyclic mechanical strain.

The airway epithelium may be damaged by inhalation of noxious agents, in response to pathogens, or during endotracheal intubation and mechanical ventilation. Maintenance of an intact epithelium is important for lung fluid balance, and the loss of epithelium may stimulate inflammatory responses. Epithelial repair in the airways following injury must occur on a substrate that undergoes cyclic elongation and compression during respiration. We have previously shown that cyclic mechanical strain inhibits wound closure in the airway epithelium (Savla and Waters, 1998b). In this study, we investigated the stimulation of epithelial wound closure by keratinocyte growth factor (KGF) in vitro and the mechanisms by which KGF overcomes the inhibition due to mechanical strain. Primary cultures of normal human bronchial epithelial cells (NHBE) and a cell line of human airway epithelial cells, Calu 3, were grown on Silastic membranes, and a wound was scraped across the well. The wells were then exposed to cyclic strain using the Flexercell Strain Unit, and wound closure was measured. While cyclic elongation (20% maximum) and cyclic compression (approximately 2%) both inhibited wound closure in untreated wells, treatment with KGF (50 ng/ml) significantly accelerated wound closure and overcame the inhibition due to cyclic strain. Since wound closure involves cell spreading, migration, and proliferation, we investigated the effect of cyclic strain on cell area, cell-cell distance, and cell velocity at the wound edge. While the cell area increased in unstretched monolayers, the cell area of monolayers in compressed regions decreased significantly. Treatment with KGF increased the cell area in both cyclically elongated and compressed cells. Also, when cells were treated with KGF, cell velocity was significantly increased in both static and cyclically strained monolayers, and cyclic strain did not inhibit cell migration. These results suggest that KGF is an important factor in epithelial repair that is capable of overcoming the inhibition of repair due to physiological levels of cyclic strain.     

Synchronization Modulation Increases Transepithelial Potentials in MDCK Monolayers through Na/K Pumps

Transepithelial potential (TEP) is the voltage across a polarized epithelium. In epithelia that have active transport functions, the force for transmembrane flux of an ion is dictated by the electrochemical gradient in which TEP plays an essential role. In epithelial injury, disruption of the epithelial barrier collapses the TEP at the wound edge, resulting in the establishment of an endogenous wound electric field (∼100 mV/mm) that is directed towards the center of the wound. This endogenous electric field is implicated to enhance wound healing by guiding cell migration. We thus seek techniques to enhance the TEP, which may increase the wound electric fields and enhance wound healing. We report a novel technique, termed synchronization modulation (SM) using a train of electric pulses to synchronize the Na/K pump activity, and then modulating the pumping cycles to increase the efficiency of the Na/K pumps. Kidney epithelial monolayers (MDCK cells) maintain a stable TEP and transepithelial resistance (TER). SM significantly increased TEP over four fold. Either ouabain or digoxin, which block Na/K pump, abolished SM-induced TEP increases. In addition to the pump activity, basolateral distribution of Na/K pumps is essential for an increase in TEP. Our study for the first time developed an electrical approach to significantly increase the TEP. This technique targeting the Na/K pump may be used to modulate TEP, and may have implication in wound healing and in diseases where TEP needs to be modulated.     

CFTR Delivery to 25% of Surface Epithelial Cells Restores Normal Rates of Mucus Transport to Human Cystic Fibrosis Airway Epithelium

Dysfunction of CFTR in cystic fibrosis (CF) airway epithelium perturbs the normal regulation of ion transport, leading to a reduced volume of airway surface liquid (ASL), mucus dehydration, decreased mucus transport, and mucus plugging of the airways. CFTR is normally expressed in ciliated epithelial cells of the surface and submucosal gland ductal epithelium and submucosal gland acinar cells. Critical questions for the development of gene transfer strategies for CF airway disease are what airway regions require CFTR function and how many epithelial cells require CFTR expression to restore normal ASL volume regulation and mucus transport to CF airway epithelium? An in vitro model of human CF ciliated surface airway epithelium (CF HAE) was used to test whether a human parainfluenza virus (PIV) vector engineered to express CFTR (PIVCFTR) could deliver sufficient CFTR to CF HAE to restore mucus transport, thus correcting the CF phenotype. PIVCFTR delivered CFTR to >60% of airway surface epithelial cells and expressed CFTR protein in CF HAE approximately 100-fold over endogenous levels in non-CF HAE. This efficiency of CFTR delivery fully corrected the basic bioelectric defects of Cl⁻ and Na⁺ epithelial ion transport and restored ASL volume regulation and mucus transport to levels approaching those of non-CF HAE. To determine the numbers of CF HAE surface epithelial cells required to express CFTR for restoration of mucus transport to normal levels, different amounts of PIVCFTR were used to express CFTR in 3%–65% of the surface epithelial cells of CF HAE and correlated to increasing ASL volumes and mucus transport rates. These data demonstrate for the first time, to our knowledge, that restoration of normal mucus transport rates in CF HAE was achieved after CFTR delivery to 25% of surface epithelial cells. In vivo experimentation in appropriate models will be required to determine what level of mucus transport will afford clinical benefit to CF patients, but we predict that a future goal for corrective gene transfer to the CF human airways in vivo would attempt to target at least 25% of surface epithelial cells to achieve mucus transport rates comparable to those in non-CF airways.     

CFTR Delivery to 25% of Surface Epithelial Cells Restores Normal Rates of Mucus Transport to Human Cystic Fibrosis Airway Epithelium

Dysfunction of CFTR in cystic fibrosis (CF) airway epithelium perturbs the normal regulation of ion transport, leading to a reduced volume of airway surface liquid (ASL), mucus dehydration, decreased mucus transport, and mucus plugging of the airways. CFTR is normally expressed in ciliated epithelial cells of the surface and submucosal gland ductal epithelium and submucosal gland acinar cells. Critical questions for the development of gene transfer strategies for CF airway disease are what airway regions require CFTR function and how many epithelial cells require CFTR expression to restore normal ASL volume regulation and mucus transport to CF airway epithelium? An in vitro model of human CF ciliated surface airway epithelium (CF HAE) was used to test whether a human parainfluenza virus (PIV) vector engineered to express CFTR (PIVCFTR) could deliver sufficient CFTR to CF HAE to restore mucus transport, thus correcting the CF phenotype. PIVCFTR delivered CFTR to >60% of airway surface epithelial cells and expressed CFTR protein in CF HAE approximately 100-fold over endogenous levels in non-CF HAE. This efficiency of CFTR delivery fully corrected the basic bioelectric defects of Cl⁻ and Na⁺ epithelial ion transport and restored ASL volume regulation and mucus transport to levels approaching those of non-CF HAE. To determine the numbers of CF HAE surface epithelial cells required to express CFTR for restoration of mucus transport to normal levels, different amounts of PIVCFTR were used to express CFTR in 3%–65% of the surface epithelial cells of CF HAE and correlated to increasing ASL volumes and mucus transport rates. These data demonstrate for the first time, to our knowledge, that restoration of normal mucus transport rates in CF HAE was achieved after CFTR delivery to 25% of surface epithelial cells. In vivo experimentation in appropriate models will be required to determine what level of mucus transport will afford clinical benefit to CF patients, but we predict that a future goal for corrective gene transfer to the CF human airways in vivo would attempt to target at least 25% of surface epithelial cells to achieve mucus transport rates comparable to those in non-CF airways.     

Integrin-β4 regulates the dynamic changes of phenotypic characteristics in association with epithelial-mesenchymal transition (EMT) and RhoA activity in airway epithelial cells during injury and repair

Background: In airway disease such as asthma a hyperactive cellular event of epithelial-mesenchymal transition (EMT) is considered as the mechanism of pathological airway tissue remodeling after injury to the airway epithelium. And the initiation of EMT in the airways depends on the epithelial disruption involving dissolution and/or destabilization of the adhesive structures between the cells and ECM. Previously, we have shown that integrin-β4, an epithelial adhesion molecule in bronchial epithelium is an important regulator of cell proliferation and wound repair in human airway epithelial cells. Therefore, in this study we aimed to investigate whether integrin-β4 also regulates EMT phenotypes during injury and repair in airway epithelial cells of both wild type/integrin-β4^-/- mice in vivo and cultured cells treated with integrin-β4/nonsense siRNA in vitro.Methods: We induced injury to the airway epithelial cells by either repeated exposure to ozone and mechanical scratch wound, and subsequently examined the EMT-related phenotypic features in the airway epithelial cells including biomarkers expression, adhesion and cytoskeleton reorganization and cell stiffness.Results: The results show that in response to injury (ozone exposure/scratch wound) and subsequent spontaneous repair (ozone withdrawal/wound healing) both in vivo and in vitro, the airway epithelial cells underwent dynamic changes in the epithelial and mesenchymal biomarkers expression, adhesion and cytoskeleton structures as well as cell stiffness, all together exhibiting enhanced EMT phenotypic features after injury and reversal of the injury-induced effects during repair. Importantly, these injury/repair-associated EMT phenotypic changes in airway epithelial cells appeared to be dependent on integrin-β4 expression. More specifically, when integrin-β4 was deficient in mice (integrin-β4^-/-) the repair of ozone-injured airway epithelium was impaired and the recovery of ozone-enhanced EMT biomarkers expression in the airway epithelium was delayed. Similarly, in the scratch wounded airway epithelial cells with integrin-β4 knockdown, the cells were impaired in all aspects related to EMT during wound and repair including cell proliferation, wound closure rate, adhesion and cytoskeleton protein expression (vinculin and vimentin), mesenchymal-like F-actin reorganization, cell stiffness and RhoA activation.Conclusion: Taken together, these results suggested that integrin-β4 may be essential in regulating the effects of injury and repair on EMT in airway epithelial cells via influencing both the cell adhesion to ECM and cells'' physical phenotypes through RhoA signaling pathway.     

Ionic components of electric current at rat corneal wounds

Background

Endogenous electric fields and currents occur naturally at wounds and are a strong signal guiding cell migration into the wound to promote healing. Many cells involved in wound healing respond to small physiological electric fields in vitro. It has long been assumed that wound electric fields are produced by passive ion leakage from damaged tissue. Could these fields be actively maintained and regulated as an active wound response? What are the molecular, ionic and cellular mechanisms underlying the wound electric currents?

Methodology/Principal Findings

Using rat cornea wounds as a model, we measured the dynamic timecourses of individual ion fluxes with ion-selective probes. We also examined chloride channel expression before and after wounding. After wounding, Ca²⁺ efflux increased steadily whereas K⁺ showed an initial large efflux which rapidly decreased. Surprisingly, Na⁺ flux at wounds was inward. A most significant observation was a persistent large influx of Cl⁻, which had a time course similar to the net wound electric currents we have measured previously. Fixation of the tissues abolished ion fluxes. Pharmacological agents which stimulate ion transport significantly increased flux of Cl⁻, Na⁺ and K⁺. Injury to the cornea caused significant changes in distribution and expression of Cl⁻ channel CLC2.

Conclusions/Significance

These data suggest that the outward electric currents occurring naturally at corneal wounds are carried mainly by a large influx of chloride ions, and in part by effluxes of calcium and potassium ions. Ca²⁺ and Cl⁻ fluxes appear to be mainly actively regulated, while K⁺ flux appears to be largely due to leakage. The dynamic changes of electric currents and specific ion fluxes after wounding suggest that electrical signaling is an active response to injury and offers potential novel approaches to modulate wound healing, for example eye-drops targeting ion transport to aid in the challenging management of non-healing corneal ulcers.     

Arsenic upregulates MMP-9 and inhibits wound repair in human airway epithelial cells

As part of the innate immune defense, the polarized conducting lung epithelium acts as a barrier to keep particulates carried in respiration from underlying tissue. Arsenic is a metalloid toxicant that can affect the lung via inhalation or ingestion. We have recently shown that chronic exposure of mice or humans to arsenic (10-50 ppb) in drinking water alters bronchiolar lavage or sputum proteins consistent with reduced epithelial cell migration and wound repair in the airway. In this report, we used an in vitro model to examine effects of acute exposure of arsenic (15-290 ppb) on conducting airway lung epithelium. We found that arsenic at concentrations as low as 30 ppb inhibits reformation of the epithelial monolayer following scrape wounds of monolayer cultures. In an effort to understand functional contributions to epithelial wound repair altered by arsenic, we showed that acute arsenic exposure increases activity and expression of matrix metalloproteinase (MMP)-9, an important protease in lung function. Furthermore, inhibition of MMP-9 in arsenic-treated cells improved wound repair. We propose that arsenic in the airway can alter the airway epithelial barrier by restricting proper wound repair in part through the upregulation of MMP-9 by lung epithelial cells.     

外源性电场对组织修复过程中细胞行为的影响

组织修复涉及一系列复杂的生理学、免疫学及细胞生物学过程。研究表明,受损组织周围存在着一定强度的内源性电场,类似生理强度的外源性电场能指导细胞定向迁移、控制细胞极化、调节细胞增殖和分化等一系列生物学行为。该文针对外源性电场在伤口愈合、骨组织愈合以及血管新生过程中的细胞生物学作用及其对修复过程中组织水平的影响进行综述,以期为外源性电场在今后临床中的应用提供参考。     

Tracheobronchial epithelium in the adult rhesus monkey: a quantitative histochemical and ultrastructural study

Previous studies of the intrapulmonary conducting airways of sheep and rabbit have demonstrated marked diversity in the epithelial populations lining them. Because studies of trachea and centriacinar regions of macaque monkeys suggested that primates may be even more diverse, the present study was designed to characterize the epithelial population throughout the airway tree of one primate species, the rhesus monkey. Trachea and intrapulmonary airways of the right cranial and middle lobes of glutaraldehyde/paraformaldehyde-infused lungs of five adult rhesus monkeys were microdissected following the axial pathway. Each branch was assigned a binary number indicating its specific location within the tree. The trachea and six generations of intrapulmonary airway from the right cranial lobe were evaluated for ultrastructure and quantitative histology as were those of the right middle lobe for quantitative carbohydrate histochemistry. Four cell types were identified throughout the tree: ciliated, mucous goblet, small mucous granule, and basal. The tallest epithelium lined the trachea; the shortest, the respiratory bronchiole. The most cells per unit length of basement membrane were in proximal intrapulmonary bronchi; the least, in the respiratory bronchiole. The nonciliated bronchiolar epithelial or Clara cell was restricted to respiratory bronchioles. Sulfomucins were present in the vast majority of surface goblet cells in the trachea and proximal bronchi. In proximal bronchi, neutral glycoconjugates predominated in glands and acidic glycoconjugates in surface epithelium. In terminal and respiratory bronchioles the ratio of acidic glycoconjugate to neutral glycoconjugate equaled that in proximal bronchi, although glands were not present. Sulfomucins were minimal in terminal airways. We conclude that the characteristics of the epithelial lining of the mammalian tracheobronchial airway tree are very species-specific. The lining of the rhesus monkey does not have the diversity in cell types in different airway generations observed in sheep and rabbit. Also, the populations lining these airways in the rhesus are very different from either the sheep or rabbit in number, proportions of different cell types, glycoconjugate content, and distribution of specific cell types.