Vasoactive intestinal peptide enhances wound healing and proliferation of human bronchial epithelial cells

In the present study, we investigated the effects of vasoactive intestinal peptide (VIP) on wound healing of bronchial epithelium. Wound healing of the mechanical damaged human bronchial epithelial cells (HBEC) was observed in the absence or presence of VIP. Effects of VIP on chemotactic migration, cell proliferation of HBEC were also tested. HBEC chemotaxis was assessed by the blind well chamber technique, the cell cycle was determined by flow cytometry, and cell proliferation was determined by measuring the expression of proliferating cell nuclear antigen Ki67. Effects of VIP on epithelial E-cadherins protein and mRNA were also measured by immunohistochemistry and RT-PCR. The results showed that VIP accelerated the recovery of wound area of HBEC. VIP increased the migration and proliferation of HBEC, and these effects were blocked by a VPAC1 receptor antagonist. VIP also increased the expression of E-cadherin mRNA and protein in HBEC, suggesting that protective effects of VIP on wound healing may be related to its ability to increase the expression of E-cadherin. In conclusion, VIP has protective effects against human bronchial epithelial cell damage, and the beneficial effects of VIP might be mediated, at least in part, by VPAC1, and associated with increased expression of E-cadherin.     

Mechanical compression attenuates normal human bronchial epithelial wound healing

Background

Highly pathogenic avian influenza (HPAI) H5N1 virus is entrenched in poultry in Asia and Africa and continues to infect humans zoonotically causing acute respiratory disease syndrome and death. There is evidence that the virus may sometimes spread beyond respiratory tract to cause disseminated infection. The primary target cell for HPAI H5N1 virus in human lung is the alveolar epithelial cell. Alveolar epithelium and its adjacent lung microvascular endothelium form host barriers to the initiation of infection and dissemination of influenza H5N1 infection in humans. These are polarized cells and the polarity of influenza virus entry and egress as well as the secretion of cytokines and chemokines from the virus infected cells are likely to be central to the pathogenesis of human H5N1 disease.

Aim

To study influenza A (H5N1) virus replication and host innate immune responses in polarized primary human alveolar epithelial cells and lung microvascular endothelial cells and its relevance to the pathogenesis of human H5N1 disease.

Methods

We use an in vitro model of polarized primary human alveolar epithelial cells and lung microvascular endothelial cells grown in transwell culture inserts to compare infection with influenza A subtype H1N1 and H5N1 viruses via the apical or basolateral surfaces.

Results

We demonstrate that both influenza H1N1 and H5N1 viruses efficiently infect alveolar epithelial cells from both apical and basolateral surface of the epithelium but release of newly formed virus is mainly from the apical side of the epithelium. In contrast, influenza H5N1 virus, but not H1N1 virus, efficiently infected polarized microvascular endothelial cells from both apical and basolateral aspects. This provides a mechanistic explanation for how H5N1 virus may infect the lung from systemic circulation. Epidemiological evidence has implicated ingestion of virus-contaminated foods as the source of infection in some instances and our data suggests that viremia, secondary to, for example, gastro-intestinal infection, can potentially lead to infection of the lung. HPAI H5N1 virus was a more potent inducer of cytokines (e.g. IP-10, RANTES, IL-6) in comparison to H1N1 virus in alveolar epithelial cells, and these virus-induced chemokines were secreted onto both the apical and basolateral aspects of the polarized alveolar epithelium.

Conclusion

The predilection of viruses for different routes of entry and egress from the infected cell is important in understanding the pathogenesis of influenza H5N1 infection and may help unravel the pathogenesis of human H5N1 disease.     

Activation of CFTR trafficking and gating by vasoactive intestinal peptide in human bronchial epithelial cells

Cystic fibrosis transmembrane conductance regulator (CFTR) is an apical membrane chloride channel critical to the regulation of fluid, chloride, and bicarbonate transport in epithelia and other cell types. The most common cause of cystic fibrosis (CF) is the abnormal trafficking of CFTR mutants. Therefore, understanding the cellular machineries that transit CFTR from the endoplasmic reticulum to the cell surface is important. Vasoactive intestinal polypeptide (VIP) plays an important role in CFTR-dependent chloride transport. The present study was designed to observe the affection of VIP on the trafficking of CFTR, and channel gating in human bronchial epithelium cells (HBEC). Confocal microscopy revealed CFTR immunofluorescence extending from the apical membrane deeply into the cell cytoplasm. After VIP treatment, apical extension of CFTR immunofluorescence into the cell was reduced and the peak intensity of CFTR fluorescence shifted towards the apical membrane. Western blot showed VIP increased cell surface and total CFTR. Compared with the augmented level of total CFTR, the surface CFTR increased more markedly. Immunoprecipitation founded that the mature form of CFTR had a marked increase in HBEC treated with VIP. VIP led to a threefold increase in Cl(-) efflux in HBEC. Glibenclamide-sensitive and DIDS-insensitive CFTR Cl(-) currents were consistently observed after stimulation with VIP (10(-8) mol/L). The augmentation of CFTR Cl(-) currents enhanced by VIP (10(-8) mol/L) was reversed, at least in part, by the protein kinase A (PKA) inhibitor, H-89 and the protein kinase C (PKC) inhibitor, H-7, suggesting PKA and PKC participate in the VIP-promoted CFTR Cl(-) currents.     

Mechanisms of vasoactive intestinal peptide-mediated vasodilation in human skin.

Vasoactive intestinal peptide (VIP) is known to induce histamine release in human skin and to include a nitric oxide (NO)-dependent dilation in several other vascular beds. However, the relative contribution of histamine and NO to VIP-mediated vasodilation in human skin is unknown. Forty-three subjects volunteered to participate in two studies designed to examine the mechanism of VIP-mediated vasodilation in human skin. Study 1 examined the contribution of NO in the skin blood flow response to eight doses of VIP ranging from 25 to 800 pmol. In addition, study 1 examined a specific role for NO in VIP-mediated dilation. Study 2 examined the relative contribution of NO and histamine to VIP-mediated dilation via H1 and H2 histamine receptors. Infusions were administered to skin sites via intradermal microdialysis. Red blood cell flux was measured by using laser-Doppler flowmetry (LDF), and cutaneous vascular conductance (CVC; LDF/mean arterial pressure) was calculated and normalized to maximal vasodilation. VIP-mediated vasodilation includes a NO-dependent component at doses above 100 pmol, where NO synthase inhibition significantly attenuates CVC (P < 0.05). Inhibition of H1 receptors attenuates the rise in CVC to exogenous VIP (P < 0.05); however, combined H1-receptor inhibition and NO synthase inhibition further reduced VIP-mediated vasodilation compared with either H1 inhibition or NO synthase inhibition alone (P < 0.05). In contrast to H1-receptor inhibition, H2-receptor inhibition did not affect vasodilation to exogenous VIP. Thus, in human skin, VIP-mediated vasodilation includes a NO-dependent component that could not be explained by H1- and H2-receptor activation.     

血管活性肠肽对支气管上皮细胞趋化迁移的影响及机制

为探讨肺内神经肽在气道损伤修复中的作用 ,采用blind wellBoydenchamber测定原代培养的支气管上皮细胞 (bronchialepithelialcells,BEC)趋化性 ,观察血管活性肠肽 (vasoactiveintestinalpeptide ,VIP)对BEC趋化迁移的影响及其机制 ,并测定经热应激后BEC分泌VIP及表达VIP受体 (vasoactiveintestinalpeptidereceptor,VIPR)的变化。结果显示 :(1)以胰岛素作为趋化因子所建立的BEC趋化性测定方法稳定 ,重现性好 (r =0 970 3,P <0 0 1) ;(2 )VIP (0 0 0 1～ 1μmol/L)均显示剂量依赖性地增强BEC的趋化迁移 ,其效应可被钙调蛋白阻断剂及蛋白激酶C阻断剂有效地抑制 (P <0 0 1) ;(3) 4 2℃、30min热应激后BEC分泌VIP (P <0 0 1)及表达VIPR明显增加 (P <0 0 5 )。实验表明 :肺内神经肽VIP可增强BEC的趋化迁移 ,其细胞内信号转导途径与钙调蛋白及蛋白激酶C有关。而热应激时VIP及VIPR的高表达进一步提示局部微环境的VIP可能是气道上皮损伤修复网络中的重要分子     

Rhinovirus infection induces cytotoxicity and delays wound healing in bronchial epithelial cells

Background

Human rhinoviruses (RV), the most common triggers of acute asthma exacerbations, are considered not cytotoxic to the bronchial epithelium. Recent observations, however, have questioned this knowledge. The aim of this study was to evaluate the ability of RV to induce epithelial cytotoxicity and affect epithelial repair in-vitro.

Methods

Monolayers of BEAS-2B bronchial epithelial cells, seeded at different densities were exposed to RV serotypes 1b, 5, 7, 9, 14, 16. Cytotoxicity was assessed chromatometrically. Epithelial monolayers were mechanically wounded, exposed or not to RV and the repopulation of the damaged area was assessed by image analysis. Finally epithelial cell proliferation was assessed by quantitation of proliferating cell nuclear antigen (PCNA) by flow cytometry.

Results

RV1b, RV5, RV7, RV14 and RV16 were able to induce considerable epithelial cytotoxicity, more pronounced in less dense cultures, in a cell-density and dose-dependent manner. RV9 was not cytotoxic. Furthermore, RV infection diminished the self-repair capacity of bronchial epithelial cells and reduced cell proliferation.

Conclusion

RV-induced epithelial cytotoxicity may become considerable in already compromised epithelium, such as in the case of asthma. The RV-induced impairment on epithelial proliferation and self-repair capacity may contribute to the development of airway remodeling.     

ADAM-15 inhibits wound healing in human intestinal epithelial cell monolayers

The disintegrin metalloproteases (or ADAMs) are membrane-anchored glycoproteins that have been implicated in cell-cell or cell-matrix interactions and in proteolysis of molecules on the cell surface. The expression and/or the pathophysiological implications of ADAMs are not known in intestinal epithelial cells. Therefore, our aim was to investigate the expression and the role of ADAMs in intestinal epithelial cells. Expression of ADAMs was assessed by RT-PCR, Western blot analysis, and immunufluorescence experiments. Wound-healing experiments were performed by using the electric cell substrate impedence sensing technology. Our results showed that ADAMs-10, -12, and -15 mRNA are expressed in the colonic human cell lines Caco2-BBE and HT29-Cl.19A. An ADAM-15 complementary DNA cloned from Caco2-BBE poly(A)+ RNA, and encompassing the entire coding region, was found to be shorter and to present a different region encoding the cytoplasmic tail compared with ADAM-15 sequence deposited in the database. In Caco2-BBE cells and colonic epithelial cells, ADAM-15 protein was found in the apical, basolateral, and intracellular compartments. We also showed that the overexpression of ADAM-15 reduced cell migration in a wound-healing assay in Caco2-BBE monolayers. Our data show that 1) ADAM-15 is expressed in human intestinal epithelia, 2) a new variant of ADAM-15 is expressed in a human intestinal epithelial cell line, and 3) ADAM-15 is involved in intestinal epithelial cells wound-healing processes. Together, these results suggest that ADAM-15 may have important pathophysiological roles in intestinal cells.     

Wnt signaling induces epithelial differentiation during cutaneous wound healing

Background  

Cutaneous wound repair in adult mammals does not regenerate the original epithelial architecture and results in altered skin function. We propose that lack of regeneration may be due to the absence of appropriate molecular signals to promote regeneration. In this study, we investigated the regulation of Wnt signaling during cutaneous wound healing and the consequence of activating either the beta-catenin-dependent or beta-catenin-independent Wnt signaling on epidermal architecture during wound repair.     

A mathematical model of intercellular signaling during epithelial wound healing

Recent experiments monitoring the healing process of wounded epithelial monolayers have demonstrated the necessity of MAPK activation for coordinated cell movement after damage. This MAPK activity is characterized by two wave-like phenomena. One MAPK “wave” that originates immediately after injury, propagates deep into the cell sheet, away from the edge, and then rebounds back to the wound interface. After this initial MAPK activity has largely disappeared, a second MAPK front propagates slowly from the wound interface and also continues into the cell sheet, maintaining a sustained level of MAPK activity throughout the cell sheet. It has been suggested that the first wave is initiated by Reactive Oxygen Species (ROS) generated at the time of injury. In this work, we develop a minimal mathematical model that reproduces the observed behavior. The main ingredients of our model are a competition between ligand (e.g., Epithelial Growth Factor) and ROS for the activation of Epithelial Growth Factor Receptor, and a feedback loop between receptor occupancy and MAPK activation. We explore the mathematical properties of the model and look for traveling wave solutions consistent with the experimentally observed MAPK activity patterns.     

Vasopressin-mediated mitogenic signaling in intestinal epithelial cells

The role of G protein-coupled receptorsand their ligands in intestinal epithelial cell signaling andproliferation is poorly understood. Here, we demonstrate that argininevasopressin (AVP) induces multiple intracellular signal transductionpathways in rat intestinal epithelial IEC-18 cells via aV_1A receptor. Addition of AVP to these cells induces arapid and transient increase in cytosolic Ca²⁺concentration and promotes protein kinase D (PKD) activation through aprotein kinase C (PKC)-dependent pathway, as revealed by in vitrokinase assays and immunoblotting with an antibody that recognizesautophosphorylated PKD at Ser⁹¹⁶. AVP also stimulates thetyrosine phosphorylation of the nonreceptor tyrosine kinaseproline-rich tyrosine kinase 2 (Pyk2) and promotes Src family kinasephosphorylation at Tyr⁴¹⁸, indicative of Src activation.AVP induces extracellular signal-related kinase (ERK)-1(p44^mapk) and ERK-2 (p42^mapk) activation, aresponse prevented by treatment with mitogen-activated protein kinasekinase (MEK) inhibitors (PD-98059 and U-0126), specific PKC inhibitors(GF-I and Ro-31-8220), depletion of Ca²⁺ (EGTA andthapsigargin), selective epidermal growth factor receptor (EGFR)tyrosine kinase inhibitors (tyrphostin AG-1478, compound 56), or theselective Src family kinase inhibitor PP-2. Furthermore, AVP acts as apotent growth factor for IEC-18 cells, inducing DNA synthesis and cellproliferation through ERK-, Ca²⁺-, PKC-, EGFR tyrosinekinase-, and Src-dependent pathways.

     

Intracellular signaling molecules involved in an inhibitory factor-induced decrease in fetal-type AChR expression

The innervation-induced down-regulation of fetal-type acetylcholine receptor (AChR) expression in developing muscle fibers has largely been attributed to nerve-evoked muscle activity; however, there is increasing evidence that a neural trophic factor also contributes to this receptor down-regulation. Previous studies from this laboratory have shown that neural extracts contain a factor which decreases fetal-type AChR expression in skeletal muscle cell lines and therefore may account for the proposed inhibitory neurotrophic influence. The current study investigated possible intracellular signaling molecules involved in this receptor down-regulation and demonstrated that activation of protein kinase C and p70(S6k) appeared to be important in receptor down-regulation. Decreases in AChR density were independent of myogenin. In addition, the receptor down-regulation was independent of neuregulin, which also induces p70(S6k) activity. These studies demonstrate that neural extracts contain an inhibitory factor which can down-regulate fetal-type AChR expression independently of nerve-evoked muscle activity through intracellular signaling molecules which are known to regulate AChR expression.     

Activin B promotes epithelial wound healing in vivo through RhoA-JNK signaling pathway

Background

Activin B has been reported to promote the proliferation and migration of keratinocytes in vitro via the RhoA-JNK signaling pathway, whereas its in vivo role and mechanism in wound healing process has not yet been elucidated.

Principal Findings

In this study, we explored the potential mechanism by which activin B induces epithelial wound healing in mice. Recombinant lentiviral plasmids, with RhoA (N19) and RhoA (L63) were used to infect wounded KM mice. The wound healing process was monitored after different treatments. Activin B-induced cell proliferation on the wounded skin was visualized by electron microscopy and analyzed by 5′-bromodeoxyuridine (BrdU) incorporation assay. Protein expression of p-JNK or p-cJun was determined by immunohistochemical staining and immunoblotting analysis. Activin B efficiently stimulated the proliferation of keratinocytes and hair follicle cells at the wound area and promoted wound closure. RhoA positively regulated activin B-induced wound healing by up-regulating the expression of p-JNK and p-cJun. Moreover, suppression of RhoA activation delayed activin B-induced wound healing, while JNK inhibition recapitulated phenotypes of RhoA inhibition on wound healing.

Conclusion

These results demonstrate that activin B promotes epithelial wound closure in vivo through the RhoA-Rock-JNK-cJun signaling pathway, providing novel insight into the essential role of activin B in the therapy of wound repair.     

Interaction of vasoactive intestinal peptide with rat small intestinal epithelial cells after intestinal resection

Specific binding of vasoactive intestinal peptide (VIP) and VIP-stimulated c y c l i c AMP accumulation were studied in small intestinal epithelial cells (both of crypt and villous levels) 3, 7 and 14 d after a 60% resection of the small intestine . The affinity, but not the binding capacity, of VIP receptors decreased during the adaptive hyperplastic response. Basal cyclic AMP levels were similar in cells of both control and resected rats. Resection induced a decrease of potency, but not of efficiency, of VIP on the stimulation of cyclic AMP accumulation.     

Exosomes derived from human amniotic epithelial cells accelerate wound healing and inhibit scar formation

Wound healing is a highly orchestrated physiological process consisting of a complex events, and scarless wound healing is highly desired for the development and application in clinical medicine. Recently, we have demonstrated that human amniotic epithelial cells (hAECs) promoted wound healing and inhibited scar formation through a paracrine mechanism. However, exosomes (Exo) are one of the most important paracrine factors. Whether exosomes derived from human amniotic epithelial cells (hAECs-Exo) have positive effects on scarless wound healing have not been reported yet. In this study, we examined the role of hAECs-Exo on wound healing in a rat model. We found that hAECs, which exhibit characteristics of both embryonic and mesenchymal stem cells, have the potential to differentiate into all three germ layers. hAECs-Exo ranged from 50 to 150 nm in diameter, and positive for exosomal markers CD9, CD63, CD81, Alix, TSG101 and HLA-G. Internalization of hAECs-Exo promoted the migration and proliferation of fibroblasts. Moreover, the deposition of extracellular matrix (ECM) were partly abolished by the treatment of high concentration of hAECs-Exo (100 μg/mL), which may be through stimulating the expression of matrix metalloproteinase-1 (MMP-1). In vivo animal experiments showed that hAECs-Exo improved the skin wound healing with well-organized collagen fibers. Taken together, These findings represent that hAECs-Exo can be used as a novel hope in cell-free therapy for scarless wound healing.     

Changes in epithelial cells in Hirudo medicinalis during wound healing.

In the leech, Hirudo medicinalis, reepithelialization is an event which takes place early in the wound healing process, immediately after the formation of the pseudoblastema, 4-8 hr postinjury. Epithelial cells on the wound margins move into the wound, modifying their phenotypic characteristics. Cells lose their columnar shape and become flattened. Dermal junctions disrupt and tonofilaments regroup around the nucleus. Then, the epithelial cell sheet moves over the newly formed pseudoblastema by extending filopodia, formed by the cells on the edge, following the so-called "sliding model." When the wound is fully covered by the new epithelium, about 24 hr postinjury, a reorganization of the cytoskeleton occurs and the basal dermal junctions are reconstructed. Six days postinjury, the epidermal cells return to their original columnar shape.     

FAK regulates intestinal epithelial cell survival and proliferation during mucosal wound healing

Background

Following damage to the intestinal epithelium, restoration of epithelial barrier integrity is triggered by a robust proliferative response. In other tissues, focal adhesion kinase (FAK) regulates many of the cellular processes that are critical for epithelial homeostasis and restitution, including cell migration, proliferation and survival. However, few studies to date have determined how FAK contributes to mucosal wound healing in vivo.

Methodology and Principal Findings

To examine the role of FAK in intestinal epithelial homeostasis and during injury, we generated intestinal epithelium (IE)-specific conditional FAK knockout mice. Colitis was induced with dextran-sulfate-sodium (DSS) and intestinal tissues were analyzed by immunohistochemistry and immunoblotting. While intestinal development occurred normally in mice lacking FAK, FAK-deficient animals were profoundly susceptible to colitis. The loss of epithelial FAK resulted in elevated p53 expression and an increased sensitivity to apoptosis, coincident with a failure to upregulate epithelial cell proliferation. FAK has been reported to function as a mechanosensor, inducing cyclin D1 expression and promoting cell cycle progression under conditions in which tissue/matrix stiffness is increased. Collagen deposition, a hallmark of inflammatory injury resulting in increased tissue rigidity, was observed in control and FAK knockout mice during colitis. Despite this fibrotic response, the colonic epithelium in FAK-deficient mice exhibited significantly reduced cyclin D1 expression, suggesting that proliferation is uncoupled from fibrosis in the absence of FAK. In support of this hypothesis, proliferation of Caco-2 cells increased proportionally with matrix stiffness in vitro only under conditions of normal FAK expression; FAK depleted cells exhibited reduced proliferation concomitant with attenuated cyclin D1 expression.

Conclusions

In the colon, FAK functions as a regulator of epithelial cell survival and proliferation under conditions of mucosal injury and a mechanosensor of tissue compliance, inducing repair-driven proliferation in the colonic epithelium through upregulation of cyclin D1.     

Decreased binding of vasoactive intestinal peptide to intestinal epithelial cells from hypothyroid rats

The binding of vasoactive intestinal peptide (VIP) and stimulation of adenylate cyclase by VIP were studied in intestinal epithelial cells during hypothyroidism. Experimental hypothyroidism was induced in rats by the administration of KC10(4). The binding capacity, but not the affinity, of VIP receptors decreased in the hypothyroid rats. Besides, the stimulation of cyclic AMP production by VIP was also diminished in cells from hypothyroid rats. These observations indicate a decrease of the responsiveness of intestinal epithelial cells to VIP in the hypothyroid status, suggesting a role of the peptide in the pathophysiologic mechanism of intestinal manifestations during hypothyroidism.