Vibration, a treatment for migraine, linked to calpain driven changes in actin cytoskeleton.

Understanding how a human cell reacts to external physical stimuli is essential to understanding why vibration can elicit localized pain reduction. Stimulation of epithelial cells with external vibration forces has been shown to change cell shape, particularly in regards to structures involved in non-muscle cell motility. We hypothesized that epithelial cells respond to vibration transduction by altering proteins involved in remodeling cytoskeleton. Epithelial cells were exposed to vibration and assessed by microscopy, cytoskeletal staining, immunoblotting and quantitative RT-PCR. Here, we report that epithelial cell lines exposed to 15 minutes of vibration retract filopodia and concentrate actin at the periphery of the cell. In particular, we show an increased expression of the calcium-dependent, cysteine protease, calpain. The discovery that cell transitions are induced by limited exposure to natural forces, such as vibration, provides a foundation to explain how vibrational treatment helps migraine patients.

A cell-based screening assay to identify pharmaceutical compounds that enhance the regenerative quality of corneal repair.

The goal of this study was to develop and validate a simple but quantitative cell-based assay to identify compounds that might be used pharmaceutically to give tissue repair a more regenerative character. The cornea was used as the model, and some specific aspects of repair in this organ were incorporated into assay design. A quantitative cell-based assay was developed based on transcriptional promoter activity of fibrotic marker genes ACT2A and TGFB2. Immortalized corneal stromal cells (HTK) or corneal epithelial cells (HCLE) were tested and compared to primary corneal stromal cells. Cells were transiently transfected with constructs containing the firefly luciferase reporter gene driven by transcriptional promoters for the selected fibrotic marker genes. A selected panel of seven chemical test compounds was used, containing three known fibrosis inhibitors: lovastatin (LOV), tyrphostin AG 1296 (6,7-dimethoxy-3-phenylquinoxaline) and SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole), and four potential fibrosis inhibitors: 5-iodotubercidin (4-amino-5-iodo-7-(ß-D-ribofuranosyl)-pyrrolo(2,3-d)pyrimidine), anisomycin, DRB (5,6-dichloro-1-ß-D-ribofuranosyl-benzimidazole) and latrunculin B. Transfected cells were treated with TGFB2 in the presence or absence of one of the test compounds. To validate the assay, compounds were tested for their direct effects on gene expression in the immortalized cell lines and primary human corneal keratocytes using RT-PCR and immunohistochemistry. Three "hits" were validated LOV, SB203580 and anisomycin. This assay, which can be applied in a high throughput format to screen large libraries of uncharacterized compounds, or known compounds that might be repurposed, offers a valuable tool for identifying new treatments to address a major unmet medical need. Anisomycin has not previously been characterized as antifibrotic, thus, this is a novel finding of the study.

Mechanisms for PDGF, a serum cytokine, stimulating loss of corneal keratocyte crystallins.

PURPOSE: As corneal stromal cells (keratocytes) become activated before transition to the fibroblastic repair phenotype in response to injury (in situ) or serum (in culture), the corneal crystallins, transketolase (TKT) and aldehyde dehydrogenase (ALDH1A1), are lost. The authors previously showed that the serum cytokine platelet-derived growth factor-BB (PDGF), but not transforming growth factor beta2 (TGF-beta2), stimulates TKT loss. The goal of this study was to further define the molecular mechanisms for PDGF-stimulated loss of crystallins to elucidate the pathway for keratocyte activation. METHODS: Freshly isolated rabbit corneal keratocytes were plated in serum-free medium with or without PDGF and/or specific inhibitors of the PDGF-relevant signal pathway components, PDGF receptor, PI3K/AKT, or ras-initiated MAPK proteins. Intracellular TKT protein levels were quantified by immunoblotting. Ubiquitinated TKT levels were assessed by immunoprecipitation, and TKT messenger RNA (mRNA) levels were quantified by quantitative reverse transcription-polymerase chain reaction. RESULTS: PDGF treatment at the same time as inhibition of PDGF receptor, Akt, JNK, and ubiquitin-proteasome pathway prevented PDGF-induced TKT protein loss. In contrast, treatment with PDGF did not affect TKT mRNA levels. CONCLUSIONS: The results suggest that PDGF-stimulated TKT loss is mediated through cross talk between PI3K-independent Akt and JNK. This signaling pathway leads to the degradation of existing TKT protein but does not compromise the accumulation of TKT mRNA. Therefore, cells retain the potential to reaccumulate TKT protein that is enabled by PDGF removal. These findings suggest that targeting PDGF signaling could improve repair outcomes after surgical procedures in the cornea.

Diesel exhaust particles induce aberrant alveolar epithelial directed cell movement by disruption of polarity mechanisms.

Disruption of the respiratory epithelium contributes to the progression of a variety of respiratory diseases that are aggravated by exposure to air pollutants, specifically traffic-based pollutants such as diesel exhaust particles (DEP). Recognizing that lung repair following injury requires efficient and directed alveolar epithelial cell migration, this study's goal was to understand the mechanisms underlying alveolar epithelial cells response to DEP, particularly when exposure is accompanied with comorbid lung injury. Separate mechanistic steps of directed migration were investigated in confluent murine LA-4 cells exposed to noncytotoxic concentrations (0-100 µg/cm(2)) of either automobile-emitted diesel exhaust particles (DEP(A)) or carbon black (CB) particles. A scratch wound model ascertained how DEP(A) exposure affected directional cell migration and BCECF ratio fluorimetry-monitored intracellular pH (pHi). Cells were immunostained with giantin to assess cell polarity, and with paxillin to assess focal cell adhesions. Cells were immunoblotted for ezrin/radixin/moesin (ERM) to assess cytoskeletal anchoring. Data demonstrate herein that exposure of LA-4 cells to DEP(A) (but not CB) resulted in delayed directional cell migration, impaired de-adhesion of the trailing edge cell processes, disrupted regulation of pHi, and altered Golgi polarity of leading edge cells, along with modified focal adhesions and reduced ERM levels, indicative of decreased cytoskeletal anchoring. The ability of DEP(A) to disrupt directed cell migration at multiple levels suggests that signaling pathways such as ERM/Rho are critical for transduction of ion transport signals into cytoskeletal arrangement responses. These results provide insights into the mechanisms by which chronic exposure to traffic-based emissions may result in decrements in lung capacity.

Nitric oxide and superoxide mediate diesel particle effects in cytokine-treated mice and murine lung epithelial cells--implications for susceptibility to traffic-related air pollution.

BACKGROUND: Epidemiologic studies associate childhood exposure to traffic-related air pollution with increased respiratory infections and asthmatic and allergic symptoms. The strongest associations between traffic exposure and negative health impacts are observed in individuals with respiratory inflammation. We hypothesized that interactions between nitric oxide (NO), increased during lung inflammatory responses, and reactive oxygen species (ROS), increased as a consequence of traffic exposure ─ played a key role in the increased susceptibility of these at-risk populations to traffic emissions. METHODS: Diesel exhaust particles (DEP) were used as surrogates for traffic particles. Murine lung epithelial (LA-4) cells and BALB/c mice were treated with a cytokine mixture (cytomix: TNFα, IL-1ß, and IFNγ) to induce a generic inflammatory state. Cells were exposed to saline or DEP (25 µg/cm(2)) and examined for differential effects on redox balance and cytotoxicity. Likewise, mice undergoing nose-only inhalation exposure to air or DEP (2 mg/m(3) × 4 h/d × 2 d) were assessed for differential effects on lung inflammation, injury, antioxidant levels, and phagocyte ROS production. RESULTS: Cytomix treatment significantly increased LA-4 cell NO production though iNOS activation. Cytomix + DEP-exposed cells incurred the greatest intracellular ROS production, with commensurate cytotoxicity, as these cells were unable to maintain redox balance. By contrast, saline + DEP-exposed cells were able to mount effective antioxidant responses. DEP effects were mediated by: (1) increased ROS including superoxide anion (O(2)(·-)), related to increased xanthine dehydrogenase expression and reduced cytosolic superoxide dismutase activity; and (2) increased peroxynitrite generation related to interaction of O(2)(·-) with cytokine-induced NO. Effects were partially reduced by superoxide dismutase (SOD) supplementation or by blocking iNOS induction. In mice, cytomix + DEP-exposure resulted in greater ROS production in lung phagocytes. Phagocyte and epithelial effects were, by and large, prevented by treatment with FeTMPyP, which accelerates peroxynitrite catalysis. CONCLUSIONS: During inflammation, due to interactions of NO and O(2)(·-), DEP-exposure was associated with nitrosative stress in surface epithelial cells and resident lung phagocytes. As these cell types work in concert to provide protection against inhaled pathogens and allergens, dysfunction would predispose to development of respiratory infection and allergy. Results provide a mechanism by which individuals with pre-existing respiratory inflammation are at increased risk for exposure to traffic-dominated urban air pollution.

Comprehensive gene expression profiling and functional analysis of matrix metalloproteinases and TIMPs, and identification of ADAM-10 gene expression, in a corneal model of epithelial resurfacing.

This study provides a comprehensive expression analysis for the entire matrix metalloproteinase (MMP) gene family during the process of epithelial resurfacing following corneal abrasion injury in the mouse. The mRNA levels for all known MMP genes expressed in mouse, the related enzyme ADAM-10, and the known tissue inhibitors of metalloproteinases (TIMPs) were determined semi-quantitatively by reverse transcriptase-polymerase chain reaction (RT-PCR) in the uninjured epithelium, and in the epithelial tissue resurfacing the abraded area or residing in its periphery at two time points: during the epithelial migration phase and immediately following wound closure. The mRNA levels for MMP-1a, -1b, -9, -10, -12, and -13 as well as TIMP-1 were significantly up-regulated in the migrating corneal epithelium. After wound resurfacing, the mRNA levels for all of these MMPs were down-regulated, although MMP-1a, -1b, and -13 remained significantly elevated in comparison to the uninjured epithelium. The only gene found to be down-regulated was TIMP-3, which occurred throughout the wound-healing process. During resurfacing, MMP-9 was localized to the front of the migrating epithelium, MMP-10 and -13 were localized throughout the migrating epithelium, and MMP-13 could also be found in the periphery. Following epithelial closure, immunoreactive MMPs-9 and -10 became undetectable, but MMP-13 continued to be found throughout the epithelium. Functional analysis of MMP-10 revealed no effects on epithelial migration or cell proliferation. In conclusion, distinct MMP temporal-spatial profiles define the uninjured corneal epithelium and the corneal epithelium at different stages of regeneration. An extensive review of the literature is also provided in the discussion.

A hyperlipidemic rabbit model provides new insights into pulmonary zinc exposure effects on cardiovascular health.

This study ascertains the effects of zinc, a major component of particulate matter, on pulmonary and systemic endpoints using hyperlipidemic rabbits to model diet-induced human atherosclerosis. New Zealand White rabbits were fed a normal or cholesterol-enriched diet and then were intratracheally instilled 1x/week for 4 weeks with saline or 16 microg/kg of zinc, equal parts sulfate and oxide. Physiologic responses, blood after each exposure, and terminal bronchoalveolar lavage (BAL) were assessed. Rabbits fed a cholesterol-rich diet developed hyperlipidemia and had consistently higher circulating leukocyte counts than rabbits fed normal chow. Within minutes after zinc instillation, saturation of peripheral oxygen was decreased in hyperlipidemic rabbits and heart rate was increased in hyperlipidemic rabbits with total serum cholesterol levels greater than 200 mg/dl. Total circulating leukocytes levels were increased 24 h after the first zinc instillation, but upon repeated exposures this effect was attenuated. After repeated zinc exposures, BAL fluid (BALF) N-acetylglucosaminidase activity was increased regardless of hyperlipidemic state. Hyperlipidemic rabbits had an increase in BALF-oxidized glutathione and a decrease in serum nitrite. The study elucidates mechanisms by which the zinc metal component of PM drives cardiovascular health effects, as well as the possible susceptibility induced by hyperlipidemia. Furthermore, the study exemplifies the benefits of monitoring circulatory physiology during exposure as well as after exposure.

Inhibition of human corneal epithelial production of fibrotic mediator TGF-beta2 by basement membrane-like extracellular matrix.

PURPOSE: Transforming growth factor (TGF)-beta2 is a major epithelial mediator of fibrotic marker expression during corneal repair in mice. Production of TGF-beta2 protein by cultured rabbit corneal epithelial cells is reduced by plating on a basement membrane-like extracellular matrix extract (Matrigel; BD Biosciences, Bedford, MA). The goal of the present study was to understand further the nature of Matrigel regulation. METHODS: TGF-beta2 protein, mRNA, and gene transcriptional promotion were characterized in cultured human corneal epithelial cells. RESULTS: TGF-beta2 production was inhibited by Matrigel at the level of mRNA accumulation and activity of the gene transcriptional promoter. This effect of Matrigel was not explained by (1) growth factor contaminants, as growth-factor reduced Matrigel also inhibited TGF-beta2; (2) independent matrix components, as the pure forms of the major ECM components laminin and collagen IV did not reproduce the effect; or (3) inhibition of a constitutive TGF-beta2 autocrine feedback loop, as addition of exogenous TGF-beta2 increased p-Smad3 and restored TGF-beta2 mRNA levels. In addition, Matrigel's ability to reduce TGF-beta2 was not explained by its geometry, as TGF-beta2 production was not inhibited by plating cells on a synthetic nanofiber matrix with a three-dimensional topography similar to Matrigel. Matrigel caused a reduction of ezrin, a member of the ezrin-radixin-moesin (ERM) family, which plays a role in establishing polarity of epithelial cells in tissues through the Rho signaling pathway. CONCLUSIONS: These findings indicate that Matrigel inhibits TGF-beta2 gene expression and point to a mechanism dependent on Matrigel composition and structure. The capacity of Matrigel to reduce ezrin is consistent with this idea and directs the focus of future studies toward the ERM/Rho pathway.

Immune accessory functions of human endothelial cells are modulated by overexpression of B7-H1 (PDL1).

B7-H1 (PDL1) is a B7-related protein that inhibits T-cell responses. Human endothelial cells (EC), which can support polyclonal stimulation (by anti-CD3 or Phytohemagglutinin (PHA)) or direct alloantigen stimulation of T cells, basally express B7-H1 and increase expression in response to IFN-gamma or coculture with allogeneic T cells. Previous studies have suggested that endogenous B7-H1 on EC reduces T-cell responses. We engineered overexpression of B7-H1 in EC (B7H1-EC) to evaluate whether this manipulation could reduce T-cell responses even further. Compared with green fluorescent protein-transduced EC (GFP-EC), B7H1-EC support less anti-CD3 or PHA-induced proliferation of CD4+ memory T cells; naive CD4+ T-cell or CD8+ T-cell responses were less inhibited. The effect of transduced B7H1-EC was more apparent when the EC were fixed prior to coculture, a manipulation that reduces the strength of costimulation and prevents upregulation of the endogenous B7-H1 molecule. T-cell activation markers, including CD25, CD62L, CD152 (CTLA-4), and CD154 (CD40L), were not altered by EC overexpression of B7-H1, whereas there was a reduction in CD69. B7-H1 reduced secretion of IL-2 and IL-10 by memory T cells. B7H1-EC were less able to stimulate allogeneic proliferation of CD4+ memory T cells than control EC. These data suggest that B7-H1 overexpression may be a useful approach for reducing allogeneic CD4+ memory T-cell responses to EC.