Human three-dimensional fibroblast cultures express angiogenic activity

Human neonatal fibroblasts were cultured on a lactate-glycollate copolymer scaffold for 12-16 days to form a three-dimensional dermal equivalent tissue. The cellular content of vascular endothelial growth factor (VEGF) mRNA in these three-dimensional cultures was 22-fold greater than that observed in the same fibroblasts grown as monolayers. No induction was shown by hepatocyte growth factor (HGF) or angiopoietin 1 indicating that the effect was specific to VEGF. The predominant VEGF splice variant, detected by RT-PCR corresponded to the 121 amino acid form, with less of the 165 amino acid form. The cell-associated forms (189 and 206 amino acids) comprised less than 1% of the total VEGF mRNA. VEGF and HGF proteins, determined by ELISA, were secreted in physiologically significant amounts, 0.5-4 ng per 24 h/10(6) cells. Conditioned medium from the three-dimensional cultures stimulated proliferation of endothelial cells in a dose-dependent manner and induced cellular expression of integrin alpha(v)beta(3). Conditioned medium from the same dermal fibroblasts cultured in monolayer showed little angiogenic activity in any of these assays. Using the chorioallantoic membrane (CAM) angiogenesis assay, the cultures stimulated blood vessel production 2.8-fold over scaffold alone. VEGF-neutralizing antibody reduced the vessel development in the CAM to the level in the scaffold control. Anti-HGF antibody had no significant effect. In conclusion, three-dimensional cultures of dermal equivalent tissue express angiogenic activity to a greater extent than monolayer cultures, some of which can be assigned to VEGF.     

Proliferating satellite cells express acidic fibroblast growth factor during in vitro myogenesis.

Recent in vitro studies have indicated that the proliferation of satellite cells, which are involved in muscular regeneration in vivo, is stimulated by exogenous addition of fibroblast growth factor (FGF). We present evidence that satellite cell cultures produce acidic, but not basic FGF. Acidic or basic FGF content was measured by enzyme immunoassay on cellular extracts after partial purification by heparin-Sepharose chromatography. During maximal cell proliferation, the level of acidic fibroblast growth factor (aFGF) was increased over fivefold from the values obtained before plating. aFGF content drastically dropped at the postmitotic stage to almost the threshold of detection, and remained weak as differentiation was completed. The immunolocalization of aFGF using highly purified anti-aFGF antibodies confirmed these results and indicated that aFGF was cytoplasma- or membrane-associated. Our work suggests that an endogenous production of aFGF by satellite cells may trigger cell proliferation by an intra- or autocrine mechanism, and therefore play an important role in muscular regeneration.     

Human keratin hydrogels support fibroblast attachment and proliferation in vitro

Human hair keratins have a strong potential for development as clinically relevant biomaterials because they are abundant and bioactive and are a realistic source of autologous proteins. Specifically, keratins have the propensity to polymerize in an aqueous environment to form hydrogels. In order to evaluate the suitability of keratin hydrogels as substrates for cell culture, we have fabricated hydrogels using keratins extracted from human hair by inducing polymerization with Ca²⁺; these hydrogels exhibit highly branched and porous micro-architectures. L929 murine fibroblasts have been used in a preliminary cell culture study to compare the in vitro biocompatibility of the keratin hydrogels with collagen type 1 hydrogels of similar viscoelastic properties. Our results reveal that keratin hydrogels are comparable with collagen hydrogels in terms of the promotion of cell adhesion, proliferation and the preservation of cell viability. Interestingly, cells remain clustered in proliferative colonies within the keratin hydrogels but are homogeneously distributed as single cells in collagen hydrogels. Collectively, our results demonstrate that keratin hydrogels can be used as substrates for cell culture. Such gels might find applications as templates for soft tissue regeneration.     

Unscheduled apoptosis during acute inflammatory lung injury

Apoptosis is a mode of cell death currently thought to occur in the absence of inflammation. In contrast, inflammation follows unscheduled events such as acute tissue injury which results in necrosis, not apoptosis. We examined the relevance of this paradigm in three distinct models of acute lung injury; hyperoxia, oleic acid, and bacterial pneumonia. In every case, it was found that apoptosis is actually a prominent component of the acute and inflammatory phase of injury. Moreover, using strains of mice that are differentially sensitive to hyperoxic lung injury we observed that the percent of apoptotic cells was well correlated with the severity of lung injury. These observations suggest that apoptosis may be one of the biological consequences during acute injury and the failure to remove these apoptotic cells may also contribute to the inflammatory response.     

Morphological and biochemical changes in human fibroblast lines induced by anthracyclines during apoptosis

We show that treating human trisomic fibroblasts with anthracyclines - aclarubicin, daunorubicin and idarubicin - leads to certain changes in these cells; namely the activation of caspase 3, morphological changes and an increase in the level of intracellular calcium. These results suggest that anthracycline drugs are also able to induce apoptosis in pathological, trisomic cells.     

Modelling in vitro lung branching morphogenesis during development

It has been shown experimentally that lung epithelial explants have an ability to undergo branching morphogenesis without mesenchyme. However, the mechanisms of this phenomenon remain to be elucidated. In the present study, we construct a mathematical model that can reproduce the dynamics of in vitro branching morphogenesis. We show that the system is essentially governed by three variables--c(0) which is the initial fibroblast growth factor (FGF) concentration, D which is the diffusion coefficient of FGF, and beta which describes the mechanical strength of the cytoskeleton. It is confirmed by numerical simulations that this model can reproduce the experimentally obtained patterns qualitatively. Finally, we experimentally verify two predictions from the model: effects of very high FGF concentration and effects of small mechanical contributions of the cytoskeleton. The theoretical predictions match well with the experimental results.     

Hesperetin mitigates acrolein-induced apoptosis in lung cells in vitro and in vivo

Objectives: A number of studies have suggested that acrolein-induced lung injury and pulmonary diseases are associated with the depletion of antioxidants and the production of reactive oxygen species. Therefore, compounds that scavenge reactive oxygen species may exert protective effects against acrolein-induced apoptosis. Because hesperetin, a natural flavonoid, has been reported to have an antioxidant activity, we investigated the effect of hesperitin against acrolein-induced apoptosis of lung cells.

Methods: We evaluated the protective role of hesperetin in acrolein-induced lung injury using Lewis lung carcinoma (LLC) cells and mice.

Results: Upon exposure of LLC cells and mice to acrolein, hesperetin ameliorated the lung inbjury through attenuation of oxidative stress.

Conclusion: In the present report, we demonstrate that hesperetin exhibits a protective effect against acrolein-induced apoptosis of lung cells in both in vitro and in vivo models. Our study provides a useful model to investigate the potential application of hesperetin for the prevention of lung diseases associated with acrolein toxicity.     

Human osteoclasts express oxytocin receptor

Increasing evidences demonstrated many new targets for the hypothalamic hormone oxytocin, as the regulation of food balance and in some cases of leptin secretion. Considering that leptin is a potent inhibitor of bone formation and that oxytocin receptors (OTR) were detected in normal human osteoblasts, we investigated if OTR was expressed by human osteoclasts (hOCs) and the effect of the hormone on these cells. Here, we demonstrate by immunofluorescence and by Western blot analysis the expression of OTR by fully differentiated hOCs and by their precursors (pOCs). We also show that the receptor is functional, as OT treatment induces an increase of [Ca(2+)](i), and that the hormone may affect osteoclastogenesis, since it increases the number of pre-osteoclasts.     

Cytoprotective activity of annphenone against oxidative stress-induced apoptosis in V79-4 lung fibroblast cells

We have elucidated the cytoprotective effect of annphenone (2,4-dihyroxy-6-methoxy-acetophenone 4-O-beta-d-glucopyranoside) against oxidative stress-induced apoptosis. Annphenone scavenged intracellular reactive oxygen species (ROS) and increased antioxidant enzyme activities. It thereby prevented lipid peroxidation and DNA damage, which was demonstrated by the inhibition of the formation of thiobarbituric acid reactive substance (TBARS), inhibition of the comet tail and decreased phospho-H2A.X expression. Annphenone protected Chinese hamster lung fibroblast (V79-4) cells from cell death via the inhibition of apoptosis induced by hydrogen peroxide (H(2)O(2)), as shown by decreased apoptotic nuclear fragmentation, decreased sub-G(1) cell population and inhibited mitochondrial membrane potential (Deltapsi) loss. Taken together, these findings suggest that annphenone exhibits antioxidant properties by inhibiting ROS generation and thus protecting cells from H(2)O(2)-induced cell damage.     

Human mesenchymal stem cells express a myofibroblastic phenotype in vitro: comparison to human cardiac myofibroblasts

Cardiac fibrosis accompanies a variety of myocardial disorders, and is induced by myofibroblasts. These cells may be composed of a heterogeneous population of parent cells, including interstitial fibroblasts and circulating progenitor cells. Direct comparison of human bone marrow-derived mesenchymal stem cells (BM-MSCs) and cardiac myofibroblasts (CMyfbs) has not been previously reported. We hypothesized that BM-MSCs readily adopt a myofibroblastic phenotype in culture. Human primary BM-MSCs and human CMyfbs were isolated from patients undergoing open heart surgery and expanded under standard culture conditions. We assessed and compared their phenotypic and functional characteristics by examining their gene expression profile, their ability to contract collagen gels and synthesize collagen type I. In addition, we examined the role of non-muscle myosin II (NMMII) in modulating MSC myogenic function using NMMII siRNA knockdown and blebbistatin, a specific small molecule inhibitor of NMMII. We report that, while human BM-MSCs retain pluripotency, they adopt a myofibroblastic phenotype in culture and stain positive for the myofibroblast markers α-SMA, vimentin, NMMIIB, ED-A fibronectin, and collagen type 1 at each passage. In addition, they contract collagen gels in response to TGF-β1 and synthesize collagen similar to human CMyfbs. Moreover, inhibition of NMMII activity with blebbistatin completely attenuates gel contractility without affecting cell viability. Thus, human BM-MSCs share and exhibit similar physiological and functional characteristics as human CMyfbs in vitro, and their propensity to adopt a myofibroblast phenotype in culture may contribute to cardiac fibrosis.     

Human mast cells express intracellular TRAIL

Recently we demonstrated that human mast cells (MC) express functional TRAIL death receptors. Here we assessed the expression of TRAIL on both mRNA and protein level in cord blood derived MC (CBMC) and HMC-1. The TRAIL release either spontaneous or induced by LPS, IFN-γ and IgE-dependent activation, was evaluated as well. The protein location was restricted to the intracellular compartment in CBMC, but not in HMC-1. The intracellular TRAIL was not localized inside the granules. The treatment with IFN-γ and LPS up-regulated intracellular TRAIL expression in CBMC, but did not induce its release. These in vitro data show that human MC can produce and express intracellular TRAIL whose location could not be altered by different stimuli.     

Production of ceramides causes apoptosis during early neural differentiation in vitro

To investigate signal transduction pathways leading to apoptosis during the early phase of neurogenesis, we employed PCC7-Mz1 cells, which cease to proliferate and begin to differentiate into a stable pattern of neurons, astroglial cells, and fibroblasts upon incubation with retinoic acid (RA). As part of lineage determination, a sizable fraction of RA-treated cultures die by apoptosis. Applying natural long-chain C(16)-ceramides as well as membrane-permeable C(2)/C(6)-ceramide analogs caused apoptosis, whereas the biologically nonactive C(2)-dihydroceramide did not. Treating PCC7-Mz1 stem cells with a neutral sphingomyelinase or with the ceramidase inhibitor N-oleoylethanolamine elevated the endogenous ceramide levels and concomitantly induced apoptosis. Addition of RA caused an increase in ceramide levels within 3-5 h, which reached a maximum (up to 3.5-fold of control) between days 1 and 3 of differentiation. Differentiated PCC7-Mz1 cells did not respond with ceramide formation and apoptosis to RA treatment. The acidic sphingomyelinase contributed only weakly and the neutral Mg(2+)-dependent and Mg(2+)-independent sphingomyelinases not at all to the RA-mediated production of ceramides. However, ceramide increase was sensitive to the ceramide synthase inhibitor fumonisin B(1), suggesting a crucial role for the de novo synthesis pathway. Enzymatic assays revealed that ceramide synthase activity remained unaltered, whereas serine palmitoyltransferase (SPT), a key enzyme in ceramide synthesis, was activated approximately 2.5-fold by RA treatment. Activation of SPT seemed to be mediated via a post-translational mechanism because levels of the mRNAs coding for the two SPT subunits were unaffected. Expression of marker proteins shows that ceramide regulates apoptosis, rather than differentiation, during early neural differentiation.     

Differential induction of apoptosis by cigarette smoke extract in primary human lung fibroblast strains: implications for emphysema

Cigarette smoke is the principal cause of emphysema. Recent attention has focused on the loss of alveolar fibroblasts in the development of emphysema. Fibroblasts may become damaged by oxidative stress and undergo apoptosis as a result of cigarette smoke exposure. Not all smokers develop lung diseases associated with tobacco smoke, a fact that may reflect individual variation among human fibroblast strains. We hypothesize that fibroblasts from different human beings vary in their ability to undergo apoptosis after cigarette smoke exposure. This could account for emphysematous changes that occur in the lungs of some but not all smokers. Primary human lung fibroblast strains were exposed to cigarette smoke extract (CSE) and assessed for viability, morphological changes, and mitochondrial transmembrane potential as indicators of apoptosis. We also examined the generation of intracellular reactive oxygen species (ROS), 4-hydroxy-2-nonenal, and changes in glutathione (GSH) and glutathione disulfide (GSSG) levels. Each human lung fibroblast strain exhibited a differential sensitivity to CSE as judged by changes in mitochondrial membrane potential, viability, ROS generation, and glutathione production. Interestingly, the thiol antioxidants N-acetyl-L-cysteine and GSH eliminated CSE-induced changes in fibroblast morphology such as membrane blebbing, nuclear condensation, and cell size and prevented alterations in mitochondrial membrane potential and the generation of ROS. These findings support the concept that oxidative stress and apoptosis are responsible for fibroblast death associated with exposure to tobacco smoke. Variations in the sensitivity of fibroblasts to cigarette smoke may account for the fact that only some smokers develop emphysema.     

Adherence of Pneumocystis carinii in lung cells during in vitro cultivation

This study describes an approach to cultivation of Pneumocystis carinii (Pc) on 2 cell lines derived from lung (A549, human and L2, rat) with emphasis on the organisms which adhered to the cells. Immunofluorescent staining was used for growth assays.     

Up-regulation of THY1 attenuates interstitial pulmonary fibrosis and promotes lung fibroblast apoptosis during acute interstitial pneumonia by blockade of the WNT signaling pathway

Acute interstitial pneumonia (AIP) is an idiopathic pulmonary disease featuring rapid progressive dyspnea and respiratory failure. These symptoms typically develop within several days or weeks in patients without any pre-existing lung disease or external chest disease. Thymocyte differentiation antigen-1 (THY1) has been reported to have an effect on lung fibroblast proliferation and fibrogenic signaling. In this study, the mechanism of THY1 in AIP in influencing pulmonary fibrosis in terms of lung fibroblast proliferation and apoptosis was examined. An AIP mouse model with the pathological changes of lung tissues observed was established to identify the role of THY1 in the pathogenesis of AIP. The expression of THY1, a key regulator of the WNT pathway β-catenin and fibroblasts markers MMP-2, Occludin, α-SMA and Vimentin were determined. Lung fibroblasts of mice were isolated, in which THY1 expression was altered to identify roles THY1 plays in cell viability and apoptosis. A TOP/TOPflash assay was utilized to determine the activation of WNT pathway. Decrement of pulmonary fibrosis was achieved through THY1 up-regulation. The expression of MMP-2, Occludin, α-SMA, Vimentin and β-catenin, and the extent of β-catenin phosphorylation, significantly decreased, thereby indicating that THY1 overexpression inactivated WNT. Cell proliferation was inhibited and apoptosis was accelerated in lung fibroblasts transfected with vector carrying overexpressed THY1. Altogether, this study defines the potential role of THY1 in remission of AIP, via the upregulation of THY1, which renders the WNT pathway inactive. This inactivation of the WNT signaling pathway could alleviate pulmonary fibrosis by reducing lung fibroblast proliferation in AIP.

Abbreviations: AIP: Acute interstitial pneumonia; ILDs: interstitial lung diseases; DAD: diffuse alveolar damage; SPF: specific-pathogen-free; NC: negative control; HCMV: human cytomegalovirus; HE: Hematoxylin-eosin; RIPA: radio-immunoprecipitation assay; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; BSA: bovine serum albumin; HRP: horseradish peroxidase; ECL: electrochemiluminescence; FBS: fetal bovine serum; DMSO: dimethyl sulfoxide; OD: optical density