Production Of Extracellular Matrix Components Research Articles

Функциональная активность перитонеальных фибробластов как объект изучения фармакодинамики недигидропиридиновых блокаторов медленных кальциевых каналов

Исследовали влияние блокаторов медленных кальциевых каналов на культуру клеток, содержащих перитонеальные фибробласты крыс с и без гемоперитонеума. Установлено, что верапамил (0,1 мг/мл) оказывает выраженный эффект в отношении избыточной активности перитонеальных фибробластов, снижая уровень пролиферации клеток, нормализуя их коллагенсинтетическую функцию, уменьшая избыточную продукцию гиалуроновой кислоты. Дилтиазем (0,1 мг/мл) оказывает менее значимое, чем верапамил (в среднем на 35,6 % p < 0,05), нормализующее влияние на функциональную активность перитонеальных фибробластов и избыточную продукцию компонентов межклеточного матрикса. У нифедипина (0,1 мг/мл) данный эффект отсутствует. Таким образом, функциональная активность перитонеальных фибробластов может служить тест-системой для изучения фармакодинамики недигидропиридиновых блокаторов медленных кальциевых каналов.

Importance of the Exopolysaccharide Matrix in Antimicrobial Tolerance of Pseudomonas aeruginosa Aggregates.

Pseudomonas aeruginosa is an opportunistic pathogen that can infect the lungs of cystic fibrosis (CF) patients and persist in the form of antibiotic-tolerant aggregates in the mucus. It has recently been suggested that such aggregates are formed due to restricted bacterial motility independent of the production of extracellular matrix components, and that they do not rely on an extracellular matrix for antimicrobial tolerance. However, we show here that biofilm matrix overexpression, as displayed by various clinical isolates, significantly protects P. aeruginosa aggregates against antimicrobial treatment. Alginate-overproducing mucA mutant bacteria growing in aggregates showed highly increased antibiotic tolerance compared to wild-type bacteria in aggregates. Deletion of algD in the mucA mutant strain abrogated alginate production and reversed the antibiotic tolerance displayed by the aggregates to a level similar to that observed for aggregates formed by the wild type. The P. aeruginosa ΔwspF and ΔyfiR mutant strains both overproduce Pel and Psl exopolysaccharide, and when these bacteria grew in aggregates, they showed highly increased antibiotic tolerance compared to wild-type bacteria growing in aggregates. However, the ΔwspF and ΔyfiR mutant strains, deficient in Pel/Psl production due to additional ΔpelA ΔpslBCD deletions, formed aggregates that displayed antibiotic tolerance levels close to those of wild-type aggregates. These results suggest that biofilm matrix components, such as alginate, Pel, and Psl, do play a role in the tolerance toward antimicrobials when bacteria grow as aggregates.

Bioengineered three-dimensional diseased intervertebral disc model revealed inflammatory crosstalk.

Without an appropriate disease model, the understanding of the pathophysiology of intervertebral disc degeneration and inflammation is limited. The lack of understanding limits the potential discovery of therapeutic targets as viable treatment options. Here, we report a versatile method to develop a three-dimensional intervertebral disc (IVD) model to study the response of nucleus pulposus (NP) and annulus fibrosus (AF) cells to inflammatory (IL-1β-induced) stimulation. The cell shape regulated IVD model was engineered by modulating the crosslinking of a self-assembled collagen hydrogel. The developed model has provided us with an understanding of the molecular changes that occur at genetic level which modulate the production of extracellular matrix components and key inflammatory pathways in the inflamed IVD. We have identified the role of the suppressor of cytokine proteins (SOCS) family in combating detrimental effects of pro-inflammatory cytokines in degenerated human NP tissue as predicted by the developed diseased model. The model could also provide an understanding of the expression of glycans implicated in the diseased IVD.

Hypoxic Expansion of Human Mesenchymal Stem Cells Enhances Three-Dimensional Maturation of Tissue-Engineered Intervertebral Discs.

Culture of three-dimensional (3D) constructs in hypoxic conditions (1-5% O2) has been shown to increase production of extracellular matrix components in primary intervertebral disc (IVD) cells and drive chondrogenesis of human mesenchymal stem cells (hMSCs). Growing evidence suggests that two-dimensional (2D) expansion under hypoxic conditions may have an even greater influence on chondrogenesis in MSCs. This study aims to determine the effects of hypoxia during 2D expansion and subsequent 3D culture on the in vitro maturation of tissue-engineered IVDs (TE-IVDs) made with hMSCs, using a previously developed TE-IVD system. hMSCs were expanded in either hypoxic (5% O2) or normoxic (21% O2) conditions before construction of TE-IVDs. Discs were cultured in 3D in either hypoxic or normoxic conditions to create four experimental groups. Discs made from MSCs expanded in hypoxia were up to 141% stiffer than those made with normoxia-expanded MSCs. Similar patterns were seen in all mechanical properties. Increases in glycosaminoglycan content and collagen content in the nucleus pulposus (NP) were associated with 3D hypoxic culture. A boundary region between the manufactured fibrosus and NP regions developed by 2 weeks and mimicked the organization of the native disc. Hypoxic conditions in both 2D expansion and subsequent 3D culture improved the maturation of TE-IVDs made with hMSCs.

Selective targeting of CREB-binding protein/β-catenin inhibits growth of and extracellular matrix remodelling by airway smooth muscle.

Asthma is a heterogeneous chronic inflammatory disease, characterized by the development of structural changes (airway remodelling). β-catenin, a transcriptional co-activator, is fundamentally involved in airway smooth muscle growth and may be a potential target in the treatment of airway smooth muscle remodelling. We assessed the ability of small-molecule compounds that selectively target β-catenin breakdown or its interactions with transcriptional co-activators to inhibit airway smooth muscle remodelling in vitro and in vivo. ICG-001, a small-molecule compound that inhibits the β-catenin/CREB-binding protein (CBP) interaction, strongly and dose-dependently inhibited serum-induced smooth muscle growth and TGFβ1-induced production of extracellular matrix components in vitro. Inhibition of β-catenin/p300 interactions using IQ-1 or inhibition of tankyrase 1/2 using XAV-939 had considerably less effect. In a mouse model of allergic asthma, β-catenin expression in the smooth muscle layer was found to be unaltered in control versus ovalbumin-treated animals, a pattern that was found to be similar in smooth muscle within biopsies taken from asthmatic and non-asthmatic donors. However, β-catenin target gene expression was highly increased in response to ovalbumin; this effect was prevented by topical treatment with ICG-001. Interestingly, ICG-001 dose-dependently reduced airway smooth thickness after repeated ovalbumin challenge, but had no effect on the deposition of collagen around the airways, mucus secretion or eosinophil infiltration. Together, our findings highlight the importance of β-catenin/CBP signalling in the airways and suggest ICG-001 may be a new therapeutic approach to treat airway smooth muscle remodelling in asthma.

0226 : Neutrophil gelatinase – associated lipocalin mediates the profibrotic effects of aldosterone in human cardiac fibroblasts

Aldosterone (Aldo) through Mineralcorticoid Receptor (MR) plays an important pathophysiological role in cardiovascular remodeling and diseases. Cardiac remodeling is characterized by changes in the extracellular matrix (ECM), which favors the development of myocardial fibrosis, and finally could compromises cardiac function and contributes to heart failure. Neutrophil gelatinase-associated lipocalin (NGAL) is a primary target of Aldosterone (Aldo)/MR in the cardiovascular system. However, the role of NGAL in cardiac remodeling is still unclear. We investigated the effects of NGAL on the production of ECM components in human cardiac fibroblasts (HCF) and whether NGAL could be a mediator of Aldo-induced ECM components in HCF. Collagen I protein levels were measured in response to Aldo (10- 10-10-8M) and NGAL (5-500 ng/ml) in HCF for 12-24 hours. Protein and gene expression of different profibrotic mediators such as galectin-3 (Gal-3), cardiotrophin-1 (CT-1), transforming growth factor-beta (TGF-β) and metalloproteinase (MMP) activities were studied in response to NGAL and Aldo. Finally, in order to study the role of NGAL on the profibrotic actions of Aldo, NGAL-silenced HCF were treated with Aldo In HCF, Aldo and NGAL increased collagen I protein expression in a dose-dependent manner, reaching maximal values at 24 hours. At this time, Aldo increased NGAL protein expression. NGAL and Aldo enhanced profibrotic mediators such as Gal-3, CT-1 and TGF-β, as well as MMP-1, MMP-2 and MMP-9 activities. In NGAL silenced cells, Aldo was not able to modify collagen I expression as well as fibronectin, galectin-3, cardiotrophin- 1 and TGF-β levels. Our results indicated that NGAL exerts profibrotic effects in HCF. Furthermore, NGAL emerges as a potential mediator of Aldo-induced cardiac fibrosis and could be consider as a biotarget for novel pharmacological approaches, especially in diseases where Aldo/MR pathway is involved. The author hereby declares no conflict of interest

Low dose short duration pulsed electromagnetic field effects on cultured human chondrocytes: An experimental study.

Background:Pulsed electromagnetic field (PEMF) is used to treat bone and joint disorders for over 30 years. Recent studies demonstrate a significant effect of PEMF on bone and cartilage proliferation, differentiation, synthesis of extracellular matrix (ECM) and production of growth factors. The aim of this study is to assess if PEMF of low frequency, ultralow field strength and short time exposure have beneficial effects on in-vitro cultured human chondrocytes.Materials and Methods:Primary human chondrocytes cultures were established using articular cartilage obtained from knee joint during joint replacement surgery. Post characterization, the cells were exposed to PEMF at frequencies ranging from 0.1 to 10 Hz and field intensities ranging from 0.65 to 1.95 μT for 60 min/day for 3 consecutive days to analyze the viability, ECM component synthesis, proliferation and morphology related changes post exposure. Association between exposure doses and cellular effects were analyzed with paired't’ test.Results:In-vitro PEMF exposure of 0.1 Hz frequency, 1.95 μT and duration of 60 min/day for 3 consecutive days produced the most favorable response on chondrocytes viability (P < 0.001), ECM component production (P < 0.001) and multiplication. Exposure of identical chondrocyte cultures to PEMFs of 0.65 μT field intensity at 1 Hz frequency resulted in less significant response. Exposure to 1.3 μT PEMFs at 10 Hz frequency does not show any significant effects in different analytical parameters.Conclusions:Short duration PEMF exposure may represent a new therapy for patients with Osteoarthritis (OA).

Inhibition of Contractile Function in Human Joint Capsule Myofibroblasts by Targeting the TGF-β1 and PDGF Pathways.

BackgroundContractile myofibroblasts (MFs) accumulate in the joint capsules of patients suffering from posttraumatic joint stiffness. MF activation is controlled by a complex local network of growth factors and cytokines, ending in the increased production of extracellular matrix components followed by soft tissue contracture. Despite the tremendous growth of knowledge in this field, inconsistencies remain in practice and prevention.Methods and FindingsIn this in vitro study, we isolated and cultured alpha-smooth muscle actin (α-SMA) positive human joint capsule MFs from biopsy specimens and investigated the effect of profibrotic and antifibrotic agents on MF function. Both TGF-β1 and PDGF significantly induced proliferation and increased extracellular matrix contraction in an established 3D collagen gel contraction model. Furthermore, both growth factors induced α-SMA and collagen type I gene expression in MFs. TGF-β1 down-regulated TGF-β1 and TGF-β receptor (R) 1 and receptor (R) 2 gene expression, while PDGF selectively down-regulated TGF-β receptor 2 gene expression. These effects were blocked by suramin. Interestingly, the anti-oxidant agent superoxide dismutase (SOD) blocked TGF-β1 induced proliferation and collagen gel contraction without modulating the gene expression of α-SMA, collagen type I, TGF-β1, TGF-β R1 and TGF-β R2.ConclusionsOur results provide evidence that targeting the TGF-β1 and PDGF pathways in human joint capsule MFs affects their contractile function. TGF-β1 may modulate MF function in the joint capsule not only via the receptor signalling pathway but also by regulating the production of profibrotic reactive oxygen species (ROS). In particular, anti-oxidant agents could offer promising options in developing strategies for the prevention and treatment of posttraumatic joint stiffness in humans.

Clinicopathological correlations at the vitreoretinal interface

Clinicopathological studies of the vitreoretinal interface (VRI) improve our understanding of the pathogenesis of vitreal maculopathy, facilitate differential diagnoses and help to develop new treatment strategies. The aim of the study was to provide a comprehensive overview on clinicopathological correlations of the VRI. A semi-structured literature search was performed in the Medline and Embase databases for relevant original studies on clinicopathological correlations of vitreal maculopathy, in addition to the latest books and review articles. Age-related vitreous changes with persistent vitreomacular adhesions on the retinal surface promote cellular migration and proliferation onto the vitreal side of the internal limiting membrane (ILM), thereby cementing the vitreomacular adhesions and strengthening the traction forces on retinal layers. Cellular or fibrocellular proliferation at the vitreomacular interface can be seen in all vitreal maculopathies. Furthermore, vitreoschisis in the context of anomalous posterior vitreous detachment causes the presence of vitreous cortex collagen fibrils on the vitreal side of the ILM which is associated with epiretinal membrane formation. Glial cells, hyalocytes and myofibroblasts represent the major cell types in the epiretinal cell proliferation. Glial cells and hyalocytes are capable of transdifferentiation into myofibroblasts which possess strong contractive properties and are well known for the production of extracellular matrix components. Removing vitreomacular adhesions and vitreous cortex collagen fibrils from the retinal surface is most important for successful treatment. In cases with epiretinal cell proliferation, however, removal of the ILM during macular surgery is mandatory to avoid reproliferation and recurrence. Improving the detection of epiretinal cell proliferation and cell distribution in patient eyes by optical coherence tomography or by introduction of new technologies should be addressed in the future.

Effect of glucosamine and its peptidyl-derivative on the production of extracellular matrix components by human primary chondrocytes

Aim of this study is to investigate the effects of Glucosamine (GlcN) and its peptidyl-derivative, 2-(N-Acetyl)-L-phenylalanylamido-2-deoxy-β-D-glucose (NAPA), on extracellular matrix (ECM) synthesis in human primary chondrocytes (HPCs). Dose-dependent effect of GlcN and NAPA on Glycosaminoglycan (GAG), Collagen type II (Col2) and Small Leucine-Rich Proteoglycans (SLRPs) was examined by incubating HPCs, cultured in micromasses (3D), with various amounts of two molecules, administered as either GlcN alone or NAPA alone or GlcN plus NAPA (G + N). Immunohystochemical and immunofluorescent staining and biochemical analysis were used to determine the impact of the two molecules on ECM production. Gene expression analysis was performed by TaqMan Real-Time Polymerase Chain Reaction (PCR) assays. The lowest concentration to which GlcN and NAPA were able to affect ECM synthesis was 1 mM. Both molecules administered alone and as G + N stimulated GAGs and SLRPs synthesis at different extent, NAPA and mainly G + N stimulated Col2 production, whereas GlcN was not effective. Both molecules were able to induce Insulin Growth Factor-I (IGF-I) and to stimulate SOX-9, whereas NAPA and G + N were able to up-regulate both Hyaluronic Acid Synthase-2 and Hyaluronic acid. Very interesting is the synergistic effect observed when chondrocyte micromasses were treated with G + N. The observed anabolic effects and optimal concentrations of GlcN and NAPA, in addition to beneficial effects on other cellular pathways, previously reported, such as the inhibition of IKKα, could be useful to formulate new cartilage repair strategies.

Recent advances in pancreatology.

Recent advances in pancreatology.

The potential role of leptin in the vascular remodeling associated with obesity

Extracellular matrix (ECM) participates in the vascular remodeling associated with obesity. We investigated the effects of leptin on the production of ECM components in primary cultured vascular smooth muscle cells (VSMCs) and whether leptin could be a mediator of obesity-induced vascular remodeling. T he effects of leptin (100 ng ml(-1)) on ECM components and superoxide anion production (O(2)(.-)) were evaluated in presence or absence of the antioxidant melatonin (10(-)(3) mmol l(-1)) or the inhibitor of phosphatidylinositol 3'-kinase (PI3K), LY294002 (2 × 10(-)(4) mmol l(-1)) in VSMCs from adult rats in order to explore the role of both oxidative stress and the participation of PI3K/Akt pathway in the effects of leptin. ECM components and O(2)(.-) were quantified in the aortic media of male Wistar rats fed a high-fat diet (HFD; 33.5% fat), or a standard diet (CT; 3.5% fat) for 6 weeks. In VSMCs, leptin enhanced gene and protein levels of collagen I, fibronectin, transforming growth factor (TGF)-β and connective tissue growth factor (CTGF) but did not change those of collagen III and galectin-3. Leptin also increased O(2)(.-) and Akt phosphorylation in VSMCs. These effects were prevented by the presence of either melatonin or LY294002, except O(2)(.-) production in the case of PI3K inhibition. The increase in body weight in HFD rats was accompanied by aorta thickening due to an increase in media area. The aortic fibrosis observed in HFD rats was associated with high levels of leptin, collagen type I, fibronectin, TGF-β, CTGF, phosphorylated Akt and O(2)(.-). Aortic leptin levels were positively correlated with total collagen, collagen I, TGF-β and CTGF levels. No differences were observed in the levels of collagen III, elastin or galectin-3 between both the groups. Leptin could participate in the vascular remodeling and stiffness associated with obesity by ECM production in VSMCs through the activation of oxidative stress-PI3K/Akt pathway and the production of the profibrotic factors TGF-β and CTGF.

Endothelial dysfunction in patients with primary hypertension and hyperhomocysteinemia

It is widely accepted that endothelial dysfunction is the basis of the development of cardiovascular diseases, including hypertension. With regard to hypertension, endothelial dysfunction is concerned mainly with impaired vascular expansion; however, it is also related to the intensity of the development of atherosclerosis and thrombosis. Among the factors that cause damage to the endothelium, along with classic risk factors, is hyperhomocysteinemia. Hyperhomocysteinemia promotes the formation of oxygen radicals, lowering the oxidation-reduction potential, adversely affects the biosynthesis and function of vasodilator factors in the vascular wall, contributes to the inhibition of endothelial cell division with intense myocyte proliferation and migration, and impairs production of extracellular matrix components in the vascular wall. In addition, high levels of homocysteine and its derivatives contribute to the modification of LDL and HDL particles, inflammation and disorders in coagulation and fibrinolysis. Biochemical effects of the impact of hyperhomocysteinemia on endothelium can lead to damage of endothelial cells, dysfunction of diastolic function of vessels and reduction of their flexibility through its influence on vascular wall remodeling. These changes lead to an increase in blood pressure, strengthening the development of hypertension and target organ damage in patients with this disease.

Differential response of human gingival fibroblasts to titanium‐ and titanium‐zirconium‐modified surfaces

Gingival fibroblasts are responsible for the constant adaptation, wound healing and regeneration of gingival connective tissue. New titanium-zirconium (TiZr) abutment surfaces have been designed to improve soft tissue integration and reduce implant failure compared with titanium (Ti). The aim of the present study was first to characterize a primary human gingival fibroblast (HGF) model and secondly to evaluate their differential response to Ti and TiZr polished (P), machined (M) and machined + acid-etched (modMA) surfaces, respectively. HGF were cultured on tissue culture plastic or on the different Ti and TiZr surfaces. Cell morphology was evaluated through confocal and scanning electron microscopy. A wound healing assay was performed to evaluate the capacity of HGF to close a scratch. The expression of genes was evaluated by real-time RT-PCR, addressing: (i) extracellular matrix organization and turnover; (ii) inflammation; (iii) cell adhesion and structure; and (iv) wound healing. Finally, cells on Ti/TiZr surfaces were immunostained with anti-ITGB3 antibodies to analyze integrin β3 production. Matrix metalloproteinase-1 (MMP1) and inhibitor of metallopeptidases-1 (TIMP1) production were analyzed by enzyme-linked immunosorbent assays. On tissue culture plastic, HGF showed no differences between donors on cell proliferation and on the ability for wound closure; α-smooth muscle actin was overexpressed on scratched monolayers. The differentiation profile showed increased production of extracellular matrix components. Ti and TiZr showed similar biocompatibility with HGF. TiZr increased integrin-β3 mRNA and protein levels, compared with Ti. Cells on TiZr surfaces showed higher MMP1 protein than Ti surfaces, although similar TIMP1 protein production. In this in vitro experiment, P and M surfaces from both Ti and TiZr showed better HGF growth than modMA. Taking into account the better mechanical properties and bioactivity of TiZr compared with Ti, the results of the present study show that TiZr is a potential clinical candidate for soft tissue integration and implant success.

Developing a tissue engineered repair material for treatment of stress urinary incontinence and pelvic organ prolapse-which cell source?

Synthetic non-absorbable meshes are widely used to augment surgical repair of stress urinary incontinence (SUI) and pelvic organ prolapse (POP); however, there is growing concern such meshes are associated with serious complications. This study compares the potential of two autologous cell sources for attachment and extra-cellular matrix (ECM) production on a biodegradable scaffold to develop tissue engineered repair material (TERM). Human oral fibroblasts (OF) and human adipose-derived stem cells (ADSC) were isolated and cultured on thermo-annealed poly-L-lactic acid (PLA) scaffolds for two weeks under either unrestrained conditions or restrained (either with or without intermittent stress) conditions. Samples were tested for cell metabolic activity (AlamarBlue® assay), contraction (serial photographs analyzed with image J software), total collagen production (Sirius red assay), and production of ECM components (immunostaining for collagen I, III, and elastin; and scanning electron microscopy) and biomechanical properties (BOSE tensiometer). Differences were statistically tested using two sample t-test. Both cells showed good attachment and proliferation on scaffolds. Unrestrained scaffolds with ADSC produced more total collagen and a denser homogenous ECM than OF under same conditions. Restrained conditions (both with and without intermittent stress) gave similar total collagen production, but improved elastin production for both cells, particularly OF. The addition of any cell onto scaffolds led to an increase in biomechanical properties of scaffolds compared to unseeded scaffolds. OF and ADSC both appear to be suitable cell types to combine with biodegradable scaffolds, in the development of a TERM for the treatment of SUI and POP. Neurourol. Urodynam. 33:531-537, 2014. © 2013 Wiley Periodicals, Inc.

Large Scale Expansion of Human Umbilical Cord Cells in a Rotating Bed System Bioreactor for Cardiovascular Tissue Engineering Applications

Widespread use of human umbilical cord cells for cardiovascular tissue engineering requires production of large numbers of well-characterized cells under controlled conditions. In current research projects, the expansion of cells to be used to create a tissue construct is usually performed in static cell culture systems which are, however, often not satisfactory due to limitations in nutrient and oxygen supply. To overcome these limitations dynamic cell expansion in bioreactor systems under controllable conditions could be an important tool providing continuous perfusion for the generation of large numbers of viable pre-conditioned cells in a short time period. For this purpose cells derived from human umbilical cord arteries were expanded in a rotating bed system bioreactor for up to 9 days. For a comparative study, cells were cultivated under static conditions in standard culture devices.Our results demonstrated that the microenvironment in the perfusion bioreactor was more favorable than that of the standard cell culture flasks. Data suggested that cells in the bioreactor expanded 39 fold (38.7 ± 6.1 fold) in comparison to statically cultured cells (31.8 ± 3.0 fold). Large-scale production of cells in the bioreactor resulted in more than 3 x 108 cells from a single umbilical cord fragment within 9 days. Furthermore cell doubling time was lower in the bioreactor system and production of extracellular matrix components was higher. With this study, we present an appropriate method to expand human umbilical cord artery derived cells with high cellular proliferation rates in a well-defined bioreactor system under GMP conditions.

Syndecan-2 is upregulated in colorectal cancer cells through interactions with extracellular matrix produced by stromal fibroblasts

BackgroundThe extracellular matrix (ECM) influences the structure, viability and functions of cells and tissues. Recent evidence indicates that tumor cells and stromal cells interact through direct cell-cell contact, the production of ECM components and the secretion of growth factors. Syndecans are a family of transmembrane heparan sulfate proteoglycans that are involved in cell adhesion, motility, proliferation and differentiation. Syndecan-2 has been found to be highly expressed in colorectal cancer cell lines and appears to be critical for cancer cell behavior. We have examined the effect of stromal fibroblast-produced ECM on the production of proteoglycans by colorectal cancer cell lines.ResultsOur results showed that in a highly metastatic colorectal cancer cell line, HCT-116, syndecan-2 expression is enhanced by fibroblast ECM, while the expression of other syndecans decreased. Of the various components of the stromal ECM, fibronectin was the most important in stimulating the increase in syndecan-2 expression. The co-localization of syndecan-2 and fibronectin suggests that these two molecules are involved in the adhesion of HCT-116 cells to the ECM. Additionally, we demonstrated an increase in the expression of integrins alpha-2 and beta-1, in addition to an increase in the expression of phospho-FAK in the presence of fibroblast ECM. Furthermore, blocking syndecan-2 with a specific antibody resulted in a decrease in cell adhesion, migration, and organization of actin filaments.ConclusionsOverall, these results show that interactions between cancer cells and stromal ECM proteins induce significant changes in the behavior of cancer cells. In particular, a shift from the expression of anti-tumorigenic syndecans to the tumorigenic syndecan-2 may have implications in the migratory behavior of highly metastatic tumor cells.

Sulfadiazine modified PDMS as a model material with the potential for the mitigation of posterior capsule opacification (PCO)

Cataract surgery, while the most common surgical procedure performed, leads to posterior capsule opacification in approximately 30% of cases. Transforming growth factor beta 2 (TGF-β2) and matrix metalloproteinases (MMPs) have been shown to play important roles in the cellular processes leading to posterior capsule opacification. Delivery of inhibitors to MMPs may have the potential to inhibit the initial cascade of events that lead to PCO. However, delivery of these molecules via tethering has proven difficult. In this work, sulfadiazine was tethered to polydimethylsiloxane (PDMS) via a polyethylene glycol (PEG) spacer as a potential MMPI mimic. Surface characterization using a variety of methods demonstrated successful modification with the antibiotic. The surfaces were examined with lens epithelial cells to determine their effect on these cellular processes, including cell transdifferentiation and production of extracellular matrix components. The presence of TGF-β2 in the cell culture media was found to stimulate the production of ECM components such as collagen, fibronectin, and laminin, as well as alpha smooth muscle actin (α-SMA), and the migration marker Rho by HLE-B3 and FHL124 cells. In all cases, these effects were decreased but not completely eradicated by the presence of sulfadiazine on the PDMS surfaces. While the level of inhibition necessary for inhibition of PCO in vivo is unknown, these results suggest that IOL surface modification with sulfadiazine has the potential to reduce cellular changes associated with PCO. Furthermore, the results demonstrate for the first time that changes consistent with inhibition of fibrosis may be elicited by surfaces modified with sulfadiazine.

Transforming Growth Factor-β1 Regulates Cdk5 Activity in Primary Sensory Neurons

In addition to many important roles for Cdk5 in brain development and synaptic function, we reported previously that Cdk5 regulates inflammatory pain signaling, partly through phosphorylation of transient receptor potential vanilloid 1 (TRPV1), an important Na(+)/Ca(2+) channel expressed in primary nociceptive afferent nerves. Because TGF-β regulates inflammatory processes and its receptor is expressed in TRPV1-positive afferents, we studied the cross-talk between these two pathways in sensory neurons during experimental peripheral inflammation. We demonstrate that TGF-β1 increases transcription and protein levels of the Cdk5 co-activator p35 through ERK1/2, resulting in an increase in Cdk5 activity in rat B104 neuroblastoma cells. Additionally, TGF-β1 enhances the capsaicin-induced Ca(2+) influx in cultured primary neurons from dorsal root ganglia (DRG). Importantly, Cdk5 activity was reduced in the trigeminal ganglia and DRG of 14-day-old TGF-β1 knock-out mice, resulting in reduced Cdk5-dependent phosphorylation of TRPV1. The decreased Cdk5 activity is associated with attenuated thermal hyperalgesia in TGF-β1 receptor conditional knock-out mice, where TGF-β signaling is significantly reduced in trigeminal ganglia and DRG. Collectively, our results indicate that active cross-talk between the TGF-β and Cdk5 pathways contributes to inflammatory pain signaling.

The Pseudomonas aeruginosa Transcriptome in Planktonic Cultures and Static Biofilms Using RNA Sequencing

In this study, we evaluated how gene expression differs in mature Pseudomonas aeruginosa biofilms as opposed to planktonic cells by the use of RNA sequencing technology that gives rise to both quantitative and qualitative information on the transcriptome. Although a large proportion of genes were consistently regulated in both the stationary phase and biofilm cultures as opposed to the late exponential growth phase cultures, the global biofilm gene expression pattern was clearly distinct indicating that biofilms are not just surface attached cells in stationary phase. A large amount of the genes found to be biofilm specific were involved in adaptation to microaerophilic growth conditions, repression of type three secretion and production of extracellular matrix components. Additionally, we found many small RNAs to be differentially regulated most of them similarly in stationary phase cultures and biofilms. A qualitative analysis of the RNA-seq data revealed more than 3000 putative transcriptional start sites (TSS). By the use of rapid amplification of cDNA ends (5′-RACE) we confirmed the presence of three different TSS associated with the pqsABCDE operon, two in the promoter of pqsA and one upstream of the second gene, pqsB. Taken together, this study reports the first transcriptome study on P. aeruginosa that employs RNA sequencing technology and provides insights into the quantitative and qualitative transcriptome including the expression of small RNAs in P. aeruginosa biofilms.