Analysis of tissue transglutaminase function in the migration of Swiss 3T3 fibroblasts:the active-state conformation of the enzyme does not affect cell motility but is important for its secretion

Increasing evidence suggests that tissue transglutaminase (tTGase; type II) is externalized from cells, where it may play a key role in cell attachment and spreading and in the stabilization of the extracellular matrix (ECM) through protein cross-linking. However, the relationship between these different functions and the enzyme's mechanism of secretion is not fully understood. We have investigated the role of tTGase in cell migration using two stably transfected fibroblast cell lines in which expression of tTGase in its active and inactive (C277S mutant) states is inducible through the tetracycline-regulated system. Cells overexpressing both forms of tTGase showed increased cell attachment and decreased cell migration on fibronectin. Both forms of the enzyme could be detected on the cell surface, but only the clone overexpressing catalytically active tTGase deposited the enzyme into the ECM and cell growth medium. Cells overexpressing the inactive form of tTGase did not deposit the enzyme into the ECM or secrete it into the cell culture medium. Similar results were obtained when cells were transfected with tTGase mutated at Tyr(274) (Y274A), the proposed site for the cis,trans peptide bond, suggesting that tTGase activity and/or its tertiary conformation dependent on this bond may be essential for its externalization mechanism. These results indicate that tTGase regulates cell motility as a novel cell-surface adhesion protein rather than as a matrix-cross-linking enzyme. They also provide further important insights into the mechanism of externalization of the enzyme into the extracellular matrix.

Gravity spun polycaprolactone fibres for soft tissue engineering:interaction with fibroblasts and myoblasts in cell culture

Poly(ε-caprolactone) (PCL) fibres were produced by wet spinning from solutions in acetone under low shear (gravity flow) conditions. As-spun PCL fibres exhibited a mean strength and stiffness of 7.9 MPa and 0.1 GPa, respectively and a rough, porous surface morphology. Cold drawing to an extension of 500% resulted in increases in fibre strength (43 MPa) and stiffness (0.3 GPa) and development of an oriented, fibrillar surface texture. The proliferation rate of Swiss 3T3 mouse fibroblasts and C2C12 mouse myoblasts on as-spun, 500% cold-drawn and gelatin-modified PCL fibres was determined in cell culture to provide a basic measure of the biocompatibility of the fibres. Proliferation of both cell types was consistently higher on gelatin-coated fibres relative to as-spun fibres at time points below 7 days. Fibroblast growth rates on cold-drawn PCL fibres exceeded those on as-spun fibres but myoblast proliferation was similar on both substrates. After 1 day in culture, both cell types had spread and coalesced on the fibres to form a cell layer, which conformed closely to the underlying topography. The high fibre compliance combined with a potential for modifying the fibre surface chemistry with cell adhesion molecules and the surface architecture by cold drawing to enhance proliferation of fibroblasts and myoblasts, recommends further investigation of gravity-spun PCL fibres for 3-D scaffold production in soft tissue engineering. © 2005 Elsevier Ltd. All rights reserved.

Consumption of bilberries controls gingival inflammation

Bioactive molecules in berries may be helpful in reducing the risk of oral diseases. The aim of this study was to determine the effect of bilberry consumption on the outcome of a routine dental clinical parameter of inflammation, bleeding on probing (BOP), as well as the impact on selected biomarkers of inflammation, such as cytokines, in gingival crevicular fluid (GCF) in individuals with gingivitis. Study individuals who did not receive standard of care treatment were allocated to either a placebo group or to groups that consumed either 250 or 500 g bilberries daily over seven days. The placebo group consumed an inactive product (starch). A study group, receiving standard of care (debridement only) was also included to provide a reference to standard of care treatment outcome. Cytokine levels were assayed using the Luminex MagPix system. The mean reduction in BOP before and after consumption of test product over 1 week was 41% and 59% in the groups that consumed either 250 or 500 g of bilberries/day respectively, and was 31% in the placebo group, and 58% in the standard of care reference group. The analysis only showed a significantreduction in cytokine levels in the group that consumed 500 g of bilberries/day. A statistically significant reduction was observed for IL-1β (p = 0.025), IL-6 (p = 0.012) and VEGF (p = 0.017) in GCF samples in the group that consumed 500 g of bilberries daily. It appears that berry intake has an ameliorating effect on some markers of gingival inflammation reducing gingivitis to a similar extent compared to standard of care.

Importance of tissue transglutaminasenitrosylation in fibroblasts migration and MMPregulation

As an extracellular second messenger, nitric oxide (NO) mediates the modification of proteins through nitrosylation of cysteine andtyrosine residues. Tissue Transglutaminase (TG2) is a Ca2+ activated, sulfhydryl rich protein with 18 free cysteine residues, which catalyzes ε-(γ glutamyl)lysine crosslink between extracellular and intracellular proteins. NO can nitrosylate up to 15 of the cysteine residues in TG2, leading to the irreversible inactivation of the enzyme activity. The interplay between these two agents was revealed for the first time by our study showing that NO inhibited the TG2-induced transcriptional activation of TGFb1and extracellular matrix (ECM) protein synthesis by nitrosylating TG2 in an inactive confirmation with inert catalytic activity. However, nitrosylated TG2 was still able to serve as a novel cell adhesion protein. In the light of our previous findings, in this study we aim to elucidate the NO modified function of TG2 in cell migration using an in vitro model mimicking the tissue matrix remodeling phases of wound healing. Using transfected fibroblasts expressing TG2 under the control of the tetracycline-off promoter, we demonstrate that upregulation of TG2 expression and activity inhibited the cell migration through the activation of TGFβ1. Increased TG2 activity led to arise in the biosynthesis and activity of the gelatinases, MMP-2 andMMP-9, while decreasing the biosynthesis and activity of the col-lagenases MMP-1a and MMP-13. NO donor S-Nitroso-N-acetyl-penicillamine (SNAP) treatment relieved the TG2 obstructed-cellmigration by blocking the TG2 enzyme activity. In addition,decrease in TG2 activity due to nitrosylation led to an inhibition of TGFβ1, which in turn affected the pattern of MMP activation. Recent evidence suggests that, once in complex with fibronectin in the ECM, TG2 can interact with syndecan-4 or integrinβ-1and regulate the cell adhesion. In the other part of this study, the possible role of nitrosylated TG2 on the regulation of cell migration during wound healing was investigated with respect to its interactions with integrin β1 (ITGβ1) and syndecan-4 (SDC4). Treatment with TG2 inhibitor Z-DON resulted in a 50% decrease in the TG2 interaction with ITGB1 and SDC4, while increasing concentrations of SNAP firstly led to a substantial decrease and then completely abolished the TG2/ITGβ1 and TG2/SDC4 complex formation on the cell surface. Taken together, data obtained from this study suggests that nitrosylation of TG2 leads to a change not only in the binding partners of TG2 on cell surface but also in TGFβ1-dependent MMP activation, which give rise to an increase in the migration potential of fibroblasts.

Mechanical stretch up-regulates the B-type natriuretic peptide system in human cardiac fibroblasts:a possible defense against transforming growth factor-β mediated fibrosis

BACKGROUND: Mechanical overload of the heart is associated with excessive deposition of extracellular matrix proteins and the development of cardiac fibrosis. This can result in reduced ventricular compliance, diastolic dysfunction, and heart failure. Extracellular matrix synthesis is regulated primarily by cardiac fibroblasts, more specifically, the active myofibroblast. The influence of mechanical stretch on human cardiac fibroblasts' response to pro-fibrotic stimuli, such as transforming growth factor beta (TGFβ), is unknown as is the impact of stretch on B-type natriuretic peptide (BNP) and natriuretic peptide receptor A (NPRA) expression. BNP, acting via NPRA, has been shown to play a role in modulation of cardiac fibrosis.

METHODS AND RESULTS: The effect of cyclical mechanical stretch on TGFβ induction of myofibroblast differentiation in primary human cardiac fibroblasts and whether differences in response to stretch were associated with changes in the natriuretic peptide system were investigated. Cyclical mechanical stretch attenuated the effectiveness of TGFβ in inducing myofibroblast differentiation. This finding was associated with a novel observation that mechanical stretch can increase BNP and NPRA expression in human cardiac fibroblasts, which could have important implications in modulating myocardial fibrosis. Exogenous BNP treatment further reduced the potency of TGFβ on mechanically stretched fibroblasts.

CONCLUSION: We postulate that stretch induced up-regulation of the natriuretic peptide system may contribute to the observed reduction in myofibroblast differentiation.

Hypoxia-induced DNA hypermethylation in human pulmonary fibroblasts is associated with Thy-1 promoter methylation and the development of a pro-fibrotic phenotype

BACKGROUND: Pulmonary fibrosis is a debilitating and lethal disease with no effective treatment options. Understanding the pathological processes at play will direct the application of novel therapeutic avenues. Hypoxia has been implicated in the pathogenesis of pulmonary fibrosis yet the precise mechanism by which it contributes to disease progression remains to be fully elucidated. It has been shown that chronic hypoxia can alter DNA methylation patterns in tumour-derived cell lines. This epigenetic alteration can induce changes in cellular phenotype with promoter methylation being associated with gene silencing. Of particular relevance to idiopathic pulmonary fibrosis (IPF) is the observation that Thy-1 promoter methylation is associated with a myofibroblast phenotype where loss of Thy-1 occurs alongside increased alpha smooth muscle actin (α-SMA) expression. The initial aim of this study was to determine whether hypoxia regulates DNA methylation in normal human lung fibroblasts (CCD19Lu). As it has been reported that hypoxia suppresses Thy-1 expression during lung development we also studied the effect of hypoxia on Thy-1 promoter methylation and gene expression.

METHODS: CCD19Lu were grown for up to 8 days in hypoxia and assessed for global changes in DNA methylation using flow cytometry. Real-time PCR was used to quantify expression of Thy-1, α-SMA, collagen I and III. Genomic DNA was bisulphite treated and methylation specific PCR (MSPCR) was used to examine the methylation status of the Thy-1 promoter.

RESULTS: Significant global hypermethylation was detected in hypoxic fibroblasts relative to normoxic controls and was accompanied by increased expression of myofibroblast markers. Thy-1 mRNA expression was suppressed in hypoxic cells, which was restored with the demethylating agent 5-aza-2'-deoxycytidine. MSPCR revealed that Thy-1 became methylated following fibroblast exposure to 1% O2.

CONCLUSION: These data suggest that global and gene-specific changes in DNA methylation may play an important role in fibroblast function in hypoxia.

Interactions between Spider Silk and Cells - NIH/3T3 Fibroblasts Seeded on Miniature Weaving Frames

Background: Several materials have been used for tissue engineering purposes, since the ideal matrix depends on the desired tissue. Silk biomaterials have come to focus due to their great mechanical properties. As untreated silkworm silk has been found to be quite immunogenic, an alternative could be spider silk. Not only does it own unique mechanical properties, its biocompatibility has been shown already in vivo. In our study, we used native spider dragline silk which is known as the strongest fibre in nature. Methodology/Principal Findings: Steel frames were originally designed and manufactured and woven with spider silk, harvesting dragline silk directly out of the animal. After sterilization, scaffolds were seeded with fibroblasts to analyse cell proliferation and adhesion. Analysis of cell morphology and actin filament alignment clearly revealed adherence. Proliferation was measured by cell count as well as determination of relative fluorescence each after 1, 2, 3, and 5 days. Cell counts for native spider silk were also compared with those for trypsin-digested spider silk. Spider silk specimens displayed less proliferation than collagen-and fibronectin-coated cover slips, enzymatic treatment reduced adhesion and proliferation rates tendentially though not significantly. Nevertheless, proliferation could be proven with high significance (p<0.01). Conclusion/Significance: Native spider silk does not require any modification to its application as a biomaterial that can rival any artificial material in terms of cell growth promoting properties. We could show adhesion mechanics on intracellular level. Additionally, proliferation kinetics were higher than in enzymatically digested controls, indicating that spider silk does not require modification. Recent findings concerning reduction of cell proliferation after exposure could not be met. As biotechnological production of the hierarchical composition of native spider silk fibres is still a challenge, our study has a pioneer role in researching cellular mechanics on native spider silk fibres.

Comparative transcriptome profiling of human foreskin fibroblasts infected with the Sylvio and Y strains of Trypanosoma cruzi

Trypanosoma cruzi, the causative agent of Chagas Disease, is phylogenetically distributed into nearly identical genetic strains which show divergent clinical presentations including differences in rates of cardiomyopathy in humans, different vector species and transmission cycles, and differential congenital transmission in a mouse model. The population structure of these strains divides into two groups, which are geographically and clinically distinct. The aim of this study was to compare the transcriptome of two strains of T. cruzi, Sylvio vs. Y to identify differences in expression that could account for clinical and biochemical differences. We collected and sequenced RNA from T. cruzi-infected and control Human Foreskin Fibroblasts at three timepoints. Differential expression analysis identified gene expression profiles at different timepoints in Sylvio infections, and between Sylvio and Y infections in both parasite and host. The Sylvio strain parasite and the host response to Sylvio infection largely mirrored the host-pathogen interaction seen in our previous Y strain work. IL-8 was more highly expressed in Sylvio-infected HFFs than in Y-infected HFFs.

Técnica quirúrgica de Edland Mejchar para corrección de recesión gingival

Las técnicas quirúrgicas periodontales correctivas se diversifican ampliamente en la actualidad por la necesidad de restablecer un periodonto sano y lograr una estética aceptable, en los últimos tiempos la casuística de las alteraciones gingivales va en aumento quizás por el advenimiento de la búsqueda de ideales de perfección por parte de los pacientes, o por la instauración de tratamientos odontológicos como la ortodoncia que se relacionan etiológicamente con estos defectos. La retracción gingival y la atrofia gingival son dos de las anomalías de la encía más prevalentes dentro de la población y, aunque la inducción más común de su aparición es una mala higiene oral, se presentan casos fenotípicos relacionados con la deficiencia de lámina ósea, grosor de encía deficiente, frenillos en inserción penetrante o vestíbulos poco profundos que ejercen fuerzas traccionales patológicas; el tratamiento de estas patologías se decide de acuerdo con las necesidades correctivas y consideraciones de cada caso. A continuación se describe la técnica mucogingival de Edland Mejchar, la cual se realizó concomitantemente con tratamiento ortodóncico, en ella hay profundización de vestíbulo, como solución a defectos de recesión gingival vestibular en sector anteroinferior causada por fuerzas traccionales ofreciendo una solución al defecto al aliviar la tensión generada en la zona por las fibras musculares y por la creación de una banda de encía fibrosa que detiene la migración apical del margen gingival.

The effect of chaperone compounds on glucocerebrosidase stability and activity in fibroblasts from Gaucher Disease patients with different genotypes

Dissertação de mestrado, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2014

Cited2 in mouse embryonic fibroblasts reprogramming

Dissertação de Mestrado, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2014

Ex vivo investigation of novel wound healing therapies and development of a 3-D human skin equivalent wound model

It has previously been found that complexes comprised of vitronectin and growth factors (VN:GF) enhance keratinocyte protein synthesis and migration. More specifically, these complexes have been shown to significantly enhance the migration of dermal keratinocytes derived from human skin. In view of this, it was thought that these complexes may hold potential as a novel therapy for healing chronic wounds. However, there was no evidence indicating that the VN:GF complexes would retain their effect on keratinocytes in the presence of chronic wound fluid. The studies in this thesis demonstrate for the first time that the VN:GF complexes not only stimulate proliferation and migration of keratinocytes, but also these effects are maintained in the presence of chronic wound fluid in a 2-dimensional (2-D) cell culture model. Whilst the 2-D culture system provided insights into how the cells might respond to the VN:GF complexes, this investigative approach is not ideal as skin is a 3-dimensional (3-D) tissue. In view of this, a 3-D human skin equivalent (HSE) model, which reflects more closely the in vivo environment, was used to test the VN:GF complexes on epidermopoiesis. These studies revealed that the VN:GF complexes enable keratinocytes to migrate, proliferate and differentiate on a de-epidermalised dermis (DED), ultimately forming a fully stratified epidermis. In addition, fibroblasts were seeded on DED and shown to migrate into the DED in the presence of the VN:GF complexes and hyaluronic acid, another important biological factor in the wound healing cascade. This HSE model was then further developed to enable studies examining the potential of the VN:GF complexes in epidermal wound healing. Specifically, a reproducible partial-thickness HSE wound model was created in fully-defined media and monitored as it healed. In this situation, the VN:GF complexes were shown to significantly enhance keratinocyte migration and proliferation, as well as differentiation. This model was also subsequently utilized to assess the wound healing potential of a synthetic fibrin-like gel that had previously been demonstrated to bind growth factors. Of note, keratinocyte re-epitheliasation was shown to be markedly improved in the presence of this 3-D matrix, highlighting its future potential for use as a delivery vehicle for the VN:GF complexes. Furthermore, this synthetic fibrin-like gel was injected into a 4 mm diameter full-thickness wound created in the HSE, both keratinocytes and fibroblasts were shown to migrate into this gel, as revealed by immunofluorescence. Interestingly, keratinocyte migration into this matrix was found to be dependent upon the presence of the fibroblasts. Taken together, these data indicate that reproducible wounds, as created in the HSEs, provide a relevant ex vivo tool to assess potential wound healing therapies. Moreover, the models will decrease our reliance on animals for scientific experimentation. Additionally, it is clear that these models will significantly assist in the development of novel treatments, such as the VN:GF complexes and the synthetic fibrin-like gel described herein, ultimately facilitating their clinical trial in the treatment of chronic wounds.