Post‐transcriptional regulation of α‐smooth muscle actin determines the contractile phenotype of Dupuytren's nodular cells

The objective was to study Dupuytren's myofibroblast cells in constrained collagen matrices in order to more closely emulate their in vivo environment and, to correlate their contractility with α‐smooth muscle actin (α‐SMA) expression and determine if dermal fibroblasts regulate Dupuytren's myofibroblast phenotype. Isotonic and isometric force contraction by cells isolated from Dupuytren's nodules, palmar and non‐palmar skin fibroblasts was measured in collagen matrices. The effect of co‐culturing nodule cells with dermal fibroblasts on isometric contraction was examined. Isometric contraction was correlated with levels of α‐SMA mRNA by pcr and protein by Western blotting, and α‐SMA distribution assessed by immunofluorescence. Dupuytren's nodule cells exhibited similar levels of isotonic contraction to both palmar and non‐palmar dermal fibroblasts. However, nodule cells generated high levels of isometric force (mean: 3.5 dynes/h), which continued to increase over 24 h to a maximum of 173 dynes. In contrast, dermal fibroblasts initially exhibited low levels of contraction (mean: 0.5 dynes/h) and reached tensional homeostasis on average after 15 h (range: 4–20 h), with a maximum force of 52 dynes. Although all three cell types had similar α‐SMA mRNA levels, increased levels of α‐SMA protein were observed in nodule cells compared to dermal fibroblasts. α‐SMA localised to stress fibres in 35% (range: 26–50%) of nodule cells compared to only 3% (range:0–6%) of dermal fibroblasts. Co‐cultures of Dupuytren's cells and dermal fibroblasts showed no contractile differences. The contractile phenotype of Dupuytren's myofibroblasts is determined by increased α‐SMA protein distributed in stress fibres, not by cellular mRNA levels. Dupuytren's cell contractility is not influenced by dermal fibroblasts. J. Cell. Physiol. 224: 681–690, 2010. © 2010 Wiley‐Liss, Inc.     

Evidence from a Ghanaian population of known African descent to support the proposition that hemochromatosis is a Caucasian disorder

Mutations in the HFE gene on chromosome 6 are believed to cause the iron overload disorder hemochromatosis, the most common single gene disorder in northern Europeans. Two mutations have been described previously: C282Y, with an allele frequency of between 3% and 10% in the caucasian population, and H63D, which has an allele frequency of 16%. Published data shows that C282Y appears to be causative in the homozygous state, while the frequency of H63D/C282Y compound heterozygotes is much greater than expected in patient groups. There also appears to be a slightly elevated risk for H63D homozygotes. Hemochromatosis has been thought to be primarily a caucasian disorder. We have studied 97 healthy, black Ghanaian subjects, whose parents and grandparents were also African, to find the frequency of the two mutations. C282Y was absent, while H63D occurred in 2 individuals. These differences are significant at the 0.05 and 0.001 levels, respectively. The prevalence of H63D homozygotes in this population at 1 in 10,000 is clearly of no use in studying the effect of this genotype on phenotype. However, this study suggests an absence of the C282Y mutation in African populations, and the possibility that other populations might provide different genotypes and hence an analysis of H63D risk. A possible heterozygote advantage for the mutation is discussed.     

Heart valve and arterial tissue engineering

Abstract.  In the industrialized world, cardiovascular disease alone is responsible for almost half of all deaths. Many of the conditions can be treated successfully with surgery, often using transplantation techniques; however, autologous vessels or human-donated organs are in short supply. Tissue engineering aims to create specific, matching grafts by growing cells on appropriate matrices, but there are many steps between the research laboratory and the operating theatre. Neo-tissues must be effective, durable, non-thrombogenic and non-immunogenic. Scaffolds should be bio-compatible, porous (to allow cell/cell communication) and amenable to surgery. In the early days of cardiovascular tissue engineering, autologous or allogenic cells were grown on inert matrices, but patency and thrombogenicity of grafts were disappointing. The current ethos is toward appropriate cell types grown in (most often) a polymeric matrix that degrades at a rate compatible with the cells' production of their own extracellular matrical proteins, thus gradually replacing the graft with a living counterpart. The geometry is crucial. Computer models have been made of valves, and these are used as three-dimensional patterns for mass-production of implant scaffolds. Vessel walls have integral connective tissue architecture, and application of physiological level mechanical forces conditions bio-engineered components to align in precise orientation. This article reviews the concepts involved and successes achieved to date.     

Enhanced fibroblast contraction of 3D collagen lattices and integrin expression by TGF-beta1 and -beta3: mechanoregulatory growth factors?

Generation of contractile forces as fibroblasts attach and migrate through collagenous substrates is a fundamental behavior, yet its regulation and consequences are obscure. Although the transforming growth factor-betas (TGF-beta) are similarly important in fibrosis and tissue repair, their role in contraction is controversial. Using a quantitative, 3D collagen culture model we have measured the effects of TGF-beta1 and -beta3 on contractile forces generated by human dermal fibroblasts. Maximal stimulation was between 7.5 and 15 ng/ml of TGF-beta1. Higher doses were inhibitory (30 ng/ml), giving a bell-shaped dose response. The initial rate of force generation was increased sevenfold (15 ng/ml). A similar response pattern was seen with TGF-beta3 alone. However, the addition of both isoforms together stimulated a biphasic increase in force generation, suggesting that there was a distinct temporal cooperativity between the two isforms. This very early onset (10-20 min) of stimulation suggested that TGF-beta might act through cell attachment and integrin function and the effect of TFG-beta on expression of fibronectin (FnR) and vitronectin (VnR) integrin receptors was monitored over the same time scale. TGF-beta1 dramatically up-regulated VnR expression, relative to FnR, over time but the optimal time for this was 2-4 h later than that of force stimulation. It is concluded that TGF-beta1 and -beta3 behave here primarily as mechanoregulatory growth factors and that stimulation of integrin expression may be a consequence of the altered cell stress.     

The action of structural analogues of ethidium bromide on the mitochondrial genome of yeast

We have studied the effects on the yeast mitochondrial genome of four analogues of ethidium bromide, in which the phenyl moiety has been replaced by linear alkyl chains of lengths varying from seven to fifteen carbon atoms. These analogues are more efficient than ethidium bromide in inducing petite mutants inSaccharomyces cerevisiae. The drugs also cause a loss of mtDNA from the cellsin vivo; however these analogues are in fact less effective inhibitors of mitochondrial DNA replicationper se, as shown by directin vitro studies. It is concluded that these analogues are more efficient than ethidium bromide in causing the fragmentation of mitochondrial DNA inS. cerevisiae.     

Quantitative analysis of collagen gel contractile forces generated by dermal fibroblasts and the relationship to cell morphology

The force generated in granulation tissue during wound contraction is thought to be cell mediated; however, it is unclear whether contractile forces are generated by fibroblast locomotion or contraction of myofibroblasts. To help clarify this question the force of this contraction can now be determined accurately in a human dermal fibroblast collagen lattice system using a novel instrument known as a Culture Force Monitor. Three distinct phases of contraction of such collagen gels could be identified over the first 24 hours. Most of the force generated by human dermal fibroblasts was produced during the first stage in parallel with cell attachment and associated changes in cell shape, and the appearance of cell processes. During this initial 24 hours no evidence could be found for the presence of myofibroblasts, but stereoscopic and electron microscopic analysis at a range of time points indicated that migratory fibroblasts were present in the system. Comparison of the contraction profiles of cells extracted from other tissues (tendon and articular cartilage), and extracted by different means from the same tissue specimen, indicated that different populations of fibroblasts can be distinguished on the basis of their pattern of contractions. It would seem that most of the force generated in this model is a result of fibroblast attachment and movement within the collagen lattice. Furthermore, different groups of fibroblasts, even within the same tissue, may vary in their contraction (hence locomotory) activity. © 1996 Wiley-Liss, Inc.     

Plasticizers increase adhesion of the deteriogenic fungus Aureobasidium pullulans to polyvinyl chloride.

Initial adhesion of fungi to plasticized polyvinyl chloride (pPVC) may determine subsequent colonization and biodeterioration processes. The deteriogenic fungus Aureobasidium pullulans was used to investigate the physicochemical nature of adhesion to both unplasticized PVC (uPVC) and pPVC containing the plasticizers dioctyl phthalate (DOP) and dioctyl adipate (DOA). A quantitative adhesion assay using image analysis identified fundamental differences in the mechanism of adhesion of A. pullulans blastospores to these substrata. Adhesion to pPVC was greater than that to uPVC by a maximum of 280% after a 4-h incubation with 10(8) blastospores ml(-1). That plasticizers enhance adhesion to PVC was confirmed by incorporating a dispersion of both DOA and DOP into the blastospore suspension. Adhesion to uPVC was increased by up to 308% in the presence of the dispersed plasticizers. Hydrophobic interactions were found to dominate adhesion to uPVC because (i) a strong positive correlation was observed between substratum hydrophobicity (measured by using a dynamic contact angle analyzer) and adhesion to a range of unplasticized polymers including uPVC, and (ii) neither the pH nor the electrolyte concentration of the suspension buffer, both of which influence electrostatic interactions, affected adhesion to uPVC. In contrast, adhesion to pPVC is principally controlled by electrostatic interactions. Enhanced adhesion to pPVC occurred despite a relative reduction of 13 degrees in the water contact angle of pPVC compared to that of uPVC. Furthermore, adhesion to pPVC was strongly dependent on both the pH and electrolyte concentration of the suspension medium, reaching maximum levels at pH 8 and with an electrolyte concentration of 10 mM NaCl. Plasticization with DOP and DOA therefore increases adhesion of A. pullulans blastospores to pPVC through an interaction mediated by electrostatic forces.     

Analysis of Human Proteome Organization Plasma Proteome Project (HUPO PPP) reference specimens using surface enhanced laser desorption/ionization-time of flight (SELDI-TOF) mass spectrometry: multi-institution correlation of spectra and identification of biomarkers

We report on a multicenter analysis of HUPO reference specimens using SELDI-TOF MS. Eight sites submitted data obtained from serum and plasma reference specimen analysis. Spectra from five sites passed preliminary quality assurance tests and were subjected to further analysis. Intralaboratory CVs varied from 15 to 43%. A correlation coefficient matrix generated using data from these five sites demonstrated high level of correlation, with values >0.7 on 37 of 42 spectra. More than 50 peaks were differentially present among the various sample types, as observed on three chip surfaces. Additionally, peaks at approximately 9200 and approximately 15,950 m/z were present only in select reference specimens. Chromatographic fractionation using anion-exchange, membrane cutoff, and reverse phase chromatography, was employed for protein purification of the approximately 9200 m/z peak. It was identified as the haptoglobin alpha subunit after peptide mass fingerprinting and high-resolution MS/MS analysis. The differential expression of this protein was confirmed by Western blot analysis. These pilot studies demonstrate the potential of the SELDI platform for reproducible and consistent analysis of serum/plasma across multiple sites and also for targeted biomarker discovery and protein identification. This approach could be exploited for population-based studies in all phases of the HUPO PPP.     

Endothelin-1 promotes myofibroblast induction through the ETA receptor via a rac/phosphoinositide 3-kinase/Akt-dependent pathway and is essential for the enhanced contractile phenotype of fibrotic fibroblasts

The endothelins are a family of endothelium-derived peptides that possess a variety of functions, including vasoconstriction. Endothelin-1 (ET-1) is up-regulated during tissue repair and promotes myofibroblast contraction and migration, hence contributing to matrix remodeling during tissue repair. Here, we show that addition of ET-1 to normal lung fibroblasts induces expression of proteins that contribute to a contractile phenotype, including alpha-smooth muscle actin (alpha-SMA), ezrin, moesin, and paxillin. We confirm that ET-1 enhances the ability of lung fibroblasts to contract extracellular matrix, a function essential for tissue repair, through induction of de novo protein synthesis. Blockade of the Akt/phosphoinositide 3-kinase (PI3-kinase) pathway with LY294002 and wortmannin prevents the ability of ET-1 to induce alpha-SMA, ezrin, paxillin, and moesin and to promote matrix contraction. Dominant negative rac and Akt blocked the ability of ET-1 to promote formation of alpha-SMA stress fibers. Using specific ET-1 receptor inhibitors, we show that ET-1 induces collagen matrix contraction through the ETA, but not the ETB, receptor. Relative to normal pulmonary fibroblasts, fibroblasts cultured from scars of patients with the fibrotic disease systemic sclerosis (scleroderma) show enhanced ET-1 expression and binding. Systemic sclerosis lung fibroblasts show increased ability to contract a collagen matrix and elevated expression of the procontractile proteins alpha-SMA, ezrin, paxillin, and moesin, which are greatly reduced by antagonizing endogenous ET-1 signaling. Thus, blocking ET-1 or the PI3-kinase/Akt cascades might be beneficial in reducing scar formation in pulmonary fibrosis.