The Scar-in-a-Jar: studying potential antifibrotic compounds from the epigenetic to extracellular level in a single well.

BACKGROUND AND PURPOSE: Fibrosis, a pathological accumulation of collagen in tissues, represents a major global disease burden. Effective characterization of potential antifibrotic drugs has been constrained by poor formation of the extracellular matrix in vitro, due to tardy procollagen processing by collagen C-proteinase/BMP-1, and difficulties in relating this matrix to cell numbers in experimental samples. EXPERIMENTAL APPROACH: The Scar-in-a-Jar model provided, in vitro, the complete biosynthetic cascade of collagen matrix formation including complete conversion of procollagen by C-proteinase/BMP-1, its subsequent extracellular deposition and lysyl oxidase-mediated cross-linking, achieved by applying the biophysical principle of macromolecular 'crowding'. Collagen matrix deposition, velocity and morphology can be controlled using negatively charged 'crowders' in a rapid (2 days) mode or a mixture of neutral 'crowders' in an accelerated (6 days) mode. Combined with quantitative optical bioimaging, this novel system allows for in situ assessment of the area of deposited collagen(s) per cell. KEY RESULTS: Optical evaluation of known and novel antifibrotic compounds effective at the epigenetic, post-transcriptional/translational/secretional level correlated excellently with corresponding biochemical analyses. Focusing on quantitation of deposited collagen, the Scar-in-a-Jar was most effective in assessing novel inhibitors that may have multiple targets, such as microRNA29c, found to be a promising antifibrotic agent. CONCLUSIONS AND IMPLICATIONS: This novel screening system supersedes current in vitro fibroplasia models, as a fast, quantitative and non-destructive technique. This method distinguishes a reduction in collagen I deposition, excluding collagen cross-linking, and allows full evaluation of inhibitors of C-proteinase/BMP-1 and other matrix metalloproteinases.

Collagen solubility testing, a quality assurance step for reproducible electro-spun nano-fibre fabrication. A technical note.

Collagen is the main component of the extra-cellular matrix and has been utilised for numerous clinical applications in many forms and products. However, since collagen remains a natural animal-derived biopolymer, variation between batches should be addressed and minimised to ensure reproducibility of the fabrication process. Recently, electro-spinning of collagen has been introduced as a leading technique for the production of bio-mimetic nano-scale scaffolds for tissue-engineering applications. However, no protocols are available that would allow comparisons of the quality of different collagen raw materials prior to the electro-spinning process. In order to bridge this gap we assessed the solubility of various freeze-dried collagens in 0.5 M acetic acid and analysed the solved collagen by gel electrophoresis. We show that raw material of limited solubility in acetic acid will not render high quality electro-spun nano-fibres using hexafluoropropanol. In particular, insoluble collagen directly failed to produce nano-fibres, collagen of reduced solubility produced fused nano-fibres with limited inter-nano-fibre space, whilst purified type-I collagen of high solubility produced smooth, reproducible nano-fibres. Gel electrophoresis confirmed the amount of solubility, as well as qualitative differences in terms of collagen cross-links and collagen types. We recommend this simple and fast step to save costs and to enhance control over the electro-spinning process of collagen. Furthermore, we believe that the solubility test should be introduced prior to any collagenous matrix preparation in order to ensure reproducibility and accuracy.

Role of apoptosis in functional luteolysis in the pregnant rabbit corpus luteum: evidence of a role for placental-derived factors in promoting luteal cell survival.

Corpora lutea (CL) were isolated from one rabbit ovary on days 4, 8, 16 (peak luteal function), 28 (functional regression) and 30 of pregnancy and processed for biochemical analysis of DNA integrity. Analysis of DNA integrity revealed the presence of oligonucleosomal fragments in day 28 and day 30 CL but not in day 16 CL. The extent of low molecular weight (<15 kb) DNA labeling was 6.6 +/- 0.84 fold higher in day 30 as compared to day 16 CL (mean +/- SEM; n = 4, P < 0.01). In a second series of experiments, healthy CL collected from day 16 pregnant rabbits were incubated for 2 h in the absence or presence of 250 microg/ml of placental extract (PE) obtained from day 16 and/or day 30 placentas. Analysis of DNA integrity revealed that extensive apoptosis occurred in CL incubated in medium alone and in medium containing day 30 PE. In contrast, day 16 PE significantly suppressed apoptosis vs control (70 +/- 4%). In a third series of experiments, expression of mRNA for bcl-x and bax was measured by Northern analysis of CL treated without and with day 16 PE using cRNA probes for bcl-x and bax developed in our laboratory by RT-PCR. Treatment with PE significantly reduced bax mRNA levels but did not change bcl-x mRNA levels. These studies provide evidence that functional luteolysis in the pregnant rabbit CL is correlated with the occurrence of apoptosis. The data suggest that a factor(s) derived from the placenta may be responsible for the prolongation of CL life span during pregnancy by its ability to alter the bax:bcl -x rheostat and suppress apoptosis.

Inhibition of nitric oxide synthesis potentiates apoptosis in the rabbit corpus luteum.

To determine if nitric oxide (NO) plays a role in corpus luteum (CL) physiology by affecting progesterone secretion or luteal apoptosis, an in-vitro pseudopregnant rabbit ovarian perfusion system was used to measure the effects of an inhibitor of NO synthesis, NG-nitro-L-arginine methyl ester (L-NAME), on progesterone secretion and corpus luteal apoptosis as measured by internucleosomal DNA breakdown. Pseudopregnant rabbit ovaries perfused in vitro with L-NAME did not demonstrate any significant differences compared with control ovaries in progesterone secretion. However, apoptosis, as measured by internucleosomal breakdown, was significantly increased in L-NAME-perfused CL compared with controls. While NO does not appear to directly affect progesterone secretion, there does appear to be a role for NO in CL maintenance, or a role for inhibition of NO production in CL regression.

Role of DDC-4/sFRP-4, a secreted frizzled-related protein, at the onset of apoptosis in mammary involution.

Using differential display, we isolated DDC-4, a secreted frizzled-related protein (sFRP), which is induced in the physiological apoptosis of hormonally regulated, reproductive tissues such as mammary gland, prostate, corpus luteum and uterus. The role of this gene in apoptosis was studied in animals overexpressing ectopic DDC-4/sFRP-4. Transgenic mice bearing the DDC-4/sFRP-4 cDNA under the control of the MMTV-LTR promoter showed lactational insufficiency and many apoptotic cells in the alveoli between day 19 of pregnancy and day 4 of lactation as demonstrated by TUNEL reaction and the presence of activated caspase-3. We performed a PKB/Akt kinase assay and studied several of its substrates using phosphorylation-specific antibodies to show reduced phosphorylation in PKB/Akt itself, as well as in glycogen synthetase kinase-3beta (GSK-3beta), BAD, and Forkhead. Taken together, our results show a role for DDC-4/sFRP-4 in abrogating an epithelial cell survival pathway at the onset of mammary gland involution.

Fas and Fas ligand messenger ribonucleic acid and protein expression in the rat corpus luteum during apoptosis-mediated luteolysis.

Apoptosis has been found to occur during regression of the corpus luteum (CL) in many species. The Fas (APO-1/CD95) receptor, a transmembrane protein that induces apoptosis in the cell when bound to Fas ligand (FasL), may be involved. This study established and quantitated the presence and regulation of Fas receptor and FasL in the rat CL during pregnancy and postpartum. Using immunohistochemistry, FasL was localized in CL during pregnancy and postpartum. Fas was localized at Day 1 of pregnancy and at the time of luteolysis. Both Fas and FasL mRNA were found to be expressed throughout pregnancy and postpartum using reverse transcription-polymerase chain reaction (RT-PCR). Relative quantitative RT-PCR established that expression of FasL mRNA increased significantly at Day 22 of pregnancy and decreased by Day 3 postpartum. Spontaneous apoptosis of rat CL placed in an in vitro culture model with serum-free medium was examined by analysis of extracted DNA using 3' end-labeling. Treatment with an anti-rat Fas monoclonal antibody demonstrated a reduction in the occurrence of spontaneous apoptosis. These data support a role for Fas receptor and FasL in rat CL apoptosis during luteolysis.

Rapid and sensitive genotyping of hepatitis C virus by single-strand conformation polymorphism.

There is an increasing demand for genotyping hepatitis C virus (HCV) isolates due to the rapidly expanding list of distinct HCV genotypes and the mounting evidence of genotype-specific clinical consequences. We describe an SSCP-based assay for determining genotypes in HCV infections. HCV RNA extracted from serum was amplified by a sensitive nested-PCR assay producing a 287 bp fragment of the conserved 5' non-coding region (NCR) and analysed by non-denaturing polyacrylamide gel electrophoresis. Following empirical optimisation of the SSCP assay we identified distinct conformation polymorphisms (characteristic band patterns) corresponding to types 1a, 1b, 2a, 2b, 2c, 3 and 4 found in the Western Australian population. Seventy-three HCV RNA-positive samples were used to evaluate the SSCP genotyping assay for accuracy and efficiency by comparison with the previously established genotyping methods of manual direct sequencing and dideoxy fingerprinting. SSCP genotyping was in concord with control methods while performing more rapidly and at a fraction of the cost. Moreover, SSCP detected two co-infected samples that were not shown by the control methods. The PCR-SSCP assay provides an accurate and rapid method for genotyping of HCV RNA-positive samples at the 5' NCR by type-specific sequence polymorphisms which is applicable to large-scale screening.

Rapid genotyping of hepatitis C virus isolates by dideoxy fingerprinting.

A number of distinct hepatitis C virus (HCV) types and subtypes have been identified by DNA sequencing of multiple genome regions. It has been postulated that these might also reflect phenotypic differences in the nature of HCV infection. Recent evidence suggests a relationship between HCV genotype and alpha-interferon response in patients with chronic hepatitis C. A simplified method of genotyping in comparison to direct DNA sequencing was investigated with the intention of providing a rapid, less labour-intensive method for routine genotyping. HCV RNA was extracted from serum by a modified guanidinium/acid-phenol extraction and peripheral blood lymphocytes using RNAzol B (Cinna-Biotecx). The RNA was reverse transcribed and a 287 bp segment of the 5' non-coding region (5' NCR) amplified using a nested-PCR reaction. PCR products were purified using Qiaquick spin columns. Products were directly sequenced by cycle sequencing. Dideoxy termination analysis was carried out by cyclic extension of a 33P-labelled primer by Tth polymerase with termination by dideoxy thiamine (ddT) or cytosine (ddC). Reaction products were analysed by electrophoresis on denaturing 7 M urea/6% acrylamide gels followed by autoradiography. Computer aided sequence analysis indicated that conserved 5' NCR sequence variation alone was sufficient to identify HCV types 1a, 1b, 2a, 2b, 3 and 4. Dideoxy fingerprinting improved greatly the efficiency of genotyping with an approximate four-fold increase in throughput. In addition, the results were very easily analysed although it was essential to run appropriate controls for each genotype. Reactions incorporating ddT distinguished types 1, 2a, 2b, 3 (provisionally 1a & 1b); a ddC reaction confirmed 1a and 1b typing.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitation of muscle precursor cell activity in skeletal muscle by Northern analysis of MyoD and myogenin expression: Application to dystrophic (mdx) mouse muscle.

The regeneration of skeletal muscle is dependent upon proliferation and fusion of activated mononuclear muscle precursor cells. Early and specific markers of this population of activated cells are the transcription factors MyoD and myogenin. Northern analysis was used to determine levels of MyoD and myogenin mRNA in (i) muscles regenerating after experimental crush injury and (ii) in limb muscles of dystrophic mdx mice at various ages in comparison to controls. In crush-injured muscle, MyoD and myogenin mRNA increased at 24 h, peaked between 2 to 6 days, and returned to uninjured control levels by 15 days after injury. In both mdx and control mice, MyoD and myogenin mRNA levels were high in fetal muscles and decreased rapidly during the 2 weeks after birth. In mdx muscles, the mRNA levels increased significantly from about 21 days, remained high until around 40 days, and then decreased to a relatively constant yet elevated level when compared to control muscles. The elevated levels persisted to 420 days of age. The results show that this technique can be used to provide sensitive quantitative information on the size of the population of activated precursor cells in skeletal muscle. As such, it represents a novel and convenient means of measuring regenerative activity in vivo in whole muscles.