Epidermal growth factor-induced epithelio-mesenchymal transition in human breast carcinoma cells

PMC42-LA cells display an epithelial phenotype: the cells congregate into pavement epithelial sheets in which E-cadherin and beta-catenin are localized at cell-cell borders. They abundantly express cytokeratins, although 5% to 10% of the cells also express the mesenchymal marker vimentin. Stimulation of PMC42-LA cells with epidermal growth factor (EGF) leads to epithelio-mesenchymal transition-like changes including up-regulation of vimentin and down-regulation of E-cadherin. Vimentin expression is seen in virtually all cells, and this increase is abrogated by treatment of cells with an EGF receptor antagonist. The expression of the mesenchyme-associated extracellular matrix molecules fibronectin and chondroitin sulfate proteoglycan also increase in the presence of EGF. PMC42-LA cells adhere rapidly to collagen I, collagen IV, and laminin-1 substrates and markedly more slowly to fibronectin and vitronectin. EGF increases the speed of cell adhesion to most of these extracellular matrix molecules without altering the order of adhesive preference. EGF also caused a time-dependent increase in the motility of PMC42-LA cells, commensurate with the degree of vimentin staining. The increase in motility was at least partly chemokinetic, because it was evident both with and without chemoattractive stimuli. Although E-cadherin staining at cell-cell junctions disappeared in response to EGF, beta-catenin persisted at the cell periphery. Further analysis revealed that N-cadherin was present at the cell-cell junctions of untreated cells and that expression was increased after EGF treatment. N- and E-cadherin are not usually coexpressed in human carcinoma cell lines but can be coexpressed in embryonic tissues, and this may signify an epithelial cell population prone to epithelio-mesenchymal-like responses.

Changes in the composition of the extracellular matrix in patellar tendinopathy

Objective: To compare the chemical levels and mRNA expression of proteoglycan and collagen in normal human patellar tendons and tendons exhibiting chronic overuse tendinopathy.

Methods: Sulfated glycosaminoglycan and hydroxyproline content were investigated by spectrophotometric measurement using papain-digested samples. Deglycosylated proteoglycan core proteins were analysed by Western blot using specific antibodies. Total mRNA isolated from samples of frozen tendons was assayed by relative quantitative RT-PCR for decorin, biglycan, fibromodulin, versican, aggrecan, and collagens Type I, II and III and normalised to glyceraldehyde-3-phosphate dehydrogenase.

Results: There was a significant increase in sulfated glycosaminoglycan content in pathologic tendons compared to normal. This was attributed to an increased deposition of the large aggregating proteoglycans versican and aggrecan and the small proteoglycans biglycan and fibromodulin, but not decorin. Aggrecan and versican were extensively degraded in both normal and pathologic tendons, biglycan was more fragmented in the pathologic tendons while predominantly intact fibromodulin and decorin were present in normal and pathologic tendons. There was a greater range in total collagen content but no change in the level of total collagen in pathologic tendons. There were no significant differences between the pathologic and normal tendon for all genes, however p values close to 0.05 indicated a trend in downregulation of Type I collagen and fibromodulin, and upregulation in versican and Type III genes in pathologic tissue.

Conclusion: The changes in proteoglycan and collagen levels observed in patellar tendinopathy appear to be primarily due to changes in the metabolic turnover of these macromolecules. Changes in the expression of these macromolecules may not play a major role in this process.

Three-month bilateral hopping intervention is ineffective in initiating bone biomarker response in healthy elderly men.

In animal studies, bone adaptation has been initiated successfully without the transient force spike associated with high impact exercises. Consequently, a 12-week bilateral hopping on the balls of the feet intervention was conducted. 25 elderly men (age 72(SD4) years, height 171(6) cm, weight 75(9) kg) were randomly assigned into exercise and control groups. Ten subjects in each group completed the study. Carboxyterminal propeptide of type I collagen (CICP), bone-specific alkaline phosphatase (bALP) and carboxyterminal telopeptide of type I collagen (CTx) were measured from venous blood samples at baseline, at 2 weeks and at the end of the intervention. Maximal ground reaction force (GRF), osteogenic index (OI) and jump height (JH) were determined from bilateral hopping test and balance was assessed with velocity of center of pressure (COPvelocity) while standing on the preferred leg with eyes open. The intervention consisted of 5–7 sets of 10 s timed bilateral hopping exercise at 75–90% intensity three times/week. There was no significant group 9 time interaction for GRF, OI and JH (P = 0.065). GRF (11% change from baseline vs. 4%), OI (15 vs. 6%) and COPvelocity (-10 vs. -1%) were not influenced by the intervention (P[0.170), while the control group improved JH (P = 0.031) (2 vs. 18%). For the biomarkers, no effect was observed in MANOVA (P = 0.536) or in univariate analyses (P = 0.082 to P = 0.820) (CICP -2 vs. -3%, CTx 8 vs. -12%, bALP 0 vs. -3.7%). Allowing transient impact force spikes may be necessary to initiate a bone response in elderly men as the intervention was ineffective.

Collagen type-I leads to in vivo matrix mineralization and secondary stabilization of Mg-Zr-Ca alloy implants

Biodegradable magnesium-zirconia-calcium (Mg-Zr-Ca) alloy implants were coated with Collagen type-I (Coll-I) and assessed for their rate and efficacy of bone mineralization and implant stabilization. The phases, microstructure and mechanical properties of these alloys were analyzed using X-ray diffraction (XRD), optical microscopy and compression test, respectively, and the corrosion behavior was established by their hydrogen production rate in simulated body fluid (SBF). Coll-I extracted from rat tail, and characterized using fourier transform infrared (FT-IR) spectroscopy, was used for dip-coating the Mg-based alloys. The coated alloys were implanted into the femur bones of male New Zealand white rabbits. In vivo bone formation around the implants was quantified by measuring the bone mineral content/density (BMC/BMD) using dual-energy X-ray absorptiometry (DXA). Osseointegration of the implant and new bone mineralization was visualized by histological and immunohistochemical analysis. Upon surface coating with Coll-I, these alloys demonstrated high surface energy showing enhanced performance as an implant material that is suitable for rapid and efficient new bone tissue induction with optimal mineral content and cellular properties. The results demonstrate that Coll-I coated Mg-Zr-Ca alloys have a tendency to form superior trabecular bone structure with better osteoinduction around the implants and higher implant secondary stabilization, through the phenomenon of contact osteogenesis, compared to the control and uncoated ones in shorter periods of implantation. Hence, Coll-I surface coating of Mg-Zr-Ca alloys is a promising method for expediting new bone formation in vivo and enhancing osseointegration in load bearing implant applications.

Strontium content and collagen-I coating of magnesium-zirconia-strontium implants influence osteogenesis and bone resorption.

Our objective was to study the role of Collagen type-I (Col-I) coating on Magnesium-Zirconia (Mg-Zr) alloys, containing different quantities of Strontium (Sr), in enhancing the in vitro bioactivity and in vivo bone-forming and mineralisation properties of the implants.

Computer-assisted image analysis of liver collagen: relationship to Ishak scoring and hepatic venous pressure gradient

Histopathological scoring of disease stage uses descriptive categories without measuring the amount of fibrosis. Collagen, the major component of fibrous tissue, can be quantified by computer-assisted digital image analysis (DIA) using histological sections. We determined relationships between DIA, Ishak stage, and hepatic venous pressure gradient (HVPG) reflecting severity of fibrosis. One hundred fifteen patients with hepatitis C virus (HCV) who had undergone transplantation had 250 consecutive transjugular liver biopsies combined with HVPG (median length, 22 mm; median total portal tracts, 12), evaluated using the Ishak system and stained with Sirus red for DIA. Liver collagen was expressed as collagen proportionate area (CPA). Median CPA was 6% (0.2-45), correlating with Ishak stage (stage 6 range, 13%-45%), and with HVPG (<i>ri> = 0.62; <i>Pi> < 0.001). Median CPA was 4.1% when HVPG was less than 6 mm Hg and 13.8% when HVPG was 6 mm Hg or more (<i>Pi> < 0.0001) and 6% when HVPG was less than 10 mm Hg and 17.3% when HVPG was 10 mm Hg or higher (<i>Pi> < 0.0001). Only CPA, not Ishak stage/grade, was independently associated by logistic regression, with HVPG of 6 mm Hg or more [odds ratio, 1.206; 95% confidence interval (CI), 1.094-1.331; <i>Pi> < 0.001], or HVPG of 10 mm Hg or more (odds ratio, 1.105; 95% CI, 1.026-1.191; <i>Pi> = 0.009). CPA increased by 50% (3.6%) compared with 20% in HVPG (1 mm Hg) in 38 patients with repeated biopsies. Conclusion: CPA assessed by DIA correlated with Ishak stage scores and HVPG measured contemporaneously. CPA was a better histological correlate with HVPG than Ishak stage, had a greater numerical change when HVPG was low, and resulted in further quantitation of fibrosis in cirrhosis.