A central bioactive region of LTBP-2 stimulates the expression of TGF-β1 in fibroblasts via akt and p38 signalling pathways

Latent transforming growth factor-β-1 binding protein-2 (LTBP-2) belongs to the LTBP-fibrillin superfamily of extracellular proteins. Unlike other LTBPs, LTBP-2 does not covalently bind transforming growth factor-β1 (TGF-β1) but appears to be implicated in the regulation of TGF-β1 bioactivity, although the mechanisms are largely unknown. In experiments originally designed to study the displacement of latent TGF-β1 complexes from matrix storage, we found that the addition of exogenous LTBP-2 to cultured human MSU-1.1 fibroblasts caused an increase in TGF-β1 levels in the medium. However, the TGF-β1 increase was due to an upregulation of TGF-β1 expression and secretion rather than a displacement of matrix-stored TGF-β1. The secreted TGF-β1 was mainly in an inactive form, and its concentration peaked around 15 h after addition of LTBP-2. Using a series of recombinant LTBP-2 fragments, the bioactivity was identified to a small region of LTBP-2 consisting of an 8-Cys motif flanked by four epidermal growth factor (EGF)-like repeats. The LTBP-2 stimulation of TGF-β expression involved the phosphorylation of both Akt and p38 mitogen-activated protein kinase (MAPK) signalling proteins, and specific inactivation of each protein individually blocked TGF-β1 increase. The search for the cell surface receptor mediating this LTBP-2 activity proved inconclusive. Inhibitory antibodies to integrins β1 and αVβ5 showed no reduction of LTBP-2 stimulation of TGF-β1. However, TGF-β1 upregulation was partially inhibited by anti-αVβ3 integrin antibodies, suggestive of a direct or indirect role for this integrin. Overall, the study indicates that LTBP-2 can directly upregulate cellular TGF-β1 expression and secretion by interaction with cells via a short central bioactive region. This may be significant in connective tissue disorders involving aberrant TGF-β1 signallin

Function of latent transforming growth factor-β binding protein-2 (LTBP-2) in elastinogenesis and modulation of growth factor storage, expression and activity in normal and fibrotic tissues

LTBP-2 is tightly associated with fibrillin microfibrils and elastic fibres in a range of tissues mainly in the lung, heart, skeletal muscle, placenta, liver and the aorta. LTBP-2 belongs to the fibrillin-LTBP superfamily of extracellular matrix proteins. Unlike other LTBPs, LTBP-2 does not covalently bind TGF-beta and its molecular function remains unclear. LTBP-2 complexes with fibulin-5, an elastin-chaperone protein critical for normal elastic fibre assembly, and it has been suggested that LTBP-2 may preferentially direct fibulin-5-elastin globules onto fibrillin-1 (rather than fibrillin-2) microfibrils during elastinogenesis. However, we have now shown that LTBP-2 inhibits rather than enhances the interaction of tropoelastin with fibulin-5 in vitro. In addition LTBP-2 inhibited elastic fibre assembly in ear cartilage chondrocyte cultures largely at the stage of elastin deposition onto the fibrillin microfibril scaffold. In parallel experiments, LTBP-2 was shown to significantly inhibit the binding of heparin to tropoelastin suggesting LTBP-2 may compete with tropoelastin for binding to certain cell surface HSPGs and contribute to controlling the release of elastin microassemblies from the cell surface. Confocal microscopy showed strong co-distribution of LTBP-2 with fibulin-5 and fibrillin-1 and partial co-distribution with HSPGs, perlecan and syndecan-4 in fibroblast matrix Thus it is evident that LTBP-2 is a negative modulator of elastinogenesis and that LTBP-2 levels may regulate the rate and extent of elastinogenesis in some tissues. A recent study has linked LTBP-2 gene mutations to recessive form of Weill-Marchesani syndrome which is characterised by short stature, thick fibrotic skin and ectopia lentis. Since fibrillin-1 mutations can also cause this syndrome it is now clear that LTBP-2 is linked to fibrillin biology, growth factor regulation and fibrosis. To investigate growth factor binding to LTBP-2, our laboratory screened a number of cytokines involved in the pathogenesis of fibrotic disorders and identified a very strong specific interaction of FGF-2. The activity was confined to a central region of the LTBP-2 consisting of 6 EGF-like repeats, suggesting a single binding sequence. The finding presented in this thesis found that 5-fold molar excess LTBP-2 can completely block FGF-2 stimulation of fibroblast proliferation via its receptor. In addition increased levels and extensive co-localisation of LTBP-2 and FGF-2 were observed and quantitated in human hypertrophic scars and keloids. Furthermore, qPCR confirmed consistent elevation of LTBP-2 and FGF-2 expression in samples of these fibrotic tissues. The results support the concept that increased LTBP-2 expression in fibrotic disorders may increase FGF-2 binding and reduce FGF-2 activity, inhibiting normal repair processes. Previously we have shown that LTBP-2 competes with LTBP-1 for binding to fibrillin in vitro, suggesting that LTBP-2 may modulate TGF-β storage and activation. In experiments designed to measure displacement of TGF-β complexes from fibrillin microfibrils, our laboratory discovered addition of LTBP-2, or a small bioactive fragment LTBP-2C F3 to MSU 1.1 skin fibroblasts resulted in a large increase in TGF-β levels in culture medium. However the increase in TGF-β the medium was cycloheximide sensitive indicating elevated cellular expression and secretion of TGF-β rather than release of matrix-stored TGF-β. Exogenous LTBP-2 or fragment F3 significantly increased levels of latent TGF-β in the medium after 9h peaking at 15h. The signalling mechanism appears to involve the PI3K/Akt and p38 MAPK pathways, as incubation of cells with LTBP-2 (10μg/ml) elevated Akt 1/2/3 Ser473 and P38 D-8 phosphorylation and inhibition of each pathway completely blocked the synthesis of TGF-β. Investigation of the cell surface receptor for the bioactive fragment of LTBP-2 was less informative. Inhibitory antibody to β1 integrins did not affect the TGF-β upregulation but it was partially inhibited by an antibody to the integrin αVβ3 receptor, suggesting it may be involved in LTBP-2-cell interaction(s) resulting in elevated TGF-β expression. In conclusion, these findings are consistent with LTBP-2 having novel regulatory functions in elastinogenesis, growth factor modulation and fibrosis which may lead to novel therapy development for fibrotic diseases and tissue repair.Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, School of Medicine, 2016

Association between physical exercise and stress level among first year students of Kulliyyah of Allied Health Sciences in International Islamic University Malaysia Kuantan campus

Stress may affect students physically and psychologically. As much as stress is affecting students’ life in many ways, physical exercise can be used in managing stress. This research sought to identify the association between physical exercise and stress level among first year students of Kulliyyah of Allied Health Sciences (KAHS) intheInternational Islamic University Malaysia (IIUM) Kuantan Campus. A total of 111 subjects consistingof only first year students of Kulliyyah of Allied Health Science (KAHS) participated in this study. Physical exercise was assessed by basic exercise questionnaire, while thestress level was measured using the Perceived Stress Scale(PSS) questionnaire.The data acquired were then analyzed using SPSS version 20.0. The findings indicated that 55.9% participants perform exercise while 44.1% of the participants do not perform regular exercise. As for the association between the physicalexercise and stress level, there is a significant association found (p < 0.001). While for type, frequency and duration of exercise with the stress level also shown a significant association p value less than 0.05. The study has provided evidence of an association between physical exercise and stress level among subjects. This finding is hoped to bring more awareness regarding the use of exercise as a mean to manage stress among university students

Current trends in gene-enhanced tissue engineering for articular cartilage regeneration in animal model

The aim of this review was to summarize and discuss the patterns and trends of the combination of cartilage tissue engineering and gene transfer approaches in in vivo preclinical (or, animal) studies. Electronic literature search of articles published in the Web of Science (WOS) and SCOPUS databases was done based on the following keywords: “tissue engineering”, “articular cartilage”, “animal OR vivo” and “gene”. Two levels of screening were used i.e. based on selected themes and full-text availability of the articles. Inclusion criteria were original full-text papers of preclinical studies using tissue engineering application and gene transfer strategy for articular cartilage regeneration. While, exclusion criteria were conference and review papers, and drug delivery based studies. The search yielded a total of 632 articles (WOS=211; SCOPUS=421). Out of 632, only 612 articles were selected after the removal of 20 overlapping articles from both WOS and SCOPUS databases. After further screening had completed, only 35 articles were included based on their relevancy to this study. Eight (8) articles were then excluded because full-text version of publications was not available. With that, only 27 articles left and included in this present review. The trend in animal studies shows the use of TGF-β (33.33%) and SOX9 (14.81%) transfections enhance chondrogenic differentiation, whilst TGF-β and ChM-1 (3.70%) maintain the chondrocytic phenotype. The co-transfection of TGF-β and BMP (40.74%) promote the integration between tissue engineered implant and the adjacent host cartilage in animal models. Other genes such as IGF-1 (7.41%), CTGF (3.70%) and Bcl-Xl (3.70%) have been reported to enhance cartilage regeneration. In conclusion, the use of genes transfer approach for cartilage tissue engineering in various animal models promotes articular cartilage regeneration which may be useful for future clinical setting

Characterization of 3d (65:35) poly(lactic-co-glycolic acid) incorporated with fibrin and atelocollagen scaffolds using scanning electron microscopy, porosity and swelling tests

Scaffolds play a role as a temporary framework and an extracellular matrix substitute for cultured cells. They provide cells growth substrate and promote mechanical integrity for the newly formed tissues. Previous studies indicated that there were many limitations when natural or synthetic scaffolds material is applied individually. To overcome this, hybrid scaffolds have been introduced for tissue regeneration by studying cellular interactions with relevant scaffolds. However, this present study only focused on fabrication and characterization of three-dimensional (3D) poly(lactic-co-glycolic acid) (PLGA) incorporated with fibrin (PF), atelocollagen (PA) and both fibrin and atelocollagen (PFA) scaffolds materials. The PLGA (mole ratio 65:35) scaffolds were fabricated using solvent casting and salt leaching method. The PA and PFA were crosslinked using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 20mM N-hyroxysuccinimide (NHS). The interaction of incorporated scaffolds materials with PLGA were demonstrated through the notable peaks of amide bonds, as shown by the attenuated total reflectance Fourier transform infrared (ATR-FTIR). Other evaluations included the observation using scanning electron microscopy (SEM), the interconnection of pore structures (porosity), and water uptake capacity (swelling) of the scaffolds. The SEM showed the interconnection between pores in the scaffolds. This is supported by the increased of total porosity in PLGA after the incorporation of fibrin, atelocollagen and both fibrin and atelocollagen. Despite its hydrophobicity, PLGA alone group exhibited the highest percentage of water uptake compared to other hybrid scaffolds namely PF, PA and PFA. Based on the preliminary results, the PLGA based scaffolds may have potential to be used in tissue engineering application

Co-localization of LTBP-2 with FGF-2 in fibrotic human keloid and hypertrophic scar

We have recently shown that Latent transforming growth factor-beta 1 binding protein-2 (LTBP-2) has a single high-affinity binding site for fibroblast growth factor-2 (FGF-2) and that LTBP-2 blocks FGF-2 induced cell proliferation. Both proteins showed strong co-localisation within keloid skin from a single patient. In the current study, using confocal microscopy, we have investigated the distribution of the two proteins in normal and fibrotic skin samples including normal scar tissue, hypertrophic scars and keloids from multiple patients. Consistently, little staining for either protein was detected in normal adult skin and normal scar samples but extensive co-localisation of the two proteins was observed in multiple examples of hypertrophic scars and keloids. LTBP-2 and FGF-2 were co-localised to fine fibrous elements within the extracellular matrix identified as elastic fibres by immunostaining with anti-fibrillin-1 and anti-elastin antibodies. Furthermore, qPCR analysis of RNA samples from multiple patients confirmed dramatically increased expression of LTBP-2 and FGF-2, similar TGF-beta 1, in hypertrophic scar compared to normal skin and scar tissue. Overall the results suggest that elevated LTBP-2 may bind and sequester FGF-2 on elastic fibres in fibrotic tissues and modulate FGF-2’s influence on the repair and healing processes

Evaluation of thresholding techniques on 3d fractal dimension MRI images

Currently, there are few textural measures being used to characterize the textual memorization of brain structures. Fractal Analysis (FA) application in medical field has been used to measure the occurrence of changes in the brain complexity for some diseased and normal aging brain. Within FA, there is Fractal Dimension (FD), which is an index of structural complexity. FD methods were classified into three major categories which are box-counting method, variance method and spectral method. Here in this study we used box-counting method to measure our FD due to its directness and automatic computability capabilities. We evaluated the impact of different thresholding techniques when quantifying the FD. We applied three different thresholding techniques on our brain MRI images: Otsu’s method, midpoint method, and hysteresis method. A total of 27 subjects (14 males and 13 females) aged ranging between 21-25 years old were voluntarily participated. The process of thresholding the images and computation of the FD values were done under MATLAB. There was a statistically significant difference between groups of thresholding techniques as determined by Friedman test with χ2(2)=48.667 and p-value was less than 0.001. Post-hoc Wilcoxon-Signed Ranked test with Bonferroni correction done where the p-value was set to 0.017. All three group pairs (midpoint vs Otsu’s, midpoint vs hysteresis and hysteresis vs Otsu’s) were statistically significant different with p-value less than 0.001. Pearson’s correlation showed moderate correlation (r=0.446, p-value = 0.02) between hysteresis and Otsu’s. Spearman correlation showed weak correlation which is not significant (r=0.256, p=0.198) for midpoint vs Otsu’s and (r=0.252, p=0.204) for midpoint vs hysteresis. In conclusion, different thresholding techniques do have impact on FD values, but with moderate correlation between them. Keywords: Box-counting; Thresholding; Fractal dimension; Brain; Magnetic resonance imaging; Textual memorizatio