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

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

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 signalling

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

LTBP-2 Has a Single High-Affinity Binding Site for FGF-2 and Blocks FGF-2-Induced Cell Proliferation

<div><p>Latent transforming growth factor-beta-1 binding protein-2 (LTBP-2) belongs to the fibrillin-LTBP superfamily of extracellular matrix proteins. LTBPs and fibrillins are involved in the sequestration and storage of latent growth factors, particularly transforming growth factor β (TGF-β), in tissues. Unlike other LTBPs, LTBP-2 does not covalently bind TGF-β, and its molecular functions remain unclear. We are screening LTBP-2 for binding to other growth factors and have found very strong saturable binding to fibroblast growth factor-2 (FGF-2) (Kd = 1.1 nM). Using a series of recombinant LTBP-2 fragments a single binding site for FGF-2 was identified in a central region of LTBP-2 consisting of six tandem epidermal growth factor-like (EGF-like) motifs (EGFs 9–14). This region was also shown to contain a heparin/heparan sulphate-binding site. FGF-2 stimulation of fibroblast proliferation was completely negated by the addition of 5-fold molar excess of LTBP-2 to the assay. Confocal microscopy showed strong co-localisation of LTBP-2 and FGF-2 in fibrotic keloid tissue suggesting that the two proteins may interact in vivo. Overall the study indicates that LTBP-2 is a potent inhibitor of FGF-2 that may influence FGF-2 bioactivity during wound repair particularly in fibrotic tissues.</p></div

FGF-2 has a single binding domain in the central region of LTBP-2.

<p><b>A</b>. Three recombinant fragments spanning the LTBP-2 molecule were tested for binding to FGF-2 in a solid phase assay. Full length LTBP-2(H), fragments LTBP-2 NT (H), LTBP-2C (H), LTBP-2 CT (H) or BSA control were coated onto wells at 100 ng/ml, followed by incubation with FGF-2 (100ng/ml) for 3h at 37°C. Strong specific binding to central fragment LTBP-2C(H) was detected as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135577#pone.0135577.g002" target="_blank">Fig 2A</a>. Mean values ± S.D. from triplicate wells are shown. <b>B</b>. A binding curve was produced for the FGF-2 interaction with fragment LTBP-2C(H) following the protocol described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135577#pone.0135577.g002" target="_blank">Fig 2</a>, with 400 ng/well (4.8 pmol) of LTBP-2C (H) or BSA control coated on the wells incubated with increasing concentrations FGF-2 (0–1.5 nM). The Kd for binding of FGF-2 to fragment LTBP-2C (H) was calculated as 1.02 ± 0.19 nM. Mean values ± S.D. from triplicate determinations are shown. <b>C</b>. Three sub-fragments F1, F2 and F3 spanning fragment LTBP-2 C(H) were produced and tested for FGF-2 binding as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135577#pone.0135577.g002" target="_blank">Fig 2</a>. LTBP-2C (H) (200 ng/well, 2.4 pmol) or sub-fragment (F1, F2 or F3) (66ng/well, 2.4 pmol) or BSA control was coated on the wells and incubated with FGF-2 (100 ng/ml). Strong specific binding of FGF-2 to sub-fragment LTBP-2C F2 was detected. Mean values ± S.D. from triplicate wells are shown. <b>D</b>. Subsequently binding curves were obtained for sub-fragments F1 (solid squares), F2 (open circles), F3 (solid circles) (35 ng/well, 1.2 pmol) coated on the wells and incubated with increasing concentrations of FGF-2 (0–30 ng / ml). Note specific FGF-2 binding to sub-fragment LTBP-2C F2 but no binding of fragments F1 and F3 above the BSA control (triangles). Mean values ± S.D. from triplicate determinations are shown. <b>E</b>. The Kd for the FGF-2 interaction with sub-fragment LTBP-2C F2 was calculated as 1.03 ± 0.10 nM which is similar to the Kds calculated for the interactions of FGF-2 with full-length LTBP-2 and fragment LTBP2C. Mean values ± S.D. from triplicate determinations are shown.</p

Recombinant LTBP-2 Fragments.

<p><b>A</b>. Schematic diagram of recombinant LTBP-2 fragments. Protein fragments generated specifically for this study (LTBP-2C(H) F1, F2 and F3) are highlighted within the blue box. FGF-2 binding was confined to a single central region of the LTBP-2 molecule consisting of 6 EGF-like repeats (fragment LTBP-2C(H) F2).<b>B</b>. SDS-PAGE of purified recombinant LTBP-2 fragments. Samples of purified fragments LTBP-2 C(H), LTBP-2 C(H) F1, LTBP-2 C(H) F2 and LTBP-2 C(H) F3 were analyzed on a 12% gel under non-reducing conditions and stained with Coomassie blue. The relative mobilities of protein standards are indicated by arrows.</p

LTBP-2 specifically binds FGF-2 but not VEGF, BMP-4, BMP-7 or TGF-beta.

<p><b>A</b>. Microtitre wells were coated with rLTBP-2 (black columns) or BSA(shaded columns) (100 ng/ well). After blocking, triplicate wells were incubated at 37°C for 2h with TGF-beta (13 ng / well), VEGF (21 ng / well), BMP-7 (4 ng/well), BMP-4 (4 ng / well) or FGF-2 (10 ng / well). Growth factor binding was detected using specific biotinylated antibodies from Duoset kits as described in material and methods. Mean values ± S.D. from triplicate wells are shown. <b>B</b>. Microtitre wells were coated with rLTBP-2 (100ng/well) was coated onto microtitre plates. After blocking, triplicate wells were incubated at 37°C for 2h with (black columns) or without (cross-hatched) growth factor, (BMP-4 (4ng/well) or FGF-2 (10ng/well). Binding of growth factor to LTBP-2 was detected using biotinylated anti-BMP-4 detection antibody (0.5ug/ml) or anti-FGF-2 detection antibody (0.25ug/ml), followed by a peroxidase detection method (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135577#sec002" target="_blank">material and methods</a>). Mean values ± S.D. from triplicate wells are shown. Note the anti-BMP-4 antibody bound to the wells equally strongly in the presence or absence of added BMP-4, indicating the interaction was non-specific.</p

FGF-2 has a single binding domain in the central region of LTBP-2.

<p><b>A</b>. Three recombinant fragments spanning the LTBP-2 molecule were tested for binding to FGF-2 in a solid phase assay. Full length LTBP-2(H), fragments LTBP-2 NT (H), LTBP-2C (H), LTBP-2 CT (H) or BSA control were coated onto wells at 100 ng/ml, followed by incubation with FGF-2 (100ng/ml) for 3h at 37°C. Strong specific binding to central fragment LTBP-2C(H) was detected as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135577#pone.0135577.g002" target="_blank">Fig 2A</a>. Mean values ± S.D. from triplicate wells are shown. <b>B</b>. A binding curve was produced for the FGF-2 interaction with fragment LTBP-2C(H) following the protocol described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135577#pone.0135577.g002" target="_blank">Fig 2</a>, with 400 ng/well (4.8 pmol) of LTBP-2C (H) or BSA control coated on the wells incubated with increasing concentrations FGF-2 (0–1.5 nM). The Kd for binding of FGF-2 to fragment LTBP-2C (H) was calculated as 1.02 ± 0.19 nM. Mean values ± S.D. from triplicate determinations are shown. <b>C</b>. Three sub-fragments F1, F2 and F3 spanning fragment LTBP-2 C(H) were produced and tested for FGF-2 binding as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135577#pone.0135577.g002" target="_blank">Fig 2</a>. LTBP-2C (H) (200 ng/well, 2.4 pmol) or sub-fragment (F1, F2 or F3) (66ng/well, 2.4 pmol) or BSA control was coated on the wells and incubated with FGF-2 (100 ng/ml). Strong specific binding of FGF-2 to sub-fragment LTBP-2C F2 was detected. Mean values ± S.D. from triplicate wells are shown. <b>D</b>. Subsequently binding curves were obtained for sub-fragments F1 (solid squares), F2 (open circles), F3 (solid circles) (35 ng/well, 1.2 pmol) coated on the wells and incubated with increasing concentrations of FGF-2 (0–30 ng / ml). Note specific FGF-2 binding to sub-fragment LTBP-2C F2 but no binding of fragments F1 and F3 above the BSA control (triangles). Mean values ± S.D. from triplicate determinations are shown. <b>E</b>. The Kd for the FGF-2 interaction with sub-fragment LTBP-2C F2 was calculated as 1.03 ± 0.10 nM which is similar to the Kds calculated for the interactions of FGF-2 with full-length LTBP-2 and fragment LTBP2C. Mean values ± S.D. from triplicate determinations are shown.</p

LTBP-2 and FGF-2 co-localize in keloid tissue.

<p>Keloid tissue was also analyzed for LTBP-2 and FGF-2 by confocal microscopy. <b>A and F</b>, polyclonal anti-[human LTBP-2 peptide] antibody 3504 (2 μg/ ml) detected with anti-rabbit IgG antibody conjugated to fluor Alexa 488; <b>B and G</b>, monoclonal anti-[human FGF-2] antibody #61087 (BD Biosciences) (2.5 μg/ml) detected with anti-mouse IgG antibody conjugated to Alexa 594; <b>C</b>, A and B merged; <b>D</b>, rabbit IgG control (2 μg/ ml); <b>E</b>, mouse IgG control (2.5 μg / ml); <b>H</b>, F, and G merged; <b>I</b>, Control confocal image showing distinct immunostaining patterns for VEGF (red) and LTBP-2 (green). Magnification: top row, Bar = 100 μm; bottom row, Bar = 50 μm.</p