Selection of Phages that Inhibit vWF Interaction with Collagen under both Static and Flow Conditions

2001 ◽  
Vol 86 (08) ◽  
pp. 630-635 ◽  
Author(s):  
H. Ulrichts ◽  
H. Depraetere ◽  
J. Harsfalvi ◽  
H. Deckmyn
Keyword(s):  
Collagen Type ◽  
Collagen Type I ◽  
Phage Display Library ◽  
Type I ◽  
Flow Conditions ◽  
Coated Surface ◽  
Human Collagen ◽  
The One ◽  
Selection Of

SummaryPhages from a pentadecamer phage display library were selected for binding to vWF by affinity panning. Bound phages were selectively eluted with human collagen type I. After the third round of panning 95% of individual phage clones bound to vWF. The B8-phage inhibited the binding of collagen to vWF with an IC50 of 0.6 × 1010 phages/ml, and of vWF to collagen with an IC50 of 1.0 × 1010 phages/ml at 0.5 μg/ml vWF. Under flow conditions, 1.5 × 1011 B8-phage/ml nearly completely inhibited platelet deposition on a human collagen type I coated surface at a shear rate of 1200 s-1, while phages without an insert had no effect. The peptide corresponding to the one displayed on the B8-phage competed with the phage for binding to vWF with an IC50 of 30 μg/ml (16 μM). The peptide furthermore inhibited vWF-binding to collagen with a maximum of 40% at a concentration of 1.25 mg/ml (650 μM), higher concentrations of peptide could not improve this. We thus have selected phages that are potent vWF-binders and that can be used as tools to detect vWF, to inhibit vWF-collagen interaction and to further analyse the role of vWF-collagen binding.

The Collagen-binding Leech Products rLAPP and Calin Prevent both von Willebrand Factor and α2β1 (GPIa/IIa)-I-domain Binding to Collagen in a Different Manner

1999 ◽  
Vol 82 (09) ◽  
pp. 1160-1163 ◽  
Author(s):  
H. Deckmyn ◽  
H. Depraetere ◽  
A. Kerekes
Keyword(s):  
Binding Sites ◽  
Collagen Type ◽  
Collagen Type I ◽  
The Other ◽  
Type I ◽  
Flow Conditions ◽  
Human Collagen ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Natural Inhibitors

SummaryCalin and rLAPP are two natural inhibitors that are able to inhibit the vWF-binding and platelet adhesion to collagen both under static and flow conditions. In this study we demonstrate that both rLAPP and Calin prevent α2I-domain binding to human collagen type I with an IC50 of 5 μg/ml. However, although both vWF and α2I-domain binding to collagen is prevented by rLAPP and Calin, the latter two do not bind to the same collagen site since Calin only partially could compete with rLAPP for binding to collagen. Also vWF and the α2I-domain were unable to compete completely with each other for the binding to collagen. So the following hypothesis can be made: the binding sites of vWF and of the α2I-domain on human collagen type I are different but close to each other since rLAPP could inhibit both interactions, and thus should bind to an overlapping epitope. The Calin preparation on the other hand may still contain two active principles, one interfering with vWF-binding, the other with the α2I-domain-binding to collagen.

scholarly journals Plant Recombinant Human Collagen Type I Hydrogels for Corneal Regeneration

2021 ◽  
Author(s):  
Michel Haagdorens ◽  
Elle Edin ◽  
Per Fagerholm ◽  
Marc Groleau ◽  
Zvi Shtein ◽  
...  
Keyword(s):  
Collagen Type ◽  
Collagen Type I ◽  
Collagen Fibrils ◽  
Type I ◽  
Safe Alternative ◽  
Human Donor ◽  
Human Collagen ◽  
Donor Corneas

Abstract Purpose To determine feasibility of plant-derived recombinant human collagen type I (RHCI) for use in corneal regenerative implants Methods RHCI was crosslinked with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to form hydrogels. Application of shear force to liquid crystalline RHCI aligned the collagen fibrils. Both aligned and random hydrogels were evaluated for mechanical and optical properties, as well as in vitro biocompatibility. Further evaluation was performed in vivo by subcutaneous implantation in rats and corneal implantation in Göttingen minipigs. Results Spontaneous crosslinking of randomly aligned RHCI (rRHCI) formed robust, transparent hydrogels that were sufficient for implantation. Aligning the RHCI (aRHCI) resulted in thicker collagen fibrils forming an opaque hydrogel with insufficient transverse mechanical strength for surgical manipulation. rRHCI showed minimal inflammation when implanted subcutaneously in rats. The corneal implants in minipigs showed that rRHCI hydrogels promoted regeneration of corneal epithelium, stroma, and nerves; some myofibroblasts were seen in the regenerated neo-corneas. Conclusion Plant-derived RHCI was used to fabricate a hydrogel that is transparent, mechanically stable, and biocompatible when grafted as corneal implants in minipigs. Plant-derived collagen is determined to be a safe alternative to allografts, animal collagens, or yeast-derived recombinant human collagen for tissue engineering applications. The main advantage is that unlike donor corneas or yeast-produced collagen, the RHCI supply is potentially unlimited due to the high yields of this production method. Lay Summary A severe shortage of human-donor corneas for transplantation has led scientists to develop synthetic alternatives. Here, recombinant human collagen type I made of tobacco plants through genetic engineering was tested for use in making corneal implants. We made strong, transparent hydrogels that were tested by implanting subcutaneously in rats and in the corneas of minipigs. We showed that the plant collagen was biocompatible and was able to stably regenerate the corneas of minipigs comparable to yeast-produced recombinant collagen that we previously tested in clinical trials. The advantage of the plant collagen is that the supply is potentially limitless.

scholarly journals WNT-5A regulates TGF-β-related activities in liver fibrosis

2017 ◽  
Vol 312 (3) ◽  
pp. G219-G227 ◽  
Author(s):  
Leonie Beljaars ◽  
Sara Daliri ◽  
Christa Dijkhuizen ◽  
Klaas Poelstra ◽  
Reinoud Gosens
Keyword(s):  
Liver Fibrosis ◽  
Functional Role ◽  
Collagen Type ◽  
Collagen Type I ◽  
Matrix Proteins ◽  
Type I ◽  
Human Livers

WNT-5A is a secreted growth factor that belongs to the noncanonical members of the Wingless-related MMTV-integration family. Previous studies pointed to a connection between WNT-5A and the fibrogenic factor TGF-β warranting further studies into the functional role of WNT-5A in liver fibrosis. Therefore, we studied WNT-5A expressions in mouse and human fibrotic livers and examined the relation between WNT-5A and various fibrosis-associated growth factors, cytokines, and extracellular matrix proteins. WNT-5A gene and protein expressions were significantly increased in fibrotic mouse and human livers compared with healthy livers. Regression or therapeutic intervention in mice resulted in decreased hepatic WNT-5A levels paralleled by lower collagen levels. Immunohistochemical analysis showed WNT-5A staining in fibrotic septa colocalizing with desmin staining indicating WNT-5A expression in myofibroblasts. In vitro studies confirmed WNT-5A expression in this cell type and showed that TGF-β significantly enhanced WNT-5A expression in contrast to PDGF-BB and proinflammatory cytokines IL-1β and TNF-α. Additionally, TGF-β induces the expression of the WNT receptors FZD2 and FZD8. After silencing of WNT-5A, reduced levels of collagen type I, vimentin, and fibronectin in TGF-β-stimulated myofibroblasts were measured compared with nonsilencing siRNA-treated controls. Interestingly, the antifibrotic cytokine IFNγ suppressed WNT-5A in vitro and in vivo. IFNγ-treated fibrotic mice showed significantly less WNT-5A expression compared with untreated fibrotic mice. In conclusion, WNT-5A paralleled collagen I levels in fibrotic mouse and human livers. WNT-5A expression in myofibroblasts is induced by the profibrotic factor TGF-β and plays an important role in TGF-β-induced regulation of fibrotic matrix proteins, whereas its expression can be reversed upon treatment, both in vitro and in vivo. NEW & NOTEWORTHY This study describes the localization and functional role of WNT-5A in human and mouse fibrotic livers. Hepatic WNT-5A expression parallels collagen type I expression. In vivo and in vitro, the myofibroblasts were identified as the key hepatic cells producing WNT-5A. WNT-5A is under control of TGF-β and its activities are primarily profibrotic.

Role of catechin on collagen type I stability upon oxidation: a NMR approach

2019 ◽  
Vol 34 (1) ◽  
pp. 53-62 ◽  
Author(s):  
Massimo Lucarini ◽  
Fabio Sciubba ◽  
Donatella Capitani ◽  
Maria Enrica Di Cocco ◽  
Laura D’Evoli ◽  
...  
Keyword(s):  
Collagen Type ◽  
Collagen Type I ◽  
Type I

Flow Analysis of Von Willebrand Factor Collagen Binding Mutants

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2213-2213
Author(s):  
Thomas A J McKinnon ◽  
Agata Anna Nowak ◽  
Alina Hua ◽  
Carolyn Millar ◽  
Michael Laffan
Keyword(s):  
Shear Stress ◽  
Collagen Type ◽  
Collagen Type I ◽  
Type I ◽  
Flow Conditions ◽  
Collagen Binding ◽  
Binding Function ◽  
Static Conditions ◽  
A1 Domain ◽  
Von Willebrand

Abstract Abstract 2213 Von Willebrand Factor (VWF) binds to exposed sub-endothelial collagen at sites of vessel injury principally via its A3 domain, although some evidence suggests that the A1 domain can compensate for the A3 domain under flow conditions if the A3 domain is absent or non-functional. Recently, several naturally occurring Von Willebrand disease-causing mutations have been indentified in the A3 domain; S1731T, W1745C, S1783, H1786D and most recently M1761K, as well as one mutation in the A1 domain (I1343V) all of which have defective collagen binding. While the collagen binding function of these mutations has been assessed under static conditions it remains to be established if these affect collagen binding under shear stress. In the present study the collagen binding mutants were expressed in HEK293T cells and collagen binding function determined using an in vitro flow assay. All of the mutations were expressed at similar levels to wild type (wt) VWF and demonstrated normal multimeric patterns and binding to GPIbα under static conditions. As expected, collagen binding analysis under static conditions confirmed the collagen binding defect of all the mutants, with reduced or abolished binding to both collagens type I and III for all the mutants except S1731T which demonstrated normal binding to collagen type III and slightly reduced binding to collagen type I. Analysis of platelet capture under flow conditions confirmed that all the mutants were able to capture platelets similarly to wtVWF. Analysis of VWF mediated platelet capture to a collagen surface under flow conditions confirmed the phenotype of the collagen binding mutants. With the exception of S1731T, which demonstrated normal platelet capture on both collagens, none of the mutants were able to bind to collagen type I or III under flow conditions, or mediate platelet capture at high shear stress. The collagen binding function of these mutants under flow was partially restored when co-expressed with wtVWF. Interestingly, in contrast to a previous study, a VWF variant lacking the A3 domain (VWF-ΔA3) failed to bind to collagen under shear stress and was not able to mediate platelet capture to collagen. Together these data confirm that the major collagen binding site in VWF is located in the A3 domain and demonstrate that collagen binding mutations affect VWF mediated platelet capture under shear stress. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Hydroxylation of recombinant human collagen type I alpha 1 in transgenic maize co-expressed with a recombinant human prolyl 4-hydroxylase

2011 ◽  
Vol 11 (1) ◽  
pp. 69 ◽  
Author(s):  
Xing Xu ◽  
Qinglei Gan ◽  
Richard C Clough ◽  
Kameshwari M Pappu ◽  
John A Howard ◽  
...  
Keyword(s):  
Collagen Type ◽  
Collagen Type I ◽  
Transgenic Maize ◽  
Type I ◽  
Human Collagen

Blockade of hypoxia-reoxygenation-mediated collagen type I expression and MMP activity by overexpression of TGF-β1delivered by AAV in mouse cardiomyocytes

2007 ◽  
Vol 293 (3) ◽  
pp. H1833-H1838 ◽  
Author(s):  
Chang-Ping Hu ◽  
Abhijit Dandapat ◽  
Yong Liu ◽  
Paul L. Hermonat ◽  
Jawahar L. Mehta
Keyword(s):  
Cardiac Remodeling ◽  
Collagen Type ◽  
Transforming Growth Factor ◽  
Collagen Type I ◽  
Type I ◽  
Antioxidant Mechanism ◽  
Multifunctional Peptides ◽  
Expression And Activity ◽  
Hypoxia Reoxygenation

Transforming growth factor (TGF)-β1is one of the most pleiotropic and multifunctional peptides known. While the cardioprotective effect of TGF-β1during ischemia is well known, the specific role of TGF-β1in altering the cardiac remodeling process remains unclear. This study was designed to examine the regulation of hypoxia-reoxygenation-mediated collagen type I expression and activity of matrix metalloproteinases (MMPs) by overexpression of TGF-β1in cultured HL-1 mouse cardiomyocytes. TGF-β1was overexpressed in cardiomyocytes by transfection with adeno-associated virus (AAV)/TGF-β1Latentor with AAV/TGF-β1ACT(active TGF-β1). Twenty-four hours of hypoxia followed by 3 h of reoxygenation (H-R) markedly enhanced (pro)collagen type I expression and activity of MMPs concomitant with an increase in reactive oxygen species (ROS) release and LOX-1 expression. Overexpression of TGF-β1reduced these alterations induced by H-R. TGF-β1overexpression also blocked H-R-mediated p38 and p44/42 MAPK activation. Transfection with AAV/TGF-β1ACTwas superior to that with AAV/TGF-β1Latent. These data for the first time demonstrate that H-R induces signals for cardiac remodeling in cardiomyocytes and TGF-β1can modulate, possibly via antioxidant mechanism, these signals. These findings contribute to further understanding of the role of TGF-β1in the cardiac remodeling process.

The role of collagen type I α2 polymorphisms: intracranial aneurysms in Koreans

2009 ◽  
Vol 72 (1) ◽  
pp. 48-53 ◽  
Author(s):  
Sung-Pil Joo ◽  
Tae-Sun Kim ◽  
Il-Kwon Lee ◽  
Jung-Kil Lee ◽  
Bo-Ra Seo ◽  
...  
Keyword(s):  
Intracranial Aneurysms ◽  
Collagen Type ◽  
Collagen Type I ◽  
Type I

scholarly journals Discoidin Domain Receptor 2 regulates AT1 receptor expression in Angiotensin II-stimulated cardiac fibroblasts via fibronectin-dependent Integrin-β1 signalling

2021 ◽  
Author(s):  
Allen Sam Titus ◽  
Harikrishnan V ◽  
Mingyi Wang ◽  
Edward G Lakkatta ◽  
Shivakumar Kailasam
Keyword(s):  
Angiotensin Ii ◽  
Collagen Type ◽  
Cardiac Fibroblasts ◽  
Collagen Type I ◽  
Cardiac Fibroblast ◽  
Regulatory Role ◽  
Type I ◽  
Integrin Β1 ◽  
Fibronectin Expression

Fibronectin is an extracellular matrix glycoprotein with a regulatory role in fundamental cellular processes. Recent reports on the cardioprotective effect of fibronectin inhibition in a setting of myocardial injury suggest a role for fibronectin in cardiac fibroblast function, which remains largely unexplored. This study probed the molecular basis and functional implications of fibronectin gene expression in cardiac fibroblasts exposed to Angiotensin II, a potent pro-fibrotic factor in the myocardium. Using gene knockdown and over-expression approaches, western blotting and promoter pull-down assay, we show that collagen type I-activated Discoidin Domain Receptor 2 (DDR2) mediates Angiotensin II-stimulated transcriptional up-regulation of fibronectin expression by Yes-activated Protein in cardiac fibroblasts. Further, siRNA-mediated fibronectin knockdown attenuated Angiotensin II-dependent expression of anti-apoptotic cIAP2 and promoted cell death under oxidative stress. Fibronectin was also found to mediate Angiotensin II-stimulated collagen type I expression. Importantly, an obligate role for fibronectin was observed in Angiotensin II-stimulated expression of its receptor, AT1R, which would link ECM signalling and Angiotensin II signalling in cardiac fibroblasts. Moreover, the regulatory role of DDR2-dependent fibronectin expression in Ang II-stimulated cIAP2, collagen type I and AT1R expression was mediated by Integrin-β1-integrin-linked kinase signalling. The pro-survival role of fibronectin in cardiac fibroblasts and its regulatory role in collagen and AT1R expression, downstream of DDR2, could be critical determinants of cardiac fibroblast-mediated wound healing following myocardial injury. Our findings point to a complex mechanism of regulation of cardiac fibroblast function involving two major extracellular matrix proteins, collagen type I and fibronectin, and their receptors, DDR2 and Integrin-β1.

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