P056 Crohn’s Disease associated fibrosis modulates the expression of collagen receptors

Background Crohn’s Disease (CD) patients often develop stenotic complications as immunomodulatory treatments do not prevent the fibrogenic response in the affected tissues, where a dysregulated activation of stromal cells provokes an excessive deposition of extracellular matrix (ECM). Recent evidences support the notion that local cells can sense the consequent alterations in tissue structure and rigidity through receptors that respond to some ECM components, and this may perpetuate the fibrogenic process even in the absence of inflammation. We aim to analyse the relevance of these signalling pathways in the fibrotic process associated to CD. Methods We obtained fibrotic ileal tissues from CD patients and healthy ileal samples from colon carcinoma patients (control), and analysed the expression (RT-PCR, IHQ) of collagen receptors (integrins, discoidin domain receptors/DDR) and of markers for some stromal cells (fibroblasts, endothelial cells). The relationship between these sets of data was analysed by Pearson’s correlation and the results organized as a correlation matrix. The expression of collagen receptors was also analysed in endothelial cells (HUVEC) treated with TGFβ 2 (1ng/ml, 48h). Results Ileal samples from CD patients present a differential gene expression of collagen receptors (Fig 1), with increased levels of ITGA10, ITGA11 and DDR2, and reduced expression of DDR1. In CD tissues, the expression of ITGA11 and DDR1 showed a significant correlation, positive and negative respectively, with that of endothelial markers (Fig 2). These correlations do not occur in control tissues. Integrin-α11 was detected in endothelial cells of submucosal vessels (IHQ). In HUVEC, TGFβ 2 increased the expression of ITGA11 (8.1±0.7 fold induction, p<0.01) and reduced that of DDR1 (0.74±0.06 fold induction, p<0.01), without affecting that of the other collagen receptors. Conclusion Intestinal tissues from CD patients affected by fibrosis present an altered pattern of expression of collagen receptors, which suggests a regulatory role of the ECM in the fibrotic response, while the correlation analysis and the changes induced by the fibrotic cytokine TGFβ in endothelial cells insinuates a particular relevance of these stromal cells in this process.

The expression of two collagen receptor subfamilies, integrins and discoidin domains during osteogenic and chondrogenic differentiation of human mesenehymal stem cells

BACKGROUND: Collagen receptors are characterized by binding to and being activated by collagens. We know little about the molecular mechanism by which the integrins and discoidin domains (DDRs) recognize collagen. OBJECTIVE: The aim of this study was to investigate the expression of two main collagen receptor subfamilies, integrins and DDRs, during osteogenic and chondrogenic differentiation of human mesenehymal stem cells (hMSCs). METHODS: Using qRT-PCR, Western blots and FACS, the levels of DDR1, DDR2, integrin subunits β1, α1, α2, α10 and α11 receptors on hMSCs, were assessed upon activation by collagen type I, as well as during osteogenic and chondrogenic differentiation. RESULTS: The expression of DDR2 and integrin α11β1 was altered compared with other receptors when the cells were cultured under undifferentiated conditions. During osteogenic and chondrogenetic differentiation, DDR2 and α11 were up-regulated during early stages (6 day) of osteogenesis and chondrogenesis, respectively. The expression and activation of DDR2 was concomitant with another receptor integrin subunit β1 during osteogenetic differentiation. CONCLUSIONS: The results suggested that DDR2 was more specific for osteogenesis than chondrogenesis, while integrin α11β1 was more specific in chondrogenesis. DDR2 and α11 may play a role in the regulation of osteogenesis and chondrogenesis based on the differential expression of these receptors during lineage-dependent changes.

Receptor-mediated endocytosis is an important way for gelatin nano-particles penetration into cells

Experimental data detailing the possibilities of using gelatin nanoparticles obtained by the two-stage desolvation method without the use of surfactants for delivering pharmacologically active substances to cultured normal and human cancer cells are presented. It was shown that clathrin-dependent endocytosis is the main route of entry of such nanoparticles into normal human fibroblasts and cancer cells of the MDA-MB-231 line. Due to this type of endocytosis, more than 50 % of gelatin nanoparticles enter the cells. It was shown that in the process of clathrin-dependent endocytosis, gelatin nanoparticles specifically bind to collagen receptors.

Targeting DDR1 and DDR2 overcomes matrix-mediated melanoma cell adaptation to BRAF-targeted therapy

Resistance to BRAF and MEK inhibitors in BRAFV600E mutant melanomas remains a major obstacle that limits patient benefit. Microenvironment components including the extracellular matrix (ECM) can support tumor cell adaptation and tolerance to targeted therapies, however the underlying mechanisms remain poorly understood. Here, we investigated the process of matrix-mediated drug resistance (MM-DR) in response to BRAF inhibition in melanoma. We demonstrate that physical and structural cues from fibroblast-derived ECM abrogate anti-proliferative responses to BRAF/MEK inhibition. MM-DR is mediated by the drug-induced clustering of DDR1 and DDR2, two tyrosine kinase collagen receptors. Genetic depletion and pharmacological inhibition of DDR1 and DDR2 overcome ECM-mediated resistance to BRAF inhibition. In melanoma xenografts, targeting DDRs by Imatinib enhances BRAF inhibitor efficacy, counteracts drug-induced collagen remodeling and delays tumor relapse. Mechanistically, DDR-mediated MM-DR fosters a targetable pro-survival NIK/IKKα/NF-κB2 pathway. Our study reveals a novel role of collagen-rich matrix and DDRs in tumor cell adaptation and therapy resistance, thus providing important insights into environment-mediated drug resistance and a pre-clinical rationale for targeting DDR1/2 signaling in combination with BRAF-targeted therapy in melanoma.

Collagen-binding proteins: insights from the Collagen Toolkits

The Collagen Toolkits are libraries of 56 and 57 triple-helical synthetic peptides spanning the length of the collagen II and collagen III helices. These have been used in solid-phase binding assays to locate sites where collagen receptors and extracellular matrix components bind to collagens. Truncation and substitution allowed exact binding sites to be identified, and corresponding minimal peptides to be synthesised for use in structural and functional studies. 170 sites where over 30 proteins bind to collagen II have been mapped, providing firm conclusions about the amino acid distribution within such binding sites. Protein binding to collagen II is not random, but displays a periodicity of approximately 28 nm, with several prominent nodes where multiple proteins bind. Notably, the vicinity of the collagenase-cleavage site in Toolkit peptide II-44 is highly promiscuous, binding over 20 different proteins. This may reflect either the diverse chemistry of that locus or its diverse function, together with the interplay between regulatory binding partners. Peptides derived from Toolkit studies have been used to determine atomic level resolution of interactions between collagen and several of its binding partners and are finding practical application in tissue engineering.

Impact ofItga2-Gp6-double collagen receptor deficient mice for bone marrow megakaryocytes and platelets

ABSTRACTThe two main collagen receptors on platelets, GPVI and integrin α2β1, play an important role for the recognition of exposed collagen at sites of vessel injury, which leads to platelet activation and subsequently stable thrombus formation. Both receptors are already expressed on megakaryocytes, the platelet forming cells within the bone marrow. Megakaryocytes are in permanent contact with collagen filaments in the marrow cavity and at the basal lamina of sinusoids without obvious preactivation. The role of both collagen receptors for megakaryocyte maturation and thrombopoiesis is still poorly understood. To investigate the function of both collagen receptors, we generated mice that are double deficient forGp6andItga2. Flow cytometric analyses revealed that the deficiency of both receptors had no impact on platelet number and the expected lack in GPVI responsiveness. Integrin activation and degranulation ability was comparable to wildtype mice. By immunofluorescence microscopy, we could demonstrate that double-deficient megakaryocytes were overall normally distributed within the bone marrow. We found megakaryocyte count and size to be normal, the localization within the bone marrow, the degree of maturation, as well as their association to sinusoids were also unaltered. However, the contact of megakaryocytes to collagen type I filaments was decreased at sinusoids compared to wildtype mice, while the interaction to type IV collagen was unaffected. Our results imply that GPVI and α2β1 have no influence on the localization of megakaryocytes within the bone marrow, their association to the sinusoids or their maturation. The decreased contact of megakaryocytes to collagen type I might at least partially explain the unaltered platelet phenotype in these mice, since proplatelet formation is mediated by these receptors and their interaction to collagen. It is rather likely that other compensatory signaling pathways and receptors play a role that needs to be elucidated.

The pronounced high expression of discoidin domain receptor 2 in human interstitial lung diseases

The most typical structural feature of human interstitial lung diseases (ILDs) is the accumulation of vast amounts of collagens within the lung interstitium. The membrane receptors that are responsible for recognising collagens and then transducing signals into the cells include four members of the integrin family (α1β1, α2β1, α10β1 and α11β1) and two members of the discoidin domain receptor family (DDR1 and DDR2). However, it remains unknown whether these six collagen receptors similarly contribute to the pathogenesis of fibrotic lung diseases.Quantitative real-time PCR (qPCR) was utilised to assess the mRNA expression of the genes studied. Immunoblot experiments were performed to analyse the protein abundance and kinase activity of the gene products. The tissue location was determined by immunohistochemical staining.qPCR data showed that DDR2 mRNA displays the most dramatic difference between idiopathic pulmonary fibrosis (IPF) patients and healthy groups. The outstanding increases in DDR2 proteins were also observed in some other types of ILD besides IPF. DDR2-expressing cells in ILD tissue sections were found to exhibit spindle or fibroblastic shapes.Our investigation suggests that DDR2 might represent a major cell surface protein that mediates collagen-induced cellular effects in human ILD and, hence, is suitable for their diagnosis and therapy.

ROLE OF HUMAN DISKOIDIN DOMAIN RECEPTOR 1 IN THE PROGRESSION OF CHRONIC PYELONEPHRITIS IN CHILDREN

The purpose: to evaluate the role of collagen receptors Human Discoidin Domain Receptors (DDR1) as mediators of inflammation, proliferation and fibrosis in children with chronic pyelonephritis (CP), to reveal their relationship to the clinical form of the disease and the characteristics of its flow. Materials and methods: The levels of DDR1, transforming growth factor (TGF-β1), insulin-like growth factor (IGF-1) in the serum, β2 - microglobulin ( β2- MG) in the serum and urine were identified during the study of 40 children, ages 6 to 16 with CP in a state of clinical and laboratory remission. Results: Clinical and laboratory remission HP was associated with significant increased levels of DDR1 sera from long ill patients with frequent exacerbations, as well as 2-3 degree of activity last exacerbation, with family history. Found a strong inverse correlation between the levels of DDR1 and IGF-1, and the line with TGF-β1 and β2-MG of blood and urine. In patients with obstructive HP DDR1 level was significantly higher than in patients with non-obstructive clinical form. Conclusions: Increased serum DDR1 shows the progression of kidney damage with active fibrogenesis and inflammation in certain categories of patients with CP in a state of clinical and laboratory remission.