Lara Derycke

TGF-β signaling and collagen deposition in chronic rhinosinusitis

BACKGROUND Chronic rhinosinusitis is an inflammatory disease with distinct cytokine and remodeling patterns. OBJECTIVE The objective was to analyze the presence of TGF-beta isoforms, receptors, intracellular signaling, and collagen deposition in chronic rhinosinusitis. METHODS Sinonasal mucosal samples obtained from chronic rhinosinusitis with nasal polyps (CRSwNP; n = 13), chronic rhinosinusitis without nasal polyps (CRSsNP; n = 13), and controls (n = 10) were analyzed for TGF-beta isoforms 1 and 2 by means of ELISA and IHC, and for TGF-beta R1, 2, and 3 by RT-PCR and IHC. As downstream proteins, phospho-Smad 2 (pSmad 2) and collagen were analyzed by performing immunostaining and picrosirius red staining, respectively. RESULTS TGF-beta 1 and 2 protein concentrations, TGF-beta receptor (R) I and TGF-beta RIII mRNA expression, the number of pSmad 2-positive cells, and total collagen amount were significantly higher in CRSsNP versus controls. In CRSwNP, TGF-beta 1 protein concentration, TGF-beta RII and TGF-beta RIII mRNA expression, the number of pSmad 2-positive cells, and total collagen amount were significantly lower versus controls. Only TGF-beta 2 protein was found higher in CRSwNP versus controls. CONCLUSION A high TGF-beta 1 protein expression, increased TGF-beta RI expression, and a high number of pSmad 2-positive cells all indicate an enhanced TGF-beta signaling in CRSsNP, whereas a low TGF-beta 1 protein concentration, a decreased expression of TGF-beta RII, and a low number of pSmad 2-positive cells in CRSwNP indicate a low level of TGF-beta signaling in CRSwNP. These findings are compatible with the remodeling patterns observed, reflected by a lack of collagen in CRSwNP, and excessive collagen production with thickening of the collagen fibers in the extracellular matrix in CRSsNP.

Alternatively activated macrophages and impaired phagocytosis of S-aureus in chronic rhinosinusitis

To cite this article: Krysko O, Holtappels G, Zhang N, Kubica M, Deswarte K, Derycke L, Claeys S, Hammad H, Brusselle GG, Vandenabeele P, Krysko DV, Bachert C. Alternatively activated macrophages and impaired phagocytosis of S. aureus in chronic rhinosinusitis. Allergy 2011; 66: 396–403.

Soluble cadherins as cancer biomarkers

Molecular activities, regulating a balanced tissue organisation, are frequently disturbed during cancer progression. These include protein ectodomain shedding, a post-translational process that substantially changes the functional properties of the substrate protein. In comparison with normal epithelia, cancer cells almost invariably show diminished cadherin-mediated intercellular adhesion. This review will address cadherin ectodomain shedding and its functional consequence in normal physiology and in the tumor environment. Soluble cadherin fragments may retain specific biological activities during cancer cell invasion, angiogenesis and perineural invasion. When diffusion barriers disappear, soluble cadherins are detected in sera from cancer patients. Soluble N-(neural) cadherin may represent a novel diagnosis/prognostic biomarker showing a correlation with PSA in sera of prostate cancer patients. Furthermore, therapeutic monitoring in pancreas adenomacarcinoma revealed a correlation between circulating soluble N-cadherin and CA 19-9. A better understanding of cadherin regulation in cancer progression will likely increase our awareness of the importance of the combinatorial signals that regulate tissue integrity and eventually result in the identification of new therapeutics targeting cadherins.

Antiinvasive effect of xanthohumol, a prenylated chalcone present in hops (Humulus lupulus L.) and beer

The female inflorescences of the hop plant (Humulus lupulus L.) are essential during brewing to add taste and flavor to beer and to stabilize beer foam. Xanthohumol, the main prenylated chalcone in hops, was investigated for its antiinvasive activity on human breast cancer cell lines (MCF‐7 and T47‐D) in vitro. Xanthohumol was able to inhibit the invasion of MCF‐7/6 cells at 5 μM in the chick heart invasion assay and of T47‐D cells in the collagen invasion assay. Xanthohumol inhibited growth of MCF‐7/6 and T47‐D cells, but not of chick heart cells. Moreover, it induced apoptosis of these tumor cells as demonstrated by the cleavage of nuclear PARP after 48 hr treatment. To probe the mechanism of the antiinvasive effect of xanthohumol, involvement of the E‐cadherin/catenin invasion‐suppressor complex was investigated. An aggregation assay demonstrated stimulation of aggregation of MCF‐7/6 cells in the presence of 5 μM xanthohumol and this could be completely inhibited by an antibody against E‐cadherin. Xanthohumol upregulates the function of the E‐cadherin/catenin complex and inhibits invasion in vitro, indicating a possible role as an antiinvasive agent in vivo as well.

P-Cadherin Is Up-Regulated by the Antiestrogen ICI 182,780 and Promotes Invasion of Human Breast Cancer Cells

P-cadherin expression in breast carcinomas has been associated with tumors of high histologic grade and lacking estrogen receptor-α, suggesting a link between these proteins. In the MCF-7/AZ breast cancer cell line, blocking estrogen receptor-α signaling with the antiestrogen ICI 182,780 induced an increase of P-cadherin, which coincided with induction of in vitro invasion. Retroviral transduction of MCF-7/AZ cells, as well as HEK 293T cells, showed the proinvasive activity of P-cadherin, which requires the juxtamembrane domain of its cytoplasmic tail. This study establishes a direct link between P-cadherin expression and the lack of estrogen receptor-α signaling in breast cancer cells and suggests a role for P-cadherin in invasion, through its interaction with proteins bound to the juxtamembrane domain.

Differential impact of TGF-β and EGF on fibroblast differentiation and invasion reciprocally promotes colon cancer cell invasion

Several studies indicate that cancer-associated fibroblasts play a critical role in cancer cell invasion and metastasis, the hallmarks of malignancy. To better understand the mechanisms underlying such effects, we established a heterotypic model of human fibroblasts (primary colon fibroblasts and immortalized human dermal fibroblasts) in co-culture with human colon cancer cells (HCT-8/E11), using three-dimensional collagen type-I and Matrigel matrices. We report that TGF-beta is the unique and dominant factor to provide pro-invasive signals to HCT-8/E11 colon cancer cells from TGF-beta-treated human fibroblasts in three-dimensional collagen type I and Matrigel matrices. These effects are not mimicked or reversed by EGF or bFGF, and are associated with the TGF-beta-mediated induction of myofibroblast differentiation and functional markers, such as alpha-SMA, the haptotactic matrix molecule TNC, collagen type 1 maturation enzyme P4H, serine protease FAP, and myofibroblast contractility. Accordingly, TGF-beta induced a strong activation of RhoA and stress fiber formation in fibroblasts, with no impact on Rac1-GTP levels. In contrast, EGF down-regulated Rho-GTP levels in fibroblasts, giving permissive signals for Rac1 activation, fibroblast polarization, and invasion. Taken together, our data imply that TGF-beta and EGF exert invasive growth-promoting actions in human colon tumors through a differential and cumulative impact on the stromal and cancer cell compartments. Our data predict that inhibitors directed at this reciprocal molecular and cellular crosstalk will have therapeutic applications for targeting the invasive growth of human primary tumors and their metastatic spread.

Mixed T Helper Cell Signatures In Chronic Rhinosinusitis with and without Polyps

In chronic rhinosinusitis (CRS) different phenotypes have been reported based on cytokine profile and inflammatory cell patterns. The aim of this study was to characterize the intracytoplasmatic cytokines ofTcells infiltrating theinflamed sinonasal mucosa. Methods Infiltrated T cells and tissue homogenates from sinonasal mucosal samples of 7 healthy subjects, 9 patients with CRS without nasal polyp (CRSsNP), 15 with CRS with nasal polyps (CRSwNP) and 5cystic fibrosis patients (CF-NP) were analyzed for cytokine expression using flow cytometry and multiplex analysis respectively. Intracytoplasmic cytokinesin T cells were analyzed after stimulation of nasal polyps with Staphylococcus aureus enterotoxin B for 24 hours. Results The number of T cellsper total living cells was significantly higher in patients with CRSwNP vs. CRSsNP and controls. 85% of the CD4+ Tcells showed to be memory T cells. The effector T cells present in all tissues have apredominantTh1 phenotype. Only in CRSwNP, a significantfraction of T cellsproduced the Th2 cytokinesIL-4 and IL-5, while nasal polyps from CF patients were characterized by a higher CD4/CD8 T cell ratio and an increased number of Th17 cells. 24 h stimulation with SEB resulted in a significant induction of CD4+ T cells producing IL-10 (Tr1 cells). Conclusion T cell cytokine patternsin healthy and inflamed sinonasal mucosa revealed that Th2 cells (IL-4 and IL-5 producing cells) are significantly increased in CRSwNP mucosal inflammation. Exposure to SEB stimulates Tr1 cellsthat may contribute to the Th2 bias in CRSwNP.

The role of non-muscle myosin IIA in aggregation and invasion of human MCF-7 breast cancer cells

Human MCF-7/6 breast cancer cells differ from their MCF-7/AZ counterparts by their invasiveness in a number of assays in vitro, such as invasion of MCF-7 spheroids into embryonic chick heart fragments or type I collagen gels. Comparative proteomic analysis of these two variants revealed an identical pattern, except for a 230 kDa protein present in the invasive MCF-7/6 variant, but hardly detectable in the non-invasive MCF-7/AZ one. This protein appeared to be the non-muscle myosin IIA heavy chain (NMIIA), also coined MYH9. Experimental inhibition of NMIIA by reducing either its expression (via stable shRNA transduction) or its function (via the specific ATPase inhibitor blebbistatin) underpinned the decisive role of NMIIA in MCF-7 cell invasion. Inhibition of NMIIA indeed blocked the invasion of MCF-7/6 cells in three-dimensional invasion substrata such as embryonic chick heart fragments and type I collagen gels. Invasiveness of MCF-7/6 cells has been related to poor formation and compaction of aggregates, due to a functionally defective E-cadherin/catenin complex. Both genetic and pharmacological inhibition of NMIIA stimulated MCF-7/6 cell aggregation. Together, these data indicate that NMIIA is a decisive protein for MCF-7 cells to invade, indicating that this molecule is a candidate for targeted anti-invasive treatment.

IL-17A as a regulator of neutrophil survival in nasal polyp disease of patients with and without cystic fibrosis

UNLABELLED Nasal polyps in adults are characterized by a chronic inflammation of the upper airways and by the preferential activation of Th2 cells. In contrast, IL-17 producing Th17 cells dominate the inflammation in nasal polyps of cystic fibrosis (CF) patients. METHOD IL-17A, IL-5, IL-6, IL-8, IL-1β, ECP, MCP-1 and myeloperoxidase expression was determined in tissue homogenates of nasal polyps of non-CF and CF patients and controls. The cellular source of IL-17A was determined by immuno-histochemistry and FACS analysis. The functional role of IL-17A in the survival of neutrophils from CF and non-CF patients was tested. RESULTS A significant upregulation of IL-17A and myeloperoxidase could be observed in nasal polyps from CF-patients. The cellular sources of IL-17A in nasal polyps were mainly T-lymphocytes. IL-17A was able to modulate the survival of neutrophils in nasal polyps from non-CF patients; however the survival of neutrophils in CF patients was independent of IL-17A. CONCLUSION The present study shows that IL-17A has an impact on neutrophil survival in adult nasal polyp disease, but not in nasal polyps from CF patients.

Soluble N-cadherin fragment promotes angiogenesis

Endothelial cells express two dependent intercellular adhesion molecules: vascular endothelial (VE)-cadherin, specific for endothelial cells, and N-cadherin, also present in neuronal, lens, skeletal and heart muscle cells, osteoblasts, pericytes and fibroblasts. While there exists a vast amount of evidence that VE-cadherin promotes angiogenesis, the role of N-cadherin still remains to be elucidated. We found that a soluble 90-kDa fragment N-cadherin promotes angiogenesis in the rabbit cornea assay and in the chorioallantoic assay when cleaved enzymatically from the extracellular domain of N-cadherin. Soluble N-cadherin stimulates migration of endothelial cells in the wound healing assay and stimulates phosphorylation of extracellular regulated kinase. In vitro experiments with PD173074 and knock-down of N-cadherin and fibroblast growth factor (FGF)-receptor, showed that the pro-angiogenic effect of soluble N-cadherin is N-cadherin- and FGF-receptor-dependent. Our results suggest that soluble N-cadherin stimulates migration of endothelial cells through the FGF-receptor.