Nicky D'Haene

VEGFR1 and VEGFR2 Involvement in Extracellular Galectin-1- and Galectin-3-Induced Angiogenesis

Aim Accumulating evidence suggests that extracellular galectin-1 and galectin-3 promote angiogenesis. Increased expression of galectin-1 and/or galectin-3 has been reported to be associated with tumour progression. Thus, it is critical to identify their influence on angiogenesis. Methods We examined the individual and combined effects of galectin-1 and galectin-3 on endothelial cell (EC) growth and tube formation using two EC lines, EA.hy926 and HUVEC. The activation of vascular endothelial growth factor receptors (VEGFR1 and VEGFR2) was determined by ELISA and Western blots. We evaluated the VEGFR1 and VEGFR2 levels in endosomes by proximity ligation assay. Results We observed different responses to exogenous galectins depending on the EC line. An enhanced effect on EA.hy926 cell growth and tube formation was observed when both galectins were added together. Focusing on this enhanced effect, we observed that together galectins induced the phosphorylation of both VEGFR1 and VEGFR2, whereas galectin-1 and −3 alone induced VEGFR2 phosphorylation only. In the same way, the addition of a blocking VEGFR1 antibody completely abolished the increase in tube formation induced by the combined addition of both galectins. In contrast, the addition of a blocking VEGFR2 antibody only partially inhibited this effect. Finally, the addition of both galectins induced a decrease in the VEGFR1 and VEGFR2 endocytic pools, with a significantly enhanced effect on the VEGFR1 endocytic pool. These results suggest that the combined action of galectin-1 and galectin-3 has an enhanced effect on angiogenesis via VEGFR1 activation, which could be related to a decrease in receptor endocytosis.

Gene expression profiling of the hedgehog signaling pathway in human meningiomas

The Hedgehog (Hh) signaling pathway has an important role during embryogenesis and in adult life, regulating proliferation, angiogenesis, matrix remodeling and stem-cell renewal. Deregulation of the Hh pathway is involved in tumor development, since mutations in several components of this pathway were found in patients with basal cell carcinoma, medulloblastoma and other tumors; however, the role of Hh in meningiomas has not been studied yet. Meningiomas represent 30% of primary cranial tumors, are mostly benign and prevail in the second half of life. Novel therapies for meningiomas such as targeted molecular agents could use Hh pathway components. To provide information concerning molecular alterations, by use of real-time RT-PCR, we studied expression at the mRNA level of 32 Hh pathway and target genes in 36 meningioma specimens of different grades. mRNA levels of 16 genes, involved mainly in Hh pathway activation and cell proliferation, increased in meningiomas in comparison with normal tissue, whereas those of 7 genes, mainly related to Hh pathway repression, decreased. The most significant changes occurred in signal transduction (SMO) and GLI-transcription factor genes, and the target FOXM1 mRNA attained the highest values; their overexpression was found in aggressive and in benign tumors. Some proliferation-related genes (SPP1, IGF2) were overexpressed in higher meningioma grades. A correlation in expression between genes with a similar function was also found. Our results show a marked activation of the Hh pathway in meningiomas, which may be important for their biological and clinical characterization and would be useful for gene therapy.

Formyl Peptide Receptor-Like 2 Is Expressed and Functional in Plasmacytoid Dendritic Cells, Tissue-Specific Macrophage Subpopulations, and Eosinophils

The formyl peptide receptor (FPR) is a key player in innate immunity and host defense mechanisms. In humans and other primates, a cluster of genes encodes two related receptors, FPR-like 1 and FPR-like 2 (FPRL1 and FPRL2). Despite their high sequence similarity, the three receptors respond to different sets of ligands and display a different expression pattern in leukocyte populations. Unlike FPR and FPRL1, FPRL2 is absent from neutrophils, and two endogenous peptide agonists, F2L and humanin, were recently described. In the present work, we investigated the detailed functional distribution of FPRL2 in leukocytes by quantitative PCR, flow cytometry, immunohistochemistry, and chemotaxis assays, with the aim of raising hypotheses regarding its potential functions in the human body. We describe that FPRL2 is highly expressed and functional in plasmacytoid dendritic cells and up-regulated upon their maturation. FPRL2 is also expressed in eosinophils, which are recruited but do not degranulate in response to F2L. FPRL2 is expressed and functional in macrophages differentiated from monocytes in vitro in different conditions. However, in vivo, only specific subsets of macrophages express the receptor, particularly in the lung, colon, and skin, three organs chronically exposed to pathogens and exogenous aggressions. This distribution and the demonstration of the production of the F2L peptide in mice underline the potential role of FPRL2 in innate immunity and possibly in immune regulation and allergic diseases.

Next-generation sequencing improves the diagnosis of thyroid FNA specimens with indeterminate cytology

The assessment of thyroid nodules is a common clinical challenge. Fine‐needle aspiration (FNA) is the standard pre‐operative tool for thyroid nodule diagnosis. However, up to 30% of the samples are classified as indeterminate. This often leads to unnecessary surgery. In this study, we evaluated the added value of next‐generation sequencing (NGS) for helping in the diagnosis of FNA samples.

Requirements for the valid quantification of immunostains on tissue microarray materials using image analysis

Antibody‐based proteomics applied to tissue microarray (TMA) technology provides a very efficient means of visualizing and locating antigen expression in large collections of normal and pathological tissue samples. To characterize antigen expression on TMAs, the use of image analysis methods avoids the effects of human subjectivity evidenced in manual microscopical analysis. Thus, these methods have the potential to significantly enhance both precision and reproducibility. Although some commercial systems include tools for the quantitative evaluation of immunohistochemistry‐stained images, there exists no clear agreement on best practices to allow for correct and reproducible quantification results. Our study focuses on practical aspects regarding (i) image acquisition (ii) segmentation of staining and counterstaining areas and (iii) extraction of quantitative features. We illustrate our findings using a commercial system to quantify different immunohistochemistry markers targeting proteins with different expression patterns (cytoplasmic, nuclear or membranous) in colon cancer or brain tumor TMAs. Our investigations led us to identify several steps that we consider essential for standardizing computer‐assisted immunostaining quantification experiments. In addition, we propose a data normalization process based on reference materials to be able to compare measurements between studies involving different TMAs. In conclusion, we recommend certain critical prerequisites that commercial or in‐house systems should satisfy in order to permit valid immunostaining quantification.

Imaging and classification of congenital cystic renal diseases

OBJECTIVE The purpose of this clinical perspective is to describe a decision-tree approach to the finding of hyperechoic kidneys as signs of congenital renal cystic disease in fetuses and children. This approach takes into account the latest classification of inherited renal cystic diseases. The basis of the approach is a detailed sonographic analysis in addition to assessment of clinical data and the familial history. CONCLUSION With the decision-tree approach, typical sonographic patterns can be described and used for accurate diagnosis of isolated renal cystic diseases and polymalformative syndromes. In some cases, however, the diagnosis is not achieved, and complementary examinations are needed.

Ultrasound-guided fine-needle aspiration of thyroid nodules: stratification of malignancy risk using follicular proliferation grading, clinical and ultrasonographic features.

OBJECTIVE To evaluate the diagnostic value of fine-needle aspiration (FNA) cytology and the additive contribution brought by clinical and ultrasound (US) features. METHOD Cytological and histological diagnoses were compared in a series of 924 patients who underwent US-guided FNA before surgery. We additionally developed a grading system for follicular proliferation (FP) FNA diagnosis, and investigated its impact on the malignancy risk as well as the additive contribution of clinical and US features by means of decision tree analysis. RESULTS Excluding FP cases (n=395), our data demonstrated that strictly benign or malignant FNA diagnoses exhibit great concordance with benign or malignant histological diagnoses (97.8% accuracy). Our grading system that was applied to the 395 FP cases revealed that grades 1, 2 and 3 were associated with a 7.7, 17.7 and 45.7% incidence of malignancy respectively. Decision tree analysis resulted in a classification model which involved FP grade, patients age, serum thyroglobulin level, nodule size and nodule uniqueness. This model identified a subgroup of patients with grade 1 FP nodules who were older than 50 years, and who had a higher risk of malignancy (17.9%). In addition, high serum thyroglobulin levels were associated with a very high malignancy risk (75.0%) for patients with grade 3 FP nodules. Finally, among grade 2 FP patients, unique and large nodules were associated with a high malignancy risk of 36.1%. CONCLUSIONS The integration of FP grade, clinical and US features allows the stratification of patients with FP cytology according to their risk of malignancy.

Matrix metalloproteinase-9 interplays with the IGFBP2-IGFII complex to promote cell growth and motility in astrocytomas.

Insulin‐like growth factor II (IGFII) acts as a potent mitogen for several tumor types and has been reported to positively influence astrocytoma cell growth and motility. In the central nervous system, IGFII bioavailability is mainly modulated by insulin‐like growth factor binding protein 2 (IGFBP2), which sequestrates IGFII and therefore prevents its interaction with the type‐1 IGF receptor (IGF‐IR). Proteolysis of IGFBP2 is the predominant mechanism recognized to reduce the binding affinity of IGFBP2 for IGFII, thus favoring dissociation of IGFII from the IGFBP2–IGFII complex. It is known that certain proteases involved in astrocytoma malignancy, such as matrix metalloproteinase‐7 (MMP‐7), plasmin, and cathepsin D, are able to proteolyze IGFBP2 in vitro. The present study aims to investigate whether other proteases expressed by astrocytomas, specifically MMP‐2, MMP‐9, and membrane‐type 1 matrix metalloprotease (MT1‐MMP), are able to proteolyze the IGFBP2–IGFII complex. Our results show the following: (i) MMP‐9 proteolyzes the IGFBP2–IGFII complex in vitro, while MMP‐2 and MT1‐MMP do not; (ii) this MMP‐9‐induced IGFBP2–IGFII complex proteolysis releases free IGFII, which contributes to enhance the motility and the growth of LN229 astrocytoma cells. Furthermore, this study also highlights that the formation of the IGFBP2–IGFII complex inhibits IGFBP2s cell motility promoting effect by reducing the pool of free IGFBP2. In conclusion, MMP‐9‐induced IGFBP2 proteolysis may be regarded as an important post‐translational event involved in astrocytoma aggressiveness. These new findings support drug targeting of MMP‐9 as an interesting approach in the treatment of astrocytoma.

Gene Expression Profiling of ErbB Receptors and Ligands in Human Meningiomas

ErbB family receptors mediate major cellular functions implied in tumorigenesis, though their role in meningiomas was not thoroughly studied. Meningiomas represent 30% of primary cranial tumors, are mostly benign, and prevail in the second half of life. Tumor therapy requires information about molecular alterations, thus we studied expression of ErbB receptor and ligand genes by real-time RT-PCR in different meningioma grades. Receptors were overexpressed (ErbB1, ErbB2) or underexpressed (ErbB3, ErbB4). Ligands EGF, TGFA, AREG, DTR, BTD were underexpressed and the neuregulins were overexpressed or underexpressed. A strong ErbB1–ErbB2 correlation was found. These data might be useful for gene therapy.

Galectins and neovascularization in central nervous system tumors

Despite advances in diagnosis and treatment, the overall outcomes for patients with brain tumors remain unpredictable. New prognostic markers are still needed to identify high-risk patients for whom the standard treatment has poor outcomes and would thus be well suited for more aggressive therapies. Neovascularization has long been implicated as a salient feature of glioma progression. In fact, high-grade gliomas are among the most vascular of all solid tumors, and vascular proliferation is a pathological hallmark of glioblastomas. Galectins are known to play important roles in cancer biology, including cancer cell migration, tumor immune escape or tumor angiogenesis. Moreover, galectins were reported to be involved in glioma progression. Given the key role of angiogenesis in brain tumors, the expression of galectins in tumor-associated endothelial cells (EC) and the implication of galectins in angiogenesis, the present review will focus on the expression of galectins in ECs of normal brain and brain tumors.