Prolyl 3‐hydroxylase 2 is a molecular player of angiogenesis

Prolyl 3‐hydroxylase 2 (P3H2) catalyzes the post‐translational formation of 3‐ hydroxyproline on collagens, mainly on type IV. Its activity has never been directly associated to angiogenesis. Here, we identified P3H2 gene through a deep‐sequencing transcriptome analysis of human umbilical vein endothelial cells (HUVECs) stimulated with vascular endothelial growth factor A (VEGF‐A). Differently from many previous studies we carried out the stimulation not on starved HUVECs, but on cells grown to maintain the best condition for their in vitro survival and propagation. We showed that P3H2 is induced by VEGF‐A in two primary human endothelial cell lines and that its transcription is modulated by VEGF‐A/VEGF receptor 2 (VEGFR‐2) signaling pathway through p38 mitogen‐activated protein kinase (MAPK). Then, we demonstrated that P3H2, through its activity on type IV Collagen, is essential for angiogenesis properties of endothelial cells in vitro by performing experiments of gain‐ and loss‐of‐function. Immunofluorescence studies showed that the overexpression of P3H2 induced a more condensed status of Collagen IV, accompanied by an alignment of the cells along the Collagen IV bundles, so towards an evident pro‐angiogenic status. Finally, we found that P3h2 knockdown prevents pathological angiogenesis in vivo, in the model of laser‐induced choroid neovascularization. Together these findings reveal that P3H2 is a new molecular player involved in new vessels formation and could be considered as a potential target for anti‐angiogenesis therapy

Placental Growth Factor-1 and -2 Induce Hyperplasia and Invasiveness of Primary Rheumatoid Synoviocytes

Inflammation-mediated oncogenesis has been implicated in a variety of cancer types. Rheumatoid synovial tissues can be viewed as a tumor-like mass, consisting of hyperplastic fibroblast-like synoviocytes (FLSs). FLSs of rheumatoid arthritis (RA) patients have promigratory and invasive characteristics, which may be caused by chronic exposure to genotoxic stimuli, including hypoxia and growth factors. We tested whether a transformed phenotype of RA-FLSs is associated with placental growth factor (PlGF), a representative angiogenic growth factor induced by hypoxia. In this study, we identified PlGF-1 and PlGF-2 as the major PlGF isoforms in RA-FLSs. Global gene expression profiling revealed that cell proliferation, apoptosis, angiogenesis, and cell migration were mainly represented by differentially expressed genes in RA-FLSs transfected with small interfering RNA for PlGF. Indeed, PlGF-deficient RA-FLSs showed a decrease in cell proliferation, migration, and invasion, but an increase in apoptotic death in vitro. PlGF gene overexpression resulted in the opposite effects. Moreover, exogeneous PlGF-1 and PlGF-2 increased survival, migration, and invasiveness of RA-FLSs by binding their receptors, Flt-1 and neuropilin-1, and upregulating the expression of antiapoptotic molecules, pErk and Bcl2. Knockdown of PlGF transcripts reduced RA-FLS proliferation in a xenotransplantation model. Collectively, in addition to their role for neovascularization, PlGF-1 and -2 promote proliferation, survival, migration, and invasion of RA-FLSs in an autocrine and paracrine manner. These results demonstrated how primary cells of mesenchymal origin acquired an aggressive and transformed phenotype. PlGF and its receptors thus offer new targets for anti-FLS therapy.1177Ysciescopu

Thyroid hormone enhances angiogenesis and the warburg effect in squamous cell carcinomas

Cancer angiogenesis is required to support energetic demand and metabolic stress, particu-larly during conditions of hypoxia. Coupled to neo-vasculogenesis, cancer cells rewire metabolic programs to sustain growth, survival and long-term maintenance. Thyroid hormone (TH) signaling regulates growth and differentiation in a variety of cell types and tissues, thus modulating hyper proliferative processes such as cancer. Herein, we report that TH coordinates a global program of metabolic reprogramming and induces angiogenesis through up-regulation of the VEGF-A gene, which results in the enhanced proliferation of tumor endothelial cells. In vivo conditional depletion of the TH activating enzyme in a mouse model of cutaneous squamous cell carcinoma (SCC) reduces the concentration of TH in the tumoral cells and results in impaired VEGF-A production and atten-uated angiogenesis. In addition, we found that TH induces the expression of the glycolytic genes and fosters lactate production, which are key traits of the Warburg effect. Taken together, our results reveal a TH–VEGF-A–HIF1α regulatory axis leading to enhanced angiogenesis and glycolytic flux, which may represent a target for SCC therapy

A proteomic approach for the rapid, multi-informative and reliable identification of blood

Blood evidence is frequently encountered at the scene of violent crimes and can provide valuable intelligence in the forensic investigation of serious offences. Because many of the current enhancement methods used by crime scene investigators are presumptive, the visualisation of blood is not always reliable nor does it bear additional information. In the work presented here, two methods employing a shotgun bottom up proteomic approach for the detection of blood are reported; the developed protocols employ both an in solution digestion method and a recently proposed procedure involving immobilization of trypsin on hydrophobin Vmh2 coated MALDI sample plate. The methods are complementary as whilst one yields more identifiable proteins (as biomolecular signatures), the other is extremely rapid (5 minutes). Additionally, data demonstrate the opportunity to discriminate blood provenance even when two different blood sources are present in a mixture. This approach is also suitable for old bloodstains which had been previously chemically enhanced, as experiments conducted on a 9-year-old bloodstain deposited on a ceramic tile demonstrate

BATS: a Bayesian user-friendly software for Analyzing Time Series microarray experiments

BATS is a user-friendly software for Bayesian Analysis of Time Series microarray experiments based on the novel, truly functional and fully Bayesian approach proposed in Angelini et at. (2006). The software is specifically designed for time series data. It allows an user to automatically identify and rank differentially expressed genes and to estimate their expression profiles. BATS successfully manages various technical difficulties which arise in microarray time-course experiments, such as a small number of observations, non-uniform sampling intervals, and presence of missing or multiple data. BATS can carry out analysis with both simulated and real experimental data. It also handles data from different platforms. 1 Availability: BATS is written in Matlab and executable in Windows (Macintosh and Linux version are currently under development). It is freely available upon request from the authors.

EBP1 and DRBP76/NF90 binding proteins are included in the major histocompatibility complex class II RNA operon

Major histocompatibility complex class II mRNAs encode heterodimeric proteins involved in the presentation of exogenous antigens during an immune response. Their 3′UTRs bind a protein complex in which we identified two factors: EBP1, an ErbB3 receptor-binding protein and DRBP76, a double-stranded RNA binding nuclear protein, also known as nuclear factor 90 (NF90). Both are well-characterized regulatory factors of several mRNA molecules processing. Using either EBP1 or DRBP76/NF90-specific knockdown experiments, we established that the two proteins play a role in regulating the expression of HLA-DRA, HLA-DRB1 and HLA-DQA1 mRNAs levels. Our study represents the first indication of the existence of a functional unit that includes different transcripts involved in the adaptive immune response. We propose that the concept of ‘RNA operon’ may be suitable for our system in which MHCII mRNAs are modulated via interaction of their 3′UTR with same proteins

Differential Expression of VEGFA Isoforms Regulates Metastasis and Response to Anti-VEGFA Therapy in Sarcoma

Elevated plasma concentrations of soluble VEGFA isoforms are associated with poor prognosis in parallel with improved response to treatment with the anti-VEGFA antibody bevacizumab. To uncover the underlying mechanism to these observations, we administered anti-VEGFA therapy to mice bearing luminescent mouse fibrosarcomas expressing single VEGFA isoforms or their wild-type counterparts expressing all isoforms (fs120, fs164, fs188, or fsWT). Expression of the more soluble isoforms conferred an advantage for lung metastasis from subcutaneous tumors (fs120/164 vs. fs188/WT); fs120 cells also produced more lung colonies than fs188 cells when injected intravenously. Metastasis from subcutaneous fs120 tumors was more sensitive than fs188 to treatment with the anti-VEGFA antibody B20-4.1.1. Despite elevated plasma levels of VEGFA in fs120 tumor-bearing mice and a dependence on VEGF receptor 1 activity for metastasis to the lung, B20-4.1.1 did not affect survival in the lung on intravenous injection. B20-4.1.1 inhibited subcutaneous tumor growth and decreased vascular density in both fs120 and fs188 tumors. However, migration of fs120, but not fs188 cells, in vitro was inhibited by B20-4.1.1. The greater survival of fs120 cells in the lung was associated with VEGFR1-dependent accumulation of CD11b-positive myeloid cells and higher expression of the VEGFR1 ligand, PlGF2, by the fs120 cells in vitro and in the plasma and lungs of fs120 tumor-bearing mice. We conclude that soluble VEGFA isoform expression increases fibrosarcoma metastasis through multiple mechanisms that vary in their sensitivity to anti-VEGF/VEGFR inhibition, with VEGFA-targeted therapy suppressing metastasis through effects on the primary tumor rather than the metastatic site

HAX-1 overexpression, splicing and cellular localization in tumors

<p>Abstract</p> <p>Background</p> <p>HAX-1 has been described as a protein potentially involved in carcinogenesis and especially metastasis. Its involvement in regulation of apoptosis and cell migration along with some data indicating its overexpression in cancer cell lines and tumors suggests that HAX-1 may play a role in neoplastic transformation. Here we present the first systematic analysis of HAX-1 expression in several solid tumors.</p> <p>Methods</p> <p>Using quantitative RT-PCR, we have determined the mRNA levels of <it>HAX1 </it>splice variant I in several solid tumors. We have also analyzed by semiquantitative and quantitative RT-PCR the expression of five <it>HAX-1 </it>splice variants in breast cancer samples and in normal tissue from the same individuals. Quantitative PCR was also employed to analyze the effect of estrogen on <it>HAX1 </it>expression in breast cancer cell line. Immunohistochemical analysis of HAX-1 was performed on normal and breast cancer samples.</p> <p>Results</p> <p>The results reveal statistically important <it>HAX1 </it>up-regulation in breast cancer, lung cancer and melanoma, along with some minor variations in the splicing pattern. HAX-1 up-regulation in breast cancer samples was confirmed by immunohistochemical analysis, which also revealed an intriguing HAX-1 localization in the nuclei of the tumor cells, associated with strong ER status.</p> <p>Conclusion</p> <p>HAX-1 elevated levels in cancer tissues point to its involvement in neoplastic transformation, especially in breast cancer. The connection between HAX-1 nuclear location and ER status in breast cancer samples remains to be clarified.</p