In Vitro Identification of New Transcriptomic and miRNomic Profiles Associated with Pulmonary Fibrosis Induced by High Doses Everolimus: Looking for New Pathogenetic Markers and Therapeutic Targets.

The administration of Everolimus (EVE), a mTOR inhibitor used in transplantation and cancer, is often associated with adverse effects including pulmonary fibrosis. Although the underlying mechanism is not fully clarified, this condition could be in part caused by epithelial to mesenchymal transition (EMT) of airway cells. To improve our knowledge, primary bronchial epithelial cells (BE63/3) were treated with EVE (5 and 100 nM) for 24 h. EMT markers (\u3b1-SMA, vimentin, fibronectin) were measured by RT-PCR. Transepithelial resistance was measured by Millicell-ERS ohmmeter. mRNA and microRNA profiling were performed by Illumina and Agilent kit, respectively. Only high dose EVE increased EMT markers and reduced the transepithelial resistance of BE63/3. Bioinformatics showed 125 de-regulated genes that, according to enrichment analysis, were implicated in collagen synthesis/metabolism. Connective tissue growth factor (CTGF) was one of the higher up-regulated mRNA. Five nM EVE was ineffective on the pro-fibrotic machinery. Additionally, 3 miRNAs resulted hyper-expressed after 100 nM EVE and able to regulate 31 of the genes selected by the transcriptomic analysis (including CTGF). RT-PCR and western blot for MMP12 and CTGF validated high-throughput results. Our results revealed a complex biological network implicated in EVE-related pulmonary fibrosis and underlined new potential disease biomarkers and therapeutic targets

Interplay between transglutaminases and heparan sulphate in progressive renal scarring

Transglutaminase-2 (TG2) is a new anti-fibrotic target for chronic kidney disease, for its role in altering the extracellular homeostatic balance leading to excessive build-up of matrix in kidney. However, there is no confirmation that TG2 is the only transglutaminase involved, neither there are strategies to control its action specifically over that of the conserved family-members. In this study, we have profiled transglutaminase isozymes in the rat subtotal nephrectomy (SNx) model of progressive renal scarring. All transglutaminases increased post-SNx peaking at loss of renal function but TG2 was the predominant enzyme. Upon SNx, extracellular TG2 deposited in the tubulointerstitium and peri-glomerulus via binding to heparan sulphate (HS) chains of proteoglycans and co-associated with syndecan-4. Extracellular TG2 was sufficient to activate transforming growth factor-β1 in tubular epithelial cells, and this process occurred in a HS-dependent way, in keeping with TG2-affinity for HS. Analysis of heparin binding of the main transglutaminases revealed that although the interaction between TG1 and HS is strong, the conformational heparin binding site of TG2 is not conserved, suggesting that TG2 has a unique interaction with HS within the family. Our data provides a rationale for a novel anti-fibrotic strategy specifically targeting the conformation-dependent TG2-epitope interacting with HS

LGI1 Affects survival of neuroblastoma cells by inhibiting signalling through phosphoinositide 3-Kinase

Overexpression of the leucine-rich, glioma-inactivated 1 (LGI1) gene in neuroblastoma cells inhibited proliferation and efficiently induced apoptosis. Cell clones stably transfected with LGI1 cDNA showed greater mortality during a period of serum starvation in comparison with control cells stably transfected with empty vector. This observation suggested hindrance of the PI3K/Akt pathway, a central transducer of survival stimuli elicited by serum growth factors. Treatment with inhibitors of PI3K significantly increased the death of control cells but substantially failed to influence LGI1 cell death, which was greatest independently of the presence of inhibitors. Blockage of the PI3K/Akt pathway in LGI1 cells was confirmed by the lack of serum-induced Akt phosphorylation, in contrast with the strong response of control cells. Instead, serum-induced phosphorylation of ERK1/2 was not impaired by the expression of LGI1. This study showed that overexpression of LGI1 caused neuroblastoma cell death by blocking activation of the PI3K/Akt pathway. Thus, the possibility of upregulating LGI1 expression may be a novel strategy in suppressing oncogenesis and metastasis sustained by excessive activation of the PI3K/Akt pathway

Expression of LGI1 impairs proliferation and survival of HeLa cells

The LGI1 gene was suggested to function as tumor suppressor for its ability to reduce malignant features of glioblastoma cells. In support to this proposal was the findings that overexpression of LGI1 in neuroblastoma cells inhibited proliferation and induced apoptosis. In this study we performed stable LGI1 expression in HeLa cells to examine whether the noxious effect of LGI1 might be extended to cancer cells of diverse origin. HeLa cell clones stably expressing LGI1 exhibited a significant impairment of proliferation and a consistent increase of cell death when compared with control cells lacking expression of LGI1. Expression of LGI1 increased the activity of apoptosis effectors caspase \u20133/7, furthermore it downregulated the anti-apoptotic BCL2 gene and upregulated the pro-apoptotic BAX gene expression, suggesting that the cause of HeLa cells death might be an increased susceptibility to apoptosis induced by LGI1. The results suggested that LGI1 is capable to restrain growth and survival of adenocarcinoma cells such as HeLa

Heparanase as active player in endothelial glycocalyx remodeling

The surface of all animal cells is coated with a layer of carbohydrates linked in various ways to the outer side of the plasma membrane. These carbohydrates are mainly bound to proteins in the form of glycoproteins and proteoglycans and together with the glycolipids constitute the so-called glycocalyx. In particular, the endothelial glycocalyx that covers the luminal layer of the endothelium is composed of glycosaminoglycans (heparan sulphate -HS and hyaluronic acid -HA), proteoglycans (syndecans and glypicans) and adsorbed plasma proteins. Thanks to its ability to absorb water, this structure contributes to making the surface of the vessels slippery but at the same time acts by modulating the mechano-transduction of the vessels, the vascular permeability and the adhesion of leukocytes in thus regulating several physiological and pathological events. Among the various enzymes involved in the degradation of the glycocalyx, heparanase (HPSE) has been shown to be particularly involved. This enzyme is responsible for the cutting of heparan sulfate (HS) chains at the level of the proteoglycans of the endothelial glycocalyx whose dysfunction appears to have a role in organ fibrosis, sepsis and viral infection. In this mini-review, we describe the mechanisms by which HPSE contributes to glycocalyx remodeling and then examine the role of glycocalyx degradation in the development of pathological conditions and pharmacological strategies to preserve glycocalyx during disease pathogenesis

Heparanase as a target in cancer therapy.

Heparanase is the unique and specific functional endoglycosidase capable of cleaving heparan sulfate (HS) chains. It exerts its enzymatic activity catalyzing the cleavage of the \u3b2 (1,4)-glycosidic bond between glucuronic acid and glucosamine residue. HS cleavage results in remodelling of the extracellular matrix as well as in regulating the release of many HS-linked molecules such as growth factors, cytokines and enzymes involved in inflammation, wound healing and tumour invasion. A pro-metastatic and pro-angiogenic role for this enzyme has been widely demonstrated in many primary human tumours since high levels of heparanase correlate with lymph node and distant metastasis, elevated micro vessel density and reduced survival of cancer patients. Recently, data have been reported that heparanase regulates heparan sulfate proteoglycan syndecan-1 and promotes its shedding from the cell surface. Shed syndecan-1 in turn controls tumour growth, metastasis and neo-angiogenesis mainly by promoting growth-factor signaling in the tumour milieu. Considering that, once inactivated, there are no other molecules capable of performing the same function, it is evident how this enzyme may be an effective and attractive drug target. Several heparanase inhibitors have been developed and some of them have undergone clinical trials showing efficacy against tumours. In this mini-review we will discuss current knowledge of heparanase involvement in cancer as well as its targeted inhibition as a promising therapeutic option in tumour treatment