Cellular and molecular components of the atherosclerotic process: Primary colture and characterization of human vascular smooth muscle cells from carotid artery

Introduction The last years were characterized by several studies proceeding to understand SMC heterogeneity. Animal models of rat or rabbit IT after endothelial lesion are presently the most studied models for atherosclerotic plaque formation and have been useful in order to understand several biological features of SMCs. The disadvantages of animal models are that there is no possibility to reproduce the same history of pathogenesis in animals as that in man. There is no perfect animal model that completely replicates all stages of human atherosclerosis. Howevere, identification of particular SMCs subpopulation in human arteries represents a difficult challenge for understandable reason, such us material availability and experiment standardization. The relevance of SMC heterogenity to human disease still remains to be demonstrated. Aim Aim of this study is to obtain primary culture of human vascular smooth muscle cells (SMCs) from both atherosclerotic and no-atherosclerotic carotid artery, to investigare human SMC heterogeneity. Moreover, the possibility to isolate o SMC subpopulations, from a single human artery, could be usefull to verify the hypothesis that distinct SMC subtypes are involved in atherosclerosis pathogenesis and progression. Materials, Methods and Results Thanks to a collaboration between our laboratory and the Vascular Surgery’s group of Sant’Anna Hospital(University of Ferrara) leading by Francesco Mascoli, we had the possibility to obtain samples of atherosclerotic and noatherosclerotic human carotid artery from patients undergone to carotid endarterectomy. From these specimens we proceeded with tissue explantation in order to obtain primary colture of vascular smooth muscle cells both from no-atherosclerotic and atherosclerotic carotid artery. Cells obtained by tissue explantation were tested by immunoflurescences staining in order to verify the presence of alfa smooth muscle actin, a specific marker of vascular smooth muscle cells. Once we verified the nature of our cells through the presence of alfa smooth muscle actin we have tried to divide the atherosclerotic artery in media and plaque and than proceeding tissue explatation only with media or only with plaque or with complete lesion. We have found out that only from the complete lesion (media +plaque) we have obtained cellular outgrowth, while media and plaque alone has not gave cells at all. From these results we were started to understand which tissue, between media and plaque was the proliferative ones. In order to obtain this data we have proceeded with an experiment of cocolture using transwell insert, wich allows media and plaque to stay phisically separeted but to share the same medium. After these experiments, we have found out that the proliferative tissue was the media but plaque presence was a “condicio sine qua non” for cellular outgrowth in atherosclerotic carotid artery. We have compared cells coming from non atherosclerotic media with the ones coming from atherosclerotic artery: early, we have found out the morphological differences: cells from non atherosclerotic media was large and grew up in a monolayer even at confluence, while cells from atherosclerotic media was small and elongated and, at confluence, grew up forming a multilayer net. We have analyzed the cells also using immunofluorescences finding out that large cells(coming from no-atherosclerotic artery), had much alfa smooth muscle actin and did not shown Desmin, or S100A4(a marker of atheroma prone cells in porcine model of atherosclerosis), but show SMMHC. Small cells (coming from atherosclerotic artery), instead, had less alfa smooth muscle actin compared to large cells, and did not shown desmin and SMMHC but show an high production of S100A4. We also have tried to add plaque in tissue explantation of no-atherosclerotique media and the outgrownig from these experiments was small and by immunofluorescence shown the presences of S100A4. These are only preliminary data. The presence of the mRNA of these markers was analyzed by using RT-PCR. For further information about the differences between these two celluar populations we led a microarray experiment using biological material coming from three patiences. We discovered that there are 375 genes modulated in the two populations; these genes are involved in several biological pathways, in particular inflammation and cellular development. In conclusion we found that there are two kind of cellular populations in human artery with atherosclerotic lesion, they have some important differences that could explain why cells change phenotype and migrate in to the intima. Cells from atherosclerotic artery are strictly dependent of plaque presences that act on on them as a magnet. We identify a group of genes that can help us to understand what are the steps of phenotypical modulation. Presence of plaque nearby no-atherosclerotic artery can switch the cells from a large phenotype to a Small Phenotype (this preliminary data is confirmed by the presence in cells, outgrowed from no-atherosclerotic artery in presece of plaque, of S100A4

Cancer gene therapy 2020: highlights from a challenging year

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miRNA transported by exosomes: a key machinery in tumor microenvironment mediated chemoresistance

It is well established that the tumor microenvironment (TME) actively participates in fundamental processes such as tumor initiation, invasion and response to therapies, apart from its documented scaffold role. Ultimately, the TME can be defined as a tri-dimensional network structured by the tumor-associated extra-cellular matrix (ECM) in which tumor cells interact with various types of stromal cells in an attempt to hijack the normal organization of the host tissue and support cancer growth

IGF-I influences everolimus activity in medullary thyroid carcinoma

Context: Medullary thyroid carcinoma (MTC) is a rare tumor originating from thyroid parafollicular C cells. It has been previously demonstrated that insulin-like growth factor I (IGF-I) protects MTC from the effects of antiproliferative drugs. Everolimus, an mTOR inhibitor, has shown potent antiproliferative effects in a human MTC cell line, TT, and in two human MTC primary cultures. Objective: To verify whether IGF-I may influence the effects of everolimus in a group of human MTC primary cultures. Design: We collected 18 MTCs that were dispersed in primary cultures, treated without or with 10 nM-1 mu M everolimus and/or 50 nM IGF-I. Cell viability was evaluated after 48 h, and calcitonin (CT) secretion was assessed after a 6 h incubation. IGF-I receptor downstream signaling protein expression profile was also investigated. Results: Everolimus significantly reduced cell viability in eight MTC [by similar to 20%; P < 0.01 vs. control; everolimus-responders (E-R) MTCs], while cell viability did not change in 10 MTCs [everolimus-non-responders (E-NR) MTCs]. In E-R MTCs, IGF-I blocked the antiproliferative effects of everolimus that did not affect CT secretion, but blocked the stimulatory effects of IGF-I on this parameter. IGF-I receptor downstream signaling proteins were expressed at higher levels in E-NR MTC as compared to E-R MTCs. Conclusion: IGF-I protects a subset of MTC primary cultures from the antiproliferative effects of everolimus and stimulates CT secretion by an mTOR mediated pathway that, in turn, may represent a therapeutic target in the treatment of aggressive MTCs

EGF and IGF1 affect Sunitinib activity in BP-NEN: new putative targets beyond VEGFR?

Broncho-Pulmonary Neuroendocrine Neoplasms (BP-NENs) are neoplasms orphan of an efficient therapy. Available medical treatments derived from clinical trials are not specific for the management of this malignancy. Sunitinib is a multi-receptor tyrosine-kinases (RTKs) inhibitor that has already shown its efficacy in NENs but there are not available data about its action in BP-NENs. Therefore, our aim was to understand the effects of RTKs inhibition promoted by Sunitinib in order to evaluate new putative targets useful in malignancy treatment. Since our results underlined a role for EGFR and IGF1R in modulating Sunitinib antiproliferative action, we investigated the effects of Erlotinib, an EGFR inhibitor, and Linsitinib, an IGF1R inhibitor, in order to understand their function in regulating cells behaviour. Cell viability and caspase activation were evaluated on two immortalized human BP-NEN cell lines and primary cultures. Our results showed that after treatment with Sunitinib and/or IGF1, EGF and VEGF, the antiproliferative effect of Sunitinib was counteracted by EGF and IGF1 but not by VEGF. Therefore, we evaluated with alpha-screen technology the phosphorylated EGFR and IGF1R levels in primary cultures treated with Sunitinib and/or EGF and IGF1. Results showed a decrease of p-IGF1R after treatment with Sunitinib and an increase after co-treatment with IGF1. Then, we assessed cell viability and caspase activation on BP-NEN cell lines after treatment with Linsitinib and/or Erlotinib. Results demonstrate that these two agents have a stronger antiproliferative effect compared to Sunitinib. In conclusion, our results suggest that IGF1R and EGF1R could represent putative molecular targets in BP-NENs treatment

The multifaceted role of lemur tyrosine kinase 3 in health and disease

In the last decade, LMTK3 (lemur tyrosine kinase 3) has emerged as an important player in breast cancer, contributing to the advancement of disease and the acquisition of resistance to therapy through a strikingly complex set of mechanisms. Although the knowledge of its physiological function is largely limited to receptor trafficking in neurons, there is mounting evidence that LMTK3 promotes oncogenesis in a wide variety of cancers. Recent studies have broadened our understanding of LMTK3 and demonstrated its importance in numerous signalling pathways, culminating in the identification of a potent and selective LMTK3 inhibitor. Here, we review the roles of LMTK3 in health and disease and discuss how this research may be used to develop novel therapeutics to advance cancer treatment

Changes in chromatin accessibility and transcriptional landscape induced by HDAC inhibitors in TP53 mutated patient-derived colon cancer organoids

: Here we present the generation and characterization of patient-derived organoids (PDOs) from colorectal cancer patients. PDOs derived from two patients with TP53 mutations were tested with two different HDAC inhibitors (SAHA and NKL54). Cell death induction, transcriptome, and chromatin accessibility changes were analyzed. HDACIs promote the upregulation of low expressed genes and the downregulation of highly expressed genes. A similar differential effect is observed at the level of chromatin accessibility. Only SAHA is a potent inducer of cell death, which is characterized by the upregulation of BH3-only genes BIK and BMF. Up-regulation of BIK is associated with increased accessibility in an intronic region that has enhancer properties. SAHA, but not NKL54, also causes downregulation of BCL2L1 and decreases chromatin accessibility in three distinct regions of the BCL2L1 locus. Both inhibitors upregulate the expression of innate immunity genes and members of the MHC family. In summary, our exploratory study indicates a mechanism of action for SAHA and demonstrate the low efficacy of NKL54 as a single agent for apoptosis induction, using two PDOs. These observations need to be validated in a larger cohort of PDOs

Correction to: LMTK3 inhibition affects microtubule stability

Correction to: Mol Cancer 20, 53 (2021) https://doi.org/10.1186/s12943-021-01345-3 Following the publication of the original article [1], the authors noticed errors on the figures introduced during the production step. Below are the errors: Fig. 2a: The labels for the y- and x-axes have been translocated upwards and need to be realigned with the axes. The x-axis should read: -Log2 (fold change). Fig. 2d: the quantification values in the NUSAP1 blots, for T47D and MDA-MB-231 cells, are no longer visible. The original article has been corrected

LMTK3 inhibition affects microtubule stability

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