Characteristics of ABCC4 and ABCG2 High Expression Subpopulations in CRC-A New Opportunity to Predict Therapy Response.

BACKGROUND: Our previous findings proved that ABCC4 and ABCG2 proteins present much more complex roles in colorectal cancer (CRC) than typically cancer-associated functions as drug exporters. Our objective was to evaluate their predictive/diagnostic potential. METHODS: CRC patients' transcriptomic data from the Gene Expression Omnibus database (GSE18105, GSE21510 and GSE41568) were discriminated into two subpopulations presenting either high expression levels of ABCC4 (ABCC4 High) or ABCG2 (ABCG2 High). Subpopulations were analysed using various bioinformatical tools and platforms (KEEG, Gene Ontology, FunRich v3.1.3, TIMER2.0 and STRING 12.0). RESULTS: The analysed subpopulations present different gene expression patterns. The protein-protein interaction network of subpopulation-specific genes revealed the top hub proteins in ABCC4 High: RPS27A, SRSF1, DDX3X, BPTF, RBBP7, POLR1B, HNRNPA2B1, PSMD14, NOP58 and EIF2S3 and in ABCG2 High: MAPK3, HIST2H2BE, LMNA, HIST1H2BD, HIST1H2BK, HIST1H2AC, FYN, TLR4, FLNA and HIST1H2AJ. Additionally, our multi-omics analysis proved that the ABCC4 expression correlates with substantially increased tumour-associated macrophage infiltration and sensitivity to FOLFOX treatment. CONCLUSIONS: ABCC4 and ABCG2 may be used to distinguish CRC subpopulations that present different molecular and physiological functions. The ABCC4 High subpopulation demonstrates significant EMT reprogramming, RNA metabolism and high response to DNA damage stimuli. The ABCG2 High subpopulation may resist the anti-EGFR therapy, presenting higher proteolytical activity.

The Comparison of Serum Exosome Protein Profile in Diagnosis of NSCLC Patients.

A thorough study of the exosomal proteomic cargo may enable the identification of proteins that play an important role in cancer development. The aim of this study was to compare the protein profiles of the serum exosomes derived from non-small lung cancer (NSCLC) patients and healthy volunteers (control) using the high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS) method to identify potentially new diagnostic and/or prognostic protein biomarkers. Proteins exclusively identified in NSCLC and control groups were analyzed using several bioinformatic tools and platforms (FunRich, Vesiclepedia, STRING, and TIMER2.0) to find key protein hubs involved in NSCLC progression and the acquisition of metastatic potential. This analysis revealed 150 NSCLC proteins, which are significantly involved in osmoregulation, cell-cell adhesion, cell motility, and differentiation. Among them, 3 proteins: Interleukin-34 (IL-34), HLA class II histocompatibility antigen, DM alpha chain (HLA-DMA), and HLA class II histocompatibility antigen, DO beta chain (HLA-DOB) were shown to be significantly involved in the cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs) infiltration processes. Additionally, detected proteins were analyzed according to the presence of lymph node metastasis, showing that differences in frequency of detection of protein FAM166B, killer cell immunoglobulin-like receptor 2DL1, and olfactory receptor 52R1 correlate with the N feature according to the TNM Classification of Malignant Tumors. These results prove their involvement in NSCLC lymph node spread and metastasis. However, this study requires further investigation.

Assessment of the Influence of 5-Fluorouracil on SMAD4 and TGFB1 Gene Expression, Apoptosis Induction and DNA Damage in Human Cell Lines.

PURPOSE: Suppressor of mothers against decapentaplegic homolog 4 (SMAD family member 4, SMAD4) is involved in the adenoma-carcinoma pathway, leading to the development of colon cancer. The encoded protein is a key downstream signaling mediator in the TGFß pathway. This pathway has tumor-suppressor functions, including cell-cycle arrest and apoptosis. Its activation in late-stage cancer can promote tumorigenesis, including metastasis and chemoresistance. Most colorectal cancer patients receive chemotherapy based on 5-FU as an adjuvant treatment. However, the success of therapy is hampered by multidrug resistance by neoplastic cells. In colorectal cancer, resistance to 5-FU-based therapy is influenced by SMAD4 gene expression, as patients with decreased SMAD4 gene expression probably have a higher risk of developing 5-FU-induced resistance. The mechanism leading to the development of this phenomenon is not fully understood. Therefore, the present study assesses the possible influence of 5-FU on changes in the expression of the SMAD4 and TGFB1 genes. PATIENTS AND METHODS: The effect of 5-FU on the expression of SMAD4 and TGFB1 in colorectal cancer cells derived from the CACO-2, SW480 and SW620 cell lines was evaluated using real-time PCR. The cytotoxicity of 5-FU on colon cancer cells was assessed by the MTT method, and its effect on the induction of cell apoptosis and the initiation of DNA damage using a flow cytometer. RESULTS: Significant changes in the level of SMAD4 and TGFB1 gene expression were noted in the CACO-2, SW480 and SW620 cells treated with 5-FU at various concentrations during 24 h and 48 h exposure. The use of 5-FU at a concentration of 5 µmol/L resulted in a decrease in the expression of the SMAD4 gene in all cell lines at both exposure times, while the concentration of 100 µmol/L increased the expression of the SMAD4 gene in CACO-2 cells. The level of expression of the TGFB1 gene was higher for all cells treated with 5-FU at the highest concentrations, while the exposure time was extended to 48 h. CONCLUSION: The observed in vitro changes in CACO-2 cells caused by 5-FU may be of clinical relevance when choosing the drug concentration for treating patients with colorectal cancer. It is possible that 5-FU has a stronger effect on colorectal cancer cells at the higher concentrations. Low concentrations of 5-FU may not have a therapeutic effect and may also influence drug resistance in cancer cells. Higher concentrations and prolonged exposure time may affect SMAD4 gene expression, which may increase the effectiveness of therapy.

Platelet-derived microparticles stimulate the invasiveness of colorectal cancer cells via the p38MAPK-MMP-2/MMP-9 axis.

BACKGROUND: Metastasis is the main cause of death in patients with colorectal cancer (CRC). Apart from platelets, platelet-derived microparticles (PMPs) are also considered important factors that can modify the activity of cancer cells. PMPs are incorporated by cancer cells and can also serve as intracellular signalling vesicles. PMPs are believed to affect cancer cells by upregulating their invasiveness. To date, there is no evidence that such a mechanism occurs in colorectal cancer. It has been shown that platelets can stimulate metalloproteases (MMPs) expression and activity via the p38MAPK pathway in CRC cells, leading to their elevated migratory potential. This study aimed to investigate the impact of PMPs on the invasive potential of CRC cells of various phenotypes via the MMP-2, MMP-9 and p38MAPK axis. METHODS: We used various CRC cell lines, including the epithelial-like HT29 and the mesenchymal-like SW480 and SW620. Confocal imaging was applied to study PMP incorporation into CRC cells. The presence of surface receptors on CRC cells after PMP uptake was evaluated by flow cytometry. Transwell and scratch wound-healing assays were used to evaluate cell migration. The level of C-X-C chemokine receptor type 4 (CXCR4), MMP-2, and MMP-9 and the phosphorylation of ERK1/2 and p38MAPK were measured by western blot. MMP activity was determined using gelatine-degradation assays, while MMP release was evaluated by ELISA. RESULTS: We found that CRC cells could incorporate PMPs in a time-dependent manner. Moreover, PMPs could transfer platelet-specific integrins and stimulate the expression of integrins already present on tested cell lines. While mesenchymal-like cells expressed less CXCR4 than epithelial-like CRC cells, PMP uptake did not increase its intensity. No significant changes in CXCR4 level either on the surface or inside CRC cells were noticed. Levels of cellular and released MMP-2 and MMP-9 were elevated in all tested CRC cell lines after PMP uptake. PMPs increased the phosphorylation of p38MAPK but not that of ERK1/2. Inhibition of p38MAPK phosphorylation reduced the PMP-induced elevated level and release of MMP-2 and MMP-9 as well as MMP-dependent cell migration in all cell lines. CONCLUSIONS: We conclude that PMPs can fuse into both epithelial-like and mesenchymal-like CRC cells and increase their invasive potential by inducing the expression and release of MMP-2 and MMP-9 via the p38MAPK pathway, whereas CXCR4-related cell motility or the ERK1/2 pathway appears to not be affected by PMPs. Video Abstract.

Neuromedin U secreted by colorectal cancer cells promotes a tumour-supporting microenvironment.

BACKGROUND: Neuromedin U (NMU) was identified as one of the hub genes closely related to colorectal cancer (CRC) progression and was recently shown to be a motility inducer in CRC cells. Its autocrine signalling through specific receptors increases cancer cell migration and invasiveness. Because of insufficient knowledge concerning NMU accessibility and action in the tumour microenvironment, its role in CRC remains poorly understood and its potential as a therapeutic target is still difficult to define. METHODS: NMU expression in CRC tissue was detected by IHC. Data from The Cancer Genome Atlas were used to analyse gene expression in CRC. mRNA and protein expression was detected by real-time PCR, immunoblotting or immunofluorescence staining and analysed using confocal microscopy or flow cytometry. Proteome Profiler was used to detect changes in the profiles of cytokines released by cells constituting tumour microenvironment after NMU treatment. NMU receptor activity was monitored by detecting ERK1/2 activation. Transwell cell migration, wound healing assay and microtube formation assay were used to evaluate the effects of NMU on the migration of cancer cells, human macrophages and endothelial cells. RESULTS: Our current study showed increased NMU levels in human CRC when compared to normal adjacent tissue. We detected a correlation between high NMUR1 expression and shorter overall survival of patients with CRC. We identified NMUR1 expression on macrophages, endothelial cells, platelets, and NMUR1 presence in platelet microparticles. We confirmed ERK1/2 activation by treatment of macrophages and endothelial cells with NMU, which induced pro-metastatic phenotypes of analysed cells and changed their secretome. Finally, we showed that NMU-stimulated macrophages increased the migratory potential of CRC cells. CONCLUSIONS: We propose that NMU is involved in the modulation and promotion of the pro-metastatic tumour microenvironment in CRC through the activation of cancer cells and other tumour niche cells, macrophages and endothelial cells. Video abstract.

Isothiocyanates (ITCs) 1-(Isothiocyanatomethyl)-4-phenylbenzene and 1-Isothiocyanato-3,5-bis(trifluoromethyl)benzene-Aldehyde Dehydrogenase (ALDH) Inhibitors, Decreases Cisplatin Tolerance and Migratory Ability of NSCLC.

One of the main treatment modalities for non-small-cell lung cancer (NSCLC) is cisplatin-based chemotherapy. However, the acquisition of cisplatin resistance remains a major problem. Existing chemotherapy regimens are often ineffective against cancer cells expressing aldehyde dehydrogenase (ALDH). As such, there is an urgent need for therapies targeting ALDH-positive cancer cells. The present study compares the anticancer properties of 36 structurally diverse isothiocyanates (ITCs) against NSCLC cells with the ALDH inhibitor disulfiram (DSF). Their potential affinity to ALDH isoforms and ABC proteins was assessed using AutoDockTools, allowing for selection of three compounds presenting the strongest affinity to all tested proteins. The selected ITCs had no impact on NSCLC cell viability (at tested concentrations), but significantly decreased the cisplatin tolerance of cisplatin-resistant variant of A549 (A549CisR) and advanced (stage 4) NSCLC cell line H1581. Furthermore, long-term supplementation with ITC 1-(isothiocyanatomethyl)-4-phenylbenzene reverses the EMT phenotype and migratory potential of A549CisR to the level presented by parental A549 cells, increasing E-Cadherin expression, followed by decreased expression of ABCC1 and ALDH3A1. Our data indicates that the ALDH inhibitors DSF and ITCs are potential adjuvants of cisplatin chemotherapy.

Integrated Bioinformatics Analysis of the Hub Genes Involved in Irinotecan Resistance in Colorectal Cancer.

Different drug combinations including irinotecan remain some of the most important therapeutic modalities in treating colorectal cancer (CRC). However, chemotherapy often leads to the acquisition of cancer drug resistance. To bridge the gap between in vitro and in vivo models, we compared the mRNA expression profiles of CRC cell lines (HT29, HTC116, and LoVo and their respective irinotecan-resistant variants) with patient samples to select new candidate genes for the validation of irinotecan resistance. Data were downloaded from the Gene Expression Omnibus (GEO) (GSE42387, GSE62080, and GSE18105) and the Human Protein Atlas databases and were subjected to an integrated bioinformatics analysis. The protein-protein interaction (PPI) network of differently expressed genes (DEGs) between FOLFIRI-resistant and -sensitive CRC patients delivered several potential irinotecan resistance markers: NDUFA2, SDHD, LSM5, DCAF4, COX10 RBM8A, TIMP1, QKI, TGOLN2, and PTGS2. The chosen DEGs were used to validate irinotecan-resistant cell line models, proving their substantial phylogenetic heterogeneity. These results indicated that in vitro models are highly limited and favor different mechanisms than in vivo, patient-derived ones. Thus, cell lines can be perfectly utilized to analyze specific mechanisms on their molecular levels but cannot mirror the complicated drug resistance network observed in patients.

Neuromedin U induces an invasive phenotype in CRC cells expressing the NMUR2 receptor.

BACKGROUND: Successful colorectal cancer (CRC) therapy often depends on the accurate identification of primary tumours with invasive potential. There is still a lack of identified pathological factors associated with disease recurrence that could help in making treatment decisions. Neuromedin U (NMU) is a secretory neuropeptide that was first isolated from the porcine spinal cord, and it has emerged as a novel factor involved in the tumorigenesis and/or metastasis of many types of cancers. Previously associated with processes leading to CRC cell invasiveness, NMU has the potential to be a marker of poor outcome, but it has not been extensively studied in CRC. METHODS: Data from The Cancer Genome Atlas (TCGA) were used to analyse NMU and NMU receptor (NMUR1 and NMUR2) expression in CRC tissues vs. normal tissues, and real-time PCR was used for NMU and NMU receptor expression analysis. NMU protein detection was performed by immunoblotting. Secreted NMU was immunoprecipitated from cell culture-conditioned media and analysed by immunoblotting and protein sequencing. DNA demethylation by 5-aza-CdR was used to analyse the regulation of NMUR1 and NMUR2 expression. NMU receptor activity was monitored by detecting calcium mobilisation in cells loaded with fluo-4, and ERK1/2 kinase activation was detected after treatment with NMU or receptor agonist. Cell migration and invasion were investigated using membrane filters. Integrin expression was evaluated by flow cytometry. RESULTS: The obtained data revealed elevated expression of NMU and NMUR2 in CRC tissue samples and variable expression in the analysed CRC cell lines. We have shown, for the first time, that NMUR2 activation induces signalling in CRC cells and that NMU increases the motility and invasiveness of NMUR2-positive CRC cells and increases prometastatic integrin receptor subunit expression. CONCLUSIONS: Our results show the ability of CRC cells to respond to NMU via activation of the NMUR2 receptor, which ultimately leads to a shift in the CRC phenotype towards a more invasive phenotype.

Snail Overexpression Alters the microRNA Content of Extracellular Vesicles Released from HT29 Colorectal Cancer Cells and Activates Pro-Inflammatory State In Vivo.

During metastasis, cancer cells undergo phenotype changes in the epithelial-mesenchymal transition (EMT) process. Extracellular vesicles (EVs) released by cancer cells are the mediators of intercellular communication and play a role in metastatic process. Knowledge of factors that influence the modifications of the pre-metastatic niche for the migrating carcinoma cells is important for prevention of metastasis. We focus here on how cancer progression is affected by EVs released from either epithelial-like HT29-cells or from cells that are in early EMT stage triggered by Snail transcription factor (HT29-Snail). We found that EVs released from HT29-Snail, as compared to HT29-pcDNA cells, have a different microRNA profile. We observed the presence of interstitial pneumonias in the lungs of mice injected with HT29-Snail cells and the percent of mice with lung inflammation was higher after injection of HT29-Snail-EVs. Incorporation of EVs released from HT29-pcDNA, but not released from HT29-Snail, leads to the increased secretion of IL-8 from macrophages. We conclude that Snail modifications of CRC cells towards more invasive phenotype also alter the microRNA cargo of released EVs. The content of cell-released EVs may serve as a biomarker that denotes the stage of CRC and EVs-specific microRNAs may be a target to prevent cancer progression.

Implications of ABCC4-Mediated cAMP Eflux for CRC Migration.

Colorectal cancer (CRC) presents significant molecular heterogeneity. The cellular plasticity of epithelial to mesenchymal transition (EMT) is one of the key factors responsible for the heterogeneous nature of metastatic CRC. EMT is an important regulator of ATP binding cassette (ABC) protein expression; these proteins are the active transporters of a broad range of endogenous compounds and anticancer drugs. In our previous studies, we performed a transcriptomic and functional analysis of CRC in the early stages of metastasis induced by the overexpression of Snail, the transcription factor involved in EMT initiation. Interestingly, we found a correlation between the Snail expression and ABCC4 (MRP4) protein upregulation. The relationship between epithelial transition and ABCC4 expression and function in CRC has not been previously defined. In the current study, we propose that the ABCC4 expression changes during EMT and may be differentially regulated in various subpopulations of CRC. We confirmed that ABCC4 upregulation is correlated with the phenotype conversion process in CRC. The analysis of Gene Expression Omnibus (GEO) sets showed that the ABCC4 expression was elevated in CRC patients. The results of a functional study demonstrated that, in CRC, ABCC4 can regulate cell migration in a cyclic nucleotide-dependent manner.

Erratum: Neuromedin U: A Small Peptide in the Big World of Cancer. Cancers 2019, 11, 1312.

The authors wish to make the following corrections to this paper [...].

Neuromedin U: A Small Peptide in the Big World of Cancer.

Neuromedin U (NMU), a neuropeptide isolated from porcine spinal cord and named because of its activity as a rat uterus smooth muscle contraction inducer, is emerging as a new player in the tumorigenesis and/or metastasis of many types of cancers. Expressed in a variety of tissues, NMU has been shown to possess many important activities in the central nervous system as well as on the periphery. Along with the main structural and functional features of NMU and its currently known receptors, we summarized a growing number of recently published data from different tissues and cells that associate NMU activity with cancer development and progression. We ask if, based on current reports, NMU can be included as a marker of these processes and/or considered as a therapeutic target.

TUBB4B Downregulation Is Critical for Increasing Migration of Metastatic Colon Cancer Cells.

Tumor metastasis, the major problem for clinical oncology in colon cancer treatment, is linked with an epithelial-mesenchymal transition (EMT). The observed cellular transformation in this process is manifested by cell elongation, enhanced cell migration and invasion ability, coordinated by cytoskeleton reorganization. In the present study, we examined the role of tubulin-ß4 (TUBB4B) downregulation that occurs during EMT in colon cancer cells, in the modulation of the function of microtubules. Based on biochemical and behavioral analysis (transmigration) we posit that the decrease of the TUBB4B level is critical for microtubule-vimentin interaction and contributes to the maintenance of polarity in migrating cells. The microscopic studies revealed that TUBB4B decrease is accompanied by cell elongation and increased number of matured focal adhesion sites, which is a characteristic of the cell metastatic stage. We also demonstrated faster polymerization of microtubules in cells with a lower level of TUBB4B. Simultaneous TUBB3 upregulation, reported during EMT, acts additively in this process. Our studies suggest that the protein level of TUBB4B could be used as a marker for detection of the preinvasive stages of the colon cancer cells. We also concluded that chemotherapy enriched to increase TUBB4B level and/or to stabilize microtubule polymerization might more effectively prevent metastasis in colon cancer development.

Cathepsin B Is Upregulated and Mediates ECM Degradation in Colon Adenocarcinoma HT29 Cells Overexpressing Snail.

During tumor development and ongoing metastasis the acquisition of mesenchymal cell traits by epithelial carcinoma cells is achieved through a programmed phenotypic shift called the epithelial-to-mesenchymal transition, EMT. EMT contributes to increased cancer cell motility and invasiveness mainly through invadosomes, the adhesion structures that accompany the mesenchymal migration. The invadosomes and their associated proteases restrict protease activity to areas of the cell in direct contact with the ECM, thus precisely controlling cell invasion. Our data prove that Snail-overexpressing HT-29 cells that imitate the phenotype of colon cancer cells in the early stage of the EMT showed an increase in the expression and pericellular activity of cathepsin B. It appears that the pericellular localization of cathepsin B, also observed in colon and rectum adenocarcinoma tissue samples, plays a key role in its function.

Regulation of miRNAs by Snail during epithelial-to-mesenchymal transition in HT29 colon cancer cells.

Epithelial-to-mesenchymal transition (EMT) in cancer cells, represents early stages of metastasis and is a promising target in colorectal cancer (CRC) therapy. There have been many attempts to identify markers and key pathways induced throughout EMT but the process is complex and depends on the cancer type and tumour microenvironment. Here we used the colon cancer cell line HT29, which stably overexpressed Snail, the key transcription factor in early EMT, as a model for colorectal adenocarcinoma cells with a pro-metastatic phenotype. We investigated miRNA expression regulation during that phenotypic switching. We found that overexpression of Snail in HT29 cells triggered significant changes in individual miRNA levels but did not change the global efficiency of miRNA processing. Snail abundance repressed the expression of miR-192 and miR-194 and increased miR-205, let-7i and SNORD13 levels. These identified changes correlated with the reported transcriptomic alterations in Snail-overexpressing HT29 cells. We also investigated how Snail affected the miRNA content of extracellular vesicles (EVs) released from HT29 cells. Our data suggest that the presence of Snail significantly alters the complex mRNA/miRNA interactions in the early steps of metastasis and also has an impact on the content of EVs released from HT29 cells.

Cell Migration Related to MDR-Another Impediment to Effective Chemotherapy?

Multidrug resistance, mediated by members of the ATP-binding cassette (ABC) proteins superfamily, has become one of the biggest obstacles in conquering tumour progression. If the chemotherapy outcome is considered successful, when the primary tumour volume is decreased or completely abolished, modulation of ABC proteins activity is one of the best methods to overcome drug resistance. However, if a positive outcome is represented by no metastasis or, at least, elongation of remission-free time, then the positive effect of ABC proteins inhibition should be compared with the several side effects it causes, which may inflict cancer progression and decrease overall patient health. Clinical trials conducted thus far have shown that the tested ABC modulators add limited or no benefits to cancer patients, as some of them are merely toxic and others induce unwanted drug-drug interactions. Moreover, the inhibition of certain ABC members has been recently indicated as potentially responsible for increased fibroblasts migration. A better understanding of the complex role of ABC proteins in relation to cancer progression may offer novel strategies in cancer therapy.

Filamin A upregulation correlates with Snail-induced epithelial to mesenchymal transition (EMT) and cell adhesion but its inhibition increases the migration of colon adenocarcinoma HT29 cells.

Filamin A (FLNA) is actin filament cross-linking protein involved in cancer progression. Its importance in regulating cell motility is directly related to the epithelial to mesenchymal transition (EMT) of tumor cells. However, little is known about the mechanism of action of FLNA at this early stage of cancer invasion. Using immunochemical methods, we evaluated the levels and localization of FLNA, pFLNA[Ser2152], ß1 integrin, pß1 integrin[Thr788/9], FAK, pFAK[Y379], and talin in stably transfected HT29 adenocarcinoma cells overexpressing Snail and looked for the effect of Snail in adhesion and migration assays on fibronectin-coated surfaces before and after FLNA silencing. Our findings indicate that FLNA upregulation correlates with Snail-induced EMT in colorectal carcinoma. FLNA localizes in the cytoplasm and at the sites of focal adhesion (FA) of invasive cells. Silencing of FLNA inhibits Snail-induced cell adhesion, reduces the size of FA sites, induces the relocalization of talin from the cytoplasm to the membrane area and augments cell migratory properties. Our findings suggest that FLNA may not act as a classic integrin inhibitor in invasive carcinoma cells, but is involved in other pro-invasive pathways. FLNA upregulation, which correlates with cell metastatic properties, maybe an additional target for combination therapy in colorectal carcinoma tumor progression.

Proteases Revisited: Roles and Therapeutic Implications in Fibrosis.

Proteases target many substrates, triggering changes in distinct biological processes correlated with cell migration, EMT/EndMT and fibrosis. Extracellular protease activity, demonstrated by secreted and membrane-bound protease forms, leads to ECM degradation, activation of other proteases (i.e., proteolysis of nonactive zymogens), decomposition of cell-cell junctions, release of sequestered growth factors (TGF-ß and VEGF), activation of signal proteins and receptors, degradation of inflammatory inhibitors or inflammation-related proteins, and changes in cell mechanosensing and motility. Intracellular proteases, mainly caspases and cathepsins, modulate lysosome activity and signal transduction pathways. Herein, we discuss the current knowledge on the multidimensional impact of proteases on the development of fibrosis.

HMEC-1 adopt the mixed amoeboid-mesenchymal migration type during EndMT.

The contribution of endothelial cells to scar and fibrotic tissue formation is undisputedly connected to their ability to undergo the endothelial-to-mesenchymal transition (EndMT) towards fibroblast phenotype-resembling cells. The migration model of fibroblasts and fibroblast-resembling cells is still not fully understood. It may be either a Rho/ROCK-independent, an integrin- and MMP-correlated ECM degradation-dependent, a mesenchymal model or Rho/ROCK-dependent, integrin adhesion- and MMP activity-independent, an amoeboid model. Here, we hypothesized that microvascular endothelial cells (HMEC-1) undergoing EndMT adopt an intermediate state of drifting migration model between the mesenchymal and amoeboid protrusive types in the early stages of fibrosis. We characterized the response of HMEC-1 to TGF-ß2, a well-known mediator of EndMT within the microvasculature. We observed that TGF-ß2 induces up to an intermediate mesenchymal phenotype in HMEC-1. In parallel, MMP-2 is upregulated and is responsible for most proteolytic activity. Interestingly, the migration of HMEC-1 undergoing EndMT is dependent on both ECM degradation and invadosome formation associated with MMP-2 proteolytic activity and Rho/ROCK cytoskeleton contraction. In conclusion, the transition from mesenchymal towards amoeboid movement highlights a molecular plasticity mechanism in endothelial cell migration in skin fibrosis.

Vascular endothelial growth factor-D modulates oxidant-antioxidant balance of human vascular endothelial cells.

Vascular endothelial growth factor-D (VEGF-D) is an angiogenic and lymphangiogenic glycoprotein that facilitates tumour growth and distant organ metastasis. Our previous studies showed that VEGF-D stimulates the expression of proteins involved in cell-matrix interactions and promoting the migration of endothelial cells. In this study, we focused on the redox homoeostasis of endothelial cells, which is significantly altered in the process of tumour angiogenesis. Our analysis revealed up-regulated expression of proteins that form the antioxidant barrier of the cell in VEGF-D-treated human umbilical endothelial cells and increased production of reactive oxygen and nitrogen species in addition to a transient elevation in the total thiol group content. Despite a lack of changes in the total antioxidant capacity, modification of the antioxidant barrier induced by VEGF-D was sufficient to protect cells against the oxidative stress caused by hypochlorite and paraquat. These results suggest that exogenous stimulation of endothelial cells with VEGF-D induces an antioxidant response of cells that maintains the redox balance. Additionally, VEGF-D-induced changes in serine/threonine kinase mTOR shuttling between the cytosol and nucleus and its increased phosphorylation at Ser-2448, lead us to the conclusion that the observed shift in redox balance is regulated via mTOR kinase signalling.