Jolanta Niewiarowska

Lumican inhibits angiogenesis by interfering with α2β1 receptor activity and downregulating MMP-14 expression

INTRODUCTION Previous studies showed that lumican, a small leucine-rich proteoglycan that binds to α2 integrin I domain, is an efficient inhibitor of cell adhesion and migration. In this report, we tested its effect on angiogenesis in vitro and in vivo. MATERIALS AND METHODS Effect of lumican on angiogenesis was evaluated by in vitro capillary tube formation test performed between Fibrin II Gels or in Matrigel™ and in vivo by Matrigel(™) plug assay in BALB/c mice. Changes in matrix metalloproteinases expression caused by lumican were analyzed in endothelial cells by real-time PCR, Western immunoblotting and gelatin zymography. RESULTS In unchallenged endothelial cells, Matrigel™ induced robust capillary morphogenesis. In contrast, tube formation was dramatically reduced by lumican, and by siRNA to β1 integrin subunit mRNA but not by control siRNA. Similarly, lumican effectively inhibited neovascularization in vivo in assays using Matrigel™ plugs formed in BALB/c mice. Interestingly, lumican significantly reduced expression of matrix metalloproteinases, particularly MMP-14 that is known to activate other MMPs in close vicinity of endothelial cell membranes. CONCLUSIONS Our results provide strong evidence that lumican affects angiogenesis both by interfering with α2β1 receptor activity and downregulating proteolytic activity associated with surface membranes of endothelial cells.

Effect of lumican on the migration of human mesenchymal stem cells and endothelial progenitor cells: involvement of matrix metalloproteinase-14.

Background Increasing number of evidence shows that soluble factors and extracellular matrix (ECM) components provide an optimal microenvironment controlling human bone marrow mesenchymal stem cell (MSC) functions. Successful in vivo administration of stem cells lies in their ability to migrate through ECM barriers and to differentiate along tissue-specific lineages, including endothelium. Lumican, a protein of the small leucine-rich proteoglycan (SLRP) family, was shown to impede cell migration and angiogenesis. The aim of the present study was to analyze the role of lumican in the control of MSC migration and transition to functional endothelial progenitor cell (EPC). Methodology/Principal Findings Lumican inhibited tube-like structures formation on Matrigel® by MSC, but not EPC. Since matrix metalloproteinases (MMPs), in particular MMP-14, play an important role in remodelling of ECM and enhancing cell migration, their expression and activity were investigated in the cells grown on different ECM substrata. Lumican down-regulated the MMP-14 expression and activity in MSC, but not in EPC. Lumican inhibited MSC, but not EPC migration and invasion. The inhibition of MSC migration and invasion by lumican was reversed by MMP-14 overexpression. Conclusion/Significance Altogether, our results suggest that lumican inhibits MSC tube-like structure formation and migration via mechanisms that involve a decrease of MMP-14 expression and activity.

Downregulation of PMCA2 or PMCA3 reorganizes Ca2+ handling systems in differentiating PC12 cells

Changes in PMCA2 and PMCA3 expression during neuronal development are tightly linked to structural and functional modifications in Ca(2+) handling machinery. Using antisense strategy we obtained stably transfected PC12 lines with reduced level of PMCA2 or PMCA3, which were then subjected to dibutyryl-cAMP differentiation. Reduced level of neuron-specific PMCAs led to acceleration of differentiation and formation of longer neurites than in control PC12 line. Treatment with dibutyryl-cAMP was associated with retraction of growth cones and intensified formation of varicosities. In PMCA2-reduced cells development of apoptosis and DNA laddering were detected. Higher amounts of constitutive isoforms PMCA1 and PMCA4, their putative extended location to gaps left after partial removal of PMCA2 or PMCA3, together with increased SERCA may indicate the induction of compensatory mechanism in modified cells. Functional studies showed altered expression of certain types of VDCCs in PMCA-reduced cells, which correlated with their higher contribution to Ca(2+) influx. The cell response to PMCAs suppression suggests the interplay between transcription level of two opposite calcium-transporting systems i.e. voltage- and store depletion-activated channels facilitating Ca(2+) influx and calcium pumps responsible for Ca(2+) clearance, as well highlights the role of both neuron-specific PMCA isoforms in the control of PC12 cells differentiation.

Thymosin β4 regulates migration of colon cancer cells by a pathway involving interaction with Ku80

Aberrant expression of thymosin β4 (Tβ4) has recently been found to be associated with colorectal carcinoma (CRC) progression evidently due to an increase of the motility and invasion of tumor cells and the induction of a proangiogenic phenotype of endothelial cells. Both mechanisms depend upon matrix‐degrading proteases, particularly plasmin and matrix metalloproteinases (MMPs) that are responsible for extensive tissue remodeling. Cleavage of ECM macromolecules weakens the structural integrity of tissues and exposes cryptic domains of extracellular components, which elicit biological responses distinct from intact molecules. Interestingly, signaling via integrins (αVβ3, α5β1) in CRC cells (HT29, CX1.1) is induced by Tβ4 and VEGF‐A only when they grow in 3D fibrin gels but not in 2D ones. The cells growing in 3D fibrin gels release upon Tβ4 significant amounts of active MMPs (MMP‐2, MMP‐9, and MMP‐7) that cause extensive proteolysis in their close vicinity. As evidenced by a variety of approaches (transfection experiments, coimmunoprecipitation, gene silencing with siRNA), we found that this involves interaction of Tβ4 with Ku80, which has recently been described by us to mediate Tβ4 intracellular activity.

β-III tubulin modulates the behavior of Snail overexpressed during the epithelial-to-mesenchymal transition in colon cancer cells.

Class III β-tubulin (TUBB3) is a marker of drug resistance expressed in a variety of solid tumors. Originally, it was described as an important element of chemoresistance to taxanes. Recent studies have revealed that TUBB3 is also involved in an adaptive response to a microenvironmental stressor, e.g. low oxygen levels and poor nutrient supply in some solid tumors, independently of the microtubule targeting agent. Furthermore, it has been demonstrated that TUBB3 is a marker of biological aggressiveness associated with modulation of metastatic abilities in colon cancer. The epithelial-to-mesenchymal transition (EMT) is a basic cellular process by which epithelial cells lose their epithelial behavior and become invasive cells involved in cancer metastasis. Snail is a zinc-finger transcription factor which is able to induce EMT through the repression of E-cadherin expression. In the presented studies we focused on the analysis of the TUBB3 role in EMT-induced colon adenocarcinoma cell lines HT-29 and LS180. We observed a positive correlation between Snail presence and TUBB3 upregulation in tested adenocarcinoma cell lines. The cellular and behavioral analysis revealed for the first time that elevated TUBB3 level is functionally linked to increased cell migration and invasive capability of EMT induced cells. Additionally, the post-transcriptional modifications (phosphorylation, glycosylation) appear to regulate the cellular localization of TUBB3 and its phosphorylation, observed in cytoskeleton, is probably involved in cell motility modulation.

Senescent endothelial cells: Potential modulators of immunosenescence and ageing.

Recent studies have demonstrated that the accumulation of senescent endothelial cells may be the primary cause of cardiovascular diseases. Because of their multifunctional properties, endothelial cells actively take part in stimulating the immune system and inflammation. In addition, ageing is characterized by the progressive deterioration of immune cells and a decline in the activation of the immune response. This results in a loss of the primary function of the immune system, which is eliminating damaged/senescent cells and neutralizing potential sources of harmful inflammatory reactions. In this review, we discuss cellular senescence and the senescence-associated secretory phenotype (SASP) of endothelial cells and summarize the link between endothelial cells and immunosenescence. We describe the possibility that age-related changes in Toll-like receptors (TLRs) and microRNAs can affect the phenotypes of senescent endothelial cells and immune cells via a negative feedback loop aimed at restraining the excessive pro-inflammatory response. This review also addresses the following questions: how do senescent endothelial cells influence ageing or age-related changes in the inflammatory burden; what is the connection between ECs and immunosenescence, and what are the crucial hypothetical pathways linking endothelial cells and the immune system during ageing.

Decreased deformability in aging erythrocytes

Deformability of bovine erythrocytes separated according to density (and age) was estimated by a modified Teitels filterability test, the centrifugational test of Sirs, and viscosity measurements of cell suspensions. Both youngest and oldest erythrocytes were found to be less deformable than middle-aged cells, a result speaking against any chief role for deformability in the recognition of senescent erythrocytes and their removal from the circulation.

The ILK-MMP9-MRTF axis is crucial for EndMT differentiation of endothelial cells in a tumor microenvironment

Increasing evidence indicates that the tumor microenvironment is a critical factor supporting cancer progression, chemoresistance and metastasis. Recently, cancer-associated fibroblasts (CAFs) have been recognized as a crucial tumor stromal component promoting cancer growth and invasiveness via modulation of the extracellular matrix (ECM) structure, tumor metabolism and immune reprogramming. One of the main sources of CAFs are endothelial cells undergoing the endothelial-mesenchymal transition (EndMT). EndMT is mainly promoted by the Transforming Growth Factor-β (TGF-β) family secreted by tumor cells, though the role of particular members in EndMT regulation remains poorly understood. Our findings demonstrate that TGF-β2 induces mesenchymal transdifferentiation of human microvascular endothelial cells (HMEC-1 cells) to CAF-like cells in association with elongated cell morphology, modulation of stress fiber organization, higher α-SMA protein levels and activation of RhoA and Rac-1 pathways. Such regulation is similar to that observed in cells maintained using conditioned medium from invasive colorectal cancer cell line culture. Furthermore, TGF-β2 stimulation resulted in myocardin-related transcription factor (MRTF) activation and upregulation. Our results demonstrate for the first time that such interaction is sufficient for integrin-linked kinase (ILK) overexpression. ILK upregulation also enhanced MRTF activation via RhoA and Rac-1-MMP9 via inside-out integrin activation. Herein, we propose a new ILK-MMP9-MRTF axis that appears to be critical for EndMT differentiation of endothelial to CAF-like cells. Thus, it might be an attractive target for cancer treatment.

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

&NA; 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], &bgr;1 integrin, p&bgr;1 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. HighlightsFilamin A upregulation is observed in Snail‐transfected invasive colorectal cancer cells.Filamin A is present in full‐length form in the cytoplasm and co‐localizes with focal adhesions of invasive clones.Filamin A upregulation does not inhibit Snail‐stimulated cell migration.

Tubulin beta 3 and 4 are involved in the generation of early fibrotic stages

The endothelial-mesenchymal transition (EndMT) is a fundamental cellular mechanism that occurs under both physiological and pathological conditions and includes the fibrotic stages of numerous organs, namely, the skin, kidneys, heart, lungs and liver. Endothelial cells that undergo EndMT are one of the main source of (myo)fibroblasts in fibrotic tissues. A critical step in cellular transdifferentiation is morphological change, which is engineered by the reorganization of cytoskeletal elements such as microtubules. These dynamic structures consist of αβ-tubulin heterodimers that are also involved in cellular movement and intracellular trafficking, processes modulated during EndMT. One fundamental mechanism that underlies microtubule stabilization is the regulation of the levels of α and β-tubulin. However, little is known about the roles of specific tubulin isotypes in the development of EndMT-based diseases. This study provides the first evidence that the upregulation of TUBB3 and TUBB4 is coupled with increased cell migration in EndMT-induced HMEC-1 cells. Immunochemical analysis reveals that these tubulins are upregulated in the early stages of EndMT, and siRNA analysis indicates that they are engaged in the generation of mesenchymal behavior via the enhancement of cell migration. This modulation seems to be especially important in wound healing. Finally, cell surface analysis reveals that TUBB3 and TUBB4 are necessary for the transport and proper localization of N-cadherin within the plasma membrane. We believe that our results will be valuable for the development of effective new anti-fibrotic therapies.