Transmembrane/cytoplasmic domain-mediated membrane type 1-matrix metalloprotease docking to invadopodia is required for cellinvasion

The invasion of human malignant melanoma cells into the extracellular matrix (ECM) involves the accumulation of proteases at sites of ECM degradation where activation of matrix metalloproteases (MMP) occurs. Here, we show that when membrane type 1 MMP (MT-MMP) was overexpressed in RPMI7951 human melanoma cells, the cells made contact with the ECM, activated soluble and ECM-bound MMP-2, and degraded and invaded the ECM. Further experiments demonstrated the importance of localization of the MT-MMP to invadopodia. Overexpression of MT-MMP without invadopodial localization caused activation of soluble MMP-2, but did not facilitate ECM degradation or cell invasiveness. Up-regulation of endogenous MT-MMP with concanavalin A caused activation of MMP-2. However, concanavalin A treatment prevented invadopodial localization of MT-MMP and ECM degradation. Neither a truncated MT-MMP mutant lacking transmembrane (TM) and cytoplasmic domains (ΔTM(MT-MMP)), nor a chimeric MT-MMP containing the interleukin 2 receptor α chain (IL-2R) TM and cytoplasmic domains (ΔTM(MT-MMP)/TM(IL-2R)) were localized to invadopodia or exhibited ECM degradation. Furthermore, a chimera of the TM/cytoplasmic domain of MT-MMP (TM(MT-MMP)) with tissue inhibitor of MMP 1 (TIMP-1/TM(MT- MMP)) directed the TIMP-1 molecule to invadopodia. Thus, the MT-MMP TM/cytoplasmic domain mediates the spatial organization of MT-MMP into invadopodia and subsequent degradation of the ECM.

Progression of human breast cancer cells from hormone-dependent to hormone-independent growth both in vitro and in vivo

We have isolated a series of sublines of the hormone-dependent MCF-7 human breast cancer cell line after selection both in vivo and in vitro for growth in the presence of subphysiological concentrations of estrogens. These sublines represent a model system for study of the processes leading to hormonal autonomy. The cells form growing tumors in ovariectomized athymic nude mice in the absence of estrogen supplementation but retain some responsivity to estrogen as determined by stimulation of the rate of tumor growth in vivo and by induction of progesterone receptor. An ovarian-independent but hormone-responsive phenotype may occur early in the natural progression to hormone-independent and unresponsive growth in breast cancer. We observed no change in the affinity or decrease in the level of expression of estrogen receptors and progesterone receptors among the sublines and the parental cells. Epidermal growth factor receptors are not overexpressed in ovarian-independent cells. Thus, altered hormone receptor expression may be a late event in the acquisition of a hormone-independent and unresponsive phenotype. Sublines isolated by in vivo but not in vitro selection are more invasive than the parental cells both in vivo and across an artificial basement membrane in vitro. Thus, as yet unknown tumor-host interactions may be important in the development of an invasive phenotype. Furthermore, acquisition of the ovarian-independent and invasive phenotypes can occur independently.

Implication of collagen type I-induced membrane-type 1-matrix metalloproteinase expression and matrix metalloproteinase-2 activation in the metastatic progression of breast carcinoma

We have previously demonstrated that fibroblasts and invasive human breast carcinoma (HBC) cells specifically activate matrix metalloproteinase- 2 (MMP-2) when cultured on 3-dimensional gels of type I collagen but not a range of other substrates. We show here the constitutive expression of membrane-type 1 (MT1)-MMP in both fibroblasts, and invasive HBC cell lines, that have fibroblastic attributes presumably acquired through an epithelial- to-mesenchymal transition (EMT). Treatment with collagen type I increased the steady-state MT1-MMP mRNA levels in these cells but did not induce either MT1-MMP expression or MMP-2 activation in noninvasive breast carcinoma cell lines, which retain epithelial features. Basal MT3-MMP mRNA expression had a pattern similar to that of MT1-MMP but was not up-regulated by collagen. MT4- MMP mRNA was seen in both invasive and noninvasive HBC cell lines and was also not collagen-regulated, and MT2-MMP mRNA was not detected in any of the HBC cell lines tested. These data support a role for MT1-MMP in the collagen- induced MMP-2-activation seen in these cells. In situ hybridization analysis of archival breast cancer specimens revealed a close parallel in expression of both collagen type I and MT1-MMP mRNA in peritumoral fibroblasts, which was correlated with aggressiveness of the lesion. Relatively high levels of expression of both mRNA species were seen in fibroblasts close to invasive tumor nests and, although only focally, in certain areas close to preinvasive tumors. These foci may represent hot spots for local degradation and invasive progression. Collectively, these results implicate MT1-MMP in collagen- stimulated MMP-2 activation and suggest that this mechanism may be employed in vivo by both tumor-associated fibroblasts and EMT-derived carcinoma cells to facilitate increased invasion and/or metastasis.

Anti-proliferative and cytotoxic activity of pentadactylin isolated from Leptodactylus labyrinthicus on melanoma cells

Nowadays, the emergence of resistance to the current available chemotherapeutic drugs by cancer cells makes the development of new agents imperative. The skin secretion of amphibians is a natural rich source of antimicrobial peptides (AMP), and researchers have shown that some of these wide spectrum molecules are also toxic to cancer cells. The aim of this study was to verify a putative anticancer activity of the AMP pentadactylin isolated for the first time from the skin secretion of the frog Leptodactylus labyrinthicus and also to study its cytotoxic mechanism to the murine melanoma cell line B16F10. The results have shown that pentadactylin reduces the cell viability of B16F10 cells in a dose-dependent manner. It was also cytotoxic to normal human fibroblast cells; nevertheless, pentadactylin was more potent in the first case. The studies of action mechanism revealed that pentadactylin causes cell morphology alterations (e.g., round shape and shrinkage morphology), membrane disruption, DNA fragmentation, cell cycle arrest at the S phase, and alteration of mitochondrial membrane potential, suggesting that B16F10 cells die by apoptosis. The exact mechanism that causes reduction of cell viability and cytotoxicity after treatment with pentadactylin is still unknown. In conclusion, as cancer cells become resilient to death, it is worthwhile the discovery of new drugs such as pentadactylin that induces apoptosis.

A coupled SPH-DEM model for micro-scale structural deformations of plant cells during drying

A single plant cell was modeled with smoothed particle hydrodynamics (SPH) and a discrete element method (DEM) to study the basic micromechanics that govern the cellular structural deformations during drying. This two-dimensional particle-based model consists of two components: a cell fluid model and a cell wall model. The cell fluid was approximated to a highly viscous Newtonian fluid and modeled with SPH. The cell wall was treated as a stiff semi-permeable solid membrane with visco-elastic properties and modeled as a neo-Hookean solid material using a DEM. Compared to existing meshfree particle-based plant cell models, we have specifically introduced cell wall–fluid attraction forces and cell wall bending stiffness effects to address the critical shrinkage characteristics of the plant cells during drying. Also, a moisture domain-based novel approach was used to simulate drying mechanisms within the particle scheme. The model performance was found to be mainly influenced by the particle resolution, initial gap between the outermost fluid particles and wall particles and number of particles in the SPH influence domain. A higher order smoothing kernel was used with adaptive smoothing length to improve the stability and accuracy of the model. Cell deformations at different states of cell dryness were qualitatively and quantitatively compared with microscopic experimental findings on apple cells and a fairly good agreement was observed with some exceptions. The wall–fluid attraction forces and cell wall bending stiffness were found to be significantly improving the model predictions. A detailed sensitivity analysis was also done to further investigate the influence of wall–fluid attraction forces, cell wall bending stiffness, cell wall stiffness and the particle resolution. This novel meshfree based modeling approach is highly applicable for cellular level deformation studies of plant food materials during drying, which characterize large deformations.

Transplantation of human amnion epithelial cells reduces hepatic fibrosis in immunocompetent CCl4-treated mice

Chronic liver injury and inflammation lead to hepatic fibrosis, cirrhosis, and liver failure. Embryonic and mesenchymal stem cells have been shown to reduce experimental liver fibrosis but have potential limitations, including the formation of dysplastic precursors, tumors, and profibrogenic cells. Other stem-like cells may reduce hepatic inflammation and fibrosis without tumor and profibrogenic cell formation. To test this hypothesis we transplanted human amnion epithelial cells (hAEC), isolated from term delivered placenta, into immunocompetent C57/BL6 mice at week 2 of a 4-week regimen of carbon tetrachloride (CCl4) exposure to induce liver fibrosis. Two weeks following hAEC infusion, intact cells expressing the human-specific markers inner mitochondrial membrane protein and human leukocyte antigen-G were found in mouse liver without evidence of host rejection of the transplanted cells. Human albumin, known to be produced by hAEC, was detected in sera of hAEC-treated mice. Human DNA was detected in mouse liver and also spleen, lungs, and heart of some animals. Following hAEC transplantation, CCl4-treated animals showed decreased serum ALT levels and reduced hepatocyte apoptosis, compared to controls. hAEC-treated mouse liver had lower TNF-α and IL-6 protein levels and higher IL-10 compared to animals given CCl4 alone. Compared to CCl4 controls, hAEC-treated mice showed fewer activated collagen-producing hepatic stellate cells and less fibrosis area and collagen content. Reduced hepatic TGF-β levels in conjunction with a twofold increase in the active form of the collagen-degrading enzyme matrix metalloproteinase-2 in hAEC-treated mice compared to CCl4 controls may account for the reduction in fibrosis. hAEC transplantation into immunocompetent mice leads to cell engraftment, reduced hepatocyte apoptosis, and decreased hepatic inflammation and fibrosis.

Phosphorylation of caveolin-1 is anti-apoptotic and promotes cell attachment during oxidative stress of kidney cells

Aims: Caveolin-1 (cav1) is reported to have both cell survival and pro-apoptotic characteristics. This may be explained by its localisation or phosphorylation in injured cells. This study investigated the role of cav1 in kidney cells of different nephron origin and developmental state after oxidative stress. Methods: Renal MCDK distal tubular, HK2 proximal tubular epithelial cells and HEK293T renal embryonic cells were treated with 1mM hydrogen peroxide. Apoptosis, loss of cell adhesion, and cell survival were compared with expression of cav1 in its non-phosphorylated and phosphorylated (p-cav1) forms. Cav1 was transfected into the HEK293T cells, or caveolae were disrupted with filipin or nystatin in HK2 cells, to investigate functions of cav1 and p-cav1. Results: Oxidative stress induced more apoptosis in HK2s than MDCKs (p<0.05). HK2s had lower endogenous cav1 and p-cav1 than MDCKs (p<0.05). Both cell lines had increased p-cav1, but not cav1, with oxidative stress. This increase was greatest in MDCKs (p<0.01). Cav1 was located mainly in the plasma membrane of untreated cells and translocated to the cytoplasm with oxidative stress in both cell lines, more so in MDCKs. Disruption of caveolae caused cytoplasmic translocation of cav1 in HK2s, but did not alter high levels of oxidative stress-induced apoptosis. When HEK293Ts lacking endogenous cav1 were transfected with cav1, oxidant-induced apoptosis and loss of cell adhesion was decreased (p<0.01), and p-cav1 was induced by treatment. Conclusion: Cav1 expression and localisation in kidney cells is not anti-apoptotic, but increased expression of p-cav1 may promote cell survival after oxidative stress. © 2008 Royal College of Pathologists of Australasia.

GABAB receptors expressed in human aortic endothelial cells mediate intracellular calcium concentration regulation and endothelial nitric oxide synthase translocation

GABAB receptors regulate the intracellular Ca2+ concentration ([Ca2+]i) in a number of cells (e.g., retina, airway epithelium and smooth muscle), but whether they are expressed in vascular endothelial cells and similarly regulate the [Ca2+]i is not known. The purpose of this study was to investigate the expression of GABAB receptors, a subclass of receptors to the inhibitory neurotransmitter γ-aminobutyric acid (GABA), in cultured human aortic endothelial cells (HAECs), and to explore if altering receptor activation modified [Ca2+]i and endothelial nitric oxide synthase (eNOS) translocation. Real-time PCR, western blots and immunofluorescence were used to determine the expression of GABAB1 and GABAB2 in cultured HAECs. The effects of GABAB receptors on [Ca2+]i in cultured HAECs were demonstrated using fluo-3. The influence of GABAB receptors on eNOS translocation was assessed by immunocytochemistry. Both GABAB1 and GABAB2 mRNA and protein were expressed in cultured HAECs, and the GABAB1 and GABAB2 proteins were colocated in the cell membrane and cytoplasm. One hundred μM baclofen caused a transient increase of [Ca2+]i and eNOS translocation in cultured HAECs, and the effects were attenuated by pretreatment with the selective GABAB receptor antagonists CGP46381 and CGP55845. GABAB receptors are expressed in HAECs and regulate the [Ca2+]i and eNOS translocation. Cultures of HAECs may be a useful in vitro model for the study of GABAB receptors and vascular biology.

GABAB1 and GABAB2 receptor subunits co-expressed in cultured human RPE cells regulate intracellular Ca2+ via Gi/o-protein and phospholipase C pathways

GABAB receptors associate with Gi/o-proteins that regulate voltage-gated Ca(2+) channels and thus the intracellular Ca(2+) concentration ([Ca(2+)]i), there is also reported cross-regulation of phospholipase C. These associations have been studied extensively in the brain and also shown to occur in non-neural cells (e.g. human airway smooth muscle). More recently GABAB receptors have been observed in chick retinal pigment epithelium (RPE). The aims were to investigate whether the GABAB receptor subunits, GABAB1 and GABAB2, are co-expressed in cultured human RPE cells, and then determine if the GABAB receptor similarly regulates the [Ca(2+)]i of RPE cells and if phospholipase C is involved. Human RPE cells were cultured from 5 donor eye cups. Evidence for GABAB1 and GABAB2 mRNAs and proteins in the RPE cell cultures were investigated using real time PCR, western blots and immunofluorescence. The effects of the GABAB receptor agonist baclofen, antagonist CGP46381, a Gi/o-protein inhibitor pertussis toxin, and the phospholipase C inhibitor U73122 on [Ca(2+)]i in cultured human RPE were demonstrated using Fluo-3. Both GABAB1 and GABAB2 mRNA and protein were identified in cell cultures of human RPE; antibody staining was co-localized to the cell membrane and cytoplasm. One-hundred μM baclofen caused a transient increase in the [Ca(2+)]i of RPE cells regardless of whether Ca(2+) was added to the buffer. Baclofen induced increases in the [Ca(2+)]i were attenuated by pre-treatment with CGP46381, pertussis toxin, and U73122. GABAB1 and GABAB2 are co-expressed in cell cultures of human RPE. GABAB receptors in RPE regulate the [Ca(2+)]i via a Gi/o-protein and phospholipase C pathway.

Formation of the three-dimensional geometry of the red blood cell membrane

Red blood cells (RBCs) are nonnucleated liquid capsules, enclosed in deformable viscoelastic membranes with complex three dimensional geometrical structures. Generally, RBC membranes are highly incompressible and resistant to areal changes. However, RBC membranes show a planar shear deformation and out of plane bending deformation. The behaviour of RBCs in blood vessels is investigated using numerical models. All the characteristics of RBC membranes should be addressed to develop a more accurate and stable model. This article presents an effective methodology to model the three dimensional geometry of the RBC membrane with the aid of commercial software COMSOL Multiphysics 4.2a and Fortran programming. Initially, a mesh is generated for a sphere using the COMSOL Multiphysics software to represent the RBC membrane. The elastic energy of the membrane is considered to determine a stable membrane shape. Then, the actual biconcave shape of the membrane is obtained based on the principle of virtual work, when the total energy is minimised. The geometry of the RBC membrane could be used with meshfree particle methods to simulate motion and deformation of RBCs in micro-capillaries

Incorporation of human recombinant tropoelastin into silk fibroin membranes with the view to repairing Bruch’s membrane

Bombyx mori silk fibroin membranes provide a potential delivery vehicle for both cells and extracellular matrix (ECM) components into diseased or injured tissues. We have previously demonstrated the feasibility of growing retinal pigment epithelial cells (RPE) on fibroin membranes with the view to repairing the retina of patients afflicted with age-related macular degeneration (AMD). The goal of the present study was to investigate the feasibility of incorporating the ECM component elastin, in the form of human recombinant tropoelastin, into these same membranes. Two basic strategies were explored: (1) membranes prepared from blended solutions of fibroin and tropoelastin; and (2) layered constructs prepared from sequentially cast solutions of fibroin, tropoelastin, and fibroin. Optimal conditions for RPE attachment were achieved using a tropoelastin-fibroin blend ratio of 10 to 90 parts by weight. Retention of tropoelastin within the blend and layered constructs was confirmed by immunolabelling and Fourier-transform infrared spectroscopy (FTIR). In the layered constructs, the bulk of tropoelastin was apparently absorbed into the initially cast fibroin layer. Blend membranes displayed higher elastic modulus, percentage elongation, and tensile strength (p < 0.01) when compared to the layered constructs. RPE cell response to fibroin membranes was not affected by the presence of tropoelastin. These findings support the potential use of fibroin membranes for the co-delivery of RPE cells and tropoelastin.

Pancreatic islet basement membrane loss and remodeling after mouse islet isolation and transplantation: Impact for allograft rejection

The isolation of islets by collagenase digestion can cause damage and impact the efficiency of islet engraftment and function. In this study, we assessed the basement membranes (BMs) of mouse pancreatic islets as a molecular biomarker for islet integrity, damage after isolation, and islet repair in vitro as well as in the absence or presence of an immune response after transplantation. Immunofluorescence staining of BM matrix proteins and the endothelial cell marker platelet endothelial cell adhesion molecule-1 (PECAM-1) was performed on pancreatic islets in situ, isolated islets, islets cultured for 4 days, and islet grafts at 3-10 days posttransplantation. Flow cytometry was used to investigate the expression of BM matrix proteins in isolated islet β-cells. The islet BM, consisting of collagen type IV and components of Engelbreth-Holm-Swarm (EHS) tumor laminin 111, laminin α2, nidogen-2, and perlecan in pancreatic islets in situ, was completely lost during islet isolation. It was not reestablished during culture for 4 days. Peri- and intraislet BM restoration was identified after islet isotransplantation and coincided with the migration pattern of PECAM-1(+) vascular endothelial cells (VECs). After islet allotransplantation, the restoration of VEC-derived peri-islet BMs was initiated but did not lead to the formation of the intraislet vasculature. Instead, an abnormally enlarged peri-islet vasculature developed, coinciding with islet allograft rejection. The islet BM is a sensitive biomarker of islet damage resulting from enzymatic isolation and of islet repair after transplantation. After transplantation, remodeling of both peri- and intraislet BMs restores β-cell-matrix attachment, a recognized requirement for β-cell survival, for isografts but not for allografts. Preventing isolation-induced islet BM damage would be expected to preserve the intrinsic barrier function of islet BMs, thereby influencing both the effector mechanisms required for allograft rejection and the antirejection strategies needed for allograft survival.

Comparative proteomics of uropathogenic Escherichia coli during growth in human urine identify UCA-like (UCL) fimbriae as an adherence factor involved in biofilm formation and binding to uroepithelial cells

Uropathogenic Escherichia coli (UPEC) are the primary cause of urinary tract infection (UTI) in humans. For the successful colonisation of the human urinary tract, UPEC employ a diverse collection of secreted or surface-exposed virulence factors including toxins, iron acquisition systems and adhesins. In this study, a comparative proteomic approach was utilised to define the UPEC pan and core surface proteome following growth in pooled human urine. Identified proteins were investigated for subcellular origin, prevalence and homology to characterised virulence factors. Fourteen core surface proteins were identified, as well as eleven iron uptake receptor proteins and four distinct fimbrial types, including type 1, P, F1C/S and a previously uncharacterised fimbrial type, designated UCA-like (UCL) fimbriae in this study. These pathogenicity island (PAI)-associated fimbriae are related to UCA fimbriae of Proteus mirabilis, associated with UPEC and exclusively found in members of the E. coli B2 and D phylogroup. We further demonstrated that UCL fimbriae promote significant biofilm formation on abiotic surfaces and mediate specific attachment to exfoliated human uroepithelial cells. Combined, this study has defined the surface proteomic profiles and core surface proteome of UPEC during growth in human urine and identified a new type of fimbriae that may contribute to UTI.

GABAAα1 and GABAAρ1 subunits are expressed in cultured human RPE cells and GABAA receptor agents modify the intracellular calcium concentration.

Purpose: Gamma-aminobutyric acid A (GABAA) receptors (GABAARs), which are ionotropic receptors involving chloride channels, have been identified in various neural (e.g., mouse retinal ganglion cells) and nonneural cells (e.g., mouse lens epithelial cells) regulating the intracellular calcium concentration ([Ca(2+)]i). GABAAR β-subunit protein has been isolated in the cultured human and rat RPE, and GABAAα1 and GABAAρ1 mRNAs and proteins are present in the chick RPE. The purpose of this study was to investigate the expression of GABAAα1 and GABAAρ1, two important subunits in forming functional GABAARs, in the cultured human RPE, and further to explore whether altering receptor activation modifies [Ca(2+)]i. Methods: Human RPE cells were separately cultured from five donor eye cups. Real-time PCR, western blots, and immunofluorescence were used to test for GABAAα1 and GABAAρ1 mRNAs and proteins. The effects of the GABAAR agonist muscimol, antagonist picrotoxin, or the specific GABAAρ antagonist 1,2,5,6-tetrahydropyridin-4-yl) methylphosphinic acid (TPMPA) on [Ca(2+)]i in cultured human RPE were demonstrated using Fluo3-AM. Results: Both GABAAα1 and GABAAρ1 mRNAs and proteins were identified in cultured human RPE cells; antibody staining was mainly localized to the cell membrane and was also present in the cytoplasm but not in the nucleus. Muscimol (100 μM) caused a transient increase of the [Ca(2+)]i in RPE cells regardless of whether Ca(2+) was added to the buffer. Muscimol-induced increases in the [Ca(2+)]i were inhibited by pretreatment with picrotoxin (300 μM) or TPMPA (500 μM). Conclusions: GABAAα1 and GABAAρ1 are expressed in cultured human RPE cells, and GABAA agents can modify [Ca(2+)]i.

A coupled SPH-DEM approach to model the interactions between multiple red blood cells in motion in capillaries

Red blood cells (RBCs) are the most common type of blood cells in the blood and 99% of the blood cells are RBCs. During the circulation of blood in the cardiovascular network, RBCs squeeze through the tiny blood vessels (capillaries). They exhibit various types of motions and deformed shapes, when flowing through these capillaries with diameters varying between 5 10 µm. RBCs occupy about 45 % of the whole blood volume and the interaction between the RBCs directly influences on the motion and the deformation of the RBCs. However, most of the previous numerical studies have explored the motion and deformation of a single RBC when the interaction between RBCs has been neglected. In this study, motion and deformation of two 2D (two-dimensional) RBCs in capillaries are comprehensively explored using a coupled smoothed particle hydrodynamics (SPH) and discrete element method (DEM) model. In order to clearly model the interactions between RBCs, only two RBCs are considered in this study even though blood with RBCs is continuously flowing through the blood vessels. A spring network based on the DEM is employed to model the viscoelastic membrane of the RBC while the inside and outside fluid of RBC is modelled by SPH. The effect of the initial distance between two RBCs, membrane bending stiffness (Kb) of one RBC and undeformed diameter of one RBC on the motion and deformation of both RBCs in a uniform capillary is studied. Finally, the deformation behavior of two RBCs in a stenosed capillary is also examined. Simulation results reveal that the interaction between RBCs has significant influence on their motion and deformation.