Cell-extracellular Matrix Contacts Research Articles

Epigenetic Modifications as Antidedifferentiation Strategy for Primary Hepatocytes in Culture.

A well-known problem of cultured primary hepatocytes is their rapid dedifferentiation. During the last years, several strategies to counteract this phenomenon have been developed, of which changing the in vitro environment is the most popular one. However, mimicking the in vivo setting in vitro by adding soluble media additives or the restoration of both cell-cell and cell-extracellular matrix contacts is not sufficient and only delays the dedifferentiation process instead of counteracting it. In this chapter, new strategies to prevent the deterioration of the liver-specific phenotype of primary hepatocytes in culture by targeting the (epi)genetic mechanisms that drive hepatocellular gene expression are described.

Mechanical Checkpoint For Persistent Cell Polarization In Adhesion-Naive Fibroblasts

Cell polarization is a fundamental biological process implicated in nearly every aspect of multicellular development. The role of cell-extracellular matrix contacts in the establishment and the orientation of cell polarity have been extensively studied. However, the respective contributions of substrate mechanics and biochemistry remain unclear. Here we propose a believed novel single-cell approach to assess the minimal polarization trigger. Using nonadhered round fibroblast cells, we show that stiffness sensing through single localized integrin-mediated cues are necessary and sufficient to trigger and direct a shape polarization. In addition, the traction force developed by cells has to reach a minimal threshold of 56 ± 1.6 pN for persistent polarization. The polarization kinetics increases with the stiffness of the cue. The polarized state is characterized by cortical actomyosin redistribution together with cell shape change. We develop a physical model supporting the idea that a local and persistent inhibition of actin polymerization and/or myosin activity is sufficient to trigger and sustain the polarized state. Finally, the cortical polarity propagates to an intracellular polarity, evidenced by the reorientation of the centrosome. Our results define the minimal adhesive requirements and quantify the mechanical checkpoint for persistent cell shape and organelle polarization, which are critical regulators of tissue and cell development.

Musculoskeletal Regenerative Engineering: Biomaterials, Structures, and Small Molecules

Musculoskeletal tissues are critical to the normal functioning of an individual and following damage or degeneration they show extremely limited endogenous regenerative capacity. The future of regenerative medicine is the combination of advanced biomaterials, structures, and cues to re-engineer/guide stem cells to yield the desired organ cells and tissues. Tissue engineering strategies were ideally suited to repair damaged tissues; however, the substitution and regeneration of large tissue volumes and multi-level tissues such as complex organ systems integrated into a single phase require more than optimal combinations of biomaterials and biologics. We highlight bioinspired advancements leading to novel regenerative scaffolds especially for musculoskeletal tissue repair and regeneration. Tissue and organ regeneration relies on the spatial and temporal control of biophysical and biochemical cues, including soluble molecules, cell-cell contacts, cell-extracellular matrix contacts, and physical forces. Strategies that recapitulate the complexity of the local microenvironment of the tissue and the stem cell niche play a crucial role in regulating cell self-renewal and differentiation. Biomaterials and scaffolds based on biomimicry of the native tissue will enable convergence of the advances in materials science, the advances in stem cell science, and our understanding of developmental biology.

Merlin deficiency predicts FAK inhibitor sensitivity: a synthetic lethal relationship.

The goal of targeted therapy is to match a selective drug with a genetic lesion that predicts for drug sensitivity. In a diverse panel of cancer cell lines, we found that the cells most sensitive to focal adhesion kinase (FAK) inhibition lack expression of the neurofibromatosis type 2 (NF2) tumor suppressor gene product, Merlin. Merlin expression is often lost in malignant pleural mesothelioma (MPM), an asbestos-induced aggressive cancer with limited treatment options. Our data demonstrate that low Merlin expression predicts for increased sensitivity of MPM cells to a FAK inhibitor, VS-4718, in vitro and in tumor xenograft models. Disruption of MPM cell-cell or cell-extracellular matrix (ECM) contacts with blocking antibodies suggests that weak cell-cell adhesions in Merlin-negative MPM cells underlie their greater dependence on cell-ECM-induced FAK signaling. This provides one explanation of why Merlin-negative cells are vulnerable to FAK inhibitor treatment. Furthermore, we validated aldehyde dehydrogenase as a marker of cancer stem cells (CSCs) in MPM, a cell population thought to mediate tumor relapse after chemotherapy. Whereas pemetrexed and cisplatin, standard-of-care agents for MPM, enrich for CSCs, FAK inhibitor treatment preferentially eliminates these cells. These preclinical results provide the rationale for a clinical trial in MPM patients using a FAK inhibitor as a single agent after first-line chemotherapy. With this design, the FAK inhibitor could potentially induce a more durable clinical response through reduction of CSCs along with a strong antitumor effect. Furthermore, our data suggest that patients with Merlin-negative tumors may especially benefit from FAK inhibitor treatment.

Thermoresponsive Worms for Expansion and Release of Human Embryonic Stem Cells

The development of robust suspension cultures of human embryonic stem cells (hESCs) without the use of cell membrane disrupting enzymes or inhibitors is critical for future clinical applications in regenerative medicine. We have achieved this by using long, flexible, and thermoresponsive polymer worms decorated with a recombinant vitronectin subdomain that bridge hESCs, aiding in hESC's natural ability to form embryoid bodies (EBs) and satisfying their inherent requirement for cell-cell and cell-extracellular matrix contact. When the EBs reached an optimal upper size where cytokine and nutrient penetration becomes limiting, these long and flexible polymer worms facilitated EB breakdown via a temperature shift from 37 to 25 °C. The thermoresponsive nature of the worms enabled a cyclical dissociation and propagation of the cells. Repeating the process for three cycles (over eighteen days) provided a >30-fold expansion in cell number while maintaining pluripotency, thereby providing a simple, nondestructive process for the 3D expansion of hESC.

Real-time quantitative polymerase chain reaction and flow cytometric analyses of cell adhesion molecules expressed in human cell–multilayered periosteal sheets in vitro

Background aimsCultured human periosteal sheets more effectively function as an osteogenic grafting material at implantation sites than do dispersed periosteal cells. Because adherent cell growth and differentiation are regulated by cell-cell and cell–extracellular matrix contacts, we hypothesized that this advantage is a result of the unique cell adhesion pattern formed by their multiple cell layers and abundant extracellular matrix. To test this hypothesis, we prepared three distinct forms of periosteal cell cultures: three-dimensional cell-multilayered periosteal sheets, two-dimensional dispersed cell cultures, and three-dimensional hybrid mock-ups of cells dispersed onto collagen sponges. MethodsPeriosteal cells were obtained from human alveolar bone. Cell adhesion and extracellular matrix molecules were quantitatively determined at the messenger RNA and protein levels by means of real-time quantitative polymerase chain reaction and flow cytometry, respectively. ResultsReal-time quantitative polymerase chain reaction analysis demonstrated that regardless of culture media α1 integrin, vascular cell adhesion molecule-1, fibronectin and collagen type 1 were substantially upregulated, whereas CD44 was strongly downregulated in periosteal sheets compared with dispersed cell monolayers. With increased thickness, stem cell medium upregulated several integrins (β1, α1 and α4), CD146, vascular cell adhesion molecule-1, fibronectin and collagen type 1 in the periosteal sheets. Flow cytometric analysis revealed that the active configuration of β1 integrin was substantially downregulated in the stem cell medium–expanded cell cultures. The cell adhesion pattern found in the mock-up cultures was almost identical to that of genuine periosteal sheets. ConclusionsIntegrin α1β1 and CD44 function as the main cell adhesion molecule in highly cell-multilayered periosteal sheets and dispersed cells, respectively. This difference may account for the more potent osteogenic activity shown by the thicker periosteal sheets.

Abstract C262: Malignant mesothelioma lacking merlin shows enhanced sensitivity to the FAK inhibitor defactinib (VS-6063): Elucidation of the merlin-FAK relationship.

Abstract Malignant pleural mesothelioma (MPM) is an aggressive tumor in the lining of the lung often resulting from prior exposure to asbestos. Median overall survival with the standard of care chemotherapy is only 12 months from diagnosis. Thus, new therapeutic modalities are urgently needed to improve the prognosis of MPM patients. ∼50% of MPM patients exhibit biallelic inactivation of the NF2 tumor suppressor gene by mutation and/or deletion resulting in lack of expression of functional merlin protein. Merlin inactivation is a critical step in MPM tumorigenesis and is related, at least in part, to up-regulation of focal adhesion kinase (FAK) activity. FAK has also been demonstrated to play a role in maintenance of cancer stem cells (CSCs), a subpopulation of tumor cells associated with drug resistance and tumor recurrence. Therefore, therapeutic targeting of FAK using defactinib (VS-6063), a potent, orally active FAK kinase inhibitor, may benefit cancer patients through targeting of both CSCs and bulk tumor cells. Among a panel of MPM cell lines in 3-dimensional Matrigel culture, MPM lines lacking expression of merlin were found to be especially sensitive to defactinib. In contrast, MPM cell lines with wild-type merlin expression were less sensitive with average EC50 values of 5.1 μM. Oral dosing of defactinib induced significant tumor growth inhibition in a merlin-negative MPM model pre-implanted in the lungs of mice. Using Aldefluor activity as a marker for CSCs in MPM, we find that chemotherapeutic agents enriched for Aldefluor-positive CSCs, while the FAK inhibitor defactinib reduced the proportion of CSCs. When used in combination with pemetrexed or cisplatin, defactinib blocked the enrichment of CSCs cells by these chemotherapeutic agents. Disruption of cell-cell or cell-extracellular matrix (ECM) contacts with blocking antibodies allowed us to propose a molecular model to account for the observed merlin-FAK inhibitor synthetic lethal relationship. We find that weak cell-cell adhesions in merlin-negative MPM cells lead to a greater dependence of tumor cell survival and proliferation on cell-ECM-induced FAK signaling rendering merlin-negative cells especially vulnerable to FAK inhibitor treatment. Taken together, these data support the clinical development of defactinib in an ongoing Phase 2 registration-directed clinical trial evaluating the activity of defactinib in MPM patients (stratified by merlin status) with a confirmed response (PR or SD) following first line platinum/pemetrexed therapy. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C262. Citation Format: Irina M. Shapiro, Vihren N. Kolev, Yuwaraj Kadariya, Christian M. Vidal, Quentin G. Wright, Jennifer E. Ring, Craig Menges, Joseph R. Testa, Qunli Xu, Jonathan A. Pachter. Malignant mesothelioma lacking merlin shows enhanced sensitivity to the FAK inhibitor defactinib (VS-6063): Elucidation of the merlin-FAK relationship. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C262.

PP2A regulatory subunit Bα controls endothelial contractility and vessel lumen integrity via regulation of HDAC7

To supply tissues with nutrients and oxygen, the cardiovascular system forms a seamless, hierarchically branched, network of lumenized tubes. Here, we show that maintenance of patent vessel lumens requires the Bα regulatory subunit of protein phosphatase 2A (PP2A). Deficiency of Bα in zebrafish precludes vascular lumen stabilization resulting in perfusion defects. Similarly, inactivation of PP2A-Bα in cultured ECs induces tubulogenesis failure due to alteration of cytoskeleton dynamics, actomyosin contractility and maturation of cell-extracellular matrix (ECM) contacts. Mechanistically, we show that PP2A-Bα controls the activity of HDAC7, an essential transcriptional regulator of vascular stability. In the absence of PP2A-Bα, transcriptional repression by HDAC7 is abrogated leading to enhanced expression of the cytoskeleton adaptor protein ArgBP2. ArgBP2 hyperactivates RhoA causing inadequate rearrangements of the EC actomyosin cytoskeleton. This study unravels the first specific role for a PP2A holoenzyme in development: the PP2A-Bα/HDAC7/ArgBP2 axis maintains vascular lumens by balancing endothelial cytoskeletal dynamics and cell-matrix adhesion.

The regulation of cell polarity in the progression of lung cancer

Lung cancer is the most frequent malignant disease, since it has often metastasized to distant organs by the time of diagnosis. Epithelial-mesenchymal transition (EMT) is an important process during the progression of lung cancer. Epithelial cells lose the polarity, which contributes to uncontrolled invasion and metastasis of cancer cells. Cell polarity establishment and maintenance depends upon the three complex proteins which are par, crumbs and scribble complexes, of which are reported as tumor suppressors. The cell polarity proteins could interact with cell-cell contact and cell-extracellular matrix contact and cell-intrinsic signaling. These interactions are proved to be involved in lung cancer metastasis. However, our understanding of the mechanisms by which this occurs is poor. In this review, we will discuss the regulatory network of cell polarity in the lung cancer, especially on EMT.

An in-depth map of polyadenylation sites in cancer.

We present a comprehensive map of over 1 million polyadenylation sites and quantify their usage in major cancers and tumor cell lines using direct RNA sequencing. We built the Expression and Polyadenylation Database to enable the visualization of the polyadenylation maps in various cancers and to facilitate the discovery of novel genes and gene isoforms that are potentially important to tumorigenesis. Analyses of polyadenylation sites indicate that a large fraction (∼30%) of mRNAs contain alternative polyadenylation sites in their 3′ untranslated regions, independent of the cell type. The shortest 3′ untranslated region isoforms are preferentially upregulated in cancer tissues, genome-wide. Candidate targets of alternative polyadenylation-mediated upregulation of short isoforms include POLR2K, and signaling cascades of cell–cell and cell–extracellular matrix contact, particularly involving regulators of Rho GTPases. Polyadenylation maps also helped to improve 3′ untranslated region annotations and identify candidate regulatory marks such as sequence motifs, H3K36Me3 and Pabpc1 that are isoform dependent and occur in a position-specific manner. In summary, these results highlight the need to go beyond monitoring only the cumulative transcript levels for a gene, to separately analysing the expression of its RNA isoforms.

Cell–cell and cell–matrix interactions

Molecular Biology of the CellVol. 23, No. 6 ASCB Annual Meeting HighlightsFree AccessCell–cell and cell–matrix interactionsKris A. DeMali, and Josephine C. AdamsKris A. DeMaliDepartment of Biochemistry, University of Iowa, Iowa City, IA 52242-1109Search for more papers by this author, and Josephine C. AdamsSchool of Biochemistry, University of Bristol, Bristol BS8 1TD, United KingdomSearch for more papers by this authorPublished Online:13 Oct 2017https://doi.org/10.1091/mbc.e11-12-0967AboutSectionsView PDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmail Cell–cell and cell–extracellular matrix (ECM) interactions are fundamental to the complexity of multicellularity achieved in the metazoa. These interactions share many conceptual similarities, the most prominent being a dependence on transmembrane adhesion receptors, binding of adhesion receptors to specific extracellular ligand partners, and the linkage of receptor cytoplasmic domains to intracellular cytoskeletal systems via protein complexes. In both cases, these protein complexes also integrate intracellular signaling, cytoskeletal organization, and regulation of multiple cellular functions.The 2011 Minisymposium on Cell–Cell and Cell–Matrix Interactions brought together current topics and model systems within this broad area of cell biology.Molecular mechanisms that coordinate assembly and function of cell–cell and cell–ECM adhesionsTwo presentations addressed how integrin- and cadherin-dependent adhesions intersect. Kris DeMali (University of Iowa) showed that the function of vinculin, a shared cytoplasmic component of both adhesions, is differentially regulated in cell–cell and cell–ECM adhesions. Data were presented showing that ablation of a single tyrosine residue in vinculin resulted in opposite effects on cell–cell and cell–ECM adhesions. Fangliang Zhang from Anna Kashina's laboratory (University of Pennsylvania) presented his research on talin, a protein that historically has been thought to be present only in integrin-based adhesions as a proximal mediator of integrin activation and of focal adhesion assembly in integrin outside–in signaling. Data were presented on a novel proteolytic processing of talin that is associated with its recruitment and function in cadherin-mediated adhesions.Roles of cell protrusions as initial and dynamic mediators of cell–cell and cell–ECM interactionsFilopodia have well-established roles in sampling the local environment and making initial cell–ECM contacts at the leading edge of many cell types. Filopodial-like structures, termed “epithelial zippers,” can mediate initial contacts between epithelial cells that culminate in assembly of cell–cell junctions. However, the molecular composition of these structures and their relationship to filopodia has remained unclear. Katy Liu from Richard Cheney's laboratory (University of North Carolina at Chapel Hill) showed localization of myosin-X in initial, filopodial contacts between epithelial cells. Whereas myosin-X has a known role in filopodial assembly, the data presented revealed additional important functions in assembly of cell–cell junctions and epithelial morphogenesis. Brad Davidson (University of Arizona) presented data from Ciona intestinalis embryos, a tractable system for the analysis of cell specification in an intact organism (Cooley et al., 2011). Specification of precardiac cells is directed by polarized, invasive filopodial protrusions that localize their response to an ungraded signal.Cell–ECM mechanisms that modulate cell motilitySyndecan-4 is a transmembrane proteoglycan that functions in cell–ECM interactions through its glycosaminoglycan side chains and by activating intracellular signaling that reinforces integrin-dependent focal adhesion assembly (Couchman, 2010). John Couchman (University of Copenhagen, Denmark) presented data on a new role for syndecan-4 in the protrusive invadopodia of breast carcinoma cells that regulate invasive behavior. Jo Adams (University of Bristol, United Kingdom) described recent phylogenetic studies that show components of the ECM (unlike integrins and cadherins) to be specific to the metazoa (Ozbek et al., 2010). Among the most conserved ECM components are the thrombospondins, which participate in ECM homeostasis and remodeling processes. ECM accumulation of thrombospondin-1 facilitates migration of multiple cell types. Data were presented on l-lectin domain-dependent mechanisms that mediate ECM accumulation of thrombospondins.FOOTNOTESDOI: 10.1091/mbc.E11-12-0967REFERENCES Cooley J, Whitaker S, Sweeney S, Fraser S, Davidson B (2011). Cytoskeletal polarity mediates localized induction of the heart progenitor lineage. Nat Cell Biol 13, 952-957. Crossref, Medline, Google Scholar Couchman JR (2010). Transmembrane signaling proteoglycans. Annu Rev Cell Dev Biol 26, 89-114. Crossref, Medline, Google Scholar Ozbek S, Balasubramanian PG, Chiquet-Ehrismann R, Tucker RP, Adams JC (2010). The evolution of extracellular matrix. Mol Biol Cell 21, 4300-4305. Link, Google ScholarFiguresReferencesRelatedDetailsCited byMorphological and Functional Remodeling of Vascular Endothelium in Cardiovascular Diseases19 January 2023 | International Journal of Molecular Sciences, Vol. 24, No. 3Cardiac Patch-Based Therapies of Ischemic Heart Injuries30 June 2019 Vol. 23, No. 6 March 15, 2012965-1140 Metrics Downloads & Citations Downloads: 195Citations: 2 History Information© 2012 DeMali and Adams. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).PDF download

Ex Vivo Stretch Reveals Altered Mechanical Properties of Isolated Dystrophin-Deficient Hearts

Duchenne muscular dystrophy (DMD) is a progressive and fatal disease of muscle wasting caused by loss of the cytoskeletal protein dystrophin. In the heart, DMD results in progressive cardiomyopathy and dilation of the left ventricle through mechanisms that are not fully understood. Previous reports have shown that loss of dystrophin causes sarcolemmal instability and reduced mechanical compliance of isolated cardiac myocytes. To expand upon these findings, here we have subjected the left ventricles of dystrophin-deficient mdx hearts to mechanical stretch. Unexpectedly, isolated mdx hearts showed increased left ventricular (LV) compliance compared to controls during stretch as LV volume was increased above normal end diastolic volume. During LV chamber distention, sarcomere lengths increased similarly in mdx and WT hearts despite greater excursions in volume of mdx hearts. This suggests that the mechanical properties of the intact heart cannot be modeled as a simple extrapolation of findings in single cardiac myocytes. To explain these findings, a model is proposed in which disruption of the dystrophin-glycoprotein complex perturbs cell-extracellular matrix contacts and promotes the apparent slippage of myocytes past each other during LV distension. In comparison, similar increases in LV compliance were obtained in isolated hearts from β-sarcoglycan-null and laminin-α2 mutant mice, but not in dysferlin-null mice, suggesting that increased whole-organ compliance in mdx mice is a specific effect of disrupted cell-extracellular matrix contacts and not a general consequence of cardiomyopathy via membrane defect processes. Collectively, these findings suggest a novel and cell-death independent mechanism for the progressive pathological LV dilation that occurs in DMD.

Migfilin and Filamin as Regulators of Integrin Activation in Endothelial Cells and Neutrophils

Cell adhesion and migration depend on engagement of extracellular matrix ligands by integrins. Integrin activation is dynamically regulated by interactions of various cytoplasmic proteins, such as filamin and integrin activators, talin and kindlin, with the cytoplasmic tail of the integrin β subunit. Although filamin has been suggested to be an inhibitor of integrin activation, direct functional evidence for the inhibitory role of filamin is limited. Migfilin, a filamin-binding protein enriched at cell-cell and cell-extracellular matrix contact sites, can displace filamin from β1 and β3 integrins and promote integrin activation. However, its role in activation and functions of different β integrins in human vascular cells is unknown. In this study, using flow cytometry, we demonstrate that filamin inhibits β1 and αIIbβ3 integrin activation, and migfilin can overcome its inhibitory effect. Migfilin protein is widely expressed in different adherent and circulating blood cells and can regulate integrin activation in naturally-occurring vascular cells, endothelial cells and neutrophils. Migfilin can activate β1, β2 and β3 integrins and promote integrin mediated responses while migfilin depletion impairs the spreading and migration of endothelial cells. Thus, filamin can act broadly as an inhibitor and migfilin is a promoter of integrin activation.

A Syndecan-4 Hair Trigger Initiates Wound Healing through Caveolin- and RhoG-Regulated Integrin Endocytosis

SummaryCell migration during wound healing requires adhesion receptor turnover to enable the formation and disassembly of cell-extracellular matrix contacts. Although recent advances have improved our understanding of integrin trafficking pathways, it is not known how extracellular ligand engagement controls receptor dynamics. Using atomic force microscopy, we have measured cell avidity for fibronectin and defined a mechanism for the outside-in regulation of α5β1-integrin. Surprisingly, adhesive strength was attenuated by the syndecan-4-binding domain of fibronectin due to a rapid triggering of α5β1-integrin endocytosis. Association of syndecan-4 with PKCα was found to trigger RhoG activation and subsequent dynamin- and caveolin-dependent integrin uptake. Like disruption of syndecan-4 or caveolin, gene disruption of RhoG in mice was found to retard closure of dermal wounds due to a migration defect of the fibroblasts and keratinocytes of RhoG null mice. Thus, this syndecan-4-regulated integrin endocytic pathway appears to play a key role in tissue repair.

A secreted protein microarray platform for extracellular protein interaction discovery

Characterization of the extracellular protein interactome has lagged far behind that of intracellular proteins, where mass spectrometry and yeast two-hybrid technologies have excelled. Improved methods for identifying receptor–ligand and extracellular matrix protein interactions will greatly accelerate biological discovery in cell signaling and cellular communication. These technologies must be able to identify low-affinity binding events that are often observed between membrane-bound coreceptor molecules during cell–cell or cell–extracellular matrix contact. Here we demonstrate that functional protein microarrays are particularly well-suited for high-throughput screening of extracellular protein interactions. To evaluate the performance of the platform, we screened a set of 89 immunoglobulin (Ig)-type receptors against a highly diverse extracellular protein microarray with 686 genes represented. To enhance detection of low-affinity interactions, we developed a rapid method to assemble bait Fc fusion proteins into multivalent complexes using protein A microbeads. Based on these screens, we developed a statistical methodology for hit calling and identification of nonspecific interactions on protein microarrays. We found that the Ig receptor interactions identified using our methodology are highly specific and display minimal off-target binding, resulting in a 70% true-positive to false-positive hit ratio. We anticipate that these methods will be useful for a wide variety of functional protein microarray users.

Mechanics of cell spreading within 3D-micropatterned environments

Most tissue cells evolve in vivo in a three-dimensional (3D) microenvironment including complex topographical patterns. Cells exert contractile forces to adhere and migrate through the extracellular matrix (ECM). Although cell mechanics has been extensively studied on 2D surfaces, there are too few approaches that give access to the traction forces that are exerted in 3D environments. Here, we describe an approach to measure dynamically the contractile forces exerted by fibroblasts while they spread within arrays of large flexible micropillars coated with ECM proteins. Contrary to very dense arrays of microposts, the density of the micropillars has been chosen to promote cell adhesion in between the pillars. Cells progressively impale onto the micropatterned substrate. They first adhere on the top of the pillars without applying any detectable forces. Then, they spread along the pillar sides, spanning between the elastic micropillars and applying large forces on the substrate. Interestingly, the architecture of the actin cytoskeleton and the adhesion complexes vary over time as cells pull on the pillars. In particular, we observed less stress fibers than for cells spread on flat surfaces. However, prominent actin stress fibers are observed at cell edges surrounding the micropillars. They generate increasing contractile forces during cell spreading. Cells treated with blebbistatin, a myosin II inhibitor, relax their internal tension, as observed by the release of pillar deformations. Moreover, cell spreading on pillars coated with ECM proteins only on their tops are not able to generate significant traction forces. Taken together, these findings highlight the dynamic relationship between cellular forces and acto-myosin contractility in 3D environments, the influence of cytoskeletal network mechanics on cell shape, as well as the importance of cell-ECM contact area in the generation of traction forces.

Annexin A6 contributes to the invasiveness of breast carcinoma cells by influencing the organization and localization of functional focal adhesions

The interaction of annexin A6 (AnxA6) with membrane phospholipids and either specific extracellular matrix (ECM) components or F-actin suggests that it may influence cellular processes associated with rapid plasma membrane reorganization such as cell adhesion and motility. Here, we examined the putative roles of AnxA6 in adhesion-related cellular processes that contribute to breast cancer progression. We show that breast cancer cells secrete annexins via the exosomal pathway and that the secreted annexins are predominantly cell surface-associated. Depletion of AnxA6 in the invasive BT-549 breast cancer cells is accompanied by enhanced anchorage-independent cell growth but cell–cell cohesion, cell adhesion/spreading onto collagen type IV or fetuin-A, cell motility and invasiveness were strongly inhibited. To explain the loss in adhesion/motility, we show that vinculin-based focal adhesions in the AnxA6-depleted BT-549 cells are elongated and randomly distributed. These focal contacts are also functionally defective because the activation of focal adhesion kinase and the phosphoinositide-3 kinase/Akt pathway were strongly inhibited while the MAP kinase pathway remained constitutively active. Compared with normal human breast tissues, reduced AnxA6 expression in breast carcinoma tissues correlates with enhanced cell proliferation. Together this suggests that reduced AnxA6 expression contributes to breast cancer progression by promoting the loss of functional cell–cell and/or cell–ECM contacts and anchorage-independent cell proliferation.

Viral-Derived Serp-1 as an Adjunctive Therapy for Percutaneous Coronary Intervention

The story of Serp-1 began in 1859 when Thomas Austin imported 24 European rabbits to Australia and released them into the wild for sport. Within 30 years, the rabbits had multiplied exponentially until the population reached into the millions and they had decimated the landscape. Nearly a century later, when other methods of population control had failed, the Australians acquired the myxoma virus from South America and introduced it into the rabbit population. This species-specific virus, which is harmless to humans, infected the rabbits and decreased their numbers by ≈99.9%.1 The key to the success of the myxoma virus in eradicating the rabbit population was its ability to downregulate the rabbit host's immune and inflammatory responses to infection, in part, by synthesizing and secreting Serp-1.2, 3 Years later, it was recognized that the same properties that made Serp-1 devastating for the rabbits could also have therapeutic benefits in human inflammatory disorders. Consequently, viral-derived Serp-1 was purified for experimental testing and, ultimately, clinical use. Article see p 543 Serine protease inhibitors, also known as serpins, comprise up to 10% of plasma proteins and act as regulators of coagulation cascades, fibrinolysis, complement activation, and inflammation.4 Serp-1 is a myxoma virus–derived serpin that regulates endogenous thrombolysis by binding to and inhibiting urokinase- and tissue-type plasminogen activators and plasmin.2, 3, 5, 6 Serp-1 decreases inflammation indirectly by preventing activation of the urokinase-plasminogen activator receptor. This, in turn, inhibits inflammatory cell migration, adhesion to the endothelium, matrix metalloproteinase–mediated invasion of the vessel wall, and proliferation at sites of injury.3, 5, 6 Serp-1 also limits inflammation directly by decreasing monocyte and T-lymphocyte expression of β-integrins, which are necessary for cell-cell and cell–extracellular matrix contact, as well as increasing filamin expression to prevent cell adhesion and …

Single-cell attachment and culture method using a photochemical reaction in a closed microfluidic system.

Recently, interest in single cell analysis has increased because of its potential for improving our understanding of cellular processes. Single cell operation and attachment is indispensable to realize this task. In this paper, we employed a simple and direct method for single-cell attachment and culture in a closed microchannel. The microchannel surface was modified by applying a nonbiofouling polymer, 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer, and a nitrobenzyl photocleavable linker. Using ultraviolet (UV) light irradiation, the MPC polymer was selectively removed by a photochemical reaction that adjusted the cell adherence inside the microchannel. To obtain the desired single endothelial cell patterning in the microchannel, cell-adhesive regions were controlled by use of round photomasks with diameters of 10, 20, 30, or 50 μm. Single-cell adherence patterns were formed after 12 h of incubation, only when 20 and 30 μm photomasks were used, and the proportions of adherent and nonadherent cells among the entire UV-illuminated areas were 21.3%±0.3% and 7.9%±0.3%, respectively. The frequency of single-cell adherence in the case of the 20 μm photomask was 2.7 times greater than that in the case of the 30 μm photomask. We found that the 20 μm photomask was optimal for the formation of single-cell adherence patterns in the microchannel. This technique can be a powerful tool for analyzing environmental factors like cell-surface and cell-extracellular matrix contact.

A Cytocompatible Poly(N-isopropylacrylamide-co-glycidylmethacrylate) Coated Surface as New Substrate for Corneal Tissue Engineering

The conventional method of retrieving cells for tissue engineering to create three-dimensional functional tissues uses enzymes that may hamper cell viability and re-adhesion. Culturing cells on thermoresponsive surfaces of poly(N-isopropylacrylamide) (PNIPAAm) is a relatively new nondestructive method of creating in vitro tissues. In this study, PNIPAAm and glycidylmethacrylate (GMA)-based thermoresponsive copolymer N-isopropylacrylamide-co-glycidylmethacrylate (NGMA) were synthesized as a potential cell culture harvesting system for generating 3D synthetic tissues. The copolymer was characterized by differential scanning calorimetry, gel permeation chromatography, Fourier transform infrared spectroscopy, water contact angle, atomic force microscopy, and nuclear magnetic resonance spectroscopy. The NGMA-coated dishes were evaluated for cytotoxicity and cytocompatibility using L-929 cells. Primary rabbit corneal cultures established on NGMA surface were detached as an intact cell sheet with epithelial specific characteristics as well as maintenance of cell—cell and cell—extracellular matrix contact. The results confirmed the suitability of NGMA substrate for cell culture and temperature-induced cell sheet harvest. This is the first report on this copolymer formulation as a substrate for tissue engineering application. Hydrophobic GMA apart from modulating the lower critical solution temperature features the prospects of further modification, namely the incorporation of biomolecules through the epoxy groups.