Three-dimensional biomimetic vascular model reveals a RhoA, Rac1, and N-cadherin balance in mural cell-endothelial cell-regulated barrier function.

The integrity of the endothelial barrier between circulating blood and tissue is important for blood vessel function and, ultimately, for organ homeostasis. Here, we developed a vessel-on-a-chip with perfused endothelialized channels lined with human bone marrow stromal cells, which adopt a mural cell-like phenotype that recapitulates barrier function of the vasculature. In this model, barrier function is compromised upon exposure to inflammatory factors such as LPS, thrombin, and TNFα, as has been observed in vivo. Interestingly, we observed a rapid physical withdrawal of mural cells from the endothelium that was accompanied by an inhibition of endogenous Rac1 activity and increase in RhoA activity in the mural cells themselves upon inflammation. Using a system to chemically induce activity in exogenously expressed Rac1 or RhoA within minutes of stimulation, we demonstrated RhoA activation induced loss of mural cell coverage on the endothelium and reduced endothelial barrier function, and this effect was abrogated when Rac1 was simultaneously activated. We further showed that N-cadherin expression in mural cells plays a key role in barrier function, as CRISPR-mediated knockout of N-cadherin in the mural cells led to loss of barrier function, and overexpression of N-cadherin in CHO cells promoted barrier function. In summary, this bicellular model demonstrates the continuous and rapid modulation of adhesive interactions between endothelial and mural cells and its impact on vascular barrier function and highlights an in vitro platform to study the biology of perivascular-endothelial interactions.

Inquiry-based learning: inflammation as a model to teach molecular techniques for assessing gene expression.

This laboratory module simulates the process used by working scientists to ask and answer a question of biological interest. Instructors facilitate acquisition of knowledge using a comprehensive, inquiry-based approach in which students learn theory, hypothesis development, experimental design, and data interpretation and presentation. Using inflammation in macrophages as a model system, students perform a series of molecular biology techniques to address the biological question: "Does stimulus 'X' induce inflammation?" To ask this question, macrophage cells are treated with putative inflammatory mediators and then assayed for evidence of inflammatory response. Students become familiar with their assigned mediator and the relationship between their mediator and inflammation by conducting literature searches, then using this information to generate hypotheses which address the effect of their mediator on induction of inflammation. The cellular and molecular approaches used to test their hypotheses include transfection and luciferase reporter assay, immunoblot, fluorescence microscopy, enzyme-linked immunosorbent assay, and quantitative PCR. Quantitative and qualitative reasoning skills are developed through data analysis and demonstrated by successful completion of post-lab worksheets and the generation and oral presentation of a scientific poster. Learning objective assessment relies on four instruments: pre-lab quizzes, post-lab worksheets, poster presentation, and posttest. Within three cohorts (n = 85) more than 95% of our students successfully achieved the learning objectives.

Spatial patterning of gene expression using surface-immobilized recombinant adenovirus.

Spatially patterned gene expression drives tissue organization and is a critical determinant of tissue function. Approaches in functional tissue engineering will require not only the spatial organization of cells but also control of their gene expression patterns. We report a method to generate patterns of gene expression within a monolayer of cells by using surface-immobilized recombinant adenovirus. This study represents a new approach to engineering tissues that relies on controlling spatial patterns of gene expression, and can be used independently or in combination with positioning of different cell types. This technique may have broad applications in biotechnology including tissue engineering and targeted gene delivery.

Versatile, fully automated, microfluidic cell culture system.

There is increasing demand for automated and quantitative cell culture technology, driven both by the intense activity in stem cell biology and by the emergence of systems biology. We built a fully automated cell culture screening system based on a microfluidic chip that creates arbitrary culture media formulations in 96 independent culture chambers and maintains cell viability for weeks. Individual culture conditions are customized in terms of cell seeding density, composition of culture medium, and feeding schedule, and each chamber is imaged with time-lapse microscopy. Using this device, we perform the first quantitative measurements of the influence of transient stimulation schedules on the proliferation, osteogenic differentiation, and motility of human primary mesenchymal stem cells.

An inhibitory role for FAK in regulating proliferation: a link between limited adhesion and RhoA-ROCK signaling.

Focal adhesion kinase (FAK) transduces cell adhesion to the extracellular matrix into proliferative signals. We show that FAK overexpression induced proliferation in endothelial cells, which are normally growth arrested by limited adhesion. Interestingly, displacement of FAK from adhesions by using a FAK-/- cell line or by expressing the C-terminal fragment FRNK also caused an escape of adhesion-regulated growth arrest, suggesting dual positive and negative roles for FAK in growth regulation. Expressing kinase-dead FAK-Y397F in FAK-/- cells prevented uncontrolled growth, demonstrating the antiproliferative function of inactive FAK. Unlike FAK overexpression-induced growth, loss of growth control in FAK-/- or FRNK-expressing cells increased RhoA activity, cytoskeletal tension, and focal adhesion formation. ROCK inhibition rescued adhesion-dependent growth control in these cells, and expression of constitutively active RhoA or ROCK dysregulated growth. These findings demonstrate the ability of FAK to suppress and promote growth, and underscore the importance of multiple mechanisms, even from one molecule, to control cell proliferation.

E-cadherin engagement stimulates proliferation via Rac1.

E-cadherin has been linked to the suppression of tumor growth and the inhibition of cell proliferation in culture. We observed that progressively decreasing the seeding density of normal rat kidney-52E (NRK-52E) or MCF-10A epithelial cells from confluence, indeed, released cells from growth arrest. Unexpectedly, a further decrease in seeding density so that cells were isolated from neighboring cells decreased proliferation. Experiments using microengineered substrates showed that E-cadherin engagement stimulated the peak in proliferation at intermediate seeding densities, and that the proliferation arrest at high densities did not involve E-cadherin, but rather resulted from a crowding-dependent decrease in cell spreading against the underlying substrate. Rac1 activity, which was induced by E-cadherin engagement specifically at intermediate seeding densities, was required for the cadherin-stimulated proliferation, and the control of Rac1 activation by E-cadherin was mediated by p120-catenin. Together, these findings demonstrate a stimulatory role for E-cadherin in proliferative regulation, and identify a simple mechanism by which cell-cell contact may trigger or inhibit epithelial cell proliferation in different settings.

T cell-to-T cell clustering enhances NF-kappaB activity by a PI3K signal mediated by Cbl-b and Rho.

Full activation of T cells requires the binding of antigen to the T cell receptor and stimulation of the CD28 molecule, a process which typically occurs when T cells bind to an antigen presenting cell. The transcription factor, NF-kappaB, is an integration point for these two signals and its activation is critical for T cell function. Using antibodies to the TCR and CD28 molecules to activate Jurkat T cells, we show that cells that were permitted to aggregate into multi-cellular clusters increased NF-kappaB activity compared to unclustered cells. Inhibition of PI3K signaling with wortmannin decreased the clustering-mediated NF-kappaB signal. Over-expression of a dominant negative form of Cbl-b, an endogenous inhibitor of PI3K, in unclustered cells rescued NF-kappaB activation to the same levels caused by cell clustering. Inhibiting signaling through Rho with dominant negative RhoA abrogated both clustering-mediated and dominant negative Cbl-b-mediated NF-kappaB inactivation, but not TCR/CD28 mediated NF-kappaB activation. Taken together, these results suggest that in addition to pathways stimulated by classical T cell-APC interactions, another signal arising from T cell clustering can enhance activation.

Vascular endothelial-cadherin regulates cytoskeletal tension, cell spreading, and focal adhesions by stimulating RhoA.

Changes in vascular endothelial (VE)-cadherin-mediated cell-cell adhesion and integrin-mediated cell-matrix adhesion coordinate to affect the physical and mechanical rearrangements of the endothelium, although the mechanisms for such cross talk remain undefined. Herein, we describe the regulation of focal adhesion formation and cytoskeletal tension by intercellular VE-cadherin engagement, and the molecular mechanism by which this occurs. Increasing the density of endothelial cells to increase cell-cell contact decreased focal adhesions by decreasing cell spreading. This contact inhibition of cell spreading was blocked by disrupting VE-cadherin engagement with an adenovirus encoding dominant negative VE-cadherin. When changes in cell spreading were prevented by culturing cells on a micropatterned substrate, VE-cadherin-mediated cell-cell contact paradoxically increased focal adhesion formation. We show that VE-cadherin engagement mediates each of these effects by inducing both a transient and sustained activation of RhoA. Both the increase and decrease in cell-matrix adhesion were blocked by disrupting intracellular tension and signaling through the Rho-ROCK pathway. In all, these findings demonstrate that VE-cadherin signals through RhoA and the actin cytoskeleton to cross talk with cell-matrix adhesion and thereby define a novel pathway by which cell-cell contact alters the global mechanical and functional state of cells.

Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment.

Commitment of stem cells to different lineages is regulated by many cues in the local tissue microenvironment. Here we demonstrate that cell shape regulates commitment of human mesenchymal stem cells (hMSCs) to adipocyte or osteoblast fate. hMSCs allowed to adhere, flatten, and spread underwent osteogenesis, while unspread, round cells became adipocytes. Cell shape regulated the switch in lineage commitment by modulating endogenous RhoA activity. Expressing dominant-negative RhoA committed hMSCs to become adipocytes, while constitutively active RhoA caused osteogenesis. However, the RhoA-mediated adipogenesis or osteogenesis was conditional on a round or spread shape, respectively, while constitutive activation of the RhoA effector, ROCK, induced osteogenesis independent of cell shape. This RhoA-ROCK commitment signal required actin-myosin-generated tension. These studies demonstrate that mechanical cues experienced in developmental and adult contexts, embodied by cell shape, cytoskeletal tension, and RhoA signaling, are integral to the commitment of stem cell fate.

Strategies for engineering the adhesive microenvironment.

Cells exist within a complex tissue microenvironment, which includes soluble factors, extracellular matrix molecules, and neighboring cells. In the breast, the adhesive microenvironment plays a crucial role in driving both normal mammary gland development as well tumor initiation and progression. Researchers are designing increasingly more complex ways to mimic the in vivo microenvironment in an in vitro setting, so that cells in culture may serve as model systems for tissue structures. Here, we explore the use of microfabrication technologies to engineer the adhesive microenvironment of cells in culture. These new tools permit the culture of cells on well-defined surface chemistries, patterning of cells into defined geometries either alone or in coculture scenarios, and measurement of forces associated with cell-ECM interactions. When applied to questions in mammary gland development and neoplasia, these new tools will enable a better understanding of how adhesive, structural, and mechanical cues regulate mammary epithelial biology.

Cells lying on a bed of microneedles: an approach to isolate mechanical force.

We describe an approach to manipulate and measure mechanical interactions between cells and their underlying substrates by using microfabricated arrays of elastomeric, microneedle-like posts. By controlling the geometry of the posts, we varied the compliance of the substrate while holding other surface properties constant. Cells attached to, spread across, and deflected multiple posts. The deflections of the posts occurred independently of neighboring posts and, therefore, directly reported the subcellular distribution of traction forces. We report two classes of force-supporting adhesions that exhibit distinct force-size relationships. Force increased with size of adhesions for adhesions larger than 1 microm(2), whereas no such correlation existed for smaller adhesions. By controlling cell adhesion on these micromechanical sensors, we showed that cell morphology regulates the magnitude of traction force generated by cells. Cells that were prevented from spreading and flattening against the substrate did not contract in response to stimulation by serum or lysophosphatidic acid, whereas spread cells did. Contractility in the unspread cells was rescued by expression of constitutively active RhoA. Together, these findings demonstrate a coordination of biochemical and mechanical signals to regulate cell adhesion and mechanics, and they introduce the use of arrays of mechanically isolated sensors to manipulate and measure the mechanical interactions of cells.