Notch1 and Notch3 coordinate for pericyte-induced stabilization of vasculature.

The Notch pathway regulates complex patterning events in many species and is critical for the proper formation and function of the vasculature. Despite this importance, how the various components of the Notch pathway work in concert is still not well understood. For example, NOTCH1 stabilizes homotypic endothelial junctions, but the role of NOTCH1 in heterotypic interactions is not entirely clear. NOTCH3, on the other hand, is essential for heterotypic interactions of pericytes with the endothelium, but how NOTCH3 signaling in pericytes impacts the endothelium remains elusive. Here, we use in vitro vascular models to investigate whether pericyte-induced stabilization of the vasculature requires the cooperation of NOTCH1 and NOTCH3. We observe that both pericyte NOTCH3 and endothelial NOTCH1 are required for the stabilization of the endothelium. Loss of either NOTCH3 or NOTCH1 decreases the accumulation of VE-cadherin at endothelial adherens junctions and increases the frequency of wider, more motile junctions. We found that DLL4 was the key ligand for simulating NOTCH1 activation in endothelial cells and observed that DLL4 expression in pericytes is dependent on NOTCH3. Altogether, these data suggest that an interplay between pericyte NOTCH3 and endothelial NOTCH1 is critical for pericyte-induced vascular stabilization.

Harnessing Mechanobiology for Tissue Engineering.

A primary challenge in tissue engineering is to recapitulate both the structural and functional features of whole tissues and organs. In vivo, patterning of the body plan and constituent tissues emerges from the carefully orchestrated interactions between the transcriptional programs that give rise to cell types and the mechanical forces that drive the bending, twisting, and extensions critical to morphogenesis. Substantial recent progress in mechanobiology-understanding how mechanics regulate cell behaviors and what cellular machineries are responsible-raises the possibility that one can begin to use these insights to help guide the strategy and design of functional engineered tissues. In this perspective, we review and propose the development of different approaches, from providing appropriate extracellular mechanical cues to interfering with cellular mechanosensing machinery, to aid in controlling cell and tissue structure and function.

Uncovering mutation-specific morphogenic phenotypes and paracrine-mediated vessel dysfunction in a biomimetic vascularized mammary duct platform.

The mammary gland is a highly vascularized tissue capable of expansion and regression during development and disease. To enable mechanistic insight into the coordinated morphogenic crosstalk between the epithelium and vasculature, we introduce a 3D microfluidic platform that juxtaposes a human mammary duct in proximity to a perfused endothelial vessel. Both compartments recapitulate stable architectural features of native tissue and the ability to undergo distinct forms of branching morphogenesis. Modeling HER2/ERBB2 amplification or activating PIK3CA(H1047R) mutation each produces ductal changes observed in invasive progression, yet with striking morphogenic and behavioral differences. Interestingly, PI3KαH1047R ducts also elicit increased permeability and structural disorganization of the endothelium, and we identify the distinct secretion of IL-6 as the paracrine cause of PI3KαH1047R-associated vascular dysfunction. These results demonstrate the functionality of a model system that facilitates the dissection of 3D morphogenic behaviors and bidirectional signaling between mammary epithelium and endothelium during homeostasis and pathogenesis.

Multiple roles of lymphatic vessels in peripheral lymph node development.

The mammalian lymphatic system consists of strategically located lymph nodes (LNs) embedded into a lymphatic vascular network. Mechanisms underlying development of this highly organized system are not fully understood. Using high-resolution imaging, we show that lymphoid tissue inducer (LTi) cells initially transmigrate from veins at LN development sites using gaps in venous mural coverage. This process is independent of lymphatic vasculature, but lymphatic vessels are indispensable for the transport of LTi cells that egress from blood capillaries elsewhere and serve as an essential LN expansion reservoir. At later stages, lymphatic collecting vessels ensure efficient LTi cell transport and formation of the LN capsule and subcapsular sinus. Perinodal lymphatics also promote local interstitial flow, which cooperates with lymphotoxin-ß signaling to amplify stromal CXCL13 production and thereby promote LTi cell retention. Our data unify previous models of LN development by showing that lymphatics intervene at multiple points to assist LN expansion and identify a new role for mechanical forces in LN development.

Biomimetic Model of Tumor Microenvironment on Microfluidic Platform.

The "Tumor microenvironment" (TME) is a complex, interacting system of the tumor and its surrounding environment. The TME has drawn more attention recently in attempts to overcome current drug resistance and the recurrence of cancer by understanding the cancer and its microenvironment systematically, beyond past reductionist approaches. However, a lack of experimental tools to dissect the intricate interactions has hampered in-depth research into the TME. Here, a biomimetic TME model using a microfluidic platform is presented, which enables the interaction between TME constituents to be studied in a comprehensive manner. Paracrine interactions of cocultured tumor cell lines (SK-OV-3, MKN-74, and SW620) with primary fibroblasts show marked morphological changes in the tumor cells, depending on the type of tumor cells, and, importantly, the composition of the extracellular matrix. Furthermore, this model allows direct observation of angiogenesis induced by the tumor-stroma interaction. Finally, reconstituting simultaneous angiogenesis and lymphangiogenesis induced by the tumor-stromal interaction with TME mimicking extrinsic factors is enabled. It is believed that the in vitro biomimetic model and the experimental concepts described will help to shed light on the complex biology of the TME.

Microvessels-on-a-Chip to Assess Targeted Ultrasound-Assisted Drug Delivery.

Microbubbles have been used in ultrasound-assisted drug delivery to help target solid tumors via blood vessels in vivo; however, studies to understand the phenomena at the cellular level and to optimize parameters for ultrasound or microbubbles in vivo are challenging and expensive to perform. Here, we utilize microfluidic microvessels-on-a-chip that enable visualization of microbubble/ultrasound-dependent drug delivery to microvasculature. When exposed to pulsed ultrasound, microbubbles perfused through microvessels-on-a-chip were observed to stably oscillate. Minimal cellular damage was observed for both microbubbles and untargeted doxorubicin-encapsulating liposomes (DOX-liposomes) perfused through chip microvessels. In contrast, passive and ultrasound-assisted perfusion of integrin-targeted DOX-liposomes induced cytotoxicity, which was only significantly enhanced for ultrasound-assisted perfusion when microbubbles were coperfused. These results suggest that stably oscillating microbubbles enhance targeted DOX-liposome internalization/cytotoxicity largely by stimulating integrin receptor endocytosis. Furthermore, our study demonstrates the utility of our microvessels-on-a-chip as a screening platform for optimizing drug dosage, targeting ligands and drugs.

Interstitial flow regulates the angiogenic response and phenotype of endothelial cells in a 3D culture model.

A crucial yet ill-defined phenomenon involved in the remodeling of vascular networks, including angiogenic sprouting, is flow-mediated endothelial dynamics and phenotype changes. Despite interstitial flow (IF) being ubiquitously present in living tissues surrounding blood capillaries, it is rarely investigated and poorly understood how endothelial cells respond to this flow during morphogenesis. Here we develop a microfluidic 3D in vitro model to investigate the role of IF during vasculogenic formation and angiogenic remodeling of microvascular networks. In the presented model, human blood endothelial cells co-cultured with stromal fibroblasts spontaneously organize into an interconnected microvascular network and then further expand to adjacent avascular regions in a manner of neovessel sprouting. We found that in the presence of IF, vasculogenic organization of the microvascular network was significantly facilitated regardless of the flow direction, whereas angiogenic sprouting was promoted only when the directions of flow and sprouting were opposite while angiogenic activity was suppressed into the direction of flow. We also observed that the vasculatures switch between active angiogenic remodeling and quiescent/non-sprouting state in the contexts provided by IF. This regulatory effect can be utilized to examine the role of anti-angiogenic compounds, clearly distinguishing the differential influences of the compounds depending on their mechanisms of action. Collectively, these results suggest that IF may serve as a critical regulator in tissue vascularization and pathological angiogenesis.

Establishment and antitumor effects of dasatinib and PKI-587 in BD-138T, a patient-derived muscle invasive bladder cancer preclinical platform with concomitant EGFR amplification and PTEN deletion.

Muscle-invasive bladder cancer (MIBC) consists of a heterogeneous group of tumors with a high rate of metastasis and mortality. To facilitate the in-depth investigation and validation of tailored strategies for MIBC treatment, we have developed an integrated approach using advanced high-throughput drug screening and a clinically relevant patient-derived preclinical platform. We isolated patient-derived tumor cells (PDCs) from a rare MIBC case (BD-138T) that harbors concomitant epidermal growth factor receptor (EGFR) amplification and phosphatase and tensin homolog (PTEN) deletion. High-throughput in vitro drug screening demonstrated that dasatinib, a SRC inhibitor, and PKI-587, a dual PI3K/mTOR inhibitor, exhibited targeted anti-proliferative and pro-apoptotic effects against BD-138T PDCs. Using established patient-derived xenograft models that successfully retain the genomic and molecular characteristics of the parental tumor, we confirmed that these anti-tumor responses occurred through the inhibition of SRC and PI3K/AKT/mTOR signaling pathways. Taken together, these experimental results demonstrate that dasatinib and PKI-587 might serve as promising anticancer drug candidates for treating MIBC with combined EGFR gene amplification and PTEN deletion.

Three-dimensional biomimetic model to reconstitute sprouting lymphangiogenesis in vitro.

Formation of new lymphatic vessels, termed lymphangiogenesis, is central for diverse biological processes during development, inflammation and tumor metastasis. However, reliable in vitro model is still under demand for detailed elucidation of how sprouting lymphangiogenesis is initiated and coordinated. Here, we describe a microfluidic platform optimized for close reconstitution of lymphangiogenesis, achieved by on-chip integration of salient constituents of lymphatic microenvironment found in vivo. With flexible and precise control over the factors that include biochemical cues, interstitial flow (IF), and endothelial-stromal interactions, we found that orchestrated efforts of multiple environmental factors are necessary for robust lymphatic sprouting in 3D extracellular matrix. Especially, we demonstrate that IF serves as a central regulatory cue which defines lymphangiogenic responses and phenotypes of lymphatic endothelial cells. When synergized with pro-lymphangiogenic factors, IF significantly augmented initiation and outgrowth of lymphatic sprouts toward upstream of the flow while suppressing downstream-directed sprouting. In an appropriate synergism, lymphatic sprouts exhibited structural, molecular signatures and cellular phenotypes that closely approximate sprouting lymphatic neovessels in vivo, and precisely reflected the modulatory effects of pro- and anti-lymphangiogenic stimuli. Our study not only reveals critical but unappreciated role of mechanical cue that regulates lymphangiogenic sprouting, but also provides a novel biomimetic model that may leverage further biological studies as well as phenotypic drug screening.

Microfluidic vascularized bone tissue model with hydroxyapatite-incorporated extracellular matrix.

Current in vitro systems mimicking bone tissues fail to fully integrate the three-dimensional (3D) microvasculature and bone tissue microenvironments, decreasing their similarity to in vivo conditions. Here, we propose 3D microvascular networks in a hydroxyapatite (HA)-incorporated extracellular matrix (ECM) for designing and manipulating a vascularized bone tissue model in a microfluidic device. Incorporation of HA of various concentrations resulted in ECM with varying mechanical properties. Sprouting angiogenesis was affected by mechanically modulated HA-extracellular matrix interactions, generating a model of vascularized bone microenvironment. Using this platform, we observed that hydroxyapatite enhanced angiogenic properties such as sprout length, sprouting speed, sprout number, and lumen diameter. This new platform integrates fibrin ECM with the synthetic bone mineral HA to provide in vivo-like microenvironments for bone vessel sprouting.

Engineering of a Biomimetic Pericyte-Covered 3D Microvascular Network.

Pericytes enveloping the endothelium play an important role in the physiology and pathology of microvessels, especially in vessel maturation and stabilization. However, our understanding of fundamental pericyte biology is limited by the lack of a robust in vitro model system that allows researchers to evaluate the interactions among multiple cell types in perfusable blood vessels. The present work describes a microfluidic platform that can be used to investigate interactions between pericytes and endothelial cells (ECs) during the sprouting, growth, and maturation steps of neovessel formation. A mixture of ECs and pericytes was attached to the side of a pre-patterned three dimensional fibrin matrix and allowed to sprout across the matrix. The effects of intact coverage and EC maturation by the pericytes on the perfused EC network were confirmed using a confocal microscope. Compared with EC monoculture conditions, EC-pericyte co-cultured vessels showed a significant reduction in diameter, increased numbers of junctions and branches and decreased permeability. In response to biochemical factors, ECs and pericytes in the platform showed the similar features with previous reports from in vivo experiments, thus reflect various pathophysiological conditions of in vivo microvessels. Taken together, these results support the physiological relevancy of our three-dimensional microfluidic culture system but also that the system can be used to screen drug effect on EC-pericyte biology.

Snail1 induced in breast cancer cells in 3D collagen I gel environment suppresses cortactin and impairs effective invadopodia formation.

Although an in vitro 3D environment cannot completely mimic the in vivo tumor site, embedding tumor cells in a 3D extracellular matrix (ECM) allows for the study of cancer cell behaviors and the screening of anti-metastatic reagents with a more in vivo-like context. Here we explored the behaviors of MDA-MB-231 breast cancer cells embedded in 3D collagen I. Diverse tumor environmental conditions (including cell density, extracellular acidity, or hypoxia as mimics for a continuous tumor growth) reduced JNKs, enhanced TGFß1/Smad signaling activity, induced Snail1, and reduced cortactin expression. The reduced JNKs activity blocked efficient formation of invadopodia labeled with actin, cortactin, or MT1-MMP. JNKs inactivation activated Smad2 and Smad4, which were required for Snail1 expression. Snail1 then repressed cortactin expression, causing reduced invadopodia formation and prominent localization of MT1-MMP at perinuclear regions. MDA-MB-231 cells thus exhibited less efficient collagen I degradation and invasion in 3D collagen I upon JNKs inhibition. These observations support a signaling network among JNKs, Smads, Snail1, and cortactin to regulate the invasion of MDA-MB-231 cells embedded in 3D collagen I, which may be targeted during screening of anti-invasion reagents.

ß-Amyloid is transmitted via neuronal connections along axonal membranes.

OBJECTIVE: ß-amyloid plaque is a critical pathological feature of Alzheimer disease. Pathologic studies suggest that neurodegeneration may occur in a retrograde fashion from axon terminals near ß-amyloid plaques, and that plaque may spread through brain regions. However, there is no direct experimental evidence to show transmission of ß-amyloid. METHODS: Microscopic imaging data of ß-amyloid transmission was acquired in cortical neuron cultures from Sprague-Dawley rat embryos using polydimethylsiloxane (PDMS) microfluidic culture chambers and in brain sections from in vivo ß-amyloid injection. RESULTS: We present direct imaging evidence in cultured cortical neurons, using PDMS microfluidic culture chambers, that ß-amyloid is readily absorbed by axonal processes and retrogradely transported to neuronal cell bodies. Transmission of ß-amyloid via neuronal connections was also confirmed in mouse brain. ß-Amyloid absorbed by distal axons accumulates in axonal swellings, mitochondria, and lysosomes of the cell bodies. Interestingly, dynasore, an inhibitor of dynamin, which is a protein indispensable for endocytosis, did not prevent retrograde transport of ß-amyloid, indicating that ß-amyloid is absorbed onto axonal membranes and transmitted via them to the cell body. Dynasore did decrease the transneuronal transmission of ß-amyloid, suggesting that this requires the internalization and secretion of ß-amyloid. INTERPRETATION: Our findings provide direct in vitro and in vivo evidence for spreading of ß-amyloid through neuronal connections, and suggest possible therapeutic approaches to blocking this spread.

A bioengineered array of 3D microvessels for vascular permeability assay.

Blood vessels exhibit highly regulated barrier function allowing selective passage of macromolecules. Abnormal vascular permeability caused by disorder in barrier function is often associated with various pathological states such as tumor progression or pulmonary fibrosis. There are no realistic in vitro models for measuring vascular permeability as most models are limited to mimicking anatomical structural properties of in vivo vessel barriers. This paper presents a reliable microfluidic-based chip for measuring permeability by engineering tubular perfusable microvessels. This platform is compatible with high resolution, live-cell time-lapse imaging and high throughput permeability measurements. The microvessels were formed by natural angiogenic process and thus exhibit reliable barrier properties with permeability coefficient of 1.55×10(-6)cm/s (for 70kDa FITC-dextran). The bioengineered microvessels showed properties similar to in vivo vessels in terms of cell-cell junction expression (ZO-1, Claudin-5 and VE-cadherin) and response to agonists such as histamine and TNF-α. We showed that hyperpermeability of the tumor microvessel could be normalized with anti-VEGF (bevacizumab) treatment, consistent with the mechanism of action for bevacizumab. The method developed here provides a relatively simple, robust technique for assessing drug effects on permeability of microvessels with a number of potential applications in fundamental vascular biology as well as drug screening.

Engineering of functional, perfusable 3D microvascular networks on a chip.

Generating perfusable 3D microvessels in vitro is an important goal for tissue engineering, as well as for reliable modelling of blood vessel function. To date, in vitro blood vessel models have not been able to accurately reproduce the dynamics and responses of endothelial cells to grow perfusable and functional 3D vascular networks. Here we describe a microfluidic-based platform whereby we model natural cellular programs found during normal development and angiogenesis to form perfusable networks of intact 3D microvessels as well as tumor vasculatures based on the spatially controlled co-culture of endothelial cells with stromal fibroblasts, pericytes or cancer cells. The microvessels possess the characteristic morphological and biochemical markers of in vivo blood vessels, and exhibit strong barrier function and long-term stability. An open, unobstructed microvasculature allows the delivery of nutrients, chemical compounds, biomolecules and cell suspensions, as well as flow-induced mechanical stimuli into the luminal space of the endothelium, and exhibits faithful responses to physiological shear stress as demonstrated by cytoskeleton rearrangement and increased nitric oxide synthesis. This simple and versatile platform provides a wide range of applications in vascular physiology studies as well as in developing vascularized organ-on-a-chip and human disease models for pharmaceutical screening.

Application of a new wall-less plate technology to complex multistep cell-based investigations using suspension cells.

Despite significant progresses, cell-based assays still have major limitations part to because of their plate format. Here, we present a wall-less plate technology based on unique liquid dynamics named DropArray that takes advantage of hydrophobic and hydrophilic surface properties. Liquid velocities within the DropArray plate were quantified through fluid dynamics simulation and complete retention of suspension cells experimentally demonstrated within the range of simulated shear stresses. Subsequently, we compared the DropArray technology with conventional microtiter plates in a cell-based protein-binding assay. Although the wall-less plate produced similar results with adherent cells, the advantage of the DropArray technology was absolutely clear when semiadherent or suspension cells were used in this multistep experimental procedure. The technology also was evaluated for the cell viability assay and generated similar results to conventional plate format while enabling significant reduction in toxic reagent use. Finally, we developed a DropArray cell-based assay to evaluate a bispecific antibody designed to engage cytotoxic T cells and trigger tumor cell killing. This assay enables for the first time the visualization and quantification of the specific killing events and represents a very powerful tool to further investigate functional aspects of the cancer immunotherapy.

Biological applications of microfluidic gradient devices.

Molecular gradients play an important role in diverse physiological and pathological phenomena such as immune response, wound healing, development and cancer metastasis. In the past 10 years, engineering tools have been increasingly used to develop experimental platforms that capture important aspects of cellular microenvironments to allow quantitative and reproducible characterization of cellular response to gradients. This review discusses the emergence of microfluidics-based gradient generators and their applications in enhancing our understanding of fundamental biological processes such as chemotaxis and morphogenesis. The principles and applications of microfluidic gradient generation in both 2D and 3D cellular microenvironments are discussed with emphasis on approaches to manipulate spatial and temporal distribution of signaling molecules.

Structural requirements for the neuroprotective effects of aspirin analogues against N-methyl-D-aspartate and zinc ion neurotoxicity.

In order to elucidate the structural requirements for the dual neuroprotective activity of aspirin against N-methyl-D-aspartate (NMDA) and zinc ion neurotoxicity, various aspirin analogues and derivatives, modified at the carboxylic group, the acetyl group, and the chain length between the carboxylic acid moiety and phenyl ring, were synthesized. Replacement of the carboxylic acid group with alkyl groups (compounds 2c and 2d) resulted in a dramatic increase in neuroprotective activity against NMDA neurotoxicity, while reduction of the carboxylic acid group to the alcohol (compound 2g) completely abolished this activity. In contrast to NMDA neurotoxicity, compounds that are devoid of the carboxylic acid group did not show any activity against zinc ion neurotoxicity. Replacement of the acetyl group with a propionyl (compound 5a) or butyryl group (compound 5b) did not significantly change the activity against NMDA neurotoxicity, but replacement of the acetyl group with a propionyl group (compound 5a) resulted in a slight decrease in activity against zinc ion neurotoxicity. Compound 12, which has ethylene units between the carboxylic acid moiety and phenyl ring in the structure of aspirin, exhibited greater neuroprotective activity against NMDA neurotoxicity than the compared compounds (aspirin, compound 9 and compound 17), which have different chain lengths. A similar trend was also observed in the neuroprotective activity against zinc ion neurotoxicity. These results indicate that the carboxylic acid group in aspirin is not indispensable for the inhibitory effect against NMDA neurotoxicity, but is essential for the inhibitory effect against zinc ion neurotoxicity. The acetyl group and ethylene unit's distance are favourable for the inhibitory effect against NMDA neurotoxicity as well as zinc ion neurotoxicity.