Confluent Endothelial Cell Monolayers Research Articles

Bi-layer blood vessel mimicking microfluidic platform for antitumor drug screening based on co-culturing 3D tumor spheroids and endothelial layers.

Two-dimensional (2D) cell culture is not ideal for traditional drug screening, because 2D culture does not accurately mimic the physiological microenvironment of tumor cells. Thus, a drug-screening system which more closely mimics the microenvironment of in vivo tumors is necessary. Here, we present a biomimicking bilayer microfluidic device that can facilitate antitumor drug screening. The microfluidic device consists of two polydimethylsiloxane (PDMS) pieces with channels which are separated by a semipermeable membrane to allow water, oxygen, and nutrition supply, while preventing cell migration. The channels embedded on the two PDMS pieces overlap each other over a long distance to ensure a larger exchange area to mimic the blood vessel-tumor model. High concentrations of endothelial cells (EC) are first seeded onto the membrane through the apical channel, and after a two-day culture, a confluent EC monolayer forms. Tumor spheroid-laden Matrigel is then seeded into the basal channel. After the Matrigel is cured, the device is ready for drug testing. Paclitaxel is used as the model drug for testing. Confocal microscopy and ImageJ are used to assess the efficacy of different concentrations of paclitaxel, and optical coherence tomography (OCT) is employed to determine the tumor volumetric change after the drug treatment. The results indicate that the proposed bilayer microfluidic device in combination with confocal and OCT optical characterization provide an efficient platform for antitumor drug testing.

Differential outcomes of venous and arterial tissue engineered vascular grafts highlight the importance of coupling long-term implantation studies with computational modeling

Electrospinning is commonly used to generate polymeric scaffolds for tissue engineering. Using this approach, we developed a small-diameter tissue engineered vascular graft (TEVG) composed of poly-ε-caprolactone-co-l-lactic acid (PCLA) fibers and longitudinally assessed its performance within both the venous and arterial circulations of immunodeficient (SCID/bg) mice. Based on in vitro analysis demonstrating complete loss of graft strength by 12 weeks, we evaluated neovessel formation in vivo over 6-, 12- and 24-week periods. Mid-term observations indicated physiologic graft function, characterized by 100% patency and luminal matching with adjoining native vessel in both the venous and arterial circulations. An active and robust remodeling process was characterized by a confluent endothelial cell monolayer, macrophage infiltrate, and extracellular matrix deposition and remodeling. Long-term follow-up of venous TEVGs at 24 weeks revealed viable neovessel formation beyond graft degradation when implanted in this high flow, low-pressure environment. Arterial TEVGs experienced catastrophic graft failure due to aneurysmal dilatation and rupture after 14 weeks. Scaffold parameters such as porosity, fiber diameter, and degradation rate informed a previously described computational model of vascular growth and remodeling, and simulations predicted the gross differential performance of the venous and arterial TEVGs over the 24-week time course. Taken together, these results highlight the requirement for in vivo implantation studies to extend past the critical time period of polymer degradation, the importance of differential neotissue deposition relative to the mechanical (pressure) environment, and further support the utility of predictive modeling in the design, use, and evaluation of TEVGs in vivo. Statement of SignificanceHerein, we apply a biodegradable electrospun vascular graft to the arterial and venous circulations of the mouse and follow recipients beyond the point of polymer degradation. While venous implants formed viable neovessels, arterial grafts experienced catastrophic rupture due to aneurysmal dilation. We then inform a previously developed computational model of tissue engineered vascular graft growth and remodeling with parameters specific to the electrospun scaffolds utilized in this study. Remarkably, model simulations predict the differential performance of the venous and arterial constructs over 24 weeks. We conclude that computational simulations should inform the rational selection of scaffold parameters to fabricate tissue engineered vascular grafts that must be followed in vivo over time courses extending beyond polymer degradation.

Endothelial cell migration, adhesion and proliferation on different polymeric substrates.

The formation of a functionally-confluent endothelial cell (EC) monolayer affords proliferation of EC, which only happens in case of appropriate migratory activity. The migratory pathway of human umbilical endothelial cells (HUVEC) was investigated on different polymeric substrates. Surface characterization of the polymers was performed by contact angle measurements and atomic force microscopy under wet conditions. 30,000 HUVEC per well were seeded on polytetrafluoroethylene (PTFE) (θadv = 119°±2°), on low-attachment plate LAP (θadv = 28°±2°) and on polystyrene based tissue culture plates (TCP, θadv = 22°±1°). HUVEC tracks (trajectories) were recorded by time lapse microscopy and the euclidean distance (straight line between starting and end point), the total distance and the velocities of HUVEC not leaving the vision field were determined. On PTFE, 42 HUVEC were in the vision field directly after seeding. The mean length of single migration steps (SML) was 6.1±5.2 μm, the mean velocity (MV) 0.40±0.3 μm·min-1 and the complete length of the trajectory (LT) was 710±440 μm. On TCP 82 HUVEC were in the vision field subsequent to seeding. The LT was 840±550 μm, the SML 6.1±5.2 μm and the MV 0.44±0.3 μm·min-1. The trajectories on LAP differed significantly in respect to SML (2.4±3.9 μm, p < 0.05), the MV (0.16±0.3 μm·min-1, p < 0.05) and the LT (410±300 μm, p < 0.05), compared to PTFE and TCP. Solely on TCP a nearly confluent EC monolayer developed after three days. While on TCP diffuse signals of vinculin were found over the whole basal cell surface organizing the binding of the cells by focal adhesions, on PTFE vinculin was merely arranged at the cell rims, and on the hydrophilic material (LAP) no focal adhesions were found. The study revealed that the wettability of polymers affected not only the initial adherence but also the migration of EC, which is of importance for the proliferation and ultimately the endothelialization of polymer-based biomaterials.

Towards non-invasive characterisation of re-endothelialisation and restenosis following coronary stenting : an in vitro investigation using impedance spectroscopy

Following the permanent implantation of a coronary stent, optimal arterial wall healing is characterised by re-endothelialisation, the regrowth of a functional Endothelial Cell (EC) monolayer over the exposed stent surface, which reduces the risk of thrombosis. However restenosis, arising from the proliferation and migration of medial Smooth Muscle Cells (SMCs) can cause luminal narrowing to reoccur. Previous research has suggested that the stent itself could be used as an electrode and, when combined with non-invasive impedance spectroscopy techniques, monitor post stenting recovery. This could then inform clinicians on cell regrowth without the need for invasive imaging techniques. In this study we investigated the feasibility of this concept using two in-vitro models representing the cellular regrowth scenarios: re-endothelialisation and restenosis. Primary porcine ECs and SMCs were seeded onto platinum electrodes and electrical impedance spectroscopy measurements were made for up to 10 days in the frequency range 1 KHz to 100 KHz. Endothelium function was assessed through the measurement of the impedance response of confluent EC monolayers to the addition of a gap junction enhancer, dipyridamole, or an inhibitor (heptanol or carbenoxolone). Our results show that confluent, stent surface comparable populations of SMCs and ECs give rise to distinct impedance signatures, providing a novel method of non-invasively characterising these cell types. Gap junction inhibition of EC monolayers dose dependently reduced total impedance. Conversely dipyridamole’s enhancing effect on gap junction formation caused an increase in total impedance. These novel findings show the importance of intercellular gap junction communication in maintaining EC barrier function. Our current work is focused on the translation of this technology towards in-vivo monitoring of in-stent restenosis and recovery of a functional endothelium. Acknowledgements this study was funded by the UK Engineering and Physical Sciences Research Council (EP/F50036X/1) and is the subject of granted and pending patents at the University of Strathclyde.

Migration versus proliferation as contributor to in vitro wound healing of vascular endothelial and smooth muscle cells

Wound closure, as a result of collective cell growth, is an essential biological response to injury. In the field of vascular biology, the response of vascular smooth muscle cells (SMCs) and endothelial cells (ECs) to injury and substrate surface is important in therapeutic clinical treatment interventions such as angioplasty and atherectomy. Specifically, the mechanism by which cells close wounds (i.e. proliferation versus migration) in response to injury stimuli is of interest to better modulate recurrent vascular stenosis, prevent thrombus formation, occlusion, and life-threatening cardiovascular events. Here, we examine growth extent and temporal sequence of events following wound or gap introduction to a confluent monolayer of vascular SMCs or ECs. Significant differences in the preferred mechanisms of these cells to close wounds or gaps were observed; after 48 h, 73% of SMC wound closure was observed to be due to proliferation, while 75% of EC wound closure resulted from migration. These mechanisms were further modulated via addition or removal of extracellular matrix substrate and injury, with ECs more responsive to substrate composition and less to injury, in comparison to SMCs. Our results indicate that ECs and SMCs heal wounds differently, and that the time and mode of injury and associated substrate surface all impact this response.

Red Blood Cell Adhesion to Heme-Activated Endothelial Cells Reflects Clinical Phenotype in Sickle Cell Disease.

In sickle cell disease (SCD), 'disease severity' associates with increased RBC adhesion to quiescent endothelium, but the impact on activated endothelium is not known. Increased concentrations of free heme result from intravascular hemolysis in SCD. Heme is essential for aerobic metabolism, and plays an important role in numerous biological processes. Excess free heme induces reactive oxygen species generation and endothelial activation, which are associated with cardiovascular disorders including atherosclerosis, hypertension, and thrombosis. Here, we utilized an endothelialized microfluidic platform (Endothelium-on-a-chip) to assess adhesion of sickle hemoglobin-containing red blood cells (HbS RBCs), from adults with homozygous SCD, to heme-activated human endothelial cells (EC) in vitro. Confluent EC monolayers in microchannels were treated with pathophysiologically relevant levels of heme in order to simulate the highly hemolytic intravascular milieu seen in SCD. RBC adhesion to heme-activated ECs varied from subject to subject, and was associated with plasma markers of hemolysis (LDH) and reticulocytosis, thereby linking those RBCs that are most likely to adhere with those that are most likely to hemolyze. These results re-emphasize the critical contribution made by heterogeneous adhesive HbS RBCs to the pathophysiology of SCD. We found that adhesion of HbS RBCs to heme-activated ECs varied amongst individuals in the study population, and associated with biomarkers of hemolysis and inflammation, age, and a recent history of transfusion. Importantly, the microfluidic approach described herein holds promise as a clinically feasible Endothelium-on-a-chip platform with which to study complex heterocellular adhesive interactions in SCD. This article is protected by copyright. All rights reserved.

Abstract 4661: Magnetic nanoparticles (MNPs) for cancer drug delivery: The value of in vitro modeling

Abstract BACKGROUND: Magnetic nanoparticles (MNPs) have attracted great interest for use as delivery vehicles for cancer and other diseases due to their ability to be functionalized and localized using external magnets. In the presence of a magnetic field, individual MNPs form aggregates, which in turn can be made to spin and surface walk, in response to rotation of the field. Here, we present data from the use of a in vitro model system, which is useful in mimicking the various conditions and distances that MNP-drug combinations may encounter in vivo. METHODS: A sterilizable acrylic tray was designed, which has 1/8th inch wide lanes and can be used with or without the addition of cultured cells. The tray is compatible with standard plate readers, and the lanes can be modified to mimic various conduits within the body. In studies described here, glioma cell lines and normal vascular endothelial cells were used in the tray. In order to test the effect of fluid viscosity on MNP velocity in response to the rotating magnetic field, lanes were filled with 1mL of PBS, culture medium, serum, or whole blood. Pre-magnetized and unmagnetized MNPs were aliquoted into the lanes at volumes from 10 - 100 uL. T-PA and trypan blue were used as a model drugs. Velocities of MNP aggregates were determined by videography at five different tray positions relative to the magnet: centered, offset, push, pull, and below. Particle adhesion to cells could be quantified using ImageJ. RESULTS: Using the model system, greater MNP velocities were achieved with pre-magnetization, and larger aliquots. For example, 100ul aliquots had a total average velocity 1.27± 0.21 times that of 20ul. MNP velocity was found to be inversely -related to fluid viscosity, but particles could be moved magnetically even through whole blood. Test drugs could be successfully delivered by convection, even without prior binding to MNPs. MNP velocity also varied according to magnet position, with speeds up to 0.75 +/- 0.05 cm/sec in the pull (fastest) position. In the offset position, a distance of 20 cm above the magnet produced the greatest velocity. MNPs moved more slowly over confluent monolayers of endothelial or glioma cells (with greater adhesion), but a mean velocity of 0.25 cm/sec +/- 0.03 cm was typically observed. CONCLUSIONS: In vitro modeling is extremely helpful in predicting the behavior of particles intended for clinical use in magnetically-enhanced drug delivery. The velocity and cell surface adhesion of MNP aggregates can be quantified, and the effect of factors - such as fluid viscosity, cell type, and position with respect to the magnet - analyzed in order to better understand the advantages and limitations of this technology. Citation Format: Sebastian P. Pernal, Alexander J. Willis, Herbert H. Engelhard. Magnetic nanoparticles (MNPs) for cancer drug delivery: The value of in vitro modeling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4661.

Deciphering the relative contribution of vascular inflammation and blood rheology in metastatic spreading.

Vascular adhesion of circulating tumor cells (CTCs) is a key step in cancer spreading. If inflammation is recognized to favor the formation of vascular "metastatic niches," little is known about the contribution of blood rheology to CTC deposition. Herein, a microfluidic chip, covered by a confluent monolayer of endothelial cells, is used for analyzing the adhesion and rolling of colorectal (HCT-15) and breast (MDA-MB-231) cancer cells under different biophysical conditions. These include the analysis of cell transport in a physiological solution and whole blood over a healthy and a TNF-α inflamed endothelium with a flow rate of 50 and 100 nl/min. Upon stimulation of the endothelial monolayer with TNF-α (25 ng/ml), CTC adhesion increases from 2 to 4 times whilst cell rolling velocity only slightly reduces. Notably, whole blood also enhances cancer cell deposition from 2 to 3 times, but only on the unstimulated vasculature. For all tested conditions, no statistically significant difference is observed between the two cancer cell types. Finally, a computational model for CTC transport demonstrates that a rigid cell approximation reasonably predicts rolling velocities while cell deformability is needed to model adhesion. These results would suggest that, within microvascular networks, blood rheology and inflammation contribute similarly to CTC deposition, thereby facilitating the formation of metastatic niches along the entire network, including the healthy endothelium. In microfluidic-based assays, neglecting blood rheology would significantly underestimate the metastatic potential of cancer cells.

ZEB1 REGULATES MULTIPLE ASPECTS OF ENDOTHELIAL QUIESCENCE DURING VASCULAR REMODELLING

Generation of new blood vessels is essential for vascular remodeling in exercise to provide greater blood flow to developing muscles. As blood vessels grow, the sprouting endothelial tips need to recognise each other, and move from a motile, angiogenic phenotype to a maturing, quiescent phenotype,, form tubes and allow blood flow to occur and regulate their development. They grow in response to cytokine signalling such as VEGF and other angiogenic factors, but they mature as a consequence of cell contact and flow. As they do so, they change their metabolic state from growing, glycolytic, to quiescent oxidative metabolism. The mechanisms behind the transcriptional control of this phenotype are unknown, but recent work has demonstrated the dynamic interplay between ‘quiescence’ factors (such as FOXO1 expression) and expression of glycolytic enzymes such as PFKFB3 through c‐MYC. We investigated potential drivers of this process and identified Zeb1 (a transcription factor with known functions in cancer progression) to be expressed by quiescent (non‐angiogenic) endothelial cells and reduced in angiogenic endothelial cells in the developing murine retina. In vitro analyses demonstrates that Zeb1 is expressed by confluent monolayers of Human Vein Umbilical Cord Endothelial cells (HUVECs) but is downregulated in actively growing cells. Zeb1 knockdown promotes an angiogenic EC phenotype: including enhanced PFKFB3, c‐myc and VEGFR2 and reduced FOXO1, Tie2 and VE‐cadherin expression. Consistently, Zeb1 knockdown EC were metabolically more active (both through oxidative and glycolytic pathways). Furthermore, loss of Zeb1 promoted EC migration and proliferation in vitro. In vivo shRNA knockdown of Zeb1 led to altered vessel formation and hypercellularity. In conclusion, Zeb1 appears to be linked to key quiescence regulating signaling pathways and furthermore regulates aspects of these pathways. Zeb1 expression appears to be a critical component for endothelial cells to generate functional vasculatures that can support enhanced muscle blood flow, by its control of FOXO1, PFKFB3 and Myc.Support or Funding InformationThis work was supported by the British Heart Foundation (grant PG/11/67/29067), the University of Nottingham Cancer Research Priority Area, and the Medical Research Council Newton Fund (MR/N01247X/1)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract P244: Characterization of Exosomes From Vascular Endothelial and Smooth Muscle Cells

Exosomes are small nanovesicles derived from endosomal compartments termed multivesicular bodies. These multivesicular bodies fuse with the plasma membrane, release exosomes into extracellular space, allowing them to transport their cargo of functional nucleic acids and proteins. Despite their well-documented existence, not much is known about exosomes in vascular biology. The luminal surface of the intima consists of a confluent monolayer of endothelial cells (ECs), while the medial layer is characterized by presence of vascular smooth muscle cells (VSMCs). We characterized the exosomes released by these cell types in culture to better understand the packaging of these vesicles and their capacity to participate in cell-to-cell communication. Rat aortic ECs exosomes were generated in serum-free media for 48 hours while rat aortic VSMC exosomes were generated in serum-free media for 72 hours. Media harvested from both was ultracentrifuged at 10,000xg to remove microparticles followed by centrifugation of the supernatant at 100,000xg to pellet exosomes. Exosome pellet was re-suspended in PBS, 0.22 μm filtered and spun at 100,000xg to remove contaminating particles. Transmission electron microscopic analysis of VSMC exosomes confirmed particle morphology with diameters ranging from 60-150 nm. Particle size and concentration was determined using NTA NanoSight. The modal size of EC exosomes and VSMC exosomes were 107.2±5.4 nm and 116.6±7.5 nm, respectively. Western blotting analysis was performed to characterize common and specific markers for vascular exosomes. We confirmed common exosome markers such as CD81, CD63, caveolin-1, and flotillin-1 in both populations. ECs specifically expressed VE-cadherin and TSG-101 whereas VSMCs expressed syntenin-1 and smooth muscle actin. Cell-to-cell signaling in vitro assays revealed that EC-derived exosomes can stimulate hypoxia-inducible factor 1-alpha expression in VSMCs in a dose-dependent and time-dependent manner. Maximal induction was seen at 6 hours of stimulation and using 5x10 8 particles/mL. From these data, we conclude that exosomes are released from these cell types and play a potential role in cell-to-cell communication to maintain homeostasis and/or promote pathology.

Potential Influence of Endothelial Adsorption on the Delayed Time to Maximum Concentration of Biopharmaceuticals.

Background and objectivesMaximum plasma concentration of biopharmaceuticals sometimes occurs long after completion of intravenous infusion. The objective of this research was to study the hypothetical adsorption of biopharmaceuticals to endothelium and infusion material, which may theoretically explain this phenomenon.MethodsInfusion procedures were mimicked in an artificial vessel covered with a confluent monolayer of endothelial cells. Three monoclonal antibodies (MAbs) and C1 inhibitor were studied.ResultsAdsorption of MAbs to endothelium was observed followed by release when the vessel was subsequently perfused with buffer. Adsorption to infusion material also occurred to various degrees and in a seemingly random fashion, with a loss of up to 15% during a single flush of the line, but release from the line was not seen.ConclusionsOur results indicate that adsorption of biopharmaceuticals to endothelium can occur. This observation can explain the increase in plasma concentration after completion of intravenous administration.

Effect of shear stress on iPSC-derived human brain microvascular endothelial cells (dhBMECs)

BackgroundThe endothelial cells that form the lumen of capillaries and microvessels are an important component of the blood–brain barrier. Cell phenotype is regulated by transducing a range of biomechanical and biochemical signals in the local microenvironment. Here we report on the role of shear stress in modulating the morphology, motility, proliferation, apoptosis, and protein and gene expression, of confluent monolayers of human brain microvascular endothelial cells derived from induced pluripotent stem cells.MethodsTo assess the response of derived human brain microvascular endothelial cells (dhBMECs) to shear stress, confluent monolayers were formed in a microfluidic device. Monolayers were subjected to a shear stress of 4 or 12 dyne cm−2 for 40 h. Static conditions were used as the control. Live cell imaging was used to assess cell morphology, cell speed, persistence, and the rates of proliferation and apoptosis as a function of time. In addition, immunofluorescence imaging and protein and gene expression analysis of key markers of the blood–brain barrier were performed.ResultsHuman brain microvascular endothelial cells exhibit a unique phenotype in response to shear stress compared to static conditions: (1) they do not elongate and align, (2) the rates of proliferation and apoptosis decrease significantly, (3) the mean displacement of individual cells within the monolayer over time is significantly decreased, (4) there is no cytoskeletal reorganization or formation of stress fibers within the cell, and (5) there is no change in expression levels of key blood–brain barrier markers.ConclusionsThe characteristic response of dhBMECs to shear stress is significantly different from human and animal-derived endothelial cells from other tissues, suggesting that this unique phenotype that may be important in maintenance of the blood–brain barrier. The implications of this work are that: (1) in confluent monolayers of dhBMECs, tight junctions are formed under static conditions, (2) the formation of tight junctions decreases cell motility and prevents any morphological transitions, (3) flow serves to increase the contact area between cells, resulting in very low cell displacement in the monolayer, (4) since tight junctions are already formed under static conditions, increasing the contact area between cells does not cause upregulation in protein and gene expression of BBB markers, and (5) the increase in contact area induced by flow makes barrier function more robust.

Abstract 1889: Formation and merger of H69 and H69AR small cell lung cancer (SCLC) tumor cell clusters (TCCs) in vitro

Abstract A novel manifestation of tumor cell invasiveness, termed mutual cellular pervasion (MCP), was observed in vitro. It involved TCCs formed by SCLC cells. Suspension cultures of H69 and H69AR cells were grown in flavin-free RPMI 1640 medium with 10% fetal bovine serum as the only source of flavins. Experiments and subcultivations were performed under light with wavelengths above 520 nm. TCC morphology was studied by confocal microscopy and transmission and scanning electron microscopy. Interactions of TCCs were followed by video microscopy in phase contrast and fluorescence modes. Data were analyzed using the Definiens software suite. The interaction of H69AR TCCs with confluent monolayers of human umbilical vascular endothelial cells (HUVEC) was visualized. Freshly subcultivated, singularized tumor cells rapidly formed aggregates. After two days, solid TCCs prevailed whose surface cells were connected by desmosome-like junctions. Apoptosis but not necrosis became obvious in central parts of clusters with diameters above 100 to 150 μm. TCCs enlarged through both cell proliferation and serial cluster mergers. In the course of TCC merger, four stages were distinguishable: (1) directed cluster movement leading to contact of clusters, (2) formation of a near circular, symmetric structure termed contact disc. It consists of planar surface areas to which both TCCs involved contribute equally. (3) Merger of TCCs took place in the contact disc, and was accompanied by active movement of adjacent cells. (4) Starting from the merger zone, major surface areas of newly formed clusters were mutually pervaded by cells from both participating cell clusters. After numerous merger events, only low numbers of TCCs reaching sizes of up to 2 to 3 mm were present at subcultivation. No spontaneous bursting, shrinkage, degeneration, or decay of TCCs has been observed. However, when TCCs of H69AR cells were confronted with confluent layers of HUVEC, drastic changes occurred in both formations. First, intense oscillatory cellular movements were triggered on TCC surfaces, preceding the release of cell aggregates which moved away from TCCs to the HUVEC layer. At the same time, HUVEC were disconnected from each other and started moving either randomly or in an oscillatory fashion. Individual tumor cells attached to the much larger HUVEC and spread on their surface, resulting in full covering which secluded HUVEC from the medium. To our knowledge, neither formation nor merger of malignant TCCs in vitro have been described so far. TCC merger involves mutual cellular penetration of the involved TCC surfaces, i. e. MCP. Contrary to the invasion of tumor cells into non-tumor tissue, pervasion does not lead to destruction but results in TCC growth. However, key features of tumor cell invasion in vivo, such as chemotaxis, spreading and TCC formation as well as disruption of endothelial layers, manifest under conditions in vitro, thus facilitating related experimental studies. Citation Format: Nico O. Ruprecht, Martin Hungerbuehler, Philipp Kellmann, Nathalie Harder, Günter Schmidt, Carlos Wotzkow, Beat Haenni, Fabian Blank, Hendrik Von Tengg-Kobligk, Christof Granzow. Formation and merger of H69 and H69AR small cell lung cancer (SCLC) tumor cell clusters (TCCs) in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1889. doi:10.1158/1538-7445.AM2017-1889

Biomimetic modification of polyurethane-based nanofibrous vascular grafts: A promising approach towards stable endothelial lining

The emerging demand for small caliber vascular grafts to replace damaged vessels has attracted research attention. However, there is no perfect replacement in clinical use yet, mainly due to low patency rate of synthetic small caliber grafts. The main pathology behind low patency rate include thrombosis and graft/vessel hemodynamic mismatch, leading to intimal hyperplasia. Rapid in-situ endothelialization of vascular grafts is considered as one of the best strategies to overcome these complications. In the present study, Heparin and VEGF were immobilized via self-polymerization and deposition of polydopamine (PDA) on polyurethane (PU) nanofibrous scaffolds to improve endothelialization. Polyurethane nanofibrous scaffold (PUNF) that mimics vascular extracellular matrix (ECM) was chosen owing to its biocompatibility, biodegradability. Scanning electron microscopy (SEM), water contact angle (CA) measurement and Raman spectroscopy were used to characterize the surface, and tensile test was used to analyze mechanical properties before and after surface modification of the scaffolds. It was found that tensile strength and young's modulus were significantly increased after PDA coating on PUNF membranes. The hemocompatibility tests revealed that surface heparinization significantly inhibited the adhesion of platelet on the scaffolds. Immobilization of VEGF on the scaffolds significantly enhanced the proliferation of human umbilical vein endothelial cells (HUVECs) through enhanced cells adhesion and improved cell-scaffold interactions. The results suggest that dual-factor immobilization resulted in not only confluent monolayer of endothelial cells but also conferred excellent antithrombotic properties to the surface. This method of surface modification (immobilization of Heparin, VEGF by PDA layer) is suggested as a promising modification technique to increase hemocompatibility of small-diameter vascular grafts.

Continuously Grooved Stent Struts for Enhanced Endothelial Cell Seeding

PurposeImplantation of pre-endothelialized stents could enhance cellular recovery of a damaged vessel wall provided attached cells remain viable, functional and are present in sufficient numbers after deployment. The purpose of this study was to evaluate the feasibility of grooved stainless steel (SS) stents as a primary endothelial cell (EC) carrier with potentially enhanced EC protection upon stent deployment.Materials and MethodsAttachment and behavior of enzymatically harvested human adult venous ECs seeded onto gelatin-coated vascular stents were evaluated in an in vitro setting. Smooth and grooved SS stents and smooth nitinol stents were studied.ResultsAll cells expressed EC markers vWF and CD31. Using rotational seeding for a period of 16–24 h, ECs attached firmly to the stents with sufficient coverage to form a confluent EC monolayer. The grooved SS wire design was found to enable attachment of three times the number of cells compared to smooth wires. This also resulted in an increased number of cells remaining on the stent after deployment and after pulsatile flow of 180 ml/min for 24 h, which did not result in additional EC detachment.ConclusionsThe grooved stent provides a potential percutaneous means to deliver sufficient numbers of viable and functional cells to a vessel segment during vascular intervention. The grooves were found to offer a favorable surface for EC attachment and protection during stent deployment in an in vitro setting.

Laquinimod enhances central nervous system barrier functions

Laquinimod is currently being tested as a therapeutic drug in multiple sclerosis. However, its exact mechanism of action is still under investigation. Tracking of fluorescently-tagged encephalitogenic T cells during experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis, revealed that laquinimod significantly reduces the invasion of pathogenic effector T cells into the CNS tissue. T-cell activation, differentiation and amplification within secondary lymphoid organs after immunization with myelin antigen, their migratory capacity and re-activation within the nervous tissue were either only mildly affected or remained unchanged. Instead, laquinimod directly impacted the functionality of the CNS vasculature. The expression of tight junction proteins p120 and ZO-1 in human brain endothelial cells was up-regulated upon laquinimod treatment, resulting in a significant increase in the transendothelial electrical resistance of confluent monolayers of brain endothelial cells. Similarly, expression of the adhesion molecule activated leukocyte cell adhesion molecule (ALCAM) and inflammatory chemokines CCL2 and IP-10 was suppressed, leading to a significant reduction in the migration of memory TH1 and TH17 lymphocytes across the blood brain barrier (BBB). Our data indicate that laquinimod exerts its therapeutic effects by tightening the BBB and limiting parenchymal invasion of effector T cells, thereby reducing CNS damage.

Abstract A15: Development of an in vitro 3D vessel-spheroid model for investigating cancer metastasis

Abstract During metastasis, cells break free from the primary tumor, migrate through the stroma and enter either the vascular or lymph systems to disseminate into secondary sites. To better understand this process, many groups have developed in vitro models of cancer metastasis using microfluidic based platforms to mimic the tumor microenvironment and the intravasation/extravasation of tumor cells. While these models have provided significant new insights into the mechanisms by which metastatic cells move across the endothelium, they are most often focused on the movement of single cells. Notably, there is significant clinical evidence to suggest that tumor cells can travel collectively though the stroma and can circulate as cell aggregates in the vasculature. Recent advances in the understanding of cancer metastasis have shown that cancer may move more aggressively as a collection of cell when traversing the extracellular matrix (ECM) of the tumor microenvironment as opposed to more studied singular cellular motif. To better mimic these collective movements, we have developed a model that incorporates the following essential elements that exist in the tumor microenvironment: a cylindrical channel lined with a confluent monolayer of endothelial cells, a metastatic tumor analog in close proximity to the channel, the ability to incorporate flow with tunable properties to simulate shear stress due to blood flow, and a tunable collagen microenvironment. Briefly, in a 3D printed cube, Type 1 collagen is added at physiological stiffness. As the collagen is polymerizing in the device, a metastatic tumor analog (spheroid) consisting of labeled MDA-MB-231 cells is placed in proximity of the vessel that is lined with endothelial cells. The platform allows for the visualization of metastatic cell intravasation using confocal microscopy. Notably, this model is easy to build and implement with fairly accessible tools and without the use of a clean room or microfabrication facilities. Importantly, going forward this model has the ability to study the cellular behavior post-intravasation due to the incorporation of physiological flows within the channel. Citation Format: Adam Munoz, Joseph Miller, Cynthia A. Reinhart-King. Development of an in vitro 3D vessel-spheroid model for investigating cancer metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr A15.

Real-time quantification of endothelial response to shear stress and vascular modulators.

Quiescence is commonly used to describe the inactive state of endothelial cells (ECs) in monolayers that have reached homeostasis. Experimentally quiescence is usually described in terms of the relative change in cell activity (e.g. turnover, speed, etc.) in response to a perturbation (e.g. solute, shear stress, etc.). The objective of this study is to provide new insight into EC quiescence by quantitatively defining the morphology and activity of confluent cell monolayers in response to shear stress and vascular modulators. Confluent monolayers of human umbilical vein ECs (HUVECs) were subjected to a range of shear stresses (4-16 dyne cm-2) under steady flow. Using phase contrast, time-lapse microscopy and image analysis, we quantified EC morphology, speed, proliferation, and apoptosis rates over time and detected differences in monolayer responses under various media conditions: basal media supplemented with growth factors, interleukin-8, or cyclic AMP. In all conditions, we observed a transition from cobblestone to spindle-like morphology in a dose-dependent manner due to shear stress. Cyclic AMP enhanced the elongation and alignment of HUVECs due to shear stress and reduced steady state cell speed. We observed the lowest proliferation rates below 8 dyne cm-2 and found that growth factors and cyclic AMP reduced proliferation and apoptosis; interleukin-8 similarly decreased proliferation, but increased apoptosis. We have quantified the response of ECs in confluent monolayers to shear stress and vascular modulators in terms of morphology, speed, proliferation and apoptosis and have established quantifiable metrics of cell activity to define vascular quiescence under shear stress.

Intercellular Adhesion Molecule-1 and Vascular Cell Adhesion Molecule Are Induced by Ionizing Radiation on Lymphatic Endothelium

The goal of this study was to assess the effects of ionizing radiation on the expression of the integrin ligands ICAM-1 and VCAM that control leucocyte transit by lymphatic endothelial cells. Confluent monolayers of primary human lymphatic endothelial cells (LEC) were irradiated with single dose of 2, 5, 10 or 20 Gy, with 6 MeV-x-rays using a Linear-Accelerator. ICAM-1 and VCAM expression was determined by flow cytometry. Human tissue specimens received a single dose of 20 Gy with 15 MeV-x-rays. MC38, B16-OVA or B16-VEGF-C tumors grown in C57BL/6 mice were irradiated with single dose of 20Gy using a Linear-Accelerator fitted with a 10mm Radiosurgery collimator. Clinical samples were obtained from patients previous and 4 weeks after complete standard radiotherapy. ICAM-1 and VCAM expression was detected in all tissue specimens by confocal microscopy. To understand the role of TGFβ in this process anti-TGFβ blocking mAb were injected i.p. 30min before radiotherapy. Cell adhesion to irradiated LEC was analyzed in adhesion experiments performed in the presence or in the absence of anti- TGFβ and /or anti-ICAM1 blocking mAb. We demonstrate that lymphatic endothelial cells in tumor samples experience induction of surface ICAM-1 and VCAM when exposed to ionizing radiation in a dose- and time-dependent manner. These effects can be recapitulated in cultured LEC, and are in part mediated by TGFβ. These data are consistent with increases in ICAM-1 and VCAM expression on LYVE-1+ endothelial cells in freshly explanted human tumor tissue and in mouse transplanted tumors after radiotherapy. Finally, ICAM-1 and VCAM expression accounts for enhanced adherence of human T lymphocytes to irradiated LEC. Our results show induction of ICAM-1 and VCAM on LVs in irradiated lesions and offer a starting point for elucidating the biological and therapeutic implications of targeting leukocyte traffic in combination to immunotherapy.

Circulating Plasma Extracellular Microvesicle MicroRNA Cargo and Endothelial Dysfunction in Children with Obstructive Sleep Apnea.

Obese children are at increased risk for developing obstructive sleep apnea (OSA), and both of these conditions are associated with an increased risk for endothelial dysfunction (ED) in children, an early risk factor for atherosclerosis and cardiovascular disease. Although weight loss and treatment of OSA by adenotonsillectomy improve endothelial function, not every obese child or child with OSA develops ED. Exosomes are circulating extracellular vesicles containing functional mRNA and microRNA (miRNA) that can be delivered to other cells, such as endothelial cells. To investigate whether circulating exosomal miRNAs of children with OSA differentiate based on endothelial functional status. Obese children (body mass index z score >1.65) and nonobese children were recruited and underwent polysomnographic testing (PSG), and fasting endothelial function measurements and blood draws in the morning after PSG. Plasma exosomes were isolated from all subjects. Isolated exosomes were then incubated with confluent endothelial cell monolayer cultures. Electric cell-substrate impedance sensing systems were used to determine the ability of exosomes to disrupt the intercellular barrier formed by confluent endothelial cells. In addition, immunofluorescent assessments of zonula occludens-1 tight junction protein cellular distribution were conducted to examine endothelial barrier dysfunction. miRNA and mRNA arrays were also applied to exosomes and endothelial cells, and miRNA inhibitors and mimics were transfected for mechanistic assays. Plasma exosomes isolated from either obese children or nonobese children with OSA were primarily derived from endothelial cell sources and recapitulated ED, or its absence, in naive human endothelial cells and also in vivo when injected into mice. Microarrays identified a restricted signature of exosomal miRNAs that readily distinguished ED from normal endothelial function. Among the miRNAs, expression of exosomal miRNA-630 was reduced in children with ED and normalized after therapy along with restoration of endothelial function. Conversely, transfection of exosomes from subjects without ED with an miRNA-630 inhibitor induces the ED functional phenotype. Gene target discovery experiments further revealed that miRNA-630 regulates 416 gene targets in endothelial cells that include the Nrf2, AMP kinase, and tight junction pathways. These observations elucidate a novel role of exosomal miRNA-360 as a putative key mediator of vascular function and cardiovascular disease risk in children with underlying OSA and/or obesity, and identify therapeutic targets.