The role of nitric oxide in cellular response to hyperbaric conditions

Nitric oxide (NO) acts as a regulator in cell proliferation and expression of growth factors and forms peroxynitrite (ONOO⁻) in oxidative conditions. The aim of the study was to investigate the role of NO in cellular response to hyperbaric oxygen (HBO). NO and nitrotyrosine (NT), biochemical marker for ONOO⁻, cell proliferation and growth factors, were ex-vivo studied in cell cultures under HBO and normobaric (NOR) conditions. A549 (epithelial), L929 (fibroblast) and SVEC (endothelial) were exposed to 100% O₂, at P = 280 kPa for t = 60 min, once daily for five sessions. Cell proliferation was determined as the incorporation of bromodeoxyuridine (BrdU) into cells and NO as nitrates/nitrites (NO₃ ⁻/ NO₂ ⁻) Gries reaction product in cell culture supernatant (CCSP). NT, vascular endothelial growth factor (VEGF) and transforming growth factor-beta 1 (TGFb1) were measured with enzyme-inked immunosorbent assay (ELISA) in CCSP. The time course of total NO was opposite to that of cell proliferation in HBO conditions, peaking after the second HBO session, while cell proliferation showed a reverse trend, minimizing at the same time, suggesting a reverse and transient anti-proliferative effect. Released growth factors were significantly increased in late HBO sessions. NT peaked after second treatment, indicating the formation of ONOO⁻. In control cultures (NOR), proliferation rate was downward and no significant differences were found for the other parameters. In conclusion, the data suggested a key role for NO in the beneficial HBO action, depending on its concentration, which fluctuated with the time of HBO exposure and the activation of oxidant–antioxidant (REDOX) mechanisms, regardless of cell type.     

Towards microfabricated biohybrid artificial lung modules for chronic respiratory support

We have utilized soft lithography techniques to create three-dimensional arrays of blood microchannels and gas pathways in poly(dimethylsiloxane) (PDMS) that approach the microvascular scale of the natural lung. The blood microchannels were lined with endothelial cells in an effort to provide a non-thrombogenic surface that might ultimately reduce the need for systemic anticoagulation. A novel design and fabrication method were developed to create prototype modules for gas permeance and cell culture testing. The gas permeance modules contained 6 layers, four gas and two blood, while the modules for cell culture testing contained two layers of blood channels. The gas permeance of the modules was examined and maximum values of 9.16 × 10⁻⁶ and 3.55 × 10⁻⁵ mL/s/cm²/cmHg, for O₂ and CO₂ respectively, were obtained. Finally, endothelial cells were seeded and dynamically cultured in prototype cell culture modules. Confluent and viable cell monolayers were achieved after 10 days of perfusion.     

Peptide Interfacial Biomaterials Improve Endothelial Cell Adhesion and Spreading on Synthetic Polyglycolic Acid Materials

Resorbable scaffolds such as polyglycolic acid (PGA) are employed in a number of clinical and tissue engineering applications owing to their desirable property of allowing remodeling to form native tissue over time. However, native PGA does not promote endothelial cell adhesion. Here we describe a novel treatment with hetero-bifunctional peptide linkers, termed “interfacial biomaterials” (IFBMs), which are used to alter the surface of PGA to provide appropriate biological cues. IFBMs couple an affinity peptide for the material with a biologically active peptide that promotes desired cellular responses. One such PGA affinity peptide was coupled to the integrin binding domain, Arg-Gly-Asp (RGD), to build a chemically synthesized bimodular 27 amino acid peptide that mediated interactions between PGA and integrin receptors on endothelial cells. Quartz crystal microbalance with dissipation monitoring (QCMD) was used to determine the association constant (K _A 1 × 10⁷ M⁻¹) and surface thickness (~3.5 nm). Cell binding studies indicated that IFBM efficiently mediated adhesion, spreading, and cytoskeletal organization of endothelial cells on PGA in an integrin-dependent manner. We show that the IFBM peptide promotes a 200% increase in endothelial cell binding to PGA as well as 70–120% increase in cell spreading from 30 to 60 minutes after plating.     

Enhanced adhesion of endothelial cells onto a polypropylene hollow-fiber membrane by plasma discharge treatment and high inoculum cell density

The effect of plasma discharge treatment of a microporous polypropylene hollow-fiber membrane commonly used for gas exchange in a conventional artificial lung on the adhesion of endothelial cells was investigated with the aim of constructing a hybrid artificial lung bearing endothelial cells on the membrane. The initial adhesion density and growth rate of the cells on the membrane were markedly increased following plasma discharge treatment (13.56 MHz, 30W) of the membrane for 5 to 20 min in the presence of 0.05 mmHg of various gases, such as ammonia, oxygen, and water vapor. Treatment of the membrane with ammonia for 5 min resulted in the highest increase in the cell adhesion density on 5 days from 1.4×10² to 2.0×10³ cells/cm², and the cell density reached 5.0×10³ cells/cm² after cultivation for 14 days. Increasing the inoculum cell concentration from 3.3×10⁵ to 3.2×10⁶ cells/ml resulted in an initial cell adhesion of 0.9×10⁵ cells/cm², even after 1 day. It was observed under a microscope that the cells were distributed uniformly to cover almost all of the surface area of the membrane. After the plasma discharge treatment, the permeability of the membrane to water increased to 9% of that of a polyethylene hollow fiber having pore diameters larger than 0.4 μm.     

Endothelins Mediate Neutrophil Activation, ProMMP-9 Release and Endothelial Cell Detachment

Neutrophils isolated from human peripheral blood added to a monolayer of human endothelial cells (ECV-304 cell line) stimulated with LPS (100 ng ml⁻¹) resulted in: (a) neutrophil activation, measured by spreading and release of leukotriene B₄ (LTB₄); (b) neutrophil degranulation, measured by release of matrix pro-metalloproteinase-9 (proMMP-9) and (c) loss of the monolayer integrity due to detachment of the endothelial cells. Stimulation of endothelial cells with tumor necrosis factor-α (TNF-α 10 ng ml⁻¹) or interleukin-1 (IL-1; 10 ng ml⁻¹) induced a similar dose-dependent increase in the neutrophil activation and endothelial cell detachment. Pre-treatment of LPS-activated ECV-304 cells with [Phe22]BigET-1(19–37) (10⁻⁹ M; an inhibitor of endothelin converting enzyme (ECE)) or addition of BQ-123 (10⁻⁶ M; a selective endothelin A (ET_A) receptor antagonist) to the co-cultures, significantly reduced neutrophil spreading (50–70% inhibition) as well as the levels of LTB₄ (70–100% inhibition) and proMMP-9 (40–50% inhibition) in the co-culture supernatants. In addition, the detachment of endothelial cells was also reduced (60–75% inhibition). Moreover, the exogenous addition of ET-1 (10⁻⁹ M) to neutrophil suspensions induced neutrophil spreading and release of LTB₄ and proMMP-9. Taken together, these findings indicate that neutrophils added to stimulated endothelial cells in the co-culture system employed in this study, get activated by products of these cells and degranulate. In parallel, the detachment of endothelial cell monolayer from the culture plates, possibly by the action of neutrophil granule-derived gelatinases, is observed. Endothelins (ETs) produced by the endothelial cells are suggested to play an essential role in these phenomena.     

Spatial variation of aortic wall oxygen diffusion coefficient from transient polarographic measurements

Polarographic current transients following a voltage step (turn-on transient) were measured with bare cathodes (25 μm diameter) and shallowly recessed oxygen microelectrodes (<5μm diameter). Except for the initial part of the current transient, the experimental measurements were in excellent agreement with simple models in the literature, which predict an inverse relationship with . Turn-on transients were measured in aqueous solutions with known physical properties, and in aortic wall tissue from three different species (n=6 rabbits, n=3 dogs, and n=1 miniature pig). Oxygen diffusion coefficients (D) were determinedin vitro by comparing time constants measured by the same microelectrode in saline and in strips of aortic wall tissue at 37°C. On the inner side (endothelium and intima) of the aorta, D averaged (±S.E.) 7.0 (±0.8)×10⁻⁶ cm²/s in 6 rabbits, 6.4 (±1.0)×10⁻⁶ cm²/s in 3 dogs, and was 4.6×10⁻⁶ cm²/s in the pig. On the adventitial side, D was 9.5×10⁻⁶ cm²/s in 1 rabbit, 11.4 (±1.2)×10⁻⁶ cm²/s in 3 dogs, and 8.1×10⁻⁶ cm²/s in the pig. For every aortic strip on which D was measured from both sides, D for the inner wall was always lower, overall by a little more than one third (p<0.001). The lower D on the endothelial side may limit oxygen transport to the vascular wall and play a role in atherogenesis.     

Oral Tolerization with Cardiac Myosin Peptide (614–629) Ameliorates Experimental Autoimmune Myocarditis: Role of Stat 6 Genes in BALB/CJ Mice

Introduction  Experimental autoimmune myocarditis (EAM) is mediated by myocardial infiltration by myosin-specific T cells secreting inflammatory cytokines. Materials and methods  To clarify the role of cytokines in EAM, we compared STAT 6-deficient (^−/−) with STAT 4^−/− and wild-type (BALB/CJ) mice following immunization with cardiac myosin peptide (614–629). Results  Wild-type mice developed severe disease with a small increase in severity in STAT 6^−/− mice, while STAT 4^−/− mice were resistant to EAM. STAT 6^−/− mice had increased splenocyte proliferation and INF-γ production versus wild type, while STAT 4^−/− mice had decreased proliferation and INF-γ. Following oral administration of myosin (614–629), tolerization was induced in wild-type mice evidenced by amelioration of myocarditis and up-regulation of IL-4. Adoptive transfer of splenocytes from orally tolerized mice resulted in inhibition of disease in STAT 6^−/− mice. Conclusion  These results demonstrate that oral tolerization ameliorates EAM in BALB/CJ mice and indicate a down-regulatory role for STAT 6 genes.     

Shear Stress Induced Release of Von Willebrand Factor and Thrombospondin-1 in Uvec Extracellular Matrix Enhances Breast Tumour Cell Adhesion

Endothelial cells in vivo are exposed to blood shear forces and flow perturbations induce their activation. Such modifications of hemodynamic can be observed in patients with cancer. We have submitted endothelial cells (HUVEC) to shear stress (13 dynes/cm²) and isolated their extracellular matrix (ECM) prior perfusion with breast adenocarcinoma cells (MDA-MB-231) in whole blood at a shear rate of 1500 s⁻¹. Exposure of HUVEC to 13 dynes/cm² (τ₁₃) for 2 h enhanced the secretion of von Willebrand factor (vWF) and thrombospondin-1 (TSP-1) in the ECM. Moreover, MDA-MB-231 cell adhesion was enhanced to such treated-ECM. This over-adhesion was inhibited by pre-incubating the ECM with anti-vWF or anti-TSP-1 antibodies, or by blocking tumour cell α_vβ₃ integrin. Although blood platelets were involved in tumour cell adhesion to ECM, blockade of platelet GPIb or α_IIbβ₃ receptors did not specifically inhibit the enhanced tumour cell adhesion observed on τ₁₃. ECM. These findings indicate that shear stress can modulate the expression of adhesive proteins in ECM, which favours direct tumour cell adhesion via α_vβ₃ and other receptors.     

Regulation of Antioxidants and Phase 2 Enzymes by Shear-Induced Reactive Oxygen Species in Endothelial Cells

Exposure of vascular endothelial cells (ECs) to steady laminar shear stress activates the NF-E2-related factor 2 (Nrf2) which binds to the antioxidant response element (ARE) and upregulates the expression of several genes. The onset of shear is known to increase the EC reactive oxygen species (ROS) production, and oxidative stress can activate the ARE. ARE-regulated genes include phase 2 enzymes, such as glutathione-S-transferase (GST) and NAD(P)H:quinone oxidoreductase 1 (NQO1), and antioxidants, such as glutathione reductase (GR), glutathione peroxidase (GPx) and catalase. We examined how shear stress affects the antioxidant/phase 2 enzyme activities and whether ROS mediate these effects. ROS production, measured by dichlorofluorescin fluorescence, depended on level and time of shear exposure and EC origin, and was inhibited by either an endothelial nitric oxide synthase (eNOS) inhibitor or a superoxide dismutase (SOD) mimetic and peroxynitrite (ONOO⁻) scavenger. Shear stress (10 dynes/cm², 16 h) significantly increased the NQO1 activity, did not change significantly the glutathione (GSH) content, and significantly decreased the GR, GPx, GST and catalase activities in human umbilical vein ECs. Either eNOS inhibition or superoxide radical (O₂^•−)/ONOO⁻ scavenging differentially modulated the shear effects on enzyme activities suggesting that the intracellular redox status coordinates the shear-induced expression of cytoprotective genes.     

Endothelial Cell Layer Subjected to Impinging Flow Mimicking the Apex of an Arterial Bifurcation

Little is known about endothelial responses to the impinging flow hemodynamics that occur at arterial bifurcation apices, where intracranial aneurysms usually form. Such hemodynamic environments are characterized by high wall shear stress (WSS >40 dynes/cm²) and high wall shear stress gradients (WSSG >300 dynes/cm³). In this study, confluent bovine aortic endothelial cells were exposed to impinging flow in a T-shaped chamber designed to mimic a bifurcation. After 24–72 h under flow, cells around the stagnation point maintained polygonal shapes but cell density was reduced, whereas cells in adjacent downstream regions exposed to very high WSS and WSSG were elongated, aligned parallel to flow, and at higher density. Such behavior was not blocked by inhibiting proliferation, indicating that cells migrated downstream from the stagnation point in response to impinging flow. Furthermore, although the area of highest cell density moved downstream and away from the impingement point over time, it never moved beyond the WSS maximum. The accumulation of cells upstream of maximal WSS and downstream of maximal WSSG suggests that positive WSSG is responsible for the observed migration. These results demonstrate a unique endothelial response to aneurysm-promoting flow environments at bifurcation apices.     

Haptoglobin genotype and endothelial function in diabetes mellitus: a pilot study

Endothelial function (EnF) is impaired in patients with diabetes mellitus (DM) due in large part to an increase in oxidative stress. Haptoglobin (Hp) is a potent antioxidant protein which is encoded by two different alleles (1 and 2) with the Hp 1 protein being a superior antioxidant to the Hp 2 protein. We hypothesized that DM individuals with the Hp 2-2 genotype would have greater endothelial dysfunction as compared to DM individuals with the Hp 1-1 genotype. We studied EnF in 16 Hp 2-2, 14 Hp 1-1 DM individuals and 14 healthy subjects. DM patients’ groups were matched in terms of age, cardiovascular risk factors and metabolic characteristics. EnF was assessed using post-ischemic reactive hyperemia and strain gauge plethysmography and expressed either as the maximal flow after the ischemic period or as the area under the flow–time curve (AUC). We showed that EnF indices, AUC and maximal flow, were also higher in the healthy and Hp 1-1 groups compared with Hp 2-2 genotype group (615 ± 60 and 600 ± 40 vs. 450 ± 50 ml dl⁻¹, 29 ± 2.6 and 25 ± 3 vs. 14 ± 1.8 ml min⁻¹ dl⁻¹, P < 0.003 and P < 0.05, for AUC and maximal flow, one-way ANOVA, respectively). We concluded that Hp 2-2 diabetic patients had a worse EnF than controls and Hp 1-1 diabetic subjects.     

Inflammatory Pseudotumor of the Thyroid Gland Showing Prominent Fibrohistiocytic Proliferation. A Case Report

Inflammatory pseudotumor of the thyroid gland (IPT) appears to be exceedingly rare. Histologically, 14 previously reported cases demonstrated plasma cell granuloma variant. We report here an IPT showing a predominantly fibrohistiocytic proliferation that occurred in a 75-year-old Japanese woman. Histologically, the lesion was characterized by haphazardly arranged spindle cells, histiocytes having foamy cytoplasm containing intracytoplasmic brown pigments, and small lymphocytes. Immunohistochemical study demonstrated that the spindle cells were vimentin+, desmin−, muscle-specific actin+, cytokeratin−, endomysial antibody−, anaplastic lymphoma kinase−, CD34-− CD68+/−, CD99−, cyclin D1−, bcl-2−, and antifollicular dendritic cell antibody−. IPT showing a predominant fibrohistiocytic proliferation should be differentiated from various nonneoplastic or neoplastic disorders showing spindle cell proliferation and/or exuberant fibrosis. They include Riedel’s thyroiditis, fibrous variant of chronic thyroiditis, papillary carcinoma with exuberant nodular fasciitis-like stroma, paucicellular variant of anaplastic thyroid carcinoma, and solitary fibrous tumor.     

The control of endothelial cell adhesion and migration by shear stress and matrix-substrate anchorage

Endothelial cells constitute the natural inner lining of blood vessels and possess anti-thrombogenic properties. This characteristic is frequently used by seeding endothelial cells on vascular prostheses. As the type of anchorage of adhesion ligands to materials surfaces is known to determine the mechanical balance of adherent cells, we investigated herein the behaviour of endothelial cells under physiological shear stress conditions. The adhesion ligand fibronectin was anchored to polymer surfaces of four physicochemical characteristics exhibiting covalent and non-covalent attachment as well as high and low hydrophobicity. The in situ analysis combined with cell tracking of shear stress-induced effects on cultured isolated cells and monolayers under venous (0.5 dyn/cm²) and arterial (12 dyn/cm²) shear stress over a time period of 24 h revealed distinct differences in their morphological and migratory features. Most pronounced, unidirectional and bimodal migration patterns of endothelial cells in or against flow direction were found in dependence on the type of substrate-matrix anchorage. Combined by an immunofluorescent analysis of the actin cytoskeleton, cell-cell junctions, cell-matrix adhesions, and matrix reorganization these results revealed a distinct balance of laminar shear stress, cell-cell contacts and substrate-matrix anchorage in affecting endothelial cell fate under flow conditions. This analysis underlines the importance of materials surface parameters as well as primary and secondary adhesion ligand anchorage in the context of artificial blood vessels for future therapeutic devices.     

Ruthenium(II) polypyridyl complexes as mitochondria-targeted two-photon photodynamic anticancer agents

Clinical acceptance of photodynamic therapy is currently hindered by poor depth efficacy and inefficient activation of the cell death machinery in cancer cells during treatment. To address these issues, photoactivation using two-photon absorption (TPA) is currently being examined. Mitochondria-targeted therapy represents a promising approach to target tumors selectively and may overcome the resistance in current anticancer therapies. Herein, four ruthenium(II) polypyridyl complexes (RuL1–RuL4) have been designed and developed to act as mitochondria-targeted two-photon photodynamic anticancer agents. These complexes exhibit very high singlet oxygen quantum yields in methanol (0.74–0.81), significant TPA cross sections (124–198 GM), remarkable mitochondrial accumulation, and deep penetration depth. Thus, RuL1–RuL4 were utilized as one-photon and two-photon absorbing photosensitizers in both monolayer cells and 3D multicellular spheroids (MCSs). These Ru(II) complexes were almost nontoxic towards cells and 3D MCSs in the dark and generate sufficient singlet oxygen under one- and two-photon irradiation to trigger cell death. Remarkably, RuL4 exhibited an IC₅₀ value as low as 9.6 μM in one-photon PDT (λ_irr = 450 nm, 12 J cm⁻²) and 1.9 μM in two-photon PDT (λ_irr = 830 nm, 800 J cm⁻²) of 3D MCSs; moreover, RuL4 is an order of magnitude more toxic than cisplatin in the latter test system. The combination of mitochondria-targeting and two-photon activation provides a valuable paradigm to develop ruthenium(II) complexes for PDT applications.     

Endothelial cell proliferation follows the mid-cycle luteinizing hormone surge, but not human chorionic gonadotrophin rescue, in the human corpus luteum

The corpus luteum is essential for the maintenance of early pregnancy in women. Angiogenesis may be one factor involved in luteal rescue. The aim of this study was to determine the changes in endothelial cell proliferation throughout the luteal phase and in human chorionic gonadotrophin (HCG)-simulated early pregnancy. Human corpora lutea obtained throughout the luteal phase and in simulated early pregnancy were immunostained with antibodies for endothelial and proliferating cells. Number and distribution of endothelial and proliferating cells were examined. Endothelial cells were least abundant in the early luteal phase, increasing in the mid-luteal phase (P < 0.03). Endothelial numbers did not differ significantly between the late and the rescued corpora lutea. Endothelial cell proliferation was greatest in the early luteal phase and continued at a lower level during later stages. Simulated early pregnancy resulted in no change in endothelial cell proliferation. These results showed that a high degree of endothelial cell proliferation is associated with formation of the human corpus luteum. Unchanging levels of proliferation following HCG treatment (for 5-8 days from day 12 to day 16 post-ovulation, at 125 IU to 16,000 IU, following a daily doubling of dose) suggest that alternative processes are involved during luteal rescue.      

Interaction of bis-β-chlorethylamine estrogen derivatives with human ovarian adenocarcinoma cells

Antiproliferative effect of bis-β-chlorethylamine estrogen derivatives on human ovarian adenocarcinoma CaOV cells depends on the dose of the drug. In concentrations of 10⁻⁵ and 5×10⁻⁶ M they inhibit, while in a dose of 5×10⁻⁷ M stimulate cell proliferation. It is assumed that CaOV cells express estradiol receptors. The interaction of these cytostatics with estrogen-binding sites on CaOV cells is demonstrated. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 127, No. 3, pp. 302–304, March, 1999     

The endothelial microparticle response to a high fat meal is not attenuated by prior exercise

Triglyceride-rich postprandial lipoproteins are known to activate endothelial cells in vitro, contributing to atherosclerosis. Endothelial microparticles (EMP) are membranous vesicles released into the circulation from vascular endothelial cells that permit cell activation to be monitored in vivo. The objective of the study was to examine changes in EMP following a high fat meal, consumed with and without prior exercise. Eight recreationally active young men underwent two oral fat tolerance tests following either 100 min exercise at 70% VO₂peak (EX trial) or no exercise (CON trial) on the previous evening. Postprandial triglycerides were reduced (1.97 ± 0.31 vs. 1.17 ± 0.13 mmol L⁻¹, p < 0.05) and HDL-cholesterol (HDL-C) increased (1.20 ± 0.07 vs. 1.30 ± 0.08 mmol L⁻¹, p < 0.05) in the EX compared to CON trial. EMP (CD31+/42b−) increased postprandially (p < 0.05). However, counts were not different between trials (postprandial CON and EX trial counts × 10³ μL⁻¹, 3.10 ± 0.14 vs. 3.26 ± 0.37). There were no changes in sICAM-1 or sVCAM-1 postprandially and no differences between trials. Interleukin-6 (IL-6) and leukocytes increased postprandially (p < 0.05). IL-6 values were not different between trials. Leukocytes were higher at 0 h in the EX trial with CON and EX trial values similar at 6 h. EMP, but not sICAM-1 or sVCAM-1, increase in response to a high fat meal. However, EMP are not attenuated by acute exercise, despite a considerable reduction in postprandial lipemia and an increase in HDL-C. M. Harrison and R. P. Murphy contributed equally to this work.     

Membrane fragments from cultured endothelial cells for use in screening anti-FGF receptor antibodies

Summary Endothelial cell properties may be studied by either detecting their functional activity or immunologic reactivity. Antibodies against the fibroblast growth factor (FGF) receptor may help delineate signal transduction pathways involved in the proliferation of endothelial cells and angiogenesis. This paper describes membrane fragment preparation from cultured coronary venular endothelial cells for obtaining intact FGF receptors for the assay of the anti-receptor antibodies.     

Endothelial cell behaviour within a microfluidic mimic of the flow channels of a modular tissue engineered construct

To study the effect of disturbed flow patterns on endothelial cells, the channels found within a modular tissue engineering construct were reproduced in a microfluidic chip and lined with endothelial cells whose resulting phenotype under flow was assessed using confocal microscopy. Modular tissue engineered constructs formed by the random packing of sub-millimetre, cylindrically shaped, endothelial cell-covered modules into a larger container creates interconnected channels that permit the flow of fluids such as blood. Due to the random packing, the flow path is tortuous and has the potential to create disturbed flow, resulting in an activated endothelium. At an average shear stress of 2.8 dyn cm ⁻², endothelial cells within channels of varying geometries showed higher amounts of activation, as evidenced by an increase in ICAM-1 and VCAM-1 levels with respect to static controls. VE-cadherin expression also increased, however, it appeared discontinuous around the perimeter of the cells. An increase in flow (15.6 dyn cm ⁻²) was sufficient to reduce ICAM-1 and VCAM-1 expression to a level below that of static controls for many disturbed flow-prone channels that contained branches, curves, expansions and contractions. VE-cadherin expression was also reduced and became discontinuous in all channels, possibly due to paracrine signaling. Other than showing a mild correlation to VE-cadherin, which may be linked through a cAMP-initiated pathway, KLF2 was found to be largely independent of shear stress for this system. To gauge the adhesiveness of the endothelium to leukocytes, THP-1 cells were introduced into flow-conditioned channels and their attachment measured. Relative to static controls, THP-1 adhesion was reduced in straight and bifurcating channels. However, even in the presence of flow, areas where multiple channels converged were found to be the most prone to THP-1 attachment. The microfluidic system enabled a full analysis of the effect of the tortuous flow expected in a modular construct on endothelial cell phenotype.