Parallel Plate Flow Chamber Research Articles

Disturbed flow disrupts eNOS and caveolin‐1 expression and colocalization via glycocalyx degradation

Atherosclerosis is the main cause of cardiovascular diseases such as coronary artery and carotid artery disease and is known to occur in specific, atheroprone areas of the vasculature characterized by disturbed flow patterns. The endothelial glycocalyx, a mechanotransductive carbohydrate layer that lines the endothelium, helps maintain vascular homeostasis through flow‐induced regulation of vasculoprotective molecules such as nitric oxide. Areas of atherosclerosis development have been shown to contain diminished glycocalyx expression; however, it is unclear whether glycocalyx degradation contributes to atherosclerosis or is a result of its progression. Here, we investigated the effects of disturbed flow on glycocalyx‐mediated nitric oxide regulation in rat fat pad endothelial cells, which robustly express glycocalyx components. Using a custom parallel‐plate flow chamber, we found that disturbed flow (DF) patterns characteristic of atheroprone regions of the vasculature decrease glycocalyx coverage and thickness in comparison to uniform flow (UF). This decrease was accompanied by a reduction in endothelial nitric oxide synthase (eNOS) expression. For the first time, we discovered that DF also decreased the expression of caveolin‐1 (cav‐1), a core protein of caveolae microvesicles that assist in eNOS activation. Additionally, fluorescence microscopy also demonstrated decreased cav‐1 membrane and upstream distribution as well as decreased eNOS‐cav‐1 colocalization in areas of disturbed flow. Finally, UF exposure upon enzymatic degradation of heparan sulfate, a major glycocalyx component, resulted in DF‐like expression colocalization of eNOS and cav‐1, implicating the glycocalyx in eNOS and cav‐1 regulation. These findings provide new evidence to the body of information on glycocalyx‐mediated endothelial regulation, which collectively suggests that glycocalyx degradation may contribute to atherosclerosis development.Support or Funding InformationThis study was funded by National Institute of Health Grant K01 HL125499, National Science Foundation Grant DGE 1451070, Northeastern University Provost Start‐up funds, and an Engineering Dean's Fellowship provided by Northeastern University.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mechanical regulation of breast cancer migration and apoptosis via direct and indirect osteocyte signaling.

Bone metastases, the migration of cancers to bone, occur in 65-80% of patients with advanced breast cancer. Metastasized cancer cells interact with cells such as the bone-resorbing osteoclasts to alter bone remodeling. Exercise, often suggested as an intervention for cancer patients, regulates bone remodeling via osteocytes. Osteocytes also signal to endothelial cells, which may affect cancer cell extravasation. Therefore, we hypothesize that mechanically stimulated osteocytes can regulate processes in breast cancer bone metastasis. To test this, we exposed osteocytes to oscillatory fluid flow in vitro using parallel-plate flow chambers. We observed that conditioned medium from flow-stimulated osteocytes increased migration (by 45%) and reduced apoptosis (by 12%) of breast cancer cells. Conditioned medium from osteoclasts conditioned in flowed osteocytes' conditioned medium reduced migration (by 47%) and increased apoptosis (by 55%) of cancer cells. Cancer cell trans-endothelial migration was reduced by 34% toward flowed osteocytes' conditioned medium. This difference was abolished with ICAM-1 or IL-6 neutralizing antibodies. Conditioned medium from endothelial cells conditioned in flowed osteocytes' conditioned medium increased cancer cell apoptosis by 29%. To summarize, this study demonstrated mechanically stimulated osteocytes' potential to affect breast cancer cells not only through direct signaling, but also through osteoclasts and endothelial cells. The anti-metastatic potential of the indirect signalings is particularly exciting since osteocytes are further away from metastasizing cancer cells than osteoclasts and endothelial cells. Future studies into the effect of bone mechanical loading on metastases and its mechanism will assist in designing cancer intervention programs that lowers the risk for bone metastases.

Wall Shear Stress Determination in a Small-Scale Parallel Plate Flow Chamber Using Laser Doppler Velocimetry Under Laminar, Pulsatile and Low-Reynolds Number Turbulent Flows

The parallel plate flow chamber (PPFC) has gained popularity due to its applications in fields such as biological tissue engineering. However, most of the studies using PPFC refer to theoretical relations for estimating the wall shear stress (WSS) and, hence, the accuracy of such quantifications remains elusive for anything other than steady laminar flow. In the current study, a laser Doppler velocimetry (LDV) method was used to quantify the flow in a PPFC (H = 1.8 mm × W = 17.5 mm, Dh = 3.26 mm, aspect ratio = 9.72) under steady Re = 990, laminar pulsatile (carotid Re0-mean = 282 as well as a non-zero-mean sinusoidal Re0-mean = 45 pulse) and low-Re turbulent Re = 2750 flow conditions. A mini-LDV probe was applied, and the absolute location of the LDV measuring volume with the respect to the wall was determined using a signal monitoring technique with uncertainties being around ±27 μm. The uniformity of the flow across the span of the channel, as well as the WSS assessment for all the flow conditions, was measured with the uncertainties all being less than 16%. At least two points within the viscous sublayer of the low-Re turbulent flow were measured (with the y+ for the first point < 3) and the WSS was determined using two methods with the differences between the two methods being within 5%. This paper for the first time presents the experimental determination of WSS using LDV in a small-scale PPFC under various flow conditions, the challenges associated with each condition, and a comparison between the cases. The present data will be useful for those conducting biological or numerical modeling studies using such devices.

Laminar shear stress promotes mitochondrial homeostasis in endothelial cells.

Vascular endothelial cells (ECs) are constantly subjected to flow-induced shear stress that is crucial for endothelial functions. Laminar shear stress (LSS) exerts atheroprotection to ECs. Mitochondrial homeostasis is essential for cellular survival. However, the effects of LSS on mitochondrial homeostasis in ECs remain unclear. Mitochondrial homeostasis in ECs exposed to LSS was examined. Cultured human umbilical vein ECs were subjected to LSS (12 dynes/cm2 ) generated by a parallel-plate flow chamber system. ECs subjected to LSS demonstrated an increment of mitochondria in tubular form coupled with the increase of fusion proteins (Mfn2, OPA1) and the decrease of fission protein (Fis1). An increase of both long- and short- OPA1 along with a higher protease YME1L level were observed. LSS triggered a rapid phosphorylation on S637 but a decrease on S616 of fission-controlled protein Drp1. Consistently, Drp1 translocation to mitochondria was decreased in sheared ECs, suggesting that LSS promotes mitochondrial fusion. Enhanced mitochondrial biogenesis in sheared ECs was shown by the increase of mitochondrial mass and its regulatory proeins (PGC1α, TFAM, Nrf1). LSS enhances the expression of mitochondrial antioxidant enzymes and improves mitochondrial functions indicated by the increase of mitochondrial membrane potential (ΔΨm) and ATP generation. TNFα treatment decreased mitochondrial tubular network and its functions in ECs. LSS mitigated TNFα-induced mitochondrial impairments in ECs. Our results clearly indicate that LSS promotes mitochondrial homeostasis and attenuates inflammation-induced mitochondrial impairments in ECs. Our results provide novel insights into the manner of mitochondrial dynamics and functions modulated by LSS that contribute to endothelial integrity.

Deformability of breast cancer cells in correlation with surface markers and cell rolling

Although the cancer stem cell (CSC) hypothesis has been around for many years, the reliability of cell-surface markers to classify CSCs has remained debatable. The finding that cancerous cells are significantly more deformable than healthy ones has provided motivation to consider mechanical properties as a possible biomarker for stemness. In this study, using the micropipette aspiration technique, mechanical properties of multiple breast cancer cell lines were investigated and correlated with breast cancer stem cell (BCSC) marker, CD44+/CD24-/ALDH1+. The results indicated that Hs578T and MDA-MB-231 cell lines with CD44+/CD24-/ALDH1+ phenotype were significantly more deformable than the MDA-MB-468 cell line, which did not express the BCSC marker. The BT-20 cell line with intermediate deformability did not express any CD44+/CD24- phenotype, but it expressed aldehyde dehydrogenase-1 activity. In addition, more-deformable cell lines were found to roll with shear-independent velocities on E-selectin-coated substrates in a parallel-plate flow chamber, which might be a mediating factor for firm adhesion of CSCs to endothelium during metastasis. Our results indicate that rheological properties can be considered as a biomechanical marker in addition to, or as a complement of, surface markers to find more-definitive evidence of CSC characteristics within tumors.-Mohammadalipour, A., Burdick, M. M., Tees, D. F. J. Deformability of breast cancer cells in correlation with surface markers and cell rolling.

Endothelial cell monolayer-based microfluidic systems mimicking complex in vivo microenvironments for the study of leukocyte dynamics in inflamed blood vessels.

In response to inflammatory signals, leukocytes circulating in blood vessels undergo dynamic interaction with endothelium lining blood vessel walls, known as leukocyte adhesion cascade, to leave the blood vessels, and infiltrate the inflamed tissue. During leukocyte extravasation, leukocytes recognize and respond to various biophysical and biochemical cues present in the complex microenvironments of inflamed blood vessels to find optimal pathways. Although advances in intravital imaging of live animals have enabled us to observe leukocyte dynamics during extravasation, in vitro model systems mimicking complex in vivo microenvironments are still needed for mechanistic studies. A parallel-plate flow chamber assembled by placing a fluidic chamber on an endothelial cell (EC) monolayer has been widely used as an in vitro model to study leukocyte dynamics in inflamed blood vessels. Although this is a simple yet powerful model providing well-defined flow conditions, a parallel-plate flow chamber lacks the complex microenvironments of inflamed blood vessels. In this article, we first describe the basic design, assembly, and operation principles of a parallel-plate flow chamber. Then, we present methods of incorporating various features of in vivo microenvironments into parallel-plate flow chambers, including EC alignment using nanogrooved surfaces, insertion of a stenotic structure for complex flow generation, and extension to 3D blood vessel/inflamed tissue models.

The Effect of Fluid Shear Stress on the In Vitro Release Kinetics of Sirolimus from PLGA Films.

Drug-carrying coatings of stents implanted in blood vessels are exposed to various blood flows. This study investigated the effect of fluid shear stress on the in vitro release kinetics of sirolimus from poly(lactic-co-glycolic acid) (PLGA) films. The homemade parallel plate flow chamber was used to exert quantitative shear stress on the sirolimus-carrying film. By adjusting the flow rate of the release media in the chamber, three levels of shear stress (3.6, 12.0, and 36.0 dyn/cm2) were respectively applied. For each level of shear stress employed, the release kinetics of sirolimus from the PLGA films exhibited a four-phase profile: an initial burst release phase (Phase I), a lag phase (Phase II), a second burst release phase (Phase III), and a terminal release phase (Phase IV). During Phases I and II, sirolimus was released slowly and in small amounts (<10%); however, during Phases III and IV, the drug release increased considerably. Comparisons of different shear stresses indicated that greater shear stress resulted in earlier and faster sirolimus release, with more cumulative drug release observed. PLGA film degradations (molecular weight reduction, mass loss, and surface topographical variations) were also investigated to better explain the observed drug release behavior. Consequently, fluid shear stress was found to significantly accelerate the release of sirolimus from the PLGA matrices. Therefore, this study could provide a practical method for evaluating the in vitro drug release from polymer matrices under uniform shear stress, and might help improve the design of biodegradable coatings on drug-eluting stents.

Open Fluidics: A Cell Culture Flow System Developed Over Wettability Contrast-Based Chips.

Biological tissues are recurrently exposed to several dynamic mechanical forces that influence cell behavior. On this work, the focus is on the shear stress forces induced by fluid flow. The study of flow-induced effects on cells leads to important advances in cardiovascular, cancer, stem cell, and bone biology understanding. These studies are performed using cell culture flow (CCF) systems, mainly parallel plate flow chambers (PPFC), and microfluidic systems. Here, it is proposed an original CCF system based on the open fluidics concept. The system is developed using a planar superhydrophobic platform with hydrophilic paths. The paths work as channels to drive cell culture medium flows without using walls for liquid confinement. The liquid streams are controlled just based on the wettability contrast. To validate the concept, the effect of the shear stress stimulus in the osteogenic differentiation of C2C12 myoblast cells is studied. Combining bone morphogenic protein (specifically BMP-2) stimulation with this mechanical stimulus, a synergistic effect is found on osteoblast differentiation. This effect is confirmed by the enhancement of alkaline phosphatase activity, a well-known early marker of osteogenic differentiation. The suggested CCF system combines characteristics and advantages of both the PPFC and microfluidic systems.

The anti-apoptotic effect of fluid mechanics preconditioning by cells membrane and mitochondria in rats brain microvascular endothelial cells.

Exercise preconditioning is a simple and effective way to prevent ischemia. This paper further provided the mechanism in hemodynamic aspects at the cellular level. To study the anti-apoptotic effects of fluid mechanics preconditioning, Cultured rats brain microvascular endothelial cells were given fluid intervention in a parallel plate flow chamber before oxygen glucose deprivation. It showed that fluid mechanics preconditioning could inhibit the apoptosis of endothelial cells, and this process might be mediated by the shear stress activation of Tie-2 on cells membrane surface and Bcl-2 on the mitochondria surface.

ROLE OF REDUCING CELL LEAKAGE IN CELL CULTURE USING LARGE PORE SIZE PERMEABLE MEMBRANE

Permeable membranes are widely used in many in vitro studies using cell culture. Some cell leakage are often occurs when using permeable membrane with large pore size. Unfortunately the use of permeable membrane with smaller pore size in permeability studies faces some difficulties due to its small pore size and pore density. Recent study provides the protocol in using permeable membrane with large pore size with smaller cell leakage. Vero cell line (CCL-81, ATCC) was used and culture on polyester permeable membrane with 3 µm pore size. Visualization using inverted microscope was used to analized the cell leakage on the permeable membrane. Parallel plate flow chamber was used to analized the permeability performance of the Vero cell cultured on the permeable membrane. The result showed that the current technique is significant in reducing cell leakage of the cell cultured on large pore size of permeable membrane. The same results were found in using polyester and polycarbonate permeable membrane.

Platelet behaviour on von Willebrand Factor changes in pregnancy: Consequences of haemodilution and intrinsic changes in platelet function

Platelet function in pregnancy is poorly understood. Previous studies of platelet function in pregnancy have used non-physiological assays of platelet function with conflicting results. This study using a physiological assay of platelet function investigated platelet interactions with von Willebrand Factor (VWF) in blood from healthy pregnant women and healthy non-pregnant controls. Blood samples (200 µl) from third-trimester pregnancies (n = 21) and non-pregnant controls (n = 21) were perfused through custom-made parallel-plate flow chambers coated with VWF under arterial shear (1,500 s−1). Multi-parameter measurements of platelet interactions with the immobilized VWF surface were recorded by digital-image microscopy and analysed using custom-designed platelet-tracking software. Platelet interactions with VWF decreased in healthy third-trimester pregnant participants relative to controls. This effect is most likely due to haemodilution which occurs physiologically during pregnancy. Interestingly, platelets in blood from pregnant participants translocated more slowly on VWF under arterial-shear conditions. These decreases in platelet translocation speed were independent of haemodilution, suggesting intrinsic changes in platelet function with pregnancy.

P 7 Fluid shear stress promotes placental growth factor upregulation in human syncytiotrophoblast through the camp-pka signaling pathway

The effects of fluid shear stress (FSS) on the human syncytiotrophoblast and its biological functions have never been studied. During pregnancy, the syncytiotrophoblast is the main source of placental growth factor (PlGF), a proangiogenic factor involved in the placental angiogenesis and the vascular adaptation to pregnancy. The role of FSS in regulating PlGF expression in syncytiotrophoblasts is unknown. We investigated the impact of FSS on the production and secretion of the PlGF by the human syncytiotrophoblasts in primary cell culture. Laminar and continuous FSS (1 dyn cm−2) was applied to human syncytiotrophoblasts cultured in a parallel-plate flow chambers. Secreted levels of PlGF, sFlt-1 (soluble fms-like tyrosin kinase-1), and prostaglandin E2 were tested by immunologic assay. PlGF levels of mRNA and intracellular protein were examined by RT-PCR and Western blot, respectively. Intracellular cAMP levels were examined by time-resolved fluorescence resonance energy transfer cAMP accumulation assay. Production of cAMP and PlGF secretion was significantly increased in FSS conditions compared with static conditions. Western blot analysis of cell extracts exposed to FSS showed an increased phosphorylation of protein kinase A substrates and cAMP response element-binding protein on serine 133. FSS-induced phosphorylation of cAMP response element-binding protein and upregulation of PlGF were prevented by inhibition of protein kinase A with H89 (3 μmol/L). FSS also triggers intracellular calcium flux, which increases the synthesis and release of prostaglandin E2. The enhanced intracellular cAMP in FSS conditions was blocked by COX1/COX2 (cyclooxygenase) inhibitors, suggesting that the increase in prostaglandin E2 production could activate the cAMP/protein kinase A pathway in an autocrine/paracrine fashion. FSS activates the cAMP/protein kinase A pathway leading to upregulation of PlGF in human syncytiotrophoblast.

Measuring Leukocyte Adhesion to (Primary) Endothelial Cells after Photon and Charged Particle Exposure with a Dedicated Laminar Flow Chamber.

The vascular endothelium interacts with all types of blood cells and is a key modulator of local and systemic inflammatory processes, for example, in the adhesion of blood leukocytes to endothelial cells (EC) and the following extravasation into the injured tissue. The endothelium is constantly exposed to mechanical forces caused by blood flow, and the resulting shear stress is essential for the maintenance of endothelial function. Changes in local hemodynamics are sensed by EC, leading to acute or persistent changes. Therefore, in vitro assessment of EC functionality should include shear stress as an essential parameter. Parallel-plate flow chambers with adjustable shear stress can be used to study EC properties. However, commercially available systems are not suitable for radiation experiments, especially with charged particles, which are increasingly used in radiotherapy of tumors. Therefore, research on charged-particle-induced vascular side effects is needed. In addition, α-particle emitters (e.g., radon) are used to treat inflammatory diseases at low doses. In the present study, we established a flow chamber system, applicable for the investigation of radiation induced changes in the adhesion of lymphocytes to EC as readout for the onset of an inflammatory reaction or the modification of a pre-existing inflammatory state. In this system, primary human EC are cultured under physiological laminar shear stress, subjected to a proinflammatory treatment and/or irradiation with X-rays or charged particles, followed by a coincubation with primary human lymphocytes (peripheral blood lymphocytes (PBL)). Analysis is performed by semiautomated quantification of fluorescent staining in microscopic pictures. First results obtained after irradiation with X-rays or helium ions indicate decreased adhesion of PBL to EC under laminar conditions for both radiation qualities, whereas adhesion of PBL under static conditions is not clearly affected by irradiation. Under static conditions, no radiation-induced changes in surface expression of adhesion molecules and activation of nuclear factor kappa B (NF-κB) signaling were observed after single cell-based high-throughput analysis. In subsequent studies, these investigations will be extended to laminar conditions.

Lateral trapezoid microfluidic platform for investigating mechanotransduction of cells to spatial shear stress gradients

Microfluidic platforms enable the influence of a variety of chemical and physical gradients on single or multiple cells to be examined and monitored in real-time. Research into chemical gradients has been more prevalent in literature; however, with the interest in mechanotransduction, research investigating the influence of physical gradients, such as force from shear stress, are appearing. Shear stress studies, to date have been focused on using parallel plate flow chambers, or shear stress gradients spanning the length of the microchannel. In this paper, we designed a Trapezoid microchannel, which enables production of a unique lateral spatial shear stress gradient, in order to examine the responses it evokes in cells, using microscopy. We investigated the effect of a lateral spatial shear stress gradient on intracellular calcium signalling in HEK-293-TRPV4 cells. The Trapezoid microchannel was designed to produce a spatial shear stress gradient, spanning the width of the microchannel. Additionally, we investigated the effect of temporal shear stress using the microfluidic platform, to further examine the complexity of the transduction of shear stress forces, which mimic pathological and physiological conditions into cellular signalling. We show that the temporal pattern of shear stress, modulated intracellular calcium signalling, with slower response times (responding to stimulus and reaching maximum Calcium influx), compared to those observed in the absence of temporal shear stress. Our experiments highlight the importance of considering the nature of shear stress under physiological and pathological conditions – with microfluidic platforms providing a useful means to do so.

Microfluidic co-culture platform for investigating osteocyte-osteoclast signalling during fluid shear stress mechanostimulation

Bone cells exist in a complex environment where they are constantly exposed to numerous dynamic biochemical and mechanical stimuli. These stimuli regulate bone cells that are involved in various bone disorders, such as osteoporosis. Knowledge of how these stimuli affect bone cells have been utilised to develop various treatments, such as pharmaceuticals, hormone therapy, and exercise. To investigate the role that bone loading has on these disorders in vitro, bone cell mechanotransduction studies are typically performed using parallel plate flow chambers (PPFC). However, these chambers do not allow for dynamic cellular interactions among different cell populations to be investigated. We present a microfluidic approach that exposes different cell populations, which are located at physiologically relevant distances within adjacent channels, to different levels of fluid shear stress, and promotes cell-cell communication between the different channels. We employed this microfluidic system to assess mechanically regulated osteocyte-osteoclast communication. Osteoclast precursors (RAW264.7 cells) responded to cytokine gradients (e.g., RANKL, OPG, PGE-2) developed by both mechanically stimulated (fOCY) and unstimulated (nOCY) osteocyte-like MLO-Y4 cells simultaneously. Specifically, we observed increased osteoclast precursor cell densities and osteoclast differentiation towards nOCY. We also used this system to show an increased mechanoresponse of osteocytes when in co-culture with osteoclasts. We envision broad applicability of the presented approach for microfluidic perfusion co-culture of multiple cell types in the presence of fluid flow stimulation, and as a tool to investigate osteocyte mechanotransduction, as well as bone metastasis extravasation. This system could also be applied to any multi-cell population cross-talk studies that are typically performed using PPFCs (e.g. endothelial cells, smooth muscle cells, and fibroblasts).

The Impact of Laminar and Oscillatory Shear Stress on Cellular Adhesion Molecule Expression in HUVEC

PURPOSE: To investigate the influence of acute laminar shear stress (LSS) on the expression of VCAM-1 and fractalkine on human umbilical vein endothelial cells (HUVEC) following prolonged exposure to oscillatory shear stress (OSS) in vitro. METHODS: Cultured EC were stimulated with TNF-α for 24 hr and immediately exposed to shear experiments using a parallel-plate flow chamber: (i) 24 hr of LSS or OSS, and (ii) 24 hr of OSS followed by 30 min of LSS. Cells were collected and incubated with primary antibodies for surface VCAM-1 and fractalkine. Samples were analyzed via standard fluorescence-activated cell sorting. RESULTS: Prolonged LSS and OSS significantly reduced the TNF-α-induced elevation in the % of gated cells expressing VCAM-1 (p<0.001) and fractalkine (p=0.001); however, the reduction in VCAM-1 caused by LSS was shown to be significantly greater than the reduction elicited by OSS (p=0.006). Interestingly, the mean fluorescent intensity (MFI) of TNF-α-induced VCAM-1 was significantly elevated by prolonged OSS (1.60±0.01 fold [p<0.001]). Whereas, prolonged LSS had no impact on the MFI of TNF-α induced VCAM-1 (p=0.931). Prolonged LSS and OSS elicited a significant elevation in the MFI of TNF-α induced fractalkine (p=0.017). Lastly, acute LSS following prolonged OSS had no effect on the % of gated or MFI of cells expressing VCAM-1 and fractalkine. CONCLUSIONS: Prolonged OSS may increase markers of vascular inflammation. However, an acute period of LSS, utilized as a model of physical activity, does not appear to alter the OSS-induced inflammatory state.

Effect of matrix rigidity on organ-specific capture of tumor cells by flow.

Accumulating evidences have suggested that tumor metastasis exists prominent organ discrepancy. In thisprogression, the capture of intravascular tumor cells (TCs) to endothelium in distant tissues and organs plays a decisive role in the organ-specific metastasis formation. However, the mechanism of tumor cells preferentially arrest and adhere to endothelial cells (ECs) of target organ still remains elusive. By using the parallel plate flow chamber and the polyacrylamide gels with different matrix stiffness, we here explored the combined effects of matrix rigidity, shear stress, and chemokine SDF-1 on the capture of circulating tumor cells to ECs in thebloodstream. In addition, the expression and the role of integrin β1 on the tumor cells surface were also detected by SDF-1 treatment. The results show that breast tumor cells MDA-MB-231 display an increasing number of adherent cells on the preferred substrate, which is similar to the matrix rigidity of breast cancer tissue (about 5kPa), under a certain shear stress. Moreover, ECs exacerbates the preferred capture of tumor cells compared with the FN-coated substrate alone. Besides, SDF-1 upregulates the number of adherent tumor cells by responding to matrix stiffness via promoting the expression of integrin β1, which is abolished by blocking of integrin β1. These results may provide a novel point of view for the mechanism of "organ specificity" phenomenon in tumor metastasis, which in turn contribute to a rational development of new drugs for cancer.

Effect of surface conditioning with cellular extracts on Escherichia coli adhesion and initial biofilm formation

Bacterial adhesion and subsequent biofilm formation start with surface conditioning by molecules originating from the surrounding medium and from cell lysis. Different cell extracts e.g. total cell extract (TCE), cytoplasm with cellular debris (CCDE) and periplasmic extract (PE) were tested in agitated 96-well microtiter plates and in a flow cell. Crystal violet assay demonstrated that a polystyrene substratum conditioned with TCE or CCDE decreased initial biofilm formation, however cell adhesion generally increased when PE was used. These results were dependent on conditioning film concentration. Using a parallel plate flow chamber, the use of optimal conditioning film concentrations resulted in all the different cellular extracts reducing biofilm formation. Multifractal analysis was used to generate quantitative data on the number of cell clusters. Surface conditioning with cellular components affected the amount and clustering of bacteria on polystyrene surfaces and their propensity to induce biofilm formation. To the best of our knowledge, this is the first study addressing the effect of cellular surface conditioning of cellular compartments on Escherichia coli adhesion and initial biofilm formation. This work leads to a greater understanding of the factors that influence biofilm formation under flow conditions which are prevalent in food industry.

Effect of Clopidogrel on Thrombus Formation in an Ex Vivo Parallel Plate Flow Chamber Model Cannot Be Reversed by Addition of Platelet Concentrates or vWF Concentrate.

Hemorrhage is the most important complication of antithrombotic therapy with P2Y12 receptor blockers. The administration of platelet concentrates (PCs) and von Willebrand factor (vWF) concentrates are common procedures to normalize impaired primary hemostasis in bleeding patients. We tested whether this strategy reverses the effect of clopidogrel using a parallel plate flow chamber model. Whole blood from patients, who received a loading dose of clopidogrel with 600 mg and an ongoing dual antiplatelet therapy with 75 mg/d clopidogrel and 100 mg/d acetyl salicylic acid, compared with blood from healthy volunteers was examined in a collagen-coated parallel plate flow chamber. Blood was perfused by suction at a shear rate of 300/s, which is equivalent to 14 dynes/cm to resemble shear stress in conduit arteries. Platelet-covered area, individual thrombus size, and the average thrombus size were assessed morphometrically. The equivalent of 2 or 5 units of PC and/or 2 U/mL of vWF concentrate were used in an attempt to restore coagulation capacity in blood samples of clopidogrel-treated patients. In this model, clopidogrel reduced the increase of thrombus size. The equivalent of 2 U of PC or 2 U/mL of vWF alone did not show any significant changes in thrombus size. 5 U of PC increased thrombus size in clopidogrel-treated patients (P < .05). Thrombus size in clopidogrel blood was increased by combined PC and vWF treatment (by 50%, P < .05), but this increase did not reach control levels (P < .05). This flow chamber model is suitable for detection of the antiplatelet effect of clopidogrel. Ex vivo addition of PC or vWF does not overcome the effects of clopidogrel in this model, but the combination of both shows a mild and significant improvement in thrombus size.

Probing the Dynamics of Clot-Bound Thrombin at Venous Shear Rates

In closed system models of fibrin formation, exosite-mediated thrombin binding to fibrin contributes to clot stability and is resistant to inhibition by antithrombin/heparin while still susceptible to small, active-site inhibitors. Each molecule of fibrin can bind ∼1.6 thrombin molecules at low-affinity binding sites (Kd = 2.8 μM) and ∼0.3 molecules of thrombin at high-affinity binding sites (Kd = 0.15 μM). The goal of this study is to assess the stability of fibrin-bound thrombin under venous flow conditions and to determine both its accessibility and susceptibility to inhibition. A parallel-plate flow chamber (7 × 50 × 0.25 mm) for studying the stability of thrombin (0–1400 nM) adhered to a fibrin matrix (0.1–0.4 mg/mL fibrinogen, 10 nM thrombin) under a variety of venous flow conditions was developed using the thrombin-specific, fluorogenic substrate SN-59 (100 μM). The flow within this system is laminar (Re < 1) and reaction rates are driven by enzyme kinetics (Pe = 100, Da = 7000). A subpopulation of active thrombin remains stably adhered to a fibrin matrix over a range of venous shear rates (46–184 s−1) for upwards of 30 min, and this population is saturable at loads >500 nM and sensitive to the initial fibrinogen concentration. These observations were also supported by a mathematical model of thrombin adhesion to fibrin, which demonstrates that thrombin initially binds to the low-affinity thrombin binding sites before preferentially equilibrating to higher affinity sites. Antithrombin (2.6 μM) plus heparin (4 U/mL) inhibits 72% of the active clot-bound thrombin after ∼10 min at 92 s−1, while no inhibition is observed in the absence of heparin. Dabigatran (20 and 200 nM) inhibits (50 and 93%) clot-bound thrombin reversibly (87 and 66% recovery). This model illustrates that clot-bound thrombin stability is the result of a constant rearrangement of thrombin molecules within a dense matrix of binding sites.