High-Throughput Biofilm Assay to Investigate Bacterial Interactions with Surface Topographies.

The specific topography of biomaterials plays an important role in their biological interactions with cells and thus the safety of medical implants. Antifouling materials can be engineered with topographic features to repel microbes. Meanwhile, undesired topographies of implants can cause complications such as breast implant-associated anaplastic large cell lymphoma (BIA-ALCL). While the cause of BIA-ALCL is not well understood, it is speculated that textured surfaces are prone to bacterial biofilm formation as a contributing factor. To guide the design of safer biomaterials and implants, quantitative screening approaches are needed to assess bacterial adhesion to different topographic surface features. Here we report the development of a high-throughput microplate biofilm assay for such screening. The assay was used to test a library of polydimethylsiloxane (PDMS) textures composed of varying sizes of recessive features and distances between features including those in the range of breast implant textures. Outliers of patterns prone to bacterial adhesion were further studied using real-time confocal fluorescence microscopy. The results from these analyses revealed that surface area itself is a poor predictor for adhesion, while the size and spacing of topographic features play an important role. This high-throughput biofilm assay can be applied to studying bacteria-material interactions and rational development of materials that inhibit bacterial colonization.

Putative mechanobiological impact of surface texture on cell activity around soft-tissue implants undergoing micromotion.

Recent reports of adverse health effects (e.g., capsular contracture, lymphoma) linked to the absence or presence of texture on soft-tissue implants (e.g., breast implants) suggest surface topography may have pathological impact(s). We propose that surface texture influences the transfer of displacements, experienced by an implant undergoing micromotion, to surrounding interfacial extracellular matrix, which in turn impacts the activity of the resident cells and is based on degree of tissue integration. We hypothesize that transfer of displacements due to micromotion promotes interstitial fluid movement that imposes hydrodynamic stresses (pressures, shear stresses) on cells residing in the interfacial tissues and impacts their activity. To address this, we developed a computer simulation to approximate hydrodynamic stresses in the interstitial environment of saturated poroelastic tissues (model soft-tissue implantation sites) generated from oscillatory implant micromotion as a function of the magnitude of translational displacement, direction of motion, degree of tissue integration, and surface roughness of the implant. Highly integrated implants were predicted to generate the highest fluid shear stresses within model tissues, with oscillatory fluid shear stresses up to 80 dyn/cm2 for a 20-µm displacement. Notably, application of oscillatory 80 dyn/cm2 shear stress to cultured human fibroblasts elicited cell death after 20 h compared to cells maintained under static conditions or exposed to 80 dyn/cm2 steady, unidirectional shear. These results indicate that oscillatory interstitial fluid stresses generated by micromotion of an integrated implant may influence the activity of the surrounding cells and play a role in the body's fibrotic response to textured soft-tissue implants.

Chemical Characterization and Non-targeted Analysis of Medical Device Extracts: A Review of Current Approaches, Gaps, and Emerging Practices.

The developers of medical devices evaluate the biocompatibility of their device prior to FDA's review and subsequent introduction to the market. Chemical characterization, described in ISO 10993-18:2020, can generate information for toxicological risk assessment and is an alternative approach for addressing some biocompatibility end points (e.g., systemic toxicity, genotoxicity, carcinogenicity, reproductive/developmental toxicity) that can reduce the time and cost of testing and the need for animal testing. Additionally, chemical characterization can be used to determine whether modifications to the materials and manufacturing processes alter the chemistry of a patient-contacting device to an extent that could impact device safety. Extractables testing is one approach to chemical characterization that employs combinations of non-targeted analysis, non-targeted screening, and/or targeted analysis to establish the identities and quantities of the various chemical constituents that can be released from a device. Due to the difficulty in obtaining a priori information on all the constituents in finished devices, information generation strategies in the form of analytical chemistry testing are often used. Identified and quantified extractables are then assessed using toxicological risk assessment approaches to determine if reported quantities are sufficiently low to overcome the need for further chemical analysis, biological evaluation of select end points, or risk control. For extractables studies to be useful as a screening tool, comprehensive and reliable non-targeted methods are needed. Although non-targeted methods have been adopted by many laboratories, they are laboratory-specific and require expensive analytical instruments and advanced technical expertise to perform. In this Perspective, we describe the elements of extractables studies and provide an overview of the current practices, identified gaps, and emerging practices that may be adopted on a wider scale in the future. This Perspective is outlined according to the steps of an extractables study: information gathering, extraction, extract sample processing, system selection, qualification, quantification, and identification.

Nitinol Release of Nickel under Physiological Conditions: Effects of Surface Oxide, pH, Hydrogen Peroxide, and Sodium Hypochlorite.

Nitinol is a nickel-titanium alloy widely used in medical devices for its unique pseudoelastic and shape-memory properties. However, nitinol can release potentially hazardous amounts of nickel, depending on surface manufacturing yielding different oxide thicknesses and compositions. Furthermore, nitinol medical devices can be implanted throughout the body and exposed to extremes in pH and reactive oxygen species (ROS), but few tools exist for evaluating nickel release under such physiological conditions. Even in cardiovascular applications, where nitinol medical devices are relatively common and the blood environment is well understood, there is a lack of information on how local inflammatory conditions after implantation might affect nickel ion release. For this study, nickel release from nitinol wires of different finishes was measured in pH conditions and at ROS concentrations selected to encompass and exceed literature reports of extracellular pH and ROS. Results showed increased nickel release at levels of pH and ROS reported to be physiological, with decreasing pH and increasing concentrations of hydrogen peroxide and NaOCl/HOCl having the greatest effects. The results support the importance of considering the implantation site when designing studies to predict nickel release from nitinol and underscore the value of understanding the chemical milieu at the device-tissue interface.

Fluid shear stress-mediated mechanotransduction in circulating leukocytes and its defect in microvascular dysfunction.

Leukocytes (neutrophils, monocytes) in the active circulation exhibit multiple phenotypic indicators for a low level of cellular activity, like lack of pseudopods and minimal amounts of activated, cell-adhesive integrins on their surfaces. In contrast, before these cells enter the circulation in the bone marrow or when they recross the endothelium into extravascular tissues of peripheral organs they are fully activated. We review here a multifaceted mechanism mediated by fluid shear stress that can serve to deactivate leukocytes in the circulation. The fluid shear stress controls pseudopod formation via the FPR receptor, the same receptor responsible for pseudopod projection by localized actin polymerization. The bioactivity of macromolecular factors in the blood plasma that interfere with receptor stimulation by fluid flow, such as proteolytic cleavage in the extracellular domain of the receptor or the membrane actions of cholesterol, leads to a defective ability to respond to fluid shear stress by actin depolymerization. The cell reaction to fluid shear involves CD18 integrins, nitric oxide, cGMP and Rho GTPases, is attenuated in the presence of inflammatory mediators and modified by glucocorticoids. The mechanism is abolished in disease models (genetic hypertension and hypercholesterolemia) leading to an increased number of activated leukocytes in the circulation with enhanced microvascular resistance and cell entrapment. In addition to their role in binding to biochemical agonists/antagonists, membrane receptors appear to play a second role: to monitor local fluid shear stress levels. The fluid shear stress control of many circulating cell types such as lymphocytes, stem cells, tumor cells remains to be elucidated.

An ex vivo model of medical device-mediated bacterial skin translocation.

The skin is a barrier and part of the immune system that protects us from harmful bacteria. Because indwelling medical devices break this barrier, they greatly increase the risk of infection by microbial pathogens. To study how these infections can be prevented through improved clinical practices and medical device technology, it is important to have preclinical models that replicate the early stages of microbial contamination, ingress, and colonization leading up to infection. At present, there are no preclinical ex vivo models specifically developed to simulate conditions for indwelling medical devices. Translocation of pathogens from outside the body across broken skin to normally sterile internal compartments is a rate-limiting step in infectious pathogenesis. In this work, we report a sensitive and reproducible ex vivo porcine skin-catheter model to test how long antimicrobial interventions can delay translocation. Skin preparation was first optimized to minimize tissue damage. The presence of skin dramatically decreased bacterial migration time across the polyurethane catheter interface from > 96 h to 12 h. Using visual colony detection, fluorescence, a luminescent in vitro imaging system, and confocal microscopy, the model was used to quantify time-dependent differences in translocation for eluting and non-eluting antimicrobial catheters. The results show the importance of including tissue in preclinical biofilm models and help to explain current gaps between in vitro testing and clinical outcomes for antimicrobial devices.

Cell Death Persists in Rapid Extrusion of Lysis-Resistant Coated Cardiac Myoblasts.

As the demand for organ transplants continues to grow faster than the supply of available donor organs, a new source of functional organs is needed. High resolution high throughput 3D bioprinting is one approach towards generating functional organs for transplantation. For high throughput printing, the need for increased print resolutions (by decreasing printing nozzle diameter) has a consequence: it increases the forces that cause cell damage during the printing process. Here, a novel cell encapsulation method provides mechanical protection from complete lysis of individual living cells during extrusion-based bioprinting. Cells coated in polymers possessing the mechanical properties finely-tuned to maintain size and shape following extrusion, and these encapsulated cells are protected from mechanical lysis. However, the shear forces imposed on the cells during extrusion still cause sufficient damage to compromise the cell membrane integrity and adversely impact normal cellular function. Cellular damage occurred during the extrusion process independent of the rapid depressurization.

Characterization of lymphatic malformations using primary cells and tissue transcriptomes.

Lymphatic malformations (LMs) are disfiguring congenital anomalies characterized by aberrant growth of lymphatic vessels. They are broadly categorized histopathologically as macrocystic and microcystic. Although sclerotherapy has shown some success in the treatment of macrocystic malformations, there has been less progress with developing treatment strategies for microcystic malformations. In this study, we characterized lymphatic endothelial cells isolated from lymphatic and lymphaticovenous malformations. When compared to cells from normal lymphatic vessels, we found that the primary cultured malformed cells are morphologically different and also exhibited differences in binding, proliferation, migration and tube formation. Transcriptome analysis identified several genes whose expression was substantially higher in malformed compared to normal lymphatic endothelium, including DIRAS3 and FOXF1. Further analysis of LM tissue samples revealed distinguishing gene expression patterns that could pave the way to understanding the molecular pathogenesis of LMs. Based on gene expression signatures, we propose a new hypothesis that the subtype of localized LMs could be formed because of disruptions in lymph node development.

Fluid shear-induced cathepsin B release in the control of Mac1-dependent neutrophil adhesion.

There is compelling evidence that circulatory hemodynamics prevent neutrophil activation, including adhesion to microvessels, in the microcirculation. However, the underlying mechanism or mechanisms by which that mechanoregulation occurs remain unresolved. Here, we report evidence that exposure to fluid shear stress (FSS) promotes neutrophils to release cathepsin B (ctsB) and that this autocrine regulatory event is antiadhesive for neutrophils on endothelial surfaces through Mac1-selective regulation. We used a combined cell-engineering and immunocytochemistry approach to find that ctsB was capable of cleaving Mac1 integrins on neutrophils and demonstrated that this proteolysis alters their adhesive functions. Under no-flow conditions, ctsB enhanced neutrophil migration though a putative effect on pseudopod retraction rates. We also established a flow-based cell detachment assay to verify the role of ctsB in the control of neutrophil adhesion by fluid flow stimulation. Fluid flow promoted neutrophil detachment from platelet and endothelial layers that required ctsB, consistent with its fluid shear stress-induced release. Notably, compared with leukocytes from wild-type mice, those from ctsB-deficient (ctsB -/- ) mice exhibited an impaired CD18 cleavage response to FSS, significantly elevated baseline levels of CD18 surface expression, and an enhanced adhesive capacity to mildly inflamed postcapillary venules. Taken together, the results of the present study support a role for ctsB in a hemodynamic control mechanism that is antiadhesive for leukocytes on endothelium. These results have implications in the pathogenesis of chronic inflammation, microvascular dysfunction, and cardiovascular diseases involving sustained neutrophil activation in the blood and microcirculation.

Flow-Through Electroporation of HL-60 White Blood Cell Suspensions using Nanoporous Membrane Electrodes.

A flow-through electroporation system, based on a novel nanoporous membrane/electrode design, for the delivery of cell wall-impermeant molecules into model leukocytes, HL-60 promyelocytes, was demonstrated. The ability to apply low voltages to cell populations, with nm-scale concentrated electric field in a periodic array, contributes to high cell viability. With applied biases of 1-4V, delivery of target molecules was achieved with 90% viability and up to 65% transfection efficiency. More importantly, the system allowed electrophoretic pumping of molecules from a microscale reservoir across the membrane/electrode system into a microfluidic flow channel for transfection of cells, a design that can reduce reagent amount by eightfold compared to current strategies. The flow-through system, which forces intimate membrane/electrode contact by using a 10µm channel height, can be easily scaled-up by adjusting the microfluidic channel geometry and/or the applied voltage pulse frequency to control cell residence times at the cell membrane/electrode interface. The demonstrated system shows promise in clinical applications where low-cost, high cell viability and high volume transfection methods are needed without the risk of viral vectors. In particular genetic modification of freely mobile white blood cells to either target disease cells or to express desired protein/enzyme biomolecules is an important target platform enabled by this device system.

Dependence of macrophage superoxide release on the pulse amplitude of an applied pressure regime: a potential factor at the soft tissue-implant interface.

Failure of soft tissue implants has been largely attributed to the influence of biomaterial surface properties on the foreign body response, but some implant complications, e.g. macrophage accumulation and necrosis, are still not effectively addressed with surface treatments to minimize deleterious biomaterial effects. We explored an alternative explanation for implant failure, linking biocompatibility with implant micromotion-induced pressure fluctuations at the tissue-biomaterial interface. For this purpose, we used a custom in vitro system to characterize the effects of pressure fluctuations on the activity of macrophages, the predominant cells at a healing implant site. Initially, we quantified superoxide production by HL60-derived macrophage-like cells under several different pressure regimes with means of 5-40 mmHg, amplitudes of 0-15 mmHg and frequencies of 0-1.5 Hz. All pressure regimes tested elicited significantly (p < 0.05) reduced superoxide production by macrophage-like cells relative to parallel controls. Notably, pressure-sensitive reductions in superoxide release correlated (r(2) = 0.74; p < 0.01) only with pulse pressures. Based on the connection between superoxide production and cell viability, we also explored the influence of cyclic pressure on macrophage numbers and death. Compared to controls, adherent macrophage-like cells exposed to 7.5/2.5 mmHg cyclic pressures for 6 h exhibited significantly (p < 0.01) reduced cell numbers, independent of cell death. A similar effect was observed for cells treated with 10 U/ml superoxide dismutase. Collectively, our results suggest that pressure pulses are a putative regulator of macrophage adhesion via a superoxide-related effect. Pressure fluctuations, e.g. due to implant micromotion, may, therefore, potentially modulate macrophage-dependent wound healing.

Protective Polymer Coatings for High-Throughput, High-Purity Cellular Isolation.

Cell-based therapies are emerging as the next frontier of medicine, offering a plausible path forward in the treatment of many devastating diseases. Critically, current methods for antigen positive cell sorting lack a high throughput method for delivering ultrahigh purity populations, prohibiting the application of some cell-based therapies to widespread diseases. Here we show the first use of targeted, protective polymer coatings on cells for the high speed enrichment of cells. Individual, antigen-positive cells are coated with a biocompatible hydrogel which protects the cells from a surfactant solution, while uncoated cells are immediately lysed. After lysis, the polymer coating is removed through orthogonal photochemistry, and the isolate has >50% yield of viable cells and these cells proliferate at rates comparable to control cells. Minority cell populations are enriched from erythrocyte-depleted blood to >99% purity, whereas the entire batch process requires 1 h and <$2000 in equipment. Batch scale-up is only contingent on irradiation area for the coating photopolymerization, as surfactant-based lysis can be easily achieved on any scale.

In Vitro Evaluation of the Link Between Cell Activation State and Its Rheological Impact on the Microscale Flow of Neutrophil Suspensions.

Activated neutrophils have been reported to affect peripheral resistance, for example, by plugging capillaries or adhering to the microvasculature. In vivo and ex vivo data indicate that activated neutrophils circulating in the blood also influence peripheral resistance. We used viscometry and microvascular mimics for in vitro corroboration. The rheological impact of differentiated neutrophil-like HL-60 promyelocytes (dHL60s) or human neutrophil suspensions stimulated with 10 nM fMet-Leu-Phe (fMLP) was quantified using a cone-plate rheometer (450 s(-1) shear rate). To evaluate their impact on microscale flow resistance, we used 10-µm Isopore® membranes to model capillaries as well as single 200 × 50 µm microchannels and networks of twenty 20 × 50 µm microfluidic channels to mimic noncapillary microvasculature. Stimulation of dHL60 and neutrophil populations significantly altered their flow behavior as evidenced by their impact on suspension viscosity. Notably, hematocrit abrogated the impact of leukocyte activation on blood cell suspension viscosity. In micropore filters, activated cell suspensions enhanced flow resistance. This effect was further enhanced by the presence of erythrocytes. The resistance of our noncapillary microvascular mimics to flow of activated neutrophil suspensions was significantly increased only with hematocrit. Notably, it was elevated to a higher extent within the micronetwork chambers compared to the single-channel chambers. Collectively, our findings provide supportive evidence that activated neutrophils passing through the microcirculation may alter hemodynamic resistance due to their altered rheology in the noncapillary microvasculature. This effect is another way neutrophil activation due to chronic inflammation may, at least in part, contribute to the elevated hemodynamic resistance associated with cardiovascular diseases (e.g., hypertension and hypercholesterolemia).

Characterization of molecular transport in ultrathin hydrogel coatings for cellular immunoprotection.

PEG hydrogels are routinely used in immunoprotection applications to hide foreign cells from a host immune system. Size-dependent transport is typically exploited in these systems to prevent access by macromolecular elements of the immune system while allowing the transport of low molecular weight nutrients. This work studies a nanoscale hydrogel coating for improved transport of beneficial low molecular weight materials across thicker hydrogel coatings while completely blocking transport of undesired larger molecular weight materials. Coatings composed of PEG diacrylate of molecular weight 575 and 3500 Da were studied by tracking the transport of fluorescently labeled dextrans across the coatings. The molecular weight of dextran at which the transport is blocked by these coatings are consistent with cutoff values in analogous bulk PEG materials. Additionally, the diffusion constants of 4 kDa dextrans across PEG 575 coatings (9.5 × 10(-10)-2.0 × 10(-9) cm(2)/s) was lower than across PEG 3500 coatings (5.9-9.8 × 10(-9) cm(2)/s), and these trends and magnitudes agree with bulk scale models. Overall, these nanoscale thin PEG diacrylate films offer the same size selective transport behavior of bulk PEG diacrylate materials, while the lower thickness translates directly to increased flux of beneficial low molecular weight materials.

Shear-sensitive regulation of neutrophil flow behavior and its potential impact on microvascular blood flow dysregulation in hypercholesterolemia.

OBJECTIVE: Shear stress-induced pseudopod retraction is an anti-inflammatory measure that minimizes neutrophil activity and is regulated by membrane cholesterol. We tested the hypothesis that a hypercholesterolemic impairment of shear mechanotransduction alters the neutrophil flow behavior leading to microvascular dysfunction. APPROACH AND RESULTS: We examined the shear effects on the flow behavior of human leukocytes. When subjected to shearing during cone-plate viscometry, leukocyte suspensions exhibited parallel time-dependent reductions in viscosity and pseudopod activity. Shear-induced reductions in suspension viscosity were attenuated by membrane cholesterol enrichment. We also showed that enhanced pseudopod activity of leukocyte suspensions in 10% hematocrit significantly (P<0.05) raised the flow resistance of microvascular mimics. These results implicate an impaired neutrophil pseudopod retraction response to shear in hypercholesterolemic microvascular dysfunction. We confirmed this using near-infrared diffuse correlation spectroscopy to assess skeletal muscle blood flow regulation in the hindlimbs of mice subjected to reactive hyperemia. Using a custom protocol for the mouse, we extrapolated an adjusted peak flow and time to adjusted peak flow to quantify the early phase of the blood flow recovery response during reactive hyperemia when shear mechanobiology likely has a maximal impact. Compared with mice on normal diet, hypercholesterolemic mice exhibited significantly (P<0.05) reduced adjusted peak flow and prolonged time to adjusted peak flow which correlated (r=0.4 and r=-0.3, respectively) with neutrophil shear responsiveness and were abrogated by neutropenia. CONCLUSIONS: These results provide the first evidence that the neutrophils contribute to tissue blood flow autoregulation. Moreover, a deficit in the neutrophil responsiveness to shear may be a feature of hypercholesterolemia-related microvascular dysfunction.

Noninvasive quantification of postocclusive reactive hyperemia in mouse thigh muscle by near-infrared diffuse correlation spectroscopy.

Many vasculature-related diseases affecting skeletal muscle function have been studied in mouse models. Noninvasive quantification of muscle blood flow responses during postocclusive reactive hyperemia (PORH) is often used to evaluate vascular function in human skeletal muscles. However, blood flow measurements during PORH in small skeletal muscles of mice are rare due to the lack of appropriate technologies coupled with the challenge of measurement setup resulting from the lack of large enough test sites. In this study, we explored adapting diffuse correlation spectroscopy (DCS) for noninvasive measurement of the relative changes of blood flow (rBF) in mouse thigh muscles during PORH. A small fiber-optic probe was designed and glued on the mouse thigh to reduce the motion artifact induced by the occlusion procedure. Arterial occlusion was created by tying a polyvinyl chloride (PVC) tube around the mouse thigh while the muscle rBF was continuously monitored by DCS to ensure the success of the occlusion. After 5 min, the occlusion was rapidly released by severing the PVC tube using a cautery pen. Typical rBF responses during PORH were observed in all mice (n=7), which are consistent with those observed by arterial-spin-labeled magnetic resonance imaging (ASL-MRI) as reported in the literature. On average, rBF values from DCS during occlusion were lower than 10% (3.1±2.2%) of the baseline values (assigning 100%), indicating the success of arterial occlusion in all mice. Peak values of rBF during PORH measured by the DCS (357.6±36.3%) and ASL-MRI (387.5±150.0%) were also similar whereas the values of time-to-peak (the time duration from the end of occlusion to the peak rBF) were quite different (112.6±35.0 s versus 48.0±27.0 s). Simultaneous measurements by these two techniques are needed to identify the factors that may cause such discrepancy. This study highlights the utility of DCS technology to quantitatively evaluate tissue blood flow responses during PORH in mouse skeletal muscles. DCS holds promise as valuable tool to assess blood flow regulation in mouse models with a variety of vascular diseases (e.g., hypercholesterolemia, diabetes, peripheral artery disease).

Integrin subtype-dependent CD18 cleavage under shear and its influence on leukocyte-platelet binding.

Previous studies showed that exposure of neutrophils to shear stress induces cysteine protease-mediated shedding of surface CD18 integrins involved in leukocyte-platelet interactions. Based on this, we hypothesized that, under noninflamed conditions, shear-induced CD18 cleavage is a control mechanism to minimize spontaneous leukocyte-platelet binding. For this purpose, we characterized the influence of shear on CD18 surface expression and platelet binding by the different leukocyte subsets. Shear stress elicited magnitude- (between 0 and 5 dyn/cm(2)) and time-dependent reductions in CD18 surface expression. This response was integrin- and cell type-specific, with neutrophils and monocytes exhibiting Mac-1 proteolysis but lymphocytes displaying LFA-1 shedding. Correspondingly, platelet binding, through CD18-fibrinogen interactions, was also influenced by shear exposure in a leukocyte-dependent manner. After treatment with cysteine protease inhibitor E64, neutrophils, but neither monocytes nor lymphocytes, exhibited significantly (P<0.05) enhanced platelet binding and CD18 surface expression under shear. Furthermore, shear exposure significantly (P<0.05) inhibited binding of naïve but not E64-treated neutrophils to fibrinogen. Combined, we provide first evidence that the CD18-cleavage responses of neutrophils to shear interfere with fibrinogen binding and platelet adhesion. These findings have implications as it relates to the efficiency of leukocyte passage through the microcirculation.

Fluid pressure is a magnitude-dependent modulator of early endothelial tubulogenic activity: implications related to a potential tissue-engineering control parameter.

A significant barrier to the success of engineered tissues is the inadequate transport of nutrients and gases to, and waste away from, cells within the constructs, after implantation. Generation of microtubular networks by endothelial cells in engineered constructs to mimic the in vivo transport scheme is essential for facilitating tissue survival by promoting the in vitro formation of microvessels that integrate with host microvasculature, after implantation. Previously, we reported that select pressures stimulate endothelial proliferation involving protubulogenic molecules such as fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor-C (VEGF-C). Based on this, we investigated fluid pressure as a selective modulator of early tubulogenic activity with the intent of assessing the potential utility of this mechanical stimulus as a tissue-engineering control parameter. For this purpose, we used a custom pressure system to expose two-dimensional (2D) and three-dimensional (3D) cultures of endothelial cells to static pressures of 0 (controls), 20, or 40 mmHg for 3 days. Compared to controls, 2D endothelial cultures exposed to 20, but not 40 mmHg, exhibited significantly (p<0.05) enhanced cell growth that depended on VEGF receptor-3 (VEGFR-3), a receptor for VEGF-C. Moreover, endothelial cells grown on microbeads and suspended in 3D collagen gels under 20 mmHg, but not 40 mmHg, displayed significantly (p<0.05) increased sprout formation. Interestingly, pressure-dependent proliferation and sprout formation occurred in parallel with pressure-sensitive upregulation of VEGF-C and VEGFR-3 expression and were sensitive to local FGF-2 levels. Collectively, the results of the present study provided evidence that early endothelial-related tubulogenic activity depends on local hydrostatic pressure levels in the context of local growth factor conditions. In addition to relevance to microvascular diseases associated with interstitial hypertension (e.g., cancer and glaucoma), these findings provided first insight into the potential utility of hydrostatic pressure as a fine-tune control parameter to optimize microvascularization of tissue-engineering constructs in the in vitro setting before their implantation.

Membrane cholesterol modulates the fluid shear stress response of polymorphonuclear leukocytes via its effects on membrane fluidity.

Continuous exposure of polymorphonuclear leukocytes (PMNLs) to circulatory hemodynamics points to fluid flow as a biophysical regulator of their activity. Specifically, fluid flow-derived shear stresses deactivate leukocytes via actions on the conformational activities of proteins on the cell surface. Because membrane properties affect activities of membrane-bound proteins, we hypothesized that changes in the physical properties of cell membranes influence PMNL sensitivity to fluid shear stress. For this purpose, we modified PMNL membranes and showed that the cellular mechanosensitivity to shear was impaired whether we increased, reduced, or disrupted the organization of cholesterol within the lipid bilayer. Notably, PMNLs with enriched membrane cholesterol exhibited attenuated pseudopod retraction responses to shear that were recovered by select concentrations of benzyl alcohol (a membrane fluidizer). In fact, PMNL responses to shear positively correlated (R(2) = 0.96; P < 0.0001) with cholesterol-related membrane fluidity. Moreover, in low-density lipoprotein receptor-deficient (LDLr(-/-)) mice fed a high-fat diet (a hypercholesterolemia model), PMNL shear-responses correlated (R(2) = 0.5; P < 0.01) with blood concentrations of unesterified (i.e., free) cholesterol. In this regard, the shear-responses of PMNLs gradually diminished and eventually reversed as free cholesterol levels in blood increased during 8 wk of the high-fat diet. Collectively, our results provided evidence that cholesterol is an important component of the PMNL mechanotransducing capacity and elevated membrane cholesterol impairs PMNL shear-responses at least partially through its impact on membrane fluidity. This cholesterol-linked perturbation may contribute to dysregulated PMNL activity (e.g., chronic inflammation) related to hypercholesterolemia and causal for cardiovascular pathologies (e.g., atherosclerosis).

Receptor cleavage reduces the fluid shear response in neutrophils of the spontaneously hypertensive rat.

Physiological fluid shear stress evokes pseudopod retraction in normal leukocytes by a mechanism that involves the formyl peptide receptor (FPR) as mechanosensor. In hypertensives, such as the spontaneously hypertensive rat (SHR), leukocytes lack the normal fluid shear response. The increased activity of matrix metalloproteinases (MMPs, including MMP-9) in SHR plasma is associated with cleavage of several cell membrane receptors. We hypothesize that the attenuated fluid shear response in leukocytes (neutrophils) of the SHR is due to extracellular proteolytic cleavage of the FPR. We show that suspended SHR neutrophils in whole blood sheared in a cone-and-plate device or individual neutrophils adherent to a glass surface and subject to fluid shear exhibited reduced pseudopod retractions compared with neutrophils of control Wistar-Kyoto (WKY) rats. SHR neutrophils and naïve Wistar rat neutrophils exposed to SHR plasma also exhibited impaired fluid shear responses as shown by their inability to project pseudopods with fluid shear. Labeling of extracellular FPR revealed that the FPR density in SHR neutrophils is on average 27% reduced compared with those of the WKY rats. Exposure of Wistar rat neutrophils to the gelatinase MMP-9 (final concentration 5 nM) led to attenuation of fluid shear response and decrease in extracellular FPR density. Chronic treatment of the SHR with a broad-acting MMP inhibitor, doxycycline, significantly improved the fluid shear response and increased the FPR extracellular density of SHR neutrophils. These results suggest that proteolytic cleavage of the FPR may interfere with normal fluid shear-induced pseudopod retractions in SHR neutrophils.