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Apicomplexa is a large phylum of intracellular parasites that are notable for the diseases they cause, including toxoplasmosis, malaria, and cryptosporidiosis. A conserved motile system is critical to their life cycles and drives directional gliding motility between cells, as well as invasion of and egress from host cells. However, our understanding of this system is limited by a lack of measurements of the forces driving parasite motion. We used a laser trap to measure the function of the motility apparatus of living Toxoplasma gondii by adhering a microsphere to the surface of an immobilized parasite. Motion of the microsphere reflected underlying forces exerted by the motile apparatus. We found that force generated at the parasite surface begins with no preferential directionality but becomes directed toward the rear of the cell after a period of time. The transition from nondirectional to directional force generation occurs on spatial intervals consistent with the lateral periodicity of structures associated with the membrane pellicle and is influenced by the kinetics of actin filament polymerization and cytoplasmic calcium. A lysine methyltransferase regulates both the magnitude and polarization of the force. Our work provides a novel means to dissect the motile mechanisms of these pathogens., (© 2017 Stadler et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Despite the life-preserving function blood clotting serves in the body, inadequate or excessive blood clot stiffness has been associated with life-threatening diseases such as stroke, hemorrhage, and heart attack. The relationship between blood clot stiffness and vascular diseases underscores the importance of quantifying the magnitude and kinetics of blood's transformation from a fluid to a viscoelastic solid. To measure blood plasma clot stiffness, we have developed a method that uses ultrasound acoustic radiation force (ARF) to induce micron-scaled displacements (1-500 μm) on microbeads suspended in blood plasma. The displacements were detected by optical microscopy and took place within a micro-liter sized clot region formed within a larger volume (2 mL sample) to minimize container surface effects. Modulation of the ultrasound generated acoustic radiation force allowed stiffness measurements to be made in blood plasma from before its gel point to the stage where it was a fully developed viscoelastic solid. A 0.5 wt % agarose hydrogel was 9.8-fold stiffer than the plasma (platelet-rich) clot at 1 h post-kaolin stimulus. The acoustic radiation force microbead method was sensitive to the presence of platelets and strength of coagulation stimulus. Platelet depletion reduced clot stiffness 6.9 fold relative to platelet rich plasma. The sensitivity of acoustic radiation force based stiffness assessment may allow for studying platelet regulation of both incipient and mature clot mechanical properties.

Endothelial cell (EC) alignment to directional flow or stretch supports anti-inflammatory functions, but mechanisms controlling polarized structural adaptation in response to physical cues remain unclear. This study aimed to determine whether factors associated with early actin edge ruffling implicated in cell polarization are prerequisite for stress fiber (SF) reorientation in response to cyclic uniaxial stretch. Time-lapse analysis of EGFP-actin in confluent ECs showed that onset of either cyclic uniaxial or equibiaxial stretch caused a non-directional increase in edge ruffling. Edge activity was concentrated in a direction perpendicular to the stretch axis after 60 min, consistent with the direction of SF alignment. Rho-kinase inhibition caused reorientation of both stretch-induced edge ruffling and SF alignment parallel to the stretch axis. Arp2/3 inhibition attenuated stretch-induced cell elongation and disrupted polarized edge dynamics and microtubule organizing center reorientation, but it had no effect on the extent of SF reorientation. Disrupting localization of p21-activated kinase (PAK) did not prevent stretch-induced SF reorientation, suggesting that this Rac effector is not critical in regulating stretch-induced cytoskeletal remodeling. Overall, these results suggest that directional edge ruffling is not a primary mechanism that guides SF reorientation in response to stretch; the two events are coincident but not causal.

In January of 2011, the Biomedical Engineering Society (BMES) and the Society for Physical Regulation in Biology and Medicine (SPRBM) held its inaugural Cellular and Molecular Bioengineering (CMBE) conference. The CMBE conference assembled worldwide leaders in the field of CMBE and held a very successful Round Table discussion among leaders. One of the action items was to collectively construct a white paper regarding the future of CMBE. Thus, the goal of this report is to emphasize the impact of CMBE as an emerging field, identify critical gaps in research that may be answered by the expertise of CMBE, and provide perspectives on enabling CMBE to address challenges in improving human health. Our goal is to provide constructive guidelines in shaping the future of CMBE.

Spatial asymmetry of actin edge ruffling contributes to the process of cell polarization and directional migration, but mechanisms by which external cues control actin polymerization near cell edges remain unclear. We designed a quantitative image analysis strategy to measure the spatiotemporal distribution of actin edge ruffling. Time-lapse images of endothelial cells (ECs) expressing mRFP-actin were segmented using an active contour method. In intensity line profiles oriented normal to the cell edge, peak detection identified the angular distribution of polymerized actin within 1 µm of the cell edge, which was localized to lamellipodia and edge ruffles. Edge features associated with filopodia and peripheral stress fibers were removed. Circular statistical analysis enabled detection of cell polarity, indicated by a unimodal distribution of edge ruffles. To demonstrate the approach, we detected a rapid, nondirectional increase in edge ruffling in serum-stimulated ECs and a change in constitutive ruffling orientation in quiescent, nonpolarized ECs. Error analysis using simulated test images demonstrate robustness of the method to variations in image noise levels, edge ruffle arc length, and edge intensity gradient. These quantitative measurements of edge ruffling dynamics enable investigation at the cellular length scale of the underlying molecular mechanisms regulating actin assembly and cell polarization.

Leukocyte rolling on the blood vessel wall represents the first step in the process of inflammation. In this study, nanofabricated substrates were designed with two different sets of feature size and spacing to mimic the expected distribution of discrete molecular adhesion patches on the surfaces of endothelial cells lining the blood vessel wall. P-selectin was attached to these nanopatterned dots, and the rolling behaviour of HL60 cells was analysed as a function of wall shear stress. When wall shear stress was less than 1 dyne/cm 2 , rolling velocity was independent of substrate patterning. However, when wall shear stress was higher than 2 dyne/cm 2 , rolling velocity was increased on the patterned substrates compared with the unpatterned sample, and rolling velocity increased with nanodot spacing distance. The influence of pattern spacing on the waiting time, the duration of zero-velocity pauses during rolling, also increased for wall shear stresses greater than 2 dyne/cm 2 . Additionally, the variance of instantaneous rolling velocities increased among substrates when the shear stress was greater than 6 dyne/cm 2 , indicating that the spatial arrangement of the nanodot pattern influenced not only the average velocity with which the cells rolled but also the saltatory nature of rolling. These results suggest that nanodot substrates represent a tool to investigate the biophysical and biochemical mechanisms regulating dynamic adhesion of leukocytes to the blood vessel wall.

Several custom-built and commercially available devices are available to investigate cellular responses to substrate strain. However, analysis of structural dynamics by microscopy in living cells during stretch is not readily feasible. We describe a novel stretch device optimized for high-resolution live-cell imaging. The unit assembles onto standard inverted microscopes and applies constant magnitude or cyclic stretch at physiological magnitudes to cultured cells on elastic membranes. Interchangeable modular indenters enable delivery of equibiaxial and uniaxial stretch profiles. Strain analysis performed by tracking fluorescent microspheres adhered onto the substrate demonstrated reproducible application of stretch profiles. In endothelial cells transiently expressing enhanced green fluorescent protein (EGFP)-vimentin and paxillin-DsRed2 and subjected to constant magnitude equibiaxial stretch, the two-dimensional strain tensor demonstrated efficient transmission through the extracellular matrix and focal adhesions. Decreased transmission to the intermediate filament network was measured, and a heterogeneous spatial distribution of maximum stretch magnitude revealed discrete sites of strain focusing. Spatial correlation of vimentin and paxillin displacement vectors provided an estimate of the extent of mechanical coupling between the structures. Interestingly, switching the spatial profile of substrate strain reveals that actin-mediated edge ruffling is not desensitized to repeated mechanostimulation. These initial observations show that the stretch device is compatible with live-cell microscopy and is a novel tool for measuring dynamic structural remodeling under mechanical strain.

Cobblestone-shaped endothelial cells in confluent monolayers undergo triphasic mechanotaxis in response to steady unidirectional shear stress, but cells that are elongated and aligned on micropatterned substrates do not change their migration behavior in response to either perpendicular or parallel flow. Whether mechanotaxis of micropatterned endothelial cell layers is suppressed by elongated cytoskeletal structure or limited availability of adhesion area remains unknown. In this study, cells were examined on wide (100-200 μm) micropatterned lines after onset of shear stress. Cells in center regions of the lines exhibited cobblestone morphology and triphasic mechanotaxis behavior similar to that in unpatterned monolayers, whereas cells along the edges migrated parallel to the line axis regardless of the flow direction. When scratch wounds were created perpendicular to the micropatterned lines, the cells became less elongated before migrating into the denuded area. In sparsely populated lines oriented perpendicular to the flow direction, elongated cells along the upstream edge migrated parallel to the edge for 7 h before migrating parallel to the shear stress direction, even though adhesion area existed in the downstream direction. Thus, cytoskeletal structure and not available adhesion area serves as the dominant factor in determining whether endothelial mechanotaxis occurs in response to shear stress.

Purpose: It has been proposed that quantum dots (QDs) can be used to excite conjugated photosensitizers and produce cytotoxic singlet oxygen. To study the potential of using such a conjugate synergistically with radiotherapy to enhance cell killing, we investigated the energy transfer from megavoltage (MV) X-rays to a photosensitizer using QDs as the mediator and quantitated the enhancement in cell killing., Methods and Materials: The photon emission efficiency of QDs on excitation by 6-MV X-rays was measured using dose rates of 100-600 cGy/min. A QD-Photofrin conjugate was synthesized by formation of an amide bond. The role of Förster resonance energy transfer in the energy transferred to the Photofrin was determined by measuring the degree of quenching at different QD/Photofrin molar ratios. The enhancement of H460 human lung carcinoma cell killing by radiation in the presence of the conjugates was studied using a clonogenic survival assay., Results: The number of visible photons generated from QDs excited by 6-MV X-rays was linearly proportional to the radiation dose rate. The Förster resonance energy transfer efficiency approached 100% as the number of Photofrin molecules conjugated to the QDs increased. The combination of the conjugate with radiation resulted in significantly lower H460 cell survival in clonogenic assays compared with radiation alone., Conclusion: The novel QD-Photofrin conjugate shows promise as a mediator for enhanced cell killing through a linear and highly efficient energy transfer from X-rays to Photofrin.

Vascular endothelial cell migration is critical in many physiological processes including wound healing and stent endothelialization. To determine how preexisting cell morphology influences cell migration under fluid shear stress, endothelial cells were preset in an elongated morphology on micropatterned substrates, and unidirectional shear stress was applied either parallel or perpendicular to the cell elongation axis. On micropatterned 20-microm lines, cells exhibited an elongated morphology with stress fibers and focal adhesion sites aligned parallel to the lines. On 115-microm lines, cell morphology varied as a function of distance from the line edge. Unidirectional shear stress caused unpatterned cells in a confluent monolayer to exhibit triphasic mechanotaxis behavior. During the first 3 h, cell migration speed increased in a direction antiparallel to the shear stress direction. Migration speed then slowed and direction became spatially heterogeneous. Starting 11-12 h after the onset of shear stress, the unpatterned cells migrated primarily in the downstream direction, and migration speed increased significantly. In contrast, mechanotaxis was suppressed after the onset of shear stress in cells on micropatterned lines during the same time period, for the cases of both parallel and perpendicular flow. The directional persistence time was much longer for cells on the micropatterned lines, and it decreased significantly after flow onset. Migration trajectories were highly correlated among micropatterned cells within a three-cell neighborhood, and shear stress disrupted this spatially correlated migration behavior. Thus, presetting structural morphology may interfere with mechanisms of sensing local physical cues, which are critical for establishing mechanotaxis in response to hemodynamic shear stress.

Hemodynamic shear stress guides a variety of endothelial phenotype characteristics, including cell morphology, cytoskeletal structure, and gene expression profile. The sensing and processing of extracellular fluid forces may be mediated by mechanotransmission through the actin cytoskeleton network to intracellular locations of signal initiation. In this study, we identify rapid actin-mediated morphological changes in living subconfluent and confluent bovine aortic endothelial cells (ECs) in response to onset of unidirectional steady fluid shear stress (15 dyn/cm(2)). After flow onset, subconfluent cells exhibited dynamic edge activity in lamellipodia and small ruffles in the downstream and side directions for the first 12 min; activity was minimal in the upstream direction. After 12 min, peripheral edge extension subsided. Confluent cell monolayers that were exposed to shear stress exhibited only subtle increases in edge fluctuations after flow onset. Addition of cytochalasin D to disrupt actin polymerization served to suppress the magnitude of flow-mediated actin remodeling in both subconfluent confluent EC monolayers. Interestingly, when subconfluent ECs were exposed to two sequential flow step increases (1 dyn/cm(2) followed by 15 dyn/cm(2) 12 min later), actin-mediated edge activity was not additionally increased after the second flow step. Thus, repeated flow increases served to desensitize mechanosensitive structural dynamics in the actin cytoskeleton.

We determined the effects of peroxynitrite (ONOO-) on cardiac myosin, actin, and thin filaments in order to more clearly understand the impact of this reactive compound in ischemia/reperfusion injury and heart failure. Actin filaments, native thin filaments, and alpha-cardiac myosin from rat hearts were exposed to ONOO- in the presence of 2 mM bicarbonate. Filament velocities over myosin, calcium sensitivity, and relative force generated by myosin were assessed in an in vitro motility assay in the absence of reducing agents. ONOO- concentrations > or =10 microM significantly reduced the velocities of thin filaments or bare actin filaments over alpha-cardiac myosin when any of these proteins were exposed individually. These functional deficits were linearly related to the degree of tyrosine nitration, with myosin being the most sensitive. However, at 10 microM ONOO- the calcium sensitivity of thin filaments remained unchanged. Cotreatment of myosin and thin filaments, analogous to the in vivo situation, resulted in a significantly greater functional deficit. The load supported by myosin after ONOO- exposure was estimated using mixtures experiments to be increased threefold. These data suggest that nitration of myofibrillar proteins can contribute to cardiac contractile dysfunction in pathologic states in which ONOO- is liberated.

Hemodynamic shear stress regulates endothelial cell biochemical processes that govern cytoskeletal contractility, focal adhesion dynamics, and extracellular matrix (ECM) assembly. Since shear stress causes rapid strain focusing at discrete locations in the cytoskeleton, we hypothesized that shear stress coordinately alters structural dynamics in the cytoskeleton, focal adhesion sites, and ECM on a time scale of minutes. Using multiwavelength four-dimensional fluorescence microscopy, we measured the displacement of rhodamine-fibronectin and green fluorescent protein-labeled actin, vimentin, paxillin, and/or vinculin in aortic endothelial cells before and after onset of steady unidirectional shear stress. In the cytoskeleton, the onset of shear stress increased actin polymerization into lamellipodia, altered the angle of lateral displacement of actin stress fibers and vimentin filaments, and decreased centripetal remodeling of actin stress fibers in subconfluent and confluent cell layers. Shear stress induced the formation of new focal complexes and reduced the centripetal remodeling of focal adhesions in regions of new actin polymerization. The structural dynamics of focal adhesions and the fibronectin matrix varied with cell density. In subconfluent cell layers, shear stress onset decreased the displacement of focal adhesions and fibronectin fibrils. In confluent monolayers, the direction of fibronectin and focal adhesion displacement shifted significantly toward the downstream direction within 1 min after onset of shear stress. These spatially coordinated rapid changes in the structural dynamics of cytoskeleton, focal adhesions, and ECM are consistent with focusing of mechanical stress and/or strain near major sites of shear stress-mediated mechanotransduction.

The aims of this study were to develop a method for deriving purified populations of contractile smooth muscle cells (SMCs) from embryonic stem cells (ESCs) and to characterize their function. Transgenic ESC lines were generated that stably expressed a puromycin-resistance gene under the control of either a smooth muscle alpha-actin (SMalphaAlpha) or smooth muscle-myosin heavy chain (SM-MHC) promoter. Negative selection, either overnight or for 3 days, was then used to purify SMCs from embryoid bodies. Purified SMCs expressed multiple SMC markers by immunofluorescence, immunoblotting, quantitative reverse transcription-polymerase chain reaction, and flow cytometry and were designated APSCs (SMalphaAlpha-puromycin-selected cells) or MPSCs (SM-MHC-puromycin-selected cells), respectively. Both SMC lines displayed agonist-induced Ca(2+) transients, expressed functional Ca(2+) channels, and generated contractile force when aggregated within collagen gels and stimulated with vasoactive agonists, such as endothelin-1, or in response to depolarization with KCl. Importantly, subcutaneous injection of APSCs or MPSCs subjected to 18 hours of puromycin selection led to the formation of teratomas, presumably due to residual contamination by pluripotent stem cells. In contrast, APSCs or MPSCs subjected to prolonged puromycin selection for 3 days did not form teratomas in vivo. These studies describe for the first time a method for generating relatively pure populations of SMCs from ESCs which display appropriate excitation and contractile responses to vasoactive agonists. However, studies also indicate the potential for teratoma development in ESC-derived cell lines, even after prolonged differentiation, highlighting the critical requirement for efficient methods of separating differentiated cells from residual pluripotent precursors in future studies that use ESC derivatives, whether SMC or other cell types, in tissue engineering applications.

Nanotechnology-based materials are beginning to emerge as promising platforms for biomedical analysis, but measurement and control at the cell-chip interface remain challenging. This idea served as the basis for discussion in a focus group at the recent National Academies Keck Futures Initiative. In this Perspective, we first outline recent advances and limitations in measuring nanoscale mechanical, biochemical, and electrical interactions at the interface between biomaterials and living cells. Second, we present emerging experimental and conceptual platforms for probing living cells with nanotechnology-based tools in a microfluidic chip. Finally, we explore future directions and critical needs for engineering the cell-chip interface to create an integrated system capable of high-resolution analysis and control of cellular physiology.

The final stage of lung development in humans and rodents occurs principally after birth and involves the partitioning of the large primary saccules into smaller air spaces by the inward protrusion of septae derived from the walls of the saccules. Several observations in animal models implicate angiogenesis as critical to this process of alveolarization, but all anti-angiogenic treatments examined to date have resulted in endothelial cell (EC) death. We therefore targeted the function of platelet endothelial cell adhesion molecule, (PECAM-1), an EC surface molecule that promotes EC migration and has been implicated in in vivo angiogenesis. Administration of an anti-PECAM-1 antibody that inhibits EC migration, but not proliferation or survival in vitro, disrupted normal alveolar septation in neonatal rat pups without reducing EC content. Three-dimensional reconstruction of lungs showed that pups treated with a blocking PECAM-1 antibody had remodeling of more proximal branches resulting in large tubular airways. Subsequent studies in PECAM-1-null mice confirmed that the absence of PECAM-1 impaired murine alveolarization, without affecting EC content, proliferation, or survival. Further, cell migration was reduced in lung endothelial cells isolated from these mice. These data suggest that the loss of PECAM-1 function compromises postnatal lung development and provide evidence that inhibition of EC function, in contrast to a loss of viable EC, inhibits alveolarization.

Essentially all organisms from bacteria to humans are mechanosensitive. Physical forces regulate a large array of physiological processes, and dysregulation of mechanical responses contributes to major human diseases. A survey of both specialized and widely expressed mechanosensitive systems suggests that physical forces provide a general means of altering protein conformation to generate signals. Specialized systems differ mainly in having acquired efficient mechanisms for transferring forces to the mechanotransducers.

The endothelium at the interface between blood and tissue acts as a primary transducer of local hemodynamic forces into signals that maintain physiological function or initiate pathological processes in vessel walls. Rapid intracellular spatial gradients of structural dynamics and signaling molecule activity suggest that mechanical cues at the molecular level guide cellular mechanotransduction and adaptation to shear stress profiles.

Both mechanical and chemical stimuli guide tissue function. In a recent paper, Tschumperlin et al. proposed that pressure acting on airway epithelium elicits mechanotransduction not by directly altering biochemical signaling but by regulating extracellular fluid volume to modulate ligand-receptor interactions.

A central aspect of cellular mechanochemical signaling is a change of cytoskeletal tension upon the imposition of exogenous forces. Here we report measurements of the spatiotemporal distribution of mechanical strain in the intermediate filament cytoskeleton of endothelial cells computed from the relative displacement of endogenous green fluorescent protein (GFP)-vimentin before and after onset of shear stress. Quantitative image analysis permitted computation of the principal values and orientations of Lagrangian strain from 3-D high-resolution fluorescence intensity distributions that described intermediate filament positions. Spatially localized peaks in intermediate filament strain were repositioned after onset of shear stress. The orientation of principal strain indicated that mechanical stretching was induced across cell boundaries. This novel approach for intracellular strain mapping using an endogenous reporter demonstrates force transfer from the lumenal surface throughout the cell.

Descriptive and quantitative analyses of microstimuli in living endothelial cells strongly support an integrated mechanism of mechanotransduction regulated by the spatial organization of multiple structural and signaling networks. Endothelial responses to blood flow are regulated at multiple levels of organization extending over scales from vascular beds to single cells, subcellular structures, and individual molecules. Microstimuli at the cellular and subcellular levels exhibit temporal and spatial complexities that are increasingly accessible to measurement. We address the cell and subcellular physical interface between flow-related forces and biomechanical responses of the endothelial cell. Live cell imaging and computational analyses of structural dynamics, two important approaches to microstimulation at this scale, are briefly reviewed.

Cytoskeletal proteins play important roles in regulating cellular morphology, cytokinesis and intracellular signaling. In this report, we describe a developmentally regulated gene encoding a novel cell lineage-restricted cytoskeletal protein, designated SM22beta. SM22beta shares high-grade sequence identity with the smooth muscle cell (SMC)-specific protein, SM22alpha, the neuron-specific protein, NP25, and the Drosophila melanogaster flight muscle-specific protein, mp20. The mouse SM22beta cDNA encodes a 199-amino acid polypeptide that contains a single conserved calponin-like repeat domain. During mouse embryonic development, the SM22beta gene is expressed in a temporally and spatially regulated pattern in the tunica media of arteries and veins, endocardium and compact layer of the myocardium, bronchial epithelium and mesenchyme of the lung, gastrointestinal epithelium and cartilaginous primordia. During postnatal development, SM22beta is co-expressed with SM22alpha in arterial and venous SMCs. In addition, SM22beta is expressed at high levels in the bronchial epithelium and lung mesenchyme, gastrointestinal epithelial cells and in the cartilagenous and periosteal layer of bones. Three-dimensional deconvolution microscopic analyses of A7r5 SMCs revealed that SM22beta co-localizes with SM22alpha to cytoskeletal actin filaments. Taken together, these data demonstrate that SM22beta is a novel actin-associated protein with a unique cell lineage-restricted pattern of expression.

The endothelium, a single layer of cells that lines all blood vessels, is the focus of intense interest in biomechanics because it is the principal recipient of hemodynamic shear stress. In arteries, shear stress has been demonstrated to regulate both acute vasoregulation and chronic adaptive vessel remodeling and is strongly implicated in the localization of atherosclerotic lesions. Thus, endothelial biomechanics and the associated mechanotransduction of shear stress are of great importance in vascular physiology and pathology. Here we discuss the important role of the cytoskeleton in a decentralization model of endothelial mechanotransduction. In particular, recent studies of four-dimensional cytoskeletal motion in living cells under external fluid mechanical forces are summarized together with new data on the spatial distribution of cytoskeletal strain. These quantitative studies strongly support the decentralized distribution of luminally imposed forces throughout the endothelial cell.

The completion of the Human Genome Project and ongoing sequencing of mouse, rat and other genomes has led to an explosion of genetics-related technologies that are finding their way into all areas of biological research; the field of biorheology is no exception. Here we outline how two disparate modern molecular techniques, microarray analyses of gene expression and real-time spatial imaging of living cell structures, are being utilized in studies of endothelial mechanotransduction associated with controlled shear stress in vitro and haemodynamics in vivo. We emphasize the value of such techniques as components of an integrated understanding of vascular rheology. In mechanotransduction, a systems approach is recommended that encompasses fluid dynamics, cell biomechanics, live cell imaging, and the biochemical, cell biology and molecular biology methods that now encompass genomics. Microarrays are a useful and powerful tool for such integration by identifying simultaneous changes in the expression of many genes associated with interconnecting mechanoresponsive cellular pathways.

Atherosclerosis originates at predictable focal and regional sites that are associated with complex flow disturbances and flow separations in large arteries. The spatial relationships associated with hemodynamic shear stress forces acting on the endothelial monolayer are considered in experiments that model regions susceptible to atherosclerosis (flow disturbance) and resistant to atherosclerosis (undisturbed flow). Flow disturbance in vitro induced differential expression at the single gene level as illustrated for the intercellular communication gene and protein, connexin 43. Transcription profiles of individual endothelial cells isolated from both disturbed and undisturbed flow regions exhibited more expression heterogeneity in disturbed than in undisturbed flow. We propose that within highly heterogeneous populations of endothelial cells located in disturbed flow regions, proatherosclerotic gene expression may occur within the range of expression profiles induced by the local hemodynamics. These may be sites of initiation of focal atherosclerosis. Mechanisms are proposed to account for heterogeneous endothelial responses to shear stress by reference to the decentralized model of endothelial mechanotransduction. Length scales ranging from centimeters to nanometers are useful in describing regional, single cell, and intracellular mechanotransduction mechanisms.

SM22alpha is a 22-kDa smooth muscle cell (SMC) lineage-restricted protein that physically associates with cytoskeletal actin filament bundles in contractile SMCs. To examine the function of SM22alpha, gene targeting was used to generate SM22alpha-deficient (SM22(-/-LacZ)) mice. The gene targeting strategy employed resulted in insertion of the bacterial lacZ reporter gene at the SM22alpha initiation codon, permitting precise analysis of the temporal and spatial pattern of SM22alpha transcriptional activation in the developing mouse. Northern and Western blot analyses confirmed that the gene targeting strategy resulted in a null mutation. Histological analysis of SM22(+/-LacZ) embryos revealed detectable beta-galactosidase activity in the unturned embryonic day 8.0 embryo in the layer of cells surrounding the paired dorsal aortae concomitant with its expression in the primitive heart tube, cephalic mesenchyme, and yolk sac vasculature. Subsequently, during postnatal development, beta-galactosidase activity was observed exclusively in arterial, venous, and visceral SMCs. SM22alpha-deficient mice are viable and fertile. Their blood pressure and heart rate do not differ significantly from their control SM22alpha(+/-) and SM22alpha(+/+) littermates. The vasculature and SMC-containing tissues of SM22alpha-deficient mice develop normally and appear to be histologically and ultrastructurally similar to those of their control littermates. Taken together, these data demonstrate that SM22alpha is not required for basal homeostatic functions mediated by vascular and visceral SMCs in the developing mouse. These data also suggest that signaling pathways that regulate SMC specification and differentiation from local mesenchyme are activated earlier in the angiogenic program than previously recognized.

The distribution of hemodynamic shear stress throughout the arterial tree is transduced by the endothelium into local cellular responses that regulate vasoactivity, vessel wall remodeling, and atherogenesis. Although the exact mechanisms of mechanotransduction remain unknown, the endothelial cytoskeleton has been implicated in transmitting extracellular force to cytoplasmic sites of signal generation via connections to the lumenal, intercellular, and basal surfaces. Direct observation of intermediate filament (IF) displacement in cells expressing green fluorescent protein-vimentin has suggested that cytoskeletal mechanics are rapidly altered by the onset of fluid shear stress. Here, restored images from time-lapse optical sectioning fluorescence microscopy were analyzed as a four-dimensional intensity distribution function that represented IF positions. A displacement index, related to the product moment correlation coefficient as a function of time and subcellular spatial location, demonstrated patterns of IF displacement within endothelial cells in a confluent monolayer. Flow onset induced a significant increase in IF displacement above the nucleus compared with that measured near the coverslip surface, and displacement downstream from the nucleus was larger than in upstream areas. Furthermore, coordinated displacement of IF near the edges of adjacent cells suggested the existence of mechanical continuity between cells. Thus, quantitative analysis of the spatiotemporal patterns of flow-induced IF displacement suggests redistribution of intracellular force in response to alterations in hemodynamic shear stress acting at the lumenal surface.

Hemodynamic shear stress at the endothelial cell surface induces acute and chronic intracellular responses that regulate vessel wall biology. The cytoskeleton is implicated by acting both as a direct connector to local surface deformation and as a distribution network for mechanical forces throughout the cell; however, direct observation and measurement of its position during flow have only recently become possible. In this study, we directly demonstrate rapid deformation of the intermediate filament (IF) network in living endothelial cells subjected to changes in hemodynamic shear stress. Time-lapse optical sectioning and deconvolution microscopy were performed within the first 3 minutes after the introduction of flow (shear stress, 12 dyn/cm(2)). Spatial and temporal dynamics of green fluorescent protein-vimentin IFs in confluent endothelial cells were analyzed. The imposition of shear stress significantly increased the variability of IF movement throughout the cell in the x-, y-, and z-directions compared with the constitutive dynamics noted in the absence of flow. Acute polymerization and depolymerization of the IF network were absent. The magnitude and direction of flow-induced IF displacement were heterogeneous at the subcellular level. These qualitative and quantitative data demonstrate that shear stress acting at the luminal surface of the endothelium results in rapid deformation of a stable IF network.

An obstacle to real-time in vitro measurements of endothelial cell responses to hemodynamic forces is the inaccessibility of the cells to instruments of measurement and manipulation. We have designed a parallel plate laminar flow chamber that permits access to adherent cells during exposure to flow. The "minimally invasive flow device" (MIF device) has longitudinal slits (1 mm wide) cut in the top plate of the chamber to allow insertion of a recording, measurement, or stimulating instrument (e.g., micropipette) into the flow field. Surface tension forces at the slit openings are sufficient to counteract the hydrostatic pressure generated in the chamber and thus prevent overflow. The invasive probe is brought near to the cell surface, makes direct contact with the cell membrane, or enters the cell. The slits provide access to a large number (and choice) of cells. The MIF device can maintain physiological levels of shear stress (<1-15 dyn/cm2) without overflow in the absence and presence of fine instruments such as micropipettes used in electrophysiology, membrane aspiration, and microinjection. Microbead trajectory profiles demonstrated negligible deviations in laminar flow near the surface of target cells in the presence of microscale instruments. Patch-clamp electrophysiological recordings of flow-induced changes in membrane potential were demonstrated. The MIF device offers numerous possibilities to investigate real-time endothelial responses to well-defined flow conditions in vitro including electrophysiology, cell surface mechanical probing, local controlled chemical release, biosensing, microinjection, and amperometric techniques.

The initiation and progression of focal atherosclerotic lesions has long been known to be associated with regions of disturbed blood flow. Improved precision in experimental models of spatially defined flow has recently been combined with regional and single-cell gene-expression profiling to investigate the relationships linking haemodynamics to vessel-wall pathobiology.

In spite of the relatively small number of leukocytes in the circulation, they have a significant influence on the perfusion of such organs as skeletal muscle or kidney. However, the underlying mechanisms are incompletely understood. In the current study a combined in vivo and computational approach is presented in which the interaction of individual freely flowing leukocytes with erythrocytes and its effect on apparent blood viscosity are explored. The skeletal muscle microcirculation was perfused with different cell suspensions with and without leukocytes or erythrocytes. We examined a three-dimensional numerical model of low Reynolds number flow in a capillary with a train of erythrocytes (small spheres) in off-axis positions and single larger leukocytes in axisymmetric positions. The results indicate that in order to match the slower axial velocity of leukocytes in capillaries, erythrocytes need to position themselves into an off-axis position in the capillary. In such off-axis positions at constant mean capillary velocity, erythrocyte axial velocity matches on average the axial velocity of the leukocytes, but the apparent viscosity is elevated, in agreement with the whole organ perfusion observations. Thus, leukocytes influence the whole organ resistance in skeletal muscle to a significant degree only in the presence of erythrocytes.

Despite the small number of leukocytes relative to erythrocytes in the circulation, leukocytes contribute significantly to organ blood flow resistance. The present study was designed to investigate whether interactions between leukocytes and erythrocytes affect the pressure-flow relationship in a hemodynamically isolated rat gracilis muscle. At constant arterial flow rate, arterial pressure was increased significantly when relatively few physiological counts of leukocytes were added to a suspension containing erythrocytes at physiological hematocrits. However, the arterial pressure after perfusion of similar numbers of isolated leukocytes without erythrocytes was only slightly increased. An increase in resistance was also observed when leukocytes were replaced with 6-micron microspheres. We propose a new mechanism for increasing the hemodynamic resistance that involves hydrodynamic interactions between leukocytes and erythrocytes. In the presence of larger and less deformable leukocytes, erythrocytes move through capillaries more slowly than without leukocytes. Therefore erythrocytes are displaced from their axial positions. Slowing and radial displacement of erythrocytes serve to increase the relative apparent viscosity attributable to erythrocytes, thereby causing a significant elevation of organ blood flow resistance.