Analysis of SM22alpha-deficient mice reveals unanticipated insights into smooth muscle cell differentiation and function.

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.

Spatiotemporal analysis of flow-induced intermediate filament displacement in living endothelial cells.

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.

Hemodynamics and the focal origin of atherosclerosis: a spatial approach to endothelial structure, gene expression, and function.

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.

Rapid displacement of vimentin intermediate filaments in living endothelial cells exposed to flow.

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.

A chamber to permit invasive manipulation of adherent cells in laminar flow with minimal disturbance of the flow field.

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.

A spatial approach to transcriptional profiling: mechanotransduction and the focal origin of atherosclerosis.

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.

A mechanism for erythrocyte-mediated elevation of apparent viscosity by leukocytes in vivo without adhesion to the endothelium.

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.

Mechanisms for increased blood flow resistance due to leukocytes.

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.