© 2000 American Heart Association, Inc.
Cellular Biology |
Rapid Displacement of Vimentin Intermediate Filaments in Living Endothelial Cells Exposed to Flow
From the Institute for Medicine and Engineering (B.P.H., P.F.D.), Department of Pathology and Laboratory Medicine (P.F.D.), and Department of Bioengineering (B.P.H., P.F.D.), University of Pennsylvania, Philadelphia, Pa; Department of Cell and Molecular Biology (R.D.G.), Northwestern University Medical School, Chicago, Ill.
Correspondence to Peter F. Davies, PhD, Institute for Medicine and Engineering, University of Pennsylvania, 1010 Vagelos Research Labs, 3340 Smith Walk, Philadelphia, PA 19104-6383. E-mail pfd{at}pobox.upenn.edu
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Abstract |
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Abstract—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/cm2). 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.
Key Words: mechanotransduction • endothelium • green fluorescent protein
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Introduction |
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Vascular endothelial cells (ECs) are highly specialized to respond to local changes in blood flow, particularly hemodynamic shear stress forces that act at the luminal cell surface in the direction of flow.1 2 The mechanisms responsible for endothelial mechanotransduction have physiological and pathological relevance. Acute dilation or constriction of arteries in response to changes of flow is controlled by the endothelium through the release of nitric oxide, prostaglandins, and related molecules.3 4 5 Furthermore, chronic changes in local flow characteristics stimulate EC-dependent structural remodeling of the artery wall,6 and flow is implicated in the localization of atherosclerotic lesions.7 Both acute and chronic changes appear to be regulated principally by shear stress.2
ECs respond to shear stress by initiating a cascade of intracellular events that begins with the generation of second messengers and is quickly followed by transcriptional, synthetic, and structural changes (reviewed in References 2 , 8 , and 9 ). Initiation of mechanosignaling events has been measured within seconds of exposure to flow; however, the mechanism(s) by which shear stress acting on the luminal cell surface is converted to specific cellular responses is unclear. Shearing forces may directly deform the cell surface10 to generate local biochemical responses arising from undefined sensor proteins11 and/or deformation of the lipid bilayer.12 Signal transduction, however, may not be limited exclusively to the luminal surface. In a decentralization model,2 forces acting on the cell surface evoke responses at other locations within the cell, perhaps via force transmission through the cytoskeleton. Apical shear stress induces directional movement of basal focal adhesion contacts and phosphorylation of proteins concentrated at these sites.13 14 Thus, instead of operating through a single sensing pathway for mechanical forces, cellular mechanotransduction may result from an integrated response of multiple signaling networks that are spatially organized throughout the surfaces and interior of the ECs.
Although extensive slow remodeling of the endothelial cytoskeleton in response to shear stress has been inferred from fixed-cell studies,1 15 16 the use of green fluorescent protein (GFP) now allows direct observation of spatiotemporal dynamics in living cells.17 18 In the present study, we have used a GFP-vimentin fusion protein19 expressed in ECs to evaluate changes in position of the intermediate filament (IF) cytoskeleton during a step change in hemodynamic shear stress.
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Materials and Methods |
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Cell Culture and Transfection
Bovine aortic ECs were transfected for 2.5 hours with 1 to 2 µg pEGFP-hVIM-Myc19 using a liposomal method (Lipofectin, Life Technologies) according to the manufacturer’s recommendations. Cells were allowed to recover overnight in complete growth medium. Red fluorescent microspheres, 0.1 µm in diameter (Molecular Probes), were dried onto sterile No. 1.5 thickness glass coverslips (Bioptechs). Microspheres served as fiducial markers that allowed removal of coverslip movements during time-lapse image acquisition (see below). Transfected ECs were plated onto the coverslips over the microspheres and were grown to confluence.
To establish cell lines with stable expression of GFP-vimentin, ECs were maintained in growth medium containing 400 µg/mL G418 (Life Technologies) beginning 2 days after transfection. Because the plasmid contained a kanamycin/neomycin resistance gene,19 only cells expressing GFP-vimentin survived the selection pressure. Groups of cells expressing similar levels of fluorescence intensity were isolated and grown to confluence.
Deconvolution Microscopy
Wide-field fluorescence optical sections were acquired
using a DeltaVision system (Applied Precision). Spatial and temporal
normalization of the illumination intensity allowed quantitative
analysis of GFP-vimentin fluorescence
intensity.20 21 A quantitative method for image
restoration improved the spatial precision of IF locations over that
typically measured by wide-field microscopy. Stacks of optical sections
spaced 0.1 to 0.5 µm apart were acquired. A 3D point spread
function was measured experimentally, and a constrained iterative
deconvolution algorithm20 was applied to arrays of optical
sections. Resolution in restored images was 317 nm in the
xy-plane and 758 nm along the z-axis. Maximum
intensity volume projections were computed using DeltaVision
software.
Time-Lapse Imaging of GFP-Vimentin in Living
Cells
Coverslips containing ECs expressing GFP-vimentin were assembled
into a temperature-controlled parallel plate flow chamber (FCS2,
Bioptechs) and maintained at 37°C. Dual-wavelength 3D image stacks
with 0.5-µm spacing between optical sections were acquired every 90
seconds for 20 to 30 minutes. A step change in flow was imposed so that
wall shear stress was 12 dyn/cm2, and image
acquisition continued at the same rate for an additional 20 to 30
minutes. A red fluorescent microsphere on the coverslip
was chosen as a fiducial marker, and its 3D position in the image stack
was determined at each time point. Single fluorescence optical
sections from regions of interest were chosen so that image position
along the x-, y-, and z-axes was
normalized relative to that of the microsphere. In this manner,
motion of GFP-vimentin in the time-lapse measurements was attributable
only to IF displacement and not to movement of the coverslip.
Three-dimensional analysis was performed on images of 1 to 5
cells from each of 4 experiments.
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Results |
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Visualization of GFP-Vimentin Networks in ECs
The 3D distribution of IFs in living ECs was observed using GFP-vimentin (Figure 1
). Deconvolved
optical sections at 2-µm intervals near the base, middle, and apex of
transiently (Figure 1A) and stably (Figure 1B)
transfected confluent ECs are shown in the first 3 panels of the
figure. Maximum-intensity volume projections in the fourth panels
were constructed from stacks of optical sections acquired at 0.1- to
0.5-µm intervals. Typically, a prominent perinuclear ring was
visible, and IFs extended above and below the nucleus. A mesh network
of IFs radiated throughout the cytoplasm from the perinuclear ring, and
thick circumferential IF bundles were sometimes present near the
cell edge or reaching into regions containing lamellipodia. In some
cells, a higher concentration of GFP-vimentin–labeled IFs was
associated with juxtanuclear centers. The distribution of IFs was
similar after transient or stable transfection. The spatial patterns of
GFP-vimentin IFs were consistent with immunocytochemically
defined IF distribution in
endothelium22 23 and other mammalian
cells.19 24 25 Furthermore, indirect
immunofluorescence labeling of vimentin with a
monoclonal antibody colocalized with GFP-vimentin (data not shown),
indicating that GFP-vimentin was incorporated into the preexisting
endogenous vimentin network as previously
demonstrated.19 The 0.1-µm red fluorescent
microspheres visible in the last panel of Figure 1B are
fiducial markers on the glass surface used to normalize for coverslip
displacement during flow.
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IF Motion During a Change in Shear Stress
Time-lapse movies of deconvolved optical sections demonstrated
that IFs do not form a static architecture but are dynamic in living
ECs, even in the absence of externally imposed forces (see
http://www.circresaha.org for online time-lapse movies). During
constitutive motion, the number of filaments remained constant, and
inter-IF connections were unchanged.
ECs were exposed to unidirectional laminar steady flow (wall shear stress, 12 dyn/cm2), and IF positions were compared in fluorescence optical sections from 6 time points at 3-minute intervals before (t1, t2, t3) and after (t4, t5, t6) a step change in flow. For consecutive time points, images from the beginning and end of the intervals were false-colored red and green, respectively. Therefore, in merged color images, yellow represented no change in GFP-vimentin position.
In contrast to the apparently random fluctuations in IF position under
no-flow conditions, significant directional displacement of IFs (by
nearly 1 µm) in regions of the cell occurred within 3 minutes of
the onset of shear stress (Figure 2A),
and increased displacement continued during exposure to flow. This was
not attributable to cell migration; the position of each cell within
the confluent monolayer remained unchanged throughout the observation
period (up to 45 minutes). Flow-induced IF displacements were spatially
heterogeneous within the same cell. Correlation plots of
GFP-vimentin fluorescence intensity at each pixel for
consecutive time points were measured. In such plots, larger scatter
relative to the diagonal corresponded to fewer yellow pixels in the
image (ie, decreased overlap in IF positions) and increased IF
displacement during the interval. The correlation coefficient, 
,
quantitatively describes the degree of overlap between red and green
images. In the field of view showing an entire cell (Figure 2A),
regional displacement after flow onset caused a decreased correlation
during the flow-step interval that was maintained during the subsequent
flow interval, demonstrating that displacement of IF position was
increased coincident with and after the onset of flow.
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At higher magnification, flow-induced alteration of IF motion was
clearly visible (Figure 2B
). Within 3 minutes of flow onset, IFs
were displaced directionally, as indicated by less overlap (yellow
color) in color-merged images. Furthermore, was significantly
decreased during the flow-step interval
(t3–t4) compared with the
preceding no-flow interval. During the succeeding interval in the
continued presence of shear stress, directional motion proceeded at a
slower rate, as reflected by less separation between overlapping
images. The correlation coefficient only partially recovered the value
computed during the no-flow interval, indicating that IFs continued to
move more actively than in the absence of shear stress but less
actively than after the initial onset of flow.
Further illustrations of flow-induced IF displacement are shown in
Figure 3. During no-flow, most filaments
appeared primarily yellow with patches of red and green, indicating
random "wiggling" of individual filament bundles. During the
flow-step interval, significant displacement of filaments was observed
both in the same focal plane (arrowheads) and out of the plane of focus
(red filaments without corresponding green positions at the end of the
interval). Displacement often continued at a slower rate (a smaller
distance of red-green separation) during the succeeding 3-minute flow
interval.
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The correlation of GFP-vimentin distribution was measured for
consecutive time points before (t1,
t2, t3) and after
(t4, t5,
t6) flow onset (Figure 4); was computed for fields of view
containing 1 to 5 cells (•, solid line) or for several subregions
(
, dashed line) in which IF motion was significantly altered by
flow. For consecutive no-flow intervals, remained constant,
indicating a steady-state constitutive motion of IFs. For fields of
view containing whole cells or multiple cells, decreased slightly
on average at the onset of flow; however, larger and more variable
decreases were computed in the subregions, reflecting subcellular
heterogeneity of IF redistribution. Partial recovery of
in these regions during the subsequent flow intervals suggested
that IFs continue to move more actively than in the absence of flow.
These data demonstrate that significant initial displacement of IF
occurs in response to shear stress and that distribution of
flow-induced IF movement is heterogeneous within the
cell.
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Spatial Measurements of IF Displacement
To evaluate quantitatively the spatial redistribution of
IFs, fluorescence intensity line profiles were obtained from
optical sections. During a 3-minute no-flow interval, small IF
displacements were observed in color-merged optical sections acquired
at the beginning (red) and end (green) of the interval (Figure 5A). In intensity profiles measured at
the beginning (red) and end (green) of the no-flow interval (Figure 5B), most peaks did not change intensity or position (arrows),
indicating zero displacement of the corresponding filaments along the
x-axis. Several peaks were displaced along the optical axis,
as indicated by a change in intensity at constant x-axis
position (open arrow). In contrast, red-green filament pairs and fewer
yellow pixels showed that some IFs were significantly displaced during
the succeeding flow-step interval (Figure 5C). Comparison of
intensity profiles before and after flow onset (Figure 5D)
confirmed that some IFs were significantly displaced along the flow
axis (arrowhead) whereas others moved into or out of the focal plane
(open arrows), as indicated by a change in peak intensity without
displacement along the x-axis. A few peaks did not change
intensity or average x-position (arrows), corresponding to
zero displacement. Thus, filaments only a few micrometers
apart exhibited different displacement responses to the onset of shear
stress.
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IF network motion was analyzed in 3 dimensions (Figure 6). Nodes connecting a minimum of 3
filaments were chosen from 34 randomly selected positions in 5 cells
selected from 3 confluent monolayers. The positions of the nodes
projected on the x-, y-, and
z-axes were tracked relative to reference positions
(x0, y0,
z0) just before flow onset (time=0
minutes). Step movements in the z-direction were larger than
those in the xy-plane due to lower resolution along the
optical axis. Before the start of flow, movement consisted of
fluctuations around a constant average position along the
x-, y-, and z-axes (time
0 minutes).
After the onset of flow along the x-axis (time>0 minutes),
a number of nodes underwent significant directional movement. These
nodes moved rapidly during the first 3 minutes of flow and then
continued to fluctuate around a new average position. Nodes throughout
the cytoplasm moved in various magnitudes and directions, but mean
relative positions of nodes located higher in the cell were more likely
to be affected by flow than nodes near the basal surface. Furthermore,
the change in IF distribution measured after flow onset was primarily
due to initial displacement rather than altered spatial fluctuations of
IF position. Thus, the variability in IF positions increased
significantly, suggesting rapid force redistribution through the cell
within 3 minutes of the onset of shear stress.
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Discussion |
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These experiments represent the first spatiotemporal measurements of cytoskeletal motion in response to a controlled mechanical environment with physiological relevance (flow). The decentralization model of mechanotransduction2 proposes that the dynamic response of the cytoskeleton to an instantaneous change in the distribution of extracellular applied forces plays a key role in the integration of fast signaling responses. The early changes in IF displacement observed in response to shear stress suggest that 3D force redistribution throughout the cell acts rapidly at sites micrometers away from the luminal surface.
ECs contain an extensive interconnecting cytoplasmic network of vimentin IFs. Several physiological roles for IFs have been proposed,26 including determination and maintenance of cell shape, transmission of mechanical stress, targeting of molecules between the nucleus and cytoplasm, and regulation of nuclear position and morphology. The network has traditionally been regarded as a static architecture because of the low solubility and detergent extractability of IFs. However, recent studies have revealed a dynamic IF structure during cell spreading24 and division26 that depends to some extent on the state of vimentin phosphorylation.27 These properties of overall network stability combined with spatial dynamics make IFs an interesting candidate for cytoskeletal force responses.
The flexing and bending behavior of IFs without flow was similar to IF motion reported by other investigators.19 24 25 Thin IFs reaching into the cytoplasm flexed more than thicker filament bundles and organizing centers, network nodes moved in apparently random directions, and the interconnecting filament segments flexed or changed length accordingly. GFP-vimentin IFs maintained their connections so that assembly or disassembly of network segments was not observed as individual mesh units changed shape during these relatively short time intervals.
A complicated 3D distribution of IF movement was frequently observed. For example, IF unidirectional movement in one focal plane was accompanied by variable direction of motion in a plane lower in the cell. Fung and Liu28 have proposed that shear stresses are transmitted over the cell surface to junctions and basal adhesion sites, largely bypassing the cytoskeleton. Although this mechanism may contribute to force redistribution, our studies support a broader interpretation of force transmission through transcellular cytoskeletal displacement.
The distribution of initial network strain in response to applied shear stress may be related to IF physical properties, which are distinctly different from those of microfilament and microtubule networks.29 Polymerized vimentin in vitro has higher elasticity and a lower shear modulus than actin. However, vimentin hardens at high strain without breaking, unlike microtubules. In the present studies, disassembly and reassembly of IF network connections were not observed during the short periods analyzed. This IF network stability may be important during reorganization of microfilament and/or microtubule networks by a mechanism that requires breaking of those network connections. The combined strain-hardening and elastic properties of the vimentin IF network may therefore confer mechanical stability on the cell. Vimentin knockout mice provide further evidence for the role of IFs in mechanical stability. Although these mice grow to maturity and lack an obvious phenotype,30 a more careful evaluation has revealed regional defects at the cellular level that suggest disruption of cellular functions that depend on an intact mechanical organization.31 32 Cell-wide force distribution and mechanical properties depend on a composite material description of IFs, microfilaments, and microtubules. Thus, interactions between IFs and other cytoskeletal elements will play a role in determining the dynamic response to shear stress.33 34 Potential crossbridge elements include nestin35 and a plectin-like 300-kDa protein.23 36 37 Rapid changes in IF distribution near the cell base may also participate in a mechanism whereby the dynamics of focal adhesion sites are altered by shear stress.38 Furthermore, because the perinuclear ring of vimentin IFs may be directly or indirectly linked to the nuclear lamina,26 force redistribution under flow may also affect the karyoskeleton, consistent with other mechanical perturbations.39 Through interactions between nuclear IF proteins, the nuclear lamins, DNA, and histones,40 changes in gene expression may be directly mediated by flow.
In summary, both qualitative and quantitative spatial analyses in living ECs revealed rapid regional IF displacement in response to shear stress. Although such measurements do not exclude a role for local deformation in mechanical signaling, they suggest an integrated mechanism of mechanotransduction in which spatial organization of multiple structural and signaling networks regulates cellular responses to an altered hemodynamic environment.
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Acknowledgments |
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This work was supported by National Institutes of Health (NIH) National Research Service Award grant HL10058 (to B.P.H.), NIH Method to Extend Research in Time (MERIT) grant GM36806 (to R.D.G.), and NIH MERIT grant HL36049 (to P.F.D.). We thank F. Flitney, S. Heidemann, P. Janmey, J. Murray, and S. Zigmond for critical reading of the manuscript.
Received December 27, 1999; accepted January 31, 2000.
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 B. P. Helmke Molecular Control of Cytoskeletal Mechanics by Hemodynamic Forces Physiology, February 1, 2005; 20(1): 43 - 53. [Abstract] [Full Text] [PDF]
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 M. M. Thi, J. M. Tarbell, S. Weinbaum, and D. C. Spray The role of the glycocalyx in reorganization of the actin cytoskeleton under fluid shear stress: A "bumper-car" model PNAS, November 23, 2004; 101(47): 16483 - 16488. [Abstract] [Full Text] [PDF]
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S. Hu, J. Chen, B. Fabry, Y. Numaguchi, A. Gouldstone, D. E. Ingber, J. J. Fredberg, J. P. Butler, and N. Wang Intracellular stress tomography reveals stress focusing and structural anisotropy in cytoskeleton of living cells Am J Physiol Cell Physiol, November 1, 2003; 285(5): C1082 - C1090. [Abstract] [Full Text] [PDF]
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 S. Mochizuki, H. Vink, O. Hiramatsu, T. Kajita, F. Shigeto, J. A. E. Spaan, and F. Kajiya Role of hyaluronic acid glycosaminoglycans in shear-induced endothelium-derived nitric oxide release Am J Physiol Heart Circ Physiol, July 11, 2003; 285(2): H722 - H726. [Abstract] [Full Text] [PDF]
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 P. Fiorina, F. Folli, F. Bertuzzi, P. Maffi, G. Finzi, M. Venturini, C. Socci, A. Davalli, E. Orsenigo, L. Monti, et al. Long-Term Beneficial Effect of Islet Transplantation on Diabetic Macro-/Microangiopathy in Type 1 Diabetic Kidney-Transplanted Patients Diabetes Care, April 1, 2003; 26(4): 1129 - 1136. [Abstract] [Full Text] [PDF]
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B. P. Helmke, A. B. Rosen, and P. F. Davies Mapping Mechanical Strain of an Endogenous Cytoskeletal Network in Living Endothelial Cells Biophys. J., April 1, 2003; 84(4): 2691 - 2699. [Abstract] [Full Text] [PDF]
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P. F. Davies, J. Zilberberg, and B. P. Helmke Spatial Microstimuli in Endothelial Mechanosignaling Circ. Res., March 7, 2003; 92(4): 359 - 370. [Abstract] [Full Text] [PDF]
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 K. Matrougui, L. B. Tanko, L. Loufrani, D. Gorny, B. I. Levy, A. Tedgui, and D. Henrion Involvement of Rho-Kinase and the Actin Filament Network in Angiotensin II-Induced Contraction and Extracellular Signal-Regulated Kinase Activity in Intact Rat Mesenteric Resistance Arteries Arterioscler. Thromb. Vasc. Biol., August 1, 2001; 21(8): 1288 - 1293. [Abstract] [Full Text] [PDF]
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Z. Ungvari, D. Sun, A. Huang, G. Kaley, and A. Koller Role of endothelial [Ca2+]i in activation of eNOS in pressurized arterioles by agonists and wall shear stress Am J Physiol Heart Circ Physiol, August 1, 2001; 281(2): H606 - H612. [Abstract] [Full Text] [PDF]
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 D. Riveline, E. Zamir, N. Q. Balaban, U. S. Schwarz, T. Ishizaki, S. Narumiya, Z. Kam, B. Geiger, and A. D. Bershadsky Focal Contacts as Mechanosensors: Externally Applied Local Mechanical Force Induces Growth of Focal Contacts by an mDia1-dependent and ROCK-independent Mechanism J. Cell Biol., June 4, 2001; 153(6): 1175 - 1186. [Abstract] [Full Text] [PDF]
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K. H. Yoon, M. Yoon, R. D. Moir, S. Khuon, F. W. Flitney, and R. D. Goldman Insights into the Dynamic Properties of Keratin Intermediate Filaments in Living Epithelial Cells J. Cell Biol., April 24, 2001; 153(3): 503 - 516. [Abstract] [Full Text] [PDF]
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P. J. Butler, G. Norwich, S. Weinbaum, and S. Chien Shear stress induces a time- and position-dependent increase in endothelial cell membrane fluidity Am J Physiol Cell Physiol, April 1, 2001; 280(4): C962 - C969. [Abstract] [Full Text] [PDF]
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 J. C. L. Zhang, S. Kim, B. P. Helmke, W. W. Yu, K. L. Du, M. M. Lu, M. Strobeck, Q.-C. Yu, and M. S. Parmacek Analysis of SM22{alpha}-Deficient Mice Reveals Unanticipated Insights into Smooth Muscle Cell Differentiation and Function Mol. Cell. Biol., February 15, 2001; 21(4): 1336 - 1344. [Abstract] [Full Text]
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