Novel Optical Methodologies in Studying Mechanical Signal

Herein we briefly review the current knowledge on mechanical signal transduction in mammalian cells, focusing on the application of novel optical tech...
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Ind. Eng. Chem. Res. 1999, 38, 601-609

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Novel Optical Methodologies in Studying Mechanical Signal Transduction in Mammalian Cells Georgios N. Stamatas and Larry V. McIntire* Cox Laboratory for Biomedical Engineering, Institute of Biosciences and Bioengineering, MS-142, Rice University, Houston, Texas 77251-1892

For the last 3 decades evidence has been accumulating that some types of mammalian cells respond to their mechanically active environment by altering their morphology, growth rate, and metabolism. The study of such responses is very important in understanding physiological and pathological conditions ranging from bone formation to atherosclerosis. Obtaining this knowledge has been the goal for an active research area in bioengineering termed cell mechanotransduction. The advancement of optical methodologies used in cell biology research has given the tools to elucidate cellular mechanisms that would otherwise be impossible to visualize. Combined with molecular biology techniques, they give engineers invaluable tools in understanding the chemical pathways involved in mechanotransduction. Herein we briefly review the current knowledge on mechanical signal transduction in mammalian cells, focusing on the application of novel optical techniques in the ongoing research. Introduction Mammalian cells alter their morphology, growth rate, and metabolism not only in response to chemical signals (such as hormones, growth factors, toxins, etc.) but also in response to mechanical stimuli. Blood flow, interstitial fluid flow, gravity, and interconnections with the surrounding mechanically active environment (extracellular matrix or other cells) are some examples of sources that may exert shearing and/or stretching inducing forces on the membranes of mammalian cells. These forces can modulate alterations in the cellular physiology that are highly tissue specific. For example, when endothelial cells are exposed to flow-induced shear stress, they align in the direction of flow, whereas vascular smooth muscle cells do not. Most of the research so far has been focused on mechanotransduction in vascular cells, and the results will be discussed in the following paragraphs. However, at this point it is noteworthy to mention that other types of mammalian cells have been shown to respond to mechanical stresses as well. Osteoblasts, the bone-forming cells, increase their levels of 3′,5′-cyclic monophosphate (cAMP),1 regulate their intracellular concentration of 1,4,5-triphosphate (IP3), and stimulate their rostaglandin E2 synthesis after exposure to shear stress.2 Baby hamster kidney fibroblasts increase their intracellular cAMP concentration after 15 min of exposure to fluid shear stress. When human embryonic epithelial kidney cells are exposed to fluid flow, they align in the direction of flow and they increase urokinase release.3 Cells from different tissues in our bodies are subjected to different mechanical stimuli depending on their environment. Before our discussion on mechanotransduction in vascular cells, the nature of the forces experienced by these cells will be presented. Vascular Cell Mechanotransduction Forces Experienced by Vascular Cells. There are three distinct histological layers that makeup the * To whom correspondence should be addressed. E-mail: [email protected].

vascular walls.4,5 The innermost layer, the tunica intima, is a monolayer lining of endothelial cells (EC), with their luminal side being exposed to blood flow and their abluminal side adherent on the elastic lamina. The intermediate layer, the tunica media, is composed of several layers of smooth muscle cells (SMC) embedded in extracellular matrix components. Finally, the outermost layer, the tunica adventitia, contains SMC, fibroblasts, and adipocytes, as well as areas of relatively sparsely populated connective tissue. These layers are more distinct in arteries and less in veins. A factor that plays an important role in vascular physiology and pathology is the mechanically active environment, which among eliciting other responses modulates the intracellular chemical crosstalk between endothelial and SMC (for example, through the secretion of vasoactive compounds and growth factors). An example of a physiologic response is that large blood vessels can undergo dramatic adaptations (dilation or constriction) in response to both chronic and acute alterations in blood flow, a response that is SMCmediated as well as EC-dependent.6 An example from vascular pathology is that atherosclerotic lesions have been observed to be localized in regions that share common flow profiles.6 As structural elements of the vascular wall in a mechanically active environment, the vascular cells experience normal and tangential forces on their membranes, all of which may be represented as elements of a stress tensor. Here we use a cylindrical coordinate system where z is the longitudinal, r is the radial, and θ is the circumferential direction (Figure 1). The first subscript of each stress component denotes the surface where the stress is applied, and the second subscript denotes the direction of the corresponding force. Thus, τrr, τθθ, and τzz are normal stresses corresponding to the radial, circumferential, and longitudinal directions. More specifically, τrr derives from blood pressure, whereas τθθ and τzz result from circumferential and longitudinal vessel wall stretching. All of the other components of the stress tensor are tangential stresses. For EC, τrz is the fric-

10.1021/ie980426a CCC: $18.00 © 1999 American Chemical Society Published on Web 02/09/1999

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Figure 1. Normal and tangential forces experienced by vascular cells.

tional shear stress due to blood flow, and the rest are shear stresses due to contact with neighboring cells or the basal lamina. It has been shown recently that for SMC significant tangential stresses may also arise from interstitial fluid flow driven by differences in transmural pressure.7,8 Both stretch-induced normal stresses and flowinduced tangential stresses have been shown to affect vascular cell responses in different ways.9 Here we focus on the effects of shear forces. Vascular Cell Responses to Fluid Shear Stress. During the last 25 years our knowledge of the endothelium has evolved from considering it a passive antithrombotic permeability barrier to the circulating blood to understanding its role as a dynamic and complex modulator of diverse physiological and pathophysiological functions ranging from vascular tone, hemostasis, inflammation, fibrinolysis, and angiogenesis to atherosclerosis, thrombosis, tumor metastasis, and intimal thickening. Many if not all of the EC functions have been shown to be strongly modulated by hemodynamic factors. Extensive research has been performed to study the effects of the flow-induced shear forces on endothelial metabolism, growth, gene regulation, and morphology. In Table 1 a partial list of the endothelial responses to fluid shear stress is presented. For a more complete coverage the reader is urged to refer to a number of review papers that summarize the current knowledge in this field.6,10-18 Despite the large amount of information we have gained in this area, there are still many unanswered questions regarding the mechanochemical signal transduction involved in such cellular processes. Although responses of the endothelium to flow have been extensively investigated, studies of the effects of flow on vascular SMC have been very sparse, mostly due to the fact that these cells are not normally exposed directly to blood flow. However, recent modeling studies have suggested that vascular SMC can be exposed to levels of shear stress in the range 1-10 dyn/cm2 on their cell membranes because of interstitial fluid flow.7,8 This flow is driven by the transmural pressure gradient across the arterial wall. Vascular SMC may also be subjected to higher shear stress levels when exposed to blood flow following endothelium denudation. Endothelium injury and denudation are common after bal-

loon angioplasty treatment of occluded arteries and may be an important component of restenosis.19-21 The importance of studying the vascular SMC derives from the crucial role of these cells in physiologic responses such as vascular tone regulation, as well as their implication in pathological conditions such as hypertension and atherosclerosis and in the frequent failure of restorative surgical procedures such as angioplasty and bypass grafting due to restenosis.21,22 Exposure of arterial SMC to fluid shear stress has been shown to affect their growth rate and metabolism both in vivo and in vitro.23-29 The vascular SMC responses to fluid shear stress are summarized in Table 2. Shear stress has been found to decrease the proliferation rate of both bovine27,28 and human23 aortic smooth muscle cells (HASMC). In the case of HASMC, the reduction of cell number was not due to cell injury and the cultures were not growth-arrested 24 h after exposure to shear stress.23 The flow effect on the cell growth rate was dependent on the magnitude of the shear stress value. Higher levels of shear stress resulted in a greater decrease of the cell growth rate. Furthermore, Papadaki et al.24 have measured an increase in the production of nitric oxide, an important vasoactive compound and regulator of SMC proliferation, when HASMC were exposed to shear stress. Moreover, in contrast to what happens to EC, shear stress does affect the morphology of SMC. Finally, it has been shown that shear stress differentially modulates the expression of thrombin receptor and tissue plasminogen activator (tPA) in HASMC.30 High shear stress upregulates tPA and downregulates the thrombin receptor expression, whereas low shear stress causes the reverse effects. These findings may correlate with the observation that thrombosis usually localizes at areas exposed to low shear. Fluid Shear Stress-Induced Mechanotransduction. There are two general ways that a mechanical signal from the surrounding environment may be transmitted from the cell membrane to the nucleus to account for the observed shear-induced gene regulation (Figure 2). One way is through diffusible second messengers, initially released locally close to the membrane, that are able to activate a chemical reaction cascade, which eventually leads to transcription factor activation and translocation to the nucleus. The second general pathway involves direct mechanical force transmission through cytoskeletal structures from the cell membrane to other parts of the cell, mainly the nucleus, intercellular junctions, and focal adhesion sites. What is generally believed is that both the cytoskeleton and the second messenger systems play important roles (for extensive reviews refer to refs 11-13, 15, 17, and 18). Table 3 summarizes the signaling molecules shown to be involved in vascular cell mechanotransduction. Again almost all of the data with only few exceptions have been derived from experiments on EC. It is obvious that the mosaic of mechanotransduction pathways is complex and there are still missing pieces to the puzzle. In the past, basic techniques such as tissue culture and video microscopy have been invaluable tools to study cell responses under controlled environments.31 Such traditional techniques have been coupled to carefully designed devices such as stretchers, flow chambers, and viscometers that attempt to recreate in vitro the mechanically active environment that cells experience in vivo.32 Recently, novel optical methodolo-

Ind. Eng. Chem. Res., Vol. 38, No. 3, 1999 603 Table 1. Flow-Induced Endothelial Cell Responsesa effect

significance

ref

hyperpolarization NO production increase ATP release prostacyclin release increase in c-myc, c-fos, and c-jun mRNA VCAM-1 downregulation, ICAM-1 upregulation PDGF mRNA increase increase in pinocytosis TGF-β1 upregulation redistribution of microtubule organizing center and Golgi apparatus endothelin-1 downregulation tPA upregulation proliferation inhibition cytoskeleton remodeling and cell alignment to flow

vasorelaxation vasorelaxation autocrine regulation antithrombotic, regulator of vascular tone immediate early growth response genes leukocyte adhesion SMC growth factor membrane vesicle formation inhibits SMC growth organelle translocation

48 77-80 81 82-84 85-87 88 6 and 89 90 91 55

vasoconstriction fibrinolysis regulation of reendothelialization reduction of shear stress gradients on cell surface

92 93 94 and 95 58, 94, and 96-103

a Abbreviations: ATP, inositol 1,4,5-triphosphate; VCAM-1, vascular cell adhesion molecule-1; ICAM-1, intercellular adhesion molecule1; PDGF, platelet-derived growth factor; TGF-β1, transforming growth factor beta-1; tPA, tissue plasminogen activator.

Table 2. Flow-Induced Vascular Smooth Muscle Cell Responsesa effect proliferation inhibition bFGF release bFGF receptor mRNA upregulation PDGF release prostaglandin and prostacyclin release NO production increase tPA expression modulation thrombin receptor expression modulation

significance

ref

regulation of intimal thickening growth factor growth regulation

23 and 28

growth factor antithrombotics, vascular tone regulation vasorelaxation thrombosis thrombosis

29 30 29 104 24 30 30

a Abbreviations: BFGF, basic fibroblast growth factor; PDGF, platelet-derived growth factor; tPA, tissue plasminogen activator.

Figure 2. Possible mechanotransduction pathways in vascular cells. Putative mechanoreceptors (“stress sensors”) on the luminal cell membrane may alter their conformation in response to mechanical stimuli, activating a signal transduction cascade involving diffusible second messengers. This chemical reaction cascade leads to activation of gene transcription in the nucleus. Alternatively, mechanotransduction may be decentralized by means of the cytoskeleton. Mechanical forces may be transmitted through the cytoskeletal elements from the luminal cell membrane to focal adhesion sites, intercellular junctions, or directly to the nucleus. Translation of the mechanical force to a chemical signal that would eventually lead to gean regulation may occur at these sites.

gies have evolved that appear to be exceptionally promising for revealing further local details of intracellular mechanochemical signal transduction.

New Optical Methodologies Fluorescence Microscopy, Fluorescent Probes, and Ratio Imaging. Fluorescence microscopy allows for selective examination of a particular component of complex biomolecular assemblies, including experiments utilizing live cells.33 The commercial availability of fluorescent stains sensitive to the local concentrations of intracellular second messengers such as calcium, pH, and membrane potential made possible the study of their involvement in cell mechanotransduction. An interesting aspect of some fluorescent probes is that they are available in a membrane-permeable form. This provides for dye loading without introducing membrane perturbations, which are inevitable when using microinjection. The membrane-permeable dyes are usually introduced to cultured cells as a nonfluorescent ester form. The ester may readily cross the cell membrane and then is hydrolyzed within the cell by indogenous esterases to produce the free (charged) form of the dye. The dye molecule changes its fluorescent properties upon binding to the substance being studied. Using fluorescent dyes and video microscopy, investigators have studied the effect of fluid shear stress on intracellular calcium,34-44 pH,45-47 and membrane potential48 in vascular cells. Fluorescent indicators that exhibit an excitation or emission spectral shift upon binding with the substance to be labeled can be used for quantitative measurements of the substance concentration by ratio fluorescence microscopy.49 It has been shown that the ratio of the fluorescence intensities measured at two different wavelengths is nearly independent of various artifacts in the fluorescence signal.50-52 Briefly, the intensity emitted by a fluorescent probe, I, may be approximated by

I ) ΦFI0bc

(1)

where ΦF is the quantum yield of the fluorophore, I0 is the excitation intensity,  is the extinction coefficient of the probe, b is the optical path length, and c is the concentration of the probe. In other words, the emitted intensity is affected by artifacts such as fluctuations in excitation intensity (I0), differences in cell thickness (b), dye leakage, photobleaching, and uneven dye loading (all incorporated in c). If the probe’s excitation spectrum shifts upon binding, all of these artifacts may be normalized by ratioing the emission intensities obtained at two different excitation wavelengths.

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Table 3. Signal Transduction Pathways Involved in Flow-Induced Vascular Cell Mechanotransductiona effect K+

channel activation G-protein activation pH: acidification in EC and alkalinization in SMC IP3 transient elevation Ca2+ transients (?) cGMP increases stimulation of MAPK activation of NFκB induction of c-fos and c-jun remodeling of focal adhesion sites protein kinase C DAG

pathway

ref

mechanosensitive channel G-protein coupled receptors pH regulation of metabolism and growth

105 and 106 79 45-47

phosphoinositide metabolism calcium/calmodulin system activation cGMP as second messenger protein kinases, ras-ERK1/2, and ras/JNK pathways transcription factors transcription factors cytoskeleton force transmission and focal adhesion site involvement in mechanotransduction protein kinase C pathway phosphoinositide metabolism

36, 82, and 84 34-44 107 108-110 87 and 111 87 56, 57, and 60 89 and 92 84

a Abbreviations: IP , inositol 1,4,5-triphosphate; cGMP, cyclic guanosine monophosphate; MAPK, mitogen-activated protein kinase; 3 NFκB, nuclear factor kappa B; DAG, diacyl glycerol.

The ratio of intensities from two excitation wavelengths with opposite ion-sensitive responses (such as the ratio of 340/380 nm excitation for calcium measurements with the dye Fura-2) results in the largest possible dynamic range of ratio signals.49 Alternatively, the ratio of an ion-sensitive intensity to an ion-insensitive intensity (spectral isosbestic point) can be used (like the ratio of 495/440 nm excitation for pH measurements with BCECF). Indirect Immunofluorescence. The availability of a wide variety of monoclonal antibodies against intracellular proteins has provided the tools for protein localization by using indirect immunofluorescence coupled to confocal microscopy. This is an “end point” technique in the sense that the specimen needs to be fixed and permeabilized before staining. This means that protein localization may not be monitored dynamically. Furthermore, there are concerns of whether the fixation procedures affect the results acquired. However, indirect immunofluorescence has been used extensively in cell mechanotransduction studies to demonstrate flowinduced responses such as stress fiber formation,53,54 preferential positioning of the Golgi apparatus and the microtubule organizing center,55 remodeling of focal adhesion sites, and translocation of related proteins to these sites.56,57 Atomic Force Microscopy. To localize putative mechanotransducive sites in endothelial cells, it is important to understand the three-dimensional (3D) force distribution in and around the cells. The information on the 3D structure of the cell is critical for understanding both the characteristics of the hemodynamic forces acting on the cells and the putative force transmission from the cell surface to other mechanotransducive sites. The first attempt to map the detailed 3D geometry of endothelial monolayers was performed using atomic force microscopy (AFM).58 AFM has been used widely over the past decade, principally in material characterization and solid-state chemistry, for submicroscopic imaging of 3D surfaces.59 Although not completely an “optical” method, it has been classified as mechano-optical.59 The basic principle is that a sharp microtip scans the surface of the specimen. Forces between the tip and the scanned surface deflect the tip and cantilever causing an incident laser beam to reflect proportionately. Specially positioned photodetectors sense the changes of the reflected beam, and their output is used to keep the tip force constant.60 Barbee et al.58 used AFM to scan cultured endothelial monolayers under stationary conditions and after 24 h

of flow application. These authors reported that undulations between the apex of the cell and the intercellular junctions were reduced by about 1.6 µm. It has been proposed that this height reduction may be an adaptation to the flow conditions in order to reduce the shear stress gradients experienced on the luminal cell membrane. AFM has the advantage of spatial resolution unparalleled by optical microscopes. In the paper mentioned above,58 the authors report the appearance of ≈100 nm thick stress fibers on the cell surface after flow application that would not be able to be observed with any optical method. The major disadvantage of this technique is that a measurement may not be performed on a specimen under flow conditions. Therefore, it may not be used for the study of dynamic responses. Only conditions before and after mechanical stimulation may be compared. Confocal Microscopy. There are two general ways to perform three-dimensional fluorescence microscopy: confocal scanning microscopy and optical sectioning microscopy. In confocal scanning microscopy a volume of the specimen is scanned by an excitation light beam (usually provided by a laser) and the emitted light is collected through a small pinhole. The light intensities are registered in the 3D volume, and a digitized 3D image may be reconstructed. This technique has been used widely in cell biology for high-resolution 3D imaging of intracellular components principally for imaging cytoskeleton and DNA of fixed specimens. For mechanotransduction studies, confocal microscopy has been used in a tandem scanning mode to image the 3D topography of the abluminal surface of endothelial cells and its dynamic alterations under shear stress.60 This technique revealed adhesion site alignment toward the direction of flow within several minutes after flow initiation. Laser confocal fluorescence microscopy provides images with very high spatial resolution. However, the disadvantage is that it is used primarily with fixed specimens. This means that measurements may only be performed before and after mechanical stimulation and for irreversible or very slowly reversible responses. Furthermore, the experience of reliable ultraviolet laser sources and the technical difficulties arising from the need for dual wavelength excitation for ratio imaging render this technique quite difficult for quantitative intracellular ion measurements. 3D Fluorescence Microscopy: Optical Sectioning Coupled to Deconvolution. An alternative tech-

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Here * symbolizes convolution, and o(x,y,z) is the observed (acquired) 3D image. After Fourier transformation, eq 2 can be simplified to 3D (Toeplitz) matrix multiplication:

O)I×S

(3)

where O, I, and S are the Fourier transforms of o(x,y,z), i(x,y,z), and s(x,y,z), respectively, and × symbolizes matrix multiplication. The Fourier transform of the point spread function is called the optical transfer function (OTF). Assuming that S is nonsingular, eq 3 may be rewritten as

I ) O × S-1

Figure 3. Principles of optical sectioning. The coverslip with the fluorescently labeled cells is mounted on the flow chamber and placed on the stage of the microscope. By moving the microscope’s objective in the z axis by a computer-controlled stepper motor, we can acquire images at different focal planes. By digitally stacking the planes on top of each other, we can obtain a 3D image of the specimen.

nique to confocal microscopy that may be used to acquire three-dimensional information from fluorescently labeled specimens is the use of a optical sectioning coupled to numerical deconvolution. This technique allows for live cell imaging as well as ratio imaging. Optical sectioning of the specimen may be performed by acquiring images at different focal planes (Figure 3). This may be achieved by automatically moving the objective in the z direction usually with the use of a high-speed, computer-controlled, z axis stepper motor mounted on the microscope’s fine focus control knob. The images of the series of optical sections may be stacked on top of each other to create a 3D representation of the specimen. Commercially available software may be used for scientific visualization of the 3D image. Ideally the series of optical sections would look like a series of physical sections. However, because of its wavelike nature, light emitted from the specimen undergoes constructive and destructive interference as it travels through the optical path of the microscope and especially through the objective. The result is that the light collected from each optical section contains information originating not only from the section in focus but also from its neighboring planes. This extra information is the cause of blurring of the acquired images and needs to be removed from the plane in focus and to be accounted for at the planes it originated from. The light diffraction pattern of a point light source is called the point spread function (PSF) and is the impulse response of the optical system (microscope). The PSF either may be calculated by using mathematical models61,62 or may be determined experimentally by optically sectioning fluorescent beads with diameter lower than the resolution limit of the microscope.41,63 Under the assumption of a linear and shift invariable system the blurring phenomenon may be mathematically expressed by a convolution of the original true image, i(x,y,z), with the point spread function, s(x,y,z):64,65

o(x,y,z) ) i(x,y,z)*s(x,y,z)

(2)

(4)

However, because the large number of zeros within S, the matrix is usually nearly singular. Also the noise that is present in microscopy images introduces small lowfrequency values in S, which, when inverted, amplify noise artifacts. Thus, the problem of calculating the true image i(x,y,z) through I, also referred to as “deconvolution”, is ill-posed and requires the implementation of special numerical algorithms. The simplest solution that has been proposed for this problem is the application of a Wiener filter:65

I ) O × S × (S2 + λ)-1

(5)

where λ is a parameter that may be constant or a function of the signal-to-noise ratio of s(x,y,z). This algorithm often results in a poor quality image that is distorted with “ripples” building up around strong features and causing weak features to be obscured. Other algorithms have been proposed, with the most successful being the nearest neighbor,64-68 which takes into account only information from adjacent sections, and the constrained iterative deconvolution with nonnegativity constraints.64-66,69-71 The best results for low-contrast images, such as those acquired in intracellular ionic measurements, have been obtained by using an iterative regularization scheme with nonnegativity constraints and a solution based on projections onto closed convex sets. This algorithm was first proposed by Butler et al.72 and then refined by Carrington et al.73-76 Briefly, the basic idea is that i(x,y,z) can be calculated by minimizing the following expression:

min {|o(x,y,z) - s(x,y,z)*i(x,y,z)|2 + R|i(x,y,z)|22} (6)

i(x,y,z)g0

Here, R is a regularization parameter which depends on the amount of noise in the image and assumes a value of zero for noiseless images. The first term of the above expression is the mean-square error of the solution i(x,y,z) as compared to the observed image o(x,y,z). The second term is the “energy” of the solution and accounts for the noise inherent in the observed image. This algorithm has the advantages of a strong mathematical basis, guaranteed convergence, and the ability to be used with both high- and low-contrast images.41 To extract accurate 3D information, the number of the optical sections to be acquired has to be considerably larger than the size of the specimen in the z direction. This number has to be at least equal to the height of the specimen plus twice the size of the point spread function to account for light scattered above and below

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the specimen. The distance between two adjacent optical sections, controlled by the size of each step of the stepper motor, has to be larger than the depth of field (DOF) of the microscope given by

DOF )

λ 4n sin2(θ/2)

(7)

where λ is the wavelength of the emitted fluorescence, n is the refractive index of the objective’s immersion medium, and θ is the half angle (rad) of the cone of light that is captured by the objective. After the number of the required optical sections and the step size in the z direction are determined, the position of the most in-focus plane is located and arbitrarily assigned number zero. The optical sections above the most in-focus plane may be assigned negative numbers and those below it positive numbers. A major advantage of the optical sectioning method is that live cells may be imaged during mechanical (or chemical) stimulation and dynamic responses may be recorded. One of the concerns, however, is the temporal resolution. Depending on the number of optical sections and the required camera exposure time for each optical section, acquisition of a complete 3D image may be timeconsuming (on the order of a few minutes). Thus, there appears to be a tradeoff between temporal resolution and 3D spatial information. We recently applied this 3D fluorescence microscopy method to study rapid flow-induced responses in endothelial cells.63 More specifically, we applied the technique to study putative calcium signals localized within the 3D cell architecture.34-44 The effect of deconvolution in calcium imaging is shown in Figure 4. Parts A and B of Figure 4 show the same optical section of the same endothelial cell before and 3 min after application of shear stress (13 dyn/cm2). No significant differences in the intracellular calcium concentration may be observed. It appears that calcium is distributed uniformly within the cell. Parts C and D of Figure 4 show the same optical sections after Wiener filtering. The noise of the images has been reduced significantly, but the intensities are still very homogeneous and possible heterogeneities have been smoothed out. Parts E and F of Figure 4 show the same optical sections after deconvolution with the algorithm described above (eq 6). This time high intensities are apparent at the central area of the cell, which corresponds to the nucleus. Moreover, it appears that flow increased further the intensities at the nuclear region. We concluded that fluid shear stress increases nuclear calcium, a phenomenon that is not evident with conventional 2D microscopy (i.e., without 3D image processing, as in Figure 4A,B) because of contamination from light coming from neighboring planes. We further applied the optical sectioning technique coupled to deconvolution for studying flow-induced rapid alterations of the 3D endothelial cell and nuclear morphology.63 Figure 5 depicts processed xy and xz cross sections of a single endothelial cell stained with a dye specific for the nucleus before and 3 min after flow application. The xz cross sections show that the cell nucleus decreases its height in response to flow, while the xy cross section is correspondingly larger after flow exposure. Similar results have been observed for the whole cell height.63 Furthermore, we showed that the nuclear response is dependent on the microtubule integrity but not on the microfilament or intermediate

Figure 4. Ratio fluorescence images of a single endothelial cell stained for intracellular calcium by the dye Fura-2. Images acquired at the excitation wavelengths 340 and 380 nm have been ratioed, and the ratio values correspond to calcium concentration values (pseudocolored according to the colorbar at the bottom). All images correspond to the same optical section of a series of sections constituting the 3D image of the cell. (A) Raw image acquired under stationary conditions. (B) Raw image acquired under flow conditions. (C and D) The same as parts A and B but after Wiener filtering of the 3D volume. (E and F) The same as parts A and B but after deconvolution with the algorithm described in the text.

filament integrity. This indicates that microtubules may be important for direct force transmission from the cell membrane to the nucleus.63 Concluding Remarks As noted before, much of the interest in fluid mechanotransduction in vascular cells stems from the possible role of local hemodynamics as a principal factor for explanation of the observed atherosclerosis localization in humans. Much has been learned during the last 3 decades on the subject. However, a complete understanding of all of the details of mechanotransduction is still a distant goal. In comparison to many biochemical methods, optical methods have the advantage of allowing for better spatial resolution of the intracellular details at the micron or even smaller levels. In addition, the saying that an image says more than a thousand words applies perfectly here.

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Figure 5. Cross sections of an endothelial cell stained for its nucleus by the dye Hoechst 33342. (A) xy cross section of the cell under stationary conditions. (B) xy cross section of the same cell under flow conditions. (C) xz cross section under stationary conditions. (D) xz cross section under flow conditions. All images have been processed by 3D deconvolution. In addition, all images are obtained without fixation, both under stationary and flow conditions.

In this brief review, we presented a few of the new optical methodologies that have been shown to be very promising as useful tools in vascular biology research. Before we apply such methodologies, it is important to understand the advantages and limitations of each of them. Future research should be directed toward applying the current techniques to other aspects of mechanotransduction (e.g., 3D cytoskeleton dynamics under flow), advancing the existing technologies (e.g., optical sectioning coupled to deconvolution may be used for superresolution), and devising novel methodologies to overcome their limitations (e.g., the very recently developed multiphoton microscopy greatly reduces photodamage caused by laser confocal microscopy). Literature Cited (1) Reich, K. M.; Gay, C. V.; Frangos, J. A. Fluid Shear Stress as a Mediator of Osteoblast Cyclic Adenosine Monophosphate Production. J. Cell. Physiol. 1990, 143, 100. (2) Reich, K. M.; Frangos, J. A. Effect of Flow on Prostaglandin E2 and Inositol Triphosphate Levels in Osteoblasts. Am. J. Physiol. 1991, 261, C428. (3) Stathopoulos, N. A.; Hellums, J. D. Shear Stress Effects on Human Embryonic Kidney Cells In Vitro. Biotechnol. Bioeng. 1985, 27, 1021. (4) Di Fiore, M. S. H. Atlas of Human Histology, 4th ed.; Lea and Febiger: Philadelphia, PA, 1974. (5) Jensen, D. The Principles of Physiology, 2nd ed.; Appleton Century Crofts: New York, 1980. (6) Resnick, N.; Gimbrone, M. A., Jr. Hemodynamic Forces are Complex Regulator of Endothelial Gene Expression. FASEB J. 1995, 9, 874. (7) Huang, Z. J.; Wang, D. M.; Tarbell, J. M. Modeling Interstitial Flow and Transport in the Artery Wall. Adv. Bioeng. 1995, 29, 191. (8) Wang, D. M.; Tarbell, J. M. Modeling Interstitial Flow in an Artery Wall Allows Estimation of Wall Shear Stress on Smooth Muscle. J. Biomech. Eng. 1995, 117, 358. (9) Carosi, J. A.; McIntire, L. V.; Eskin, S. G. Modulation of Secretion of Vasoactive Materials from Human and Bovine Endothelial Cells by Cyclic Strain. Biotechnol. Bioeng. 1994, 43, 615.

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Received for review June 25, 1998 Revised manuscript received October 16, 1998 Accepted October 22, 1998 IE980426A