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J Appl Physiol 93: 1907-1917, 2002. First published August 30, 2002; doi:10.1152/japplphysiol.00988.2001
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Vol. 93, Issue 6, 1907-1917, December 2002

Smooth muscle cells contract in response to fluid flow via a Ca2+-independent signaling mechanism

Mete Civelek, Kristy Ainslie, Jeff S. Garanich, and John M. Tarbell

Biomolecular Transport Dynamics Laboratory, Departments of Chemical Engineering and Bioengineering, The Pennsylvania State University, University Park, Pennsylvania 16802


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Smooth muscle cells (SMC) are exposed to fluid shear stress because of transmural (interstitial) flow across the arterial wall. This shear stress may play a role in the myogenic response and flow-mediated vasomotion. We, therefore, examined the effects of fluid flow on contraction of rat aortic SMC. SMC that had been serum-starved to induce a contractile phenotype were plated on quartz slides and exposed to controlled shear stress levels in a flow chamber. The area of the cells was quantified, and reduction in the cell area was reported as contraction. At 25 dyn/cm2, significant area reduction was apparent 3 min after the onset of flow and exceeded 30% at 30 min. At 1 dyn/cm2, significant contraction was not observed at 30 min. The threshold for significant shear-induced contraction appeared to be 11 dyn/cm2. The signal transduction mechanism was studied at 25 dyn/cm2. Intracellular calcium was imaged by using the calcium-sensitive fluorescent dye fura 2-AM. There was no detectable change in intracellular calcium during 10 min of exposure to shear stress, even though the cells displayed a significant calcium response to thapsigargin, calcium ionophore, and KCl. Further studies using pathway inhibitors provided evidence that the most important signal transduction pathway mediating calcium-independent contraction in response to fluid flow is the Rho-kinase pathway, although there was a suggestion that protein kinase C plays a secondary role.

shear stress; vascular smooth muscle; myogenic response; calcium-independent contraction


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

SMOOTH MUSCLE CELLS (SMC) are not normally exposed directly to the shear stresses of flowing blood in the vascular system because the endothelial cell (EC) layer, which lines all blood vessels, provides the contacting surface for blood flow and shields underlying SMC. In cases of endothelial injury and denudation, however, SMC may be exposed directly to the shear stress of flowing blood. This occurs, for example, at sites of vessel repair by angioplasty, at the anastomoses of vascular grafts with arteries, and in other types of cardiovascular interventions (40, 47). Another more subtle mechanism by which SMC are exposed to fluid shear stress is associated with the interstitial flow across the vessel wall, which is driven by the transmural pressure differential. Although the superficial velocity is typically very low (10-5-10-6 cm/s), the interstitial spaces in the tissue are small and the shear stress on SMC can be significant. Wang and Tarbell (58) estimated shear stresses of 1-3 dyn/cm2 at physiological transmural pressures in rabbit aortas for cells not influenced by the internal elastic lamina (IEL). These levels are nearly the same order of magnitude as wall shear stresses imposed by blood flow on the endothelium. In arteries and arterioles, the SMC bordering the subendothelial intima are affected by the presence of the IEL that contains a system of pores or fenestrae. Yuan et al. (61) suggested that the extracellular matrix filling the fenestral pores is much less dense than the elastin fibers comprising the IEL and that, therefore, transmural water flow would be funneled through the pore system as it enters the SMC-laden media. Tada and Tarbell (52) estimated the effect of this entrance condition and determined that the average shear stress around the most superficial layer of SMC just below the IEL could be 30 times higher than that around the second SMC layer.

Interstitial flow is influenced by the hemodynamic state in the blood vessel of interest. According to Starling's law, in arteries and arterioles where blood pressure, which drives transmural flow, is much higher than oncotic pressure, which resists it, interstitial flow shear stress on SMC is approximately proportional to blood pressure. Accordingly, an increase in blood pressure is expected to induce a proportionate increase of transmural flow shear stress on SMC. In some blood vessels, increases in blood flow and associated shear stress on the endothelial layer lead to increases in the hydraulic conductivity of this layer (39, 48). This, in turn, elevates transmural flow and the shear stress on SMC.

In arteries in which the endothelium is intact, it is widely believed that increases in flow induce the release of vasodilatory agents from ECs, which relax SMC and result in dilation of the blood vessel (30, 46). A number of studies have shown, however, that changes in flow can, in fact, induce vessel constriction under certain circumstances. For example, Bevan and Joyce (6), using intact rabbit resistance arteries mounted ex vivo, showed that increased flow induced vessel contraction at low levels of vessel tone. The same vessels could be induced to relax in response to an increase in flow by increasing the level of vessel tone with norepinephrine. Similar experiments with arteries denuded of endothelium showed constriction in response to flow at low tone and dilation at high tone (7). These studies, which have been reviewed by Bevan and Henrion (5), suggest that both EC and SMC produce vasoconstrictors and vasodilators in response to changes in flow and that the balance of production rates and SMC responsiveness depends on vessel tone. These studies, however, have not been interpreted in light of the possibility that changes in transmural flow associated with altered levels of SMC tone and hydraulic conductivity of the endothelium could affect shear stress on SMC, which may have a direct effect on SMC contraction. Indeed, transmural flow has not been measured or controlled in studies of this type. High levels of tone (induced by norepinephrine, not elevated pressure) would be expected to reduce transmural flow (3, 54) and, in turn, the shear stress on SMC. The converse would be expected at low levels of tone. Thus, if increased shear stress on SMC induces contraction, high levels of tone (reduced interstitial shear stress) would be expected to reduce the direct contraction effect on SMC, which would favor dilation as observed. In addition, an increase in SMC shear stress associated with an increase in transmural pressure suggests a potential role for fluid shear stress in mediating the myogenic response, wherein a blood vessel contracts in response to an increase in vascular pressure (4). In the present study, therefore, to elucidate the direct effects of fluid flow on the SMC contraction response, we used an in vitro culture system in which we applied controlled levels of shear stress on SMC.

It is widely believed that the rise and fall of intracellular free Ca2+ concentration ([Ca2+]i) are the principal mechanisms that initiate, respectively, contraction and relaxation in smooth muscles (42). In the myogenic response, the initial stretch driven by a step increase in pressure induces a [Ca2+]i transient, which stimulates phosphorylation of the 20-kDa light chain of myosin (MLC20) by a Ca2+/calmodulin-dependent myosin light-chain kinase (MLCK) leading to SMC contraction. The [Ca2+]i transient may be mediated by stretch-activated ion channels, voltage-gated calcium channels, or Ca2+ release from intracellular stores mediated by inositol phosphates (13, 25, 29, 41, 44). Recent studies, however, indicate that the steady-state contraction response to both step and ramp increases in pressure is not accompanied by a statistically significant increase in [Ca2+]i (26). It is, therefore, important to consider Ca2+-independent pathways that may mediate SMC contraction.

A considerable and growing body of evidence suggests that several factors, in addition to the level of MLC20 phosphorylation, are responsible for regulating sustained contraction in SMC. Two putative pathways have been identified. The involvement of protein kinase C (PKC) was first suggested by the observation that phorbol esters, known to activate PKC, induce slow sustained contractions in several types of SMC (10, 32, 33). In some cases, phorbol ester-induced contractions were observed in the absence of changes in [Ca2+]i or phosphorylation of MLC20 (49). Unlike myosin regulation by MLCK, PKC regulates actin by phosphorylating calponin and caldesmon, inhibiting binding of these two proteins to actin. When actin is free of calponin and caldesmon, it can readily bind to myosin, leading to SMC contraction. The Ca2+-independent PKC isoform PKC-epsilon appears to mediate the contractile response (28). PKC-epsilon phosphorylates calponin and regulates caldesmon activity through extracellular signal-regulated kinase/mitogen-activated protein kinase, leading to caldesmon phosphorylation (17). The second Ca2+-independent contraction pathway involves the G protein RhoA, which stimulates Rho kinase, which, along with arachidonic acid, inhibits myosin light-chain phosphatase (MLCP), which dephosphorylates MLC20 during the contraction cycle. The phosphorylation state of MLC20 is under dual control of MLCK and MLCP. The inhibition of MLCP results in an increase in MLC20 phosphorylation without a change in [Ca2+]i (23, 27).

In the present study, we conducted experiments to identify the principal signal transduction pathways (Ca2+-dependent vs. Ca2+-independent contraction signaling mechanisms) in the smooth muscle contraction response to fluid shear stress.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Chemicals were purchased from Sigma Chemical (St. Louis, MO), unless otherwise noted.

SMC culture. Rat aortic SMC were enzymatically isolated from the thoracic aortas of adult male Sprague-Dawley rats (6-7 wk old, ~150 g), as described elsewhere (2). The cell isolation protocol was reviewed and approved by The Institutional Animal Care and Use Committee at the Pennsylvania State University. SMC were positively identified by their characteristic "hill-and-valley" morphology and their positive staining with specific antismooth muscle alpha -actin stain.

Flow experiments. Cells were grown to confluency in DMEM-F12 supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin (1% P/S) and 10% fetal bovine serum (FBS) in culture flasks. To induce the contractile phenotype, the cells were starved of FBS in culture flasks containing DMEM-F12 + 1% P/S for 2-5 days before flow experiments. The cells were then detached with trypsin-EDTA (0.07%) and plated on 35 × 75-mm, 1.7-mm-thick quartz slides (Friedrich & Dimmock, Millville, NJ) with DMEM-F12 + 1% P/S at a density of 150,000 cells/slide. Once plated, the cells were starved for 2 additional days in petri dishes. All experiments were conducted 2 days postplating, and only passages 4-9 were used. In some experiments, the quartz slides were coated with 1 µg/ml fibronectin. In other experiments, cells were grown and plated in the presence of FBS to maintain the proliferative phenotype.

The parallel plate flow chamber was a modification of the design of Frangos et al. (21). The quartz slide with attached preconfluent cells formed the bottom plate of the flow chamber, and a polycarbonate plate formed the top. A Silastic gasket (SF Medical, Hudson, MA) was used to maintain a uniform gap between the two parallel plates. All three components were held together by an applied vacuum. The chamber was inverted and placed onto the stage of a microscope (Olympus IMT-2). A field of view with ample isolated cells was chosen to be recorded for the time course of the experiment. The image of the cells was recorded for 30 s before the flow was started and then for 30 min of flow. In some experiments, flow was stopped after 30 min, and the cells were recorded for an additional 30 min.

During flow experiments, the chamber was placed in a closed continuous-flow loop circulating DMEM-F12 + 1% P/S or Dulbecco's phosphate buffer solution without calcium or magnesium (PBS) at room temperature (equilibrated with 95% air-5% CO2). The flow rate and the gap width of the chamber could be adjusted to expose the cells to different levels of shear stress. Wall shear stress was calculated, assuming fully developed laminar flow between infinite parallel plates, by using the following equation: tau  = 6µQ/bh2, where tau  is the wall shear stress, µ is the viscosity, Q is the fluid flow rate, b is the width of the flow channel, and h is the height of the flow channel (21). For signal transduction pathway blocker experiments, cells were preincubated with inhibitors for 20-30 min in petri dishes at 37°C in a 5% CO2 incubator before being submitted to shear stress.

The microscope was interfaced to a charged-coupled device camera, which was connected to a VCR and TV to record all experiments. The image processing software, Bioscan OPTIMAS or Image-Pro Express, was used to gather data from videotapes by calculating the area of each cell at 0 min (before the onset of flow) and then at 1-min intervals up to 5 min and at additional 5-min intervals up to 30 min. In order for the software to calculate cell area, the outline of a cell had to be manually traced by using the computer's mouse. Cell areas at each time point were calculated by the software, and reduction in cell area over time was used as the criterion for contraction. Cell area has been used previously as a measure of contraction (8, 19). To determine the angle of orientation of a cell with respect to the flow direction (0° aligned; 90° perpendicular), the major axis of a cell was drawn manually with the computer's mouse, and its angle with respect to a reference line indicating the flow direction was determined by the software.

Individual cell areas at each time point were normalized with respect to their area at 0 min to account for the differences in cell size. By this method, each cell had a normalized area of 1 at 0 min; thus the percent area reduction was calculated at subsequent time points. Each data set is presented as average normalized cell area ± SE of the mean.

[Ca2+]i imaging. In Ca2+ experiments, cell plating was identical to the previously described method. The cell culture media used in the experiments was phenol red free DMEM-F12 + 1% P/S. In experiments in which cells were stimulated with calcium ionophore (Ca IO; 4-bromo-A-23187), thapsigargin (TG) (both from Molecular Probes, Eugene, OR), or potassium chloride (KCl), the cells, after 2-5 days of starvation in culture flasks, were cultured onto 25-mm round glass coverslips (pretreated with 0.1% gelatin solution overnight) and maintained in DMEM-F12 + 1% P/S for 2 additional days.

The procedures for the digital Ca2+ ratiometric assay are detailed elsewhere (57). In brief summary, preconfluent cells on quartz slides or coverslips were washed with PBS and then incubated with 1 µM fura 2-AM (Molecular Probes) (60) dissolved in pluronic acid F12 and DMSO (20% solution in DMSO, Molecular Probes) (1:1 vol/vol) solution for 30 min at 37°C. The cells were then washed with fresh PBS, and the slide was mounted on a parallel-plate chamber, or the coverslip was mounted on a specially designed chamber (Molecular Probes). The chamber or coverslip was placed on an inverted fluorescence microscope (Olympus IMT-2) and left undisturbed for 10 min. The cells were illuminated with ultraviolet excitation light from a 75-W mercury lamp, and a Lambda 10 optical filter changer was used to control a filter wheel, which rotated in front of the light source and alternately placed 340- and 380-nm filters in the light path. The light was reflected up to the cells through a long distance ×10 fluorescence objective by a dichroic mirror. Emitted light passed through the objective and a 510-nm filter and was detected by an intensified charged-coupled device camera. Axon Imaging Workbench 2.1 software (Axon Instruments, Foster City, CA) was used to sample and record the emitted light from the cells in the field of view once every 4 s, and background fluorescence was subtracted from each image. The same software was used to outline and calculate the 340-to-380-nm ratio (340:380 nm) for each cell in the field of view, which reflects [Ca2+]i.

Data were collected for 2 min at the start of each experiment in the absence of flow, TG, Ca IO, or KCl to establish a steady baseline for each cell. An average of the time points taken for 2 min before application of flow or addition of chemicals was used to calculate the baseline 340:380 nm. All Ca2+ data for each cell were transferred to a Microsoft Excel spreadsheet for analysis. A macro was constructed to determine peak wavelength ratio during the flow period. This value was divided by the average baseline wavelength ratio and converted to a percentage. Thus data were expressed as percent increase in wavelength ratio over baseline for each cell in a field of view.

Modification of the Ca2+ environment. Experiments were performed in which the [Ca2+]i and extracellular Ca2+ concentrations were manipulated. In one set of experiments, Ca2+-free PBS was employed as the flow loop media. In another set of experiments, the cell-permeable agent 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM (Molecular Probes) was used to chelate [Ca2+]i. BAPTA-AM was dissolved in DMSO and added to the culture medium to reach a final concentration of 10 µM. The cells were incubated in the 10 µM BAPTA-AM solution for 20 min before exposure to shear stress in the parallel plate flow chamber. The circulating flow loop media was DMEM-F12 + 1% P/S with 10 µM BAPTA-AM.

Signaling pathways. We used bisindolylmaleimide I (Calbiochem, San Diego, CA), which is a highly selective cell-permeable PKC inhibitor (inhibitor constant = 10 nM) that is structurally similar to staurosporine. It acts as a competitive inhibitor for the ATP-binding site of PKC. Bisindolylmaleimide I was dissolved in DMSO and added to culture medium at a final concentration of 10 µM (56). The cells were incubated in a 5% CO2-95% air, 37°C incubator for 30 min. Later, they were washed with fresh culture medium at 37°C and mounted in the parallel plate flow chamber for contraction studies using DMEM-F12 + 1% P/S flow media.

We used an exoenzyme of Clostridium botulinum, named C3, fused with glutathionine S-transferase (C3-GST). C3-GST was a gift of Dr. Cheng Dong from the Pennsylvania State Department of Bioengineering. C3 ADP ribosylates RhoA, which in turn cannot phosphorylate Rho kinase, which is needed for the dephosphorylation of MLC20. C3-GST was dissolved in culture medium to a final concentration of 10 µg/ml (27). The cells were incubated in a 5% CO2, 37°C incubator for 4 h in the presence of C3-GST to permeate the cells. Later they were washed with fresh culture medium at 37°C and were mounted in the parallel-plate flow chamber for contraction studies.

Statistical analysis. Significant differences between group means were analyzed by a two-way (time and treatment) repeated-measure ANOVA by using statistical analysis software (SAS) incorporating a Bonferroni correction. Time was the repeated factor. P < 0.05 was used as the significance level for the statistical analysis. The Bonferroni correction gives a conservative significance level of P/m, where m is the number of comparisons to be performed. For example, if two groups were compared, the P value of 0.05 was replaced by 0.05/2 = 0.025.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of flow rate on SMC contraction response. Figure 1 illustrates the SMC contraction response after exposure to different levels of shear stress for 30 min. At 1 dyn/cm2 shear stress, no significant contraction relative to control cells was observed after 30 min of exposure. The threshold for shear-induced contraction after 30 min of exposure appeared to be 11 dyn/cm2. When cells were exposed to 11 dyn/cm2 shear stress, a significant contraction relative to control was observed after 15 min of flow. The area reduction reached 11.2 ± 1.8% after 30 min of exposure. When the cells were subjected to 25 dyn/cm2 shear stress, significant area reduction relative to unsheared controls was apparent 3 min after the onset of flow. The area reduction was 30.8 ± 1.4% after 30 min of exposure. When shear stress was removed after 30 min, the cells did not relax (increase their area) within an additional 30 min (Fig. 2).


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  		Fig. 1.   Change in normalized cell area over 30 min in response to 1, 11, and 25 dyn/cm2 fluid shear stress. Control cells were mounted in the flow loop but not subjected to fluid flow. Values are means ± SE; n, total no. of cells. Statistically significant from control (no flow): * P < 0.05, ** P < 0.01.



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  		Fig. 2.   Change in normalized cell area over 60 min of exposure to 25 dyn/cm2 shear stress. Solid bars, control cells, which were not subjected to flow; open bars, cells that were observed for 60 min, but, at the end of 30 min, fluid flow was stopped (arrow). Values are means ± SE; n, total no. of cells. Statistically significant from control (no flow): * P < 0.05, ** P < 0.01.

Figure 3 shows a representative video image of cultured SMC exposed to 25 dyn/cm2 shear stress. The cells were randomly oriented relative to the flow direction. The images were taken under ×10 magnification, and it is possible to follow the changes in area of individual cells in successive frames. Note in Fig. 3 that the angle of cell orientation with respect to the flow direction does not change during the contraction and that contraction is characterized by shortening of a cell. In one set of experiments, we determined the cell area reduction (contraction) as a function of the cell orientation. After 30 min of exposure to 25 dyn/cm2 shear stress, cells with orientation angle 0 ± 15° contracted 26 ± 10% (n = 8), 30 ± 15° contracted 33 ± 7% (n = 13), 60 ± 15° contracted 30 ± 8% (n = 9), 90 ± 15° contracted 44 ± 6% (n = 13), 120 ± 15° contracted 38 ± 10% (n = 13), and 150 ± 15° contracted 26 ± 8% (n = 9). These data indicate significant contraction at all orientation angles, and there is the suggestion that 90° (perpendicular) gives the greatest contraction (lowest area at most time points). However, the differences in normalized area between 90° cells and any other orientation are not statistically significant.


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  		Fig. 3.   Representative video image of cultured smooth muscle cells subjected to 25 dyn/cm2 shear stress. The images were taken under ×10 magnification. The flow direction is indicated with a large white arrow in the 0-min frame. Angles with respect to flow are reported for selected cells. Small arrows in the 0-min frame denote cell for reported angle. The angle of the cell was calculated at all time points, unless the cell became circular in shape.

To rule out the possibility that the cells are detaching from the slide when subjected to flow, we incubated cells on quartz slides coated with fibronectin, which prevents the detachment of SMC from the substrate surface. We did not observe any significant difference in the contraction response between cells that were grown on uncoated slides and cells that were grown on slides coated with fibronectin. At the end of 30 min, the cells on uncoated slides contracted by 34.8 ± 2.1%, and the cells on fibronectin-coated slides contracted 32.1 ± 1.2% (data not shown). We repeated experiments at 25 dyn/cm2 with cells grown continuously in serum and compared their contraction response to serum-starved cells. At the end of 30 min, cells grown in serum contracted only 12.0 ± 1.8%, whereas serum-starved cells contracted 35.1 ± 1.2%. This difference was statistically significant (P < 0.01).

Effect of shear stress on [Ca2+]i. A major objective of this study was to investigate the signal transduction mechanisms mediating the contraction response of SMC on exposure to shear stress. The rise and fall in [Ca2+]i are generally considered to be the principal mechanisms that initiate, respectively, contraction and relaxation in smooth muscles. Because we observed that 25 dyn/cm2 shear stress induced a substantial contraction response in SMC, we measured the [Ca2+]i changes in response to fluid flow at this level of shear stress. Figure 4 illustrates the effect of 25 dyn/cm2 shear stress on [Ca2+]i. Although 25 dyn/cm2 shear stress induced a substantial level of contraction, surprisingly, it did not elicit a significant Ca2+ response. The cells were capable of demonstrating Ca2+ response to known calcium agonist 10 µM TG and 2 µM Ca IO.


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  		Fig. 4.   Effect of 10 µM thapsigargin (TG), 2 µM calcium ionophore (Ca IO), and 25 dyn/cm2 shear stress on intracellular Ca2+ concentration in smooth muscle cells. Values are means ± SE. Each trace represents the average intracellular Ca2+ response from n cells. The arrow indicates the addition of chemicals or onset of flow.

In addition, the range of [Ca2+]i stimulated by a Ca2+-dependent contraction agonist was observed when cells were stimulated by 51 mM KCl. A peak normalized fluorescence ratio of 1.79 ± 0.07 with an elevated steady-state ratio, after 100 s of stimulus, of 1.56 ± 0.06 was observed. Cells exposed to 51 mM KCl contracted significantly (49.9 ± 2.1% area reduction after 30 min).

Effects of modifying the calcium environment. To further test whether the SMC contraction in response to shear was calcium independent, we measured the contraction response in the absence of extracellular Ca2+ (PBS) or in the presence of a cell-permeable, Ca2+-chelating agent (BAPTA-AM). Figure 5 illustrates the SMC contraction response to 25 dyn/cm2 shear stress over the course of 30 min, with PBS as the shearing media or 10 µM BAPTA-AM, with DMEM-F12 and 1% P/S as the shearing media. Both treatments resulted in nearly the same significant contraction response beyond 5-min exposure to shear stress. The data in Figs. 4 and 5 taken together lead us to conclude that the SMC contraction response to shear stress is mediated by a Ca2+-independent transduction mechanism.


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  		Fig. 5.   Change in normalized cell area over 30 min in response to 25 dyn/cm2 fluid shear stress under modified Ca2+ conditions. Solid bars, response of cells exposed to 25 dyn/cm2 shear stress with DMEM-F12 with 100 U/ml penicillin and 100 µg/ml streptomycin as the shearing media (control and same media as in Fig. 2). Open bars, response of cells that were exposed to 25 dyn/cm2 shear stress with a 20-min preincubation in 10 µM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM and DMEM-F12 with 100 U/ml penicillin and 100 µg/ml streptomycin as the shearing media. Shaded bars, response of cells exposed to 25 dyn/cm2 shear stress with PBS as the shearing media. Values are means ± SE; n, total no. of cells. Statistically significant from controls (25 dyn/cm2): * P < 0.05, ** P < 0.01.

Signaling pathways mediating the contraction response. Next, we studied Ca2+-independent contraction pathways in SMC. Two putative pathways have been identified: the PKC and Rho kinase pathways. In the first set of experiments, the cells were incubated with a PKC inhibitor, bisindolylmaleimide I, at a final concentration of 10 µM, before exposure to 25 dyn/cm2 shear stress. We observed a slight inhibition of contraction at the end of 15 min (Fig. 6). However, 30 min after the onset of flow, there was ~18% inhibition of the contraction response. The cells exposed to 25 dyn/cm2 reduced their area by 37.6 ± 2.0%, whereas bisindolylmaleimide-treated cells reduced their area by 30.8 ± 3.5% at the end of 30 min (Fig. 6).


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  		Fig. 6.   Effect of 5 µM bisindolylmaleimide I (Bis I) on smooth muscle cell contraction on exposure to 25 dyn/cm2 shear stress. Solid bars, response of cells that were exposed to 25 dyn/cm2 shear stress without incubation in Bis I (control); open bars, response of cells that were incubated in 5 µM Bis I for 30 min before exposure to 25 dyn/cm2 shear stress. Values are means ± SE; n, total no. of cells. Statistically significant from control (25 dyn/cm2), * P < 0.05.

To study the Rho kinase pathway, we used exoenzyme C3 as an inhibitor. We observed a suppression of contraction response when the cells were incubated with 10 µg/ml exoenzyme C3 before exposure to 25 dyn/cm2 (Fig. 7). At 4 min, the area reduction of cells that were incubated in C3 was 4.3 ± 1.4%, which was significantly different than the area reduction of cells that were not incubated in C3 (13.2 ± 1.2%). After 30 min of exposure to 25 dyn/cm2 shear stress, there was 38.6 ± 2.3% contraction in control cells, but only 18.1 ± 4.5% contraction in cells treated with C3.


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  		Fig. 7.   Effect of 10 µg/ml exoenzyme C3 on smooth muscle cell contraction on exposure to 25 dyn/cm2 shear stress. Solid bars, response of cells that were exposed to 25 dyn/cm2 shear stress without incubation in C3 (control); open bars, response of cells that were incubated in 10 µg/ml exoenzyme C3 for 4 h before exposure to 25 dyn/cm2 shear stress. Values are means ± SE; n, total no. of cells. Statistically significant from control (25 dyn/cm2): * P < 0.05, ** P < 0.01.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

SMC exhibit two distinct phenotypes in the vasculature: synthetic (proliferative/migratory) and quiescent (differentiated/contractile). In numerous investigations using cultured SMC, 10-20% serum has been added to the medium to maintain viable cells. The proliferative response of subcultured SMC to serum is concentration dependent with extended logarithmic growth phases and higher final densities at higher serum concentrations. The cells remain in a quiescent state in serum-free, defined media (11). Tagami et al. (53) were able to detect spontaneous contractions of rat aortic SMC cultured in serum-free medium for up to 8 days, whereas SMC cultured in serum-containing medium showed no detectable signs of contractile activity. They concluded that SMC cultured for 7-8 days without serum were completely differentiated and were morphologically the same as SMC in vivo. Recently, Flaherty and Grushkin-Lerner (20) constructed cell culture environments optimized for synthetic and differentiated (contractile) phenotypes and observed that cells had to be cultured in serum-free media to stabilize the contractile form. Therefore, we maintained cells in serum-free media for 4-7 days before exposure to fluid flow.

Detection of the contractile response of subcultured SMC after exposure to contraction agonists has been assessed qualitatively by using cells grown on deformable substrates (e.g., polydimethyl siloxane) (43) by observing the wrinkling of the flexible surface. In other studies, cells were plated on rigid surfaces at subconfluent densities, and the surface area of individual cells was used as a measure of the contractile state (8, 53). In these quantitative studies, up to a 30% reduction in cell surface area was observed in response to contraction agonists. Thus we quantitated cell contraction by the reduction in cell area.

Our experiments show that SMC that are serum starved to induce the contractile phenotype do contract in response to elevated levels of shear stress (Fig. 1) and that this response does not alter the global [Ca2+]i of the cells (Fig. 4), as was also shown by Hill et al. (26) in response to both step and ramp increases in pressure (comparing basal and steady-state [Ca2+]i levels). However, it should be noted that other studies (12, 62) did show significant elevation in steady-state [Ca2+]i levels in response to step increases in pressure. Contracting cells tended to maintain their orientation with respect to the shear direction throughout their exposure to flow. Cells oriented perpendicular to the shear direction contracted more than cells in other orientations, although the differences in contraction with orientation angle were not statistically significant. Because SMC in a vessel wall are oriented perpendicular to the transmural flow direction, our data suggest that they may be in an optimal orientation to respond to transmural flow. Cells grown in serum, to induce the proliferative phenotype, did not contract appreciably in response to shear stress, demonstrating our ability to manipulate the phenotype in vitro.

We also observed that, when shear stress was removed, the cells did not show signs of relaxing back to their initial area within 30 min (Fig. 2). This differs from muscle cell behavior in a three-dimensional tissue. In the tissue environment, the muscle cell is restored to its initial state after the removal of a contraction agonist by the elastic recoil forces of the tissue matrix in which it is embedded. In the two-dimensional surface model that we have employed, there are no elastic restoring forces to reextend the cells after removal of the contraction stimulus (shear stress). Cells on a rigid two-dimensional surface increase their area by spreading, which involves the formation of adhesion bonds with the substrate. The spreading of SMC on unprepared surfaces without adhesion molecules deposited has been reported to be minimal after 60 min of surface contact (24). This would seem to explain why we did not observe an increase in cell area 30 min after removal of shear stress.

The notion that shear stress on SMC induces contraction was suggested by Kuo et al. (35), who observed that coronary venules dilated in response to increased flow when the endothelium was intact but actually constricted when the endothelium was removed. They hypothesized that the response after removal of the endothelium was indicative of a direct contraction response of SMC to an increase in flow (shear stress). Thorin-Trescases and Bevan (55) have presented more convincing evidence in favor of the hypothesis that there is a direct contraction response of SMC in response to an increase in shear stress. Using intact rabbit cerebral arteries ex vivo, they demonstrated that the vasodilatory response to an increase in luminal flow, which was apparent at a vascular pressure of 40 mmHg, was transformed into a vasoconstrictor response at higher pressures (60 and 80 mmHg). When the same experiments were conducted after endothelium removal, the vasodilator response to flow was greatly attenuated at 40 mmHg, and the vasoconstrictor response to flow was enhanced at 60 mmHg and maintained at 80 mmHg. These observations are consistent with a mechanism in which elevated pressure drives increased transmural flow and associated shear stress on SMC, which, in turn, increases myogenic tone. This mechanism also requires that an increase in blood flow leads to an elevation of transmural flow on the underlying SMC. Although this effect was not measured directly in the experiments, other studies (9, 48) have demonstrated an increase in endothelial hydraulic conductivity with increasing shear stress, which implies an increase in transmural flow with an increase in luminal flow. The attenuation of the vasodilatory response and amplification of the vasoconstrictor response in vessels denuded of endothelium are consistent with the removal of vasodilatory mediators produced by the endothelium and increased transmural flow shear stress on SMC (inducing contraction) due to removal of the hydraulic resistance of the endothelium.

The myogenic response is usually associated with the contraction of a blood vessel after an increase in vascular pressure, as originally described by Bayliss (4). This is an important local mechanism in the control of blood flow that has been studied extensively (see Refs. 38 and 41 for reviews). Most studies, with the exception of those using vessels isolated from the brain, have shown that the myogenic response is independent of the endothelium (18, 50). However, when flow (wall shear stress) and pressure changes occur simultaneously, there is interaction between the flow response (which is endothelium dependent) and the pressure response, which can be interpreted as endothelial modulation of the myogenic response (37, 45).

The mechanical forces that mediate the myogenic response are not well established in the literature. To date, there have been two prominent ideas for the involvement of mechanical factors: stretch and tension (16, 22). Circumferential stretch of SMC during the initial transient response to a step increase in pressure is characteristic of the myogenic response, and it has been shown that stretch can lead to SMC contraction in the absence of flow (31). This initial stretch, however, is not sustained during the later phases of the myogenic contraction when the vessel diameter is reduced below its initial value. It is also possible to sustain a myogenic contraction without an initial increase in diameter (stretch) and to generate a myogenic response without overt vessel/cell stretch if the pressure increases in ramp rather than step fashion. It should be noted, however, that, even when there is no overall stretch of the vessel segment, it is possible that an element within the cell membrane or the cell interior remains stretched, as suggested by Johnson (34). In addition to stretch, there is a rise in wall tension, proposed by Johnson, to be the controlled variable in the myogenic response that tends to reach a steady-state value elevated above the initial state of tone. This increase in tension may drive the sustained contraction. A third mechanical factor, which may play a role in the myogenic response, is fluid shear stress driven by transmural interstitial flow. When vascular pressure is increased, an increase in transmural flow is driven by classic Starling forces, which, in turn, increase fluid shear stress on SMC. It is possible that interstitial fluid shear stress remains elevated during the myogenic response or that it is attenuated during the contraction, if hydraulic conductivity is reduced in the contracted vessel. Because transmural flow has not, to our knowledge, ever been measured during the myogenic response, we do not know whether interstitial shear stress remains elevated or is regulated during the response. It seems certain, however, that this shear stress is initially elevated as the pressure rises. Thus, consistent with the findings of this study, the increase in shear stress may induce SMC contraction, which reduces the vessel diameter. This view is consistent with a role for interstitial flow shear stress in the myogenic response, wherein SMC contract when vascular pressure is increased.

One could predict, based on the above arguments, that the myogenic response would be affected by removal of the endothelium, because transmural flow might be expected to increase, due to loss of endothelial hydraulic resistance. However, Kuo et al. (36) have shown that there is no significant difference in the myogenic response of arterioles in the presence or absence of an intact endothelium. However, because the hydraulic resistance (conductivity) of arterioles with and without endothelium has not, to our knowledge, been measured, we do not know whether transmural flow across arterioles would be affected by the endothelium. It is plausible, but not proven, that hydraulic resistance of arterioles is dominated by the SMC layers with little contribution from the endothelial layer. Additional studies are required to assess this hypothesis. It should be noted that the time course of contraction (approximately 3 min needed to detect significant contraction at 25 dyn/cm2; Fig. 1) may differ somewhat from observations in vivo for smaller arterioles. The cells used in the present study were harvested from aortas, and others have observed significant vessel contraction in active arterioles within 1 min, both in vivo (14) and ex vivo (15). Also, others (59) have shown significant differences in SMC response to shear stress in vitro for cells embedded in a three-dimensional gel compared with cells on a two-dimensional surface, as in the present work. Therefore, the dynamic response time and threshold level (11 dyn/cm2) for contraction observed in the present study are only suggestive of the values for contraction in vivo.

The intracellular signaling mechanisms that mediate the myogenic response are not well established either. Ca2+-dependent and -independent mechanisms of SMC contraction have been reported (51). We studied the mechanism of shear-induced contraction at 25 dyn/cm2 because maximum contraction was observed at this level of shear stress. [Ca2+]i was imaged under shear stress conditions in the parallel plate flow chamber by using the Ca2+-sensitive fluorescent dye fura 2-AM. There was no detectable change in [Ca2+]i during 10 min of exposure to shear stress (Fig. 4), even though the cells did display a significant Ca2+ response to 10 µM TG, which releases Ca2+ from intracellular stores, 2 µM Ca IO, which increases transport of Ca2+ from the extracellular medium, and 51 mM KCl, which is known to contract SMC in a Ca2+-dependent manner. In addition, removal of either extracellular or intracellular Ca2+ had no effect on shear-dependent contraction (Fig. 5). We, therefore, concentrated on the calcium-independent pathways of contraction and studied two proposed pathways: the PKC and Rho kinase pathways. The PKC inhibitor bisindolylmaleimide I inhibited the contraction response by 18% (Fig. 6), whereas the Rho kinase inhibitor exoenzyme C3 inhibited the contraction response by 53% (Fig. 7). Thus we concluded that the most important signal transduction pathway mediating Ca2+-independent contraction in response to fluid flow is the Rho kinase pathway, although there is a suggestion that PKC plays a secondary role.

The Rho kinase and PKC pathways are also known to be involved in cell spreading and the formation of focal adhesions. Inhibition of Rho kinase in SMC led to cell rounding (weakening of adhesion) (1), and downregulation of PKC isoforms alpha  and epsilon  resulted in significant inhibition of SMC spreading (24). Therefore, our observations that inhibition of Rho kinase and PKC suppressed SMC contraction in response to shear cannot be interpreted as an influence on cell adhesion, because inhibition of adhesion would have been expected to enhance area reduction in response to shear.

This was the first in vitro study of SMC contraction in response to controlled fluid shear stress. The results provide indirect evidence suggesting that interstitial flow shear stress on SMC plays a role in the myogenic response. Hill et al. (26) showed that steady-state myogenic tone could be generated by using a ramp in pressure that did not induce vessel (SMC) stretch and did not elevate [Ca2+]i in a statistically significant manner. In light of this study, an interpretation of the study by Hill et al. would be that a ramp in vascular pressure ramps the interstitial shear stress on SMC, which induces SMC contraction in a Ca2+-independent manner. Further studies are required to evaluate this hypothesis more directly.


    ACKNOWLEDGEMENTS

The authors extend thanks to Louis Hodgson for technical expertise in calcium experiments and Dr. Cheng Dong for use of Ca2+ imaging facilities.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grant HL-35549, and National Aeronautics and Space Administration Grant NAG3-2746.

Address for reprint requests and other correspondence: J. M. Tarbell, 155 Fenske Laboratory, Univ. Park, PA 16802 (E-mail: jmt{at}psu.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

August 30, 2002;10.1152/japplphysiol.00988.2001

Received 26 September 2001; accepted in final form 3 August 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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