- Email: [email protected]
Effect of cyclic axial stretch of rat arteries on endothelial cytoskeletal morphology and vascular reactivity
Journal of Biomechanics 36 (2003) 653–659
Effect of cyclic axial stretch of rat arteries on endothelial cytoskeletal morphology and vascular reactivity Pieter Sipkemaa,*, Peter J.W. van der Lindena, Nico Westerhofa, Frank C.P. Yinb a
Laboratory for Physiology, Institute for Cardiovascular Research-VU, Vrije University, Van der Boechorststraat 7, Amsterdam 1081 BT, The Netherlands b Department of Biomedical Engineering, Washington University, St. Louis, MO, USA Accepted 25 November 2002
Abstract Pulsatile fluid shear stress and circumferential stretch are responsible for the axial alignment of vascular endothelial cells and their actin stress fibers in vivo. We studied the effect of cyclic alterations in axial stretch independent of flow on endothelial cytoskeletal organization in intact arteries and determined if functional alterations accompanied morphologic alterations. Rat renal arteries were axially stretched (20%, 0.5 Hz) around their in vivo lengths, for up to 4 h. Actin stress fibers were examined by immunofluorescent staining. We found that cyclic axial stretching of intact vessels under normal transmural pressure in the absence of shear stress induces within a few hours realignment of endothelial actin stress fibers toward the circumferential direction. Concomitant with this morphologic alteration, the sensitivity (log(EC50)) to the endothelium-dependent vasodilator (acetylcholine) was significantly decreased in the stretched vessels (after stretching –5.1570.79 and before stretching –6.7170.78, resp.), while there was no difference in sodium nitroprusside (SNP) sensitivity. There was no difference in sensitivity to both acetylcholine and SNP in time control vessels. Similar to cultured cells, endothelial cells in intact vessels subjected to cyclic stretching reorganize their actin filaments almost perpendicular to the stretching direction. Accompanying this morphological alteration is a loss of endothelium-dependent vasodilation but not of smooth muscle responsiveness. r 2003 Elsevier Science Ltd. All rights reserved. Keywords: Mechanics; Cytoskeleton; Imaging; Vasodilation
1. Introduction In most arteries, endothelial cells are ellipsoidal and oriented with their long axes parallel to the flow direction and perpendicular to the hoop stress (due to circumferential stretch), except near branches. The actin cytoskeleton in endothelial cells of intact vessels consists of a cortical web connected to a system of individual filaments and bundles of filaments called actin filaments. If present, most cytoskeletal actin filaments are aligned in the flow direction. Studies using cultured endothelial cells have shown that fluid shear stress induces endothelial cells to elongate with their actin cytoskele*Corresponding author. Tel.: +31-20-4448117; fax: +31-204448255. E-mail address: [email protected] (P. Sipkema).
tons aligned in the flow direction (Kataoka et al., 1998; Nerem, 1985), whereas cyclic stretching causes the cells and their cytoskeletons to be oriented perpendicular to the stretching direction (Shirinsky et al., 1989; Takemasa et al., 1997; Wang et al., 1995). When applied simultaneously, interaction was found between endothelial morphology and cytoskeletal orientation (Zhao et al., 1995). However, the artificial cell culture conditions preclude definitive conclusions about the individual roles and interactions between these mechanical forces in intact arteries. To further extend our understanding on the role of mechanics on endothelial cell morphology and function in intact arteries, it is desirable to impose an intervention to which they have not previously been exposed. Consequently, we chose to apply cyclic axial stretch in the absence of flow to isolated, intact rat renal arteries to
0021-9290/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0021-9290(02)00443-8
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P. Sipkema et al. / Journal of Biomechanics 36 (2003) 653–659
study the adaptive response of endothelial cells to stretching alone. This choice makes it possible to study the adaptation to a new situation. If the endothelial cells in these intact vessels respond like cultured cells, they and their cytoskeletons are expected to align about perpendicular to the stretching direction. Additionally, since function of enzymes is closely related to attachment to or transport along the cytoskeleton, it is expected that endothelium-dependent vasodilation may be altered. The present study was designed to address these issues.
2. Methods 2.1. Preparation and setup The investigation conforms to the Guide for the Care and Use of laboratory animals published by the US National institutes of health (NIH Publication # 85-23). The ethics committee for animal experiments (DEC) at the Vrije Universiteit of Amsterdam approved the procedures. Wistar rats weighing 306718 g (n ¼ 14) were anesthetized with sodium pentobarbital (70 mg/kg i.p.). Two similar-sized first-order side branches of the right renal artery, pointing cranially and caudally, respectively, were dissected free and a segment (1.5–2 mm in length, 0.6 mm inner diameter) was cut from each vessel. The arteries were dissected at 41C in MOPS buffer consisting of (in mM) 145 NaCl, 5 KCl, 2 CaCl2, 1 MgSO4, 1 Na2H2PO4, 5 dextrose, 2 pyruvate, 0.02 EDTA and 3 mM 3-(N-morpholino)propanesulphonic acid. Both ends of the segments were tied around glass cannulas (outer diameter approximately 0.6 mm) in a pressure myograph consisting of a temperature-controlled, glass-covered chamber, a thermistor and a heating coil for temperature control (Hoogerwerf et al., 1989; Sipkema et al., 1989). One cannula was connected to a reservoir to pressurize the vessel. The other cannula was connected to an apparatus that induced cyclic, axial stretch of the vessel segment (see Fig. 1). The amplitude and frequency of the stretching were adjustable. The vessels were filled and superfused with a physiological Krebs solution consisting of (in mM) 110 NaCl, 5 KCl, 2.5 CaCl2, 1 MgSO4, 24 NaHCO3, 1 KH2PO4, 0.02 EDTA and 10 dextrose which was equilibrated with 95% air, 5% CO2 at 371C to yield pH of 7.4, pO2 of 150 mmHg and pCO2 of 35 mmHg. Throughout the protocol, the transmural (inside minus outside) pressure was maintained at 100 cm H2O (75 mm Hg) and flow through the vessel was zero. Segments were equilibrated at their in vivo length for 30 min before starting the experimental protocols. One branch was cyclically stretched (0.5, 2 and 4 h) in the axial direction with a strain amplitude of 20% from
Fig. 1. Experimental setup to stretch cannulated vessel segments cyclically and to determine the diameter changes during vasoactive responses. The vessel segment is mounted on two cannulas. The left cannula is connected to a stepper motor. A wave generator controls the movement of the cannula and the stretch of the vessel. The right cannula is connected to a fluid column to set the pressure in the vessel. Experiments were done at 371C.
its in vivo length at a frequency of 0.5 Hz. Strain is defined as Dl=lo; where Dl is the amplitude of cyclic strain and lo is the in-vivo length of the vessel. The other branch was mounted identically and kept at its in vivo length for the same time periods to serve as a control (time control experiment). 2.2. Study design 2.2.1. Structural responses At the end of the experiment, the vessels were perfusion fixed for 30 min at a pressure of 100 cm H2O with 2% formaldehyde in phosphate-buffered saline (PBS) consisting of 137 NaCl, 8.3 Na2PO4, 1.5 KH2PO4 and 2.7 KCL (pH 7.4), then perfused with Triton X-100 (0.05% in PBS) for 2 min to permeabilize the endothelial cell membrane. The permeabilized endothelium was stained for 1 h with 0.264 mM Rhodamine Phalloidin (R415, Molecular Probes Europe BV, Leiden, The Netherlands) and then washed again with PBS. The fixation and staining were done at room temperature. To visualize the stained endothelial cells, the vessels were cut open longitudinally and pinned to a silicone rubber substrate with the endothelial side facing upward. Small variations in the flatness of the specimen in some cases resulted in not all of the cells being in focus in one photomicrograph. Photomicrographs were obtained with a Zeiss (473028) fluorescence microscope equipped with a mercury light source and appropriate filters (red/green: excitation at 530 nm and emission at 590 nm) and a Planapo oil objective (40/1.0, Zeiss 5098226). NIH image software (version: 1.62b7) running
P. Sipkema et al. / Journal of Biomechanics 36 (2003) 653–659
on a MacIntosh computer (8500/180) was used to analyze the images. The portions of the vessel segment near the ties (about 10% of the length) were not used in the analysis because of possible damage of the endothelium. 2.2.2. Functional responses Vasodilatory responses were assessed by measuring diameter changes of the vessels before and after 2 h of the same stretching protocol as described above as well as in time-matched control vessels. A video camera on a microscope was used to visualize the vessel and a video caliper system was used to monitor the outer diameter continuously (Hoogerwerf et al., 1989; Sipkema et al., 1989). All drugs were added to the superfusate and not recirculated. Vessels were pre-constricted with 0.1 mM phenylephrine and then the vasodilatory responses to an endothelium-dependent dilator (acetylcholine; Ach 0.1–10 mM) and an endothelium-independent dilator (sodium nitroprusside; SNP, 0.01–1 mM) were obtained. All salts and MOPS were of analytical grades and purchased from Merck, Inc. (Darmstadt, Germany). Pyruvate, phenylepinephrine, acetylcholine and SNP were obtained from Sigma Chemicals (St. Louis, MO, USA). 2.2.3. Pilot studies We performed a few pilot studies to address some methodological issues prior to performing the main sets of studies. We first verified that the orientations of the actin filaments of the endothelial cells of the branches did not differ. These vessels were prepared as described above. The actin filament orientation and number were assessed as described below. The homogeneity of strain along the vessel was determined by tracking the motion of plastic microspheres (15 mm) adhering to the vessel wall as it was stretched. The mean value of the local strains determined from microspheres in the region encompassing 10–90% of the vessel length was determined. Since it took about an hour to determine the vasodilatory responses of the vessel, we ascertained that the structural changes induced by cyclic stretching were stable for at least this amount of time. For this purpose, we performed one stretching protocol for 2 h, then waited for 1 h with the vessel held at its in vivo length before fixing and staining the endothelial cells. Finally, to verify that any differences in functional response after stretching were not due to major stretching-induced damage to endothelial cells, we stained vessels with 1 M ethidium homodimer-1 (EthD-1, Molecular Probes # E-1169; Leiden, The Netherlands) and compared the number of stained, i.e. nonviable cells, in an unstretched and stretched vessel.
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2.2.4. Data analysis From photomicrographs of the cells, the directions of the actin filaments relative to the axial (stretching) direction were determined. Fibers oriented within 7301 of the stretching direction were denoted as ‘‘axial’’ while fibers oriented more than 501 away from the stretching direction were denoted as ‘‘oblique’’. The rationale for dividing the orientations into two groups is the following. Based on studies using cultured endothelial cells, the actin filaments are expected to align in parallel arrays along the direction of minimal deformation (Wang et al., 1995; Wang, 2000). With our stretching apparatus, the tissue in the direction perpendicular to stretching direction is unconstrained. That is, as the length of the vessel is increased, the diameter is decreased and vice versa. Therefore, the direction of minimum deformation is not circumferential but rather about 701 (oblique) from the axial direction. In the photomicrographs of each experiment, we counted the total number of axial and oblique fibers in all cells and divided the number of fibers by the number of cells (approximately 50 per specimen). Dose-dependent responses obtained to the endothelium-dependent dilator acetylcholine and the endothelium-independent dilator SNP were fitted with a sigmoidal dose response relation (Hill equation) using Graph-Pad Prism Version 3.00 and the sensitivity (log(EC50)) was determined. EC50 is the concentration where 50% of the maximal response is found. Values before and after stretching and from the time control experiments were compared using the paired t-test. Linear regression analysis was performed on the number of fibers per cell versus the duration of stretching. Statistical significance was taken at the p ¼ 0:05 level.
3. Results 3.1. Pilot studies In the vessels studied immediately after dissection (n ¼ 3), there was no significant difference in the number of axial actin fibers between the two side branches. The average numbers of axial fibers in the two branches were 2.7570.63 and 3.1670.60 (p ¼ 0:67). The number of perpendicular fibers was negligible. Thus the choice of the vessel to be stretched should not affect our results. The homogeneity of strain was tested in one vessel segment. The mean value of the local strains (19.670.3%) in the region encompassing 10–90% of the vessel length did not differ from the overall vessel strain (19.4%). Therefore, the strains along the length of the vessel were homogeneous and accurately represented the deformation imposed at the vessel ends.
P. Sipkema et al. / Journal of Biomechanics 36 (2003) 653–659
3.2. Main study Fig. 2 shows fluorescent images from a control (left) and a stretched (right) vessel from the same animal. In the control vessel, the actin filaments are mainly aligned in the axial direction. In the vessel stretched for 2 h, the fibers are predominantly oblique. Figs. 3 and 4 summarize the effect of stretching on actin filament alignment. Fig. 3 shows the number of axial and oblique actin filaments as a function of duration of stretching. The slope of the linear regression of the relation for axial fibers is 0.008 (fibers per minute of stretching) and the intercept is 2.93 (number of fibers at time zero). The slope for oblique fibers is 0.038 and the intercept is 0.77. The difference in slopes is highly significant (po0:0001) but the difference in intercepts is not (p ¼ 0:24). Fig. 4 shows the ratio of oblique to axial fibers as a function of the duration of stretch. The slope of the regression line of this ratio versus duration is 0.025 (po0:0001) and the intercept is 0.34. The square symbol in Fig. 4 denotes the ratio of oblique to axial fibers of a vessel cyclically stretched for 2 h and held for 1 h at the in vivo length before staining. This ratio is similar to the results obtained from the other vessels that were stained immediately after the
12
# of axial fibers # of oblique fibers
10
# of fibers per cell
Examination of the ethidium homodimer-1 staining revealed, that on the inner surface between the cannulae, there were only nine fluorescent spots in both the stretched and the unstretched specimen. However, as expected, many damaged cells were found near where the vessels were tied to the cannulae. Thus, stretching did not increase the number of damaged cells in the region where the functional responses were examined.
8 6 4 2 0
0
50
100
150
200
250
300
Duration of cyclic stretch (min) Fig. 3. The average number of axial and ‘oblique’ stress fibers per cell as a function of duration of cyclic axial stretching (n ¼ 9). The slopes of the linear regression of the relation for axial and for oblique fibers is 0.008 and 0.038, resp., and significantly (po0:0001) different.
8 7 6
oblique/axial
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5 4 3 2 1 0 0
50
100
150
200
250
300
Duration of cyclic stretch (min) Fig. 4. The ratio of oblique to axial fibers as a function of the duration of cyclic stretch (slope: 0.025). The ratio of one vessel stretched for 2 h and held for 1 h before staining is shown as the square.
Fig. 2. Images of rhodamine phalloidin stained endothelial cells from a specimen in the control state (left) and one cyclically stretched for two hours (right). The vessel axis and flow direction is from bottom to top. Notice the change in the direction of the actin fibers between the left and right panels.
experiment. This is evidence that, in the time needed for the functional studies, there was no discernable change in the structural alterations induced by the stretching. Fig. 5 shows the log(EC50) values for acetylcholine and SNP for vessels (n ¼ 5) cyclically stretched for 2 h and for their time-matched controls, both before stretching (begin) and after stretching (end). Values for acetylcholine after stretching are significantly different, but the SNP response is not affected.
P. Sipkema et al. / Journal of Biomechanics 36 (2003) 653–659
Sodium Nitroprusside
Acetylcholine begin end
657
begin end
begin end
begin end
control
stretched
log (EC50)
-4
-6
*
-8 control
stretched
Fig. 5. Sensitivities (n ¼ 5) to acetylcholine and SNP. In vessels cyclically stretched for 2 h, the sensitivity to acetylcholine was significantly decreased. Sensitivity to acetylcholine in unstretched vessels and to SNP in stretched and unstretched vessels was not different.
4. Discussion
4.1. Structural responses
We found that cyclic axial stretching of intact vessels under normal transmural pressure in the absence of shear stress induces within a few hours realignment of endothelial actin actin filaments toward the circumferential direction. Concomitant with this morphologic alteration, the sensitivity to the endothelium-dependent vasodilator (acetylcholine) was significantly decreased in the stretched vessels compared to the unstretched (time-matched control) vessels. We first mention some issues related to the mechanical stimuli on cells in intact blood vessels. Fluid shear stress and (circumferential) stretch are the major mechanical stimuli on endothelial cells in intact blood vessels. The role of hydrostatic pressure (compression), has received less attention than the other two. In this study, we chose to examine the effect of stretching, but in the axial direction. To enhance the possibility of elucidating a response, we chose a branch of the renal artery because, unlike arteries supplying muscular organs, it is not normally subject to much axial stretching. We subjected the arteries to 20% stretch around their in vivo lengths—a change that is on the order of that experienced by vessels adjacent to or embedded in muscle, e.g. coronary arteries, that normally undergo cyclic axial stretching. The number of axially oriented actin filaments per cell in the renal artery is not known, because most authors score the percentage of cells that show at least one fiber. In a study by Yoshida and Sugimoto (1996) the number of axial fibers per cell in a control femoral artery was approximately 3, which is very similar to our findings in the unstretched renal artery branches. This is shown in Fig. 3 at 0 min duration of cyclic stretch (intercept is 2.93) and the pilot studies.
The realignment of actin filaments is significant from several standpoints. First, while cytoskeletal rearrangement has been reported in many studies using cultured cells on artificial substrates (Dartsch and Betz, 1989; Kada et al., 1999; Kim et al., 1989; Naruse et al., 1998; Shirinsky et al., 1989; Takemasa et al., 1997), to our knowledge, the present study is the first in intact vessels to show realignment of the actin fibers in endothelial cells due to cyclic stretching. Our results agree with Takemasa et al. (1997), who found oblique orientation of actin filaments in cultured endothelial cells. Second, the finding that normally adherent endothelial cells can also be induced to undergo such changes suggests that culture conditions may mimic at least some physiological conditions better than generally appreciated. This provides more compelling arguments for the utility of culture systems for the study of mechanical stress-induced changes in cells. Third, the demonstration of the dynamic nature of endothelial cell morphology in intact vessels suggests that, in regions of blood vessels where the synergistic effects of fluid shear and circumferential stretch are absent, the effects of other mechanical stimuli, such as axial stretch may become important. Cells might then respond with both morphologic and functional responses, such as altered permeability (Meyer et al., 1996) that, together with low shear, might enhance conditions favoring atherosclerosis (Ku et al., 1985; Moore et al., 1994). The fact that cells in regions of disturbed flow near branching points tend to be rounded is further indirect evidence for the possible importance of a precise balance between the various mechanical factors in determining cell morphology and function. Several studies in cultured cell preparations subjected to either fluid shear stress or cyclic stretching have
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shown that cytoskeletal rearrangement occurs within minutes after the onset of the mechanical stimulus and precedes cell realignment (Ookawa et al., 1993; Shirinsky et al., 1989; Wang et al., 1995). In contrast, we found that the cytoskeletal rearrangement took hours in intact vessels subjected to stretching. Additionally, within the time frame of our studies, we did not observe any reorientation of the cells. It would not be surprising, however, that endothelial cell reorganization is considerably slower in a confluent layer of cells attached to a normal substrate than under the artificial cell culture conditions. 4.2. Functional responses The altered endothelium-dependent response, together with the cytoskeletal reorganization after stretching, suggests that the alterations in cytoskeletal organization may have an influence on this aspect of endothelial cell function. Our results showed no differences in the number of nonviable cells between stretched and unstretched vessels; staining of the cytoskeleton of the endothelium showed that cells remained in place and furthermore the number of oblique actin filaments is increasing over time. Thus the reduced acetylcholine response after stretching is unlikely due to damage of endothelial cells resulting from the stretching. We found results consistent with a decrease in eNOS activity, while Awolesi (Awolesi et al., 1994, 1995) found an increase. This difference might be attributed to our vessels starting with all the endothelial cells aligned whereas the cells in the Alowesi study started from random orientation. If, as our results suggest, cytoskeletal organization and eNOS activity are related, then it might not be surprising to find differences depending upon the details of how and from what state the cytoskeleton reorganizes. Additionally, it could be that cyclic stretching alters the coupling between acetylcholine receptor mechanism and eNOS or the number of acetylcholine receptors. Stretching could also be a critical determinant of oxidative stress in endothelial cells opposing the effect of endothelium-mediated dilation (nitric oxide). It was found (Silacci et al., 2001) that pulsatility of flow, but not cyclic stretch, was a determinant of flow-induced increase of superoxide anion production. Further studies addressing these issues are necessary. In contrast to the alterations in endothelium-dependent function, our results suggest that direct smooth muscle vasodilation is not affected by cyclic stretching. A study in which smooth muscle cells were cyclically stretched (Numaguchi et al., 1999) found increased extracellular signal-regulated kinases (ERK) with a peak response at 20 min, but vasoactive responses were not studied.
In summary, we found that cyclic axial stretching of intact vessels, like in cultured cells, caused endothelial cell cytoskeletal reorientation away from the stretching direction. Accompanying this structural response is an altered endothelium-dependent vasodilatation. These results suggest that the organization of the actin endothelial cytoskeleton is critically dependent upon the local mechanical environment and this organization, in turn, is accompanied by influences on endotheliumdependent vasodilation.
References Awolesi, M.A., Sessa, W.C., Sumpio, B.E., 1995. Cyclic strain upregulates nitric oxide synthase in cultured bovine aortic endothelial cells. Journal of Clinical Investment 96, 1449–1454. Awolesi, M.A., Widmann, M.D., Sessa, W.C., Sumpio, B.E., 1994. Cyclic strain increases endothelial nitric oxide synthase activity. Surgery 116, 439–444. Dartsch, P.C., Betz, E., 1989. Response of cultured endothelial cells to mechanical stimulation. Basic Research in Cardiology 84, 268–281. Hoogerwerf, N., van der Linden, P.W.J., Sipkema, P., 1989. Effects of oxygen and flow on the diameter of the femoral artery of the rabbit. Blood Vessels 26, 360–367. Kada, K., Yasui, K., Naruse, K., Kamiya, K., Kodama, I., Toyama, J., 1999. Orientation change of cardiocytes induced by cyclic stretch stimulation: time dependency and involvement of protein kinases. Journal of Molecular and Cellular Cardiology 31, 247–259. Kataoka, N., Ujita, S., Sato, M., 1998. Effect of flow direction on the morphological responses of cultured bovine aortic endothelial cells. Med. Biol. Eng. Comput. 36, 122–128. Kim, D.W., Gotlieb, A.L., Langille, B.L., 1989. In vivo modulation of endothelial F-actin microfilaments by experimental alterations in shear stress. Arteriosclerosis 9, 439–445. Ku, D.N., Giddens, D.P., Zarins, C.K., Glagov, S., 1985. Pulsatile flow and atherosclerosis in the human carotid bifurcation: positive correlation between plaque location and low-oscillating shear stress. Arteriosclerosis 5, 293–302. Meyer, G., Merval, R., Tedgui, A., 1996. Effects of pressure induced stretch and convection on low-density lipoprotein and albumin uptake in the rabbit aortic wall. Circulation Research 79, 532–540. Moore Jr., J.E., Xu, C., Glagov, S., Zarins, C.K., Ku, D.N., 1994. Fluid wall shear stress measurements in a model of the human abdominal aorta: oscillatory behavior and relationship to atherosclerosis. Atherosclerosis 110, 225–240. Naruse, K., Yamada, T., Sokabe, M., 1998. Involvement of SA channels in orienting response of cultured endothelial cells to cyclic stretch. American Journal of Physiology (Heart and Circulation Physiology) 43, H1532–1538. Nerem, R.M., 1985. Hemodynamics and vascular endothelium. Transactions of the ASME 115, 510–514. Numaguchi, K., Eguchi, S., Yamakawa, T., Motley, E.D., Inagami, T., 1999. Mechanotransduction of rat aortic vascular smooth muscle cells requires Rho-A and intact filaments. Circulation Research 85, 5–11. Ookawa, K., Sato, M., Ohshima, N., 1993. Time course changes in cytoskeletal structures of cultured endothelial cells exposed to shear stress. Frontiers of Medical and Biological Engineering 5, 121–125. Shirinsky, V.P., Antonov, A.S., Birukov, K.G., Sobolevsky, A.V., Romanov, Y.A., Kabaeva, N.V., Antonova, G.N., Smirnov, V.N., 1989. Mechano-chemical control of human endothelium orientation and size. Journal of Cell Biology 109, 331–339.
P. Sipkema et al. / Journal of Biomechanics 36 (2003) 653–659 Silacci, P., Desgeorges, A., Mazzolai, L., Chambaz, C., Hayoz, D., 2001. Flow pulsatility is a critical determinant of oxidative stress in endothelial cells. Hypertension 38, 1162–1166. Sipkema, P., van der Linden, P.J.W., Hoogerwerf, N., Westerhof, N., 1989. Does the endothelium play a role in flow-dependent constriction? Blood Vessels 26, 368–376. Takemasa, T., Sugimoto, K., Yamashita, K., 1997. Amplitudedependent stress fiber reorientation in early response to cyclic strain. Experimental Cell Research 230, 407–410. Wang, H.C., Ip, W., Boissy, R., Grood, E.S., 1995. Cell orientation response to cyclically de-formed substrates: experimental validation of a cell model. Journal of Biomechanics 28, 1543–1552.
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Wang, J.H., 2000. Substrate deformation determines actin cytoskeleton reorganization: a mathematical modeling and experimental study. Journal of Theoretical Biology 202, 33–41. Yoshida, K., Sugimoto, K., 1996. Morphological and cytoskeletal changes in endothelial cells of vein grafts under arterial hemodynamic conditions in vivo. Journal of Electron Microscopy 45, 428–435. Zhao, S., Suciu, A., Ziegler, T., Moore Jr., J.E., Burki, E., Meister, J.J., Brunner, H.R., 1995. Synergistic effects of fluid shear stress and cyclic circumferential stretch on vascular endothelial cell morphology and cytoskeleton. Arteriosclerosis Thrombosis and Vascular Biology 15, 1781–1786.
Effect of cyclic axial stretch of rat arteries on endothelial cytoskeletal morphology and vascular reactivity Pieter Sipkemaa,*, Peter J.W. van der Lindena, Nico Westerhofa, Frank C.P. Yinb a
Laboratory for Physiology, Institute for Cardiovascular Research-VU, Vrije University, Van der Boechorststraat 7, Amsterdam 1081 BT, The Netherlands b Department of Biomedical Engineering, Washington University, St. Louis, MO, USA Accepted 25 November 2002
Abstract Pulsatile fluid shear stress and circumferential stretch are responsible for the axial alignment of vascular endothelial cells and their actin stress fibers in vivo. We studied the effect of cyclic alterations in axial stretch independent of flow on endothelial cytoskeletal organization in intact arteries and determined if functional alterations accompanied morphologic alterations. Rat renal arteries were axially stretched (20%, 0.5 Hz) around their in vivo lengths, for up to 4 h. Actin stress fibers were examined by immunofluorescent staining. We found that cyclic axial stretching of intact vessels under normal transmural pressure in the absence of shear stress induces within a few hours realignment of endothelial actin stress fibers toward the circumferential direction. Concomitant with this morphologic alteration, the sensitivity (log(EC50)) to the endothelium-dependent vasodilator (acetylcholine) was significantly decreased in the stretched vessels (after stretching –5.1570.79 and before stretching –6.7170.78, resp.), while there was no difference in sodium nitroprusside (SNP) sensitivity. There was no difference in sensitivity to both acetylcholine and SNP in time control vessels. Similar to cultured cells, endothelial cells in intact vessels subjected to cyclic stretching reorganize their actin filaments almost perpendicular to the stretching direction. Accompanying this morphological alteration is a loss of endothelium-dependent vasodilation but not of smooth muscle responsiveness. r 2003 Elsevier Science Ltd. All rights reserved. Keywords: Mechanics; Cytoskeleton; Imaging; Vasodilation
1. Introduction In most arteries, endothelial cells are ellipsoidal and oriented with their long axes parallel to the flow direction and perpendicular to the hoop stress (due to circumferential stretch), except near branches. The actin cytoskeleton in endothelial cells of intact vessels consists of a cortical web connected to a system of individual filaments and bundles of filaments called actin filaments. If present, most cytoskeletal actin filaments are aligned in the flow direction. Studies using cultured endothelial cells have shown that fluid shear stress induces endothelial cells to elongate with their actin cytoskele*Corresponding author. Tel.: +31-20-4448117; fax: +31-204448255. E-mail address: [email protected] (P. Sipkema).
tons aligned in the flow direction (Kataoka et al., 1998; Nerem, 1985), whereas cyclic stretching causes the cells and their cytoskeletons to be oriented perpendicular to the stretching direction (Shirinsky et al., 1989; Takemasa et al., 1997; Wang et al., 1995). When applied simultaneously, interaction was found between endothelial morphology and cytoskeletal orientation (Zhao et al., 1995). However, the artificial cell culture conditions preclude definitive conclusions about the individual roles and interactions between these mechanical forces in intact arteries. To further extend our understanding on the role of mechanics on endothelial cell morphology and function in intact arteries, it is desirable to impose an intervention to which they have not previously been exposed. Consequently, we chose to apply cyclic axial stretch in the absence of flow to isolated, intact rat renal arteries to
0021-9290/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0021-9290(02)00443-8
654
P. Sipkema et al. / Journal of Biomechanics 36 (2003) 653–659
study the adaptive response of endothelial cells to stretching alone. This choice makes it possible to study the adaptation to a new situation. If the endothelial cells in these intact vessels respond like cultured cells, they and their cytoskeletons are expected to align about perpendicular to the stretching direction. Additionally, since function of enzymes is closely related to attachment to or transport along the cytoskeleton, it is expected that endothelium-dependent vasodilation may be altered. The present study was designed to address these issues.
2. Methods 2.1. Preparation and setup The investigation conforms to the Guide for the Care and Use of laboratory animals published by the US National institutes of health (NIH Publication # 85-23). The ethics committee for animal experiments (DEC) at the Vrije Universiteit of Amsterdam approved the procedures. Wistar rats weighing 306718 g (n ¼ 14) were anesthetized with sodium pentobarbital (70 mg/kg i.p.). Two similar-sized first-order side branches of the right renal artery, pointing cranially and caudally, respectively, were dissected free and a segment (1.5–2 mm in length, 0.6 mm inner diameter) was cut from each vessel. The arteries were dissected at 41C in MOPS buffer consisting of (in mM) 145 NaCl, 5 KCl, 2 CaCl2, 1 MgSO4, 1 Na2H2PO4, 5 dextrose, 2 pyruvate, 0.02 EDTA and 3 mM 3-(N-morpholino)propanesulphonic acid. Both ends of the segments were tied around glass cannulas (outer diameter approximately 0.6 mm) in a pressure myograph consisting of a temperature-controlled, glass-covered chamber, a thermistor and a heating coil for temperature control (Hoogerwerf et al., 1989; Sipkema et al., 1989). One cannula was connected to a reservoir to pressurize the vessel. The other cannula was connected to an apparatus that induced cyclic, axial stretch of the vessel segment (see Fig. 1). The amplitude and frequency of the stretching were adjustable. The vessels were filled and superfused with a physiological Krebs solution consisting of (in mM) 110 NaCl, 5 KCl, 2.5 CaCl2, 1 MgSO4, 24 NaHCO3, 1 KH2PO4, 0.02 EDTA and 10 dextrose which was equilibrated with 95% air, 5% CO2 at 371C to yield pH of 7.4, pO2 of 150 mmHg and pCO2 of 35 mmHg. Throughout the protocol, the transmural (inside minus outside) pressure was maintained at 100 cm H2O (75 mm Hg) and flow through the vessel was zero. Segments were equilibrated at their in vivo length for 30 min before starting the experimental protocols. One branch was cyclically stretched (0.5, 2 and 4 h) in the axial direction with a strain amplitude of 20% from
Fig. 1. Experimental setup to stretch cannulated vessel segments cyclically and to determine the diameter changes during vasoactive responses. The vessel segment is mounted on two cannulas. The left cannula is connected to a stepper motor. A wave generator controls the movement of the cannula and the stretch of the vessel. The right cannula is connected to a fluid column to set the pressure in the vessel. Experiments were done at 371C.
its in vivo length at a frequency of 0.5 Hz. Strain is defined as Dl=lo; where Dl is the amplitude of cyclic strain and lo is the in-vivo length of the vessel. The other branch was mounted identically and kept at its in vivo length for the same time periods to serve as a control (time control experiment). 2.2. Study design 2.2.1. Structural responses At the end of the experiment, the vessels were perfusion fixed for 30 min at a pressure of 100 cm H2O with 2% formaldehyde in phosphate-buffered saline (PBS) consisting of 137 NaCl, 8.3 Na2PO4, 1.5 KH2PO4 and 2.7 KCL (pH 7.4), then perfused with Triton X-100 (0.05% in PBS) for 2 min to permeabilize the endothelial cell membrane. The permeabilized endothelium was stained for 1 h with 0.264 mM Rhodamine Phalloidin (R415, Molecular Probes Europe BV, Leiden, The Netherlands) and then washed again with PBS. The fixation and staining were done at room temperature. To visualize the stained endothelial cells, the vessels were cut open longitudinally and pinned to a silicone rubber substrate with the endothelial side facing upward. Small variations in the flatness of the specimen in some cases resulted in not all of the cells being in focus in one photomicrograph. Photomicrographs were obtained with a Zeiss (473028) fluorescence microscope equipped with a mercury light source and appropriate filters (red/green: excitation at 530 nm and emission at 590 nm) and a Planapo oil objective (40/1.0, Zeiss 5098226). NIH image software (version: 1.62b7) running
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on a MacIntosh computer (8500/180) was used to analyze the images. The portions of the vessel segment near the ties (about 10% of the length) were not used in the analysis because of possible damage of the endothelium. 2.2.2. Functional responses Vasodilatory responses were assessed by measuring diameter changes of the vessels before and after 2 h of the same stretching protocol as described above as well as in time-matched control vessels. A video camera on a microscope was used to visualize the vessel and a video caliper system was used to monitor the outer diameter continuously (Hoogerwerf et al., 1989; Sipkema et al., 1989). All drugs were added to the superfusate and not recirculated. Vessels were pre-constricted with 0.1 mM phenylephrine and then the vasodilatory responses to an endothelium-dependent dilator (acetylcholine; Ach 0.1–10 mM) and an endothelium-independent dilator (sodium nitroprusside; SNP, 0.01–1 mM) were obtained. All salts and MOPS were of analytical grades and purchased from Merck, Inc. (Darmstadt, Germany). Pyruvate, phenylepinephrine, acetylcholine and SNP were obtained from Sigma Chemicals (St. Louis, MO, USA). 2.2.3. Pilot studies We performed a few pilot studies to address some methodological issues prior to performing the main sets of studies. We first verified that the orientations of the actin filaments of the endothelial cells of the branches did not differ. These vessels were prepared as described above. The actin filament orientation and number were assessed as described below. The homogeneity of strain along the vessel was determined by tracking the motion of plastic microspheres (15 mm) adhering to the vessel wall as it was stretched. The mean value of the local strains determined from microspheres in the region encompassing 10–90% of the vessel length was determined. Since it took about an hour to determine the vasodilatory responses of the vessel, we ascertained that the structural changes induced by cyclic stretching were stable for at least this amount of time. For this purpose, we performed one stretching protocol for 2 h, then waited for 1 h with the vessel held at its in vivo length before fixing and staining the endothelial cells. Finally, to verify that any differences in functional response after stretching were not due to major stretching-induced damage to endothelial cells, we stained vessels with 1 M ethidium homodimer-1 (EthD-1, Molecular Probes # E-1169; Leiden, The Netherlands) and compared the number of stained, i.e. nonviable cells, in an unstretched and stretched vessel.
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2.2.4. Data analysis From photomicrographs of the cells, the directions of the actin filaments relative to the axial (stretching) direction were determined. Fibers oriented within 7301 of the stretching direction were denoted as ‘‘axial’’ while fibers oriented more than 501 away from the stretching direction were denoted as ‘‘oblique’’. The rationale for dividing the orientations into two groups is the following. Based on studies using cultured endothelial cells, the actin filaments are expected to align in parallel arrays along the direction of minimal deformation (Wang et al., 1995; Wang, 2000). With our stretching apparatus, the tissue in the direction perpendicular to stretching direction is unconstrained. That is, as the length of the vessel is increased, the diameter is decreased and vice versa. Therefore, the direction of minimum deformation is not circumferential but rather about 701 (oblique) from the axial direction. In the photomicrographs of each experiment, we counted the total number of axial and oblique fibers in all cells and divided the number of fibers by the number of cells (approximately 50 per specimen). Dose-dependent responses obtained to the endothelium-dependent dilator acetylcholine and the endothelium-independent dilator SNP were fitted with a sigmoidal dose response relation (Hill equation) using Graph-Pad Prism Version 3.00 and the sensitivity (log(EC50)) was determined. EC50 is the concentration where 50% of the maximal response is found. Values before and after stretching and from the time control experiments were compared using the paired t-test. Linear regression analysis was performed on the number of fibers per cell versus the duration of stretching. Statistical significance was taken at the p ¼ 0:05 level.
3. Results 3.1. Pilot studies In the vessels studied immediately after dissection (n ¼ 3), there was no significant difference in the number of axial actin fibers between the two side branches. The average numbers of axial fibers in the two branches were 2.7570.63 and 3.1670.60 (p ¼ 0:67). The number of perpendicular fibers was negligible. Thus the choice of the vessel to be stretched should not affect our results. The homogeneity of strain was tested in one vessel segment. The mean value of the local strains (19.670.3%) in the region encompassing 10–90% of the vessel length did not differ from the overall vessel strain (19.4%). Therefore, the strains along the length of the vessel were homogeneous and accurately represented the deformation imposed at the vessel ends.
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3.2. Main study Fig. 2 shows fluorescent images from a control (left) and a stretched (right) vessel from the same animal. In the control vessel, the actin filaments are mainly aligned in the axial direction. In the vessel stretched for 2 h, the fibers are predominantly oblique. Figs. 3 and 4 summarize the effect of stretching on actin filament alignment. Fig. 3 shows the number of axial and oblique actin filaments as a function of duration of stretching. The slope of the linear regression of the relation for axial fibers is 0.008 (fibers per minute of stretching) and the intercept is 2.93 (number of fibers at time zero). The slope for oblique fibers is 0.038 and the intercept is 0.77. The difference in slopes is highly significant (po0:0001) but the difference in intercepts is not (p ¼ 0:24). Fig. 4 shows the ratio of oblique to axial fibers as a function of the duration of stretch. The slope of the regression line of this ratio versus duration is 0.025 (po0:0001) and the intercept is 0.34. The square symbol in Fig. 4 denotes the ratio of oblique to axial fibers of a vessel cyclically stretched for 2 h and held for 1 h at the in vivo length before staining. This ratio is similar to the results obtained from the other vessels that were stained immediately after the
12
# of axial fibers # of oblique fibers
10
# of fibers per cell
Examination of the ethidium homodimer-1 staining revealed, that on the inner surface between the cannulae, there were only nine fluorescent spots in both the stretched and the unstretched specimen. However, as expected, many damaged cells were found near where the vessels were tied to the cannulae. Thus, stretching did not increase the number of damaged cells in the region where the functional responses were examined.
8 6 4 2 0
0
50
100
150
200
250
300
Duration of cyclic stretch (min) Fig. 3. The average number of axial and ‘oblique’ stress fibers per cell as a function of duration of cyclic axial stretching (n ¼ 9). The slopes of the linear regression of the relation for axial and for oblique fibers is 0.008 and 0.038, resp., and significantly (po0:0001) different.
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Duration of cyclic stretch (min) Fig. 4. The ratio of oblique to axial fibers as a function of the duration of cyclic stretch (slope: 0.025). The ratio of one vessel stretched for 2 h and held for 1 h before staining is shown as the square.
Fig. 2. Images of rhodamine phalloidin stained endothelial cells from a specimen in the control state (left) and one cyclically stretched for two hours (right). The vessel axis and flow direction is from bottom to top. Notice the change in the direction of the actin fibers between the left and right panels.
experiment. This is evidence that, in the time needed for the functional studies, there was no discernable change in the structural alterations induced by the stretching. Fig. 5 shows the log(EC50) values for acetylcholine and SNP for vessels (n ¼ 5) cyclically stretched for 2 h and for their time-matched controls, both before stretching (begin) and after stretching (end). Values for acetylcholine after stretching are significantly different, but the SNP response is not affected.
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Sodium Nitroprusside
Acetylcholine begin end
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begin end
begin end
begin end
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stretched
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-4
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Fig. 5. Sensitivities (n ¼ 5) to acetylcholine and SNP. In vessels cyclically stretched for 2 h, the sensitivity to acetylcholine was significantly decreased. Sensitivity to acetylcholine in unstretched vessels and to SNP in stretched and unstretched vessels was not different.
4. Discussion
4.1. Structural responses
We found that cyclic axial stretching of intact vessels under normal transmural pressure in the absence of shear stress induces within a few hours realignment of endothelial actin actin filaments toward the circumferential direction. Concomitant with this morphologic alteration, the sensitivity to the endothelium-dependent vasodilator (acetylcholine) was significantly decreased in the stretched vessels compared to the unstretched (time-matched control) vessels. We first mention some issues related to the mechanical stimuli on cells in intact blood vessels. Fluid shear stress and (circumferential) stretch are the major mechanical stimuli on endothelial cells in intact blood vessels. The role of hydrostatic pressure (compression), has received less attention than the other two. In this study, we chose to examine the effect of stretching, but in the axial direction. To enhance the possibility of elucidating a response, we chose a branch of the renal artery because, unlike arteries supplying muscular organs, it is not normally subject to much axial stretching. We subjected the arteries to 20% stretch around their in vivo lengths—a change that is on the order of that experienced by vessels adjacent to or embedded in muscle, e.g. coronary arteries, that normally undergo cyclic axial stretching. The number of axially oriented actin filaments per cell in the renal artery is not known, because most authors score the percentage of cells that show at least one fiber. In a study by Yoshida and Sugimoto (1996) the number of axial fibers per cell in a control femoral artery was approximately 3, which is very similar to our findings in the unstretched renal artery branches. This is shown in Fig. 3 at 0 min duration of cyclic stretch (intercept is 2.93) and the pilot studies.
The realignment of actin filaments is significant from several standpoints. First, while cytoskeletal rearrangement has been reported in many studies using cultured cells on artificial substrates (Dartsch and Betz, 1989; Kada et al., 1999; Kim et al., 1989; Naruse et al., 1998; Shirinsky et al., 1989; Takemasa et al., 1997), to our knowledge, the present study is the first in intact vessels to show realignment of the actin fibers in endothelial cells due to cyclic stretching. Our results agree with Takemasa et al. (1997), who found oblique orientation of actin filaments in cultured endothelial cells. Second, the finding that normally adherent endothelial cells can also be induced to undergo such changes suggests that culture conditions may mimic at least some physiological conditions better than generally appreciated. This provides more compelling arguments for the utility of culture systems for the study of mechanical stress-induced changes in cells. Third, the demonstration of the dynamic nature of endothelial cell morphology in intact vessels suggests that, in regions of blood vessels where the synergistic effects of fluid shear and circumferential stretch are absent, the effects of other mechanical stimuli, such as axial stretch may become important. Cells might then respond with both morphologic and functional responses, such as altered permeability (Meyer et al., 1996) that, together with low shear, might enhance conditions favoring atherosclerosis (Ku et al., 1985; Moore et al., 1994). The fact that cells in regions of disturbed flow near branching points tend to be rounded is further indirect evidence for the possible importance of a precise balance between the various mechanical factors in determining cell morphology and function. Several studies in cultured cell preparations subjected to either fluid shear stress or cyclic stretching have
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shown that cytoskeletal rearrangement occurs within minutes after the onset of the mechanical stimulus and precedes cell realignment (Ookawa et al., 1993; Shirinsky et al., 1989; Wang et al., 1995). In contrast, we found that the cytoskeletal rearrangement took hours in intact vessels subjected to stretching. Additionally, within the time frame of our studies, we did not observe any reorientation of the cells. It would not be surprising, however, that endothelial cell reorganization is considerably slower in a confluent layer of cells attached to a normal substrate than under the artificial cell culture conditions. 4.2. Functional responses The altered endothelium-dependent response, together with the cytoskeletal reorganization after stretching, suggests that the alterations in cytoskeletal organization may have an influence on this aspect of endothelial cell function. Our results showed no differences in the number of nonviable cells between stretched and unstretched vessels; staining of the cytoskeleton of the endothelium showed that cells remained in place and furthermore the number of oblique actin filaments is increasing over time. Thus the reduced acetylcholine response after stretching is unlikely due to damage of endothelial cells resulting from the stretching. We found results consistent with a decrease in eNOS activity, while Awolesi (Awolesi et al., 1994, 1995) found an increase. This difference might be attributed to our vessels starting with all the endothelial cells aligned whereas the cells in the Alowesi study started from random orientation. If, as our results suggest, cytoskeletal organization and eNOS activity are related, then it might not be surprising to find differences depending upon the details of how and from what state the cytoskeleton reorganizes. Additionally, it could be that cyclic stretching alters the coupling between acetylcholine receptor mechanism and eNOS or the number of acetylcholine receptors. Stretching could also be a critical determinant of oxidative stress in endothelial cells opposing the effect of endothelium-mediated dilation (nitric oxide). It was found (Silacci et al., 2001) that pulsatility of flow, but not cyclic stretch, was a determinant of flow-induced increase of superoxide anion production. Further studies addressing these issues are necessary. In contrast to the alterations in endothelium-dependent function, our results suggest that direct smooth muscle vasodilation is not affected by cyclic stretching. A study in which smooth muscle cells were cyclically stretched (Numaguchi et al., 1999) found increased extracellular signal-regulated kinases (ERK) with a peak response at 20 min, but vasoactive responses were not studied.
In summary, we found that cyclic axial stretching of intact vessels, like in cultured cells, caused endothelial cell cytoskeletal reorientation away from the stretching direction. Accompanying this structural response is an altered endothelium-dependent vasodilatation. These results suggest that the organization of the actin endothelial cytoskeleton is critically dependent upon the local mechanical environment and this organization, in turn, is accompanied by influences on endotheliumdependent vasodilation.
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