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Am J Physiol Heart Circ Physiol 284: H1839-H1847, 2003. First published January 16, 2003; doi:10.1152/ajpheart.00470.2002
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Vol. 284, Issue 5, H1839-H1847, May 2003

Degradation of alpha -actin filaments in venous smooth muscle cells in response to mechanical stretch

Jeremy Goldman, Lin Zhong, and Shu Q. Liu

Biomedical Engineering Department, Northwestern University, Evanston, Illinois 60208-3107


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Mechanical stretch has been shown to induce the degradation of alpha -actin filaments in smooth muscle cells (SMC) of experimental vein grafts. Here, we investigate the possible role of ERK1/2 and p38 MAPK in regulating this process using an ex vivo venous culture model that simulates an experimental vein graft. An exposure of a vein to arterial pressure induced a significant increase in the medial circumferential strain, which induced rapid alpha -actin filament disruption, followed by degradation. The percentage of SMC alpha -actin filament coverage was reduced significantly under arterial pressure (91 ± 1%, 43 ± 13%, 51 ± 5%, 28 ± 3%, and 19 ± 5% at 1, 6, 12, 24, and 48 h, respectively), whereas it did not change significantly in specimens under venous pressure at theses times. The degradation of SMC alpha -actin filaments paralleled an increase in the relative activity of caspase 3 (3.0 ± 0.7- and 1.7 ± 0.4-fold increase relative to the control level at 6 and 12 h, respectively) and a decrease in SMC density (from the control level of 1,368 ± 66 cells/mm2 at time 0 to 1,205 ± 90, 783 ± 129, 845 ± 61, 637 ± 55, and 432 ± 125 cells/mm2 at 1, 6, 12 , 24, and 48 h of exposure to arterial pressure, respectively). Treatment with a p38 MAPK inhibitor (SB-203580) significantly reduced the stretch-induced activation of caspase 3 at 6 h (from 3.0 ± 0.7- to 2.2 ± 0.3-fold) in conjunction with a significant rescue of alpha -actin filament degradation (from 43 ± 13% to 69 ± 15%) at the same time. Treatment with an inhibitor for the ERK1/2 activator (PD-98059), however, did not induce a significant change in the activity of caspase 3 or the percentage of SMC alpha -actin filament coverage. These results suggest that p38 MAPK and caspase 3 may mediate stretch-dependent degradation of alpha -actin filaments in vascular SMCs.

mitogen-activated protein kinases; caspase 3; vascular grafts


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE VASCULAR SYSTEM is subject to mechanical stretch due to blood pressure. An increase in mechanical stretch has been shown to induce a number of changes in the structure and function of blood vessels, including enhanced smooth muscle cell (SMC) proliferation (3, 59), overproduction of the extracellular matrix (25, 48, 60), increased actin synthesis (7), upregulation of growth factors and growth factor receptors (5, 6, 12, 21, 38, 53, 54, 61), and vessel hypertrophy (11, 30), whereas a decrease in mechanical stretch elicits opposite effects (31). Further studies have shown that stretch-induced vascular changes are mediated by signaling and transcriptional molecules, including MAPKs, G proteins, adenylyl cyclase, cAMP, diacylglycerol, protein kinase A, Jun NH2-terminal kinase, protein kinase C, cAMP response element binding protein, activating protein-1, and nuclear factor-kappa B in cultured endothelial cells and SMCs (9, 13, 15, 16, 19, 28, 43, 45, 49, 50, 55, 58). These previous investigations have provided convincing evidence demonstrating the role of mechanical stretch in regulating the activities of vascular cells.

Vein grafts have been commonly used to replace malfunctioned arteries. However, vein grafts fail due to intimal hyperplasia induced by surgical trauma, mechanical stretch, and disturbed blood flow (33-35). Experimental vein grafts have been used to study the role of mechanical stretch in regulating vascular adaptation and pathogenesis (32, 34, 36, 37). A vein graft is subject to arterial blood pressure, causing a sudden increase in tensile stretch in the vessel wall. Several recent studies have shown that the average circumferential tensile stress in the graft wall can be increased by 140 times or higher compared with that in a native vein (32, 37, 42). Mechanical stretch due to such a stress is associated with rapid disruption and degradation of alpha -actin filaments in SMCs, in conjunction with endothelial cell and SMC death, within the first day after vein grafting surgery (32, 36). These initial changes in experimental vein grafts may contribute to subsequent SMC proliferation and vascular hypertrophy (36), pathological processes contributing to vein graft failure (1, 33, 35, 40). In the present study, we investigate the mechanisms of stretch-dependent degradation of venous SMC alpha -actin filaments.

In an experimental vein graft model, the role of mechanical stretch may be obscured, unfortunately, because several other factors, including surgical trauma, blood-borne factors, and vortex blood flow, contribute to vein graft injury and remodeling (1, 18, 33, 35). To resolve this issue, we developed an ex vivo venous culture model that can be used to simulate an experimental vein graft model. In such a model, a cultured vein is perfused with a pulsatile flow under a desired pressure without the influence of surgical injury, blood-borne factors, and vortex flow. In this study, this model was used to simulate an experimental vein graft and to investigate the role of mechanical stretch in regulating the degradation of venous SMC alpha -actin filaments.

Previous studies have shown that two signaling protein kinases, p38 MAPK and ERK1/2, play a critical role in the transduction of mechanical stretch signals (20, 26). These protein kinases exert distinct effects on cell activities. p38 MAPK regulates vascular cell apoptosis, whereas ERK1/2 regulates vascular cell proliferation after activation by mechanical stretch (4, 27, 29, 40, 44, 56). While the mechanisms of MAPK-related regulatory processes are not entirely clear, recent studies have demonstrated that p38 MAPK may mediate cell death by activating caspase 3 (39, 51, 63), which cleaves proteins including actin filaments (17), whereas ERK1/2 exerts an opposing effect (24, 62). On the basis of these studies, we hypothesized that the p38 MAPK-caspase 3 signaling mechanism may mediate stretch-dependent degradation of SMC alpha -actin filaments, whereas ERK1/2 may prevent such a process. This study was designed to provide evidence for this hypothesis.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Specimen preparation. Rats (Sprague-Dawley, male, 300-350 g) were anesthetized with an intraperitoneal injection of pentobarbital sodium (50 mg/kg body wt). Heparin (200 units in PBS) was injected into the bloodstream. The inferior vena cava was excised together with a fraction of the diaphragm and the right atrium. The diaphragm and cardiac tissue were physically manipulated during the excision and cannulation process to avoid injury to the vena cava. The vessel was carefully placed in either 100 µM papaverine (Sigma) in 0.9% saline solution at 37°C to inhibit SMC contraction for a zero-stress mechanical analysis or placed directly into culture media in preparation for perfusion and inflation. The Animal Care and Use Committee of Northwestern University approved the experimental procedures used in this study.

Vena cava culture and perfusion. The harvested vena cava was placed in a petri dish, submerged under DMEM supplemented with 15% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.5 U/ml heparin (14), and cannulated at both ends onto a pair of fittings that approximately matched the inflated vena cava diameter at arterial [fitting outer diameter (OD) 3.0 mm, inner diameter (ID) 2.6 mm] or venous (fitting OD 1.96 mm, ID 1.47 mm) pressure to reduce vortex flow. To avoid surgical damage, the vessel was cannulated with cardiac tissue onto one fitting and with diaphragm tissue to the other fitting. In cases where a portion of the vessel from the diaphragm end was removed for zero-stress circumferential strain analysis, the vessel was cannulated on venous tissue at the edge of the vessel. The cannulated vessel was placed inside an incubator at 37°C in humidified air with 5% CO2 and attached to a pulsatile cardiac blood pump (model 1407, Harvard Apparatus) and a media source (Fig. 1). For selected specimens, pharmacological inhibitors for ERK1/2 (10 µM PD-98059, BioMol Research Labs) and for p38 MAPK (10 µM SB-203580, Sigma) were added to the media in the petri dish that contained the submerged vessel. The physiological flow rate and pressure in the rat abdominal aorta were used to simulate a vein graft at this location. Vena cava specimens were subjected to either arterial (mean pressure ~120 mmHg) or venous (mean pressure ~1 mmHg) pulsatile pressure. The shear stress for each vessel was estimated to be ~2 dyn/cm2, based on the mean flow rate. At the specified time point, the vessel was either fixed under pressure with 4% formaldehyde in PBS or removed from the system and placed in ice-cold PBS. Under a microscope, the cardiac and diaphragm tissues were removed, and the remaining vessel was used for analysis.


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  		Fig. 1.   Schematics of the experimental setup and mechanical strain measurements. A: the rat vena cava is connected to a pulsatile circulatory system and cultured in an incubator at 37°C with 5% CO2 in humidified air. The arrows represent the direction of flow. B: the vena cava was marked at two axial locations for strain measurements in situ after the chest wall was opened (a). The vessel was removed with a fraction of the right atrium and diaphragm (b). For selected specimens, a short segment (0.5 mm) was removed (c) for establishing a stress-free reference for strain measurements (d). The remaining specimen was cannulated to glass fittings at both ends (e), stretched to its physiological length (f), and perfused at either arterial or venous pressure.

Circumferential strain assessment. In certain cases, a small segment of the vena cava on the diaphragm side (~10% of the total vessel length) was severed. The remaining vessel was placed into the perfusion system to be fixed after 1, 6, 12, 24, or 48 h of experiments. The removed segment was used for obtaining vessel dimensions at zero stress, which were used for strain analysis. Such a segment was cut once in the axial direction to remove residual circumferential strain and embedded in 5% gelatin-PBS to maintain zero-stress geometry. The gelatin-embedded specimen was fixed with 4% formaldehyde-PBS and cut into 10-µm-thick transverse sections using a cryo microtome. The remaining vessel placed in the perfusion system was fixed under a specified pressure for 20 min and cut into 10-µm-thick transverse sections using a cryo microtome. Specimen sections collected at zero-stress and inflated pressure from the same vessel were incubated with an anti-SMC alpha -actin antibody for 1 h, washed in PBS for 30 min, incubated with a secondary rhodamine-conjugated antibody, and observed using a fluorescence microscope. The SMC alpha -actin-containing layer was identified as the media.

The average circumferential length of the media was measured from specimens at zero stress and perfusion pressure. The circumferential stretch ratio (lambda theta theta ) was calculated with the following equation
&lgr;<SUB>&thgr;&thgr;</SUB> = <FR><NU><IT>l</IT><SUB>&thgr;&thgr;</SUB></NU><DE><IT>L</IT><SUB>&thgr;&thgr;</SUB></DE></FR> (1)
where ltheta theta and Ltheta theta are circumferential lengths at a selected pressure and zero stress, respectively. The average circumferential strain of the media was calculated with the following equation
E<SUB>&thgr;&thgr;</SUB> = <FR><NU>&lgr;<SUP>2</SUP><SUB>&thgr;&thgr;</SUB> − 1</NU><DE>2</DE></FR> (2)
where Etheta theta is the circumferential Green's strain.

Axial strain assessment. To measure axial strain, two markers were placed on the vena cava and aligned in the axial direction, and the distance between the two markers was measured before the vessel was harvested from a rat. After excision, the vessel was placed in either papaverine-saline solution (for strain analysis) or cell culture media (for perfusion). The axial stretch ratio (lambda zz) was calculated with the following equation
&lgr;<SUB>zz</SUB>=<FR><NU>l<SUB>zz</SUB></NU><DE>L<SUB>zz</SUB></DE></FR> (3)
where lzz and Lzz are the axial lengths measured under inflation pressure and at zero stress, respectively. The axial strain was calculated with the following equation
E<SUB>zz</SUB>=<FR><NU>&lgr;<SUP>2</SUP><SUB>zz</SUB>−1</NU><DE>2</DE></FR> (4)
where Ezz is the axial Green's strain.

It was found that with one end of the vessel tied off and with the vessel subject to an internal hydrostatic pressure of 120 mmHg, the vena cava naturally extended to an axial strain of 2.44 ± 0.25. This axial strain is not statistically different from the physiological axial strain of the vena cava, 2.47 ± 0.37 (P > 0.5). Therefore, all vessels were restretched to the physiological axial length before perfusion. Axial strains were measured for specimens exposed to venous and arterial pressures.

Actin filament and cell nuclei staining. Selected specimens were removed from each fixed vena cava for en face examination of SMC alpha -actin filaments and cell density, as described previously (36). These specimens were incubated in 0.5% Triton X-100-PBS for 30 min and then with a mixture of 200 nM fluorescein-phalloidin (Molecular Probes), 1% BSA-PBS, and 200 nM Hoechst 33528 for 1 h at 37°C. The specimen was washed three times in PBS for 15 min each, placed on a slide with the endothelium up, and observed en face under an Olympus BX40 fluorescence microscope. In a vein, SMCs form a thin, sheetlike medial layer ~1-3 cells thick. Thus a measurement of the coverage area of SMCs in en face preparations reflects the total amount of actin filaments (32). In this study, we measured the percentage of the SMC alpha -actin filament-covered area with respect to the total area of a selected image in the same specimen by using a MetaMorph imaging system. For each specimen, five images were randomly selected for data measurement. Results were compared between specimens subject to arterial and venous pressures.

In this preparation, endothelial actin filaments were also labeled with phalloidin. But these filaments are oriented along the longitudinal axis of blood vessels, which is perpendicular to the alignment of SMC alpha -actin filaments. The SMC alpha -actin filaments can be easily distinguished from those of endothelial cells.

Hoechst 33258-labeled specimens were used for the measurement of SMC density. For each specimen, SMC and endothelial cell nuclei were both labeled with Hoechst 33258. The SMC nuclei could be distinguished from endothelial nuclei based on different shapes and alignments (32). The SMC nucleus appears a long rodlike structure and is aligned in the circumferential direction of the vessel, whereas the endothelial cell nucleus is oval in shape and is aligned in the vessel axial direction. For each selected image, the total number of SMC nuclei was counted for the calculation of SMC density (number per unit area).

Caspase 3 activity assay. A caspase 3 colorimetric activity assay kit (Chemicon International) was used to measure the activity of caspase 3. For this assay, p-nitroaniline (pNA) is detected with a spectrophotometer after caspase 3 cleaves pNA from the provided substrate, DEVD-pNA. Vessels were perfused under desired pressure for either 6 or 12 h, and the manufacturer's protocol was followed for activity analysis (note that specimens for caspase 3 assays were different from those for alpha -actin assays). Vessel specimens were placed on ice and ground in the provided cell lysis buffer. Cell lysates were centrifuged for 5 min at 10,000 g, and the protein concentration in the supernatant was detected by using the Bradford method (2). An amount of 15 µg of total protein from each specimen was added to the provided assay buffer and incubated in a 96-well plate for 90 min in the presence of the caspase 3 substrate DEVD-pNA. Samples were read at 410 nm in a microtiter plate reader. The fold increase in caspase 3 activity was determined by comparing optical density readings from induced samples to control samples.

Statistical analysis. Means, SDs, and SEs were calculated for the measured parameters. Student's t-test was used to determine the significance of difference between two groups. One-way ANOVA was used to determine the significance of difference among data collected at different times. A difference was considered statistically significant at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Mechanical analysis. The axial strain for vessels exposed to arterial and venous pressure, estimated as shown in Fig. 1B, is presented for the 1-, 12-, 24-, and 48-h groups in Fig. 2A. There is no significant difference of the axial strain for these groups shown (P > 0.5). For all the investigated time points, vessels exposed to arterial pressure experienced a significant increase in the circumferential strain compared with vessels exposed to venous pressure (P < 0.05; Fig. 2B). There was no significant difference in the circumferential strain among different times at the same pressure (P > 0.5). The circumferential and axial strains for each vessel were measured relative to their particular zero-stress conditions (Fig. 1).


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  		Fig. 2.   Changes in circumferential and axial strains at arterial and venous pressures. A: axial strain of the vena cava at 1, 12, 24, and 48 h. Note that the axial strain was preset to the physiological level (solid bar). B: circumferential strain of the vena cava under arterial and venous internal pressure at 1, 12, 24, and 48 h. Means and SDs are presented (n = 3 for axial strain measurements in physiological and static pressure groups, n = 4 for all other groups). * P < 0.05.

Degradation of SMC alpha -actin filaments. Under the mechanical conditions described above, we found extensive alpha -actin filament degradation in vessels exposed to arterial pressure but not in vessels exposed to venous pressure (Fig. 3). A significant decrease in the density of SMCs was also observed in specimens exposed to arterial pressure (Fig. 4). As early as 1 h, we measured a significant difference in SMC density (P < 0.05) but not in alpha -actin filament percentage (P > 0.5) between the two pressure levels. At all other observation times, there was a large statistical difference between the two pressure levels in both the alpha -actin percentage and SMC density (P < 0.005).


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  		Fig. 3.   Changes in the coverage of smooth muscle cell (SMC) alpha -actin filaments. A-E: en face micrographs showing fluorescein-phalloidin-labeled SMC alpha -actin filaments in a fresh specimen without perfusion (A), under venous pressure at 6 and 24 h (B and C, respectively), and under arterial pressure at 6 and 24 h (D and E, respectively). The circumferential direction of the vessel is from the top to the bottom of the micrograph. Bar = 50 µm. F: time course of the SMC alpha -actin filament percentage under arterial pressure compared with that under venous pressure. Means and SDs are presented (n = 5 for the 12- and 24-h groups under 120 mmHg of pressure, n = 4 for all other groups). Differences between the two pressure levels were significant at all observation times (P < 0.01 at 12, 24, and 48 h).



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  		Fig. 4.   Changes in the density of SMCs. A-E: en face micrographs showing Hoechst 33258-labeled SMC nuclei in a fresh specimen without perfusion (A), under venous pressure at 6 and 24 h (B and C, respectively), and under arterial pressure at 6 and 24 h (D and E, respectively). The circumferential direction of the vessel is from the top to the bottom of the micrograph. Bar = 50 µm. F: time course of the SMC density under arterial pressure compared with that under venous pressure. Means and SDs are presented (n = 5 for the 12- and 24-h groups under 120 mmHg of pressure, n = 4 for all other groups). Differences between the two pressure levels were significant at all observation times (P < 0.05 at 1 h and P < 0.005 at other times).

To assess the influence of the culture conditions on the structure of the SMCs in the experimental graft, we conducted an ANOVA statistical test on the data collected for the alpha -actin filament percentage and SMC density (Fig. 3F and 4F) in the vessels exposed to venous pressure for 1, 6, 12, 24, and 48 h. For these vessels, which were exposed to a physiological axial strain, there was no significant difference over time for the alpha -actin percentage or SMC density (P > 0.5). However, there was a significant decrease over time for the alpha -actin filament percentage and SMC density in vessels mechanically stretched under arterial pressure (P < 0.005).

Influence of p38 MAPK and ERK1/2 on stretch-dependent degradation of SMC alpha -actin filaments. To determine the role of MAPKs in mediating the degradation of alpha -actin filaments and SMC death, we exposed arterial pressure-stretched vessels to either an inhibitor of p38 MAPK (SB-203580) or ERK1/2 (PD-98059). A significant rescue of the alpha -actin filaments was found at 6, 12, and 24 h in the presence of SB-203580 (10 µM, P < 0.05; Fig. 5), but no significant change was observed with PD-98059 (10 µM). The presence of SB-203580 also rescued a significant number of SMCs (P < 0.05), but there was no significant change in the SMC density in the presence of the inhibitor of ERK1/2 (Fig. 6).


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  		Fig. 5.   Influence of SB-203580 and PD-98059 on the coverage of SMC alpha -actin filaments. Means and SDs are presented (n = 5 for the 12- and 24-h groups under 120 mmHg of pressure both with and without inhibitors, n = 4 for all other groups). *P < 0.05.



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  		Fig. 6.   Influence of SB-203580 and PD-98059 on the density of SMCs. Means and SDs are presented (n = 5 for the 12- and 24-h groups under 120 mmHg of pressure both with and without inhibitors, n = 4 for all other groups). *P < 0.05.

Changes in the activity of caspase 3. It was found that the activity of caspase 3 in vessels stretched under arterial pressure at 6 h increased threefold when compared with vessels under venous pressure at the same time (P < 0.005; Fig. 7A). At 12 h, the activity of caspase 3 in vessels under arterial pressure diminished to a 1.7-fold increase relative to vessels under venous pressure (P < 0.05). Inhibition of p38 MAPK with SB-203580 reduced the activity of caspase 3 by ~30% at 6 h (P < 0.05) and 20% at 12 h, although the influence was not statistically significant at 12 h. In contrast, treatment with PD-98059 did not significantly change the activity of caspase 3 (Fig. 7B).


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  		Fig. 7.   Changes in the activity of caspase 3. A: caspase 3 activity at 6 and 12 h after exposure to arterial and venous pressure. B: influence of SB-203580 and PD-98059 on the activity of caspase 3 at 6 and 12 h after exposure to arterial pressure. Error bars represent SEs (n = 4 and 5 at 6 h with and without inhibitors, respectively; n = 8 at 12 h for all groups). *P < 0.05; **P < 0.005.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Role of mechanical stretch in the degradation of SMC alpha -actin filaments. An experimental vein graft is subject to a significantly increased mechanical stretch due to exposure to arterial blood pressure. Previous studies have suggested a role for such a stretch in the induction of the degradation of SMC alpha -actin filaments (32, 36). However, the role of mechanical stretch has not been verified because of the coexistence of other factors such as surgical trauma and vortex blood flow, potential factors initiating vascular cell injury, death, and remodeling (1, 18, 33, 35). The present study was designed to assess the role of mechanical stretch without the influence of surgical trauma and vortex flow by using an in vitro venous perfusion system, which simulates an experimental vein graft.

SMCs and alpha -actin filaments are oriented in the circumferential direction and have the physiological function of contracting and dilating the circumference of blood vessels, suggesting that the SMCs are more sensitive to changes in circumferential than axial strain. Thus this study was focused on the influence of the circumferential strain, whereas the axial strain was preset at the physiological level. As shown in the present results, the vena cava became significantly stretched in the circumferential direction upon exposure to an arterial blood pressure. This mechanical stretch induced a progressive decrease in the percentage of SMC alpha -actin filaments over a 2-day period that was significant when compared with vessels under venous pressure. By 48 h of stretch under arterial pressure, <20% of the vein was covered with alpha -actin filaments. This remodeling process is similar to observations in an experimental vein graft model (32, 36). Our results verify that mechanical stretch plays a critical role in the degradation of SMC alpha -actin filaments.

Possible mechanism of stretch-dependent degradation of SMC alpha -actin filaments. One of the goals of this study is to understand whether mechanical stretch induces the degradation of SMC alpha -actin filaments directly or through intracellular signaling mechanisms. The clarification of such a mechanism has the potential to provide targets for preventing excessive degradation of SMC alpha -actin filaments in bypass vein grafts. Previous studies have shown that MAPKs, including p38 MAPK and ERK1/2, possibly regulate stretch-dependent cellular activities (4, 20, 26, 27, 29, 40, 44, 56). Here, we used the selective pharmacological inhibitors SB-203580 and PD-98059, specific to p38 MAPK and the ERK1/2 activator MEK1/2, respectively, to assess the role of these molecules in mediating stretch-dependent degradation of SMC alpha -actin filaments.

As shown in this study, a treatment with SB-203580 significantly prevented stretch-dependent degradation of SMC alpha -actin filaments, suggesting that p38 MAPK possibly mediated such a process. Although the downstream mechanism by which p38 MAPK mediates cell death is not well understood, previous investigations have shown that p38 MAPK regulates the activity of caspase 3, which cleaves a variety of target proteins, including actin (17). Previous studies have also suggested an important role for caspase 3 during vascular cell death in vein grafts (37, 42). In these studies, DEVD-CHO, a tetrapeptide aldehyde that inhibits the activity of caspase 3 (46, 57), significantly inhibited vascular cell death when delivered to a vein graft. These results suggested that caspase 3 possibly mediated mechanical stretch-induced cell death. We explored a potential role for caspase 3 in the present model. Our results show that caspase 3 was significantly activated by mechanical stretch (Fig. 7A). With the use of a p38 MAPK inhibitor, SB-203580, we showed that the activity of caspase 3 was downregulated (Fig. 7B) in conjunction with partially rescued degradation of SMC alpha -actin filaments (Fig. 5). These results suggest a possible regulatory mechanism for the degradation of SMC alpha -actin filaments, which involves mechanical stretch, p38 MAPK, and caspase 3.

We did not measure a statistically significant change in the density of SMCs or the percentage of SMC alpha -actin filament coverage in the presence of PD-98059, although numbers of studies have shown an opposing relationship between ERK1/2 and p38 MAPK (24, 62), and there is considerable evidence that activated ERK1/2 prevents cell death (23, 41, 52). In the present model, a rapid, large mechanical stretch may mainly activate the p38-caspase 3 signaling pathway without a significant influence on the ERK1/2 pathway during the early stage of exposure to arterial blood pressure. Thus the effect of p38 MAPK may dominate over that of ERK1/2.

Significance of the degradation of SMC alpha -actin filaments. Vascular SMCs develop for the regulation of the diameter and tone of blood vessels, and thus the rate of blood flow. A change in the structure and function of SMC alpha -actin filaments influences the contractility of SMCs and blood vessels. As shown in a previous study (7), treatment with cytochalasin B, a substance inhibiting actin polymerization, induced a significant dilatation of cerebral arteries at a given pressure (>120 mmHg), indicating that SMC contractility reduced and the vessel was not able to withstand mechanical stretch under such given pressure. In the present study, the degradation of SMC alpha -actin filaments would likely impair the contractility of blood vessels.

Analyses of mechanical stretch. In a blood vessel, the degree of mechanical stretch can be described by using tensile stress or strain. Tensile stress is often assessed for a cylindrical blood vessel based on the Laplace or force balance principle (34). Such an approach has been used for the determination of the average hoop stress in the wall of blood vessels. In the present case, SMCs reside in the media of blood vessels, and it is necessary to assess the mechanical stretch directly applied to the media. Because of the distinct mechanical properties of media and adventitia (the adventitia is more rigid than the media under arterial blood pressure), the distribution of tensile stress is highly nonuniform across different layers (the adventitia carry more load than the media under arterial blood pressure, especially, for a vein graft). Thus the average tensile stress is not an adequate parameter for the description of mechanical stretch in the media. To date, few analytic approaches have been developed for the analysis of tensile stress in the different layers of a blood vessel.

Tensile strain is a measure of deformation or a change in length. For a blood vessel, the dimensions of different layers in the circumferential direction can be measured under various blood pressure or stress levels using a combined biomechanical and histological method, providing data for the assessment of tensile strain in each of the layers. Thus tensile strain in the media of experimental vein grafts was measured and used to represent mechanical stretch in the present study. With given material constants or mechanical properties for the media, tensile stress can be estimated on the basis of the constitutive stress-strain relationship.

In addition to the circumferential stretch, the tensile component in the axial direction of a blood vessel may contribute to the regulation of vascular adaptation and pathogenesis. In the present case, the axial stretch of perfused veins under arterial blood pressure was similar to that observed in vivo. Thus the axial tensile strain was preset at a level identical to that in vivo for specimen perfusion at both arterial and venous pressure levels. Such an approach allowed an analysis of the influence of the circumferential stretch.


    ACKNOWLEDGEMENTS

This research was supported by a predoctoral fellowship from the American Heart Association and by grants from the American Heart Association and the National Science Foundation.


    FOOTNOTES

Address for reprint requests and other correspondence: J. Goldman, Biomedical Engineering Dept., Northwestern Univ., 2145 North Sheridan Rd., Evanston, IL 60208-3107 (E-mail: jgo821{at}hecky.acns.nwu.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.

First published January 16, 2003;10.1152/ajpheart.00470.2002

Received 10 June 2002; accepted in final form 9 January 2003.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 284(5):H1839-H1847
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