Stretch-Dependent Modulation of Contractility and Growth in Smooth Muscle of Rat Portal Vein
Abstract—Increased intraluminal pressure of the rat portal vein in vivo causes hypertrophy and altered contractility in 1 to 7 days. We have used organ cultures to investigate mechanisms involved in this adaptation to mechanical load. Strips of rat portal vein were cultured for 3 days, either undistended or loaded by a weight. Length-force relations were shifted toward longer length in stretched cultured veins compared with freshly dissected veins, whereas the length-force relations of unstretched cultured veins were shifted in the opposite direction. This occurred after culture either with or without 10% FCS to promote growth. The wet weight of loaded veins increased by 56% in the presence of FCS, whereas that of undistended control veins increased by 24%. No weight increase was seen in serum-free culture. The dry/wet weight ratio decreased during culture with FCS but was not affected by stretch. Electron microscopy revealed increased cell cross-sectional area in stretched relative to unstretched veins, and protein contents were greater, as were [3H]thymidine and [3H]leucine incorporation rates. Growth responses were associated with the activation of stretch-sensitive extracellular signal–regulated kinases 1 and 2 and were inhibited by herbimycin A and PD 98059, inhibitors of extracellular signal–regulated kinases 1 and 2. The results demonstrate that by culture of whole vascular tissue, smooth muscle cells are maintained in the contractile phenotype and respond to stretch with a physiological adaptation involving hypertrophy/hyperplasia and remodeling of the contractile system, similar to that in vivo. Mechanical stimulation and growth factors are both required for functionally significant growth.
Increased transmural pressure over the vascular wall in hypertension causes hypertrophy/hyperplasia of the vascular smooth muscle, which in itself contributes to the progression of hypertension by increasing reactivity to vasoconstrictor stimuli.1 Pressure-dependent growth is seen in all vascular beds, including small vessels at pressures close to the capillary pressure.
The portal vein is exposed to pressure that is intermediate between the pressures in precapillary resistance vessels and systemic veins, because it is inserted between 2 capillary networks. Partial ligation of the rat portal vein close to its entrance into the liver hilus causes increased pressure and, within 5 to 7 days, increased contractility and marked hypertrophy of the smooth muscle layer, with increased contractile and cytoskeletal protein contents.2 3
For elucidating mechanisms behind vascular adaptation and growth, in vitro culture offers an advantage in terms of experimental control, but it has proven difficult to maintain isolated smooth muscle cells in the contractile phenotype characteristic of the medial layer of the adult vessel wall. Cyclic stretch of smooth muscle cells grown on elastic media increases the proliferation rate as well as protein synthesis.4 5 Cultured smooth muscle cells do not show normal contractility and express a high level of nonmuscle myosin, which can be partially replaced by the smooth muscle–specific isoforms under the influence of cyclic strain, suggesting that strain contributes to maintenance of the contractile phenotype.6 7 8
The transduction of mechanical strain to trophic response involves cell adhesion molecules, such as integrins and extracellular matrix proteins, the roles of which have been elucidated mostly in cell culture systems.9 10 Because tissue composition and contractility are essential for the mechanical properties of the vessel wall, the influence of stretch-sensitive growth needs to be investigated in a system in which the contractile phenotype and cell-matrix interactions are maintained. Organ culture of vascular segments offers this possibility and has been used to investigate stretch-sensitive DNA and protein synthesis in the perfused rabbit aorta,11 although stretch-dependent alterations in contractility have not been investigated.
We have recently shown that rings of rat tail artery maintain contractility for several days in culture.12 These rings were cultured undistended, and there was no increase in muscle mass but, rather, a loss of proteins during the culture period. Because of its longitudinal musculature, the portal vein can be easily loaded without the need for perfusion, and contractile tone is maintained by its myogenic spontaneous activity, making it a suitable preparation for investigation of mechanical influences on growth. In addition, the existence of directly comparable in vivo data from experimental portal hypertension2 3 allows evaluation of the functional relevance of the in vitro findings.
The signal cascade for growth of vascular smooth muscle cells in response to stretch involves the activation of extracellular signal–regulated kinases (ERKs) 1 and 2, which are 44- and 42-kD mitogen-activated protein (MAP) kinases, respectively, responding to a number of stimuli, including tyrosine phosphorylation.8 13 ERK 1/2 activation by stretch of vascular tissue in vitro14 is inhibited by the Src family tyrosine kinase inhibitor herbimycin A.15 In vivo, acute elevation of blood pressure and balloon overstretch causes ERK activation.16
The present study used organ culture of the portal vein to test the hypothesis that stretch causes growth, remodeling, and increased contractility by mechanisms intrinsic to the vascular wall. The pathways involved were examined by measurements or ERK 1/2 activation and by inhibition of ERK activity by herbimycin A and by PD 98059, which specifically inhibits MAP kinase kinase and, thereby, the phosphorylation of ERK.17
Materials and Methods
Female Sprague-Dawley rats were euthanized by cervical dislocation, as approved by the Animal Ethics Committee, Lund University. The portal vein was dissected under sterile conditions and cut longitudinally into 2 halves. Short pieces of 6–0 suture silk were tied to the strips, which were then gently blotted, placed in an Eppendorf vial, and weighed.
Strips were cultured hanging in glass vials in an incubator at 37°C and 5% CO2 and attached to a 0.5-g stainless-steel weight or left unloaded. The medium was DMEM and Ham’s F12 (1:1), with 50 U/mL penicillin and 50 μg/mL streptomycin. FCS was dialyzed with a cutoff molecular mass of 6 to 8 kDa and added as indicated. After 3 days, except where noted, strips were removed, weighed as above, and mounted for force recording. The strings were cut away and weighed for correction of tissue weights. Strips were frozen and stored at −80°C for protein analysis and freeze-drying to determine dry weight. ERK 1/2 activation in response to stretch was determined in tissues preincubated in culture medium with 10% FCS for 1 hour and then loaded from 5 minutes up to 3 days until frozen. The ERK inhibitors herbimycin A (0.5 μmol/L, Calbiochem) and PD 98059 (10 μmol/L Calbiochem), when used, were present during preincubation and subsequent culture.
Strips were attached to a force transducer (AE 801, SensoNor A/S), stretched to a passive tension of 2 mN, and equilibrated for 45 minutes before experimental protocols were begun. The solution (0.4 mL, 37°C) had the following composition (mmol/L): NaCl 135.5, KCl 5.9, CaCl2 2.2, MgCl2 1.2, HEPES 11.6, and glucose 11.5. In high-K+ solution, NaCl was isosmotically replaced by KCl. The cross-sectional area of strips was determined from the length and wet weight.
For length-tension relations, a reference length under a load of 0.5 g was determined after dissection. Passive and active tension were then recorded at increasing length by using nominally Ca2+-free solution for relaxation and high-K+ solution+10 μmol/L norepinephrine for maximal activation.
Frozen samples were pulverized in liquid N2 and extracted.12 Total protein concentration was determined by using a Bio-Rad protein assay. Protein patterns were evaluated on 7.5% SDS-polyacrylamide gels (Bio-Rad Mini-Protean system), stained with Coomassie brilliant blue. ERK phosphorylation was determined by Western blot using an antibody against phosphorylated ERK 1/2 (PhosphoPlus p44/p42 MAP kinase antibody, New England Biolab) and detected by enhanced chemiluminescence.
Assay of DNA and Protein Synthesis
After 48 hours, strips in organ culture were exposed for 24 hours to [methyl-3H]thymidine or l-[4,5-3H]leucine (Amersham) at activities of 0.2 or 1 μCi/mL, respectively. Incorporation was determined as described.12
Strips were equilibrated under preload for 1 hour and then fixed for light and electron microscopy as described.2
Values are mean±SE. The Student t test was used for evaluation of statistical significance. For multiple comparisons, ANOVA was used.
Effects of Stretch During Culture on Growth and Contractility of Vascular Strips
Vascular strips from the rat portal vein were kept loaded or unloaded during culture. In physiological buffer solution, the portal vein maintains a myogenic spontaneous activity with phasic contractions elicited by bursts of action potentials.18 In culture medium, the frequency of contractions increased, whereas the amplitude decreased (data not shown). These effects were reversible. Because the Ca2+ load associated with vasoconstriction by FCS has detrimental effects on cultured vascular preparations,12 dialyzed FCS was used routinely in the present study.
Strips were weighed for evaluation of growth. Freshly dissected strips had weights in the range 0.6 to 1.2 mg. Weighing strips in a moist atmosphere in a closed vial did not impair contractility or responses to culture; thus, alterations in wet weight as well as force responses after culture could be evaluated. Stimulation by FCS (2% and 10%) for 3 days gave a concentration-dependent increase in wet weight (Figure 1A⇓). In 10% FCS, this was 56% and 24% for loaded and unloaded veins, respectively. Serum-free culture gave no weight change in loaded strips and a 9% decrease in unloaded strips.
Active force normalized to cross-sectional area (active stress) during high-K+ stimulation at optimal length was greater in loaded than in unloaded cultured strips under all conditions (Figure⇑ 1B). The normalization takes account of different degrees of growth in loaded versus unloaded strips; thus, the greater active stress in the loaded strips represents a genuine difference in force-generating ability at the tissue level, which is, however, influenced by tissue swelling due to increased water contents in the FCS-stimulated strips (see below). After serum-free culture under load, active stress was unaltered relative to fresh preparations, whereas culture with increasing concentrations of FCS causes a concomitant decrease in active stress.
Load-Dependent Shift of the Length-Force Relation
The length-force relation after culture was compared with that of fresh preparations. All strip lengths were normalized to a reference length under a load of 0.5 g, determined just after dissection. Maximal activation was achieved by high-K+ solution supplemented with noradrenaline (10−5 mol/L). The passive and active length-force relations of strips cultured unloaded in serum-free medium for 3 days were slightly left-shifted relative to those of fresh strips, whereas the passive and active length-force relations of strips cultured under load were displaced rightward toward longer strip lengths (Figure 2A⇓). The reference level of stretch (100% in Figure 2⇓) was close to the optimal length for force development. Active stress at optimal length was similar in fresh and loaded cultured strips, whereas that in unloaded strips was reduced (Figure 1B⇑).
To investigate the effects of growth stimulation on load-dependent mechanical properties, strips were cultured for 2 days with 10% FCS or for 2 days with FCS plus 1 additional day without serum, to ascertain whether alterations in contractility in response to FCS are reversible. Passive and active length-force relations of loaded and unloaded strips under these respective conditions are shown in Figures 2B and 2C. Both sets of strips show a prominent load-dependent shift of passive and active length-force relations. Maximum force values of unloaded strips were 60% and 57% of the values of loaded strips at 2 and 2+1 days, respectively, whereas the maximum force of loaded strips at 2+1 days was 85% of that at 2 days; thus, contractility is not improved by 24 hours of serum starvation.
Protein Contents After Culture
Separation on 7.5% SDS-polyacrylamide gels showed that the myosin/actin ratio was unaltered under different culture and loading conditions (fresh, 1.7±0.2; loaded+FCS, 1.9±0.2; unloaded+FCS, 2.1±0.5; and loaded−FCS, 1.7±0.2; n=4 to 11). Total protein contents were determined on whole portal veins for accuracy. Culture with 10% FCS gave increases in wet weight of 44% (loaded) and 20% (unloaded), respectively (Figure 3⇓, left). The dry/wet weight ratio was unaltered after serum-free culture, whereas in veins cultured with FCS, the dry/wet weight ratio decreased from 0.21 in fresh veins to 0.14 in loaded as well as in unloaded cultured veins (Figure 3⇓, middle). This indicates increased water contents (swelling) induced by culture with FCS.
Total protein content relative to dry weight was not significantly altered by culture with FCS, either under loaded or unloaded conditions (Figure 3⇑, middle). To evaluate changes in total protein contents during culture, the wet weight before culture was used to calculate the initial protein contents by use of the mean protein contents/wet weight determined in fresh veins. This gives increases in protein contents of 13% and 4% in loaded and unloaded veins, respectively (Figure 3⇑, right).
Morphological Effects of Stretch-Sensitive Growth
Electron micrographs of portal veins cultured under load in the absence of FCS showed a general arrangement of cells in the media similar to that of fresh veins (Figure 4D⇓). The cell area, cell profile, and the fine structure were unaltered by culture. In contrast, cells in the unloaded veins had a smaller cell area, and the cell size varied more in unloaded compared with loaded veins (Figure 4C⇓). Moreover, the space between cells was larger, indicating less smooth muscle mass in the unloaded veins after culture. Culture with FCS caused increased cell area, indicating hypertrophy. The increase was more pronounced and the space between cells was smaller in loaded (Figure⇑ 4B) compared with unloaded (Figure 4A⇓) veins. Furthermore, cell size varied more in unloaded veins. In veins cultured with FCS, the cell surface was smooth in the unloaded veins, whereas in the loaded veins, the cell surface had large invaginations and finger-shaped projections of the cell membrane toward adjacent cells. A crucial finding is that the cultured veins did not contain any dedifferentiated cells, which are usually found when isolated smooth muscle cells are cultured.
Role of ERK 1/2 Activity for Stretch-Sensitive DNA and Protein Synthesis
A time course of ERK 1/2 phosphorylation (activation) was determined on strips kept in 10% FCS for 1 hour and then loaded for different times from 5 minutes up to 3 days. Maximal phosphorylation was obtained in portal veins loaded for 1 hour. All gels were loaded with the same amount of protein, and for normalization between different blots, each gel and corresponding blot contained a sample from a strip loaded for 1 hour. This was set as 100%. In fresh portal veins, ERK 1/2 phosphorylation was very low (6.5% of maximum, Figure 5⇓). Phosphorylation in unloaded strips exposed to FCS was ≈50% of maximum and relatively constant for the first 3 hours and then declined to ≈20% of maximum after 1 and 3 days of culture. On loading, the phosphorylation of ERK 1/2 increased rapidly, with a peak (100%) at 1 hour, followed by a decrease at 3 hours, and a further decrease at 24 hours up to 3 days.
In strips exposed to herbimycin A in the presence of 10% FCS and mechanically loaded for 1 hour, ERK 1/2 phosphorylation was inhibited by 35% (Figure 6⇓). PD 98059 lowered ERK 1/2 phosphorylation under these conditions by 68%. Interestingly, in unloaded strips, herbimycin A did not inhibit ERK 1/2 activity, and PD 98059 had only a weak inhibitory effect.
The effects of the inhibitors on ERK 1/2 phosphorylation correlate with the effects on DNA and protein synthesis, as measured by [3H]thymidine and [3H]leucine incorporation. In the absence of inhibitors, incorporation of both substances was approximately twice as large in loaded as in unloaded strips (Figure 7⇓). Whereas herbimycin A and PD 98059 lowered incorporation of both labeled compounds in loaded strips, there was little effect of either inhibitor in unloaded strips, suggesting again that ERK 1/2 inhibition selectively affects stretch-sensitive growth.
The weight gain in the presence of ERK inhibitors showed a pattern similar to that of DNA and protein synthesis. The wet weight of loaded strips cultured with 10% FCS increased by 9% with herbimycin A and by 0% with PD 98059 compared with 54% in control strips without inhibitor. The dry/wet weight ratio after culture with PD 98059 and FCS was similar to that in fresh strips, unlike the reduction seen with FCS alone. In agreement with the general negative correlation between growth and contractility, PD 98059 increased force production relative to that seen with FCS alone (8.4±0.6 versus 4.8±2.0 [Figure 1⇑] mN/mm2).
To investigate whether the transient increase in ERK 1/2 activation during the first 3 hours after loading is sufficient to induce a stretch-dependent growth response, strips were loaded for 3 hours and then cultured under no load for 3 days. This caused an increase in wet weight of 27±3% (n=6), which was not significantly different from that after culture of totally unloaded strips (24±10%, n=6).
Mechanical distension during culture for 3 days of vascular smooth muscle from rat portal vein is shown in the present study to cause increased contractility and cell size, resembling the pattern seen with elevated portal venous pressure in vivo over a similar time period.2 The mechanism involves signals converging on the MAP kinase cascade as the inhibition of ERK 1/2 phosphorylation inhibits growth responses, with selectivity toward stretch-dependent effects.
Culture under load ensures that tension is maintained even when the tissue elongates under mechanical stress. The dominant smooth muscle layer in the portal vein is longitudinal, and this layer hypertrophies in portal hypertension,2 possibly because the vessel is free in the abdomen and thus experiences longitudinal stress. Although correlation with actual pressure values was not attempted, a load of 0.5 g on a half portal vein is twice the preload normally used in our experiments and stretches the strip to slightly above optimal length (Figure 2A⇑). It should thus approach the increase in load imposed by partial ligation in vivo, which raises transmural pressure by 2- to 3-fold.2 After culture under load, the length-force relation was shifted toward longer optimal length, and maximal force development was greater than after culture without distension. This demonstrates remodeling of the vascular tissue in vitro. Stretch seems to be an important stimulus for maintenance of the contractile phenotype, as demonstrated by the positive effect of intraluminal pressure on maintenance of the differentiation markers high-molecular-weight caldesmon and filamen in cultured rabbit aorta.15
Investigations of the effects of stretch on cultured cells have mainly focused on growth responses without consideration of contractility, whereas a few studies of pressurized arteries in culture have examined mechanical properties. Tonic contraction in cultured small resistance arteries under the influence of serum factors is associated with remodeling to smaller diameter at any given pressure in the relaxed as well as in the contracted state.19 The apparent difference between this response and the elongation of the tissue found in the present study is explained by the law of Laplace, which implies that constriction of the artery at constant intraluminal pressure causes decreased wall tension, favoring remodeling to smaller diameter. In accordance with this principle, pressure-diameter relations of arteries from spontaneously hypertensive rats are shifted toward smaller diameters.20 When hypertension is produced by increased intraluminal pressure rather than by vasoconstriction, the opposite response, remodeling to greater diameter, occurs, as demonstrated by the response of carotid arteries to elevated intraluminal pressure in vitro21 as well as to hypertension in humans.22
The additional growth induced by stretch in the presence of FCS was evident as an increased rate of DNA and protein synthesis and an increased gain of weight and protein contents. No mitoses were observed in electron micrographs, whereas cell size was clearly larger after culture under load. Thus, the increased rate of DNA synthesis does not necessarily imply cell division and hyperplasia but may, at least during the limited culture period, lead to increased DNA contents per cell. In fact, polyploidy of vascular cells is increased in hypertension.23 In experimental portal hypertension in rats, the DNA content of the hypertrophied portal vein was found to increase slightly, whereas the wet weight and total protein contents were almost doubled in hypertrophied compared with control veins. The net increase in DNA contents in hypertrophied veins might be due to polyploidy.3
The signals for stretch-dependent growth involve the activation of ERK 1/2, inasmuch as inhibition of ERK phosphorylation by PD 98059 decreased [3H]thymidine and [3H]leucine incorporation of stretched preparations in the presence of FCS to the level seen in unstretched control preparations. An interesting finding is that PD 98059 actually decreased ERK 1/2 phosphorylation of the stretched preparations to levels below those seen in the unstretched preparations. The reason for this is not clear at present, but it should be noted that the level of ERK phosphorylation is determined by the balance between MAP kinase kinase activity and the activities of a number of phosphatases active on ERK (for review, see Reference 24 ). Possibly, the activity of phosphatase as well as kinase is increased by stretch, causing an increased rate of turnover of phosphate on ERK. Kinase inhibition by PD 98059 would then have a greater effect on the kinase/phosphatase ratio in stretched than in unstretched preparations, leading to lower net phosphorylation in the former.
The ERK 1/2 activation after stretch is phasic, with a clear increase at 5 minutes after stretch and a maximum at ≈1 hour. Thereafter, ERK activation subsides but, after 3 days of culture, is still definitely greater than the level seen in fresh veins. The veins were exposed to 10% FCS for 1 hour before stretch was applied, and this created a sizable ERK 1/2 phosphorylation that was maintained at the same level in unstretched preparations for at least 3 hours, ie, the time during which the stretch-induced peak appeared. We found that stretch for 3 hours followed by undistended culture did not affect growth measured after 3 days; thus, the peak itself is not sufficient to induce the growth response. Birukov et al15 have shown a biphasic ERK 1/2 activation in pressurized rabbit aorta, with an early transient peak and a second phase at 24 hours. The response was unaffected by protein kinase C inhibitors but, similar to the present results, inhibited by herbimycin A.
In experimental portal hypertension, swelling of smooth muscle cells is the first response, within 1 to 2 days, followed by hypertrophy.2 Notwithstanding many differences between in vivo and in vitro conditions, it is notable that stimulation with FCS during organ culture was associated with a decreased dry/wet weight ratio. Whereas stretch during culture induced hypertrophy, the degree of tissue swelling was not affected. The reason for the swelling is unknown, but accumulation of osmotically active substances as a result of increased protein turnover is a possibility. When ERK activation was inhibited by PD 98056, there was no weight increase and no decrease in the dry/wet weight ratio. This indicates that swelling is an integral part of the growth response.
The present study demonstrates that vascular smooth muscle cells maintained in the contractile phenotype by the presence of extracellular matrix and cell-cell interactions in intact tissue respond to stretch with a physiological adaptation involving hypertrophy/hyperplasia and remodeling of the contractile system. Mechanical stimulation and growth factors are both required for functionally significant growth, and these stimuli are likely to have different but mutually dependent mechanisms of action.
This study was supported by the Swedish Medical Research Council (project 04X-28 to Dr Hellstrand and 04X-12584 to Dr Malmqvist) and by a stipend from the Swedish Institute to As’ad Zeidan.
- Received May 18, 2000.
- Accepted June 9, 2000.
- © 2000 American Heart Association, Inc.
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