Abstract
The increase in vessel wall strain in hypertension contributes to arterial remodeling by stimulating vascular smooth muscle cell (SMC) proliferation and collagen synthesis. Because L-proline is essential for the synthesis of collagen and cell growth, we examined whether cyclic strain regulates the transcellular transport of L-proline by vascular SMC.
Cultured rat aortic SMCs were subjected to mechanical strain using the Flexercell 3000 Strain Unit.
Cyclic strain increased L-proline transport in a time- and strain-degree–dependent manner that was inhibited by cycloheximide or actinomycin D. Kinetic studies indicated that cyclic strain-induced L-proline uptake was mediated by an increase in transport capacity independent of any change in the affinity for L-proline. Cyclic strain stimulated the expression of system A amino acid transporter 2 mRNA in a time-dependent fashion that paralleled the increase in L-proline transport. Cyclic strain also induced the release of transforming growth factor-beta;1 in a time- and strain-dependent manner. Moreover, conditioned media from SMCs exposed to cyclic strain stimulated the transport of L-proline in control, static SMCs and this was significantly attenuated by a transforming growth factor-β1 neutralizing antibody.
These results demonstrate that cyclic strain stimulates L-proline transport by inducing system A amino acid transporter 2 gene expression through the autocrine release of transforming growth factor-β1. The ability of cyclic strain to induce system A amino acid transporter 2 expression may promote arterial remodeling in hypertension by providing vascular SMCs with the necessary intracellular levels of L-proline required for collagen synthesis and cell growth.
The L-proline is a cyclic amino acid that is involved in numerous physiologic processes, including gluconeogenesis, lipogenesis, neurotransmission, and cell growth.1,,–4 In addition, L-proline is an essential precursor for the synthesis of many structural proteins. In this respect, L-proline and its derivative hydroxyl-L-proline account for approximately two-thirds of the collagen molecule.5 Cellular L-proline requirements are met primarily by the uptake of extracellular L-proline through specific membrane transporters. In vascular smooth muscle cells (SMCs), the uptake of L-proline is predominantly mediated by the system A amino acid carrier.6 This transport system is characterized by its high affinity for short-chain neutral amino acids, its sodium and pH dependence, and its ability to transport N-methylamino acids such as α-(methylamino)isobutyric acid.7,8 The system A amino acid carrier is the major amino acid transport system responsible for the net uptake of neutral amino acids and plays a critical role in the overall flux of amino acids into cells. In addition, system A activity is regulated in a dynamic fashion by various humoral agents and plays an integral role in cell growth and development.6,,–9
Recently, cDNA encoding three distinct system A amino acid transporters (SATs; also known as ATAs) have been cloned and functionally identified.10,,,,–15 These transport proteins show approximately 50% sequence similarity at the amino acid level and display different tissue distributions. Both SAT1 and SAT2 are high-affinity transporters (Km = 200–500 μmol/L) for neutral amino acids, whereas SAT3 is a low-affinity transporter (Km = 4 mmol/L).10,11,12,13,14,15 Although SAT2 is ubiquitously distributed in mammalian cells, SAT1 and SAT3 exhibit a more restricted expression pattern. SAT1 is predominantly expressed in the brain, whereas SAT3 is found in high concentration in the liver.10,11,12,13,14,15 We have recently demonstrated that vascular SMCs express only the SAT2 isoform.6
Vascular SMCs in the major arteries are constantly exposed to cyclic strain (2% to 18%) that arises from the periodic change in vessel diameter as a result of pulsatile blood flow. Recent studies indicate that cyclic strain may play a significant role in regulating SMC function under both normal and pathologic conditions.16,–18 In hypertension, cyclic strain increases by as much as 30%, resulting in marked alterations in gene expression that may contribute to the process of vascular remodeling by stimulating vascular SMC proliferation, hypertrophy, and the deposition of extracellular matrix proteins, particularly collagen.19,,–22 Because L-proline is essential for collagen synthesis and cell growth, the present study examined the effect of cyclic strain on the uptake of L-proline by vascular SMCs. In addition, because we recently found that transforming growth factor-β1 (TGF-β1) is a potent inducer of L-proline transport in vascular SMC,6 we examined whether cyclic strain-mediated alterations in L-proline transport involves the autocrine production and release of TGF-β1.
Methods
Materials
Collagenase, elastase, Tris, Tes, HEPES, bovine serum albumin (BSA), acrylamide, minimum essential medium, cycloheximide, and actinomycin D were purchased from Sigma (St. Louis, MO); penicillin, streptomycin, and serum were from Gibco BRL (Rockville, MD); glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA was from Ambion (Austin, TX); bicicinchoninic acid protein assay was from Pierce (Rockford, IL); TGF-β1 was from R & D Systems (Minneapolis, MN); a neutralizing monoclonal antibody directed against TGF-β1 was kindly provided by Dr. Arteaga (Vanderbilt University, Department of Medicine, Nashville, TN); [2,3,4,5-3H]L-proline (105 Ci/mmol) and [α-32P]UTP (400 Ci/mmol) were from Amersham Life Sciences (Arlington Heights, IL).
Cell culture
Vascular SMCs were isolated by elastase and collagenase digestion of rat thoracic aorta and characterized at passage 3 by morphologic and immunologic criteria, as previously described.23 Cells were cultured serially in minimum essential medium containing 10% serum, Earle's salts, 5.6 mmol/L glucose, 2 mmol/L L-glutamine, 5 mmol/L Tes, 5 mmol/L HEPES, 100 U/mL penicillin, and 100 U/mL streptomycin. Subcultured strains were used between passages 8 and 26. At these passages, SMC exhibited comparable responses to cyclic strain. When cells reached confluence, the culture media was replaced with serum-free media containing BSA (0.1%) for 24 h and then exposed to cyclic stretch.
Cyclic strain
The SMCs were plated onto six-well Bioflex plates coated with type I collagen (Flexercell, McKeesport, PA). The thickness of the silicon elastomer on the bottom of the plate varies along the diameter of the plate such that a near-homogenous strain profile is obtained throughout the membrane. Cells were subjected to mechanical deformation with the Flexercell Strain Unit (FX 3000, Flexercell). This stress unit consists of a computer-controlled vacuum unit and a baseplate to hold the culture dishes. Vacuum is repetitively applied to the bottom of the dishes through the baseplate, which is placed in a humidified incubator with 5% CO2 at 37°C. The computer system controls the frequency of deformation and the negative pressure applied to the plates. The SMCs were exposed to an equiaxial strain of 5% to 20% at a frequency of 1 Hz to mimic the heart rate-driving pulsatile flow.
L-proline transport
The L-proline transport was determined by measuring the influx of radiolabeled L-proline into SMCs, as previously described.6 Cells were washed with HEPES buffer (140 mmol/L NaCl, 5 mmol/L KCl, 0.9 mmol/L CaCl2, 1 mmol/L MgCl2, 5.6 mmol/L glucose, and 25 mmol/L HEPES at pH 7.4) and incubated for 15 min in HEPES buffer containing 50 μmol/L [3H]L-proline (1 μCi). A 15-min point for uptake experiments was used based on our previous study demonstrating that L-proline transport with time is linear at this time.6 Transport activity was terminated by aspirating the medium and washing the cells with ice-cold HEPES buffer. The cells were then solubilized by the addition of 0.2% SDS in 0.2 N NaOH. A portion of the extract was collected and radioactivity monitored by liquid scintillation counting. The remaining extract was used for protein determination using the bicinchoninic acid method with BSA as the standard. To correct for nonspecific uptake or binding to the cell surface, cells were incubated in parallel wells with HEPES buffer containing 10 mmol/L unlabeled L-proline. The fraction of radioactivity in the cells was then determined and this fraction was subtracted from each data point.
In some experiments, the dependence of L-proline transport on de novo DNA or protein synthesis was determined by treating SMCs with the transcriptional inhibitor actinomycin D (1 μg/mL) or the translational inhibitor cycloheximide (5 μg/mL), respectively, at concentrations that minimally affected cell viability.24,25
mRNA analysis
The SAT mRNA levels were determined by ribonuclease protection analysis using a commercially available kit (Ambion). In brief, total RNA (10 μg) was hybridized with ∼1 × 106 cpm of [32P]UTP-labeled antisense SAT1 (231 bp), SAT2 (202 bp), or SAT3 (187 bp) and GAPDH (316 bp) riboprobes. The SAT (202 bp) antisense RNA probes was prepared as described earlier.6 Samples were incubated for 16 h at 45°C followed by ribonuclease digestion at 22°C for 30 min. Protected RNA was analyzed by electrophoresis using 6% acrylamide/8 mmol/L urea gels. Gels were exposed overnight to X-ray film at −70°C in the presence of intensifying screens. Relative mRNA levels were quantified by scanning densitometry (LKB 2222-020 Ultrascan laser densitometer, Bromma, Sweden) and normalized with respect to GAPDH.
TGF-β1 release
The concentrations of TGF-β1 in the conditioned media of SMCs were determined by ELISA,26 according to the manufacture's instructions (R & D Systems). To quantify total TGF-β1, the latent form of TGF-β1 in the conditioned media was converted to the active form by acidification with 1 N HCl for 1 h at 4°C and then neutralized with 1 N NaOH.
Statistical analysis
Results are expressed as the mean ± SEM. Statistical analysis was performed with the use of a Student two-tailed t test and ANOVA when more than two treatments were compared. A P value of less than .05 was considered to be statistically significant.
Results
The application of cyclic strain (10%) to SMCs stimulated the transport of L-proline in a time-dependent manner (Fig. 1A). A significant increase in transport activity compared to nonstrained, static cells was observed after 24 h of mechanical strain and this was maintained during the 72 h of cyclic stretch. In addition, the strain-mediated increase in L-proline uptake was dependent on the level of cyclic strain. Although cyclic strain of 5% for 24 h failed to augment L-proline transport, elevating the magnitude of strain to 10% or 20% resulted in a progressive increase in L-proline uptake (Fig. 1B). The stimulation of L-proline transport after 24 h of 10% cyclic strain was blocked by cycloheximide (5 μg/mL) or actinomycin D (1 μg/mL) (Fig. 1C). In the absence of cyclic strain, cycloheximide and actinomycin D minimally affected L-proline transport (data not shown).
Effect of cyclic strain on L-proline transport by vascular smooth muscle cells (SMCs). A) Time course of cyclic strain (10% at 1 Hz)-induced L-proline transport expressed as percent increase over static (nonstretched) SMC. B) Strain-dependent (5% to 20% at 1 Hz) increases in L-proline transport expressed as percent increase over static (nonstretched) SMC. C) Effect of cycloheximide (CX) and actinomycin D (Act D) on cyclic strain-mediated increases in L-proline transport by vascular SMCs. SMCs were exposed to cyclic strain (10% at 1 Hz) for 24 h in the presence or absence of CX (5 μg/mL) or Act D (1 μg/mL). Results are means ± SEM of between four and five separate experiments. *Statistically significant effect of cyclic strain.
In subsequent kinetic studies, saturable uptake of L-proline was measured. Fig. 2 shows a representative Eadie-Hofstee plot demonstrating that high-affinity uptake of L-proline was mediated by a single carrier. Data from several experiments (n = 5) indicated that this transporter had a Michaelis constant (Km) of 211 ± 22 μmol/L and a maximum transport velocity (Vmax) of 177 ± 23 pg/mg protein/min. Imposition of cyclic strain (10%) for 24 h significantly (P < .05) increased the Vmax (313 ± 32 pg/mg protein/min) of L-proline transport without affecting the Km (202 ± 18 μmol/L).
Representative Eadie-Hofstee plot of saturable L-proline transport by vascular smooth muscle cells (SMCs). Specific transport of L-proline (10 to 1000 μmol/L) was measured in static control SMCs (open circle) and in SMCs (closed circle) subjected to cyclic strain (10% at 1 Hz) for 24 h. Data from one representative experiment are shown in the Figure. Similar findings were made in five separate experiments.
Ribonuclease protection assays revealed low levels of SAT2 mRNA expression in control static SMCs (Fig. 3). However, exposure of vascular SMCs to cyclic strain (10%) markedly elevated SAT2 mRNA levels. An increase in SAT2 mRNA was observed after 24 h of cyclic strain and levels remained elevated after 72 h of cyclic strain. Ribonuclease protection assays failed to detect mRNA for SAT1 or SAT3 either in control, static cells, or in cells exposed to cyclic strain (data not shown).
Effect of cyclic strain on the expression of system A amino acid transporter-2 (SAT2) mRNA by vascular smooth muscle cells (SMCs). A) Ribonuclease protection analysis of SAT2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAs after exposure of SMCs to cyclic strain (10% at 1 Hz). B) Quantification of SAT2 mRNA levels by laser densitometry after the application of cyclic strain (10% at 1 Hz). Results are means ± SEM of three separate experiments. *Statistically significant effect of cyclic strain.
Because we have recently discovered that TGF-β1 is a potent inducer of L-proline transport in vascular SMCs,6 we examined whether mechanical strain stimulated the release of TGF-β1 under our experimental conditions. Application of cyclic strain (10%) markedly increased the secretion of TGF-β1 into the culture medium after 24 h and 48 h (Fig. 4A). Moreover, augmentation of cyclic strain from 10% to 20% further increased the release of TGF-β1 from SMCs by 15.4 ± 3.9% (P < .05).
Role of transforming growth factor-β1 (TGF-β1) in cyclic strain-mediated increases in L-proline transport. A) Effect of cyclic strain on TGF-β1 release by vascular smooth muscle cells (SMCs). The SMCs were exposed to cyclic strain (10% at 1 Hz) for 4, 24, or 48 h and TGF-β1 concentration measured in the culture medium. Results are means ± SEM of between three and four separate experiments. *Statistically significant effect of cyclic strain. B) Effect of conditioned media (CM) on L-proline transport by vascular SMC. The SMCs were incubated for 24 h with CM obtained from SMC exposed to cyclic strain (10% at 1 Hz) for 24 h in the presence or absence of a specific TGF-β1 neutralizing antibody (TGF-β1AB; 20 μg/mL) or nonimmune IgG (IgG; 20 μg/mL). Results are means ± SEM of four separate experiments. *Statistically significant effect of CM. +Statistically significant effect of the TGF-β1 neutralizing antibody.
In subsequent experiments, we examined whether the strain-mediated release of TGF-β1 functions as an autocrine stimulus for the uptake of L-proline. The culture media overlying either static SMCs or SMCs exposed to chronic mechanical strain (10%) for 24 h were removed and transferred to SMCs grown on regular tissue culture plates. When compared with conditioned media from static cells, the conditioned media from strained cells caused a significant increase in L-proline transport (Fig. 4B). However, treatment of the conditioned media from SMCs exposed to cyclic strain for 24 h with a TGF-β1 neutralizing antibody (20 μg/mL) reduced the ability of the conditioned media to increase L-proline transport by >50% (Fig. 4B). In contrast, nonimmune IgG (20 μg/mL) had no effect on the TGF-β1-mediated increase in L-proline transport (Fig. 4B).
Discussion
The present study demonstrates that cyclic strain stimulates L-proline uptake in vascular SMC, possibly by inducing the expression of the SAT2 gene. Cyclic strain stimulates the transport of L-proline in a time- and strain-dependent fashion. Kinetic experiments indicate that high affinity (Km ∼200 μmol/L) L-proline transport is mediated by a single carrier and that mechanical strain selectively increases the Vmax without affecting the Km of this transport system. These kinetic data suggest that the cyclic strain-induced increase in L-proline uptake likely arises from the de novo synthesis of additional transport proteins. Consistent with this, we found that cycloheximide blocks cyclic strain-induced transport. Moreover, we observed that cyclic strain stimulates SAT2 mRNA expression in a manner that parallels the increase in L-proline uptake. Although the molecular mechanism by which cyclic strain induces SAT2 gene expression is not known, it probably involves the transcriptional activation of the SAT2 gene, as the transcriptional inhibitor actinomycin D blocks this effect. The induction of SAT2 by cyclic strain is also observed in response to osmotic shock and may provide an important adaptive mechanism by elevating the intracellular levels of L-proline and other neutral amino acids during conditions of biomechanical stress.27
The ability of cyclic strain to stimulate SMC L-proline uptake is dependent on the production of TGF-β1. Consistent with earlier studies,28,29 we found that cyclic strain induces the release of TGF-β1 into the culture media in a time- and strain-degree–dependent manner. In addition, we observed that the conditioned culture media from SMCs exposed to mechanical strain stimulates the transport of L-proline. Moreover, a specific anti-TGF-β1 antibody blocked the stimulatory activity of the strain-conditioned media, indicating that the cyclic strain-mediated release of TGF-β1 is responsible for the stimulation of L-proline transport. Interestingly, the elaboration of TGF-β1 by hemodynamic forces also contributes to it effect on SMC proliferation and extracellular matrix production,28,29 suggesting an important role for this cytokine in mediating the actions of biomechanical forces. However, other SMC-derived factors are also likely to be involved as the TGF-β1 neutralizing antibody does not fully reverse the increase in L-proline transport evoked by cyclic strain.
Recent studies suggest that cyclic strain may be a critical regulator of amino acid transport and metabolism in vascular SMC. Our finding that cyclic strain stimulates the transport of L-proline complements an earlier study demonstrating that cyclic strain induces the uptake of L-arginine in vascular SMC by inducing the expression of cationic amino acid transporter-2.30 In addition, we recently found that cyclic strain directs the metabolism of L-arginine from the formation of nitric oxide to L-ornithine by blocking the expression of inducible nitric oxide synthase and inducing the expression of arginase I.30 Thus, the capacity of cyclic strain to modulate amino acid transport and metabolism may exert an important effect on vascular tone by limiting the release of nitric oxide from vascular SMCs.
The ability of cyclic strain to stimulate SAT2 gene expression may also contribute to arterial remodeling in hypertension. Mechanical strain on the artery wall is increased by up to 30% in hypertension and is postulated to play an important role in vascular injury by stimulating SMC proliferation and collagen synthesis.19,,–22,31 Thus, our finding that cyclic strain induces SAT2 gene expression may provide a mechanism by which increased levels of precursor (L-proline) are provided to SMC during periods of collagen formation. Interestingly, we recently found that cyclic strain-mediated increases in type I collagen production are associated with a significant increase in the intracellular synthesis of L-proline by vascular SMC.30 These results suggest that cyclic strain-mediated increases in both the transcellular transport of L-proline and endogenous L-proline synthesis are coordinated to maximize the cellular capacity for collagen synthesis. Moreover, these cyclic strain actions, which promote collagen synthesis, are further amplified by the cyclic strain-mediated suppression of the type I collagen-degrading enzyme matrix metalloproteinase-1.32 Because SAT2 also mediates the uptake of other neutral amino acids, the induction of SAT2 by cyclic strain may function to provide the necessary amino acids required for the synthesis of new proteins during SMC growth. Consistent with this proposal, system A activity correlates with cell growth in numerous cells and is recognized as a permissive step in the early phases of liver regeneration.7,8,33 Thus, the ability of cyclic strain to stimulate SAT2 gene expression may contribute to its fibrogenic and mitogenic actions in hypertensive vessels.
Extrapolation of our cell monolayer experiments to the three-dimensional vessel wall requires caution. The nature of mechanical deformations in the intact blood vessel are substantially more complex than our in vitro studies owing to the nonuniformity of strain across different depths of the vessel wall as well as the variability of strain across different blood vessels. Furthermore, the response of the blood vessel to mechanical stimuli depends on the integration of responses from the different cell types that encompass the vessel wall. Although cyclic strain stimulates collagen synthesis in fibroblasts, studies examining the involvement of TGF-β in mediating this response are contradictory.34,–36 Furthermore, cyclic strain appears to inhibit the production of collagen from endothelial cells.37 Thus, the effect of cyclic strain on amino acid uptake and extracellular matrix production may be cell specific. Another possible limitation in this study concerns the use of late passage SMC (up to passage 26). Late passaged cells exhibit many biochemical and functional differences relative to freshly dispersed or early passaged SMC, and this may have impacted our results. However, we failed to detect any significant change in SMC response to cyclic strain during cell passage.
In summary, our results indicate that cyclic strain stimulates the uptake of L-proline in vascular SMC by inducing the expression of the SAT2 gene. This induction in L-proline transport by cyclic strain is mediated, in part, by autocrine TGF-β1 release. The ability of high oscillatory strain to upregulate the transport of L-proline and other neutral amino acids may contribute to arterial remodeling in hypertension by providing the necessary precursors required for vascular SMC collagen synthesis and growth.
